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HANDBOOK OF ANDROLOGV The American Society of Andrology @) Copyright © April 1995, by the American Society of Andrology 74 New Montgomery, Suite 230, San Francisco, CA 94105 Phone: (415) 764-4823; Fax: (415) 764-4915 Second printing (July 1998) made possible with financial support from Pharmacia & Upjohn and Alza Pharmaceuticals. Printed by Allen Press, Inc.; 1041 New Hampshire Street, Lawrence, KS 66044 This paper meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper). ' /'· PREFACE tion for anyone interested in Andrology. The material covered is geared toward first year medical, veterinary and graduate students and will hopefully serve as a useful teaching aid. It is clearly only a brief introduction to this complex and exciting field of research. In addition to the essential contributions of the co-authors, many individuals have made the preparation of this Handbook possible. Jon Pryor and Jacquetta Trasler have done an outstanding job of bringing together a superb collection of authors, and of editing their texts in order to facilitate the reading of the volume as a whole. Sue Grant subedited the text in a highly professional manner. The dedicated secretarial support of Gail Wolfenden provided a needed degree of cohesion to this project. The American Society of Andrology is grateful to The Upjohn Company for having kindly provided essential financial support, with no restrictions, for the preparation and publication of this Handbook. With its twentieth anniversary, the American Society of Andrology celebrates a milestone in the evolution of the field of science specifically dedicated to the the male reproductive system. Tremendous strides have been made in our understanding of each component of the male reproductive system, how these components communicate with each other, and how pathological conditions arise. We have also seen significant breakthroughs in developing effective therapies for dysfunctions of the male reproductive system. In bringing together world renown experts on most facets of andrology for the creation of this introductory Handbook, the American Society of Andrology has sought to provide introductory, up-to-date knowledge of both fundamental and clinical aspects of male reproductive function. Indeed one of the hallmarks of the American Society of Andrology is the effective communication that takes place between scientists dedicated to understanding male reproduction and treating pathological conditions of male reproduction. This Handbook is designed as an introduc- Bernard Robaire Past president American Society of Andrology 1993-1994 iii List of Contributors Co-editors Bernard Robaire, Ph.D. Department of Pharmacology & Therapeutics McGill University 3655 Drummond Street Montreal, Quebec H3G 1Y6 Canada Jon L. Pryor, M.D. Department of Urologic Surgery University of Minnesota 420 Delaware Street, SE P.O. Box 394 UMHC Minneapolis, MN 55455 Jacquetta M. Trasler, M.D., Ph.D. McGill University Montreal Children's Hospital 2300 Tupper Street Montreal, Quebec H3H 1P3 Authors Nancy J. Alexander, Ph.D.* Contraceptive Development Branch Center for Population Research, NICHD 6100 Executive Blvd., Room 8B13 Bethesda, MD 20892 Rupert P. Amann, Ph.D.* Animal Reproduction and Biotechnology Laboratory Colorado State University Fort Collins, CO 80523 Andrzej Bartke, Ph.D.* Department of Physiology Southern Illinois School of Medicine Carbondale, IL 62901 Marc R. Blackman, M.D. Johns Hopkins University School of Medicine Johns Hopkins Bayview Medical Center 4940 Eastern Avenue Baltimore, MD 21224 Gabriel Bialy, Ph.D. Contraceptive Development Branch Center for Population Research, NICHD 6100 Executive Blvd., Room 8B13 Bethesda, MD 20892 Runa Brinkmann, M.D. Department of Surgery Division of Urology Clinical Science Center University of Wisconsin, Madison 600 Highland Avenue G5/344 Madison, WI 53705 Terry R. Brown, Ph.D. Department of Population Dynamics Division of Reproductive Biology Johns Hopkins University School of Hygiene and Public Health 615 N. Wolfe Street Baltimore, MD 21205 Reginald C. Bruskewitz, M.D. Department of Surgery Division of Urology Clinical Science Center University of Wisconsin, Madison 600 Highland Avenue G5/344 Madison, WI 53705 Yves Clermont, Ph.D. Department of Anatomy and Cell Biology McGill Univesity 3640 University Street Montreal, Quebec H3A 2B2 Canada v vi Handbook of Andrology-List of Contributors Donald S. Coffey, Ph.D. Johns Hopkins School of Medicine 600 N. Wolfe St. Baltimore, MD 21205 Richard A. Fayrer-Hosken, BVS, Ph.D. Department of Large Animal Medicine University of Georgia College of Veterinary Medicine Athens, GA 30602 Michael D. Griswold, Ph.D. Program in Biochemistry Washington State University Synthesis Bldg. Room 675 Pullman, WA 99164 Barbara F. Hales, Ph.D. Department of Pharmacology & Therapeutics McGill University 3655 Drummond Street Montreal, Quebec H3G 1Y6 Canada S. Mitchell Harman, M.D., Ph.D. Gerontology Research Center National Institute on Aging, NIH 4940 Eastern Avenue Baltimore, MD 21224 Louis Hermo, Ph.D. Department of Anatomy and Cell Biology McGill University 3640 University Street Montreal, Quebec H3A 2B2 Canada Barry T. Hinton, Ph.D. Department of Cell Biology University of Virginia Health Sciences Center Charlottesville, Virginia 22908 Morten Jonler, M.D. Department of Surgery Division of Urology Clinical Science Center University of Wisconsin, Madison 600 Highland Avenue G5/344 Madison, WI 53705 David F. Katz, Ph.D. Dept. of Obstetrics/Gynecology Duke University School of Medicine Durham, NC 95616 Ronald W. Lewis, M.D. Department of Surgery Division of Urology Medical College of Georgia Augusta, GA 30912-4050 Claude J. Migeon, M.D. Department of Pediatrics Johns Hopkins University School of Medicine Pediatric Endocrine Clinic CMSC 3-110 Baltimore, MD 21205 Diana G. Myles, Ph.D. Department of Physiology University of Connecticut Health Center 263 Farmington Avenue Farmington, CT 06030 Claude M. Nagamine, Ph.D. Department of Cell Biology Vanderbilt University School of Medicine Nashville, TN 37232 Joseph E. Oesterling, M.D. Department of Urology and The Michigan Prostate Center University of Michigan 1500 East Medical Center Drive Ann Arbor, Ml 48109 vii Handbook of Andro/ogy-List of Contributors Jon L. Pryor, M.D. Department of Urologic Surgery University of Minnesota 420 Delaware Street, SE P.O. Box 394 UMHC Minneapolis, MN 55455 Bernard Robaire, Ph.D.* Department of Pharmacology & Therapeutics McGill University 3655 Drummond Street Montreal, Quebec H3G 1Y6 Canada Kenneth P. Roberts, Ph.D. Department of Urologic Surgery University of Minnesota Medical School 420 Delaware Street SE Minneapolis, MN 55455 Susan A. Rathmann, Ph.D. Fertility Solutions Inc. 13000 Shaker Blvd. Cleveland, OH 44120 Peter N. Schlegel, M.D. James Buchanan Brady Foundation The New York Hospital Cornell Medical Center 525 East 68th Street New York, NY 10021 and The Population Council Center for Biomedical Research New York, NY 10021 Richard J. Sherins, M.D.* Division of Andrology Genetics & IVF Institute 3020 Javier Road Fairfax, Virginia 22031 Donald J. Tindall, Ph.D. Urology Research Mayo Clinic Guggenheim 17 200 First Street Rochester, MN 55905 Philip Troen, M.D.*+ Department of Medicine University of Pittsburgh School of Medicine 3459 Fifth Avenue Pittsburgh, PA 15213 Christina Wang, ERACP Harbor-UCLA Medical Center Clinical Study Center 1000 West Carson Street Box 16 Torrance, CA 90509 Barry R. Zirkin, Ph.D. Division of Reproductive Biology Johns Hopkins University 615 N. Wolfe Street School of Hygiene and Public Health Baltimore, MD 21205 *Past President, American Society of Andrology +Past President, International Society of Andrology "What a piece of work is a man" Shakespeare, Hamlet, Act ii, scene 2, line 316 citing breadth of andrology and the opportunity it holds for graduate students in the biological sciences, veterinary students, and medical students. Why become an andrologist? In choosing a career one, first of all, tries to identify an area of interest. Then one looks at the opportunity to succeed and the likelihood of making a contribution. As an andrologist for 40 years, I can testify to the continued excitement and interest the discipline has held for me. As noted above, there has been a rapid escalation of growth so that our discipline now ranges from genetic studies to pubertal changes in the male and from infertility and assisted reproduction techniques to disorders of the prostate, sexual function and contraception. Advances in these and other areas have been made possible by a remarkable series of clinical studies and scientific discoveries using the classical disciplines of physiology, biochemistry, neuroscience, and molecular biology. As we have entered each new stage of understanding and science, there has been no waning of the stimulus that I and my colleagues experience. At the same time, because of the multidisciplinary nature of andrology, unsolved problems present themselves and the opportunities for advancement and success continue to expand. As Alexander Albert has noted, "Nature has experimented lavishly with the reproductive system." This fact provides both challenge and opportunity. Andrology covers a wide spectrum from before conception to aging. As you peruse this handbook, we hope you will appreciate the scope of the field and share our excitement in the study of andrology. Why a handbook of andrology? Some handbooks are published to bring together multiple aspects of a diversified subject. Some handbooks are designed to present an overview of a rapidly expanding subject for those working in the field while other handbooks are intended to codify the progress already made. Although there are elements of each of these approaches in this handbook of andrology, our main purpose is to present to scientists/clinicians early in their careers the scope, importance, and excitement of our discipline. What is andrology? Simplistically, one might say that andrology is to the male what gynecology is to the female. That is, andrology deals with matters affecting the male reproductive system. The earliest use of the term andrology, as reported by Mikko Niemi, appears to have been in 1891 in the Journal of the American Medical Association, reporting on the formation of the American Andrological Association. Little more was heard from that association and it was not until the latter half of this century that there emerged an andrology journal in 1969 and an active andrology organization, Comite lnternacional de Andrologia, in 1970. In the quarter century since, there has been a veritable explosion of journals and publications, of societies and congresses, and of workshops and symposia devoted to andrology. Thus, we appear to be on a rapidly rising growth curve of knowledge and application in andrology. The scope of modern day andrology is strikingly indicated by the range of topics discussed in this handbook. Written by distinguished leaders in their fields, these topics were chosen to indicate the ex- ix Handbook of Andrology Contents PREFACE B. Robaire LIST OF CONTRIBUTORS FOREWORD P. Troen 1. What are the components of the male reproductive system? Topics: CNS, pituitary, testis, epididymis, prostate, seminal vesicles, scrotum, penis-K.P. Roberts 1 2. What is the relationship between the various endocrine components of the male reproductive system? Topics: Hypothalamo-pituitary-testicular axis, feedback loops -B.R. Zirkin 5 3. What compounds mediate communication within the male reproductive systern? Where and how are male-associated hormones produced? Topics: Gonadotropins, steroids and their sites of synthesis -M.D. Griswold 8 4. How is communication mediated within the male reproductive system? Topics: Hormone receptors, signal transduction -D.J. Tindall 10 5. How are germ cells produced and what factors control their production? Topics: Germ cell development in the testis (including mitosis, meiosis, spermiogenesis), Sertoli cells, other cell types -L. Hermo, Y. Clermont 13 6. What are the unique chromosomal events leading to the formation of a haploid male germ cell? Topics: Stages of meiosis, chromosomal events, genetic recombination -J.L. Pryor 16 7. What does the epididymis do and how does it do it? Topics: Physiology, sperm maturation -B.T. Hinton 18 8. What is the prostate and what is its function? Topics: Physiology, function (anatomy, embryology) -D. Coffey 21 9. What is semen? How does semen analysis assist in understanding the reproductive status of the male? Topics: Semen composition and analysis (animal, human), related tests -R.P. Amann, D.F. Katz, C. Wang 25 10. What is sperm banking? When and how is it (or should it be) used in humans? Animals? Topics: Sperm banking, consequences of its use in animal and clinical practice -S.A. Rathmann 31 xi xii Handbook of Andrology-Contents 11. How does the spermatozoon make its way to the egg and how does fertilization take place? Topics: Capacitation, acrosome reaction, zona binding -D.G. Myles 35 12. What factors determine the sex of an individual? Topics: X, Y, SRY (loci, genes), sequence of events in development of normal male-C.M. Nagamine 38 13. Are there specific genetic defects affecting the male reproductive tract? What are the underlying molecular mechanisms? Topics: Androgen insensitivity, Turner's and Klinefelter's syndrome, chromosomes, gene loci -T.R. Brown 40 14. Is there a trigger for puberty in the male? Should early or delayed puberty be treated? If so, how? Topics: Early, normal, delayed puberty, treatment -C.J. Migeon 46 15. How is male infertility defined? How is it diagnosed? Topics: Epidemiology, causes, work-up (history, physical, lab tests) -R.J. Sherins 48 16. What are the existing and future therapeutic approaches for male infertility? When should IVF be used for male infertility? What is the role for psychological counselling for infertility? Topics: Treatment -medical, empirical, surgical, alternative, adoption, donor, psychological -P. Schlegel 52 17. How is fertility assessed in domestic animals? Topics: Infertility diagnosis in the different species, evaluation of the male for clinical management -R. Fayrer-Hosken, R. Amann. 56 18. What are the existing male contraceptives and what is the outlook for new ones? Topics: Androgens, GnRH antagonists, antibodies to sperm surface antigens, compounds that act on sperm maturation in the epididymis -N.J. Alexander, G. Bialy 60 19. How prevalent is erectile dysfunction? What can be done to treat it? Topics: Erectile physiology, etiology, work-up and treatment of erectile dysfunction, psychological counselling -R.W. Lewis 63 20. Can spermatozoa be targets for drugs? If so, what are the consequences of such drug exposure? Is there a need for pre-conception counselling for men? Topics: Drugs that affect sperm structure or function, male-mediated developmental toxicity, prevention, tests to detect damage to spermatozoa -B. Robaire, B. Hales 68 21. Do environmental factors affect male reproductive functions? If so, which ones and how? Topics: Season, length of day and chemical exposure effects on the male -A. Bartke 70 Handbook of Andrology-Contents xiii 22. Is there an andropause, the analog to menopause, and if so what tissues are affected and how? Topics: Fertility, androgen production and sensitivity, and sexual function in aging men -S.M. Harman, M.R. Blackman 72 23. What is BPH? Why is it so prevalent? What treatments are available? Topics: Pathophysiology, treatment -J. Oesterling 76 24. Are some men more susceptible to prostate cancer than others and why? What are the treatments and their effectiveness? What are the possibilities for improvements in therapy? Topics: Pathophysiology, present and future treatments -R. Brinkmann, M. Jonler, R.C. Bruskewitz 80 Copyright © American Society of Andrology What are the components of the male reproductive system? CNS, pituitary, testis, epididymis, prostate, seminal vesicles, scrotum, penis The male reproductive system consists of a number of individual organs acting together to produce functional spermatozoa, and to deliver these spermatozoa to the female reproductive tract. The haploid germ cells originate in the testis and continue their maturation as they transit the epididymis. The vas deferens carries the spermatozoa from the epididymis to the ampulla, provides a site for the mixing of seminal vesicle secretions, and continues as the ejaculatory duct through the prostate, emptying into the prostatic urethra (Fig. 1). The germ cells, mixed with ejaculatory secretions from the accessory sex glands (seminal vesicles, prostate, bulbourethral gland), then exit the body through the penile urethra. The entire system is dependent upon neuro-endocrine regulation from the pituitary and hypothalamus. Knowledge of the anatomy and embryological origins of each of the components of the male reproductive tract is important in developing a basic and thorough understanding of the system as a whole. Although the discussion of male reproductive anatomy and embryology in this chapter is confined to the human system, subsequent chapters will show that much of our understanding of reproductive biology has been gained from research using various experimental animal models. Testis The testis is central to the male reproductive system. It is the organ which generates the haploid germ cell by the process of spermatogenesis and it is the site of androgen production. The testis arises from the primitive gonad on the medial surface of the embryonic mesonephros. Primitive germ cells, which migrate to this region from the yolk sac, cause the coelomic epithelial cells to proliferate and form the sex cords. Formation of the sex cords gives this region a raised contour that is called the genital ridge. By the seventh week of fetal de- velopment, proliferation of the mesenchyme has separated the sex cords from the underlying coelomic epithelium. During the fourth month, the sex cords become U-shaped and their ends anastomose to form the rete testis (Fig. 2A). At this point, the primordial sex cells are referred to as pre-spermatogonia and the epithelial cells of the sex cords as Sertoli cells. The mesenchymal tissue in the interstitial space between the tubules gives rise to the Leydig cells that are the site of androgen production. The rete testis extends into the mesonephric tissue and will anastomose with some of the mesonephric tubules forming the efferent ducts which communicate with the epididymis (discussed below). The sex cords will become the seminiferous tubules, although the tubules do not develop a lumen until after birth. The testis develops abdominally and successful descent into the scrotum is essential for fertility. The scrotum is formed as coelomic epithelium penetrates the abdominal wall and protrudes into the genital swelling as the processus vaginalis. An outgrowth of each layer of the abdominal wall is carried with this epithelium, giving rise to the fascial layers of the scrotum. The testis then descends behind the processus vaginalis and the layers of fascia covering the testis on each side, with the overlying skin of the genital swelling, fuse to form the scrotum. Epididymis The epididymis, vas deferens and seminal vesicles have their origin in the mesonephric duct (or Wolffian duct). Initially formed as the early embryonic excretory system, the mesonephric system is comprised of the longitudinal duct and a series of tubules that branch from the duct toward the developing gonad. Although most will degenerate, several of these tubules persist and anastomose with the confluence of the seminiferous tubules (rete 2 Handbook of Andrology-What are the components of the male reproductive system? testrs B FIG. 2. A. The immature testis and mesonephric duct system in the fourth month of development. B. The immature mesonephric duct system after testicular descent. (From: Langman J. Medical Embryology. Baltimore. MD: Williams & Wilkins; 1969). Vas Deferens FIG. 1. The organs of the male reproductive system. (From: Kiss & Szentagothai. Atlas of Human Anatomy, Oxford, England: Pergamon Press; 1964.) testis), forming the efferent ducts (or ductuli efferentes) through which spermatozoa exit the testis (Fig. 28). The portion of the mesonephric duct closest to the ductuli efferentes elongates, becomes extensively convoluted, and forms the epididymis (Fig. 28). Because it arises from a single duct, the epididymis, unlike the testis, consists of a single tubule through which all spermatozoa must pass. The epididymis remains in close contact with the testis and descends with the testis into the scrotum. Testicular spermatozoa are non-motile and incapable of fertilization . The function of the epididymis is to bring testicular spermatozoa to maturity. How this maturation process is accomplished by the epididymis is currently unknown but is an area of active research in reproductive biology. It is known that the epididymis secretes proteins that become part of the surface architecture of the mature spermatozoa and presumably are important in postejaculatory function of the spermatozoa. That portion of the mesonephric duct extending from the caudal end of the epididymis to the seminal vesicles (discussed below) becomes thickened and muscular and forms the vas deferens (or ductus deferens). That portion of the duct which continues distal to the seminal vesicle is known as the ejaculatory duct and is contained entirely within the prostate gland. In its course the vas deferens ascends from the scrotum, with the vessels that vascularize the testis and epididymis, through the inguinal canal, over the pubic ramus, over the superior lateral aspect of the bladder medial to the ureter, and enters the posterior superior aspect of the prostate, just distal to the seminal vesicle (Fig. 1). The primary function of the vas deferens and ejaculatory duct is transport of mature spermatozoa and seminal vesicle secretions to the prostatic urethra. Seminal Vesicles The seminal vesicle develops as an outpocketing of the mesonephric duct, distal to the epididymis (Fig. 28). Consequently, this gland shares a common embryological origin with the epididymis and vas deferens. The fully developed seminal vesicle resides immediately above the prostate gland (Fig. 1). It is comprised of a series of tubular alveoli, lined with a very active secretory epithelium. In fact, • Handbook of Andrology-What are the components of the male reproductive system? the seminal vesicle contributes the majority of the fluid volume of the ejaculate. Seminal vesicle secretions are rich in fructose and prostaglandins. While fructose may be an important energy source for spermatozoa, the role of prostaglandins is unknown. The seminal vesicle also produces several androgen-dependent secretory proteins that are involved in the rapid clotting of the ejaculate. Prostate The prostate gland is located in the space below the bladder and above the urogenital diaphragm (Fig. 1). It is separated posteriorly from the rectum by the rectovesical (Denonvillier's) fascia. Its location immediately anterior to the rectum allows the prostate to be palpated and biopsied from the rectum. The prostate arises from several distinct sets of tubules that evaginate from the primitive posterior urethra. Each set of tubules develops into a separate lobe: the right and left lateral lobes, which are the largest, the middle lobe, and the very small anterior and posterior lobes. The lobes are composed of alveoli, lined with a secretory epithelium, that drain through a series of converging tubules into the prostatic urethra. Although the lobes arise independently, they are continuous in the adult with no apparent gross or morphologic distinctions. Consequently, a more useful subdivision of the prostate has been recently developed which distinguishes prostatic zones based on morphologic and functional properties (i.e., central, peripheral and transitional zones). Prostatic secretions contribute to the fluid volume of the ejaculate. These secretions are high in zinc, citric acid and choline. The function of these substances is unknown, although an anti-microbial activity for zinc has been postulated. The prostate also secretes several proteins including acid phosphatase, seminin, plasminogen activator and prostate specific antigen (PSA). The exact roles of most prostatic secretions are unknown, although they are presumed important for the function of spermatozoa during and after ejaculation. For instance, plasminogen activator and seminin are proteases involved in the liquification of 3 coagulated ejaculate. Although the function of PSA is not known, an elevated level of this protein in the blood is often diagnostic of abnormal prostatic growth such as occurs with cancer of the prostate. Penis The penis arises from the genital tubercle, a region just cranial to the cloacal folds in the embryo. Under the influence of androgens produced by the fetal testis, the cells of the genital tubercle proliferate causing elongation of the tubercle into the primitive phallus. The penile urethra is formed from the urethral folds as the phallus elongates. In the adult penis the urethra is divided into the membranous portion, which extends through the urogenital diaphragm, and the pendulous portion, which courses through the penis. Lateral to the urethra are the two corporal bodies, the corpus cavernosi, which become engorged with blood to produce the penile erection. The physiology of erection is complex and is subject to a number of clinical disorders. The importance of proper erectile function to sex and reproduction, and the common occurrence of erectile dysfunction (affecting 10-20 million men in the United States), has made erectile dysfunction a primary clinical concern in andrology. Endocrine and nervous control of the male reproductive tract The entire male reproductive tract is dependent on hormones for proper function. The pituitary produces the gonadotropins, folliclestimulating hormone (FSH) and luteinizing hormone (LH), under the control of the hypothalamus. FSH is required for spermatogenesis and LH stimulates androgen production by the testicular Leydig cells. The testis requires testosterone to maintain the process of spermatogenesis and the accessory organs are dependent on androgen for proper secretory function. The production of LH is regulated by feedback inhibition of circulating testosterone on the pituitary and hypothalamus. FSH secretion is regulated by inhibin, a peptide hormone produced by Sertoli cells, and also by 4 Handbook of Andrology-What are the components of the male reproductive system? circulating testosterone. This endocrine loop is known as the hypothalamic-pituitary-testicular axis. In addition to hormonal control, the reproductive organs are also subject to sympathetic and parasympathetic nervous control. This is particularly true for the erectile function of the penis, which is under parasympathetic control, and for ejaculation, which is under sympathetic control. Conclusion This brief introduction to the male reproductive tract demonstrates the integrated nature of the system. The seminiferous tubules are continuous with the penile urethra via the vas deferens, with the accessory organs contributing their secretions along this course. The entire system is maintained by androgens, secreted by the testis under the control of the pituitary and hypothalamus. It is important to remember that many of these structures are embryologically distinct and, consequently, that developmental abnormalities will affect these structures in different ways. Knowing the embryology and anatomy of the tract will help the student of male reproductive medicine to understand the common, and the notso-common, disorders encountered in the clinic. Suggested Reading McNeal JE. The Prostate Gland: Morphology & Pathology. In: Stamey TA, ed. Monographs in Urology, Vol 9. Princeton: Medical Directions Publishing; 1988:36-54. Langman J. Medical Embryology. Baltimore: Williams & Wilkins; 1975:160-200. Tanagho EA. Anatomy of the Lower Urinary Tract. In: Walsh PC, Retik AB, Stamey TA, Vaughan ED, Jr, eds. Campbell's Urology. Philadelphia: WB Saunders; 1992:54-69. Copyright © American Society of Andro/ogy What is the relationship between the various endocrine components of the male reproductive system? Hypothalamo-pituitary-testicular axis, feedback loops Integration of the hypothalamus, pituitary and testis is of critical importance to reproductive function. The hypothalamus comprises the lateral walls of the lower part of the third ventricle of the brain. The pituitary, an endocrine gland connected to the hypothalamus at the base of the brain (Fig. 1), is divided into two major parts: the neurohypophysis (or posterior lobe), and the adenohypophysis (or anterior lobe) (Fig. 1). The neurohypophysis, which is composed of the median eminence, infundibular stem and infundibular process, receives neural input from the brain. In contrast, the adenohypophysis, composed of the pars tuberalis, pars intermedia and pars distalis, is glandular tissue and, thus, must be regulated by factors delivered via the circulation. Some of the neurosecretory neurons from the hypothalamus send their axons down the neural stalk to terminate in the neurohypophysis (Fig. 1). When stimulated to depolarize, these neurons release the hormones vasopressin (also called antidiuretic hormone) and oxytocin from secretory granules into the bloodstream. Oxytocin causes contraction of smooth muscle, including that in the male reproductive tract, and vasopressin acts in the kidneys to cause water retention. In contrast to the neurohypophysis, the adenohypophysis is regulated by peptides and monoamines that are synthesized and secreted by specific hypothalamic neurons whose axons end, not in the adenohypophysis, but in the median eminence near the infundibular stem (Fig. 1). These hormones (hypophysiotropic hormones) are transported by the portal blood system of the pituitary from the median eminence to the adenohypophysis where they stimulate the synthesis and secretion of the adenohypophysial hormones. There are three general classes of adenohypophysical hormones synthesized and secreted by the pars distalis of the adenohy- pophysis: glycoprotein hormones, corticotropin-related peptides and somatomammotropin hormones. The glycoprotein hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), have well established effects on the testis; LH stimulates the secretion of testosterone by the Leydig cells, and FSH acts on the seminiferous tubules to promote spermatogenesis. The synthesis and secretion of LH and FSH are regulated, in part, by a decapeptide from the hypothalamus, gonadotropin-releasing hormone (GnRH). When administered to humans or to laboratory mammals, GnRH causes LH, and to a lesser extent FSH, to be secreted into the blood. Based on the known structure of GnRH, a number of analogs have been synthesized, some of which are far more potent than natural GnRH and are able to increase LH and FSH secretion when administered. GnRH antagonists or antisera block the effects of endogenous GnRH thereby reducing gonadotropin levels. LH is secreted in distinct, short-term pulses in response to pulsatile release of GnRH (Fig. 2). The importance of pulsatile secretion of GnRH is clearly illustrated by the effect of intermittent versus continuous GnRH administration on the secretion of LH and FSH in GnRH-deficient hypogonadal men; LH and FSH can be restored to normal levels in the serum when GnRH is administered intermittently, but not when it is administered continuously. Indeed, sustained hormonal signals can shut down, rather than stimulate, target cells. The episodic LH signals that are delivered to the testis via the testicular vascular system stimulate the synthesis and secretion of testosterone by Leydig cells. As is the case for LH, testosterone is secreted in pulses (Fig. 2). Simple, one-to-one, relationships between LH pulses from the adenohypophysis and testosterone pulses from the Leydig cell are often 5 6 Handbook of Andrology-What is the relationship between the various endocrine components of the male reproductive system? e 3 10.0 :§, :z: 7.5 ...J 5 .0 2 .5 '5 1 500 , . . - - - - - - - - - . . , . . - - - - - - - . ..... 0 .s (I) ..g 1000 (I) Ill -: ..... .. 