Evaluating Apoptosis, Autophagy, and Hormonal Effects on Oocyte Regulation and Cyst Breakdown

Infertility in women is caused by a depletion of eggs below the threshold for functional ovulation. Unlike men where sperm production is maintained throughout their reproductive lifespan, a women\u27s proliferation of eggs ceases prior to birth. Interestingly during development, a fetus has up to 7 million eggs and two-thirds of these eggs die via apoptosis before they are born, further signifying the importance of the quantity control of eggs to maintain fertility. The remaining eggs break apart from each other and become enclosed individually by pre-granulosa cells together forming primordial follicles. Over time, the number of oocytes will continue to decrease due to follicular atresia taking place during the ovulatory cycle or from other secondary factors such as illness or environmental consequences that can further deplete the number of eggs and eventually lead to infertility. This mysterious phenomenon of massive egg death during development has puzzled reproductive biologists and evolutionary scientists for decades. As the field continues to propose numerous reasons as to why this cell loss occurs, we have narrowed our research to understanding the intrinsic apoptotic pathway causing this death sentence. The mouse also experiences the same trend of egg depletion and primordial follicle formation making it an appropriate model organism to understand the mechanisms of apoptosis and ovarian development. By specifically analyzing the anti-apoptotic protein Myeloid Cell Leukemia-1 (MCL-1) in the mouse oocyte using the Cre Lox recombination system, we can continue our work on this protein and further understand its importance in regulating oocyte termination. Unfortunately, in this case, MCL-1 was still detected in the oocytes of the Cre- Lox recombination mice, therefore no conclusions could be made determining its importance on oocyte survival.Along with addressing the role of the MCL-1, we set forth an initiative to investigate what properties the surviving oocytes have compared to the ones designated for cell death. We turn towards autophagy to analyze whether or not certain oocytes contain more autophagic machinery in their cytoplasm compared to their connected counterparts to promote their survival. From there, we discovered an increase in the quantity of autophagic machinery in the cytoplasm of oocytes possessing a Balbiani Body, a structure that contains a collection of organelles from other oocytes. Along with this discovery, we found large lysosomal clusters localize partially within or fully within the Balbiani Bodies. Further work needs to be completed, but this may suggest a possible role that the Balbiani Body and lysosomes may work together to provide nutrients to the oocyte and promote its survival or its growth during its development. Besides addressing oocyte regulation through MCL-1 and autophagy, the increasing awareness of hormones on neonatal mouse germ cell development while oocytes are separating apart to become primordial follicles has created a surge of questions. Oocytes must break apart from each other to become primordial follicles as it is essential for fertility. If the breakdown does not occur, the pre-granulosa cells can encompass multiple oocytes by proliferating and growing around them, creating a multiple oocyte follicle (MOF), which cannot be fertilized. The presence of estradiol during oocyte breakdown in the mouse has shown to work at the membrane to inhibit the oocytes from separating, which leads to the creation of MOFs. It was here where we focused on the essential pathways - MAPK, PI3K/AKT and JAK/STAT - known to regulate gene expression during primordial follicle formation. We found estradiol has no significant effect on the activity of these pathways, suggesting estradiol may work through another membrane bound pathway or other players such as Rho-GTPases that regulate nuclear transport of proteins within these pathways. The construct of this work attempts to continue to pick apart the role of the anti-apoptotic player MCL-1 through creating and analyzing an oocyte specific knockout of MCL-1 via the Cre-Lox breeding system. On top of this assessment, we further investigated the role of autophagy through evaluating lysosomal cluster localization within oocytes connected in different sized cysts and discovered oocytes containing the Balbiani Body possess more clusters than their counterparts in the cysts that do not contain a Balbiani Body. We also discovered lysosomal clusters are either next to, partially inside and inside the Balbiani Bodies around PND3, the percent of lysosomes inside the Balbiani Body increases 30% by PND5. This event occurs days after centrosomes, mitochondria and other organelles reach and create the structure. Besides addressing oocyte quantity control, we began to address which pathways estradiol works through to inhibit oocyte breakdown for proper formation of primordial follicles required for fertility. Though we did not see a significant effect of estradiol on the MAPK, PI3K/AKT and JAK/STAT pathways, it narrows us into looking at other important pathways or certain players within these pathways that could be affected by estradiol