Transcriptome atlas of isolated bovine primordial, primary, and secondary follicles

Orchestrating in vitro the process of folliculogenesis to bring a dormant primordial follicle (PMF) to the preovulatory stage represents the most coveted goal of developing fertility preservation programs in human clinics and the livestock industry. To date, live offspring via in vitro follicle culture of the PMF reserve have only been achieved in mice but remain a proof of principle. However, the mechanisms regulating the development of PMF into the subsequent primary (PF) and secondary follicle (SF) stages still need to be resolved entirely. This study aims to elucidate the pathways guiding the transition in the bovine ovary. Individual PMF, PF, and SF pools were isolated from bovine ovaries and subjected to bulk RNA sequencing, generating 50bp paired-end reads. TrimGalore was used to remove artificial constructs and low-quality bases. Trimmed data were mapped against the Bos taurus transcriptome with Salmon, and differentially expressed genes (DEGs) were obtained with DESeq2. Functional enrichment analyses were performed on WebGestalt to identify overrepresented pathways. Gene regulatory networks of the DEGs were constructed, and hub gene identification analyses were performed to shortlist key genes guiding the PMF-to-PF and PF-to-SF transitions. PCA showed clear clustering of the samples. Between PMF-PF and PF-SF, 1083 and 4596 DEGs were identified with FDR<0.1. We report the transcriptome profiles of isolated bovine PMF, PF, and SF for the first time and shortlist key regulators of the PMF-PF-SF transition.