Laurence Gall

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.

Expression of Pluripotency Master Regulators during Two Key Developmental Transitions: EGA and Early Lineage Specification in the Bovine Embryo

Pluripotency genes are implicated in mouse embryonic genome activation (EGA) and pluripotent lineage specification. Moreover, their expression levels have been correlated with embryonic term development. In bovine, however, little information is available about dynamics of pluripotency genes during these processes. In this study, we charted quantitative and/or qualitative spatio-temporal expression patterns of transcripts and proteins of pluripotency genes (OCT4, SOX2 and NANOG) and mRNA levels of some of their downstream targets in bovine oocytes and early embryos. Furthermore, to correlate expression patterns of these genes with term developmental potential, we used cloned embryos, having similar in vitro but different full term development rates. Our findings affirm: firstly, the core triad of pluripotency genes is probably not implicated in bovine EGA since their proteins were not detected during pre-EGA phase, despite the transcripts for OCT4 and SOX2 were present. Secondly, an earlier ICM specification of transcripts and proteins of SOX2 and NANOG makes them pertinent candidates of bovine pluripotent lineage specification than OCT4. Thirdly, embryos with low term development potential have higher transcription rates; nevertheless, precarious balance between pluripotency genes is maintained. This balance presages normal in vitro development but, probably higher transcription rate disturbs it at later stage that abrogates term development.

FOXL2 is a female sex-determining gene in the goat.

The origin of sex reversal in XX goats homozygous for the polled intersex syndrome (PIS) mutation was unclear because of the complexity of the mutation that affects the transcription of both FOXL2 and several long noncoding RNAs (lncRNAs). Accumulating evidence suggested that FOXL2 could be the sole gene of the PIS locus responsible for XX sex reversal, the lncRNAs being involved in transcriptional regulation of FOXL2. In this study, using zinc-finger nuclease-directed mutagenesis, we generated several fetuses, of which one XX individual bears biallelic mutations of FOXL2. Our analysis demonstrates that FOXL2 loss of function dissociated from loss of lncRNA expression is sufficient to cause an XX female-to-male sex reversal in the goat model and, as in the mouse model, an agenesis of eyelids. Both developmental defects were reproduced in two newborn animals cloned from the XX FOXL2(-/-) fibroblasts. These results therefore identify FOXL2 as a bona fide female sex-determining gene in the goat. They also highlight a stage-dependent role of FOXL2 in the ovary, different between goats and mice, being important for fetal development in the former but for postnatal maintenance in the latter.

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.

PTGS2-Related PGE2 Affects Oocyte MAPK Phosphorylation and Meiosis Progression in Cattle: Late Effects on Early Embryonic Development

During the periovulatory period, the induction of prostaglandin G/H synthase-2 (PTGS2) expression in cumulus cells and associated prostaglandin E2 (PGE2) production are implicated in the terminal differentiation of the cumulus-oocyte complex. During the present study, the effects of the PTGS2/PGE2 pathway on the developmental competence of bovine oocytes were investigated using an in vitro model of maturation, fertilization, and early embryonic development. The specific inhibition of PTGS2 activity with NS-398 during in vitro maturation (IVM) significantly restricted mitogen-activated protein kinase (MAPK) activation in oocytes at the germinal vesicle breakdown stage and reduced both cumulus expansion and the maturation rate after 22 h of culture. In addition, significantly higher rates of abnormal meiotic spindle organization were observed after 26 h of culture. Periconceptional PTGS2 inhibition did not affect fertilization but significantly reduced the speed of embryo development. Embryo output rates were significantly decreased on Day 6 postfertilization but not on Day 7. However, total blastomere number was significantly lower in embryos obtained after PTGS2 inhibition. The addition of PGE2 to IVM and in vitro fertilization cultures containing NS-398 overrode oocyte maturation and early embryonic developmental defects. Protein and mRNA expression for the prostaglandin E receptor PTGER2 were found in oocytes, whereas the PTGER2, PTGER3, and PTGER4 subtypes were expressed in cumulus cells. This study is the first to report the involvement of PGE2 in oocyte MAPK activation during the maturation process. Taken together, these results indicate that PGE2-mediated interactions between somatic and germ cells during the periconceptional period promote both in vitro oocyte maturation and preimplantation embryonic development in cattle.

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.

Cytokeratin-like proteins in the sheep oocyte

In immunoblotting analysis of fully grown oocyte proteins separated by sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis, four polypeptides reacted specifically with cytokeratin antibodies: Mr 66,000, IEP 5.6; Mr 64,000, IEP 5.4; Mr 59,000, IEP 5.3; Mr 55,00, IEP 5.2. These proteins remained insoluble after extraction in high salt buffer and Triton X-100. In oocytes isolated from small antral follicles, only two polypeptides of Mr 66,000 and Mr 55,000 were detected. Immunofluorescence microscopy revealed a bright granular staining throughout the oocyte with an accumulation of granules in the perinuclear and cortical regions. Using electron microscopy and immunogold staining after treatment with cytokeratin antibody, gold particles were found on discrete amorphous material distributed throughout the cell. In the subcortical region of the oocyte, dense aggregates, whose diameters ranged from 3 to 8 microns, were also covered with gold particles. From these results it appears that cytokeratin-like proteins are present in sheep oocytes in a non-fibrillar form.

