The dominant follicle: the final frontier in oocyte development
The acquisition of oocyte competence, involving both cytoplasmic and nuclear maturation, is critical for successful fertilization and embryonic development in mammals. Discrepancies between in vivo and in vitro studies underline the potential impact of assisted reproductive technologies (ARTs), such as FSH stimulation and in vitro maturation, on oocyte quality and the molecular integrity of bovine oocytes. These insights underscore the importance of optimizing ART conditions to enhance reproductive success in cattle production. Therefore, this study focuses on the gene expression and DNA methylation dynamics in bovine oocytes from dominant follicles, utilising single-cell bisulfite conversion and RNA sequencing (scG&T), without ovarian stimulation. Samples were collected from synchronized heifers during three time points from 24hr to 2hr before ovulation and submitted to scG&T and compared with a dataset of >120 um in diameter oocytes collected by ovary slicing. Our findings from the single-cell RNA sequencing highlight significant changes in oxidative phosphorylation transcripts, indicating the importance of energy metabolism during oocyte capacitation. Additionally, small changes in global CpG methylation and methylation over genomic features were observed. However, modest modifications in CpG windows suggest a less stable epigenome than previously assumed in fully-grown oocytes. In conclusion, the present study brings new insights into molecular changes in bovine oocytes collected from dominant follicles, which can contribute to oocyte quality improvement in cattle production. Overall design: Oocytes from dominant follicles were collected from previous animal trials performed at University College Dublin. The oestrous cycles of 26 Crossbred nulliparous beef heifers (predominantly Limousin and Charolais crosses) were synchronized to manage the time of the ovulation, and bovine oocytes were collected from dominant follicles 24h before ovulation, 19-23 hours after estrus detection, and 2 hours before ovulation. After denudation from cumulus cells, single oocytes were snap-frozen in PCR tubes and stored at -80 ºC until processing for single-cell RNA sequencing and Bisulphite conversion.