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BIOLOGY OF REPRODUCTION 54, 1397-1404 (1996) Microtubule Organization in Porcine Oocytes during Fertilization and Parthenogenesis' Nam-Hyung Kim, 3 '4 Calvin Simerly,5 Hiroaki Funahashi, 4 Gerald Schatten,s and Billy N. Day 2' 4 Department of Animal Sciences,4 University of Missouri-Columbia, Columbia, Missouri 65211 Departmentof Zoology,5 University of Wisconsin, Madison, Wisconsin 53706 ABSTRACT Microtubule configurations inporcine oocytes after sperm penetration or after artificial activation by electrical stimulation were imaged by immunocytochemistry and laser scanning confocal microscopy. Soon after sperm penetration, an aster was seen adjacent to the incorporated sperm head. Polyspermic penetrations led to the presence of multiple sperm asters in association with each sperm. The sperm aster enlarged and, at the time of pronuclear apposition, filled the cytoplasm. After male and female gamete union, the microtubule matrix was reduced. At the mitotic metaphase stage, microtubules were detected in the spindle, which was anastral and fusiform. At anaphase, asters assembled at each spindle pole, and at telophase, large asters filled the cytoplasm. Artificial activation by electrical stimulation induced in the cytoplasm a dense network of microtubules, which seem to be involved in proper positioning of the female pronucleus. At mitotic metaphase, microtubules were concentrated around the chromatin. The results of experiments using taxol, a microtubule stabilizing agent, suggest that maternal centrosomal material is present in the mature porcine oocyte as dispersed undetectable material that can form a microtubule network after parthenogenetic activation. However, at fertilization, the paternal centrosome collects centrosomal material to form a sperm aster. These results suggest that the functional centrosome that forms during fertilization isa result of the blending of paternal and maternal centrosomal components. INTRODUCTION In most animals, the penetrating sperm introduces the centrosome, which organizes an aster of microtubules called the "sperm aster." The sperm aster appears to be involved in the process of pronuclear movement and mitosis [1-71. In contrast, in the mouse, microtubules involved in the union of the pronuclei are organized from the centrosomal foci that pre-exist in the cytoplasm of the unfertilized oocyte [8, 9]. Paternal inheritance of a functional centrosome during fertilization has been suggested in most animals; however, it is still controversial whether the sperm itself contributes the centrosome. Recent studies in bovine [10], rabbit [11], human [61, and marsupial [71 parthenotes showed that disarrayed microtubules were present in the cytoplasm soon after artificial activation. These parthenotes then formed bipolar spindles and divided normally. These results suggest that mammalian oocytes are able to form a functional centrosome without any contribution from the sperm. In sheep, rabbits, and sea urchins, cytoplasmic microtubules are undetectable in unfertilized oocytes [3, 4, 12]. Treatment of these oocytes with taxol, a drug that promotes microtubule assembly, induced numerous microtubule foci in the cytoplasm, while in fertilized zygotes, taxol treatment did not induce cytoplasmic microtubules. It has been pro'This research was funded in part by USDA NRI Competitive Grants 94-37203-1087 and 93-37202-9186. Contribution from the Missouri Agricultural Experiment Station. Joumal Set ries Number 12,349. N.-H. Kim is a recipient of a Food for the 21" Century postdoctoral fellowship at the University of Missouri. 2 Correspondence: Dr. B.N. Day, University of Missouri-Columbia, Department of Animal Sciences, 159 Animal Sciences Research Center, Columbia, MO 65211. FAX: (573) 882-6827. 