Transcriptional insights into the molecular pathways underlying oocyte developmental competence during oocyte-to-embryo transition in the ovine model

Oocyte quality is a key determinant of successful embryonic development. During oocyte growth, maternal products are stored to support the oocyte-to-embryo transition (OET), which occurs in the absence of de novo transcription until embryonic genome activation. This critical window is post-transcriptionally regulated to ensure the proper synthesis of molecules required by both the oocyte and the embryo, along with the gradual replacement of maternal transcripts by those synthesized from the embryonic genome. Alteration in these processes compromises correct embryonic genome activation, leading to developmental arrest. However, the specific genes and molecular mechanisms involved remain unclear. This study aimed to identify novel genes and molecular pathways underlying oocyte developmental competence, providing new biomarkers of oocyte quality and improving in vitro embryo production. To this aim we investigated key aspects of oocyte molecular regulation: genes involved in maternal mRNA degradation during OET; the subcortical maternal complex (SCMC); the transcriptomic landscape of the early embryo; m6A RNA methylation; a pilot functional study using RNA silencing to evaluate the effects of PADI6 downregulation. Analyses were conducted in a consolidated model of differential developmental competence consisting of sheep oocytes and in vitro produced embryos derived from adult and prepubertal donors, characterized by high and low developmental potential, respectively. Gene specific amplification was analyses by real-time PCR. M6A quantification levels were performed in cumulus cells (CCs) during vitro maturation by colorimetric assay. RNA sequencing was performed in early embryos by 75Se Illumina platform. PADI6 downregulation was assessed by microinjection of specific dsRNA during in vitro maturation. Our results showed altered expression profiles associated with low developmental potential in oocytes and early embryos. Genes related with mRNA degradation showed up-regulation of CNOT6L and ZAR1l in matured oocytes derived from prepubertal donors. SCMCs genes showed ZBED and TLE6 downregulation in GV oocytes and 2-cell stage embryos derived from prepubertal donors, respectively. The m6A RNA methylation was altered in COCs derived from prepubertal donors after in vitro maturation. Oocytes showed reduced transcripts abundance of m6A related-proteins, while CCs showed higher transcripts abundance associated with higher m6A levels. Transcriptome analysis revealed donor aged-dependent differences mainly at the 8-cell stage of early embryos. Embryos from adult donors showed 6 and 7 DEGs between the 2- and 8-cell and the 4- and 8-cell stages, respectively, compared with 1 and 7 DEGs in embryos from prepubertal donors. Direct comparison between donor groups revealed 2 DEGs at the 2-cell stage and 4 DEGs at the 8-cell stage. Gene Ontology (GO) analysis revealed that DEGs are mainly implicated in RNA translation and transcription. Preliminary RNA silencing experiments established an optimized protocol, demonstrated effective PADI6 knockdown by siRNA probes, and identified siPADI6_3 as the most effective probe. In conclusion, oocytes and embryos characterized by low developmental competence, displayed altered epigenetic regulation, perturbed expression of genes involved in RNA degradation and the SCMC, and transcriptomic difference most pronounced immediately before embryonic genome activation, mainly related in transcriptional and translational pathways. Dysregulated mRNA abundance during OET may compromise embryonic genome activation and ultimately reduce developmental potential. Overall, these findings provide new insights into the molecular basis of oocyte developmental competence and female genetic infertility.