0 500 E ;;, 0 -·()Of FIG. 1. The hypothalamus and pituitary gland. (From: © 1995 by Ciba. Reprinted with permission from Ciba Publications, illustrations by F.H. Netter, M.D. All rights reserved.) seen, although there are reports that LH pulses are not necessarily followed or preceded by a testosterone pulse. Interestingly, maximal steroidogenesis occurs at concentrations of LH that are sufficient to occupy only a small fraction of the total number of LH receptors available on the surface of Leydig cells. The significance of this relative overabundance of receptors is unclear. Fig. 3 shows testosterone production during the lifetime of the human male. Peaks of testosterone occur in the peripheral blood of the 12-18 week-old fetus, and of the 2 month-old neonate. In the prepubertal period, testosterone declines to low levels. It then increases markedly during puberty (ages 12 to 17 years) , and reaches its maximum during the second and third decades of adult life. Slow decline then occurs through the fifth decade, with more dramatic decline thereafter. Superimposed on these episodes are annual, daily and hourly fluctuations in testosterone production. ~ 0~-T~~T-~~~~T-~~~ 0 500 1000 1500 2000 2500 Time (min) FIG. 2. Profiles of serum LH and testosterone in blood samples from a healthy man drawn at 1aminute intervals. (From: Veldhuis et al, J Clin Endocrinol Metab 1987; 65:929-941.) ...... B - ~ ~' : & 600 400 I:P oe 't • I I I I I I I o I l I I ADULT ' • a 1:) '-.........! I SENESCE~ FIG. 3. Testosterone concentrations in the peripheral blood of the human male at different times of the life cycle. (From: Greep RO, ed. International Review of Physiology, Volume 22. Baltimore: University Park Press; 1980:41.) Handbook of Andrology-What is the relationship between the various endocrine components of the male reproductive system? In addition to regulation by hypothalamic Gn RH pulses, LH and, to a lesser extent, FSH are regulated by the negative feedback effects of the steroid hormones produced by the testis. Testosterone, for example, negatively feeds back at the level of the hypothalamus, slowing the GnRH pulse generator and thereby inhibiting pituitary LH pulses. Additionally, the testis is capable of metabolizing testosterone to estradiol via aromatase activity in the seminiferous tubules and interstitium. Estradiol, when present in physiological concentrations, is also able to dampen the frequency and amplitude of episodic LH release. The effect of this negative feedback is apparent in 7 men following castration; the loss of testicular steroids results in markedly increased secretion of both LH and FSH. When these men are given exogenous testosterone, LH levels in the blood diminish, and LH pulsatility returns to normal. The importance of the integration of the hypothalamic-pituitary-testicular axis is obvious in light of the critical roles of LH , FSH and testosterone in spermatogenesis. Testosterone, regulated by LH , is an absolute requirement for normal spermatogenesis. FSH plays a significant role in the initiation of spermatogenesis at puberty but its role in the adult is less certain. Suggested Reading Everett JW. Pituitary and hypothalamus: Perspective and overview. In: Knobil E and Neill JD, eds. The Physiology of Reproduction, Second Edition, New York: Raven Press, ltd.; 1994: 1509-1526. Bremner WJ, Bagatell CJ , Christensen RB and Matsumoto AM. Neuroendocrine aspects of the control of gonadotropin secretion in men. In: Whitcomb RW and Zirkin BR, eds. Understanding Male Infertility: Basic and Clinical Aspect. New York: Raven Press; 1993:29-41 . Hall PF. Testicular steroid synthesis: Organization and regulation. In: Knobil E and Nelli JD, eds. The Physiology of Reproduction, Second Edition New York: Raven Press, ltd.; 1994: 1335-1362. Sharpe AM. Regulation of spermatogenesis. In: Knobil E and Neill JD, eds. The Physiology of Reproduction, Second Edition, New York: Raven Press, Inc.; 1994:1363-1434. Copyright © American Society of Andro/ogy What compounds mediate communication within the male reproductive system? Where and how are male-associated hormones produced? Gonadotropins, steroids and their sites of synthesis receptor stimulates an intracellular signal transduction and amplification system that results in a biochemical change within the target cell. Receptors for FSH , LH and thyroid stimulating hormone constitute a closely related subfamily of G-protein coupled receptors that is distinguished by a relatively large external domain. Structurally, G-protein coupled receptors are characterized by a region of hydrophobic helices which span the membrane seven times and anchor the external portion of the protein to the plasma membrane where it can interact with its ligand. In the testis, Sertoli Production of gonadotropins Gonadal regulation begins in the hypothalamus which synthesizes and releases, in a pulsatile manner, the decapeptlde gonadotropin-releasing hormone (GnRH)(Fig. 1). GnRH acts directly on the gonadotrophs which are the specific cells in the anterior pituitary that synthesize and secrete the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH and LH are glycoproteins which share structural similarity with thyroid-stimulating hormone and chorionic gonadotropin and which are the primary hormonal mediators of testicular function. These glycoproteins are comprised of two non-covalently linked polypeptides. One pro- GnRH tein, the a-subunit, is common to both of these hormones while the [3-subunit is unique to each. Formation of the a-[3 heterodimer within the endoplasmic reticulum of the gonadotrophs is essential for the hormonal activity of the gonadotropins. Synthesis and release of the gonadotropins within the pituitary involves a complex regulatory process. Individual gonadotrophs are capable of secreting either FSH or LH , or both FSH and LH. LH is known to be released in a pulsatile fashion as a result of the pulsatile stimulus of GnRH arriving from the hypothalamus. FSH is released with less frequent and more irregular pulses that have smaller amplitudes. In some species, especially those with seasonal variations in spermatogenesis, another pituitary hormone, prolactin, may play a role in stimulating the reinitiation of spermatogenesis. -- Paracrlneo Factors Germ cells FIG 1. Components of the normal pituitary-testis axis. GnRH acts on the pituitary to stimulate the release of the gonadotropins. In this figure the testis is represented by the large oval. LH acts on the Leydig cells to stimulate testosterone (T) synthesis and secretion while FSH acts on Sertoli cells. T stimulates spermatogenesis through actions on peritubular and Sertoli cells and inhibits LH release through a classical feed-back pathway. lnhibin can negatively regulate FSH release from the pituitary while the development of germ cells to sperm is a result of the biological activities of the Sertoli cells. Sertoli cells and germ cells communicate through a number of paracrine factors. Transduction of the LH/FSH signal Glycoprotein hormones such as FSH and LH elicit responses in target cells by interacting with specific receptor proteins on the plasma membrane. Binding of the hormone to its 8 Handbook of Andro/ogy-What compounds mediate communication within the male reproductive system? cells have the membrane receptors that make them the target cells for the action of FSH. LH binds to membrane receptors on Leydig cells and stimulates the production of testosterone. The G-proteins are a large family of membrane-associated intracellular proteins that transduce the initial signal (hormone binding to receptor) into a biochemical event such as the production of cAMP and the subsequent stimulation of protein phosphorylation through kinases. Most of the changes in cellular activities that occur because of the actions of the gonadotropins are the result of phosphorylation of specific proteins. Sites of action of gonadotropins LH-stimulated Leydig cells convert cholesterol to testosterone which subsequently accumulates in the interstitium and the seminiferous tubules at relatively high concentrations. Extracellular androgens are bound to related carrier proteins such as androgen binding pro- 9 tein (ASP) produced by the Sertoli cells or testosterone binding globulin (TeBG) produced by the liver. The adrenal also produces androgens; however, the contribution of adrenal steroids to testicular function in normal males is negligible. The target cells for testosterone within the testis are the peritubular myoid cells and the Sertoli cells. There is good evidence that the germinal cells do not respond directly to androgens. In some species, a portion of the testosterone may be converted to estrogens by Sertoli or germ cells. The estrogens then feed back to reduce LH stimulation of testosterone biosynthesis by Leydig cells. FSH stimulates a variety of functions in Sertoli cells including the synthesis of secreted proteins, like transferrin, that are involved in the transfer of nutrients to germ cells. In response to FSH action Sertoli cells also produce inhibin which, along with testosterone, is involved in feedback regulation of pituitary function. lnhibin greatly reduces the release of FSH while testosterone inhibits the secretion of LH. Suggested Reading Sinha Hikim AP, Amador A, Klemcke H, Bartke A, Russell LD. Correlative morphology and endocrinology of Sertoli cells in hamster testes in active and inactive states of spermatogenesis. Endocrinology 1989;125:1829-1843. Griswold MD. Action of FSH on mammalian Sertoli cells. In: Griswold MD, Russell LD, ed. The Sertoli Cell. Clearwater, FL: Cache River Press; 1993:493-508. Salesse R., Remy JJ, M. LJ, Jallal 8, Garnier J. Towards understanding the glycoprotein hormone receptors. Biochemie 1992;73(1):109-120. Fritz I. Sites of actions of androgens and follicle stimulating hormone on cells of the seminiferous tubule. In: Litwack G, ed. Biochemical Actions of Hormones. New York: Academic Press; 1978:249-278. Cooke BA, Choi MC, Dirami G, Lopez-Ruiz MP, West AP. Control of steroidogenesis in Leydig cells. J Steroid Biochem Mol Biol1992;43(5):445-449. Copyright © American Society of Andrology How is communication mediated within the male reproductive system? Hormone receptors, signal transduction Androgenic steroids are essential for the development and maintenance of the male phenotype. Testosterone, the predominant androgenic steroid hormone, is produced in testicular Leydig cells under the control of the glycoprotein hormone, luteinizing hormone (LH). Testosterone may be metabolized to the more active androgen, 5a-dihydrotestosterone (5aDHT) or to 17[3-estradiol. Both of these metabolites play important roles in reproductive physiology: testosterone and 5a-DHT are essential for differentiation of the accessory sex organs and external genitalia; whereas 17[3estradiol is critical for differentiation of sexual dimorphic nuclei in the brain. In this review we will focus on the mechanism of action of 5aDHT in the male reproductive tract. have been demonstrated to be regulated at the transcriptional level by androgens in vitro. These include the human prostate-specific genes PSA and hKLK2, the rat prostate-specific genes probasin and the C subunit of prostatein, and the rat liver-specific gene sip. Each of these has been shown to contain a functional ARE in either the promoter or intronic regions. Evidence for post-transcriptional regulation is also available for four secretory proteins (SVP 1-4) that are the major constituents of the seminal fluid. The AR gene itself is regulated by androgens, although the exact mechanism has not been defined. In the majority of cases, steadystate levels of AR mRNA in androgen target tissues are increased following castration and decreased with androgen treatment. Moreover, changes in AR protein levels often parallel the response of mRNA to androgen availability . Recent evidence also suggests that transcription of the AR gene is modulated by agents acting through second messenger pathways such as cAMP. Androgen Receptor The biological functions of androgens are mediated through the androgen receptor (AR) protein (Fig. 1). This protein binds both testosterone and 5a-DHT, although it has a much higher affinity for the latter. Once the steroid enters the cell and binds to the AR there is a conformational change in this protein, that causes the dissociation of several accessory proteins from the AR, including heat shock proteins. This conformational change exposes the DNA binding domain on the AR, which can then interact with specific sequences of DNA termed androgen responsive elements (AREs). AREs have been identified in the promoter and intronic regions of androgen responsive genes. Binding of the AR to an ARE has the functional property of regulating transcriptional activity of the gene. Pathological actions of androgens If androgens are absent or their normal route of action is blocked, then severe deficits are observed in the male physiological state. Androgen insensitivity syndrome (AIS) is caused by functional defects in the AR protein stemming from mutations affecting predominantly the steroid-binding domain or the DNAbinding domain of the AR gene. Complete AIS is distinguished by an XIY genotypic individual with abdominal testes, absence of Wolffian derivatives and male levels of serum testosterone, who has female phenotypic characteristics including normal breast development, a blind-ending vagina and a paucity of pubic hair. This mutation emphasizes the critical necessity of a normally functioning AR in male reproductive development. Androgen Regulated Genes Although the expression of many mRNAs and proteins have been described as being under androgenic control, only a few genes 10 Handbook of Andro/ogy-How is communication mediated within the male reproductive system? 11 Mechanism of Androgen Action •······················································································ I : ~C&I'l...-:s.a::lphao~HuTc:e- 1 Cytoplasm - - - - - - 1~ I - - - - - - - Nucleus ""DHT de novo AR synthesis ARE - mRNA I ------------ -ProteiJ ~-----------~----•••••••••••••••••••••••••••••••••••••••••••••••••e•••••••••••••••••••• FIG. 1. Mechanism of androgen action. Testosterone (T) enters the cell and is converted by 5 alpha reductase to 5a-dihydrotestosterone (DHT). DHT binds to the androgen receptor (AR) leading to a conformational change in the protein and the dissociation of several accessory proteins, including heatshock proteins (HSP). Binding of the AR to the androgen response element (ARE), along with other transcription factors (TF) regulates the transcription of mRNAs. (From: Lindzey J, Kumar MV, Grossmann M, Young C, Tindall DJ. Molecular Mechanisms of Androgen Action. In: Litwack G, ed. Vitamins and Hormones. Orlando, Florida: Academic Press, Inc.; 1995;49:383-432.) Sa-Reductase deficiency is caused by a mutation in the enzyme Sa-reductase, which is responsible for converting testosterone to Sa-DHT. The absence of Sa-DHT in an X/Y genotypic individual results in a female phenotype until puberty at which time there is growth of the phallus into a penis-like organ. This phenotypic change is thought to be the result of increased availability of testosterone to bind to the AR, albeit at reduced affinity. Although prostate cancer and benign prostate hyperplasia (BPH) are both associated with androgenic influences on the prostate, there is no evidence that androgens are directly responsible for these conditions. It is likely that androgens play a supportive role in maintaining prostatic cells in such a state that environmental, dietary or genetic insults could induce mutations of key genes such as protooncogenes or tumor suppressor genes. Thus, androgens appear to be necessary but not sufficient for these pathologic conditions. Summary The development and maintenance of the male phenotype is highly dependent upon the presence of the male sex steroid testosterone and its conversion to the more active androgen Sa-DHT. Sa-DHT interacts with the androgen receptor and regulates transcriptional ac- 12 Handbook of Andrology-How is communication mediated within the male reproductive system? tivity of androgen responsive genes through an ARE sequence. This sequence of events regulates the synthesis of proteins which are critical for development of the male accessory sex tissues and for growth of the external genitalia. Suggested Reading Chan L, Johnson MP, Tindall, OJ. Steroid Hormone Action. In: Collu R, Ducharme JR, Guyda H, eds. Pediatric Endocrinology. New York: Raven Press; 1989:81-124. Griffin JE, Wilson JD. Disorders of the Testes and the Male Reproductive Tract. In: Wilson JD, Foster OW, eds. Williams Textbook of Endocrinology. Philadelphia: W.B. Saunders Co.; 1992:799-852. Lindzey JK, Grossmann, ME, Kumar MV, Tindall OJ. Regulation of the 5'-flanking region of the mouse androgen receptor gene by cAMP and androgen. Molecular Endocrinology 1993; 7:1530-1540. Lindzey J, Kumar MV, Grossmann M, Young C, Tindall OJ. Molecular Mechanisms of Androgen Action. In: Litwack G, ed. Vitamins and Hormones. Orlando, Florida: Academic Press, Inc.; 1995;49:383-432. Murtha P, Tindall OJ, Young CY-F. Androgen induction of human prostate-specific kallikrein: Characterization of an androgen response element in the 5'-promoter region of the gene. Biochemistry 1993; 32:6459-6464. Copyright © American Society of Andro/ogy How are germ cells produced and what factors control their production? Germ cell development in the testis (including mitosis, meiosis, spermiogenesis), Sertoli cells, other cell types Spermatogenesis is an elaborate process of cell differentiation starting with a non-differentiated spermatogonial germinal stem cell and terminating with a fully differentiated highly specialized motile cell called a spermatozoon (Fig. 1). The formation of spermatozoa takes place within narrow coiled seminiferous tubules which form the bulk of the testis. Each seminiferous tubule, approximately half a millimeter in diameter, may be close to one meter in length. These tubules have a central fluidfilled lumen and a wall called the seminiferous epithelium composed of germinal cells and of somatic cells, the Sertoli cells, which support and nourish the germinal·cells. Spermatogenesis may be subdivided into three main phases, each involving a class of germinal cells. First phase: The spermatogonia are immature germinal cells located at the base of the seminiferous epithelium. In man, there are three types of spermatogonia: the pale type A spermatogonia or Ap, the dark type A spermatogonia or Ad, and the type B spermatogonia. The Ap spermatogonia divide by mitosis and give rise either to new type Ap cells or to the more differentiated type B spermatogonia. Thus, the Ap cells may be thought of as self-renewing stem cells since they can produce both new Ap stem cells and a new class of type B spermatogonia. The Ad spermatogonia, which rarely divide in normal adults, are tentatively considered as dormant reserve stem cells. The type B spermatogonia produced by the Ap cells all divide by mitosis to yield differentiated spermatocytes. Thus, the spermatogonial population not only maintains itself, but continuously yields crops of spermatocytes. Second Phase: Spermatocytes are cells which are unique in undergoing two successive special cell divisions, the so-called reduc- tional or meiotic divisions, that produce, the spermatids. These cells have exactly half the number of chromosomes contained by the nuclei of cells that compose the rest of the body. Spermatids are said to be haploid while somatic cells are diploid. In man, somatic cells contain 46 chromosomes and spermatids and spermatozoa contain 23 chromosomes. The fusion of an haploid spermatozoon with an equally haploid ovum restores the diploid number of chromosomes in the cells of the embryo. Because there are two meiotic divisions, there are two generations of spermatocytes: primary and secondary. At an early or preleptotene stage, the nuclei of primary spermatocytes replicate their DNA content. Fine filamentous chromosomes subsequently appear in the nucleus and the cells are at the leptotene stage. Soon after, homologous chromosomal filaments approximate each other and form close pairs, a phenomenon called synapsis, and the cell is at the zygotene stage. Then each chromosomal pair shortens and thickens and the chromosomes assume the pachytene configuration. The spermatocytes go through an early, mid and late pachytene stage during which the cell and its nucleus progressively increase in volume. The pachytene nucleus also has a prominent nucleolus indicating that these nuclei are actively synthesizing ribosomal RNA which enters the cytoplasm and contributes to the active protein synthesis observed in these cells. Following the long pachytene stage, the primary spermatocytes rapidly complete their first meiotic division going through metaphase, anaphase and telophase during which the homologous chromosomes separate and migrate to the poles of the cell which then splits to form two daughter cells called secondary spermatocytes. These cells undergo a second matura13 14 Handlx>Ok of Andrology-How are germ cells produced and whallacJOtS ccnlrollheir prrxJIJCtlon? lion division after a short interphase which, this time, Is not accompanied by DNA repli· cation. During this division, the chromosomes (of which there is an haploid number) split in half and each half reaches the nucleus of the daughter cells, which are now referred to as the spermallds. Thus spermatocytes, through complex regulatory mechanisms and elabo· rate cell division processes. are converted to haploid spermatids. This process of meiosis is covered in more detail in the following chapter. Third phase: The newly formed spermatid, a small spheroidal cell, undergoes a dramatic metamorphosis referred to as spermiogenesis. The nucleus progressively elongates as its chromatin condenses, and gradually takes on the flaHened and pointed paddle shape that characterizes the head of human spermatozoa. The Golgi apparatus elaborates a secretory·fike granule which gradually grows to produce a cap·like structure, the acrosome. over the nuclear membrane. This structure con· tains the hydrolytic enzymes necessary for the fertilization of the ovum and partly covers the nucleus of the spermatozoon. The centrioles reach the membrane of the nucleus at the pole opposite to that occupied by the acrosome. bind to It and initiate the formation of the con· tractlle components of the tall, I.e. the microtubules that form the axoneme. Tha mitochon· dria migrate toward a segment of the growing tall and form the mitochondrial sheath, whloh constitutes the respiratory organ of the sper· matoloon. The spermatid bulk of cytoplasm is eventually discarded as the residual body, which Is phagocytosed and eliminated from the seminiferous epithelium by the Sertoll cell. Thus. the spermatozoon Is a streamlined cell 60 ~~om long with a head and a tail com· pletely encased in a cell membrane. The head is composed of a small compact nucleus cov· ered by the acrosome. The tail Is made up of the contractile axoneme associated with other complex cytoskeletal elements and is parHy covered by mitochondria. This cell will contin· ue to develop and mature during Us transit through the epididymis. FIG. 1. The various steps of spermatogenesis in man. Labels !rom the top to the bottom: Ad, dark type A spermatogonium; />9, pale type A spermat· ogonium: 8. type 8 spermatogonium; M. m1toses of Ap 01 8 spermatogonia; PI , preleptotene primal)' spermatocyte&: L, Z. EP. MP and LM correspond to the various stages ol the prophase ol primal)' spermatocytes (I.e .. leptotene. zygotene, earty, mid and tate pachytene); II, secondal)' spermatocytes. Oiv t and Dlv 2. correspond to maturation divisions of the first and seconds!)' spermatocytes; In spor· matocytes end spermalids the following elements are labelled: G, Golgl apparatus. n. nucklOius: N, nucleus. A, acrosome; c. centrioles: t, tail; m. ml· tocllondria; R8. residual body. The spermatozoon is seen in side view (left) 01 face view (right). The complexity of the whole process of spermatogenesis explains its marked sensltlv· ity to toxic substances or hormonal Imbalance. In addition, many abnormal and degenerating germinal cells are observed during spermatogenesis I n normal men. Spermatogenesis takes approximately 60 days. a duration that does not appear to vary from one individual to the next Spermatogenesis is possibly one of the most complex processes of cell differentiation taking place In the tissues of adult Individuals. Many of Its lacets remain to be studied and clarified at the molecular level. Handbook of Andrology-How are germ cells produced and what factors control their production? 15 Suggested Reading Dym M. The male reproductive system. In: Weiss L, ed. Histology. Cell and Tissue Biology, fifth edition. New York, Amsterdam, Oxford: Elsevier Biomedical; 1983:1000-1053. Desjardins C, Ewing LL, eds. Cell and Molecular Biology of the Testis. New York, Oxford: Oxford University Press, Inc., 1993. Copyright © American Society of Andrology What are the unique chromosomal events leading to the formation of a haploid male germ cell? Stages of meiosis, chromosomal events, genetic recombination Meiosis is a unique form of cell division restricted to gametes (spermatocytes and oocytes). There are two primary purposes of meiosis: MEIOSIS I 1) To have a reduction division. 2) To provide genetic variation by exchanging segments of homologous chromosomes. Prophase I Primary Spermatocyte Spermatogonia divide by mitosis and differentiate until they become primary spermatocytes. They will then stay in the primary spermatocyte stage until puberty. At puberty, primary spermatocytes begin to divide by meiosis, which consists of two divisions. In the first meiotic divisions, the prophase is long and involves so many specific nuclear events, that it is subdivided into five parts: leptotene, zygotene, pachytene, diplotene and diakinesis. In the leptotene stage, the chromosomes are evident as thin, delicate filaments which attach themselves to the nuclear envelope (Fig. 1). Each chromosome is composed of two sister chromatids. In the subsequent zygotene stage, there is intimate pairing of homologous chromosomes; in the human, 23 homologous chromosomes pair and form a trilaminar structure called the synaptonemal complex. In the pachytene stage, there is exchange of genetic material between homologous chromosomes that is mediated by the synaptonemal complex and a large recombinant nodule. This stage lasts 16 days in the human. In the diplotene stage, desynapsis occurs and the areas where there was exchange of genetic material are clearly seen at connecting sites called chiasmata. In the final stage , diakinesis, the chromosomes condense. The cells then proceed with metaphase where the paired chromosomes align at the FIG. 1. An illustration of the division of spermatocytes by meiosis, as described in the text. Each chromosome is composed of two sister chromatids in the leptotene stage, but the two sister chromatids are closely opposed at this stage and are therefore difficult to differentiate from each other until the pachytene stage of the first prophase. 16 Handbook of Andrology-What are the unique chromosomal events leading to the formation of a haploid male germ cell? equatorial plate. Chiasmata separate and the homologous chromosomes move to opposite poles of the cell during anaphase. This division is in distinction to mitosis, during which each pair of sister chromatids separates and moves to opposite poles. In telophase, cytokinesis occurs and two separate daughter cells result. At the end of this first meiotic division, the cells have differentiated to become secondary spermatocytes. There is a very short interphase between the first and second meiotic divisions, and no DNA synthesis occurs during this interphase. Almost immediately, the second division begins with the secondary spermatocyte progressing from prophase through metaphase, anaphase and telophase. The second division closely resembles mitosis where there is separation of the sister chromatids along the centromere. At the end of the second division, the secondary spermatocyte has become a spermatid. In summary (Fig. 1), at the end of the first meiotic division, one primary spermatocyte has divided into two secondary spermatocytes. At the end of the second meiotic division, each secondary spermatocyte has divided into two spermatids so that there are a total of four spermatids that were derived from the primary spermatocyte (Table 1). Interestingly, cytokinesis in both the first and second divisions is incomplete so that very small intercellular bridges form between the cells. This bridge is termed a "syncytium" and allows for simultaneous communication amongst the cells. 17 Table 1. Diploid Versus Haploid: The 4N, 2N, 1N Quandary "N" can refer to either the number of chromosomes or the amount of DNA in a cell. A. If N refers to the number of chromosomes, 2N is diploid and 1N is haploid. Since all somatic cells have 46 chromosomes or 23 homologous pairs (one paternal and one maternal), somatic cells are diploid (2N). Likewise, spermatogonia are also diploid (2N). At the end of the first meiotic division, the secondary spermatocytes have 23 double-stranded (containing a pair of daughter chromatids) unpaired chromosomes and are, therefore, haploid (1 N). At the end of the second meiotic division, there are 23 single-stranded chromosomes and these are also considered haploid (1 N). B. N can also refer to the amount of DNA in a cell. In this particular case, N refers to the minimal amount of chromosomal material which contains all of the genes. Therefore, spermatogonia prior to the Sphase are diploid and have 2N amount of DNA. After the S-phase (at the beginning of meiosis) the primary spermatocytes have doubled their amount of DNA and, therefore, are 4N. At the end of the first meiotic division, the secondary spermatocytes are haploid, but contain 2N amount of DNA since they are double-stranded chromosomes (sister chromatids). At the end of the second meiotic division, the spermatids are haploid and contain 1N amount of DNA. Copyright © American Society of Andro/ogy What does the epididymis do and how does it do it? Physiology, sperm maturation matozoa are eventually stored within the cauda region. The gross structure of the human epididymis is unique among the species studied in that it does not have a prominent cauda region. Hence the human epididymis has little capacity to store large numbers of spermatozoa as compared with many other species, for example the ram or bull. Histologically the epididymis is composed of several cell types including principal, basal, apical, halo, clear, and narrow cells, each of which vary in number and size along the epididymal duct. The principal cells in the more proximal regions of the epididymis tend to be very tall resulting in a duct with a small luminal diameter whereas, in the distal regions, the principal cells are low columnar cells and the luminal diameter much larger (Fig. 1). Such dramatic differences in the cellular architecture are primarily due to the functional roles of each cell within each epididymal region. In the proximal region there is considerable absorption of water, hence the cells take on the classical appearance of a water absorbing epithelium -large apical surface area with long stereocilia, and many mitochondria in the basal aspects. The distal epididymis is primarily a sperm storage region, hence the cells are much smaller and some cells, for example the clear cells, are specialized for removing cellular debris. Ultrastructurally, epididymal cells in general can be seen to have an extensive endoplasmic reticulum and an elaborate Golgi apparatus, reflecting the involvement of this tissue in protein synthesis. Tight junctional complexes between the epididymal cells form what is referred to as the blood-epididymis barrier, an important physiological and anatomical barrier that allows the epididymis to create a specialized fluid environment for the maturing spermatozoa. It has also been suggested that another function of the blood-epididymis barrier is immunological protection of the spermatozoa. Spermatozoa are immunogenic and must be protected from the immune "If anyone asks what the epididymis is, we shall answer that it is a vessel constituting by various twists a body affixed to the back of the testicle" (deGraaf, 1668; see Jocelyn and Setchell, 1972). Spermatozoa leave the testis neither fully motile nor able to recognize or fertilize an egg, but must traverse a long duct, the epididymis, to acquire these abilities. These transformations of spermatozoa are called sperm maturation. For a number of years, the epididymis was considered a holding-tube for the spermatozoa; the epididymis did not influence the process of sperm maturation, but was a place where spermatozoa aged. It was felt that sperm maturation was inherent to the spermatozoa and had little to do with the epididymis. Because it takes anywhere from 1 to 14 days for spermatozoa to traverse the epididymis, the aging hypothesis seemed reasonable. However, it is now very clear that the epididymis is actively involved in the sperm maturation process, not only providing an appropriate environment but also providing many of the molecules needed by the spermatozoa to allow them to fertilize an egg. Structure of the epididymis Looking at the gross structure of the epididymis of several species, the organ can be divided into a number of segments or regions: initial segment, caput (head), corpus (body), cauda (tail) and the vas deferens (Fig. 1). There has been much discussion of the precise delineation of each epididymal region as it related to the gross structure of the organ, to the physiological process within the organ and to the localization of discrete stages of sperm maturation. For example, sperm motility is generally observed as the spermatozoa pass through the caput region whereas sperm fertilizing ability is usually achieved as the spermatozoa pass through the corpus. Sper18 Handbook of Andrology-What does the epididymis do and how does it do it? initial segment caput corpus cauda FIG. 1. Schematic representation of an epididymis showing the different regions: initial segment, caput, corpus and cauda. To the right are shown cross-sectional representations of the epididymal duct at each region. Note how the luminal diameter increases and the cell height decreases from the initial segment to the cauda. system during their transit along the epididymal duct. Maturation of spermatozoa It is the fluid microenvironment within the epididymis that has been suggested to promote maturation of the spermatozoa. The fluid is hyperosmotic and distinctly different in composition from blood plasma. In the epididymis of many species the major constituents are organic solutes: L-carnitine, myo-inositol, glutamate, taurine, glycerophosphorylcholine, sialic acids, lactate, and certain steroids such as dihydrotestosterone. Concentrations of these organic solutes can reach anywhere from 20 to 90 mM depending upon the species and the epididymal region . The luminal fluid also contains several ions: sodium, potassium, chloride and bicarbonate. The fluid in the proximal 19 epididymis is quite acidic with pH values in the 6.5 range increasing 1o approximately 6.8 in the distal epididymis. The role of each organic solute and ion is not precisely known but several studies suggest they are involved in the acquisition of motility, in osmoregulation for spermatozoa and epididymal epithelial cells, and in sperm and epididymal cell metabolism. There are also several proteins found within the lumen including transferrin, albumin, clusterin (SGP-2}, immobilin, retinoid-binding proteins, metalloproteins, proenkephalin, and enzymes such as glycosyltransferases, glycosidases, glutathione peroxidase, and gammaglutamyl transpeptidase. Several of these proteins have been shown to be associated with spermatozoa suggesting a role in sperm maturation and/or sperm-egg interactions. However, the role of most of these proteins is not yet clear. Other functions of the epididymis In addition to promoting sperm maturation and providing a place for sperm storage, the epididymis plays a role in the transport of spermatozoa along the duct and protects spermatozoa from harmful substances. Spermatozoa within the epididymis of several species are held in a quiescent state by luminal fluid factors and, therefore, do not propel themselves along the duct. Transport of spermatozoa is achieved by two processes, contractions of the smooth muscle that surrounds the epididymal epithelium, and the continuous production and movement of fluid originating from the testis. Protection of spermatozoa from harmful substances such as xenobiotics and oxygen radicals is an important aspect of epididymal function. The manner by which this is achieved is unclear, but it appears that the epididymis has evolved elaborate protective mechanisms. For example, the blood-epididymis barrier regulates the entry of solutes and ions into the lumen, and the luminal fluid contains antioxidants, e.g., glutathione, and enzymes such as gamma-glutamyl transpeptidase, superoxide dismutase and glutathioneS-transferase involved in antioxidant defense and protection against xenobiotics . 