3 Current address: Animal Resource Research Center. Kon Kuk University, Seoul 143701, Korea. posed that centrosomal material exists in the cytoplasm of some mammalian oocytes as undetectable material that organizes functional microtubules after parthenogenetic activation. In contrast, during fertilization, the sperm aster introduces a strong attractant that can collect centrosomal material to form a sperm aster [10, 13]. To test the hypothesis that the centrosome in the pig is of paternal origin, we imaged the organization of microtubules after sperm penetration and parthenogenetic activation by immunocytochemistry and confocal microscopy. Szollosi and Hunter [14] have studied ultrastructural aspects of porcine fertilization by electron microscopy. They observed the appearance of clusters of electron-dense, filamentous material near the neck region of sperm nuclei, which might give the impression of a sperm aster. However, the organization of microtubules during fertilization and parthenogenesis in the pig has not been determined. In this study, we have documented microtubule assembly with the use of in vivo-fertilized embryos to determine the appearance of functional centrosomes during pronuclear formation and movement, and during mitosis. The data obtained from in vivo-fertilized zygotes were then compared with microtubule organization after in vitro fertilization and after electrical stimulus to induce activation. In addition, we treated oocytes with taxol to detect distribution of centrosomal material during fertilization and parthenogenesis. MATERIALS AND METHODS In Vitro Maturation Porcine oocyte-cumulus complexes (OCC) with uniform ooplasm and a compact cumulus cell mass were prepared 1397 1398 KIM ET AL. in HEPES-buffered Tyrode's albumin-lactate-pyruvate (TALP) medium containing 0.1% polyvinylalcohol (HEPES-TALPPVA) as described elsewhere [15]. The culture medium for in vitro maturation was BSA-free Whitten's medium [15] (OMWM) supplemented with 10% (v:v) porcine follicular fluid, 10 IU/ml eCG, and 10 IU/ml hCG. Fifty OCC were transferred to 500 gl of OMWM in a four-well culture plate, covered with paraffin oil, and then cultured for 22 h at 39C in an atmosphere of 5% CO2 in air. The OCC were then transferred to 500 tl of OMWM without hormonal supplements [16] and cultured for an additional 22 h at 39°C in 5% CO2 in air. with pH 7.2. Electrical stimulation to induce activation was delivered via a BTX Electro Cell Manipulator (Biotechnologies and Experimental Research, Inc., San Diego, CA) to a chamber with two parallel platinum wire electrodes (200 mm o.d.) spaced 1 mm apart and overlaid with electroporation medium as described above. A single DC pulse of 120 V for 30 tsec was used for electrical stimulation. After a 2min recovery, the oocytes were transferred to 500 l1of Whitten's medium and cultured at 39°C in an atmosphere of 5% CO 2 in air. The oocytes were fixed at 1.5, 3, 6, 12, 18, and 24 h after electrical stimulation for immunocytochemistry. In Vitro Fertilization Cytoskeletal Inhibitors Effects of nocodazole, a microtubule inhibitor, and cytochalasin B, a microfilament inhibitor, on pronuclear formation and movement were examined during in vitro fertilization. Stock solutions of 1 mM nocodazole (Sigma) and 5 mM of cytochalasin B in dimethyl sulfoxide were used. The stock solutions were stored at - 20°C and diluted to 10 ptM nocodazole and 5 M cytochalasin B in Whitten's medium prior to treatment of oocytes. The oocytes were treated with drugs between 6 and 12 h, and between 12 and 24 h after insemination. Statistical analyses of data from three replicate trials were carried out by analysis of variance (ANOVA) and Fisher's protected least significant difference test. Sperm-rich fractions (15 ml) were collected from boars by the gloved hand method and, after antibiotic-antimycotic solution (Gibco Labs., Grand Island, NY) was added, the semen sample was kept at 20 0C for 16 h. The semen was washed three times with 0.9% (w:v) NaCl supplemented with 1 mg/ml BSA (fraction V; Sigma Chemical Company, St. Louis, MO) by centrifugation. At the end of washing, the pellets containing spermatozoa were resuspended at 2 X 108 cells/ml in modified tissue culture Medium 199 ([15], mM199; Sigma) at pH 7.8 supplemented with 1% porcine follicular fluid. The sperm suspension was incubated for 90 min at 39°C in an atmosphere of 5% CO2 in air. Ten oocytes were washed three times with mM199 supplemented with 10 mM caffeine sodium benzoate and 4 mg/ml BSA at pH 7.4 and placed into a 50-j1d droplet of mM199 under paraffin oil. Fifty microliters of diluted preincubated sperm was added to 50 tl1of the medium containing oocytes so that a final sperm concentration of 1 X 106 cells/ml was obtained. Oocytes were cocultured with spermatozoa for 6 h at 39°C in an atmosphere of 5% CO2 in air. The oocytes were transferred to 500 pl of fresh Whitten's medium and cultured at 39°C in an atmosphere of 5% CO2 in air. The oocytes were fixed at 6, 9, 12, 24, and 36 h after insemination. In Vivo Zygotes Proestrus gilts were treated with 500 IU hCG on Day 19 or 20 of the estrous cycle in order to