Meiotic competence of in vitro grown goat oocytes.

The objective of the present study was to grow meiotically incompetent goat oocytes from early antral follicles in vitro and to render them competent to undergo germinal vesicle breakdown. Cumulus-oocyte complexes with pieces of parietal granulosa cells were isolated from follicles 0.35-0.45 mm in diameter using both mechanical and enzymatic methods. The cumulus-oocyte complexes were divided into two groups according to oocyte diameter (group A: < 95 microm; group B: > 95 microm) and cultured for 8 or 9 days on granulosa cell monolayers. Within 8 days of culture, the mean oocyte diameter increased from 86 +/- 0.4 microm to 95 +/- 0.7 microm in group Aand from 106 +/- 0.2 microm to 109 +/- 0.5 microm in group B. After 9 days of culture, the mean diameter of oocytes from groups A and B were 99 +/- 0.5 microm and 112 +/- 0.4 microm, respectively. The meiotic competence of oocytes grown in vitro was evaluated by in vitro maturation. Within 8 days of culture, only 3% of oocytes from group A and 6% of oocytes from group B acquired the ability to undergo germinal vesicle breakdown. After 9 days of culture, 7% of group A oocytes and 42% of group B oocytes were competent to resume meiosis. The expression of p34(cdc2) in oocytes grown in vitro was analysed by the western blot technique. During 9 days of culture, p34(cdc2) accumulated in both groups of growing oocytes, but its concentration was lower than in fully grown oocytes used as controls. The results showed for the first time that goat oocytes from early antral follicles can grow, accumulate p34(cdc2) and acquire the ability to resume meiosis, when cultured for 9 days on granulosa cell monolayers.

Centrosome inheritance in sheep zygotes: centrioles are contributed by the sperm.

The inheritance and duplication of the sperm centriole in the sheep zygote was studied by transmission electron microscopy. We found two centrioles at one pole and a single centriole at the opposite pole of the first mitotic spindle, in monospermic eggs, 20-21 hours postinsemination. This indicated both duplication and relocation of centrioles to opposite spindle poles during fertilization. The absence of centrioles in mature sheep oocytes was confirmed. Following activation by the calcium ionophore A 23187, mature oocytes entered mitosis and formed a bipolar spindle 18 hours later. Centrioles were not detected in the mitotic spindle of parthenogenotes. Androgenetic eggs were obtained by excision of the anaphase II/telophase II meiotic spindle of fertilized eggs. They were capable of undergoing mitosis and formed one or two bipolar spindle(s) in monospermic and dispermic eggs, respectively, 20-24 hours postinsemination. In two monospermic androgenetic eggs, two centrioles were found at one pole and a single centriole at the opposite pole of the first mitotic spindle. Three centrioles were also observed in another androgenetic egg in prometaphase of the first mitotic division, in close vicinity to the sperm neck-piece. These data provide evidence that the sperm centriole do reproduce and occupy a pivotal position on opposite spindle poles at syngamy. Altogether, the present findings suggest that centrioles of sheep zygotes are paternally derived.

In vitro binding of free cdc2 and raf kinase to membrane vesicles: a possible new regulatory mechanism for cdc2 kinase activation in Xenopus oocyte.

The G2-M transition of the cell cycle is under the control of the M-phase promoting factor (MPF) formed of cdc2 kinase and cyclin B. The Xenopus prophase-blocked oocyte contains a stockpile of cyclin B2-cdc2 complexes that are maintained inactive by a double inhibitory phosphorylation on Thr-14 and Tyr-15 of cdc2. Free cdc2 molecules that are not associated with cyclin, are present in excess as compared to cyclin B2-associated cdc2. This pool of free cdc2 is permanently recruited to associate with neosynthetized cyclin B2 in the resting prophase oocyte, to feed up the pre-MPF stockpile. During re-entry into meiosis, free cdc2 could generate with newly synthesized cyclin B a small level of active MPF, that could serve as starter to initiate the conversion of pre-MPF into MPF. It was, therefore, of high interest to investigate whether free cdc2 interacts with other proteins and what could be its intracellular localization. To address these questions, we developed an in vitro system of membrane vesicles. We demonstrate here that free cdc2 is recovered in association with the external layer of membrane vesicles, whereas cyclin B2-associated cdc2 is not. Cyclin is able to associate in vitro with cdc2-containing membrane vesicles. This association does not induce the inhibitory cdc2 phosphorylations. However, it does not lead to active complexes, suggesting that membrane vesicles prevent cdc2 activation. C-Raf1, another kinase activated during reentry into meiosis, is also totally recovered in association with the membrane vesicles.

p34cdc2 expression and meiotic competence in growing goat oocytes.

