DEVELOPMENT OF NEW OOCYTE IN VITRO CULTURE STRATEGIES TO ENHANCE THE OUTCOME OF ASSISTED REPRODUCTIVE TECHNOLOGIES

Fertility preservation has received unprecedented attention nowadays. In addition to cryopreservation and re-implantation of embryos, oocytes, and ovarian tissue pieces, in vitro culture system for follicles/oocytes has been considered an alternative strategy for fertility preservation. Reproduction strategies based on the recovery of oocytes' population from antral follicles are unsatisfactory, and the success of this approach has not exceeded 35% of embryos produced in vitro for over 30 years. The possibility of accessing the reserve of smaller follicles (primordial, secondary, and up to the preantral stage) would amplify the number of gametes available for increasing reproductive potential. Furthermore, this would open enormous prospects for the rescue of fertility in various conditions in the human clinic and genetic rescue in animal breeding and biodiversity preservation programs. However, this would require developing protocols capable of growing immature oocytes to the stage in which they can be matured and fertilized in vitro. Culture systems to achieve in vitro growth (IVG) of immature oocytes to maturity and subsequent fertilization in vitro (IVF) have been the subject of research for almost 40 years. Several systems that support the growth of later stages of follicle development from rodents have been developed, with some reporting the production of live young, but they are still at an experimental stage, and further research is required before the protocols could be clinically applied. One of the significant limitations is identifying growth factors, hormones, and nutrients necessary for each specific follicle development stage. This evidence has led to hypothesize the development of culture systems consisting of a step-by-step approach, although no reliable protocols have been developed so far. The oocyte culture at the early stages of development represents an alternative to maximize the potential source of gamete used for fertility preservation. Several attempts have been made to recreate these conditions in vitro, but no reliable protocols have been developed to date. The lack of knowledge in the mechanisms involved in the early development of the oocyte and this passage from growing to fully grown stage be one of the most critical steps during oocyte development, these still represent the significant limiting factor for this technology. The studies conducted during the doctorate program led to defining a physiological culture system that successfully differentiated growing bovine oocytes. This study used parameters predictive of oocyte differentiation to evaluate the current technique's efficiency and efficacy. Based on previous observations from our laboratory, we initially hypothesized that zinc plays a role during the latest stages of oocyte growth and differentiation, particularly in controlling transcription during the final stage of oocyte growth. This first study demonstrated that zinc supplementation improves the meiotic competence of growing oocytes, affects the global transcription activity and the global DNA methylation. This information was used in the next part to better define a culture system for growing oocytes. The subsequent study provided a 5-days protocol named L-IVCO (long in vitro culture of oocytes) to promote growing oocyte differentiation until the acquisition of meiotic and embryonic developmental competencies in a significantly higher proportion of the published protocols. This study demonstrated that a physiological medium could support a gradual transition of the oocyte from immature to mature stage, thus generating suitably quality blastocysts after fertilization. In conclusion, our studies provide an improved protocol that can increase the source of fertilizable gametes in preservation programs and gives a prospective approach in human clinics, animal breeding programs, and salvage intervention of threatened species. Moreover, our studies defined a model to perform in-depth studies of the cellular and molecular processes that regulate the acquisition of meiotic and developmental competence during oocyte differentiation.