time accurately the onset of ovulation [17]. At 39-40 h after hCG injection, the gilts were artificially inseminated. At 6, 10, and 24 h after insemination, the gilts were anesthetized for surgical recovery of oocytes. Embryos were collected by flushing the oviduct with HEPES-TALP-PVA. Oocyte Activation Before electrical stimulation, oocytes matured in vitro were denuded of cumulus cells, washed, and preincubated 5 min in electroporation medium: 0.25 M mannitol supplemented with 0.01% polyvinyl alcohol, 0.5 mM HEPES as indicated, and 100 IpM CaC12-2H20O and 25 mM MgCI 2 6H20O Taxol Treatment A stock solution of 1 mM Taxol (Sigma) in dimethyl sulfoxide was used. The stock solution was stored at -20°C and diluted to 1 tM in Whitten's medium immediately prior to treatment of oocytes. The oocytes were treated with taxol for 5 min at 39°C. The distribution of centrosomal material after treatment with taxol was examined according to the same fixing schedules followed after in vitro fertilization and electrical activation. Immunocytochemistry At specific time points, cumulus cells were removed by pipetting the oocytes through a small bore glass pipette. The zona pellucida was removed from oocytes by treatment with 0.4% pronase prepared in Dulbecco's PBS (dPBS). After 30-min recovery at 390 C, zona-free gametes were permeabilized in 25% glycerol, 50 mM KCI, 0.5 mM MgCl 2, 0.1 mM EDTA, 1 mM EGTA, 1 mM 2-mercaptoethanol, 50 mM imidazol, pH 6.7, with 4% Triton X-100 [18]. The oocytes were then affixed to polylysine-coated coverslips and fixed in methanol at - 100C. Microtubules were localized by use of monoclonal anti -tubulin antibody (Sigma). Primary antibody was detected by using a fluorescent-labeled goat anti-mouse secondary antibody (Sigma). Each antibody was applied for 45 min at 39 0C and rinsed with dPBS. DNA was MICROTUBULES DURING PORCINE FERTILIZATION AND PARTHENOGENESIS fluorescently detected after exposure to 5 ,ig/ml propidium iodide (Sigma) for 1 h. Stained oocytes were then mounted under a coverslip with antifade mounting medium (Vectashield; Vector Labs., Burlingame, CA) to retard photobleaching. Laser-scanning confocal microscopy was performed with a Bio-Rad MRC 600 equipped with a Kryptoargon ion laser (Bio-Rad Labs., Richmond, CA) for the simultaneous excitation of fluorescein for microtubules and propidium iodide for DNA. The images were recorded digitally and archived on an erasable magneto optical disk. RESULTS FertilizationIn Vivo To determine microtubule reorganization during fertilization, we collected a total of 103 in vivo-fertilized zygotes from 14 gilts at 6, 10, and 20 h after artificial insemination. At 6 h after artificial insemination, 7 unfertilized and 12 fertilized zygotes were collected in two replicates. In the unfertilized oocytes, microtubules were observed only in the second meiotic spindle (Fig. 1A). Of the total fertilized zygotes, 10 (83%) had decondensing sperm heads in the cytoplasm associated with a conspicuous aster. In two zygotes fixed at this time, compacted sperm heads were found, and microtubule asters were also found at the sperm neck area (Fig. 1B). The maternal chromatin progressed through meiosis after sperm penetration by extrusion of the second polar body and formed the midbody (Fig. 1C). The position of the incorporated sperm tail was observed to determine whether the aster was associated with the male pronucleus. In all cases, the sperm tail was found at the center of the microtubule aster. At 10 h after artificial insemination, a total of 32 zygotes were obtained from five gilts. Significant variance in developmental stage of embryos was observed both between gilts and within gilts. Of the total zygotes fixed at this time, 10 (31%) of the zygotes had decondensed male chromatin, and 17 (53%) of the zygotes had male and female pronuclei. During sperm decondensation, the microtubules elongated and filled the cytoplasm (Fig. 1D). The sperm aster was not oriented preferentially toward the female pronucleus. At 24 h after artificial insemination, a total of 59 zygotes were collected from seven gilts. Among them, pronuclear movement was found in 26 (44%) of the zygotes. During pronuclear movement, the microtubules filled the whole cytoplasm (Fig. 1E). After syngamy, the microtubules were less detectable in the cytoplasm (Fig. F). Five zygotes (8%) were at mitotic metaphase. At mitotic metaphase, the cytoplasmic microtubules had broken down completely, and a fusiform, anastral