The expression of the catalytic subunit of the maturation promoting factor (MPF), p34cdc2, was analyzed during meiosis and final growth of goat oocytes. Western blot analysis revealed the presence of p34cdc2 in fully grown oocytes (follicles > 3 mm in diameter) prior to and during meiotic maturation. p34cdc2 was present in partially competent oocytes at the germinal vesicle stage (follicles 0.5 to 0.8 mm and 1 to 1.8 mm in diameter). In contrast, p34cdc2 was not expressed in meiotically incompetent oocytes from small antral follicles (follicles < 0.5 mm in diameter). The amount of p34cdc2 increased with oocyte growth and acquistion of meiotic competence. Moreover, p34cdc2 accumulated in partially competent and incompetent oocytes within 27 hr of culture, but the level of p34cdc2 in incompetent oocytes remained very low and was not sufficient to allow spontaneous resumption of meiosis. The expression of the cdc2 gene was analyzed by polymerase-chain-reaction (PCR) on reverse transcribed mRNA. The presence of the cdc2 transcript was evidenced in both competent and incompetent oocytes at the germinal vesicle stage. These data indicate that a deficiency in the expression of p34cdc2 that could be regulated at the translational level, may be a limiting factor for meiotic competence acquistion in goat oocytes. We further investigated the mechanisms of MPF activation in competent and incompetent oocytes by using okadaic acid, a protein phosphatase inhibitor. Okadaic acid induced the premature resumption of meiosis associated with MPF activation in competent oocytes. In partially competent oocytes, okadaic acid induced premature meiosis reinitiation and MPF activation, but only after pre-culture for 10 hr. Acquisition of sensitivity to okadaic acid treatment was dependent on protein synthesis since it failed to occur when cycloheximide was added during the pre-culture period. Incompetent oocytes responded to okadaic acid treatment only after 27 hr of culture, when they had accumulated small amounts of p34cdc2. These data suggest that okadaic acid may bypass the subthreshold level of p34cdc2, provided the oocytes have synthesized some additional factors that remain to be identified.

Cyclin B1 expression in meiotically competent and incompetent goat oocytes.

To start determining the nature of meiotic incompetence in goat oocytes, we have examined the expression of one of the potential pre-MPF subunits: the cyclin B1. We have been isolating a small DNA probe encoding the goat cyclin B1 box to analyze the expression of the cyclin B1 gene in competent and incompetent goat oocytes. This probe was easily obtained by polymerase-chain-reaction (PCR) on reverse-transcribed mRNA from granulosa cells, using cyclin B specific primers derived from a bovine cDNA. The transcript corresponding to cyclin B1 in goat granulosa cells is 1.8 kb. In situ hybridization analysis indicated that competent and incompetent oocytes contained cyclin B1 mRNA, but also that active cyclin B1 mRNA synthesis occurred at the end of the growth phase, e.g., when oocytes progressed in the acquisition of meoitic competence. Western blot analysis, performed with a monoclonal anticyclin B1 antibody, revealed in competent and incompetent oocytes a polypeptide of 65 kDa corresponding to the goat cyclin B1 protein. This pattern of cyclin B1 expression further suggested that meiotic incompetence in goat oocytes could not be primarily correlated with a lack of cyclin B1 protein as potential pre-MPF subunit, but to a limiting amount of this protein.

Mitogen-activated protein kinase activity during goat oocyte maturation and the acquisition of meiotic competence.

Changes in MPF and MAPK activities during meiotic maturation of goat oocytes were investigated. Detection of MPF activity occurred concomitantly with GVBD, increased at MI, decreased during anaphase-telophase I transition, and increased thereafter in MII oocytes. The appearance of MAPK activity was delayed compared to MPF activity. MAPK activity increased after GVBD and persisted during the MI-MII transition. Whether MAPK was implicated in goat oocyte meiotic competence was also investigated by using oocytes from different follicle size categories that arrest at specific stages of the maturation process (GV, GVBD, MI, and MII). Results indicate that the ability of goat oocytes to resume meiosis is not directly related to the presence of Erk2. The ability to phosphorylate MAPK is acquired by the oocyte during follicular growth after the ability to resume meiosis. GVBD-arrested oocytes exhibited a high level of MPF activity after 27 hr of culture. However, 28% of oocytes from this group contained inactive MAPK, and 72% exhibited high MAPK activity. In addition, 29% of GVBD-arrested oocytes contained a residual interphasic network without recruitment of microtubules around the condensed chromosomes; 71% of GVBD-arrested oocytes displayed recruitment of microtubules near the condensed chromosomes and contained asters of microtubules distributed throughout the cytoplasm. These results indicate that oocytes arrested at GVBD were not exactly at the same point in the meiotic cell cycle progression, and suggest that MAPK could be implicated in the regulation of microtubule organization. The data presented here suggest that in goat oocytes, MAPK is not implicated in the early events of meiosis resumption, but rather in post-GVBD events such as spindle formation and MII arrest.

