Exploring the role of the Phosphatidylinositol-3'-Kinase (PI3K) pathway in primordial follicle activation and subsequent development
Spence, Susan Claire
Mammalian females form their germ cells (oocytes) before or shortly after birth. The oocytes interact with somatic cells to form primordial follicles, creating the quiescent population from which oocytes will be recruited to grow throughout life. A female’s fertility life span is therefore, dependant on the size of this pool and the rate at which primordial follicle are activated to grow. However, there is still much we do not know about the quiescent follicle population and the mechanisms that control their recruitment into the growing follicle population are still unclear. There is evidence that the phosphoinositide-3-kinase (PI3K) pathway is key to activation of follicle growth. The role of the PI3K pathway has been primarily explored in the rodent model and has highlighted this pathway’s importance both in the activation of quiescent follicle growth and maintaining dormancy of the quiescent follicle population. This thesis aimed to explore if the PI3K pathway played a similar role in a large mono-ovulatory species as it does in the small polyovulatory rodent species. Bovine is a mono-ovulate species, which has similar attributes in its reproduction and folliculogenesis to the human in vivo; therefore using an in vitro bovine model might be a valuable indication of the role of the PI3K pathway in the human. Initial experiments tested if the bovine was a good model for human primordial follicle activation in an in vitro environment. It was observed that the bovine and human had comparative levels of activation and subsequent increases in both the primary and secondary follicle populations within an in vitro culture system. These similarities indicate that the bovine is a relevant model for the human in vitro. It is not possible to culture the entire bovine ovary. Therefore knowing the location of the primordial follicles is important to establishing what region(s) of the ovary to use. The overall concentration of ovarian follicles was higher in the cortex and gradually declined through the consecutive inner layers of the ovary. The distribution of the ovarian follicle populations were different in each distinctive region of the ovary with the quiescent follicles representing a much larger proportion of the ovarian follicle population in the cortex compared to the inner regions of the ovary. The location an ovarian follicle in the ovary was seen to influence its health in both the quiescent and growing follicle populations, with reduced health seen in the IV inner layers of the ovary compared to the cortex. This resulted in very few healthy quiescent follicles outside of the cortex region making it the more favourable region to culture in functional studies. The role of the PI3K pathway was therefore explored using an in vitro bovine model using the pharmacological compounds bpV (HOpic) and 740Y-P, both of which caused an up-regulation of the PI3K-pathway. It was observed that up-regulation of the PI3K pathway caused an increase in the activation of the quiescent follicle population, and the resulting primary follicles were larger in size. However, there was reduced health in both the growing and quiescent follicle populations. The ill health appears to be due to a disruption in the co-ordination of growth between oocyte and granulosa cells in the ovarian follicles, leading to enlarged oocytes in both the primary follicles and quiescent follicles. Although the PI3K pathway caused an increase in quiescent follicle activation and larger primary follicles there was no increase in the number of viable large secondary follicles obtained. The growth of the secondary follicles was unaltered by the initial activation of the quiescent follicles via the PI3K pathway. These experiments show that the PI3K pathway plays a role in primordial follicle activation in large mono-ovulate species. However, up-regulating the PI3K pathway results in a decrease in health of the quiescent and primary follicle populations, thus limiting its immediate value as a therapeutic target. This study has improved our understanding of the role of the PI3K pathway in primordial follicle activation in a large mono-ovulate species. It has highlighted that the up-regulation of the PI3K pathway using both bpV (HOpic) and 740Y-P increases the activation of the bovine ovarian follicles in vitro. However, up-regulating the PI3K pathway disrupts the development of the ovarian follicles resulting in retarded growth and thereby a decrease in the survival of both the quiescent and growing follicle populations. The similarities in activation, growth and development between the bovine and the human in vitro indicate that the results observed in the bovine are a good indication of what would occur in the human. This study has also improved our understanding of the location, distribution and viability of the ovarian follicle population within the ovary.