Transcriptome profiling of bovine preantral follicles during early folliculogenesis

The ovarian reserve, primarily composed of primordial (PMF), primary (PF), and secondary follicles (SF), holds great potential for fertility preservation, yet remains inaccessible mainly due to the challenges of replicating early folliculogenesis in vitro. The molecular mechanisms governing the activation and differentiation of oocytes from the dormant PMF pool remain poorly defined. In this study, we used a bovine model to investigate early follicular development.

The transcriptome of isolated PMF, PF, and SF revealed different profiles of the follicles during early folliculogenesis. Differential gene expression analyses between PF versus PMF and SF versus PF revealed 689 and 3,206 significantly regulated genes, respectively (FDR < 0.05), highlighting key pathways including PI3K-Akt, Wnt, mTOR, and ECM-receptor interaction. Between PF versus PMF and SF versus PF, 13 and 69 DEGs were identified as transcription factors, respectively. A Likelihood Ratio Test followed by hierarchical clustering identified four major gene expression clusters across the three stages, showing significant enrichment in processes such as ECM-related functions and cell cycle. Based on the enrichment of the cell cycle pathway, we focused on RAD51, which was highly expressed in preantral follicles. Immunohistochemical analysis further showed that RAD51 protein localized in the ooplasm, along with the mitochondrial marker HSP60.

This is the first study to perform bulk RNA sequencing on isolated preantral follicles (PMF, PF, and SF) in cattle. Our findings provide new insights into the stage-specific mechanisms regulating follicle activation and growth in cattle, laying the groundwork for future in vitro fertility preservation strategies in both clinical and conservation contexts.