Meiotically incompetent and competent goat oocytes: timing of nuclear events and protein phosphorylation.

The ability of mammalian oocytes to resume meiosis and to complete the first meiotic division is acquired sequentially during their growth phase. The acquisition of meiotic competence in goat oocytes has been previously correlated with follicular size (9). Since protein phosphorylation/dephosphorylation play a key role in oocyte maturation, it could be that in meiotically incompetent oocytes, such post-translational modifications are inadequate. The aim of this study was to analyze whether changes in oocyte proteins phosphorylation occurred during the acquisition of meiotic competence. For this propose, goat oocytes were divided into 4 classes according to follicular size and meiotic competence: Class A oocytes from follicles < 0.5 mm in diameter: Class B oocytes from follicles 0.5-0.8 mm; Class C oocytes from follicles 1-1.8 mm and class D oocytes from follicles > 3 mm. The protein phosphorylation patterns of these classes of oocytes were studied at different times of in vitro maturation. After 4h of culture, when all oocytes were in the germinal vesicle stage, only the oocytes from Class D displayed the phosphoproteins at 110 kD, 31 kD and around 63 kD. In contrast to Class D oocytes Classes B and C oocytes were partially competent to mature, they underwent germinal vesicle breakdown later than fully competent Class D oocytes and remained in early prometaphase I or in metaphase I, respectively. They exhibited the phosphoprotein changes that are associated with commitment to resume meiosis; but the changes occurred later than in Class D oocytes, which were fully competent to reach metaphase II. After 27 h of culture, the phosphorylation patterns of Class B, C and D oocytes were identical, whereas the meiotic stages reached were quite different. The phosphoprotein changes associated with oocyte maturation did not occur in meiotically incompetent Class A oocytes, which were blocked at the germinal vesicle stage. From these results it can be concluded that, at the GV stage, meiotically incompetent and competent goat oocytes display different patterns of protein phosphorylation. Once oocytes are able to resume meiosis they undergo specific phosphorylation changes, but whether these changes are markers or regulators of maturation events remains to be determined.

Developmental competence of goat oocytes from follicles of different size categories following maturation, fertilization and culture in vitro.

The aim of the present study was to investigate the effect of size of from which goat oocytes originate on their subsequent ability to be fertilized and to undergo early embryonic development in vitro. Nonatretic follicles larger than 2 mm in diameter were dissected and distributed into three groups according to size (small: 2-3 mm; medium: 3.1-5 mm; large: > 5 mm). Cumulus-oocyte complexes were isolated from the follicles and only those with a compact multilayered cumulus were selected for in vitro maturation. After maturation, 70%, 83% and 97% of oocytes from small, medium and large follicles, respectively, were at metaphase II. After in vitro fertilization, no significant difference was observed in the cleavage rate 40 h after insemination between oocytes from small (46%) and medium (55%) follicles, and between oocytes from large follicles (69%) and ovulated oocytes (75%). After in vitro culture, significantly more embryos from small follicles arrested before or at the 8-16 cell stage (84% compared with 53%, 45% and 39% of embryos from medium and large follicles and ovulated oocytes, respectively). The proportion of morulae and blastocysts obtained was 10% and 6% from small follicles, 35% and 12% from medium follicles, 29% and 26% from large follicles and 20% and 41% from ovulated oocytes. Oocytes from small and medium follicles yielded a significantly lower proportion of hatched blastocysts (0% and 3%, respectively) than did those from large follicles and from ovulated oocytes (15% and 34%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Estrous sheep serum as a potent agent for ovine IVF: effect on cholesterol efflux from spermatozoa and the acrosome reaction.