20 Handbook of Andrology-What does the epididymis do and how does it do it? In summary, the epididymis promotes sperm maturation, facilitates the transport of spermatozoa along the duct, stores sperm and protects spermatozoa from harmful sub- stances. All these functions are coordinated with remarkable precision to ensure production of fully viable spermatozoa. Suggested Reading Jocelyn HD, Setchell BP. Regnier de Graaf on the human reproductive organs. J Reprod Fert 1972; Suppl. 17:1-222. Setchell BP, Brooks DE. Anatomy, vasculature, innervation, and fluids of the male reproductive tract. In: Knobil E, Neill JD, eds. The Physiology of Reproduction. New York: Raven Press Ltd.; 1988:753-836. Robaire B, Hermo L. Efferent ducts, epididymis, and vas deferens: structure, functions, and their regulation. In: Knobil E , Neill JD, eds. The Physiology of Reproduction. New York: Raven Press Ltd.; 1988:999-1080. Copyright © American Society of Andrology What is the prostate and what is its function? Physiology, function (anatomy, embryology) unknown function. The proteins from the seminal vesicle cause the ejaculate to clot and form a coagulum within a few minutes after ejaculation. Subsequently, a serine protease called prostatic specific antigen, secreted from the prostate, lyses the clot. In rodents, a hard elastic plug is formed and it is thought that the slow release of sperm from the coagulum and copulatory plug reduces reflux of sperm from the female tract. Other proteins from the sex accessory tissues coat the sperm and are believed to protect sperm from environmental shock and agglutination, and to mask sperm antigens from the female's immune system. Other proteolytic enzymes in the secretions help sperm traverse cervical mucus, while the prostaglandins stimulate the female reproductive system to transport the sperm toward the ovum. A cascade of activation of proteases and cell signalling pathways is now being studied to resolve how the sex accessory secretions work in temporal concert to assist the sperm to fertilize the ovum. The sexual accessory glands The sexual accessory glands consist of the seminal vesicles, the prostate, and Cowper's glands (Fig. 1). They are involved in maintaining the viability and motility of the sperm and in assuring the successful transfer of the sperm to the female system, ultimately to fertilize the ovum. Over 95% of the ejaculate volume originates from the sex accessory tissues and not from the testes, as is often mistakenly believed. A human male's ejaculate volume is about 3 ml and ranges from 2 to 6 mi. Of the 3 ml of an ejaculate, a very small proportion, 0.2 ml, originates from the bulbourethral gland (Cowper's gland) and 0.5 ml from the prostate gland. The largest portion, approximately 2 ml, is secreted from the seminal vesicles and appears in the latter portion of the ejaculate volume. These exocrine glands that form the ejaculate are located at the base of the bladder and empty their secretions directly into the urethra at the time of ejaculation. The growth of these glands and their secretory activity require androgen production from the testes as well as a functioning androgen receptor within the cells of the sex accessory tissues. Removal of testicular function by castration, drug action or failure of the hypothalamic-pituitarytesticular axis will result in diminished circulation of androgens in the serum. As a consequence, the sex accessory tissues will involute and markedly shrink in size and function thus drying up the ejaculate. In the normal male, the ejaculate is a rich source of proteins and enzymes (20-40 mg/ ml), as well as highly bioreactive components such as prostaglandins (200 1-Lg/ml), citrate ion (4 mg/ml), spermine (3 mg/ml) and fructose (2 mg/ml). The term prostaglandins is a misnomer since these compounds are primarily derived from the seminal vesicles, which also produce fructose. The prostate gland contributes high concentrations of citric acid, as well as spermine, a very basic organic molecule of Structure of the prostate gland So what is the prostate? It is a small gland the size of an English walnut and normally weighs 25g. The gland is located at the base of the bladder and surrounds the urethra (Fig. 1). The vas deferens, a muscular tube carrying the sperm from the testes, joins the duct from the seminal vesicles, now becoming the ejaculatory duct, and then immediately courses through the structure of the prostate to deposit the sperm and seminal vesicle fluid into the urethra, at the center of the prostate gland. The ejaculatory ducts enter the urethra at the verumontanum. In the urethra, just beyond the verumontanum, the ducts of the prostate appear allowing direct entry of the prostate secretions. There are 15-30 excretory ducts from the prostate entering the urethra as it passes through the prostate. This part of the urethra is called the prostatic urethra. Each of these 21 22 Handbook of Andrology- What is the prostate and what is its function? Lum en O Secretory g ranules ~ 0 Export~O ; ! Golgo a pp ara tus Q ~ ~ u E ~ 0£ :l , ~ ;. .. c.t> Lysosomes Q memb, _ ,•;,....- o.., , ~ ; <' i ! ~ {;RNAt~S "'!) Plj-;> c: "' -5, ~ ., _ Oct V 0 d:~ 1z f E ~n \ exporlo-ble Ac!tvotoonf Androgen receptor lfillllllllliJ 11 II -< .. ? ~ ) OHT .......,_... t Hormone~ ep~complex Ool\ydrotesto steroneiOHTI ~----·-·- -}-··-Testosterone FIG. 1 . Anatomy of the prostate and seminal ves· icles. (From: @ 1995 by Ciba. Reprinted with permission from Ciba Publications, illustrations by F.H. Netter, M.D. All rights reserved.) excretory ducts receives prostatic secretions from 4-6 prostatic lobules that contain prostatic acini surrounded by tall columnar epithelium. It is the acinar glands that respond to androgen stimulation by producing secretory proteins which are stored as viscous secretions in the ascinar spaces. During ejaculation, nerves to the prostate from the hypogastric plexus, under sympathetic stimulation, cause muscular contraction of the prostate and excretion of the ascinar contents into the ducts and out through the urethra and penis to form the ejaculate. Role of androgens in the prostate Androgens control the growth of the prostate and formation of the prostatic secretions. T estosterone, synthesized in the Leydig cells of the testes under luteinizing hormone (LH) stimula- FIG . 2. Androgen action in the prostate. Free testosterone diffuses into the prostatic cell and is converted to DHT by 5-alpha reductase. DHT binds with androgen reGertor which in turn is translocated into the nucleus, where it binds to the androgen responsive element (ARE). Binding to the ARE induces transcription of mANA which leads to translation of proteins in the cytoplasm. (From: Aumuller G. Morphologic and endocrine aspects of prostate function. The Prostate 1983;4:195-214. Reprinted by permission of John Wiley ana Sons Inc.) tion , enters the serum, is complexed to a steroid binding globulin and is transported to the prostate. The free testosterone, in equilibrium with its bound and free form in the serum, then diffuses across the epithelial and stromal cell membranes and enters the prostatic cells (Fig. 2) . The testosterone is then metabolized to a more androgenic substance called dihydrotestosterone (DHT) through reduction of a double bond at the 5-position of testosterone. The enzyme, termed 5-a reductase, forms the more potent DHT which then binds in a highly specific manner to the androgen receptor within the cell. The DHT-bound androgen receptor attaches to a promoter area on DNA at a sequence called the androgen responsive element (ARE). This binding participates in andro- 23 Handbook of Androfogy-What is the prostate and what is its function? THE PROSTATIC FUNCTIONAL UNIT (CANI NE PROSTATE ) EPITHELIUM SECRE TORY CELLS BIONATRIX Lumtn CYTOSKEl.ElON Apocal plasma M tctO'\'IIIous COf't membrane s ~crttory fermtnc.l web gro.nult5 M tcrotubules Coattd vts.clfS Condtnsong vacuoles Oesmosomes Golgo apparatus Rough tndoplasmoc tttlculu m Nud eus, Nud~tus M tcrofJioments Port comptu Prot~n network Lomtnat Motnx bound repl1somes MtiOChOf'ldrtC l ysosomes BASAl CELLS Otsmosomes Loloral and basal STROMA ptosmo mf'mbrones Basal lomtno BASAL LAMINA ConnE-cllvt elemtnts t f tbtrs~ l omtntn, f•bronechn, Ntrvf' terrn•nals praltogl(tans ) Muse It colts ( acton, myoson PIC I Ca~llan e 5 Fobrocytos Connecltvt hssue ftbt rs Smooth musct• Ground substance ~Is FIG. 3. Prostatic cell showing epithelial and stromal components. (From: Aumuller G. Morphologic and endocrine aspects of prostate function. The Prostate 1983;4:195-214. Reprinted by permission of John Wiley and Sons Inc.) gen-induced expression of genes such as prostatic specific antigen. In addition , growth factors and their receptors can be induced by DHT and there is much crosstalk between the epithelial cells that form the secretions and their surrounding stromal (connective tissue) cells (Fig. 3). Short range cell-cell interactions mediated through growth factors are termed paracrine effects and this is an important pathway for the exchange of stromal-epithelial interactions. The stromal and epithelial cells also excrete highly insoluble components forming the extracellular matrix and basement membrane that creates a physical interface and solId state support between the prostate epithelial and stromal components. The cooperation and integration of these systems during development, growth, function and pathology of the prostate is a most active area of research. We also await the definition of precise mechanisms by which the prostate influences fertility. Prostate gland disease The bad news is that the prostate is probably the leading gland in the American male for causing medical problems. One of four males will be operated on some time in their lifetime to surgically relieve benign prostatic hyperplasia (BPH) that compresses the urethra and produces urinary outflow obstruction. BPH-related operations, most commonly a transurethral resection of the prostate (TURP) , are the second leading cause of surgery in males (400,000/yr)in the United States, second only to cataract operations. Far more serious is the high incidence of malignant growths in the prostate gland. Prostate cancer is the leading cause of cancers diagnosed in the American male, now exceeding even lung cancer. The diagnosis of prostate cancer is being aided by detecting the presence of the prostatic specific antigen (PSA) protein in serum. This protein from the prostate is in high concentrations in the ejaculate (1-2 mg/ml) and, when the prostate is damaged by abnormal growth of BPH or cancer, the PSA protein inadvertently enters the serum where detection of greater than 4 mg/ml, indicates a prostate problem such as BPH or cancer. 24 Handbook of Andrology-What is the prostate and what is its function? Suggested Reading Luke MC, Coffey DS. Male Sex Accessory Tissues Structure, Androgen Action, and Physiology. In: Knobil E, Neill JD, eds. The Physiology of Reproduction, 2nd edition. New York: Raven Press; 1994. Copyright © American Society of Andrology What is semen? How does semen analysis assist in understanding the reproductive status of the male? Semen composition and analysis (animal, human), related tests cauda epididymal fluid, or even blood plasma, and others are unique products of that gland. Thus, seminal plasma includes a broad spectrum of chemical constituents contributed by the epididymis and the accessory sex glands. The relative contributions of the epididymis or different accessory sex glands to the seminal plasma of a given ejaculum are dependent on many factors including the interval of sexual abstinence, duration of foreplay, pathophysiological processes in the male, and the species. Because semen is a mixture of spermatozoa and fluids moved by emission from the cauda epididymidis and vas deferens with fluids from the accessory sex glands, the sperm to fluid ratio is quite variable. The more important attribute is the total number of normal sperm in an ejaculum rather than the concentration of sperm per unit volume. For similar reasons, in analyses of constituents of seminal plasma, the total amount of a component should be considered in parallel with its concentration. A human might ejaculate 40300 million spermatozoa, not greatly different from the number ejaculated by a rabbit (1 00300 million), but substantially less than a dog (0.2-2 billion) or horse (5-25 billion). What is semen? Semen is composed of spermatozoa (sperm), produced in the seminiferous epithelium of the testis, and seminal plasma, the components of which are secreted by the excurrent duct system and accessory sex glands. When a spermatozoon is released from the seminiferous epithelium the major structural elements are in place, but additional changes are induced by exposure to sequential milieus provided by the epididymis and mixture with fluids from the accessory sex glands at ejaculation. Typical spermatozoa of the rat, human and stallion are shown in Fig. 1 and the important elements of a spermatozoon are depicted in Fig. 2. A partial list of spermatozoal attributes essential for fertility is presented in Table 1. Collectively, these attributes depend on the normal development and function of the genomic package, the mitochondria, dense fibers and microtubular elements of the axoneme, the acrosome and enzymes therein, and the multi-compartmentalized plasma membrane. Seminal plasma is the fluid portion of an ejaculum, but only one of several distinctly different fluids to which sperm are exposed. Spermatozoa are transmitted from the seminiferous epithelium in a fluid milieu, and the solutes therein are removed and replaced within the efferent ducts and epididymis. Ultimately, sperm in cauda epididymal fluid are conveyed through the vas deferens at the time of ejaculation and mixed with fluids from the accessory sex glands, namely the prostate gland, vesicular glands (i.e., seminal vesicles), and bulbourethral glands. Some species have a complete array of accessory sex glands, including the above three types. Other species lack the bulbourethral glands or vesicular glands. Certain proteins and other molecules in the secretion of one or more accessory sex glands are identical to some components of What is the goal of seminal analysis? For a clinician, evaluation of seminal quality is linked with a desire to predict potential fertility, identify causes of infertility, or detect changes in potential fertility. The clinician is concerned with minimal requirements to achieve fertilization or contraception. For an epidemiologist or toxicologist, seminal evaluations are the basis of assessing hazards in the workplace, environmental factors, or risk assessments relating to drugs and chemicals. Detection of a significant probability of reduced fertility in a population is more important than accurate prediction of fertility for an individual. For the animal breeder, the primary 25 26 Handbook of Andrology- What is semen? Human Horse FIG. 1. 10pm Typical spermatozoa of the rat, human, and stallion as viewed by scanning electron microscopy. B. A. Plasma Mem brane ~~•+~-- ...... r.....-- Mitochondri on Plasma Membrane Mitochondrion Principal Piece ~-- Fibrous Sheath ~--Axon eme End PieceC FIG. 2. A. Major elements common to mammalian spermatozoa. B. Middle piece (top), principal (center) and end piece (bottom) of a spermatozoon viewed in cross section. 27 Handbook of Andrology-What is semen? Table 1. A Partial List of Attributes of Sperm Essential for Fertility* "Acceptable" morphology Metabolism for production of energy Progressive motility Capacity for hyperactivated motility Membrane lipids Stabilize plasma and acrosomal membranes Flippase enzyme activity Facilitate timely fusion, but not premature fusion of membranes Membrane proteins Immunosuppressive factors Attachment ligands available but masked to prevent premature binding Acrosome reaction-inhibiting factor Integral enzymes associated with fertilization Enzymes modifying membrane glycoproteins Acrosomal enzymes *In addition, the genome of the fertilizing spermatozoon may affect embryo development and apparent fertility. The genome package must contain genes needed for development, and lack lethal mutations or extra genetic material altering or inhibiting development Reprinted with permission from the Journal of Andrology 14:397406, 1993. goal is to determine which male(s) will be the most fertile of genetically superior sires. Evaluations of sperm quality and estimation of potential fertility are the basis for management decisions which might lead to production of several hundred thousand offspring from an individual sire. For each application, the implied goal is to predict accurately the potential fertility of a seminal sample from an individual male. Unfortunately, this goal is not easily achieved. Success in predicting fertility is limited by features of spermatozoa, the process of fertilization, and approaches used for evaluation in vitro of seminal quality. Also, spermatozoal attributes necessary for fertilization will depend on the methodology used to join the gametes, i.e., copulation or in vitro fertilization; on prior history of the sperm, i.e., freshly ejaculated sperm or frozen-thawed sperm; and on female factors, i.e., age or uterine and tubal environments. Sometimes the conclusion from a seminal analysis is obvious. When the semen analysis reveals azoospermia, no progressively motile spermatozoa, or a high proportion of morpho- logically abnormal spermatozoa, the fertilizing potential of the individual is poor. In most cases the challenge is more complex. The goal of a clinician or animal breeder is to predict correctly that a given male probably will be infertile or will be reasonably fertile, relative to the average value for males of that race or species, or that a given seminal sample will provide fertility similar to that previously obtained with other samples from the same male. As contrasted to lack of fertilizing capability, considered above, accurate prediction of high fertilizing capability is extremely difficult, or impossible, because a spermatozoon must retain function of each of a number of essential attributes (see Table 1) to be capable of fertilizing an oocyte. It follows that a number of spermatozoa in a sample could be incapable of fertilizing an oocyte, each for a different reason. Limitations of current approaches for evaluation of seminal quality provide great opportunity for individuals intrigued by investigating male reproductive function. How is semen evaluated? Traditionally, evaluations of seminal quality, regardless of species, include measurement of seminal volume, determination of spermatozoal concentration and, by multiplication (volume x concentration), calculation of the total number of spermatozoa in an ejaculum. This provides quantitative information which, with knowledge of the interval since the previous ejaculations by that male, and with information on testicular volume, is an indication of the capability of that individual's testes to produce sperm. Absence of spermatozoa in an ejaculate could be evidence of retrograde ejaculation, blockage of excurrent ducts, or testicular failure. There is no cut-off for the total number of sperm in an ejaculum below which fertilizing potential is reduced or eliminated. Males of most species, but less so for humans, typically ejaculate a number of sperm far in excess of that necessary for maximum fertilizing potential when deposited in the vagina or uterus by copulation. For many species, < 1% of the number of sperm in a typical ejaculation will result in maximum fertility 28 Handbook of Andrology-What is semen? when sperm are deposited by artificial insemination, provided the sperm are of good quality. Quality traditionally is considered in terms of the percentages of progressively motile sperm or morphologically normal sperm. Until recently, both were subjective evaluations and influenced by substantial observer bias. Despite these problems, the visual assessment of sperm motility and morphology is the standard method used by most clinical andrology laboratories. Computerized image analysis systems are now available for determining both percentage of motile sperm and the distribution profiles for velocity or other kinematic attributes of individual cells. Relatively simple imaging systems for objective evaluation of sperm morphology are being introduced, but have not yet gained wide acceptance. Functional tests are used to further define quality of a seminal sample in an infertility practice or research laboratory. These include capability of the spermatozoa to undergo an acrosome reaction (spontaneously or stimulated), penetrate into a heterologous oocyte or bind and penetrate into homologous zona pellucida, swim through cervical mucus, undergo motility hyperactivation, or simply swim rapidly away from a population of immotile or slow sperm. Often one or more of these tests is supplemented by immunological tests to determine if the spermatozoa or seminal plasma contains auto-antibodies associated with reduced fertility. In a research setting, one might perform more detailed analyses for the amounts of certain enzymes normally present in sperm, or analyze the presence and surface distribution of glycoproteins thought to be involved in the fertilization process. Better predictions may be possible after image or flow cytometric analysis of permeability of the sperm plasma membrane, mitochondrial function, surface properties of the plasma membrane, and/or denaturation of nuclear proteins. Finally, it is increasingly obvious that peroxidation of lipids of the plasma and acrosomal membranes of spermatozoa is associated with decreased quality. The extent of lipid peroxidation can be quantified. Many of these analyses provide only a mean value for the pop- ulation of sperm, rather than a distribution of values for the individual cells. Unfortunately, information is needed on how many cells "pass" for the full set of essential attributes. Applications of semen analysis Infertility occurs in approximately 15% of all human couples. In general, 30% of these couples have a predominant male factor, 30% have a predominant female factor, and the remainder have factors in both or no demonstrable cause. Semen analysis is the first step taken to establish a diagnosis of male factor infertility, and is performed in the initial screening tests of an infertile couple. Because of large day to day variation in the quality of the semen from an individual, at least two, and preferably three, semen analyses at least a week apart are usually performed to evaluate the male partner of an infertile couple. In general, an analysis of human semen is regarded as normal if: 1) 2) 3) 4) ejaculate volume is :::::2 ml, sperm concentration :::::20 million/ml, ::::50% of the sperm are progressively motile, and :::::30% of the sperm are morphologically normal (WHO, 1992). These assessments are performed in an andrology laboratory, usually by visual examination using a light microscope. The diagnosis is based on the semen analysis together with information from a physical examination and medical history. If a patient has azoospermia (no sperm) or very severe oligozoospermia (less than 5 million/ml), endocrine status is evaluated by measurements of serum concentrations of follicle-stimulating hormone, luteinizing hormone and testosterone. This helps in diagnosis of the underlying etiology and assessment of prognosis. For > 70% of the patients with >2-3 abnormal semen analyses, no specific cause of abnormal testicular function can be identified. With these patients, specialized tests of sperm function (Table 2) focusing upon sperm surface proteins, autoantibodies against sperm, acrosome reaction, zona-free hamster oocyte 29 Handbook of Andrology-What is semen? Table 2. Clinical Laboratory Evaluation of Human Semen Routine Specialized Seminal fluid volume, Sperm count, motility, and morphology Leukocytes in semen Sperm antibodies Sperm-cervical mucus interaction Computer-aided sperm analysis (motility, morphology, sperm hyperactivation) Acrosome reaction Semen biochemistry Sperm biochemistry Sperm membrane lipids, proteins Zona-free hamster oocyte penetration test Zona pellucida binding test penetration, human zona pellucida penetration and binding, and other functions may be required. Through a combination of these tests, more specific sites of dysfunction causing abnormality of the spermatozoa may be identified, and appropriate therapy planned. For instance, if investigations revealed that most spermatozoa in an ejaculum are unable to bind to the zona pellucida, the appropriate advice to the couple would be in vitro fertilization by subzonal injection of spermatozoa or direct intracytoplasmic injection of a spermatozoon into each oocyte. With these new andrologic techniques, fertilization and subsequent pregnancies have occurred in couples where the male partner has very severe sperm dysfunction. In veterinary medicine and animal breeding, there are two general types of semen evaluation. The first is by a clinician evaluating a male for breeding soundness and potential fertility. Typically, testicular size is measured and a single seminal sample evaluated. Animals whose testes are substantially smaller than average values for males of the same breed and age are rejected, as are males whose semen contains <80% morphologically normal sperm or <50% progressively motile sperm. Failure to meet these criteria does not mean that the male is sterile, but rather that there is a reasonable probability the male will not- be highly fertile. The second type of evaluation is used in a facility housing males, such as bulls or boars, for wide-spread commercial distribution of their spermatozoa, or a facility where dogs, stallions or males of other species are brought to enable collection and cryopreservation of a limited number of doses for artificial insemination. To enable cryopreservation, the semen is mixed with an "extender", a salt solution containing egg-yolk or milk proteins and sugars, and 4-12% glycerol, which is an essential cryoprotectant. Determination of the total number of sperm in the ejaculum is crucial to enable extension of the semen to a concentration which provides the requisite number of sperm in each insemination dose, and is linked with evaluations of sperm quality before processing. The extended semen is then sealed in a series of plastic containers which, for most species, are shaped like a drinking straw and contain 0.25, 0.5 or 4.0 ml; each straw is one insemination dose. Representative straws of cryopreserved semen are thawed and the cells evaluated immediately, and after several hours of incubation at 3rC, to establish the percentage of progressively motile sperm, their velocity, and often the percentage of sperm with a normal-appearing acrosome. Similar approaches also are utilized by individuals involved in preservation of sperm from humans or sperm from exotic animals, ranging from antelopes to zebras. What of the future? It is likely that approaches for seminal analysis in a clinical setting will remain similar to those in use today, with primary reliance on the manual counting of the number of spermatozoa and visual estimation of the percentage of motile sperm and the percentage of abnormal sperm. As a secondary screening, 30 Handbook of Andro/ogy-What is semen? these classic tests may be augmented by binding or enzyme-linked assays measuring one or more attributes of the plasma membrane or a sperm enzyme. More importantly, it is likely that some secondary and most tertiary laboratories will have access to instruments which characterize multiple attributes on several thousand individual sperm representing the population. Flow cytometers now serve this purpose. New imaging instruments and techniques likely will be developed to evaluate motion and morphology of individual sperm in a wet preparation concurrently with multiple probe assessment of biochemical attributes. Such analyses would add data for 3 to 5 functional attributes to those for 2 or 4 selected attributes of sperm motion and morphology. These newer tests may be able to replace some of the biological tests currently being used such as the zona-free hamster oocyte penetration and human zona pellucida tests which are imprecise, time consuming, technically demanding and expensive. With appropriate selection of independent attributes essential for sperm to have fertilizing capability, improved prediction of fertility should be possible. Suggested Reading Amann RP. Can the fertility potential of a seminal sample be predicted accurately? J Androl 1989; 10:89-98. Amann RP, Hammerstedt RH. In vitro evaluation of sperm quality: An opinion. J Androl1993; 14:397-406. Davis RO, Katz OF. Operational standards for CASA instruments. J Androl1993;14:385-394. Wang C, Swerdloff RS. Evaluation of testicular function. Bail/iere's Clin Endocrinol Metab 1992; 405-434. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. Cambridge, England: Cambridge University Press; 1992. Copyright © American Society of Andrology What is sperm banking? When and how is it (or should it be) used in humans? Animals? Sperm banking, consequences of its use in animal and clinical practice ease and other lymphomas, leukemia, nephrotic syndrome, diabetes, and multiple sclerosis. 