Single-cell DNA methylation profiling reveals epigenetic signatures associated with in vitro maturation of bovine oocytes and conserved patterns with porcine oocytes.

In vitro maturation (IVM) of oocytes is a key step in bovine in vitro embryo production, but concerns remain regarding its potential effects on oocyte competence and epigenetic regulation. Information on the DNA methylation profile of bovine oocytes matured in vitro is still limited and inconsistent. In this study, we applied single-cell DNA methylation sequencing to characterise methylation patterns associated with IVM in bovine oocytes. In vivo matured oocytes were included as a biological reference to contextualise methylation landscapes observed following IVM. Eighteen IVM oocytes were collected from abattoir-derived ovaries, and twenty-eight in vivo matured oocytes were obtained by ovum pick-up from Holstein and Braunvieh cows and a Holstein heifer. Single-cell whole-genome bisulfite sequencing was used to assess DNA methylation, and differentially methylated regions (DMRs; FDR < 0.05, FC > 0.1) were identified using the limma package. Genome-wide methylation profiling revealed distinct epigenetic signatures associated with IVM, affecting both gene bodies and regulatory regions. Several differentially methylated regions were linked to genes involved in RNA processing, metabolism, and stress-response pathways relevant to oocyte maturation. IVM oocytes showed lower methylation in CDKN1C and higher methylation in BEGAIN, while SEMA7A, ZNF575, MACROD1, and FGFRL1 were hypomethylated. Comparative analysis further identified a subset of conserved methylation signatures shared between bovine and porcine oocytes, suggesting evolutionary conservation of epigenetic responses to IVM conditions. This study contributes to a deeper understanding of the epigenetic mechanisms underlying oocyte maturation and supports the use of cross-species epigenomic approaches to identify candidate methylation signatures for future validations in studies of oocyte quality and developmental competence.