The aim of the present study was to evaluate the effect of heat-inactivated estrous sheep serum (ESS) on sheep IVF. When the capacitation and the fertilization media contained 20% ESS, a fertilization rate of 85% was achieved. The beneficial effect of ESS on sheep IVF was further demonstrated since the fertilization rate was null when ESS was omitted during sperm capacitation and fertilization. Estrous sheep serum supported both sperm capacitation and fertilization as shown by the results of experiments in which it was omitted during one of these steps: sperm capacitation in serum-free medium resulted in delayed sperm-oocyte penetration, while fertilization in serum-free medium significantly decreased the percentage of fertilized oocytes. To investigate the influence of serum on sperm ability to undergo the acrosome reaction, salt-stored follicular sheep oocytes were inseminated, and the acrosomal status of spermatozoa attached to zonae was examined by electron microscopy after a 4-h period of coincubation. Quantitative analysis on thin sections demonstrated that fewer acrosome-reacted spermatozoa were observed when the capacitation and insemination steps were carried out in DM-H medium without serum than in DM-H-SS supplemented with 20% ESS (0.08, [0; 0.34], (median, range)/100 microm zona vs 1.32, [0.90; 2.28]/100 microm zona; P<0.01). Since a higher number of spermatozoa attached to the zona surface in DM-H medium, the proportion of acrosome-reacted spermatozoa was much lower (0.7%, [0%; 2.2%], (median, range) vs 54%, [25%; 100%]; P<0.01) in the absence of serum. These results indicate that in our IVF system the development of the acrosome reaction depended on serum. Sperm cholesterol efflux during in vitro capacitation was measured on [3H] cholesterol labeled spermatozoa resuspended in DM-H or DM-H-SS medium. A time-dependent cholesterol removal was observed in the presence of serum (60+/-5%, mean+/-SD, after 5 h), whereas it was limited to 14+/-3% in DM-H medium; hence addition of serum to the capacitation medium efficiently supports cholesterol efflux, which is thought to be a key-event in the capacitation process.

Morphological and functional changes accompanying the acquisition of meiotic competence in ovarian goat oocyte.

In order to determine at which follicular size goat oocytes were capable of resuming and completing meiosis, we evaluated their ability to mature in vitro and measured their maturation promoting factor (MPF) activity by histone H1 kinase assay. The results indicated that goat oocyte meiotic competence developed progressively in follicles ranging from 0.5 to 3 mm; the oocytes acquired the ability to resume meiosis in follicles of 0.5-0.8 mm, to reach metaphase I (MI) in follicles of 1-1.8 mm, and to reach metaphase II (MII) in follicles larger than 3 mm. The presence of MPF activity was first observed in oocytes arrested at early prometaphase I and reached a maximum level in oocytes blocked in metaphase I (MI). In the second part of this study, RNA synthesis and nucleolar changes were analyzed during the growth period. The acquisition of meiotic competence was accompanied by nucleolar compaction and a dramatic decrease in RNA synthesis. Changes in protein patterns were also analyzed, but only slight differences were observed among oocytes from the different classes.

[Acrosome reaction and fertilization]. / Réaction acrosomique et fécondation.

The acrosome reaction that occurs after sperm capacitation, is an exocytotic event induced by a Ca++ influx. It plays an essential role during fertilization, by making spermatozoa able of penetrating the zona and capable of fusing with the egg plasma membrane. Zona pellucida is the natural inducer of the acrosome reaction. Binding of the sperm receptor to ZP3, a zona glycoprotein acting as ligand, triggers the molecular events leading to acrosomal exocytosis. G-proteins may be involved in the signal-transduction pathway during the acrosome reaction. Other known inducers of the human acrosome reaction include follicular fluid, progesterone and the calcium ionophore A23187. Progesterone acts through a receptor on the sperm plasma membrane, while the ionophore promotes non-physiological sperm Ca++ uptake. Several cytochemical procedures have been proposed for evaluating the acrosome reaction: the acrosomal status can be observed after staining or after labeling with lectins or antibodies. These methods both attempt to evaluate sperm viability and to distinguish degenerative acrosomal loss.

Microtubule distribution during fertilization in the rabbit.

The distribution of microtubules was studied during fertilization of the rabbit oocyte by immunofluorescence microscopy after staining with an anti-alpha-tubulin antibody. In ovulated oocytes, microtubules were found exclusively in the meiotic spindle. At fertilization, the paternal centrosome generated sperm astral microtubules. During pronuclear development, the sperm aster increased in size, and microtubules extended from the male pronucleus to the egg center and towards the female pronucleus. These observations indicate that microtubules emanating from the sperm centrosome were involved in the movements leading to the union of the male and female pronuclei. At late pronuclear stage, microtubules surrounded the adjacent pronuclei. The mitotic spindle that emerged from the perinuclear microtubules contained broad anastral poles.