2) Prior to elective sterilization or exposure to hazardous environments. Occupational exposure to radiation, pesticides, and chemicals can affect sperm function or genetic integrity. Men engaging in military operations where risks of death or exposure to sperm toxicants exist also are candidates for sperm storage. 3) Before participating in fertility treatments which require semen collection at a specific time. For men who develop anxiety-related impotency or emission failure, sperm banking ensures that treatment cycles can proceed as planned. Patients whose occupations require unscheduled travel also find that sperm banking reduces the risk of cancelled treatment cycles. One of the concerns often expressed by physicians about referring patients with systemic diseases for sperm banking is whether the patient's sperm are of sufficient quality and number to achieve a pregnancy. Although sperm count, motility, and physiology may be impaired before treatment is initiated, the technological advances in assisted reproduction, such as direct sperm injection into the ooplasm, often can, at the present time, or will, in the near future, be able to overcome many abnormalities present. Having many sperm stored is definitely an advantage, since it may reduce the need for in vitro fertilization and increase the chance for a successful pregnancy outcome, but the desire to bank multiple ejaculates must be balanced against the necessity of treatment initiation and financial constraints. The use of cryopreserved sperm obtained from anonymous donors as a treatment for infertility caused by absent or defective sperm is the other major medical application of sperm banking. In a 1987 survey, the United States Office of Technology Assessment es- Sperm banking, more formally referred to as sperm cryopreservation, is a process intended to preserve sperm function by freezing and storage at ultra-low temperature. Upon thawing, sperm are introduced into a suitable recipient female by insemination into either the endocervical canal or the intrauterine cavity, or are used to inseminate oocytes during in vitro fertilization. Sperm freezing originated in the late eighteenth century, but the widespread uses of sperm cryopreservation began after 1950. The discovery that glycerol had cryoprotectant properties, and the availability of liquid gases, especially liquid nitrogen, to achieve ultra low temperatures for freezing and storage, stimulated the development of many sperm banking applications. The advantages of frozen sperm over fresh sperm include the following: they can be stored almost indefinitely (at least for decades), allowing preservation of genetic characteristics that would be lost due to onset of disease, infertility, or death; they can be readily shipped anywhere in the world using small liquid nitrogen containers which can withstand the rigors of transport; and they can be placed in frozen "quarantine", while the human or animal donor can be tested for semen-borne infections or genetic problems. Human Clinical Applications of Sperm Banking An important medical use of sperm banking is patient autologous sperm cryopreservation, called client depositor sperm banking. Client depositor sperm banking is used in the following medical situations: 1) Medical disorders which inherently, or through the treatment used to cure or stabilize the disease, can impair fertility by causing decreased sperm count and function, early fetal loss, genetic mutation, or impotence. Examples include testicular cancer, Hodgkin's Dis31 32 Handbook of Andrology-What is sperm banking? timated that 30,000 births resulted from artificial insemination of donor sperm, with approximately 11 ,000 physicians providing the treatment to about 86,000 women. The practice has probably increased and the demand for fertile and safe sperm remains high. It is virtually impossible to adequately screen donors for infectious diseases with long incubation periods such as human immunodeficiency virus and hepatitis viruses or which are detected with tests that require more than a few minutes to perform, i.e., most infectious diseases. If the sperm are quarantined in the freezer, however, the donor can be examined repeatedly for disease exposure over months or years before the sperm are used. The Centers for Disease Control has cautioned that fresh anonymous donor sperm should not be used for artificial insemination, and frozen anonymous donor sperm should be used only if the donor tests negative for human immunodeficiency viruses after a minimum of 180 days quarantine. The ability to store sperm from men with many different phenotypes and genotypes increases the selection that patients have in choosing a donor, and reduces excessive use of a donor within a limited geographic area. Population statistics can allow determination of the number of pregnancies that can be achieved without increasing the risk of consanguinity in future generations. Generally, sperm from a single individual are used to achieve no more than 10-15 pregnancies in a medium-sized city (500,000 to 1,000,000 inhabitants) in the United States. In other countries where ethnic diversity and ethnic intermarriage are not as common, the number could be smaller, but depends in any case on the live birthrate and number of inhabitants. Usually, sperm banks attempt to package donor sperm in plastic vials or straws containing at least 10 million motile sperm post-thaw, which has been suggested as the minimum adequate insemination dose. Since frozenthawed sperm have shorter longevity than fresh sperm, the route and timing of insemination is critically important to achieving a successful pregnancy. Using qualitative urinary luteinizing hormone (LH) measurement to de- termine ovulation, and one or two intrauterine inseminations within 20 to 40 hours of the LH surge, approximately 70% of patients who elect donor sperm insemination conceive, the majority within six insemination cycles. The American Association of Tissue Banks (AATB) has standards for both donor and client depositor sperm banking, and accredits banks by peer inspection. The AATB also maintains a list of non-accredited sperm banks. Several states have certification programs and the Food and Drug Administration has recently begun to regulate tissue banking, including gametes. Sperm Banking in Animals Sperm cryopreservation has important uses in the livestock industry, especially in the breeding of cattle, pigs, sheep and poultry, and in animal husbandry for domesticated animals such as horses, cats and dogs. Sperm from genetically desirable or "prized" animals can be used to inseminate many females to increase the number of offspring with the desired characteristics. The ability to easily transport sperm has permitted the improvement of existing herds or the establishment of new herds in regions of the world needing development of native food sources. Sperm banking has also become an important way to perpetuate endangered or exotic species in the wild and in zoological parks. The ability to use sperm banking to preserve important research animal strains has been appreciated recently. Sperm cryopreservation could reduce the extraordinary cost of maintaining genetic lines that now must be preserved by continual breeding of the animals, increase the accessibility of various strains to researchers since frozen sperm are easier to transport than live animals, and reduce the risk of losing a valuable genetic line through catastrophic accident, impaired reproductive efficiency, genetic drift, or disease. Because the millions of sperm normally present in a single ejaculate represent millions of meiotic recombination events, cryopreserved sperm can be stored for future studies of gene recombination frequency and mapping of ge- Handbook of Andrology-What is sperm banking? 33 netic loci when new DNA probes become available. es, the solutes present in the liquid phase surrounding the sperm rapidly become concentrated. Glycerol lowers the intracellular water freezing point, thus the cells remain unfrozen and become supercooled well below their actual freezing point. In response to high extracellular solute concentration and the osmotic tendency of supercooled intracellular water to leave the cells, sperm undergo a second volume adjustment as water moves outward, and the cells become dehydrated. When extracellular water freezes and therefore solidifies, an exothermic reaction known as the "heat of fusion" occurs, which can cause serious disruption of the cells unless externally reduced by controlled cooling of the environment. Upon reaching the temperature of liquid nitrogen, -196°C, the sperm are placed in storage indefinitely, where they are presumed to reside in a quiescent state of minimal molecular motion. During thawing, the sperm are subjected to similar rapid and dramatic changes in cell volume and membrane permeability. As the extracellular ice melts and becomes liquid, solute concentrations are rapidly diluted and water rushes into the sperm. As the temperature rises, and as glycerol leaves the cells, the sperm cell volume continues to expand. In order for function to be restored, the surface area and volume must return to normal, the membrane proteins and lipids must redistribute to restore molecular structure and mobility, and bioenergetic demands must be met. For maximum functional recovery to take place, both the freezing and the thawing protocols must be optimized, a very difficult task given the paucity of data available about the processes. Research efforts to improve sperm banking techniques and post-thaw survival have intensified in the last decade and offer many career opportunities for basic and applied research. As protocols improve, the success of cryopreserved sperm applications will undoubtedly increase. The Process of Sperm Cryopreservation In spite of the important uses for cryopreserved sperm, little is known about the physical and biochemical events which occur during sperm freezing, storage, and thawing, or about methods for detecting cryogenic damage. Sperm from most species survive current cryopreservation protocols very poorly, and best efforts usually result in recovery of only about half of the original sperm motility. Sperm function is also impaired, as manifested after thawing by shorter longevity and reduced membrane stability. The goal of any sperm freezing protocol is to prevent lethal intracellular ice crystal formation, control wide fluctuations in cell volume, and reduce membrane damage that accompanies temperature-induced phase changes. The process is complicated by the biochemically and physically diverse compartments of the sperm cell (acrosome, nucleus, mitochondrial-flagellar network), all of which may respond quite differently to freezing and thawing. The sperm also are subject to damaging oxygen radical exposure during their transit through wide temperature changes. Attempts to maximize post-thaw survival have led to the development of sperm cell diluents (extenders), cryoprotectants, and various rates of temperature change to control alterations in extracellular and intracellular solvents and solutes. In most cryopreservation protocols, the ejaculated sperm are mixed with a buffered diluent that contains an energy source such as fructose or glucose, lipid, and a penetrating cryoprotectant such as glycerol. After dilution, the sperm initially undergo rapid shrinkage as intracellular water leaves the cell, then slowly return to their original volume as the glycerol enters. Rapid cooling is initiated at a rate of about -20°C per minute. Extracellular formation of ice crystals begins and, as water freez- 34 Handbook of Andrology-What is sperm banking? Suggested Reading Alvarez JG, Storey BT. Evidence for increased lipid peroxidative damage and loss of superoxide dismutase activity as a mode of sublethal cryodamage to human sperm during cryopreservation. J Andro/1992;24:232-241. Hammerstedt RH, Graham JK, Nolan JP. Cryopreservation of mammalian sperm: what we ask them to survive. J Andro/1990; 11 :73-88. Li H, Cui X, Arnheim N. Direct electrophoretic detection of the allelic state of single DNA molecules in human sperm by using the polymerase chain reaction. Proc Nat/ Acad Sci 1990; 87:4580-4585. Paige C. Freezing spermatozoa. In: Ashwood-Smith MJ, Parrant J, eds. Low Temperatures Preservation in Medicine and Biology. Pitman; 1980:45-64. Watson, P.F. Artificial insemination and the preservation of semen. In: Lamming GE, ed. Marshall's Physiology of Reproduction 4th Ed., Vol. 11. Churchill Livingstone; 1990:747-896. Watson PF, Critser JK, Mazur P. Sperm preservation: fundamental cryobiology and practical implications. In: Templeton AA, Drife JO, eds. Infertility. Springer-Verlag; 1992:101-114 Copyright © American Society of Andrology How does the spermatozoon make its way to the egg and how does fertilization take place? Capacitation, acrosome reaction, zona binding After a sperm leaves the male reproductive tract and enters the female reproductive tract it still has a long way to travel and many obstacles to overcome before it can fertilize the egg. When sperm first enter the female reproductive tract they are not capable of fertilization, but require a maturation step called capacitation. Capacitation has not been completely defined but it is thought to involve cell surface and metabolic changes. As a result of capacitation the sperm have an altered pattern of motility (called hyperactivation) and are capable of undergoing an acrosome reaction. Acrosome reaction The acrosome reaction is a Ca 2 ' -stimulated exocytosis involving reorganization of the membranes in the head of the spermatozoan. Multiple fusions occur between the outer region of the acrosomal membrane and the plasma membrane that overlies the acrosome. The hybrid vesicles which result are released into the surrounding environment along with the fluid contents of the acrosome. Loss of these anterior portions of membrane reveals the inner acrosomal membrane which, together with the original posterior head plasma membrane, form the new head cell membrane of the acrosome-reacted sperm (Fig. 1). There are specific movements of proteins from one membrane region to another during the reorganization, although mixing of the membrane components is incomplete. The acrosomal contents are a rich source of enzymes, including hyaluronidase and the protease, acrosin, that may function in penetration of the sperm through the egg investments. The hybrid vesicles released during the acrosome reaction could also carry such enzymes on their surfaces. The newly exposed inner acrosomal membrane represents a further source of molecules that could be involved in digestion of a pathway for the sperm though the egg invest- ments or in binding sperm to the zona pellucida. Where the sperm are when they acrosome react and what signal(s) causes them to acrosome react are not completely clear. Binding of sperm to the zona pellucida (an extracellular coat surrounding the oocyte) can induce the acrosome reaction, but molecules that the sperm contacts earlier in its progress through the female reproductive tract may also facilitate or induce the acrosome reaction. These include molecules in oviductal and follicular fluids (e.g. progesterone) and also components of the matrix surrounding cells of the cumulus oophorus (see below). It is possible that subpopulations of sperm acrosome react at different sites on their passage to the egg (Fig. 2). Cumulus penetration When sperm reach the egg (more correctly referred to as an oocyte because it has not yet completed meiosis) they first encounter a mass of cells, the cumulus oophorus, that surrounds the egg (Fig. 2). The cumulus cells are follicular cells that encase the oocyte and are ovulated along with the egg. Sperm swim between these cells to reach the egg, apparently dissolving the extracellular matrix that holds the cells together. The sperm carry with them in the acrosomal contents and in the plasma membrane the enzyme hyaluronidase that is required for the penetration of sperm through this cell layer rich in hyaluronic acid. Depending on whether sperm acrosome react in the cumulus mass or remain intact, either pool of hyaluronidase could be used for cumulus penetration. Zona pellucida When sperm reach the zona pellucida they recognize it and bind to it. Although there is 35 36 Handbook of Andrology-How does the spermatozoon make its way to the egg and how does fertilization take place? Outer Acrosoma! Membrane Anterior Head Inner Acrosomal Membrane Plasma Membrane Inner Acrosomal /Membrane ACROSOME REACTION ]- Equatorial Region Posterior Head Plasma Membrane Posterior Head Plasma Membrane FIG. 1. A guinea pig sperm head before and after the acrosome reaction. During the acrosome reaction, the anterior head plasma membrane (except for the posterior-most equatorial region) is lost after fusion with the outer acrosomal membrane. Acrosome intact sperm bind to the zona pellucida by the anterior head plasma membrane. Acrosome reacted sperm bind via the inner acrosomal membrane. The equatorial and/or posterior head region initiates fusion with the egg plasma membrane. The inner acrosomal membrane does not participate in the membrane fusion but is incorporated into the egg cytoplasm. (Reproduced from the Journal of Cell Biology, 1984;99:163-164, by copyright permission of the Rockefeller University Press.) not a strict species specificity in terms of which sperm will bind to a particular zona pellucida, there is often a strong preference for binding between sperm and zona of the same species. In mouse, the zona pellucida is composed of three glycoproteins called ZP1, ZP2 and ZP3. Numerous studies have indicated that acrosome-intact mouse sperm initially bind to FIG. 2. A diagrammatic representation of sperm penetration of the oocyte cumulus and zona pellucida during fertilization. the carbohydrate region of ZP3. The identity of the partner molecule on the sperm surface is not yet firmly established; however, there is excellent evidence that a galactosyl transferase on the sperm surface binds to one of its substrates (N-acetylglucosamine) on the zona and, because the second substrate (UDP galactose) is missing, the sperm remain bound. Other candidates exist that could operate instead of, or in addition to, galactosyl transferase. If sperm are acrosome-intact when they bind to the zona, they are induced to undergo the acrosome reaction as a result of binding. The acrosome reaction has been induced experimentally by the clustering of sperm surface molecules using the multivalent zona protein ZP3, or using antibodies that recognize specific sperm membrane molecules. Acrosome-reacted sperm are also able to bind to the zona pellucida. It has been shown in guinea pig that acrosome-reacted sperm can initiate binding to the zona pellucida as effectively as acrosome-intact sperm, and this may also be true of rabbit and human sperm. In all species it is presumed that, after the ac- Handbook of Andrology-How does the spermatozoon make its way to the egg and how does fertilization take place? rosome reaction, the sperm must bind or rebind at least until zona penetration has begun so that they will not be lost from the zona surface. In mouse there is some evidence that the binding of acrosome-reacted sperm occurs to ZP2. The identity of the binding partner(s) on acrosome reacted sperm is still being researched. In order for sperm to reach the egg plasma membrane they must penetrate through the zona pellucida (Fig. 2). This may involve digestion of a path through the zona and could require enzymes either released by acrosome reacting sperm, or associated with the sperm surface, including the newly inserted inner acrosomal membrane. Only acrosomereacted sperm have been observed to penetrate through the zona. Motility is maintained during penetration and the narrow penetration slit in the zona that the sperm move through may also be created, in part, by mechanical forces. When sperm penetrate the zona they come to lie in the narrow space between the inner boundary of the zona and the egg plasma 37 membrane, the perivitelline space (Fig. 2). At this stage the sperm will first bind to the egg plasma membrane and then fuse with it. There may be more than one mechanism that allows sperm to bind, because sperm from heterologous species or acrosome-intact sperm can bind without being able to fuse. The binding that is required for fusion may involve a sperm membrane protein called fertilin (or PH-30) which probably has an egg membrane integrin as an adhesion partner. If this binding step is blocked, then fusion is also blocked. One region of the fertilin/PH-30 protein that may participate in the fusion of the two lipid bilayers contains a sequence that resembles the fusion peptide of viral fusion proteins. Fusion results in confluency between the sperm and egg membranes as well as the sperm and egg cytoplasms. At the time of fertilization, the egg receives an unknown signal that results in a rise in intracellular free Ca 2 + and thereby activates the egg to initiate development of the new embryo. One of the consequences of activation is completion of meiosis, including production of the second polar body, and the initiation of mitotic divisions. Suggested Reading Florman HM, Babcock OF. Progress toward understanding the molecular basis of capacitation. In: Wassarman PM, ed. Elements of Mammalian Fertilization. Boca Raton: CRC Press; 1991:105-203. Kopf GS, Gerton GL. The mammalian sperm acrosome and the acrosome reaction. In: Wassarman PM, ed. Elements of Mammalian Fertilization. Boca Raton: CRC Press; 1991:153203. Myles DG. Molecular mechanisms of sperm-egg membrane binding and fusion in mammals. Dev Biol1993; 158:35-45. Ward C, Kopf G. Molecular events mediating sperm activation. Dev Bioi 1993;158: 9-34. Yanagimachi R. Mammalian Fertilization. In: Knobil E, Neill J, eds. The Physiology of Reproduction. New York: Raven Press, Ltd.; 1988:135-185. Copyright © American Society of Andrology What factors determine the sex of an individual? X, Y, SRY (loci, genes), sequence of events in development of normal male degenerate and the Mullerian ducts develop into the oviducts, uterus, cervix, and upper vagina. One can view mammalian sex determination as occurring in three steps. First is the establishment of chromosomal sex. This occurs at fertilization when either an X- or Ybearing sperm fertilizes an X-bearing oocyte giving rise to an XX or XY zygote. Second is the establishment of the primary sexual characteristics: the testes or ovaries. In XY fetuses, the fetal gonads differentiate into testes; in XX fetuses, ovaries form. Third is the establishment of the secondary sexual characteristics which is dependent upon hormones secreted by the gonads. What induces development of the gonads into testes or ovaries? It was initially assumed that humans had a sex determining mechanism similar to the well studied fruitfly, Drosophila, since in both species males are XY and females are XX. In the fruitfly, sex is determined by the ratio of the number of X chromosomes to autosomal sets such that an XXY individual is female and an XO is male. However, in 1959, the identification of an XXY male patient with Klinefelter syndrome, an XO female patient with Turner syndrome, and an XO female mouse suggested that, in mammals, the Y induces testes development. Cytogeneticists have since identified individuals with varying numbers of X or Y chromosomes. All individuals who had at least one Y chromosome had testes and a male phenotype, irrespective of the number of X chromosomes. The locus on the human Y that induces testes development was designated the testes-determining factor ( TDF). How does sexual differentiation occur? A fetus is initially sexually indifferent and has the primordia for both the male and female accessory sex organs, the Wolffian and Mullerian ducts, respectively. In the 1940s Jost demonstrated that the male phenotype is imposed on a fetus that would inherently develop into a female. Jost surgically removed the testes from fetal male rabbits at a stage when Wolffian and Mullerian ducts were present and then allowed fetal development to proceed in utero. When Jost examined the fetuses at a later date, the castrated males were phenotypic females (Fig. 1). Jost concluded that the fetus is programmed to develop into a female. However, if testes are present they secrete two factors that override the female program and masculinize the fetus. The first factor, secreted by Leydig cells, is testosterone which induces the Wolffian ducts to differentiate into the epididymides, vas deferens, and seminal vesicles. Male external genitalia form when the cells of the urogenital tubercle metabolize testosterone into dihydrotestosterone which induces the development of the penis and scrotum. The second factor, secreted by Sertoli cells, is Mullerian inhibiting substance (anti-Mullerian hormone), which induces the Mullerian ducts to regress. In the absence of these two factors the Wolffian ducts How does the Y chromosome control masculinization? By correlating deletions on the Y with the presence or absence of testes and by studying XX males which carry a tiny portion of the Y on one of their X chromosomes, investigators mapped TDF to a 35-kb region of the Y short arm. Cloning of this region resulted in the identification of a gene designated sex-determining region Y (SRY). Convincing evidence that SRY is the testis-determining gene was obtained when a 14.6-kb genomic sequence of the mouse SRY locus was shown to be capable of inducing XX fetuses to develop into males in transgenic experiments. The hypothesis is that SRY is a master reg38 39 Handbook of Andrology-What factors determine the sex of an individual? ~tullerian duct ¥ ~'"'~"""" Indifferent Fetal Gonad undirTerentiated / \ Ovary Testis /'\... uterus degenerating Wolman duct MIS 0 FIG. 1. Male rabbits castrated at a fetal stage at which the secondary sexual characteristics are undifferentiated, develop as phenotypic females (adapted from Jost 1947). ulatory gene that initiates a cascade of gene interactions that transforms the fetal gonad into a testis (Fig. 2). SRY encodes a member of the High Mobility Group-1/-2 (HMG) protein family whose members are characterized by an 80-amino acid DNA-binding motif called the HMG domain. Several HMG proteins, including SRY, are transcription factors that recognize and bind a specific DNA target sequence and cause the bound DNA to bend into an angle. The SRYtarget sequence has been identified in the promoter region of genes controlling sexual differentiation such as Mullerian inhibiting substance and P450 aromatase, an enzyme that converts testosterone to estradi- T PHENOTYPE FIG. 2. Model of mammalian sex determination. Male development is imposed on a fetus that would inherently develop ovaries and a female phenotype. TheY chromosome with its testis-determining gene, SRY, induces the indifferent fetal gonads to differentiate into testes. The testes secrete Mulierian inhibiting substance (MIS) and testosterone (T) which give rise to the male phenotype. ol. Furthermore it is present in the promoter region of SRY itself suggesting a positive feedback loop. It took over three decades from the recognization of the Y as testis-determining to the identification of SRY as TDF. The cloning of SRY is undoubtedly a milestone in our understanding of mammalian sex determination. The difficult job of deciphering how SRY regulates transcription and identifying the genes upstream and downstream of SRY in the sex determination cascade must now be addressed. Suggested Reading Affara NA. Sex and the single Y. BioEssays 1991;13(9):475-478. Koopman P, Gubbay J, Vivian N, Goodfellow P, Lovell-Badge R. Male development of chromosomally female mice transgenic for SRY. Nature 1991;351:117-121. Mclaren A. Sex determination in mammals. Trends in Genetics 1988;4(6): 153-157. Mclaren A. What makes a man a man? Nature 1990;346:216-217. Copyright © American Society of Andrology Are there specific genetic defects affecting the male reproductive tract? What are the underlying molecular mechanisms? Androgen insensitivity, Turner's and Klinefelter's syndrome, chromosomes, gene loci of Mullerian inhibiting substance (MIS) bytesticular tissue causes variable degrees of bilateral Mullerian duct regression, whereas local secretion of high concentrations of testosterone are required for ipsilateral development of the Wolffian ducts. If a uterus is present on one side, an associated fallopian tube is often also present. Approximately half of subjects develop a uterus, but the cervix may be absent. The external genitalia are usually ambiguous, although relatively normal male or female appearance is possible. Hypospadias, cryptorchidism or an inguinal hernia containing a gonad or Mullerian remnant may also occur. The majority of true hermaphrodites are raised as males due to the external appearance of the genitalia, even though over 50% of subjects have a 46XX karyotype. Other karyotypes, such as 46XY, 46XX/XY chimerism or various forms of mosaicism, may be present. Many 46XX true hermaphrodites do not possess the SRY gene suggesting that the etiology of 46XX true hermaphroditism differs from that of 46XX males who have a translocation of the Y chromosomal SRY gene locus. Klinefelter Syndrome: Seminiferous tubular dysgenesis occurring in 47XXY subjects with Klinefelter syndrome represents the most common cause of testicular failure, with an incidence of 1:1000 males. Prior to puberty, arm span is increased and upper-to-lower body segment ratio is decreased for age in affected subjects. They are often diagnosed as a result of personality disorders and mental retardation. Prepubertal subjects have small testes but the histology is generally normal for that age, except for a progressive tendency toward decreased numbers of spermatogonia. With the onset of puberty, gonadotropin (luteinizing hormone and follicle-stimulating hormone) concentrations in the serum increase but tes- Sex differentiation occurs as a sequential process during the first trimester of fetal life. Genetic sex (Fig. 1) is established at the time of fertilization, leading to development of gonadal sex and culminating in formation of sex phenotypes (Fig. 2). Under normal circumstances, chromosomal sex agrees with phenotypic sex; however, occasionally chromosomal sex differs or ambiguity occurs in the sex phenotype. Abnormalities of sex development are usually not life-threatening and occur at many levels. The clinical consequences of abnormalities occurring early in sex development may result in conditions of intersex whereas defects in more terminal phases of male development may be represented by isolated cryptorchidism (failure of testes to descend into the scrotum) or microphallus (normally formed, but abnormally small penis). Disorders of sex differentiation are often inherited as single gene mutations, and the analysis of these disorders has been especially informative in defining the molecular and genetic determinants of normal sex development. Gonadal Disorders True Hermaphroditism: The diagnosis of true hermaphroditism is predicated upon the presence of both testicular and ovarian tissue in the same individual. Oocytes should be present within the ovarian tissue. The majority of subjects have a testis or ovary on one side and a contralateral ovotestis containing both ovarian and testicular tissue (50%), or have a testis on one side and an ovary on the other (30%), or bilateral ovotestes, or even bilateral ovary and testis combinations (20%). The amount of functional testicular tissue determines the internal duct structures. Secretion 40 Handbook of Andro/ogy-Are there specific genetic defects affecting the male reproductive tract? ECG~SPERM GeneUc Sex • • Gonadal Sex Phenryprc~sex Internal rogenltal System r External Features External Genitalia ~ Secondary Sex Characterlsilics CNS r~ Pituitary Gonadotropin Secretion Sexual Behavior FIG. 1. Sequential events in the determination of genetic, gonadal, and phenotypic sex. tosterone levels remain relatively suppressed in accordance with the degree of testicular failure. Whereas the onset of puberty often occurs at a normal age, secondary sexual changes may not progress to the normal adult stage. Gynecomastia occurs, probably due to the increased estradiol:testosterone ratio. In all cases, seminiferous tubular function is impaired and spermatogenesis is absent. Turner Syndrome: Turner syndrome, does not strictly qualify as a disorder of sex differentiation. In the classic case, the phenotype is female, but with an absence of secondary sexual characteristics typical of puberty. Subjects lack a normal X chromosome (45X karyotype) and their ovaries degenerate into streak structures. The common anomalies of Turner syndrome include short stature, epicanthal folds, high arched palate, low nuchal hair line, webbed neck, shield-like chest, coarctation of the aorta, ventricular septal defect, renal anomalies, pigmented nevi, lymphedema, hypoplastic nails and inverted nipples. XX Males: Subjects with an apparent 46XX karyotype but male phenotype result from the translocation of a fragment of the Y chromosome containing the testicular determining, SRY, gene to another chromosome, usually the X chromosome. Subjects may have undescended testes (15%) and hypospadias (1 0%) and usually have small testes that may be soft early in life but become firm with increasing age. Testicular histology reveals no spermatogonia, a decrease in the diameter of 41 the seminiferous tubules, and Leydig cell hyperplasia, similar to that in Klinefelter syndrome. 