Transcriptomic and DNA Methylomic Defects in In Vitro Matured Lamb Oocytes and Potential Improvement of Their Development by Supplementing Phorbol Myristate Acetate During In Vitro Maturation

Juvenile in vitro embryo transfer (JIVET) presents a promising way of accelerating genetic gains in sheep breeding by reducing generational intervals. However, its application is hindered by the limited developmental competence of oocytes derived from prepubertal lambs. To elucidate the molecular underpinnings of this limitation, we conducted a comparative analysis of in vitro matured (IVM) oocytes from 4- to 6-week-old prepubertal lambs and adult ewes, focusing on developmental outcomes, transcriptomic profiles, and DNA methylation patterns. Our findings revealed that lamb oocytes exhibit reduced nuclear maturation, fertilization rates, and embryonic development compared to those from adult ewes. Enrichment analyses of differentially expressed and methylated genes highlighted significant differences in GABAergic and dopaminergic synapse pathways, as well as purine metabolism pathways, which are essential for oocyte maturation and early embryogenesis. Supplementation of the IVM medium with progesterone or phorbol myristate acetate (PMA) enhanced sheep oocyte maturation and cumulus cell function. In particular, PMA upregulated the EGFR and HAS2 expression in lamb cumulus cells and improved the developmental competence of lamb oocytes. These results underscore the transcriptional and epigenetic differences contributing to the diminished competence of lamb oocytes and suggest that targeted modulation of specific signaling pathways during IVM could enhance the JIVET efficiency in sheep breeding programs.