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.

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.

Lamb production using superovulation, embryo bisection, and transfer.

In this study, 39 embryos from donor ewes superovulated with follicle stimulating hormone-pituitary (FSH-P) were bisected to produce pairs of monozygotic twin lambs for experimentation. Each pair obtained by bisecting 8-, 9- or 10-day-old embryos was immediately transferred surgically into a recipient ewe at the same physiological stage. Of the 39 recipients which received a pair of half-embryos by transfer into the uterine horn ipsilateral to the corpus luteum, 28 (72%) lambed. Eighteen of 28 recipients lambing (64%) produced pairs, i.e., 7 male and 11 female pairs. Ten of 28 lambings produced a single lamb, i.e., six males and four females. Overall yield (the number of lambs produced in relation to the number of embryos used) was 118%. This percentage tended to increase, depending on the day of collection (Day 8, 100%; Day 9, 118%; and Day 10, 131%).

In vitro fertilization with normal development in the sheep.

Ovine tubal (n = 87) and ovarian in vitro matured oocytes (n = 99) were fertilized in vitro with ejaculated spermatozoa capacitated for 8 h in modified defined medium buffered with Hepes. High levels of fertilization were obtained as assessed by development to two- to six-cell stage within 40 h (75.8% for ovulated and 62.6% for in vitro matured oocytes). Electron microscope analysis of oocytes 20-22 h after insemination indicated that in vitro fertilization approximated the in vivo events. Embryos (two- to six-cell) were transferred surgically to the oviducts of pseudopregnant rabbits. Three days later, 42 (from ovulated oocytes) and 15 (from in vitro matured oocytes) embryos were recovered; 26 (61.9%) and 10 (66.6%), respectively, had cleaved at least once. Embryos incubated in vivo (n = 20 from ovulated oocytes; n = 9 from in vitro matured oocytes) were transferred surgically to the uteri of seven and four recipient ewes resulting in four and two pregnancies, respectively, from which three and one, respectively, have been maintained (greater than 3 months). The first lamb resulting from the in vitro fertilization of an ovulated oocyte was born. In addition, six embryos (two- to four-cell) from tubal oocytes and ten embryos (two- to six-cell) from in vitro matured oocytes were directly transferred to the oviducts of two and three ewes, respectively. Two pregnancies resulting from in vitro matured fertilized oocytes are in progress (greater than 3 months).

The development of sheep embryos when two are transferred into only one uterine horn or into each uterine horn.

In two series of experiments, two D6 morulae were surgically transferred either to each uterine horn of recipient ewes or to the same horn. When embryos were transferred to each side, the percentages of lambs born were not significantly different (68 and 62.5% respectively) whether the corpora lutea of recipient ewes were on each ovary or on the same ovary (in this case, embryos were transferred to the horn ipsilateral to the corpora lutea). When embryos were transferred to the same horn, the percentages of lambs born (71 and 92.6% respectively) were not significantly different whether the corpora lutea were on each side or on the same side. The percentages of lambs born were not significantly different whether the two morulae were transferred to each uterine horn or to the same horn.

Comparative study of preimplantation development and embryonic loss in two rabbit strains.

The prolificacy of two rabbit strains (Californian and New Zealand) has been studied in parallel with ovulation rate and embryonic development. The number of mean ovulations was 11.6 in Californians and only 9.6 in New Zealand. In spite of the higher rate in the former, the number of young born was about 8 in both strains. Californian and New Zealand blastocysts were transferred at 96 h post-coitum by injecting them into the uterus of recipient does of the same strain or of the other strain (crossed transfer). The percentage of unfertilized and delayed eggs was significantly higher (20% vs 12%) in Californians. The size of blastocysts recovered at 96 h p.c. was significantly higher in New Zealand than in Californian does (370 vs 319 micron). The difference was due to the larger proportion of small Californian blastocysts and of large New Zealand blastocysts. The larger blastocysts were placed in the right horn of recipients and the small ones in the left horn. Number of foetuses at D27 showed that the survival percentage was always higher when large blastocysts were transferred. However the survival difference with small transferred blastocysts was not significant. When transfer was within the same strain or crossed the results were not different. Since the New Zealand blastocysts were larger at D4 p.c. (one day after they arrived in the uterus), it could not be determined if this improved development was due to the conditions in the fallopian tubes or to the quality of the uterine secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Early stages of embryonic development in two rabbit genotypes.

The origin of the early embryonic loss that occurs during the first 4 days after mating in Californian doe-rabbits has been studied at 24 h or 60 h post coitum (p.c.). For comparison, 11 New Zealand doe-rabbits were slaughtered at the same times. The development stage of the embryo was determined after examination in toto at 24 h or by counting the morula nuclei at 60 h on histological sections. At 24 h p.c., 5.3% of the New Zealand eggs had not divided vs 37% of the Californian ones; this difference was highly significant. At 60 h p.c. frequency histograms of the number of morula nuclei showed that development was more variable in Californian does than in the New Zealand ones. However, the mean number of morula nuclei of each genotype was not significantly different. The higher proportion of Californian eggs (compared to New Zealand eggs) that were unfertilized or showed retarded segmentation is the main cause of the unexpectedly lower prolificacy of Californian rabbits.

Repeated superovulation and surgical recovery of embryos in the ewe.

Repeated superovulation using three treatments of pFSH at intervals of 45 to 55 days during the same breeding season was carried out in 18 Préalpes ewes. The embryos were recovered by surgery after the first two treatments and by slaughter after the last one at Day 6. Three lots of control ewes (n = 5 or 6) receiving the same superovulation treatment were slaughtered respectively at the same time. The first and third repeated superovulation treatments gave averages of 5.2 +/- 0.6 and 4.5 +/- 0.6 corpora lutea. The second one gave 3.4 +/- 0.4 corpora lutea, which was significantly lower than with treatments 1 and 3, but adhesions did not permit a perfect view of the whole surface of the ovaries. Recovery rates decreased regularly with repeated collection (88.2, 52 and 24%, respectively). The proportion of embryos at the morula/blastocyst stage also decreased from 86 and 93% to 6.7% at the third treatment. The development of post-surgical lesions probably caused variations in the apparent rate of ovulation and a decrease of egg recovery and fertilization rates. In control ewes the mean level of superovulation did not vary significantly during the breeding season and embryonic development was normal when checked at slaughter. Repeated superovulation using pFSH is worthwhile only if the eggs are recovered by perfusion with an appropriate catheter (introduced into the uterus by laparoscopy) instead of by surgery.