mitotic spindle was detected. The sperm tail was detected in association with the mitotic spindle (Fig. 1G). At anaphase (2 of 59, 3%), asters assembled at each spindle pole, and at telophase, large asters were detected (Fig. 1H). Twenty-two (37%) of the 2-cell-stage embryos were collected at this time. 1399 FertilizationIn Vitro During in vitro fertilization, sperm asters were observed between 6 h (21 of 76, 28%) and 9 h (62 of 83, 75%) after insemination. The male pronucleus was observed between 9 h (32 of 146, 22%) and 12 h (131 of 176, 74%) after insemination. Polyspermic penetration was observed in 81 (45%) zygotes at 12 h after insemination. In polyspermic zygotes, multiple sperm asters were observed in association with each penetrated sperm (Fig. 1I). During pronuclear formation, each aster became larger, and the male pronucleus had an associated sperm aster. In almost all of the unfertilized mature oocytes (135 of 146, 92%), treatment with taxol induced numerous cytoplasmic foci of microtubules (Fig. 1J). Immediately after sperm penetration, in all cases, either no cytoplasmic foci were observed or the number of cytoplasmic foci were significantly decreased (Fig. 1K). During sperm aster formation, taxol produced a larger aster but did not induce cytoplasmic microtubule foci (Fig. 1L). Microtubules extended radially from the blastomere nuclei to the cortex of the cleaved embryo (Fig. 1M). Parthenogenesis At 3 h after activation, oocytes progressed to the anaphase-II or telophase-II (65 of 179, 36%) and pre-pronuclear FIG. 1. (Page 1400) Laser scanning confocal microscope image of microtubules and chromatin in the porcine oocyte after fertilization. Green, microtubules; red, chromatin. Bar = 20 pm. t, Sperm tail. A) Microtubules are seen only in the meiotic spindle in the metaphase-ll stage oocyte. B)Shortly after sperm penetration, microtubules are found in association with the incorporated sperm head. C) Female chromatin emitted the second polar body and forms a midbody. D) Sperm aster enlarges, and no microtubules are observed in the female chromatin. E)At time of pronuclear apposition, microtubules fill the whole cytoplasm. F) After male and female chromatin union, the microtubule matrix is less detectable. G)The eccentric mitotic metaphase spindle has tightly focused anastral poles, and chromosomes are aligned on the spindle equator. H) By telophase, astral microtubules are prominent, and nuclei are reconstituted. I) During polyspermy, multiple sperm asters are observed in association with each male chromatin (M). No microtubules are observed in the female chromatin (F). J) Taxol induced numerous cytoplasmic microtubules in the matured, unfertilized oocyte. K)After sperm penetration, the number of cytoplasmic microtubule foci decreased in cytoplasm. L) Taxol treatment did not induce cytoplasmic microtubules but did induce larger sperm asters inthe dispermy zygote. M) At the 4-cell stage, microtubules appear contiguously in the cytoplasm of daughter blastomere. FIG. 2. (Page 1401) Laser scanning confocal microscope image of microtubules and chromatin in the porcine oocyte after electrical activation. Green, microtubules; red, chromatin. Bar = 20 ,um.A) Dense microtubule network is observed in the parthenote at 5 h after activation. B) In the full-grown pronuclear stage parthenote (18 h after stimulation), microtubules were less detectable. C)At pro-metaphase for mitosis, microtubules were found near the chromatin mass. D) Parthenote forms an anastral bipolar spindle at mitotic metaphase. E)Parthenotes complete mitosis and during anaphase, astral microtubules are present. F) Taxol treatment induced numerous microtubule foci that start to aggregate at 1.5 h after electrical activation. G,H) Same oocyte at different focal points. With focus at female nucleus (-40 m from surface), microtubules are found around the female chromatin (G). A dense matrix of microtubules is formed at cortex (-10 gm from surface, H). I)At 3 h after stimulation, a dense microtubule network is found in the entire cytoplasm of taxoltreated oocyte. J,K) During the pronuclear stage, taxol induced microtubules around the pronucleus. L) At mitotic pro-metaphase, a few cytoplasmic microtubules are induced by taxol treatment. Microtubules are organized at several points near the condensed chromatid (arrows). 