ABSTRACT: Unlike in the mouse, where complete folliculogenesis has been achieved from the primordial stage to the newborn, this process remains experimental in large mammals. Several studies have demonstrated proof-of-principle for the activation and growth of primordial ovarian follicles (PM) up to the primary (PF) and secondary (SF) stages. However, the transition from SF to tertiary follicles (early antral stage, EA), where the complexity of cell types and regulatory factors makes in vitro development particularly challenging, has not yet been achieved. Our ongoing research aims to identify key factors and pathways involved in the preantral (PM, PF, and SF) to EA transition in the bovine model that can be targeted to develop efficient and reliable culture protocols for assisted reproduction. PM, PF, and SF were previously collected in-house (GSE283263 and GSE309360). For this study, EA and SF were mechanically isolated from the ovarian cortex of dairy cows aged 15 to 31 months, and bulk RNA sequencing was performed. PM, PF, SF, and EA samples were batch-corrected and normalized. The data were analyzed to investigate the overall relationships within the sample. Differential gene expression analysis was performed, and genes significantly associated with increasing follicle diameter were used to build gene co-expression modules via WGCNA. Additionally, candidate biomarkers involved in the preantral-to-EA transition were identified through an integrative analysis using other publicly available NCBI datasets (refer to the paper). Moreover, the reliability of candidate biomarkers was evaluated using machine learning-based models. Our results demonstrated the robustness of the in-house datasets, which were consistent with each other and with external datasets, enabling the identification of reliable biomarkers. These biomarkers hold the potential for providing qualitative checkpoints to monitor preantral follicle progression to the antral stage during in vitro culture, in the future.
