Early signaling pathways during in vitro culture of isolated primordial follicles
The ability to grow undifferentiated oocytes in vitro from primordial follicles would increase the availability of fully grown oocytes in fertility preservation programs and other downstream applications. To date, the development of living offspring in vitro from the primordial follicle reserve has only been achieved in mice, proving the principle of the potential value of these follicle cultures as a source of competent oocytes. In certain pathophysiological conditions, such as polycystic ovarian syndrome, premature ovarian failure, or in ovarian and blood cancer where the ovarian tissue cannot be reintroduced into the patient, it is essential to isolate these follicles from the surrounding tissue and culture them in vitro. However, culture systems to produce mature oocytes from the isolated primordial follicles are still experimental. Upon isolation from the ovarian microenvironment, a critical limiting factor is follicle death after a short period of culture.  Our study defines a serum-free culture system capable of sustaining bovine primordial follicles to more than 60% viability for 24 hours. Using this system as a base, we employ RNA-Seq to advance the knowledge of the main transcriptional events and molecular factors determining follicle fate in a 2D culture system and suggest the role of ferroptosis in bringing about primordial follicle death. Future ameliorations to the culture system should not only inhibit such programmed cell death mechanisms but also ensure the physiological compliance of the genes regulating primordial follicle activation and transition to the primary stage with effective supplementation media to attain isolated primordial follicle development in vitro. Overall design: RNASeq profiling of mechanically isolated primordial follicles (3 biological replicates) that were retrieved after collection (PMF T0/PM00) or 16 hours of culture (PMF T16/PM16).