Fertilization abnormalities in human in-vitro fertilization.

Fertilization abnormalities (premature chromosome condensation of spermatozoa (PCC), triploidy, haploidy) were analysed in order to determine their origin. PCC occurs in 9% of unfertilized oocytes and seems to be the consequence of a failure of oocyte activation, leading to the continuing presence of cytoplasmic chromosome-condensing factors, causing the sperm nucleus to undergo chromosome condensation prematurely. This anomaly appears to be related to incomplete nuclear and/or cytoplasmic maturation. Triploid zygotes (6.5% of fertilized oocytes) display an original type of division: half of them divide into 3 and 6 cells, whereas at the same time diploid zygotes divide into 2 and then 4 cells. A cytological study, using both antitubulin antibodies and Hoechst dye, allowed us to demonstrate that they divide into 3 cells by means of a tripolar spindle. Triploidy seems to be correlated with four of 16 clinical or biological parameters examined: semen origin (fresh or frozen), type of stimulation treatment, number of oocytes recovered and embryo morphology. Haploid eggs (1.6% of inseminated oocytes) result from parthenogenetic activation. A correlation was found between a high number of recovered oocytes and triploid zygotes, and the occurrence of oocyte activation. These data show that increasing follicular recruitment decreases the overall oocyte quality and maturity leading to an overall 9% with impaired fertilization.

In vitro maturation and fertilization of goat oocytes.

Follicular cumulus-enclosed goat oocytes were matured in vitro in the presence of granulosa cells, follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol-17beta. While 86% of the oocytes from follicles 2 to 6 mm in diameter achieved meiotic maturation, only 24% of the oocytes from follicles 1 to 2 mm in diameter progressed to Metaphase II. Exposure of follicle-enclosed cumulus-oocyte complexes to 20 degrees C prior to culture resulted in 11.5% of the oocytes exhibiting abnormal meiotic spindle. This indicated that immature goat oocytes are particularly sensitive to temperature. Ejaculated spermatozoa were capacitated according to the technique previously proposed for ram sperm (1). The fertilization rates of ovulated and mechanically denuded in vitro-matured oocytes were 85 and 82.8%, respectively; 59.7% of ovulated and 57.1% of in vitro-matured oocytes were normally fertilized, as shown by the presence of both the female and the male pronucleus as well as by the remnants of the sperm tail in the ooplasm, 17 hours after insemination. Polyspermy was the main abnormality detected, and it affected almost 20% of the inseminated oocytes. The cleavage rate (two to fourcell stage) 41 hours after insemination of in vitro-matured and fertilized oocytes was 58%.

In vitro fertilization of sheep oocytes matured in vivo.

Incubating washed ram spermatozoa in a modified Brackett's defined medium buffered with Hepes (DM-H) containing 20% of heat-inactivated sheep serum appears to be a reliable method of capacitating sperm for in vitro fertilization. Raising the Ca(++) concentration in the fertilization medium (DM-H-SS) to 10 mM stabilized the fertilization rate of various rams (2). This study was designed to determine if the developmental competence of the oocytes fertilized under such conditions was normal. Thirty-seven ewes, treated with progestagen sponges, were superovulated with porcine follicle stimulating hormone (pFSH: 16 mg). An intramuscular injection of gonadotropin releasing hormone (GnRH: 100 mug); given 24 to 26 h after sponge removal, induced the synchronization of ovulations 24 h later. Ovulated oocytes (n = 229) recovered with flushing of the oviducts were inseminated in vitro and 17 h later either fixed in acetic/alcohol (n = 115) to evaluate fertilization or transferred (n = 114) into 38 synchronized recipients (three oocytes/recipient) to evaluate their developmental competence. Of the fixed oocytes, 82.6% were fertilized and 61.7% were monospermic. Nineteen of the recipient ewes (50%) were pregnant at Day 18, and 16 ewes produced a total of 26 live young (mean: 1.63/ewe). The results showed a high efficiency of in vitro fertilization of ovulated oocytes in sheep following a pFSH-GnRH treatment and the in vivo developmental competence of oocytes fertilized in the presence of elevated Ca(++) concentration.

Behavior of the sperm centriole during sheep oocyte fertilization.