1400 KIM ET AL. MICROTUBULES DURING PORCINE FERTILIZATION AND PARTHENOGENESIS 1401 1402 KIM ET AL. im E Female pronucleus I Male pronucleus A -r 2 8D -1-i -- 7 I-- i430 0 Cytoskeletal Inhibitors Co,ntrol Nocodawle Cytochalasin (87) (71) 85) 60 B a 50 _ dense microtubule network in the cortex, which reached to the female nucleus (Fig. 2, G and H). During pronuclear formation, microtubule matrix filled the entire cytoplasm (Fig. 21). At the late pronuclear stage, taxol induced dense microtubules around the pronucleus (Fig. 2, J and K). A few cytoplasmic microtubule foci were induced by taxol treatment after mitotic pro-metaphase (Fig. 2L). T- DISCUSSION 30 10 rb 0 I Control (59) To determine whether cytoskeletal assembly is involved in pronuclear formation and movement, oocytes were cultured in Whitten's medium containing 10 VM nocodazole or 5 jiM cytochalasin B between 6 and 12 h, and 12 and 24 h after insemination. As seen in Figure 3, treatment with either nocodazole or cytochalasin B did not inhibit pronuclear formation but did inhibit movement leading to the union of the male and female pronuclei. I . . b II Nowodazole Cytochalasin (63) (69) FIG. 3. Effects of nocodazole and cytochalasin Bon male and female pronuclear formation (A)and movement (B). Number of oocytes examined in each experimental group is given inparentheses. Bars with different letters differ (p < 0.05). stages (27 of 179, 15%). At the anaphase-II to telophase-II stage, microtubules were found in the meiotic spindle. During the pre-pronuclear stage, a dense network of microtubules was observed throughout the cytoplasm (Fig. 2A). The pronucleus was observed at 6 h (79 of 116, 68%) and 18 h (91 of 121, 75%). In most parthenotes (52 of 79, 66%) at the early pronuclear stage (6 h after stimulation), a microtubule network was present in the cytoplasm. However, microtubules were less detectable at the late pronuclear stage (79 of 91, 87%) at 18 h after stimulation (Fig. 2B). Mitosis occurred at 24 h after activation (metaphase, 24 of 82, 29%; anaphase-telophase, 9 of 82, 11%; two-cell stage, 6 of 82, 7%). During mitotic pro-metaphase, microtubules were detected in the condensed chromatin mass (Fig. 2C). At mitotic metaphase, microtubules were concentrated around the chromatin (Fig. 2D). At anaphase, the parthenotes completed mitosis, and astral microtubules were assembled (Fig. 2E). In oocytes treated with taxol immediately after stimulation, the cytoplasmic microtubule foci were aggregated with each other to form a microtubule network (Fig. 2F). During pre-pronuclear formation, taxol induced a In this study, we demonstrated that an aster of microtubules, defined as the sperm aster, became organized at the sperm neck area during fertilization in the pig. This observation of a sperm aster in fertilized pig oocytes supports the hypothesis that the sperm contributes the centrosome during fertilization. Our study also demonstrated that polyspermy results in the formation of multiple sperm asters adjacent to each male chromatin, and that the central focus of each sperm aster is toward the proximal region of the incorporated sperm tail. Polyspermy represents an experimental test of the relative parental contributions to the centrosome, since paternal contribution is multiplied while the maternal mass remains constant [13]. In most animals, including sheep [3], rabbits [4 ], cattle [5, 10], and marsupials [7], a sperm aster was observed soon after sperm penetration, and supernumerary sperm organized multiple sperm asters. In contrast, dispermic or trispermic mouse zygotes do not display sperm asters, and they divide to the two-cell stage [19], suggesting that sperm do not contribute the dominant centrosome in this rodent. In the porcine oocyte, we observed that treatment with nocodazole, a microtubule inhibitor, did not inhibit pronuclear formation but did inhibit pronuclear movement. The role of microtubules during fertilization has been studied by using microtubule inhibitors, colcemid or nocodazole, in mice and sea urchins [20]. Interestingly, microtubule assembly is required for the formation of the male and female pronuclei after fertilization in mice but not in sea urchins. However, microtubule assembly is involved in pronuclear migration in both species. Since the sperm aster is not oriented preferentially toward the female pronucleus, the female pronucleus may have receptor-like material for the microtubules. Recently, Rieder and Salmon [21] procposed that the female nucleus is covered with dynein-like, MICROTUBULES DURING PORCINE FERTILIZATION AND PARTHENOGENESIS minus-end