46XX male subjects are shorter than those (47XXY) with Klinefelter syndrome. Testosterone production is low whereas gonadotropin levels are elevated. XY Gonadal Dysgenesis: Gonadal dysgenesis may be of the "pure" or "mixed" form with the former referring to the presence of an aplastic or "streak" gonad on both sides and the latter referring most often to a unilateral streak gonad on one side and testicular tissue, usually within a dysgenetic testis, on the other side. The pure form may occur in subjects with a 46XY karyotype, whereas the mixed form commonly involves chromosomal mosaicism (45X, 46XY), but also occurs in 46XY subjects with variable degrees of functional testicular tissue in each of the gonads. The etiology may be deletion of the Y chromosome or deletion or mutation affecting the SRY gene. Female Pseudohermaphroditism Female pseudohermaphroditism occurs when the external genitalia are virilized in a female subject with a 46XX karyotype. These subjects have ovaries; virilization is caused by excessive androgen of extragonadal origin. The most common etiology is increased adrenal androgen secretion as a consequence of congenital virilizing adrenal hyperplasia (CVAH). The predominant form of CVAH is 21-hydroxylase (cytochrome P450c21) deficiency which accounts for 8090% of female pseudohermaphroditism (Fig. 3). In addition to increased adrenal androgen (dehyd roepiand rosterone and androstenedione) and reduced cortisol secretion, severe deficiencies in P450c21 also result in salt-losing nephropathy due to coincident reductions in mineralocorticoid (aldosterone and its precursors) synthesis. Less frequently, neonatal genital ambiguity may result from 11 [3-hydroxylase (P450c11 and P450c18) or 3[3-hydroxysteroid dehydrogenase/ 6 5 -6 4 isomerase (3[3-HSD) deficiency. The former condition is often accompanied by hypertension (low aldosterone, normal/high deoxycorticosterone) and the latter may involve coincident 42 Handbook of Andrology-Are there specific genetic defects affecting the male reproductive tract? UndlfferanUated Karyotype XX~ Gonad~ XV ~ I I .., TOF No Testosterone I Internal 1 ducts _ _ ~ __ 1 I Wolffian r1 --.. No Masculinization J..- Wolffian Development Mullerian Development ~e_[~l~n.J - - Testosterone I _1 / "' DHT ~Andr~en Receptor .} Gene Expre&Sion Mascullnzlatlon of Urogenital sinus & External genitalia Determinants of normal male and female sex differentiation in the human. In the presence of FIG. 2. the testis determining factor (TDF) gene the undifferentiated gonad in the male undergoes testis determination. Sertoli cells develop and secrete Mullerian inhibiting substance (MIS) that promotes regression of Mullerian structures. Leydig cells in the testis produce testosterone (T) which acts locally to promote Wolffian duct development. By four months in the female, the male ducts have disappeared and the Mullerian ducts have fused to form the fallopian tubes, uterus and upper two-thirds of the vagina. In the male, the Mullerian ducts have regressed and testosterone from the ipsilateral testis has stimulated the Wolffian ducts to proliferate, forming the epididymides, vasa deferens, and seminal vesicles. Testosterone carried by the fetal circulation is converted locally to dihydrotestosterone which interacts with androgen receptors to promote masculinization of the external genitalia. The genital tubercle grows to form the penis. The urethral folds fuse and are incorporated to form the urethra while the labioscrotal folds fuse to form the scrotum. Later in gestation, the testes descend into the scrotum. salt-losing nephropathy (low deoxycorticosterone ~aldosterone). In rare cases, excessive transplacental passage of androgen, either from an exogenous source or from pathologic maternal production, has been reported to cause masculinization of the genitalia of a female in utero. The external genitalia of females may also appear to be virilized in association with other congenital anomalies unrelated to steroid hormone effects and most often include imperforate anus, renal agenesis and malformations of the lower intestine and urinary tract. Male Pseudohermaphroditism Sexual ambiguity in the presence of symmetric gonads in a 46XY individual with testes is classified as male pseudohermaphroditism. This condition may be associated with varying degrees of incomplete external and internal virilization. Handbook of Andrology-Are there specific genetic defects affecting the male reproductive tract? 43 Cholesterol I P,.;Oscc I P450c17 Andrc>sf--5-ene--38, 17~iol 5 30-HSD /!1 - A•- Isomerase Proglsterone1 ~17~H -: 7;'-~~ne.= Prog;steroneU / 1 .-~~------P-45_0_c_2_1--~~~) 1 ~ Deoxycorticosterone ~ Corticosterone ~--~----&rR---ed--u+~~se------~--. l P450aro 11-deoxycortisol \ Androstanedione - - I P450c18 and P450c11 ~ Cortisol -'Te-fecone \ l IP450aro I ) Dihydrotestosterone f Estrone - Estradiol 18-DH Corticostemne I I P450c18 t Aldosterone FIG. 3. Schematic representation of the major steroidogenic pathways. Those enzymes essential in testosterone biosynthesis by testicular Leydig cells are discussed in the text. Cytochrome P450c21 catalyzes 21-hydroxylation in the adrenal gland and is encoded by the CYP21 8 gene. Cytochrome P450c18 mediates 11 [3-hydroxylation and the further reactions involved in the biosynthesis of aldosterone. This enzyme is encoded by the CYP11 82 gene which is expressed only in the adrenal glomerulosa. Cytochrome P450c11 is encoded by the CYP11 81 gene and exhibits exclusively 11 [3-hydroxylase activity. Cytochrome P450aro catalyzes the aromatization of li 4 -3-keto C19-steroids into estrogens and is encoded by the CYP19 gene. Sa-reductase activity is expressed predominantly in extragonadal tissues and is also discussed in the text. Disorders of Androgen Biosynthesis: These disorders affect the virilization of the internal and external genitalia of the male embryo but do not interfere with regression of the MOilerian system. These defects may be of variable severity, partial or complete, and may present at puberty as well as in the newborn period. Variable degrees of ambiguity, from complete feminization to mild hypospadias, may be present at birth. All but one of the enzymes (Fig. 3) involved in these defects are present in both the gonad and the adrenal and the primary symptoms of hypertension and/or severe renal salt loss in an affected subject may be those of congenital adrenal hyperplasia. In both cholesterol desmolase (cholesterol side chain cleavage; P450scc) and 17a-hydroxylase/17,20-lyase (P450c17) deficiencies, male infants are undervirilized due to decreased testosterone synthesis. By contrast, the occurrence of these same enzyme deficiencies in a female infant would not affect the otherwise female external genitalia. In 3[3-HSD deficiency, male infants are undervirilized whereas female infants are virilized. Severe deficiencies of P450scc and 3[3-HSD lead to extreme salt-loss due to deficits in mineralocorticoid synthesis, and diminished P450c17 activity results in hypertension. Defects of 17[3-hydroxysteroid dehydrogenase (17[3-HSD), an enzyme present in the gonad but not adrenal, result in deficient male genital 44 Handbook of Androfogy-Are there specific genetic defects affecting the male reproductive tract? development and these subjects with ambiguous genitalia may virilize at puberty. Defects in Androgen Action: Abnormalities of androgen effect can be characterized as those due to defects of the androgen receptor, both partial and complete androgen insensitivity, and to deficiency of Sa-reductase enzyme activity. a. Sa-Reductase Deficiency: Male pseudohermaphroditism may result from inadequate conversion of testosterone to dihydrotestosterone due to deficiency of steroid Sa-reductase 2 isoenzyme activity. Inadequate concentrations of dihydrotestosterone within the genital tubercle and labioscrotal folds lead to only partial masculinization of the external genitalia. Sa-Reductase activity in the fetal genital area peaks between weeks 7-12 of fetal life when masculinization of the male genitalia takes place. Later androgen exposure fails to correct any defect during this period. This condition is also referred to as pseudovaginal perineoscrotal hypospadias because of the specific anatomical ambiguity most often observed. Testicular testosterone and Mullerian inhibiting substance (MIS) production is normal so that Mullerian regression occurs and internal Wolffian structures develop to varying degrees. However, the sperm carrying ducts end blindly before the prostate gland, so even if spermatogenesis occurs, the ejaculate is azoospermic. Inheritance is autosomal recessive and is common among some ethnic groups due to consanguinity. In the undiagnosed subject or those in whom orchiectomy is not accomplished by the age of puberty, the ambiguous genitalia become further virilized with phallic growth and development of a muscular male habitus and male body hair patterns. Hormonal profiles include normal or elevated testosterone levels with low DHT levels in relation to testosterone and a high ratio of Sf3- to Sa-reduced urinary steroid metabolites. Stimulation with human chorionic gonadotropin further accentuates this altered ratio. b. Complete androgen insensitivity (CAIS) is characterized by the development of female external genitalia and failure to masculinize the Wolffian system in a subject with a 46XY karyotype. Inguinal or labial testes may be pal- pable, although they may only be discovered during exploration of an apparent inguinal hernia. The vagina is short due to secretion of MIS by testicular Sertoli cells. Increased testicular stimulation by elevated gonadotropins at puberty results in normal or elevated testosterone levels to which the subject is nonresponsive. However, the peripheral aromatization (P4SOaro; Fig. 3) of testosterone and androstenedione in skin and adipose tissue leads to normal or elevated levels of estrogens (estradiol and estrone) which promote female breast development when unopposed by androgen action. Sexual and body hair is scant. If the diagnosis is not made before puberty, primary amenhorrea or infertility may be the presenting complaint. Additional studies may include in vitro androgen receptor binding measurements, hCG stimulation of testicular androgen secretion, or assessment in vivo of testosterone effect. The abnormality lies with a molecular defect in the X-chromosomal androgen receptor gene causing an abnormality in receptor function. There is an increased risk of testicular tumors in CAIS and, therefore, orchidectomy should be performed by the end of the second decade of life, following completion of puberty. However, if there is a possibility that the subject has a partial form of androgen insensitivity (PAIS), with the risk of masculinization during puberty, the testes should be removed prior to that time. Carcinoma in situ as evidenced by abnormal morphology of germ cells has been observed in testes of a few subjects with AIS during adolescence. Later in life, adenomataus transformation of both Sertoli and Leydig cells has been reported to occur. c. Partial androgen insensitivity presents with highly variable degrees of virilization. The phenotype ranges from slightly virilized female genitalia, to penile hypospadias, undescended testes and adolescent gynecomastia, to micropenis, and to isolated infertility. Subjects who present with ambiguous genitalia or micropenis in the neonatal period may have hormonal profiles of elevated testosterone, luteinizing hormone and follicle stimulating hormone, which are characteristic of androgen insensitivity. Further diagnostic testing involves Handbook of Andrology-Are there specific genetic defects affecting the male reproductive tract? androgen stimulation in vivo. The lack of detectable or adequate penile growth in response to androgen is consistent with the diagnosis of androgen insensitivity. Marked ambiguity and biochemical evidence of severe androgen insensitivity dictates a female sex of rearing. In addition, a partial defect may allow further masculinization at puberty in response to increased testosterone secretion and therefore, gonadectomy should be performed prior to puberty to prevent this occurrence. Mutations in the androgen receptor gene are responsible for the various presentations of PAIS. d. Hypospadias or micropenis may occur as isolated phenotypic events or in association with the observance of sexual ambiguity. Hypospadias is defined as failure of complete development and incorporation of the penile ure- 45 thra within the shaft of the penis. The urethral opening may therefore be at any position on the ventral surface of the penis from the perineum to the glans. The position of the urethral opening forms the basis of classification as a glandular, coronal, distal or midshaft, penoscrotal or perineoscotal hypospadias. Because hypospadias reflects the failure of androgenstimulated midline fusion, it represents a form of ambiguous genitalia. Its estimated occurrence is 8 per 1000 males. Micropenis refers to the presence of a fully formed but small penis in the absence of other abnormalities of sex differentiation. The definition is statistical and refers to a penis which is 2.5 SO below the normal standards for age and stage of pubertal development. Normal stretched penile length for newborns is 2.8 to 4.2 em. The lower limit for 2.5 SO is 1.9 em. Suggested Reading Brown TR, Scherer PA, Chang Y-T, Migeon CJ, Ghirri P, Murano K, Zhou Z. Molecular genetics of human androgen insensitivity. Eur J Pediatr 1993;152:S62. Hum OW, Miller WL. Transcriptional regulation of human genes for steroidogenic enzymes. C/in Chem 1993;39:333. Lee MM, Donahoe PK. Mullerian inhibiting substance: a gonadal hormone with multiple functions. Endocr Rev 1993; 14:152. Migeon CJ, Berkowitz GB, Brown TR. Male sex differentiation and development. In: Kappy M, Blizzard R, Migeon CJ, eds. The Diagnosis and Treatment of Endocrine Disorders in Childhood and Adolescence. Springfield, IL: Thomas. 1994: Chap. 12. Thigpen AE, Davis DL, Milatovich A, Mendonca BB, lmperato-McKinley J, Griffin JE, Francke U, Wilson JD, Russell OW. Molecular genetics of steroid Sa-reductase deficiency. J Clin Invest 1992;90:799. Copyright © American Society of Andrology Is there a trigger for puberty in the male? Should early or delayed puberty be treated? If so, how? Early, normal, delayed puberty, treatment Physiology of puberty Precocious Puberty Reproductive function in man starts with sex differentiation in fetal life and is followed by maturation at puberty and then heterosexual intercourse in adulthood. The same basic phenomenon governs all these aspects of reproductive function. Its starting point is the "gonadotropin releasing hormone (GnRH) pulse regulator", described by Knobil, located in the arcuate nucleus of the medial basal hypothalamus. The pulsatile secretion of GnRH activates the pulsatile secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) which in turn activate the secretion of testosterone by the Leydig cells in the testes. A restraining system modulates the function of the GnRH pulse regulator. During fetal life and in the neonatal period there is very little restraint. By 4 to 6 months of age, greater (but not complete) control is imposed over the pulse regulator, and this degree of repression is maintained until the early stages of puberty at which time there is a progressive lessening of restraint. An important question remaining to be answered about the physiology of puberty is where the restraining system originates, and how it modulates the pulse regulator. Although testicular maturation or "gonadarche" is the main hormonal event in puberty, it is preceded by 6 to 12 months of adrenal androgen secretion or "adrenarche". The trigger of adrenarche is probably a pituitary peptide related to, but different from, adrenocorticotropic hormone (ACTH). This putative hormone has been termed adrenal androgen stimulation hormone (AASH). The modulator of AASH secretion is as elusive as that of the GnRH pulse regulator. Puberty begins in boys at 9 to 14 years and is completed within 3 to 4.5 years. Table 1 lists the developmental stages of external genitalia and pubic hair as described by Tanner. When the first signs of puberty occur prior to 9 years of age in boys, puberty is considered precocious. The precocity is called central if its etiology is related to an early activation of the hypothalamus and pituitary with episodic production of gonadotropins. Treatment with a long acting GnRH analogue will be effective in arresting the sexual maturation because constant levels of GnRH eventually turn off the endogenous secretion of LH. A magnetic resonance imaging scan (MRI) may show abnormalities of the central nervous system (CNS). Often precocious puberty is related to a benign hamartoma. Rarely, a malignant tumor is discovered in which case surgery is indicated. When the MRI reveals no CNS abnormality the precocity is referred to as idiopathic. Precocious puberty is called peripheral when it occurs in the absence of secretion of pituitary LH/FSH. In rare cases, a tumor (such as a hepatoma) can secrete human chorionic gonadotropin (hCG) which in turn activates the secretion of testosterone by the Leydig cells. Also rare are the tumors of Leydig cells which produce testosterone. Adrenal tumors can be virilizing. One can also encounter a mild form of congenital adrenal hyperplasia which produces signs of virilism. Finally, patients with McCune-Albright Syndrome show the triad of precocious puberty, bone fibrous dysplasia, and cafe au lait spots of the skin. The etiology of this syndrome is thought to be a mutation of the G-proteins which are coupled to various membrane receptors. Delayed Puberty Puberty is considered delayed in boys when there is no noticeable enlargement of the testes by 14 years of age. As with precocious puberty, delayed puberty can be due to central 46 Handbook of Andro/ogy-ls there a trigger for puberty in the male? 47 Table 1. Developmental stages of puberty Stage* Mean Age (years ± SD) G2 P2 11.6 + 1.1 13.4 + 1.1 Testes 2.5-3.2 em; thinning of scrotum Sparse, long G3 P3 12.8 + 1.1 13.9 + 1.5 Testes 3.3-4.0 em; pigmentation of scrotum; penis enlarged Darker, curlier, coarser G4 P4 13.7 + 1.1 14.3 + 1.1 Testes 4.1 em; further penile enlargement Extend to pubis G5 P5 14.9 + 1.6 15.1 + 1.1 Testes 4.5 em; adult penis Adult G gonadal stage, P pubic hair stage, G1: prepubertal testes, P1: no pubic hair causes (hypogonadotropic hypogonadism) or peripheral causes (hypergonadotropic hypogonadism). The lack of gonadotrophic secretion may be secondary to various CNS tumors or destructive disorders (histiocytosis, sarcoidosis, Lupus). It can also be related to congenital malformations of the brain such as pituitary aplasia, Kallmann syndrome, septo-optic dysplasia, or to head trauma with hemorrhage. In some of these patients, it might be possible to re-establish episodic LH secretion by episodic administration of GnRH. Such therapy is rather cumbersome and usually patients choose testosterone replacement treatment using an intramuscular injection of testosterone enanthate every 3-4 weeks. Primary gonadal failure is associated with hypersecretion of LH/FSH, hence the term hypergonadotropic hypogonadism. This situation can result from several abnormalities of sex chromosomes such as Klinefelter syndrome (47,XXY) and its variants including the socalled "46-XX males" in whom the Y-chromosome gene responsible for testicular determination (SRY) has been translocated to the pseudoautosomal region of an X-chromosome. Testicular failure can be due to bilateral trauma or tumor, radiation or an auto-immune disorder. Poorly explained cases of partial gonadal dysgenesis, Noonan Syndrome (46,XY,male Turner) and anarchia ("Vanishing Testes") will also result in hypergonadotropic hypogonadism. In most of these situations, the only possible therapy is testosterone replacement. Suggested Readings Germak JA, Knobil E. Control of puberty in the rhesus monkey. In: Grumback MM, Sizonenko PC, Aubert ML, eds. Control of the Onset of Puberty. Baltimore, MD: Williams and Wilkins Pub.; 1990:69. Grumbach MM, Sizonenko PC, Aubert ML, eds. Control of the Onset of Puberty. Baltimore, MD: Williams and Wilkins Pub.; 1990. Migeon CJ, Berkovitz GD, Fechner PY. Diagnosis of Pediatric Disorders in Biochemical Basis of Pediatric Disease. Soldin SJ, Rifai N, Hicks JMB, eds. Washington, D.C.: AACC Press Pub.; 1992:165. Kappy, M, Blizzard RM, Migeon CJ. Wilkins -The Diagnosis and Treatment of Endocrine Disorders in Childhood and Adolescence. 4th ed., Springfield, IL.: Charles C. Thomas Pub., 1994. Copyright © American Society of Andrology How is male infertility defined? How is it diagnosed? Epidemiology, causes, work-up (history, physical, lab tests) Defining infertility their infertility is unknown and could be congenital or acquired. Recognition of a male reproductive component in an infertile partnership is often delayed because, traditionally, women have been the primary focus of the infertility evaluation and have ready access to gynecological care; men are much more reluctant to seek advice. Men are also more apt to confuse fertility with sexual potency (the ability to have an erection), ejaculation and ability to perform sexually, and they assume that if they produce seminal fluid at orgasm then they also produce sperm. The known causes of male infertility are quite numerous but can be grouped into a moderate number of major categories (Table 1). In addition, a man may be mistakenly labelled as infertile because of failure to recognize subtle abnormalities in his sexual performance or in his partner's gynecologic function (Table 2). Infertility is defined as the inability of a sexually active, non-contracepting couple to achieve pregnancy in one year, the time in which about 90% of couples succeed. When a female is in her 20s, the average time to pregnancy is six months. This time frame reflects not only the limited few days in the middle of a woman's menstrual cycle when she ovulates and conception is possible, but also the fact that most conceptions do not survive beyond early embryonic development and are lost before a woman's next menstrual period. In addition, about 15% of couples with a clinical pregnancy go on to a spontaneous miscarriage. The female partner's reproductive age is also an important determinant of the man's ability to initiate pregnancy since the length of time required to establish pregnancy increases progressively with advancing maternal age. Fertilization of the egg is more difficult and early pregnancy loss is more frequent as a woman becomes older. As demonstrated from abortuses, chromosomal abnormalities from aging eggs are frequent among women with advancing maternal age, but there may also be uterine factors that contribute to early pregnancy loss. Among couples of reproductive age, about 10% are involuntarily infertile. Of such couples, about 30-50% are infertile because of male reproductive dysfunction and, not uncommonly, both partners have reproductive problems. An additional 40% of reproductiveage couples are infertile because of medically or surgically acquired problems, including voluntary sterilization. Thus, only about half of reproductive-age couples can easily achieve pregnancy. Clinical evaluation Considering all of the above issues, infertility requires a detailed evaluation of both partners. Meticulous attention to potential risk factors in the history plus a careful physical examination of the man can provide important clues to the origin of the problem(s) and guide the selection of laboratory tests and methods for subsequent treatment. In addition to assessing the state of virilization, presence of gynecomastia and phallic competence, the physician should also specifically document testicular size, presence of epididymis and vas deferens, prostate status, and whether a varicocele can be palpated and/or becomes evident following valsalva. With regard to testicular size, a calibrated orchidometer is recommended, rather than just length and width, as the volume of a sphere is a cubic function of the radius and a more accurate and convenient estimate of testicular mass. De- Causes of male infertility Male infertility is a multifactorial syndrome encompassing a wide variety of disorders. In more than half of infertile men, the cause of 48 49 Handbook of Andrology-How is male infertility defined? Table 1. Known causes of male infertility gonadotropin deficiency chromosome aberrations genetic disorders excurrent duct obstruction environmental toxicants alcohol/recreational drugs medications, chemotherapy, radiation systemic illnesses infectious disease (systemic and genital) neurologic disorders varicocele autoimmune disease impotence creased testicular volume and turgor (atrophy) provide important clinical clues to reduced testicular germ cell content. Laboratory evaluation Laboratory testing provides additional insight into both the extent and mechanism of testicular dysfunction (Fig. 1). The hormonal profile is essential in differentiating gonadotropin deficiency from primary testicular dysfunction. Regardless of cause, as the testicle fails, the serum follicle-stimulating hormone (FSH) level rises in proportion to the amount of spermatogenic tissue lost, while the serum luteinizing hormone (LH) level increases only when testicular dysfunction is severe. The testosterone level is maintained within the normal range, even in many men with clinical hypogonadism, because sex hormone binding globulin levels become markedly elevated in response to decreased androgen production and increased estrogen concentrations. However, the free or unbound testosterone level decreases. In contrast, disorders due to gonadotropin deficiency are characterized by a profound fall in testosterone level and a failure of reciprocal increases in FSH and LH. While prolactin concentration is elevated in the presence of a prolactin producing pituitary adenoma, and in some men with acromegaly, the production of this hormone remains unchanged in other testicular disorders. Obstruction of the excurrent ducts (epididymis, vas deferens and ejaculatory ducts) is characterized by the triad of azoospermia, Table 2. Subtle abnormalities which can lead to an erroneous diagnosis of male infertility poor coital timing retrograde ejaculation or anejaculation anovulation, despite cyclic menses corpus luteum deficiency pelvic adhesions/tubal disease recurrent early embryo loss normal testicular size and a normal serum FSH level. In this setting, a testicular biopsy is essential in order to demonstrate complete spermatogenic progression. The anatomical site of the obstruction can then be determined using a combination of procedures such as vasogram, scrotal and rectal ultrasound, and scrotal exploration with sampling of ductal fluids. In the special case of congenital absence of the vas deferens, seminal vesicles and Normal Aplasia Klinefelter Cirrhotic Castrate FIG. 1. Schematic representation of hormonal relationships with progressive degrees of testicular dysfunction. 50 Handbook of Andrology-How is male infertility defined? Table 3. Traditional semen characteristics in normal men sperm concentration volume motility forward progression morphology viability >20 x 106/ml :o>2.0 ml >50% >3 (scale 1-4) :o>30% normal forms :o>75% ejaculatory ducts, semen is uniquely characterized by a small volume of non-coagulating seminal fluid which lacks fructose. In the majority of infertile men, detailed semen analyses are required to fully characterize their reproductive dysfunction. Several important caveats are worthy of note. Semen should be collected with a consistent controlled abstinence interval (36 to 48 hours are recommended). Sperm count, motility and other characteristics change with prolonged abstinence, making comparison between samples and between different men misleading. Statistically, three semen samples are required to establish a stable estimate of values because of inherent variability of this excretory function. In addition, some noxious influences on testicular function (hot baths, viral illnesses, and toxicants) may produce transient effects on semen quality which can last for 1 to 2 sperm cycles (3 to 6 months) necessitating a moderately long term basal evaluation, especially when contemplating a therapeutic intervention. Conventionally, semen analysis includes measurement of sperm concentration, semen volume, percent of motile sperm, quality of forward progression of these motile sperm, viability and morphology (Table 3). Recently, computer-assisted sperm analysis (CASA) has become available, providing more sophisticated measures of sperm motion, such as velocity, linearity and lateral head displacement. This automated method requires considerable technical attention to semen dilution and randomized cell sampling to avoid selection bias. With regard to the various semen parameters, there is clearly a progressive increase in the frequency of male infertility as values for sperm concentration, motility and Table 4. Specialized tests of sperm function sperm autoantibodies hypo-osmotic sperm tail swelling reactive oxygen species hyperactivation of sperm motility acrosin content acrosome reaction hamster egg sperm penetration assay hemizona binding computer-assisted sperm analysis (CASA) cervical mucus penetration morphology by strict criteria morphology deteriorate. However, there are many exceptions. Some men with oligospermia (low count) can easily impregnate their partner, and other men with normal semen parameters are infertile. The above paradox has stimulated the development of a number of specialized sperm function tests which provide considerable information beyond the traditional semen parameters (Table 4). Sperm count and motility are primarily bulk parameters, while newer measures address cell membrane integrity, sperm capacitation and ability to acrosome react as well as sperm-egg interaction. With the advent of in vitro fertilization, we can now directly assess sperm fertilizing ability. We have come to recognize that male fertility involves a complex series of events, wherein abnormalities in one or more steps block the ability of that man to initiate a viable pregnancy (Table 5). Table 5. Biological events normally required to obtain pregnancy vaginal deposition of sperm at ovulation vigorous sperm motility cervical mucus penetration sperm capacitation zona pellucida binding acrosome reaction zona penetration oolemma binding ovum penetration sperm nucleus decondensation syngamy embryo development uterine implantation embryo and fetal survival Handbook of Andrology-How is male infertility defined? 