Different co-culture systems have the same impact on bovine embryo transcriptome.

During the last few years, several co-culture systems using either BOEC or VERO feeder cells have been developed to improve bovine embryo development and these systems give better results at high oxygen concentration (20%). In parallel, the SOF medium, used at 5% O2, has been developed to mimic the oviduct fluid. Since 2010s, the SOF medium has become popular in improving bovine embryo development and authors have started to associate this medium to co-culture systems. Nevertheless, little is known about the putative benefit of this association on early development. To address this question, we have compared embryo transcriptomes in four different culture conditions: SOF with BOEC or VERO at 20% O2, and SOF without feeders at 5% or 20% O2 Embryos have been analyzed at 16-cell and blastocyst stages. Co-culture systems did not improve the developmental rate when compared to 5% O2 Direct comparison of the two co-culture systems failed to highlight major differences in embryo transcriptome at both developmental stages. Both feeder cell types appear to regulate the same cytokines and growth factors pathways, and thus to influence embryo physiology in the same way. In blastocysts, when compared to culture in SOF at 5% O2, BOEC or VERO seems to reduce cell survival and differentiation by, at least, negatively regulating STAT3 and STAT5 pathways. Collectively, in SOF medium both blastocysts rate and embryo transcriptome suggest no influence of feeder origin on bovine early development and no beneficial impact of co-culture systems when compared to 5% O2.

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.