directed motors that translocate the female pronucleus from the periphery of the sperm aster towards its center. While the sperm aster enlarges, it also probably moves the decondensing male pronucleus to the center of the oocyte. The force responsible for male pronuclear movement has been suggested to be the aster ejection force, which consists of kinesin-like, plus-end directed motors [21]. The microtubules, however, may not be the only cytoskeletal element for pronuclear migration. In experiments using the microfilament inhibitor latrunculin [22], it has been shown that microfilaments also play a role in pronuclear movement. In the pig oocyte [23] as well as in the mouse [24], microfilaments become concentrated around pronuclei both after fertilization and parthenogenetic activation. In this study, we showed that a microfilament inhibitor, cytochalasin B, also inhibited pronuclear movement in the pig. These results suggest that both microtubule and microfilament assembly are necessary for the gamete union during fertilization in the pig. Further studies are required to clearly integrate the role and interaction of microtubules and microfilaments during pronuclear formation and movement and during mitosis. Previously, we reported that treatment of unfertilized pig oocytes with taxol induced numerous microtubule foci [25]. Taxol is an antitumor agent isolated from the bark of the yew, Taxous brevofola. It has been shown to promote microtubule polymerization by decreasing the critical tubulin concentration [26, 27]. The appearance of cytoplasmic microtubules in the porcine oocyte after taxol treatment indicates that centrosomal material probably exists in the cytoplasm as an undetectable component. The number of microtubule foci significantly decreased immediately after sperm penetration. After sperm aster formation, taxol did not induce cytoplasmic microtubules but larger and more dense sperm astral microtubules. Therefore, centrosomal material may be concentrated to the sperm neck area after sperm penetration, which may then form a functional centrosome for development of the sperm aster. After electrical activation, we observed that cytoplasmic microtubule foci aggregated to each other and formed a disarrayed microtubule network by connecting cytoplasmic microtubules. During pronuclear formation and migration, the microtubules were concentrated around the female pronucleus. These results indicate that the electrical activation of oocytes causes centrosomal material to form a dense network of microtubules, which seems to be involved in the proper positioning of the female pronucleus. During gamete union, after fertilization, and at the pronuclear stage after activation, the microtubules disappeared from the cytoplasm. Taxol did not induce cytoplasmic microtubule foci at this time but did induce microtubule assembly around the female pronucleus. Therefore, during the pronuclear stage, centrosomal material may be concen- 1403 trated in the pronucleus, and, at that time, the ability to organize microtubules is lost. In the mouse oocyte, Schatten et al. [2] studied the movement of centrosomes during fertilization. They observed that, after pronuclear apposition, microtubule foci migrated and aggregated to the pronuclear surface at the end of first interphase as the cytoplasmic microtubule disassembled, leaving pronuclear sheaths of microtubules. Pinto-Correia et al. [11] also observed absence of polymerized microtubules in the rabbit parthenote when the pronucleus was located in the center of the cell. The exact mechanism by which the nuclear membrane retains centrosomal material is not known. However, association of centrosomal material on the nuclear envelope seems to be a general strategy in mammalian eggs. Maro et al. [28] have suggested that centrosomal material is associated with the germinal vesicle membrane in immature mouse oocytes, which becomes dispersed as many foci within the cytoplasm at the time of germinal vesicle breakdown (GVBD). In pig oocytes, our previous study [25] showed that taxol did not induce microtubule foci at the germinal vesicle stage but did induce a few microtubule foci immediately after GVBD. After GVBD, the number of microtubule foci induced by taxol treatment increased. Therefore, the nuclear envelope at prophase, similar to that at the germinal vesicle and pronuclear stages, may have the ability to retain centrosomal material. 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