Molecular Mechanisms Regulating Neonatal Oocyte Survival and Primordial Follicle Formation in the Mouse Ovary
Date of Award
Doctor of Philosophy (PhD)
Follicle, Mouse, Oocyte, Ovary, Reproductive Biology
Cell Biology | Developmental Biology | Molecular Biology
In mammals, formation of the primordial follicle is a complex process involving the breakdown of germ cell cysts, where oocytes must separate from each other and subsequently become surrounded by somatic cells. As cysts separate, a large number of germ cells are lost by apoptosis, however the mechanisms by which cyst breakdown and germ cell death occur are not well understood. We first hypothesized that two anti-apoptotic regulators from the BCL2 family of proteins, BCL2 and MCL1, may be responsible for regulating neonatal oocyte survival. To elucidate the effects of BCL2 in the neonatal ovary, we examined ovaries of both Bcl2 overexpressing and knockout transgenic mice. When compared to wild-type mice, neither Bcl2 overexpression nor abrogation significantly altered ovarian histology. Another BCL2 family protein, MCL1, is expressed in human oocytes during ovarian development, suggesting a role for MCL1 in oocyte survival. We found that MCL1 was localized to both oocytes and somatic cells during cyst breakdown. Subsequently, we used an in vitro organ culture system to identify a role for MCL1 in oocyte survival. We found that inhibition of MCL1 with an antibody to MCL1 in culture resulted altered germ cell numbers and oocyte cyst breakdown. Our data demonstrate that while BCL2 is not likely involved in perinatal oocyte survival, MCL1 may be an important regulator of the ovarian primordial follicle reserve. Next, we hypothesized that the KIT signaling pathway may be important for oocyte survival and cyst breakdown in the neonatal ovary. The tyrosine kinase receptor, KIT, and its ligand, KITL, have been implicated in oocyte survival and follicle development in both fetal and adult ovaries but have not been well studied at the perinatal time point. To elucidate the functional role of KIT signaling in the neonatal ovary, we began by using immunohistochemistry to test the expression of KIT and KITL. We found both proteins to be expressed in the developing ovary from 17.5 dpc to PND 3, suggesting an important role for this protein in cyst breakdown or oocyte survival. To test this hypothesis, ovaries from 17.5 dpc fetal mice were cultured for 5 days in control media, or in media with the KIT blocking antibody, ACK2, or recombinant KITL. Our data demonstrated a role for KIT signaling in cyst breakdown, as inhibition and activation of the pathway altered ovarian histology. Using cell proliferation and TUNEL assays at the conclusion of culture, we identified a reduction in somatic cell proliferation when KIT signaling was inhibited and likewise, a decrease in cell death. Finally, we investigated which pathway downstream of KIT affects cyst breakdown and the effect of KIT signaling on MCL1 protein expression. After 3 days in culture with KITL supplemented media, Western blotting was used to analyze the total and phosphorylated forms of proteins from the PI3K, MAPK and JAK-STAT pathways as well as the BCL2 family protein, MCL1. We found that there was an increase in the phosphorylated forms of p44/p42 in the MAPK pathway and a downregulation of MCL1 on KIT activation. Overall our data have shown that while BCL2 may not contribute to oocyte survival during cyst breakdown, both MCL1 and KIT play important roles in formation of the primordial follicle pool.
Jones, Robin L., "Molecular Mechanisms Regulating Neonatal Oocyte Survival and Primordial Follicle Formation in the Mouse Ovary" (2012). Biology - Dissertations. 83.