FOXL2 Is Regulated During the Bovine Estrous Cycle and Its Expression in the Endometrium Is Independent of Conceptus-Derived Interferon Tau

ABSTRACT FOXL2, a winged-helix/forkhead domain transcription factor, is a key gene involved in the differentiation and biological functions of the ovary. In a recent transcriptomic analysis, we found that FOXL2 expression in bovine caruncular endometrium was different from that in intercaruncular endometrium. In order to gain new insights into FOXL2 in this tissue, we determined the expression of this transcription factor during the estrous cycle and the establishment of pregnancy in cattle. The endometrial expression of FOXL2 did not vary during maternal recognition of pregnancy (Days 16–20). Using an in vivo bovine model and primary cell cultures, we showed that FOXL2 was not an interferon-tau target gene. Both FOXL2 transcript and protein were expressed from Day 5 to Day 20 of the estrous cycle, and their levels showed a significant increase during the luteolytic phase. A 2-day progesterone supplementation in heifers led to a clear down-regulation of FOXL2 protein levels, suggesting the negative impact of progesterone on FOXL2 expression. Immunohistochemistry data revealed the localization of FOXL2 in endometrial stromal and glandular cells. FOXL2 subcellular distribution was shown to be nuclear in endometrial samples collected during the luteolytic period, while it was not detected in nuclei during the luteal phase and at implantation. Collectively, our findings provide the first evidence that FOXL2 is involved in the regulation of endometrial tissue physiology.

Co‐Expression of Cytokeratins and Vimentin in Sheep Cumulus‐Oocyte Complexes. Alteration of Intermediate Filament Distribution by Acrylamide

The association between germ cells and somatic granulosa cells persists throughout the growth of the oocyte by means of foot processes of the cumulus corona cells that cross the zona pellucida. During meiotic maturation important nuclear and cytoplasmic events occur in cumulus‐oocyte complex suggesting implication of cytoskeletal elements. Immunoblotting analysis of cytoskeletal proteins of the cumulus cells revealed the presence of vimentin polypeptide and of at least two cytokeratin polypeptides. Using immunofluorescence techniques on cryostat sections through frozen tissue, we provided evidence for the presence of cytokeratins of the simple epithelial type in addition to vimentin in sheep cumulus cells. These two types of intermediate filaments were localized throughout the cytoplasm and especially in the foot processes which cross the zona pellucida. The contact area between the two cell types was also labelled with the antibodies. Acrylamide treatment of cumulus‐oocyte complexes involved a drastic disorganization of the intermediate filament network and triggered the isolation of the oocyte from its cumulus cells. This isolation resulted in resumption of meiosis. From these results it appears that intermediate filaments could participate in the process of gap junction loss and indirectly in the control of meiosis resumption.

Nuclear remodeling in bovine somatic cell nuclear transfer embryos using MG132-treated recipient oocytes.

The early events in the nuclear reprogramming process during somatic cell nuclear transfer (SCNT) consist of morphological remodeling of the donor nucleus including premature chromosome condensation (PCC). In the present study, the objective was to increase oocyte M-Phase Promoting Factor (MPF) kinase activity and to examine the fate of the donor nucleus and the development of SCNT embryos thereafter. Indeed, in controls, recipient oocytes activated upon nuclear transfer, undergo a decrease in MPF activity, responsible for the inability to promote PCC in 77.8% of reconstituted embryos. Here we showed that exposure of the recipient oocyte to the proteasome inhibitor MG132 prior to fusion inhibited the degradation of cyclin B, which normally occurred immediately after activation by electro stimulation, and therefore sustained a high level of MPF. Treatment with MG132 also significantly increased the percentage of SCNT embryos with PCC when compared to the nontreated SCNT control embryos (94.1 vs. 22.2%, respectively, p < 0.01). The frequency of development to the blastocyst stage did not differ between MG132-treated or untreated recipient oocytes. However, we observed a significant increase of the total cells number in embryos produced after MG132 treatment. Investigation of the global nuclear organization by immunodetection of heterochromatin protein 1 (CBX1) showed that SCNT embryos derived from MG132-treated recipient oocytes displayed organization patterns similar to the ones observed in IVF embryos in contrast to the nontreated SCNT controls. Taken together, these results suggest that the PCC induced by MG132 treatment allows reorganization of the chromatin at an appropriate time potentially, leading to better reprogramming.