Molecular effect of holding equine oocytes: a road to cytoplasmic maturation?

The quality of oocytes has a direct effect on the outcome of in vitro embryo production (IVP). For successful IVP, oocytes need to undergo maturation, which requires coordinated nuclear (NM) and cytoplasmic maturation (CM). The lack of adequate cytoplasmic maturation has been proposed to be associated with a lower developmental competence of the oocyte. The improvement of in vitro techniques relies upon mirroring in vivo conditions. Therefore, acquiring a deeper knowledge of the process involved in CM and pathways leading to germinal vesicle breakdown and maturation can provide the necessary foundation to improve IVP results. Holding oocytes in commercial embryo holding media for 24hrs at room temperature has become a routine procedure for oocyte shipment and laboratory scheduling. However, the effects of this procedure on oocyte CM remain unresolved. In this study, we analyzed the changes induced by the holding period on the transcriptome of equine oocytes (OC) and the surrounding cumulus cells (CC). Cumulus-oocyte-complexes (COCs) were collected from six mares using transvaginal aspiration. Only non-expanded, non-degenerated COCs were randomly allocated to one of two groups: no holding (NH) or 24hrs holding (H). The COCs were denuded, and each single OC and CC were stored individually in RNAlater® at -20°C until further processing. RNA was extracted from 24 samples (NH/CC=6; NH/OC=6; H/CC=6; H/OC=6), followed by librarypreparation and sequencing (150 pb-PE). Reads were trimmed and mapped to the equine reference genome (Ecab 3.0). Mapped reads were quantified using featureCount and analysis of differentially expressed genes (DEGs) was performed using DESeq2 (FDR< 0.1 and log fold change >2). Overall, there were 26,077 genes expressed in CCs and 17,930 in OCs. When compared by holding time, there were 660 DEGs between the CCs (NH vs. H) and 205 between the OCs. In CC, 74 genes had a higher expression and 586 had a lower expression in H than NH groups. In OC, 7 genes were upregulated, and 198 were downregulated in H compared to NH group. Gene ontology analysis showed changes in genes related to glutathione (GSH) synthesis, AP-1 transcription factor complex and mitogen-activated protein kinase (MAPK) pathway in CC. In OC, DEGs were involved in α-tubulin detyrosination and tight junction proteins involved in magnesium transport. Overall, our results suggest modification in processes related to CM, including post-translational changes in tubulin, GSH composition, and activation of MAPK pathways during oocyte holding. Meanwhile, genes considered markers of NM and meiotic resumption remained unchanged, suggesting pre-maturation like changes occur prior to germinal vesicle breakdown. Further studies to understand the implications of this pre-maturation step in the developmental competence of oocytes can help improve the outcome of equine IVP. Funding: Center for Equine Health