51 Suggested Reading Liu, DY and Baker HWG. Tests of human sperm function and fertilization in vitro. Ferti/ Steri/ 1992;58:465-483. Clark RV, Sherins RJ. Male infertility. In: K.L. Becker, ed. Principles and Practice of Endocrinology and Metabolism. Philadelphia: J.B. Lippincott Co.; 1990:985-991. Burris AS, Clark RV, Vantman OJ, Sherins RJ. A low sperm concentration does not preclude fertility in men with isolated hypogonadotropic hypogonadism after gonadotropin therapy. Fertil Steri/ 1988;50:343-347. Calvo L, Dennison-Lagos L, Banks SM, Dorfmann A, Thorsell LP, Bustillo M, Schulman JD and Sherins RJ. Acrosome reaction inducibility predicts fertilization success at IVF. Hum Rep rod 1994;9: 1880-1886. Sherins RJ. Clinical use and misuse of automated semen analysis. New York Academy of Science 1991 ;637:424-435. Copyright © American Society of Andro/ogy What are the existing and future therapeutic approaches for male infertility? When should IVF be used for male infertility? What is the role for psychological counselling for infertility? Treatment -medical, empirical, surgical, alternative, adoption, donor, psychological tuitary tumor can also result in a lack of production of LH and FSH by the pituitary with a subsequent drop in testosterone production in the testicle and loss of sperm production. Bromocriptine suppression of a prolactin-producing tumor is highly successful in restoring both normal hormone levels and sperm production. An initial dose of 5 mg per day is gradually increased until side effects occur or there is normalization of gonadotropins and testosterone. Exogenous gonadotropin may still need to be used in these cases because the tumor, or treatment of the tumor with surgery or radiation therapy, can cause destruction of the pituitary itself. Finally, other effective specific medical treatments include eradication of infection with antibiotics and decreasing antisperm antibodies with corticosteroids. Although treating antisperm antibodies with corticosteroids is treatment for a specific problem, it needs to be emphasized that this treatment is controversial because the effectiveness is sporadic, and the steroids themselves can have serious side effects (e.g., aseptic hip necrosis). Empiric medical therapy involves administration of an agent that somehow supports the normal processes of sperm production in a man who is infertile, but who has normal hormone levels. Approaches used include estrogen receptor blockers (e.g., tamoxifen, clomiphene citrate) to stimulate the pituitary to increase LH and FSH release, with a resultant increase in intratesticular testosterone production. Chemicals known to artificially improve sperm motility or appearance in vitro, such as the protease kallikrein or the phosphodiesterase inhibitor pentoxifylline, have been given systemically in an attempt to improve sperm function. However, it must be stressed that empiric therapies are, in general, not successful in improving male fertility when evaluated in con- Infertility affects approximately 10-15% of all reproductive-age couples and a male factor is present in 40-50% of those couples. Specific interventions to treat an abnormality in the male partner is not possible for some affected couples. Fortunately, assisted reproductive techniques (ART) can help bypass the abnormality in many patients without problems amenable to specific treatment. Current treatment of male factor infertility The key to treatment of male factor infertility is identification of a specific cause of abnormal fertility. Medical treatment Specific treatment of males with hormonal abnormalities is frequently effective. For men without production of gonadotropin releasing hormone (GnRH), and the subsequent lack of pituitary release of the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH), puberty is not achieved until exogenous LH, FSH or testosterone is given. Initial treatment with testosterone injections (in oil) will activate the onset of puberty and the development of secondary sexual characteristics. When the man is interested in fertility, sperm production may be stimulated by intramuscular replacement of the pituitary hormones LH (in the form of human chorionic gonadotropin, hCG) and FSH (as human menopausal gonadotropin, hMG; or recombinant human FSH). Replacement of deficient hypothalamic hormones may also be provided with pulsatile subcutaneous GnRH given by a small portable pump, but this treatment is awkward since the patient must carry around the pump at all times. Abnormal production of prolactin by a pi52 Handbook of Andrology-What are the existing and future therapeutic approaches for male infertility? Left internal spermatic vein 53 trolled trials. A semen analysis may be abnormal for many reasons and, in addition, sperm quality is highly variable in serial semen analyses from a single man over time. An apparent improvement in sperm production temporally related to an empiric intervention is more often due to natural variability than to an actual effect of the treatment on the man's fertility. Left testicular artery FIG. 1. Illustration of the relationships between the left renal vein and left internal spermatic vein important in causation of a varicocele. Varicoceles are much less common on the right side because of the direct drainage of the right internal spermatic vein into the vena cava. Surgical intervention Surgical intervention will allow correction of obstructions of the reproductive tract or of structural abnormalities that can affect sperm production, such as a varicocele. A varicocele results from enlarged testicular veins that are thought to occur because of reflux of blood from the left renal vein down into the left internal spermatic vein (Fig. 1). The reflux manifests clinically as enlargement of the scrotal testicular veins (Fig. 2}, which may be easily seen from across the room for a man with a large varicocele in a standing position. The enlarged veins are corrected by a direct surgical dissec- FIG. 2. Schematic illustration of the external appearance of a large left varicocele and the underlying enlargement of scrotal testicular veins. I 54 Handbook of Andrology-What are the existing and future therapeutic approaches for male infertility? tion of the vessels of the spermatic cord with division of the internal spermatic veins which prevents reflux of blood in the testicular veins; outflow of blood from the testis can then occur via the external spermatic veins and vasal veins. Alternatively, the veins can be obstructed angiographically via the inferior vena cava by retrograde sclerosis. Overall, there is an improvement in the semen analysis in two-thirds of patients after treatment of a varicocele. Obstructions in the epididymis or vas deferens can be microsurgically corrected. An operating microscope is helpful to accurately identify and reconstruct these structures that are only a fraction of a millimeter in luminal diameter. Obstructions of the epididymis or vas may be congenital, due to infection, or due to iatrogenic intervention, such as a prior inguinal hernia repair. Reversal of a vasectomy is typically very successful if a second blockage or "blowout" has not occurred in the epididymis. Vasal reanastomosis (i.e., vasovasostomy) under an operating microscope will yield patency rates above 90% (and subsequent pregnancy rates of 50-70%) in experienced hands, when sperm are present in the testicular side of the vas deferens. Pregnancy is not achieved by all couples that have undergone a successful vasectomy reversal because of antisperm antibodies, female factor infertility, restricture of the vas deferens and other poorly understood factors. Obstruction of the ejaculatory duct may also occur from congenital, infectious or iatrogenic reasons. Resection of a small area of the prostate and ejaculatory duct can relieve these obstructions. Highly successful results are possible when a specific obstruction or obstructions of the male reproductive tract can be identified and corrected. Bypassing abnormal sperm quality: assisted reproduction After all factors affecting male fertility have been corrected and pregnancy has not occurred, it is appropriate to use assisted reproductive techniques (ART), which include intrauterine insemination (lUI), in vitro fertilization (IVF), IVF with micromanipulation of sperm etc., in an attempt to improve interaction between sperm and egg and, thus, increase the chance of pregnancy. In selected cases, where the female partner has an abnormality that will require ART, it may be appropriate to proceed directly to these techniques. For example, if the female partner of a man with abnormal sperm quality has obstructed fallopian tubes and IVF will be necessary, it may be indicated to proceed directly to IVF without correcting the primary cause of abnormal male fertility. On the other hand, treating a correctable male problem can result in improved semen quality and better results at the time of IVF. In general, these interventions in male and female partners must be closely coordinated to optimize chances of achieving pregnancy with a minimum of treatments. Intrauterine insemination involves processing sperm into a small volume and placement of the washed, concentrated sperm specimen directly into the female partner's uterus, timed to the woman's ovulation. After 3-4 cycles (attempts) at lUI, pregnancy is rarely achieved. The overall results with lUI are little better than natural intercourse alone for male factor infertility. Specific success rates with lUI are also dependent on sperm quality. If very poor sperm motility is present, pregnancy rates are usually less than 10% over a total of three or four cycles. With only minor impairment of semen parameters, pregnancy rates approach 50%. These numbers differ by less than 5-1 0% from expected pregnancy rates for natural intercourse over 10-12 natural cycles. IVF involves stimulation of egg production in the female partner, followed by transvaginal ultrasoundguided egg retrieval from the ovaries. Eggs and sperm are then brought together outside of the body. Up to four fertilized eggs (embryos) are returned to the uterus after 2-3 days of incubation in vitro. Overall pregnancy rates of 1015% are achieved nationwide per attempt with IVF. Optimal pregnancy rates (up to 50% per attempt) can be achieved at a select number of centers in which injection of a single sperm into the egg is performed as part of IVF, an involved and expensive process referred to as intracytoplasmic sperm injection (ICSI). Handbook of Andrology-What are the existing and future therapeutic approaches for male infertility? Substitutive treatments In some cases, a couple will elect to use sperm provided by an anonymous donor or to proceed with adoption instead of having children that are genetically their own. This is a difficult decision to make, as one's sense of gender and identity sometimes are closely related to the ability to have children. In other cases, the extremely high cost of assisted reproductive techniques and male infertility treatment are not covered by insurance, and the only option for having children is to use donor sperm or proceed with adoption. The use of donor sperm is applied in those cases where male factor infertility cannot be treated to allow for pregnancy to occur, and/or assisted reproduction is unsuccessful or not an alternative for the couple. Sperm is provided anonymously from donors who are carefully screened by history for the presence of genetic and infectious diseases. Donated sperm are frozen for a quarantine period of at least 6 months to allow serial testing of the donor for the presence of HIV antibodies. The donor is also tested for hepatitis and other sexually transmissible diseases. The donor is usually identifiable by religious, ethnic and physical characteristics, as desired by the recipient couple. In some centers sperm from a designated donor can be used. However, donation by a known donor is often discouraged because of the potential legal paternity liability that could later occur. Psychological Counselling Male infertility is often a psychologically disruptive situation, as fertility is assumed to be natural and essentially automatic. In addition, any disturbance in male sexual or fertility functions is likely to deeply affect a man's sense of 55 gender identity. Although men are unlikely to immediately verbally express their psychological difficulty with the identification of a male fertility problem, jokes, denial and other seemingly inappropriate behavior are common. In other cases, depression may occur without an apparent cause. Any suspicion that the man may be psychologically affected by the identification of male infertility is an indication for referral for psychological evaluation. In addition, any couple considering lUI with donor sperm should consider psychological counselling. The issues of masculine identity for an infertile man, and lack of genetic parenthood may arise after the "donor child" is born and cause psychological difficulty for the father if not addressed prior to donor insemination. An additional problem for the couple who chooses substitutive treatments for infertility is what to tell friends, family and the child him/herself. All of these issues should be explored and discussed openly prior to the initiation of pregnancy. Summary Male infertility is a common problem and can be addressed successfully with a number of interventions. Direct treatment of the male problem, assisted reproduction, donor insemination, and adoption are all alternatives for management of this situation. Modern technological advances, including ICSI and microsurgical correction of obstructive problems, allow many couples who were not previously treatable to successfully have children. Future developments in diagnosing and treating subtle endocrinopathies, better methods of treating antisperm antibodies, and identification of environmental causes of infertility are expected and await the next generation of andrologists. Suggested Reading Pryor JL, Howards SS. Varicocele. Ural Clin North Am 1987;14:499-513. Van Steirteghem AC, Nagy Z, Joris H, et al. Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. Report of a second series of 300 consecutive treatment cycles. Hum Rep rod 1993;8: 1061. Lipshultz Ll, Howards SS, Buch JP. Male infertility. In: Gillenwater JY, Grayhack JT, Howards SS, Duckett JW, eds. Adult & Pediatric Urology, 2nd edition. St. Louis, MO: Mosby-Year Book; 1991. Copyright © American Society of Andrology How is fertility assessed in domestic animals? Infertility diagnosis in the different species, evaluation of the male for clinical management to 5 days of this transit time are required for maturation of sperm within the caput and corpus epididymidis, and the remaining 4 to 14 days are used for maintenance and storage within the cauda epididymidis until ejaculation or voiding concomitant with urination. For the species being considered, each spermatozoon has a similar but distinctive paddle-shaped head with a compact acrosome over the rostral portion of the nucleus, under the plasma membrane. Other characteristics are as depicted in Fig. 1 of the chapter on semen. Numerous detailed analyses of sperm morphology have been completed and, at least for boars and bulls, a number of specific spermatozoal defects have been linked with the inability of that spermatozoon to fertilize an oocyte or to produce a normal embryo. Daily sperm production typically is 10-19 x 10 6/g testis in bulls, dogs and stallions and 2125 x 106/g testis in sheep and swine. Because weight of a single testis can range up to > 700g for the pig, daily sperm production per male ranges from approximately 0.4 x 109 for dogs, 5-8 x 109 for bulls, rams and stallions, to 16 x 109 for boars. For dogs, the paired epididymides contain 2-5 x 109 sperm, whereas those of the bull or stallion contain 30-80 x 109 and those of boars and rams 100-130 x 109 sperm. Frequent ejaculation reduces the number of sperm stored in the cauda epididymidis and repetitive ejaculations in a single day could remove up to 50% of the number previously present. Endocrine regulation of reproductive function involves gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), folliclestimulating hormone (FSH), testosterone and estradiol (in a negative feedback control mechanism) as in other mammals. In most domestic or pet species there is diurnal and/or seasonal variation in the frequency or amplitude of pulsatile secretion of LH, and concomitant changes in the concentration of testos- Andrological or breeding soundness examinations are common for bulls, boars, dogs, rams and stallions. This chapter describes the comparative evaluation of anatomy and physiology for a breeding soundness examination (BSE). Further, the chapter summarizes the diagnosis of common reproductive problems. Species-specific reproductive function There are subtle anatomical differences in the basic structure and architecture of the testis and epididymis among species, and all, except the dog, have a full component of accessory sex glands (seminal vesicles, prostate and bulbourethral glands). The dog has only a prostate gland which, like that in man, is bulbous, surrounds the urethra, and with age often undergoes benign prostatic hypertrophy (BPH). Anatomically, there are three types of penis. In all cases, the penis remains in a sheath when non-erect. The boar, bull and ram have a fibroelastic penis which is semirigid even when non-erect and withdrawn into an S-shaped curve; erection is a result of engorgement of the corpus cavernosum. The stallion has an erectile penis, like the human, and undergoes substantial enlargement of length and diameter upon engorgement of the corpus cavernosum. In the dog, an os penis allows intromission prior to complete erection with vascular engorgement of the corpus cavernosum. This is accompanied by engorgement of the bulbus glandis, a specialized area of the corpus spongiosum. Muscular contractions of vaginal-vestibular muscles around the engorged bulbus glandis create a mechanical lock between the bitch and stud dog. In all species except the boar, the testes hang between the back legs. In the boar the testes are located on the caudal aspect of the back legs. Depending on the species, spermatogenesis requires 39 to 61 days and transit through the epididymis an additional 7 to 17 days. Two 56 Handbook of Andrology-How is fertility assessed in domestic animals? terone as measured in peripheral blood. This seasonal variation is especially significant in horses and sheep, where the female typically is sexually receptive only during a portion of the year. There is a distinct seasonal cycle to gonadal stimulation provided by the hypothalamic-pituitary axis, and sperm production fluctuates by 30-50% annually, although sperm are produced throughout the year. This endogenous seasonality of the reproductive cycles is synchronized by the effect of photoperiod length on animals. Why a breeding soundness examination? Semen collection from domestic and pet animals is relatively easy, which is a distinct advantage to the clinician. This is especially true of cattle for which there has been tremendous utilization of genetically superior sires for artificial insemination. A clinician evaluating a male before sale/purchase or before the start of a breeding season will perform a thorough clinical examination augmented by detailed examination of the reproductive organs and collection of one or more samples of semen. The question addressed is "Is this male likely to be of low fertility or, for other reasons, not useful as a breeding male at this time?'' If the answer is yes, because the male fails to pass one or more elements of the examination (Table 1), the owner would be advised not to use him for breeding at that time. The male, however, might pass a similar examination in 1-6 months. If the male passes the BSE, it is likely that the male will be of reasonable fertility when mated to normal females, but factors not detected (or subsequent changes) may intervene. In another situation, a male is presented because the owner is suspicious or knows that fertility is reduced relative to what it had been earlier or relative to normal males of that species, or that seminal quality is "poor." This is an all too frequent occurrence with valuable stud dogs, stallions or bulls. The clinician attempts to address two questions: "Why is semen quality or fertility, or sexual behaviour of this male low?" and "What is the prognosis for improvement?" In some cases, the prognosis and treatment may be obvious, but more typ- 57 ically a limited knowledge of reproductive pathophysiology or economic conditions preclude an effective treatment other than allowing the passage of time. Semen collection and analysis There are three standard methods for collection of semen. An artificial vagina (A V) can be used with a male of each species, and semen collected by deflecting the penis into the artificial vagina as the male mounts a teaser animal or phantom. The AVis a latex-lined cylinder which contains warm water to provide heat and/or pressure. This approach produces a very physiological condition, apparently satisfying to the male, and usually provides an ejaculum most representative of that particular male. Stallions and bulls are the prime candidates for the AV and the primary limitation to its use is that the male must be accustomed to the procedure. With boars and dogs, the male is allowed to mount an estrous female or phantom and the collector simply grasps the free end of the protruded penis to mimic the spiral interior of the cervix of the female pig or the encircling vaginal vestibular muscles of the female dog, and masturbates the male. The alternative method of seminal collection, useful with bulls and rams, is electroejaculation (EE). This is achieved by placing a probe transrectally over the innervation dorsal to the intrapelvic accessory sex glands, and applying a mild electrical stimulation in a rhythmic manner. Appropriate restraint of the male is required. The resulting ejaculum typically is more dilute than that obtained by an artificial vagina, but the motility and morphology of sperm is not altered by the procedure. It is important to recognize that collection of semen, especially with an A V, can be potentially dangerous to the collector or the stud male. More importantly, although the number and quality of spermatozoa collected may be diagnostic, especially if multiple samples are collected over the course of 2 to 4 hours or at a uniform interval over several days, absence of spermatozoa in a particular ejaculum does not mean the male is permanently abnormal or infertile. Occasionally a normal male will provide 58 Handbook of Andrology-How is fertility assessed in domestic animals? Table 1. Minimal normal values for breeding soundness examination parameters for domestic species. Parameter+ Stallion Bull Ram Stud Dog Clinical examination Normal Normal Normal Normal Motility >50% >30% >30% >70% Morphology (normal cells) Specific parameters >80% Not relevant >70% Scrotal circumference >70% >80% Scrotal circumference Not relevant Insemination dose (sperm number) 500xl06 Not checked Not checked 100 X 106 +Society for Theriogenology Handbook. 1994. Society for Theriogenology, American College of Theriogenologists, Hastings, NE. seminal fluid devoid of sperm, although frequent occurrence of this will be symptomatic of a disease state. In bulls and rams, ejaculation is essentially instantaneous, occurring over a few seconds with the semen discharged in a single jet. For boars, dogs and stallions, the ejaculatory process includes a series of emissions and ejaculations, and it is possible to collect the semen as a pre-sperm fraction, a sperm-rich fraction, and a post-sperm fraction. Evaluating infertility in stud animals In order to diagnose pathology in the male, a thorough history, clinical examination and semen examination are essential. The history of a subfertile male typically reflects multiple breedings and no offspring. If clinical examination reveals gross congenital or acquired lesions then the diagnosis of infertility is simple. However, a complete BSE must usually be performed to pinpoint a problem. The normal values of BSE parameters for several species are listed in Table 1. Clinical problems in our domestic animals can be grouped into a number of primary causes. Environmental effects might be the most common cause of reproductive problems in domestic animals. Heat is probably the greatest offender, especially in stallions, bulls, and boars. Rams are less affected because they are fall breeders. Stud dogs are usually kennelled in cooler areas; however, veterinarians see more cases of heat- and stress-induced oligospermia and increased numbers of mor- phologically abnormal cells. Excessively cold temperature can also limit a male's fertility, and freezing of testicular tissue results in sterility. Temperature-induced changes are diagnosed on history and BSE results. Therapy involves reversing the initial insult. Infectious agents are common problems, especially venereal diseases. Brucellosis is a venereal disease that causes epididymitis, orchitis and azoospermia. Brucellosis is primarily a problem in cattle, sheep and dogs and usually requires euthanizing affected animals. Other bacterial infections are common in stallions and stud dogs. Diseases are diagnosed by the presence of white blood cells, culture/ sensitivities and BSE. Therapy is as indicated by the culture and sensitivity. Neoplasms are rare in domestic animals except in the dog where Sertoli cell tumors and seminomas are seen. In older stud dogs, prostatic tumors are fairly common. Non-neoplastic prostatic disease (hyperplasia or prostatitis) is also common in the stud dog. Therapy of prostatitis, prostatic hyperplasia and prostatitic neoplasia are antibiotics, castration, and ablation, respectively. Trauma is a common problem, especially in the herd situation. This is easy to diagnose clinically and therapy involves surgical intervention or medical therapy. Parasitism, metabolic problems and congenital problems are less common. These are usually diagnosed on history and clinical presentation which then guide medical treatment. Congenital problems are usually heritable and these animals should be removed from the breeding program. Finally, lack of libido can Handbook of Andrology-How is fertility assessed in domestic animals? be a significant factor that is difficult to diagnose and treat. Summary Diagnosis of fertility problems in domestic animals is fairly simple if approached correctly. Clinical problems of domestic and pet animals can be grouped by primary cause, such as environmental effects, infections, neoplasms and trauma. Idiopathic azoospermia or infertility are also common and abnormalities secondary to congenital, metabolic or parasite problems also occur. Decreased semen quality due to high environmental temperature or humidity during the summer is a frequent problem in bulls, boars, dogs and stallions. Venereal diseases, including brucellosis, are common in dogs, cattle and sheep; epididym- 59 itis, orchitis and azoospermia typically result. Dogs and stallions commonly present other bacterial infections. The diagnosis of autoimmune and endocrinological problems is rare; however, we must be ever vigilant for their presence. The acquisition of a thorough history and completion of a comprehensive BSE will usually pinpoint the problem. Failure to produce offspring is the most common manifestation of a fertility problem and can lead to substantial financial loss for the owner. Potential problems can be minimized by use of a breeding soundness examination prior to purchase of a breeding male; diagnosis of a fertility problem can often be relatively simple. Factors such as humananimal bonding and economics, as well as the underlying etiology of the infertility problem, dictate the appropriate course of action. Suggested Reading Barth AD, Oko RJ. Abnormal Morphology of Bovine Spermatozoa. Ames, lA: Iowa State University Press; 1989. Cupps PT. Reproduction in Domestic Animals (4th Ed.). New York, NY: Academic Press Inc.; 1991. McKinnon A, Voss J, eds. Equine Reproduction. Philadelphia: Lea and Febiger; 1993. Morrow DA. Current Therapy in Theriogenology. Philadelphia, PA: W.B. Saunders Company; 1986. Roberts SJ. Veterinary Obstetrics and Genital Diseases (Theriogenology). MA: Roberts Woodstock; 1986. Copyright © American Society of Andrology What are the existing male contraceptives and what is the outlook for new ones? Androgens GnRH antagonists, antibodies to sperm surface antigens, compounds that act on sperm maturation in the epididymis Hormonal-based contraceptive approach- es Production of spermatozoa in the seminiferous tubules and of the male sex hormone, testosterone, by Leydig cells, depends on pituitary gonadotropins. The two pituitary gonadotropins responsible for testicular function are luteinizing hormone (LH) and follicle-stimulating hormone(FSH). These gonadotropins are regulated by the pulsatile release of the hypothalamic decapeptide gonadotropin-releasing hormone (GnRH). There is a finely tuned relationship between the testes and the pituitary-hypothalamic axis called negative feedback. It is based on the fact that testosterone or ovarian hormones administered to the male in large quantities will suppress pituitary-hypothalamic production and the release of gonadotropins which in turn will stop spermatogenesis. Historically, many combinations of androgens, progestins and estrogens have been utilized to stop normal pituitary function, an action that has been termed "pharmacological hypophysectomy". Recently, it has been shown that men injected with large doses of testosterone have a complete absence of spermatozoa in their ejaculates. This absence is termed azoospermia and is a highly effective contraceptive method. Testosterone-induced azoospermia occurs only in 60-70% of treated Caucasian men, but in a greater proportion of Chinese and Indonesian men (>90%). Most of the men who do not develop azoospermia become oligozoospermic and have very low sperm counts. Studies are now underway to ascertain whether severe, but incomplete, suppression of sperm production renders most of these men infertile. Studies on GnRH have included development of synthetic derivatives that mimic (agonists) or oppose (antagonists) the action of 60 GnRH. Agonistic compounds, when given in large doses, initially stimulate the pituitary gland to release LH and FSH but then suppress pituitary release of these gonadotropins, a phenomenon known as down-regulation. Despite this paradoxical decline in gonadotropin levels, agonistic analogs of GnRH have not been very effective at inducing azoospermia, at least at the doses used. However, exploratory clinical studies using GnRH antagonists in combination with androgens suggest that azoospermia can be reached more readily, and in a greater proportion of volunteers, than with any of the previous hormonal approaches. This data base must be substantially expanded before the true potential of this approach is realized. Two important factors must be kept in mind concerning the GnRH antagonist approach. First of all, androgen substitution therapy is needed because the use of antagonists leads to the loss of libido. Secondly, the current generation of antagonists is too expensive to be of practical contraceptive use. In addition to hormonal drug approaches, attempts are being made to utilize vaccines based on GnRH and FSH as potential male contraceptives. These approaches are in the early stages of clinical investigation and their full utility will not be known for several years. Direct inhibition of spermatogenesis Accidental observations have shown that the rapidly dividing cells within the seminiferous epithelium are exceedingly sensitive to a variety of chemicals. Unfortunately, the effect of many of these chemicals are not testesspecific and other end organs are affected, which can result in systemic toxicity. The best known agent that directly affects the testes is gossypol, a yellow pigment found in cotton seed oil. It has been extensively Handbook of Andro/ogy-What are the existing male contraceptives and what is the outlook for new ones? 61 Secretory neurones HYPOTHALAMUS I LHRH ' • • • • • - LHRHanalogues PITUITARY [ _ _ _G_o_n_a_d_o_tr-op_h_s____] 1...1 • I••• • FSH LH 't ' Androgen-dependent target tissues T ...,. 