Sylvie Ruffini

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.

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.

Intermediate Filaments Promote Nuclear Mechanical Constraints During Somatic Cell Nuclear Transfer in the Mouse

The somatic cell nuclear transfer (SCNT) procedure requires nuclear remodeling to return differentiated somatic nuclei to the totipotent undifferentiated stage. We hypothesize that mechanical constraints might occur upon SCNT and thereby affect nuclear remodeling. Therefore, we analyzed the nuclear structures upon SCNT using as donors either wild-type fibroblasts with a dense vimentin network or vimentin-deprived cells [embryonic stem cells (ESCs) and fibroblasts invalidated for vimetin]. We demonstrated that following nuclear transfer of wild-type fibroblasts, vimentin intermediate filaments (IFs) persisted around the transplanted nuclei and 88% of them presented severe distortions. We also showed that the presence of vimentin filaments in the reconstructed embryos was correlated with DNA damage, as evidenced by γH2A.X foci. On the other hand, when ESCs or vimentin-null (Vim(-/-)) fibroblasts devoid of IFs were used as nuclear donors, no nuclear distortion and less DNA damage were observed. Altogether we believe that the introduction of vimentin into recipient oocytes during SCNT induces a mechanical constraint on the transplanted nucleus that is responsible for nuclear distortions and DNA damage. This could lead to incomplete reprogramming that would be detrimental to further embryonic development.

41 NUCLEAR REMODELING IS AFFECTED BY TREATMENT OF BOVINE OOCYTES WITH A PROTEASOME INHIBITOR BEFORE NUCLEAR TRANSFER

Complete reprogramming of somatic cell nuclei after nuclear transfer (NT) depends on extensive remodeling of chromatin by factors present in the recipient cytoplast. M-Phase Promoting Factor (MPF) activity, responsible for nuclear remodeling in metaphase II recipients, may be lowered by oocyte enucleation and handling prior to NT. Then, a partial nuclear envelope breakdown or incomplete premature chromosome condensation (PCC) may be, in turn, associated with an inefficient reprogramming. The aim of the present study was to maintain the bovine recipient cytoplast at a high level of MPF activity during the fusion procedure by using a proteasome inhibitor, MG132, and to assess the consequences on nuclear remodeling and developmental potential. Bovine COCs were in vitro-matured for 23 h. Matured oocytes were denuded, and then incubated in TCM-199 for 45 min and enucleated in the presence (treated group) or absence (control group) of 5 µm MG132. Embryos were reconstructed by fusion with adult fibroblasts and activated in 10 µg mL–1 cycloheximide and 5 µg mL–1 cytochalasin B. In Experiment 1, MPF activity was analyzed immediately after fusion/activation by measuring the phosphorylation of exogenous histone H1, and Cyclin B expression was assessed by Western blotting. In Experiment 2, microtubules revealed by immunofluorescense with anti-tubulin antibody and chromatin stained with 10 µg mL–1 propidium iodide were analyzed by confocal microscopy 1 h after fusion/activation. In Experiment 3, NT embryos activated for 5 h were cultured in vitro for 7 days. Rate of development and cell counts in both groups were then recorded at the blastocyst stage. Remarkably, in Experiment 1, a high MPF activity was found in only 50% of the control oocytes, but MG132 treatment did not enhance this rate. On the other hand, cyclin B persisted for 2 h after activation in treated oocytes whereas it had dropped in controls. Experiment 2 revealed a higher rate of PCC in the treated embryos (n = 51) than in control embryos (n = 54): 96.0% v. 24.0% (chi-square, P < 0.001). Moreover, microtubules reorganized in a metaphasic spindle in embryos undergoing PCC, whereas cytoplasmic microtubules were observed in the others. In Experiment 3, cleavage and blastocyst rates were not significantly different between the treated (n = 92) and the control groups (n = 105): 83.7% and 53.3% v. 78.1% and 50.5%, respectively. However, the mean cell number in treated embryos (n = 27) was significantly higher than in controls (n = 20): 134 ± 25 v. 109 ± 43 (P < 0.05). This study suggests that MG132 treatment improved the maintenance of oocyte factors responsible for PCC in bovine NT embryos, although it did not modify MPF activity, thus questioning the role of MPF in the induction of PCC. Accordingly, PCC may be important for blastocyst quality and nuclear reprogramming in NT embryos. Full-term development of MG132-derived embryos is under investigation.