DEVELOPMENT OF PHYSIOLOGICAL CULTURE SYSTEMS TO ACCESS UNTAPPED OVARIAN RESERVES FOR IMPROVING REPRODUCTIVE EFFICIENCY

Female fertility preservation via complete in vitro folliculogenesis is still chimerical. Recreating folliculogenesis to grow a dormant primordial follicle to the preovulatory stage in vitro remains the most coveted goal of fertility preservation programs. The ability to grow undifferentiated oocytes in vitro from primordial follicles would increase the supply of fully grown oocytes destined for downstream applications in clinical settings and the livestock industry. To date, the production of live offspring through the in vitro culture of primordial follicles has only been achieved in mice. While this demonstrates the potential value of primordial follicles as a source of fully grown oocytes, it remains a proof of principle. In large mammals, such as humans and bovine, in vitro follicle culture systems to produce mature oocytes from primordial follicles are still experimental due to follicle mortality after isolation from the surrounding tissue. While understanding the cause of follicle death is necessary to inhibit the induction of programmed cell death, it is also essential to elucidate the mechanisms responsible for steering primordial follicles toward the following stages of development, i.e., primary and secondary follicles. Mechanistically resolving both sides of the story – primordial follicle death and development - would then provide insight into crucial factors responsible for deciding the fate of the follicles and fueling their further development in culture. Compilation of the information from transcriptome studies would then inform the appropriate optimizations of culture systems and improve the growth of isolated primordial follicles to the subsequent primary and secondary stages entirely in vitro. To probe our objectives, we first laid the groundwork by developing a mechanical isolation protocol and serum-free base in vitro system to culture isolated bovine primordial follicles. We then quantitatively and qualitatively assessed the isolation efficiency of primordial, primary, and secondary follicle populations from a defined volume of the ovarian cortex, thereby providing a conservative strategy to improve assisted reproductive technologies dedicated to female fertility preservation. Following this, interdisciplinary methods were adopted to elucidate the web of interacting genes governing primordial follicle fate in vitro and in vivo. The conducted transcriptome and network analyses indicate that ferroptosis and autophagy are the elected programmed cell death mechanisms active in isolated primordial follicles cultured in vitro. Furthermore, we delineate the essential genes and pathways orchestrating the consecutive developmental transitions (primordial-to-primary and primary-tosecondary follicles) in vivo through RNA sequencing of isolated primordial, primary, and secondary follicles. To extend our findings, we investigated the conservation of functionally enriched pathways across different species. In conclusion, the studies conducted during this doctoral program, provide a high-yield protocol that accesses untapped ovarian reserves, thereby increasing the source of gametes for fertility preservation. We further define a base system for the in vitro culture of isolated bovine primordial follicles and shortlist candidate molecular targets that can be modulated to optimize physiological culture systems, thereby informing the overarching aim of this research thesis.