1 Leydig cells TESTIS Sex steroids Sertoli cells I Spermatogonia I Sperm!tocytes I ~ • • • Gossypol 1 • • • lndazole-3-carboxylic acid I spe atids 7 Spermatozoa I EPIDIDYMIS C---------------------J Spermatozoa . _ a-Chlorohydrin • • • 6-Chloro-6-deoxy sugars Sulphasalazine I Spermatozoa 1 "t • • • • FIG. 1. Condom • - Vasectomy Antibody Approaches to male contraception. studied in China and some other countries. This drug appears to have a very narrow safety margin and the effective dose must be closely controlled. Potassium metabolism and kidney function can be disturbed in individuals receiving this drug. Moreover, a relatively large proportion of men fail to regain their fertility after prolonged exposure. lndinopyridines represent a category of drugs that rapidly disrupt spermatogenesis without affecting male sex hormone production. Experiments are underway in animal models to determine the specificity and reversibility of the action of these compounds. The mechanisms by which these compounds act is not known at this time. 62 Handbook of Andrology-What are the existing male contraceptives and what is the outlook for new ones? Sperm antigen-based vaccines Interference with sperm maturation Spermatozoa contain a number of unique chemical components that are not observed in somatic cells and which can be utilized as antigens in vaccine development. Many of these antigens arise during the early stages of spermatogenesis before the fully formed spermatozoon is released into the lumen of the seminiferous tubule. When a male is immunized with these sperm components, antibodies against them penetrate into the seminiferous epithelium and react with the antigens of the early sperm cells resulting in an immunologically-mediated inflammatory process. If severe, this process destroys the seminiferous epithelium leading to permanent infertility. Women are potential recipients of antisperm vaccines. Immunized women will produce antibodies that attack spermatozoa that enter the female reproductive tract after coitus, thus preventing fertilization of the ovum. Spermatozoa acquire certain membrane components during their epididymal maturation process, after they are released from the seminiferous epithelium. These components have not been well characterized as yet, but, in theory, immunization against them should result in infertility although normal sperm production would continue. Conclusions Development of a practical male contraceptive agent is being studied by a number of investigators. Drug and vaccine approaches have had limited success to date and it appears unlikely that a male contraceptive will be available before the year 2000. A somewhat greater success may arise from methods that promote vasectomy as a contraceptive by making it more readily reversible. Suggested Reading Bernstein ME. Agents affecting the male reproductive system: effects of structure on activity. Drug Me tab Rev 1984; 15:941-996. Nieschlag E, Behre HM, Weinbauer GF. Hormonal male contraception: a real chance? In: Nieschlag E, Halbernich UF, eds. Spermatogenesis -Fertilization -Contraception: Molecular, Cellular and Endocrine Events in Male Reproduction. Heidelberg: Springer; 1992:447501. RayS, Verma P, Kumar A. Development of male fertility regulating agents. Med Res Rev 1991; 11 :437-472. Swerdloff RS, Wang C, Bhasins. Male contraception: 1988 and beyond. In: Burger HG, de Kretser DM, eds. The Testis, 2nd edn. New York: Raven Press; 1989:547-568. Waites GMH. Male fertility regulation: the challenges for the year 2000. Brit Med Bu//1993;49: 210-221. Wu FCU. Male contraception: current status and future prospects. C/in Endocrinol1988;29: 443-475. Copyright © American Society of Andrology How prevalent is erectile dysfunction? What can be done to treat it? Erectile physiology, etiology, work-up and treatment of erectile dysfunction, psychological counselling of the cavernous tissue just beneath the tunica albuginea or lining of the corpus cavernosum. These venules form a number of veins traversing the tunica albuginea called emissary veins, which usually drain into the circumflex veins on the outer surface of the tunica albuginea, that in turn drain into the deep dorsal vein of the penis located in the dorsal midline of the penile shaft (Fig. 2, 3A). Functionally, in the flaccid condition, there is a high-resistance, low-flow arterial state in the cavernous tissue, primarily regulated by the contracted smooth muscles surrounding the cavernous spaces. lntracavernous pressure in this flaccid state is usually equal to resting venous pressure. With the initiation of erection, relaxation of the sinus and arterial smooth muscle occurs and a low-resistance system is produced with blood flow increasing to 6 -10 times that of the flaccid state. As the sinus spaces expand, the subtunical venules are collapsed beneath the tunica albuginea. The emissary veins are also further collapsed by the expanding tunica albuginea so that venous efflux is markedly decreased and intracavernous pressure rises to 80 to 100 mm Hg, which is the pressure necessary for rigidity (Fig. 3B). One of the essential recommendations to come from a National Institute of Health Consensus Development Conference on Impotence held in December 1992 was to educate health care providers and the public on aspects of human sexuality, sexual dysfunction, and availability of successful treatment. Impotence, or to use the more appropriate term, erectile dysfunction, affects approximately 30 million American men. It is only over the last decade that the rigors of a more scientific approach to this dysfunction of the corporeal cavernosal tissue have begun to unlock some of the causes of and treatment for this disorder. This effort was the result of interaction between multidisciplined clinical specialists, and basic scientists. Anatomy and physiology An erection is a psychosomatic-dependent event -an integration of several mutually occurring actions in several different systems (vascular, endocrine, and neurologic). Three sets of peripheral nerves have a role in erectile function: thoracolumbar sympathetic, sacral parasympathetic and sacral somatic. The most important neurotransmitter in initiation of penile erection may be nitric oxide, also known as endothelium-derived relaxing factor. The spongy erectile tissue is located in paired cylinders, called corpora cavernosa, which are located on the dorsum of the penis. The internal pudendal artery, which generally arises from the anterior division of the hypogastric or internal iliac artery, is usually the source for the penile arteries (Fig. 1). The corpora cavernosal sinus tissue is supplied by a slightly eccentric central vessel (the cavernosal artery), which is derived from the pudendal artery, and branches to form the helicine arteries (Fig. 2). The sinus spaces drain into a system of venules that coalesce on the outer surface Diagnosis The causes of male erectile dysfunction have traditionally been separated into two broad areas, organic and psychogenic. For the most part, this is a rather difficult distinction because the impact of this disorder on the psychological state of the patient is devastating and can make treatment directed at a specific physical defect difficult. Similarly, psychological disease such as depression may result in changes in the internal chemical milieu that produce a true organic effect on the cavernosal tissue. Organic causes of impotence can be classified as hormonal, neurogenic, 63 64 Handbook of Andrology- How prevalent is erectile dysfunction? Internal pudendal a~-- - Obturator a.-- Superficial --- ---perineal a.- I I I I I Ureth~al a. I I I I ~sal penile a. FIG. 1. Diagrammatic representation of the penile arterial blood supply. vascular (arteriogenic or venogenic) , or mus- of the patient is a crucial early step in the dicular, the latter involving disease of the agnostic process. smooth muscle of the cavernous sinus or tisLaboratory studies generally obtained in all sue. patients are serum testosterone and prolactin It is impossible to discuss diagnostic steps levels and urinalysis. Other laboratory tests without stressing how this process is tailored are tailored to the patient and include: comfor each individual and his partner. Not every plete blood count and serum chemical profile step is necessary for each patient. The vari- in the patient who has not had these recently; ous diagnostic steps will be presented as in- fasting blood sugar or glycosylated hemogloformation about what is available for the mod- bin in patients with a family history of diabetes ern evaluation of this disorder. As with other or who have this disease and are unsure of disease evaluations, taking a history of the their current disease control; lipid profiles in disorder for each individual is paramount be- patients with family history of lipid disorders or fore making any further diagnostic plans (Ta- with other vascular disease history, and cerble 1). Physical evaluation should concentrate tain other endocrine evaluations when history on the genital and rectal examination (Table or physical examination suggests the possi2). An evaluation of the psychological status bility of an endocrine disease. Many consider Handbook of Andro/ogy-How prevalent is erectile dysfunction? 65 FIG. 2. Cross-section of the anatomical components of the penis. the evaluation of nocturnal erection mandatory by either a home monitoring machine or in a formal sleep laboratory. All agree that these studies are useful in patients when a psychological cause is thought to be the primary etiology of the erectile dysfunction, or a major sleep disorder is suspected from the history. Measuring the erectile response of the patient to an intracavernous injection of a smooth muscle relaxant has become an important tool in the evaluation of erectile dysfunction. A full erectile response that lasts for thirty minutes is usually indicative of no major vascular nor sinus smooth muscle disease. A lack of response does not definitely establish organicity of the erectile dysfunction. Some patients with psychologic-based impotence or patients who are apprehensive in the testing situation will not respond with a full erection. Other more sophisticated diagnostic studies, some of which are, more invasive are listed in table 3. These other tests are more important for those patients who are considered candidates for more invasive therapeutic choices (e.g., arteriography if you are considering arevascularization procedure) . As mentioned above, the diagnostic workup is directly controlled by the therapeutic goal decisions made by the patient and his partner. FIG. 3. A. Diagrammatic representation of a wedge resection of the intracavernosal tissue showing the condition of the arteries, veins and constricted sinus spaces of the penis in the flaccid state. B. Diagrammatic representation of a wedgeshaped intracavernosal space during the erect state with relaxation of the sinus smooth muscles allowing for engorgement of the sinus spaces and collapse of the subtunical veins with flattening and collapsing of the emissary veins leading to poor or little flow to the circumflex veins. Treatment Today the focus of the physician who treats erectile dysfunction is to identify the probable primary etiologies of the condition in the individual patient and to design a therapeutic regimen that will deal with the physical and psychological aspects of the disease. Successful treatment for male erectile dysfunction depends on the motivations and goals of the patient and his partner. Rarely an endocrine or hormonal primary etiology is found and this problem is best managed by the endocrinologist. Treatment may 66 Handbook of Andrology-How prevalent is erectile dysfunction? Table 1. Evaluation of impotency History Genitourinary disease or surgery Symptoms of vascular or endocrine disease Systemic debilitating disease Neurologic disease Vascular, neurologic, spinal, or inguinal surgery Genital, pelvic, or spinal trauma Table 2. Evaluation of impotency Physical Exam Secondary sex characteristics Gynecomastia Genitalia Palpation of penile corporeal tissue for plaques Neurological Perineal and penis sensation to touch and pin prick Bulbocavernosus reflex Sleep disorders Psychologic history Marital and sexual history Nocturnal, early morning, nonintercourse erections Medications Pulses Femoral Distal extremities Rectal Prostate examination Tobacco or alcohol use Other drug use or abuse consist of parenteral testosterone therapy or therapy directed at specific endocrine disease. If the patient is a diabetic with poor control of his disease, sometimes return of this control will improve erectile function. Treatment of other conditions such as prostatitis, sleep disorders or hyperlipidemia sometimes leads to improvement in sexual function. Modification of medications, such as certain antihypertensives, or avoidance of substances such as tobacco and other drugs may be the most appropriate therapeutic intervention. Success with such oral medications, such as vitamin E or yohimbine has been anecdotal or not statistically verified in placebo-controlled trials. lntraurethral delivery of vasoactive agents is currently being studied. If psychological disease or disorder is the underlying etiology, the psychiatrist or sexual therapist becomes the primary caregiver for the patient and his partner. Vacuum/constriction devices are a therapeutic choice for almost any type of erectile dysfunction. They have become very popular as the first choice of therapy with very little risk for the patient, since these are purely external devices. Proper expectations from the devices and ongoing availability of the physician to an- swer questions about the device are important for success of this treatment. Intermittent home self-injection therapy with smooth muscle relaxants is the only accepted pharmaceutical therapy directed at the cavernosal tissue. The agents most commonly used for injection therapy are papaverine with or without phentolamine, prostaglandin E-1, or a combination of the three. These agents relax arterial and sinus smooth muscle tissue converting the corpora cavernosa to a low resistance, high flow system, and mimicking natural erection. Priapism (prolonged erection) and intracavernous fibrosis are the potential complications from this type of therapy, but with proper controlled use, these complications are neither common nor serious. This therapy has Table 3. Other diagnostic tests for evaluation of erectile dysfunction Neurologic (Sensory) Biothesiometry (measurement of vibratory sensation) Bulbocavernous reflex latency time Arterial Color duplex Doppler of cavernosal artery Penile plethysmography Pelvic and penile arteriography Venous Cavernosometry Cavernosography Handbook of Andrology-How prevalent is erectile dysfunction? become quite popular worldwide and has proven effective for patients with neurogenic impotence (who are quite sensitive to very low doses of the medication), medication-associated impotence, diabetes mellitus, minor or even moderate arteriogenic erectile dysfunction, and, under some controlled situations for patients with primarily psychogenic erectile dysfunction. Penile prostheses have been available as a treatment option for almost two decades now and have been placed in approximately 250,000 men in the United States. They are basically inflatable or noninflatable rigid or semi-rigid devices. The material that makes up most of these devices is solid silicone, not gel; polyurethanes are contained in one product. They are reliable (particularly with improved engineering) and resistant to wear. Nevertheless, they are prosthetics and are not guaranteed for life. Reoperation because of mechanical or surgical complications such as infection realistically occurs in 10-15% of patients over a 5-10 year period. Since this therapy is essentially non-reversible, the patient and partner should be carefully told what the 67 prosthesis can and cannot do, and the risks of reoperation and infection. Studies of patient and partner satisfaction, although infrequently reported in the literature, indicate high satisfaction for these prosthetic devices. Vascular surgery, either arterial revascularization or venous ablation surgery, have been presented as an option for treatment in a highly selected group of patients. Long-term success with this type of surgical intervention is still lacking and, for the most part, this surgery should be performed in centers with experience in this type of procedure. The Future What we know today about erectile dysfunction has only begun to scratch the surface. Some of the basic physiology and anatomy of the corpora cavernosa tissue remains to be discovered; pathophysiological details of erectile dysfunction are needed, particularly in regards to the function and integrity of the sinusoidal smooth muscle. The epidemiology of and risk factors for erectile dysfunction are also poorly understood. Suggested Readings de Groat WC, Steers WD. Neuroanatomy and neurophysiology of penile erection. In: Tanagho EA, Lue TF, McClure RD, eds. Contemporary Management of Impotence and Infertility. Baltimore: Williams and Wilkins; 1988:3-27. Krane RJ, Goldstein I, de Tejada IS. Impotence. N Eng/ J Med 1989;321 :1648-1659. Lewis RW. Erectile dysfunction. In: Stein B, ed. Practice of Urology 1993 UPDATE. Pennsylvania: W. W. Norton & Company; 1993:21-38. Lue TF. Impotence: a patient's goal-directed approach to treatment. World J Urol1990;8:67-74. Lue TF. Tanagho EM. Physiology of erection and pharmacological management of impotence. J Urol1987; 137:829-836. Copyright © American Society of Andrology Can spermatozoa be targets for drugs? If so, what are the consequences of such drug exposure? Is there a need for pre-conception counselling for men? Drugs that affect sperm structure or function, male-mediated developmental toxicity, prevention, tests to detect damage to spermatozoa offspring, paternal smoking has been associated with low birth weight and increased perinatal mortality. In addition, an increased incidence of childhood cancer has been associated with paternal occupational exposures. The exposure of men to motor vehicle exhaust fumes or the products of combustion engines has been associated with an increase in childhood leukemia. An increased occurrence of Wilm's tumour has been reported in the children of vehicle mechanics, auto body repairmen and welders. Thus, certain paternal chemical or drug exposures, including exposure to fuel combustion products, organic solvents and metals such as lead and mercury, are consistently associated with an elevated incidence of abnormal progeny outcomes. In various studies, the increased risk of an abnormal progeny outcome associated with a particular paternal occupation has ranged from 1.5 times to as high as 5 times the risk for the control group. However, there are a number of professions which have not been associated with an increased likelihood of abnormal progeny outcome. Therapeutic drug exposures are also of concern with respect to progeny outcome. After men are treated with anticancer drugs, there is a high incidence of transient or permanent infertility. However, when these men have fathered children, the proportion of malformed children has not been higher than in control groups. The inherent limitation to most epidemiological and clinical studies is an inability to identify the specific chemicals or to control the exposures. These difficulties can be circumvented using well controlled animal studies. There is convincing evidence from animal studies that paternal exposures to specific environmental or therapeutic agents result in a higher It is well established that there are dangers to the progeny associated with maternal exposure to a variety of chemicals and drugs. Interest in this area may be traced back to the discovery in the 1960s that women exposed to thalidomide during the first trimester of pregnancy had offspring with severe limb malformations. Can exposure of men to xenobiotics (foreign chemicals) also result in an increased incidence of adverse effects on progeny? Such adverse progeny outcomes might include early or late pregnancy loss, preterm delivery or delivery of a small-for-gestational age infant, malformations, behavioral abnormalities, or cancer. Two major approaches have been taken to identify instances in which paternal exposure to xenobiotics adversely affects progeny outcome, namely epidemiological studies and animal experiments. Epidemiological studies have focused principally on determining the effects of paternal occupational exposures on fetal development and childhood cancers. Paternal occupation as a motor vehicle mechanic is associated with an increased incidence of spontaneous abortions in the spouse. Fathers employed in occupations associated with solvent exposures are more likely to have offspring with anencephaly, with painters having the highest risk. Other paternal occupations which are associated with an increased risk of having a liveborn child with a birth defect include employment as a fireman, janitor, forestry and logging worker, printer, or plywood mill worker. Further, an increased risk of stillbirth, preterm delivery, or of delivery of a small-for-gestational age infant is associated with paternal employment in the art or textile industries. Although there is no definitive evidence that life style exposures, such as paternal smoking or alcohol consumption, cause birth defects in the 68 Handbook of Andrology-Can spermatozoa be targets for drugs? incidence of adverse progeny outcomes. A wide range of environmental chemicals (e.g., lead, dibromochloropropane) and drugs (e.g., the anticancer alkylating agent, cyclophosphamide) produce abnormal progeny outcomes after paternal exposure. Drugs or environmental chemicals to which the male is exposed may be present in his seminal fluid, and thus may have direct effects on the ovulated egg, on the process of fertilization, or on embryo development. Alternatively, drugs or other chemicals may have adverse effects on the fetus by "functionally" altering male germ cells. The adverse effects on progeny outcome which have been observed include preand post-implantation loss (spontaneous abortions), physical malformations evident at birth, 69 behavioral alterations, and a higher incidence of cancer later in life. Furthermore, it is of concern that the germ cell line of the progeny may be affected, thus increasing the risk for subsequent generations. An example of an experimental approach used to demonstrate the risks to progeny due to paternal exposure is treatment with the anticancer drug cyclophosphamide during spermatogenesis. It is apparent from both epidemiological and animal studies that there are paternal exposures to chemicals that can result in abnormal progeny outcome. Men exposed to certain chemicals as a consequence of their occupation should be made aware that there is concern with respect to an increased risk of adverse progeny outcome. Suggested Reading Robaire B, Hales BF. Paternal exposure to chemicals before conception. Some children may be at risk. Brit Med J 1993;307:341-342, . Olshan AF, Mattison DR, eds. Male Mediated Developmental Toxicity, New York: Plenum Press; 1994. Copyright © American Society of Andro/ogy Do environmental factors affect male reproductive functions? If so, which ones and how? Season, length of day and chemical exposure effects on the male In the human, reproductive functions in both sexes continue throughout the year without any major or obvious changes in different seasons. However, it is important to remember that in the overwhelming majority of species inhabiting the earth, reproductive functions are restricted to a well defined and often quite short breeding season. The annual cycles of transitions between reproductive activity and quiescence are driven by environmental signals and assure arrival of the young at the time when conditions are optimal for their survival. Of the environmental signals that influence male reproductive activity in mammals inhabiting the temperate zone, the role of photoperiod is best understood and probably most important. Annual changes in the day length provide an organism with reliable information about progression of the seasons and thus, in effect, allow "prediction" of upcoming changes in temperature and availability of food. The golden (Syrian) hamster is a popular model for the study of the effects of photoperiod on reproduction. Exposure of adult male hamsters to short photoperiod inhibits the release of prolactin (PRL) and the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH)(Fig. 1). There is an associated loss of testicular LH, FSH and PRL receptors, suppression of spermatogenesis, inhibition of testicular testosterone production and sexual behavior, a drastic reduction of testicular mass and sterility which persists for several months or until the animals are again exposed to long photoperiod. These effects are mediated by the action of photoperiod on the pineal gland, altering the diurnal pattern of melatonin release. The effects of short photoperiod can be completely prevented by prior removal of the pineal gland, and mimicked by appropriately timed injections or infusions of melatonin. Melatonin acts primarily within the hypothalamus by altering the release of neurotransmitters which control pituitary hormone release. 70 In other species of small animals, photoperiod controls not only adult testicular function but also sexual maturation. Typically, increasing day lengths of the spring promote early onset of puberty while shortening photoperiod of the late summer leads to postponement of puberty until the next spring. Interestingly, regulation of puberty in these species involves transfer of information about photoperiod from the pregnant female to the developing fetuses. In effect, the juvenile animal obtains precise information about the season of the year by "comparing" the photoperiod it is exposed to after birth with the photoperiod to which its mother was exposed during pregnancy. In large species in which pregnancy lasts for several months rather than several weeks, arrival of the young in the spring is assured by breeding taking place in the fall. Thus, in male deer annual recrudescence of the testes, increase in plasma testosterone levels, conspicuous growth of neck muscles, and appearance of aggressive and sexual behavior take place in the late summer and fall in response to a short, rather than a long, photoperiod. Similar annual changes, but of lesser magnitude, occur in males of most breeds of domestic sheep. Seasonal fluctuations in male reproductive functions do not depend solely on annual changes in photoperiod. Both temperature and food availability can exert important effects and either dampen or amplify the effects of photoperiod. Reproductive functions can also be influenced by specific, often strictly seasonally available, diet components and by chemical messages received from other members of the same species. For example, male puberty in some rodents is hastened by the presence or proximity of adult females. Chemical (pheromonal) communication between the members of the same species is among many social and density dependent factors that can affect reproduction. These include territorial behavior and 71 Handbook of Andrology-Do environmental factors affect male reproductive functions? 35 D---D Pa1red Testis WI. ( Q) 30 t:r -c. nq FSH /ml Serum o--~ CJl 2.5 - 20 90 .£: 0" Q) ~ E 70 ....... - -~ CJl c (/) ~----+. 1.5 ~ _j 1.0 . '\ I I ,/I \ 'l \ '~, I I 1'I\ \ \ 05 'zh,,.... ', (:f' I ~/{+--1! J---<7' I ,J ~r::: l'f /1 I I I I I I'' ', ' E ....... CJl c 300 I (f) ~200 100 I ' 400 u... ''?---/I I 500 I I If \ I \ I I ' 30 'I' I I ~ 50 10 ~ I I 110 I nq LH/ml Serum _J 0 0 0 3 7 II 15 19 23 Weeks in LD 6:18 FIG. 1. Effect of short photoperiod (6:18) on testicular weight and on serum LH and FSH levels in adult male hamsters transferred from a long photoperiod on day 0. Means ± SE. During both regression and spontaneous recrudescence of the testes, changes in serum gonadotropins (particularly FSH) precede changes in testicular weight. (From: Biology of Reproduction 1975;13:475-481.) aggressive interactions between males that can impose major stress and interfere with access to food sources. Both stress and malnutrition can suppress reproductive development and function. In the human, seasonal fluctuations have been detected in sperm count, motility and morphology, blood levels of LH and testosterone, as well as sexual activity and are believed to be related primarily to the effects of photoperiod. However, these fluctuations are rela- tively subtle and fertility continues throughout the year. Of much greater clinical significance are effects of environmental influences unique to our own species such as occupational or accidental exposure to chemicals, use of alcohol, psychotropic drugs, prescription and over-thecounter medication, and illicit use of androgenic and anabolic steroids. Each of these factors is capable of exerting profound suppressive effects on the production of spermatozoa and androgens by the testis, on libido and on potency. Suggested Reading Reiter RJ. The pineal and its hormones in the control of reproduction. Endocr Rev 1980;1 :109131. Gilmore DP, Cook B, eds. Environmental factors in mammal reproduction. Baltimore: University Park Press; 1981. Bronson FH. Mammalian reproduction: an ecological perspective. Bioi Reprod 1985;32:1-26. Steger RW, Bartke A. Environmental modulation of neuroendocrine function. In: Gass GH, Kaplan HM, eds. Handbook of Endocrinology, Vol. II. Boca Raton: CRC Press; 1987:111-141. Bartke A, Steger RW. Seasonal changes in the function of the hypothalamic-pituitary-testicular axis in the Syrian hamster. Minireview, Proc Soc Expr Bioi Med 1992;91:139-148. Copyright © American Society of Andrology Is there an andropause, the analog to menopause, and if so what tissues are affected and how? Fertility, androgen production and sensitivity, and sexual function in aging men with age, most prominently in men with the most marked changes in seminiferous tubular morphology. There is a corresponding decrease in basal serum inhibin levels. Thus, evidence favors the hypothesis that an intrinsic age-related reduction in seminiferous tubular function leads to reduced inhibin secretion with secondary effects on pituitary function. Menopause in women is a discrete event in the life cycle, and is marked by the cessation of menses, in association with a sharp fall in circulating estradiol levels and a rise in folliclestimulating hormone (FSH). Although no single event delineates reproductive senescence in men, results of many investigations suggest that aging men experience reductions in androgen levels, virility, and fertility, along with related metabolic changes. Nonetheless, the question of a "male menopause" remains controversial, in part because of the difficulty in discriminating the effects of age-related confounding variables such as stress, nonendocrine illnesses, malnutrition, obesity and drug or medication use, from aging per se. Aging effects on sex hormone secretion and bioavailability As for sex hormone secretion, early studies demonstrated a reduction in bioassayable urinary androgen, and subsequent investigations of small numbers of older men showed decreased testicular vein testosterone levels, and reductions in both metabolic clearance and production rates of testosterone. Initially basal levels of total plasma testosterone were shown to decrease progressively after age 50. Because subjects in these studies were not always carefully screened for health factors, the confounding effects of illness, medications, etc. may have accounted for some of the results observed. Illness does affect reproductive function as is demonstrated by a report of decreased plasma free testosterone in men with benign lung disease and a reduced total and free hormone level in men with lung malignancy. In some studies of exceptionally healthy men, no age effect on circulating testosterone concentration was found. Subsequent investigations examining multiple samples over a 24 hour period demonstrated that morning peak (but not afternoon nadir) and 24 hour mean integrated testosterone levels were decreased in older men, but in a similar study no age effect on circadian levels was observed. Thus, the question whether aging per se significantly reduces morning peak testosterone secretion or total levels remains controversial. Aging of seminiferous tubules and fertility With regard to fertility, despite occasional reports of paternity in men in their 90s, there is clearly a decrease in the rate of conception in old male/young female marriages. Semen analyses in elderly men reveal normal sperm numbers, but decreased sperm motility and increases in abnormal forms. Changes in seminiferous tubules with age include thickening of the basement membrane, peritubular fibrosis, sclerotic narrowing or collapse of the lumen, patchy impairment of germ cell maturation, and immaturity or degeneration of spermatocytes, as well as increases in multinucleated Sertoli cells. Studies have also revealed a small but measurable, decrease in average testis size with advancing age, whether studied at necropsy or in vivo. Functioning seminiferous tubules exercise negative feedback control on the pituitary gonadotrope by producing inhibin, a peptide that acts to reduce FSH production. Thus, with damage or destruction of the tubules, FSH increases, even if Leydig cell testosterone secretion remains normal. In fact, basal levels of FSH increase 72 Handbook of Andrology-/s there an andropause, the analog to menopause, and if so what tissues are affected and how? The fraction of circulating testosterone bound to sex hormone binding globulin (SHBG) is considered to be biologically unavailable. Because an increase occurs with age in circulating SHBG, older men may exhibit reductions in bioavailable testosterone, and hence its effects, disproportionate to what would be expected from measurements of total testosterone. However, in healthy men the increase in plasma testosterone binding to SHBG appears insufficient to significantly alter the apparent free concentration. Although there are no large longitudinal investigations of the effects of age on sex steroids in men, two cross-sectional studies, each examining more than 1000 individuals, have recently been reported. In one study there was a significant downward trend with age in both total and non-SHBG bound testosterone concentrations, while in another no significant trend in plasma testosterone was found. Neither study demonstrated an increase with age in the number of men with truly hypogonadal androgen levels. A recent meta-analysis of studies of androgens in aging men revealed a significant inverse correlation of total plasma testosterone with age which disappeared when reports which included men with ill health were omitted. This analysis also found that investigations which included ill or institutionalized subjects consistently showed lower levels of testosterone overall. 