Single ooplasm biopsy RNA-Seq reveals important and distinct pathways linked to oocyte competence

Oocyte competence is a complex process that is directly associated with the ability of oocyte to develop a newborn. The generation of a viable gamete has a multifactorial origin directly related to follicle and oocyte growth. Several dynamic changes in the follicle have driven the oocyte to success or failure, such as storage of metabolites, epigenetic modifications, and maternal RNAs, among others. However, little is known about the precise factors of maternal RNAs associated with oocyte competence, once these RNAs are consumed during maternal embryonic transition. To investigate the oocyte transcriptome landscape, we used a biopsy method to remove ~1.5% of ooplasm followed by a Single-ooplasm-biopsy RNA-Seq approach after retrospectively tracking the oocyte fate. We identified factors driving competence and the feasibility of the technique in obtaining enough amount of sample and high-quality sequencing results, enabling the model establishment. Furthermore, we integrated data from granulosa and cumulus cell transcriptome. Besides that, the first polar body methylation showed high similarities with pathways enriched in the ooplasm-biopsy transcriptome. In granulosa and ooplasm-biopsy with high-competence, we found MAPK and Oxytocin signaling enriched, while the process of necroptosis and ferroptosis were associated with low-competence oocytes. Deep analysis in the ooplasm-biopsy transcriptome, shows MYC as the highest up-regulated gene, and GNAS as a central hub gene in the network analysis. In addition, TIMP1, APOA1, EZR1, and STAT1 were key genes in the low-competence group of oocytes. Importantly, we found that metabolic alteration in the low-low-competence group was constantly found in all transcriptomes data, suggesting a metabolic disturbance inherited since the follicle environment which is amplified in cumulus cells after oocyte maturation. Overall design: Granulosa cells from bovine oocytes were collected individually from each follicle. The respective COCs were matured in vitro where we collected an ooplasm biopsy, the first polar body, and cumulus cells. All samples were sequenced using RNA-Seq or WGBS to investigate the groups that reached the blastocyst stage or were arrested in the cleavage step.