The proximal centriole of the sperm was incorporated into the oocyte, at fertilization and a small aster of microtubules formed in the neck region containing the centriole. At late pronuclear stage, the sperm astral microtubules were located in the narrow cytoplasmic space between the apposed pronuclei. One or two centrioles were observed in/or close to the sperm neck region lying between the apposed pronuclei. Short microtubules radiated from the pericentriolar material which surrounded the centrioles. These results indicate that the proximal centriole of the sperm is not lost after incorporation into the ooplasm, as it is usually thought for mammals, but persists through the first cell cycle of the zygote. Moreover, the centriole inherited from the sperm reproduces at the pronuclear stage and migrates to one pole of the first mitotic spindle.

In vitro fertilization in the sheep: effect of elevated calcium concentration at insemination.

Oocytes (n = 273), collected from superovulated ewes, were inseminated with in vitro capacitated spermatozoa from four rams [Crozet et al., 1987]. In each experiment, parallel insemination was performed using aliquants from a single ejaculate in either our standard fertilization medium DM-H-SS (a modification of Brackett's defined medium, buffered with HEPES and containing 20% v/v sheep serum) or in the same medium supplemented with calcium lactate (DM-H-SS + Ca). The measured total calcium concentrations were Ca++ T = 2.74 mM in DM-H-SS medium and Ca++ T = 8.74 mM in DM-H-SS - Ca; the ratio of free to total calcium in DM-H-SS was Ca++ F/Ca++ T = 0.85. Fertilization was assessed at 17 hours postinsemination. Variations in the ejaculates were observed for each of the four rams tested. When DM-H-SS--CA was used, the percentages of fertilized (75% vs. 50%) and monospermic (58% vs. 41% eggs were significantly enhanced compared with use of DM-H-SS. No improvement was observed in control medium DM-H-SS + lac containing neutralized lactic acid. Supplementing the fertilization medium with calcium had no apparent effect on the incidence of polyspermy. These experiments show that the fertilization rate achievable in vitro by individual ejaculates from various rams can be increased by raising the calcium concentration in the fertilization medium to a value much higher than that present in tubal fluids from estrous ewes. Extended incubation in such a high calcium concentration is unnecessary for in vitro capacitation of ram spermatozoa.

Distribution and role of microfilaments during early events of sheep fertilization.

The distribution of actin was studied during early events of sheep fertilization by fluorescence microscopy after staining with 7-nitrobenz-2-oxal-1.3 diazole (NBD)-phallacidin and anti-actin antibody and by electron microscopy after heavy meromyosin labelling. unfertilized and fertilized eggs exhibited a continuous band of fluorescence with both NBD-phallacidin and anti-actin antibody. Unlike in mice, no high concentration of actin overlying the spindle was detected in ovulated sheep oocytes. At the site of sperm head incorporation, the fertilization cone developed above the decondensing male chromatin and was underlined by a submembranous area rich in microfilaments. A similar actin network was observed in the cortex of the second polar body. Cytochalasin D was used to investigate the role of actin during the fertilization process. This drug did not prevent sperm fusion and incorporation but inhibited polar body abstriction and fertilization cone development and retarded sperm tail incorporation. Moreover, in the presence of the drug, the anchorage of the metaphase II spindle at the surface of the egg was destroyed. The role of microfilaments in these early events is discussed.

Microtubule and centrosome distribution during sheep fertilization.

The distribution of microtubules and centrosomes was studied during sheep fertilization by electron and immunofluorescence microscopy. Tubulin and centrosomal material was identified with monoclonal anti-alpha-tubulin and MPM-2 antibodies, respectively. In ovulated eggs, microtubules were exclusively found in the meiotic spindle and centrosomal material at each of its poles. At fertilization, sperm centrosomes were incorporated into the egg and organized the sperm astral microtubules. During pronuclear development and migration, the sperm aster increased in size; microtubules of the sperm aster extended from the male pronucleus to the egg center and towards the female pronucleus. The position of the sperm aster during pronuclear migration suggests that it plays a role in this process. When the pronuclei were in apposition in the egg center, a dense array of microtubules and the centrosomal material were present between the two pronuclei. The proximal centriole of the sperm was identified by electron microscopy, between the apposed pronuclei. The centrosomal material extending around the centriole and the sperm neck and proximal mid-piece, apparently contained several foci from which microtubules radiated. These data suggest that in sheep unlike in mice, centrosomal material originating from the sperm is involved in the fertilization events.