Nicole Crozet

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.

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%.

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.

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 acquisition 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 acquisition in goat oocytes.

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. Microsc. Res. Tech. 49:445–450, 2000.

Activity of maturation promoting factor in mammalian oocytes after its dilution by single and multiple fusions

Mouse and porcine fully grown oocytes at metaphase I(MI) were fused to one or more fully grown oocytes of the same species that contained an intact germinal vesicle (GV). In fused cells containing one GV, premature chromosome condensation (PCC) was observed. In fused cells containing more than one GV, germinal vesicle breakdown (GVBD) and PCC were delayed. Fusion of an MI fully grown oocyte with a growing oocyte resulted in rapid PCC, whereas, fusion of an MI fully grown oocyte with more than one growing oocyte resulted in neither PCC nor GVBD. Moreover, MI chromosomes formed a clump of chromatin. Results of these experiments suggest that the delay in GVBD in fusions of MI oocytes with multiple GV-intact oocytes was due to dilution of maturation promoting factor (MPF) by the cytoplasm of the GV-intact oocytes and that the cytoplasm of growing oocytes can inhibit MPF present in MI oocytes.

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.

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. Microsc. Res. Tech. 45:13–30, 1999.

The Use of Isopropyl-N-phenylcarbamate as a Potential Contraceptive

The most successful contraceptive practices, exclusive of the physical methods, affect the complex hypothalamic—hypophyseal—gonadal feedback systems. The intervention could theoretically occur at any point of this feedback loop. Steroid supplementation reduces the level of gonadotrophin-secretory activity and via it gonadal steroid production. The inconveniences of this method to the user and the user’s undesirable physiological responses to this method have become known during recent decades of contraceptive practice (for a review, see Regulation de la Fecondite,Vol. 83, INSERM, Paris, 1979). Steroid-based contraceptives certainly influence gamete production and, as well, the hormone-secretory activity of the gonads themselves and various protein hormones of the central nervous system.