Morphological markers to select populations of oocytes with different cultural needs for dedicated pre-maturation protocols

Oocyte’s chromatin gradually becomes more compacted during the final stage of oocyte development and the level of chromatin compaction is considered a marker of oocyte differentiation [Luciano et al, 2014]. Moreover, several studies demonstrate that in vitro pre-maturation treatments (Pre-IVM), aimed to improve the developmental capability of immature oocytes, might behave differently depending on the oocyte metabolic status, when it is isolated from follicle [Luciano et al., 2011]. This study aims at identifying correlations between cumulus-oocyte complex (COC) morphology and oocyte chromatin configuration and secondly at testing the hypothesis that only fully grown oocytes at earlier stages of differentiation with loosely compacted chromatin  (GV1) can benefit from Pre-IVM treatment.   COCs were collected from bovine 2-6mm ovarian follicles, and further divided in three groups according to their morphology (Class-1, 2 and 3) as previously described [Blondin & Sirard, 1995]. Analysis of chromatin configuration revealed that only Class-1 COC was enriched in GV1 oocyte, while Class-2 and 3 presented a similar distribution of GV1, GV2 and GV3 oocytes, where GV2 and 3 oocytes are characterized by increased chromatin compaction [Lodde et al., 2007]. Then COCs were divided into two groups, one containing Class-1 COCs and the other containing Class-2 and 3 COCs and subjected to pre-IVM for 6 hours in presence of cilostamide and 10-4 UI/ml rhFSH. Finally, COCs underwent standard in vitro maturation (IVM) for 22 hours, in vitro fertilization and embryo culture. Blastocyst rate and embryos cell number were assessed at day 7. Pre-IVM positively affected developmental competences of Class-1, while in Classes 2 and 3 Pre-IVM had detrimental effects.In conclusion COCs morphology could be used as a non-invasive approach to select population of oocyte with different cultural needs. These data could be useful in setting-up dedicated IVM protocols considering specific genes and pathways to improve IVP efficiency

Symposium 4 "Joint CSAS/5th International Meeting on Biotechnology in Animal Reproduction"

In ARCHIV - 10841

Bovine Oocyte Transcriptome in Relation to Chromatin Configuration of the Germinal Vesicle

Various physiological contexts have been used to study the mRNA composition of bovine oocyte in relation with the potential to reach the blastocyst stage. The chromatin configuration of the oocyte at the germinal vesicle (GV) stage has been positively associated with developmental competence, where a more compacted chromatin is correlated with a better chance to get an embryo. Four distinct and progressive states of chromatin condensation can be observed and are linked with the sequential acquisition of the developmental potential in bovine species. Therefore, GV stage oocytes were collected and separated in four groups according to their degree of chromatin condensation. Transcriptomic analyses were performed with the EmbryoGENE microarray platform in order to identify transcripts modulations during the gradual chromatin remodeling. A reference design was used where GV0 stage (diffused chromatin state) was compared with the other three groups (GV1; GV2 and GV3, the latter refers to the condensed chromatin state). The number of transcripts significantly different was relatively similar between the three contrasts (an average of 300 genes, fold change > 2; p-value < 0.01) with the majority of them were presenting a reduced level in oocyte with more compacted chromatin. However, among the transcripts presenting an increased mRNA level in these contrasts, many of them are associated with histone genes, chromatin organization and transcriptional regulation. These results suggest that even in a context of transcriptional repression, there is still an accumulation of specific transcripts. Adequate storage of specific histone mRNAs could provide an optimal maternal accumulation in order to fulfill the requirement for the subsequent cell divisions until embryonic genome activation. This dataset will provide a more complete characterization of the oocyte transcriptome modulations and will help us to better understand the competence acquisition in the oocyte at the end of the follicular growth