5a-Dihydrotestosterone (DHT) is produced from testosterone and is the "activated" form which binds to cytoplasmic androgen receptor in most tissues. This testosterone metabolite can be formed in the liver and also "leaks back" from androgen target tissues, so that it circulates in plasma at about 20% of total testosterone levels. Both reduced and unaltered plasma levels of total or free DHT have been reported in older men. In one study of elderly men, many of whom had benign prostatic hyperplasia, there were high plasma levels of DHT, but subnormal levels of testosterone, suggesting an increase with age in peripheral 5a reduction of testosterone, possibly in prostate tissue. Necropsy studies have generally revealed an age-related decrease in number, and an 73 increase in morphological abnormalities, of the Leydig cells, but in some of these investigations patients had died of a malignant disease or other protracted illness. Mean basal plasma levels of luteinizing hormone (LH), as well as urinary excretion of bioassayable gonadotropins, increase progressively in men beyond the age of 50, and human chorionic gonadotropin (hCG) stimulation tests have uniformly revealed diminutions in the testosterone secretory response in older men, consistent with an age-related decrease in Leydig cell number and/or reserve secretory capacity. Thus, the evidence favors some degree of primary testicular failure in aging men. There is also evidence for an effect of aging on hypothalamic-pituitary function. For example, there is a pattern of low or normal LH in a significant fraction of older men with diminished testosterone levels. Stimulation of the pituitary with exogenous gonadotropin-releasing hormone (GnRH) has revealed decreases in the magnitude of LH and/or FSH responses in older men. Clomiphene citrate treatment also results in less gonadotropin response in older men. The finding that there is an attenuation of the amplitude of spontaneous LH secretory bursts in normal older men also provides evidence of altered hypothalamic-pituitary function. The relative contribution of hypothalamic versus pituitary dysfunction remains uncertain, but in two recent studies, repeated pulsing of GnRH appeared to restore LH secretory responsiveness in older men, suggesting that the decrease in pituitary gonadotropin secretion is mainly due to reduced hypothalamic GnRH production. Aging effects on androgen target tissues Aging might also reduce androgen effect by causing a loss of sensitivity of target tissues to testosterone or DHT. Both decreased and increased sensitivity of pituitary gonadotropin secretion to feedback regulation by androgens have been reported in older men. Binding of DHT to sex hormone responsive skin is also decreased with age, suggesting that an agerelated reduction in responsivity to androgens may result from alterations in receptor number 74 Handbook of Andrology-ls there an andropause, the analog to menopause, and if so what tissues are affected and how? or affinity. To date, there are no published reports regarding effects of aging on specific post-receptor actions of sex steroids. Reproductive aging and sexual function, body composition and metabolism It is not known whether the changes in androgen levels or action in aging men have any deleterious clinical effects. Many studies have recorded progressive declines in male sexual interest, activity and performance with age, and there is a striking increase in the prevalence of impotence to as much as 50-75% in men over 75. However, after adjustment for age and body mass index (BMI = weight/ height squared), there is no difference in bioavailable testosterone levels in potent versus impotent old men, suggesting that hypogonadism and impotence are independently distributed conditions. Because impotence in older men is likely to be due mainly to neurologic or vascular changes, the value of replacing testosterone to improve erectile function in the elderly is questionable. In young hypogonadal men reduced sex drive rather than impotence is the primary symptom of diminished androgen action. The most important predictors of sexual interest and activity in old men are their characteristic level of sexual activity in youth, their health, and the health of the spouse or partner. Nonetheless, old men with relatively high sexual activity levels have been reported to have greater total or bioavailable plasma testosterone than age-matched men with less sexual activity. Other studies have shown weak, but statistically significant, inverse correlations of free or bioavailable testosterone levels with sexual thoughts, sexual activity, and morning erections in aging men, although statistical significance may be lost when data are adjusted for the effects of age. Thus, while decreases in serum testosterone may contribute to the dimin- ished sexual activity in older men, this effect is probably minor compared with the contributions of age-related alterations in psychological, social, neurological, vascular and health factors. If it is to be used at all, testosterone replacement should probably be reserved for older men who are frankly hypogonadal. In old age, there are decreases in muscle and bone mass and increases in body fat, with fat redistribution from peripheral to central depots. The latter changes are associated with altered glucose and lipid metabolism and increased risk of diabetes mellitus and cardiovascular disease. Because men have lower levels of high density lipoprotein (HDL)-cholesterol and a greater risk of coronary vascular disease than do women, it might be expected that a decrease in serum testosterone would beneficially affect atherosclerotic risk. However, in one recent study HDL-cholesterollevels were positively correlated with serum free testosterone in men aged 30-79 years, and in another study middle-aged men treated with testosterone had decreases in intra-abdominal fat, as measured by computed tomography, and in insulin resistance, as measured by the glucose clamp technique. Testosterone treatment also produced decreases in fasting glucose, diastolic blood pressure and serum cholesterol. Although testosterone treatment has been shown to improve bone density and skeletal muscle mass and strength in older hypogonadal men, the contribution of diminished testosterone to the loss of muscle or bone mass during normal aging is unknown. Summary Although there is no inevitable event leading to age-related hypogonadism in men, there is evidence that a significant number of older men have modest reductions in androgen levels. The metabolic and clinical sequelae of this change remain to be defined as does the risk/ benefit ratio of androgen supplementation. Suggested Reading Gary A, Berlin JA, McKinlay JB, Longcope C. An examination of research design effects on the association of testosterone and male aging: results of a meta-analysis. J Clin Epidemiol 1991 ;44:671-684. Handbook of Andrology-ls there an andropause, the analog to menopause, and if so what tissues are affected and how? 75 Vermeulon A. Clinical review 24: androgens in aging male. J C/in Endocrinol Metab 1991 ;73: 221"224. Blackman MR, Elahi D, Harman SM. Endocrinology and aging. In: DeGroot LJ et al, eds. Endocrinology (3rd ed.) New York: Grune and Stratton, 1995:Chap. 147, 2702-2730. Veldhuis JD, Urban RJ, Lizarralde G, Johnson ML, lranmanesh A. Attenuation of luteinizing hormone secretory burst amplitude as a proximate basis for the hypoandrogenism of healthy aging in men. J Clin Endocrinol Metab 1992;75(52-58). Korenman SG, Morley JE, Mooradian AD. et al. Secondary hypogonadism in older men: Its relation to impotence. J Clin Endocrinol Metab 1990;71 :963-969. Marin P, Holmang S, Jonsson L, et al. The effects of testosterone treatment on body composition and metabolism in middle-aged obese men. tnt JObes 1992;16:991-997 Copyright © American Society of Andrology What is BPH? Why is it so prevalent? What treatments are available? Pathophysiology, treatment The condition Benign prostatic hyperplasia (BPH) is a nonmalignant enlargement of the prostate gland that is due to both epithelial and stromal hyperplasia. Although the exact origin of this condition is not well defined, it is thought to arise as microscopic nodules in the periurethral tissue (transition zone) of the prostate gland in men as young as their late 20s. With advancing age and the presence of androgens, this histologically identifiable hyperplastic tissue progresses to a macroscopic state, which is a palpably enlarged prostate. This enlarged prostate can cause clinically significant obstruction of the bladder outlet in many men due to constriction of the urethra as it courses from the bladder neck to the external urinary sphincter. The constellation of symptoms associated with this intravesical obstruction is known as prostatism. It is estimated that more than 16 million men in the United States suffer from prostatism or clinically significant BPH. Fifty percent of men over the age of 50 years have some degree of hyperplastic enlargement of the prostate, while 95% of men will experience symptoms related to this enlargement of the prostate by the time they reach the age of 85 years. Twenty-five percent of all patients seen by urologists suffer from BPH. Over 400,000 patients were treated surgically during 1990 in the United States, and worldwide during the same year, 1,200,000 men underwent a prostatectomy for symptomatic BPH. Although this represents only 4% of the number of patients with this condition, intervention can be expected to increase with the advent of efficacious, less invasive therapies. One source estimates that 2,250,000 interventions (medical, minimally invasive, and surgical) for BPH will be performed in the United States by the year 2000. Thus, it is safe to say that BPH is the single most prev- alent condition treated in urologic practice today. Making the diagnosis When evaluating a patient with symptoms of prostatism, it is important to: 1) make sure that the symptoms are truly due to an enlarged prostate gland, and 2) assess the severity of the condition to determine whether therapeutic intervention is necessary. With regard to the first concern, a list of potential conditions causing symptoms of prostatism is contained in Table 1. All patients with prostatism should be evaluated with both a digital rectal examination and serum prostate-specific antigen (PSA) determination to exclude a clinically significant prostate cancer. A urinalysis also should be performed to eliminate the possibility of an infectious process. In addition, it is wise to obtain a serum creatinine level to determine the baseline renal function; marked, longstanding bladder outlet obstruction due to BPH can result in kidney damage. Intravenous pyelography (IVP) should only be performed for select patients, such as men with a history of nephrolithiasis or an upper tract transitional cell carcinoma. To assess the degree of bladder outlet obstruction, the single most useful test is the American Urological Association symptom index. This is a self-administered questionnaire consisting of seven questions relating to the ability to urinate (Fig. 1). A score between 0 and 7 is consistent with mild prostatism, 8 to 18 is indicative of moderate prostatism, 19 to 35 is compatible with severe prostatism. Only patients with moderate or severe bladder outlet obstruction should be considered candidates for therapy of any type. Another diagnostic test that can provide objective information about a patient's ability to urinate is the urinary flow rate. It is an electronic recording of the velocity of the urine 76 Handbook of Andro/ogy-What is BPH? 77 Table 1. The differential diagnosis for symptoms of prostatism Benign prostatic hyperplasia Urethral stricture Prostatitis Prostate cancer Bladder neck contracture Hypotonic bladder being expelled from the bladder during micturition and represents the single best noninvasive urodynamic test to assess bladder outlet obstruction. From several investigations, it has been learned that the peak value more specifically identifies men with BPH than does the mean rate. Also of importance is the fact that flow rate is dependent upon the patient's age and the volume of urine voided; with advancing age and decreasing urine volume, the flow rate diminishes. Nevertheless, for a man in the seventh and eighth decade of life who voids 150 ml or more, a peak urinary flow rate of 15 ml/second or greater should be interpreted as appropriate. On the other hand, a peak flow rate less than 10 ml/second in the same clinical setting is consistent with significant bladder outlet obstruction. Only in select patients is it necessary to obtain a "pressure-flow" study, in which the intravesical pressure is monitored while the peak urinary flow rate is recorded. Cystoscopy should not be a part of the diagnostic evaluation unless a specific indication exists, such as hematuria. Treatment options In 1994, there are numerous medical, minimally invasive, and surgical treatments available for the management of symptomatic BPH (Table 2). It is beyond the scope of this chapter to discuss the scientific rationale, clinical The AUA Symptom Index 1. Over the past month or so, how often have you had a sensation of not emptying your bladder completely after you finished urinating? Not at all Less than 1 time in 5 Less than half the time About half the time More than half the time Almost always [!] OJ [!] ~ [!] [!] 3. Over the past month or so, how often have you found you stopped and started again several times when you urinated? 7. Over the last month, how many times did you most typically get up to urinate from the time you went to bed at night until the time you got up in the morning? [!] None OJ 1 time [!] 2 times ~ 3 times [!] 4 times [!] 5 or more times ~9A.~P~9i~•e!lmiiii~llllflll I FIG. 1. The American Urology Association symptom index. This is a self-administered questionnaire to quantitate symptoms of prostatism (From: Barry MJ, Fowler FJ and O'Leary MP, J Uro/1992;148:15491557, with permission). 78 Handbook of Andrology-What is BPH? Table 2. Treatment options for benign prostatic hyperplasia I. Medical A. Androgen deprivation therapy 1. LHRH Agonists 2. Antiandrogens 3. Sa-Reductase inhibitors B. a-Adrenergic antagonists II. Minimally invasive A. Transurethral incision of the prostate (TUIP) B. Balloon dilatation of the prostate C. Laser prostatectomy D. Microwave Therapy E. Prostatic stents F. Transurethral needle ablation of the prostate (TUNA) G. Transrectal high-intensity focused ultrasound therapy (HIFU) Ill. Surgical A. Transurethral resection of the prostate (TURP) B. Retropubic prostatectomy C. Suprapubic prostatectomy results, advantages, and disadvantages of each. In the discussion that follows, a general overview is provided. For the past 50 years, transurethral resection of the prostate (TURP) has been the mainstay of treatment for this condition. In recent times, it has been the second most common operation performed in men over 65 years of age in the United States; only cataract extraction was performed more frequently. Although TURP is an effective treatment for most patients with symptomatic BPH, it appears that approximately 20 to 25% of patients undergoing a TU RP do not obtain a satisfactory, long-term outcome. Complications do occur and include retrograde ejaculation in most men (70-75%), impotence in 5 to 10%, some degree of urinary incontinence in 2 to 4%, and postoperative urinary tract infection in 5 to 10% of patients. Based on recent investigations published in the urologic literature, the risk of blood transfusion for patients undergoing a TURPis approximately 5 to 10%, and, thus, is a significant concern to many men in this era when various types of hepatitis and AIDS are increasing in prevalence. Another concern with TURPis that the reoperation rate is approximately 15 to 20% when patients are followed for 10 years or longer (2.2% per year). Also, several large-scale investigations have shown that the life expectancy of patients undergoing a TURP is less than that for men who receive an open prostatectomy as treatment for symptomatic BPH. Because of these issues as well as the desire by men in our society to avoid surgery whenever possible, there has been tremendous interest in developing alternative treatments to TU RP for the management of symptomatic BPH (Table 2). These options include medical therapies, such as drugs that produce a state of androgen deprivation (luteinizing hormone-releasing hormone analogues, antiandrogens, and Sa-reductase inhibitors), aadrenergic antagonists which eliminate the dynamic component of BPH, and aromatase inhibitors which eliminate the biosynthesis of estrogens in the male. In 1993, finasteride (Proscar), a potent 5 a-reductase inhibitor, became the first medication approved by the Food and Drug Administration (FDA) for the treatment of BPH. Terazosin (Hytrin), also with FDA approval for the indication of BPH, and doxazisin (Cardura) are selective a-1 adrenergic antagonists that are being used for the management of BPH. Adrenergic receptors in the prostatic adenoma and capsule mediate the tension in the smooth muscle of the prostate and blocking these receptors can decrease the resistance along the prostatic urethra. Transurethral incision of the prostate (TUIP), a minimally invasive procedure, is being used with increasing frequency. When compared to TURP, TUIP is technically easier, can be performed more quickly, and has fewer side effects; patients can be discharged home the same or the following day, and the convalescence period is shorter than that for TURP. While enthusiasm for balloon dilatation of the prostate is diminishing as results of re- Handbook of Andro/ogy-What is BPH? cent studies indicate that it is not an effective, long-term treatment, there is increasing interest in laser prostatectomy, and microwave therapy. Although both of the latter procedures are still investigational and not FDA-approved for widespread clinical use, preliminary data suggest that they can improve voiding symptoms. Prostatic stents, in FDA-approved clinical trials, have been demonstrated to improve the peak urinary flow rate and decrease the obstructive symptom score to a level similar to that of TUIP and TURP. Most recently, two other therapeutic modalities, transurethral needle ablation of the prostate (TUNA) and transrectal high-intensity focused ultrasound therapy (HIFU) have been developed and are currently being investigated in clinical trials in both the United States and Europe. Summary At the present time, the management of BPH is in a state of transition. TURP is no longer the only therapeutic option available. While it is still the most efficacious with regard to relieving bladder outlet obstruction and remains the gold standard of care, there are other treatment modalities that are attractive. The single most important advantage of medical therapies is that the patient can avoid a pro- 79 cedure; however, all medical treatments must be regarded as a life-time commitment, and their efficacy is not as good as the minimally invasive treatments. The surgical procedures, on the other hand, are a one-time event, but they are associated with greater risk and a recovery period. In the future, it will be necessary to develop methods for deciding, on a scientific basis, the most appropriate treatment for each individual patient. However, once the physician has informed the patient of the benefits and risks of each therapy, the patient's preference must be considered because what is a risk to one may not be to another. With the physician and patient working together, the treatment can be tailored to the patient's specific needs and expectations. Up to this point in time, the primary focus of investigation has been to develop the "perfect" treatment for BPH. In the years ahead, it will be important to gain a better understanding of both the pathogenesis of this disease process as well as the functional interaction that exists between the prostate gland and the bladder. In this manner, it may be possible to develop methods to prevent the disease or to identify it at an early stage so that clinically manifest BPH (prostatism) does not occur. Society would benefit tremendously. Suggested Reading Oesterling JE. Benign prostatic hyperplasia: its natural history, epidemiologic characteristics, and surgical treatment. Arch Fam Med 1992; 1 :257-266. Barry MJ, Fowler FJ, Jr, O'Leary MP, Bruskewitz RC, Holtgrewe HL, Mebust WK, Cockett ATK and The Measurement Committee of the American Urological Association. The American Urological Association symptom index for benign prostatic hyperplasia. J Urol1992;148: 1549-1557. Lepor H. Medical therapy for benign prostatic hyperplasia. Urology 1993;42:483-501. McConnell JD, Barry MJ, Bruskewitz RC, et al. Benign Prostatic Hyperplasia: Diagnosis and Treatment. Quick reference guide for clinicians. No. 8. Rockville, M.D.: Department of Health and Human Services, 1994. (AHCPR publication no. 94-0583). Oesterling JE. Benign prostatic hyperplasia: medical and minimally invasive treatment options. New England Journal of Medicine 1995;332:99-1 09. Copyright © American Society of Andrology Are some men more susceptible to prostate cancer than others and why? What are the treatments and their effectiveness? What are the possibilities for improvements in therapy? Pathophysiology, present and future treatments Are some men more susceptible to prostate cancer than others and why? Prostate cancer (PC) is the most common cancer in American men and is the second leading cause of male cancer deaths with 35,000 deaths annually. Autopsy studies have demonstrated histological evidence of PC in 15% of men in the 6th decade; that rate increases to 50% in the 9th decade. Recent studies demonstrate higher rates with up to 30% of men age 30-39 showing microscopic foci of PC. This prevalence is constant worldwide so that the rate of histological PC is independent of place of birth and of race. In contrast, death rates of PC vary from country to country suggesting that environmental influences might be crucial in developing clinical PC. Studies of migrating populations have further supported this viewpoint. Men moving from Asia, where PC death is uncommon, to the United States gradually achieve the same death rates from PC as the general population in the United States. The pathogenesis of PC, is poorly understood. Men castrated before puberty almost never have clinical PC. Therefore androgens are thought to have a permissive role. Other factors influencing the rate of clinical PC are: Genetics - Prostate cancer in a first degree relative doubles the risk for PC and this risk increases as more relatives are affected. Race - African-American men in the United States are among the most susceptible in the world. Their risk for developing PC is 120 times higher than for Chinese men and 1 1/3 higher than for Caucasian men of a similar educational and socioeconomic class. Dietary fat- High intake of saturated fat from animal sources, especially red meat, elevates the relative risk for development of clinically evident PC. Some studies have demonstrated 80 a correlation with intake of dairy products, obesity and PC. Vasectomy - The overall relative risk for men of developing PC following a vasectomy is found to be elevated in some studies; however, this association is very controversial and remains under investigation. Most likely further study will show that vasectomy will have no influence on PC. What are the treatments and their effectiveness? Prostate cancer can be divided into clinical stages on the basis of tumor volume and anatomical extension (Fig. 1). Each clinical stage has different treatment options and prognosis (Table 1). The prognosis depends on the stage as well as on the histological grade of the tumor. A poorly differentiated tumor tends to metastasize more often than a well- or moderately differentiated tumor. The prognosis of PC without initial treatment is not clear, but a few studies have found that the 10 year disease-specific survival for men with a clinical diagnosis of organ-confined PC is approximately 86%, and for regional PC (not organ-confined) the 5 year survival rate is approximately 50%. Organ-confined disease Treatment is intended to be curative, which in the United States includes either radical prostatectomy or radiation therapy. A watchful-waiting policy is preferred in elderly (age > 70 or > 75 years) men with an asymptomatic organ-confined PC. In the elderly, the diseasespecific survival may approach 100% because elderly patients generally die of causes other than PC. Handbook of Andrology-Are some men more susceptible to prostate cancer than others and why? 81 c. A. Aorta--------~ Ductus Lymph node------------+-+~ deferens~ Seminal vesicle Lymph node with cancer --------... Internal lilac a. --l.,.......l,.------Arf4,.4Jt:------:::\lt'-.. Cancer--~:.... Urethra _ _----::~.,__-+ , External lilac a. ---"~--+ B. FIG. 1. Clinical stages of Prostate Cancer. A. Organ confined disease. B. Not organ confined disease with capsular penetration and/or extension into seminal vesicle. C. Metastatic disease, shown here with metastases to lymph nodes and vertebrae, pelvis, femur. Table 1. Therapy and Prognosis for Prostate Cancer Clinical Stage Organ Confined A B A B A B Treatment Life Expectancy Radical prostatectomy Radical prostatectomy 5(15)-year: 77%(40%) Radiation therapy Radiation therapy 5(15)-year: 5(15)-year: 95%(45%) 75%(28%) Watchful waiting Watchful waiting 5-year: 5-year: 75% 64% Radiation therapy Watchful waiting 5(15)-year: 5-year: 64%(20%) 50% Mono therapy Comb. androgen blockade Median: Median: 41 months 61 months Mono therapy Comb. androgen blockade Median: Median: 28 months 36 months Not Organ Confined C Metastatic PC Minimal' 01 Extensic· 02 A B PC found incidentally at prostatectomy for benign disease. PC palpable on digital rectal examination. # Metastases confined to pelvic lymph nodes. + Metastases to bone. Mono therapy bilateral orchiectomy or LH-AH analog therapy. Combined androgen therapy mono therapy (i.e. bilateral orchiectomy or an LHAH agonist) plus androgen receptor blockade. 82 Handbook of Andrology-Are some men more susceptible to prostate cancer than others and why? Radical prostatectomy has been preferred throughout the last decade after new techniques like nerve sparing surgery and autologous transfusions decreased morbidity and mortality. The mortality after radical prostatectomy is 0-2%. Morbidity primarily consists of impotence (20-80%), incontinence (5%) and urethral stricture (1 0%). Radical prostatectomy is generally reserved for men with more than 10 years life expectancy. Radiation therapy is performed most often as external beam radiation. The therapy is associated with side effects consisting primarily of impotence (1 0-20%), incontinence (1 %), rectal injury (2%), lymphedema (1 0%) and urethral stricture (6%). A major concern with radiation therapy is the fact that up to 50% of patients will have a positive prostate biopsy 18-24 months after treatment, indicating recurrent or persistent PC. The ten year survival for both therapies is approximately 60%, being perhaps slightly better in the surgically treated group. However, a prospective study randomizing patients to either radiation therapy or radical prostatectomy is not available making any comparison between the two treatment options and between treatment and watchful waiting difficult. Regional but not organ-confined disease Untreated regional PC causes local complications due to growth into adjacent structures, e.g., urethra, bladder, ureters, nerves and rectum, leading to bleeding, obstructive symptoms of bowel and bladder, and pain. A common approach to regional disease is radiation therapy, but therapy varies from watchful waiting to radical prostatectomy, radiation therapy or hormonal treatment. The 10 year survival is approximately 40% and does not differ essentially from one therapy to another. Metastatic disease Initial metastasis is often to pelvic lymph nodes. Another prime metastatic site is to bone, especially vertebrae, pelvis, ribs and femur, causing isolated or diffuse pain due to bone fractures. Neurologic symptoms also oc- cur due to compression of the spinal cord and brain metastasis. Anemia may be seen because of reduced hematopoiesis. Many patients present with metastatic disease or eventually progress to advanced stage disease. Therapy may target a specific complication, e.g., transurethral resection of the prostate to relieve urinary obstruction or hematuria, or radiation therapy to painful bony metastases. Hormonal therapy attempts to control metastatic growth by androgen depletion since growth of PC is androgen dependent in most cases. When to start androgen suppression is controversial, but recent investigations have reported a slightly better survival rate when therapy is started immediately after metastatic disease is identified, compared with when symptoms from metastases begin. Bilateral orchiectomy reduces androgen concentrations by approximately 95% with the remaining amount produced by the adrenal glands. Orchiectomy is still widely used as a safe, cost-effective method. The major concern is the permanent loss of potency and libido, and the occurrence of hot flushes in approximately 40% of patients. An alternative is therapy with a luteinizing hormone-releasing hormone (LH-RH) analog that inhibits testosterone production in the testes by decreasing (LH) release from the pituitary gland. In the past, diethylstilbestrol was widely prescribed but its association with congestive heart failure and thromboembolism has led to a decline in its use. To obtain maximum androgen blockade, it is necessary to add an antiandrogen (e.g., flutamide) to either orchiectomy or LH-RH therapy. An antiandrogen exerts its effect by competing with the remaining androgens for the intracellular androgen receptor in the target organs. This approach improves longevity by a matter of months in selected groups of men. What are the possibilities for improvements in therapy? Prostatic cancer is the most prevalent cancer in men and the second leading cause of cancer death. Despite intensified efforts to improve the diagnosis and treatment of PC, the Handbook of Andrology-Are some men more susceptible to prostate cancer than others and why? death rate has remained unchanged for decades. While it is hoped that new screening approaches, including PSA testing, and increased use of radical prostatectomy will drop the death rate, the impact of these approaches on survival will not be known for another 10-15 years. A large scale prospective clinical trial is necessary to decide the relative benefits of potentially curative treatment such as radical prostatectomy and radiation therapy versus observation. Plans for such trials are now underway. It has been 50 years since a Nobel Prize was awarded for the demonstration that PC responds to androgen deprivation, knowledge that led to treatment (orchiectomy) which has palliated and perhaps modestly improved the survival of untold thousands. Unfortunately there has been little progress in the area of treatment of metastatic disease since then, although the concept of total androgen blockade using an androgen receptor blocker has im- 83 parted some modest gains. Trials of chemotherapeutic agents applied early in the course of PC, when metastasis is first noticed, have not resulted in any improvement. Prostate cancer is a slow growing tumor and the hope for newer chemotherapeutic agents improving survival is slight. In the near future, management of PC should focus on patient selection. Patients with localized cancer which is likely to progress need to be identified and targeted for treatment. The notion that there is considerable benefit to aggressive diagnosis and treatment in men beyond the age of 70 or 75 years is not supported by the literature and this approach needs to be reassessed. Finally, a long-term study to examine the drug finasteride for prevention of prostate cancer by the reduction of prostate glandular activity has been initiated by the National Cancer Institute; however, these results are at least 5 years in the future. Suggested Reading Pienta KJ, Esper PS. Risk factors for prostate cancer. Annals of Internal Medicine. 1993;118: 793-802. Garnick MB. Prostate cancer: screening, diagnosis, and management. Annals of Internal Medicine. 1993; 118:804-818. Lynch JH. Treatment of advanced prostate cancer. The Journal of Family Practice. 1993;37(5): 448-493. Stanley T, McNeal J. Adenocarcinoma of the Prostate. In: Walsh PC, Retik AB, Stamey TA, Vaughan ED, eds. Campbell's Urology, 6th edition, Vol. 2. Philadelphia: W. d. Saunders Company; 1992:1159-1222.