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AN INTEGRATIVE APPROACH TO INVESTIGATING OVARIAN FOLLICLE DEVELOPMENT AND CYCLIC RECRUITMENT IN CHICKENS
Restricted (Penn State Only)
Integrative and Biomedical Physiology
Doctor of Philosophy
Date of Defense:
January 29, 2019
Alan L. Johnson, Dissertation Advisor
Alan L. Johnson, Committee Chair
Joy L. Pate, Committee Member
Ramesh Ramachandran, Committee Member
Robert Paulson, Outside Member
The commercial laying hen constitutes an excellent model organism to study ovarian follicle development in Galliformes and to use as a comparative model organism to other birds, reptiles and mammals. Because eggs are laid in sequences (clutches), whereby one egg is laid per day, the chicken left, and only functional ovary contains follicles at virtually all stages of development. To enable daily egg laying, the ovary contains a hierarchy of rapidly growing preovulatory follicles which are arranged by size and maturity. After almost each oviposition, the largest follicle of the preovulatory hierarchy is ovulated. The number of preovulatory follicles is, in turn, replenished by a process referred to as cyclic recruitment. During cyclic recruitment a single follicle is recruited from a pool of pre-recruitment follicles that are also arranged into a size hierarchy, to transition to the rapid growth phase and to assume the position of the smallest follicle of the preovulatory hierarchy. Recruitment of a single follicle at the appropriate time interval ensures the maintenance of the follicle hierarchy and the timely maturation of preovulatory follicles for ovulation. The process of cyclic recruitment is known to be at least partially driven by the sensitization of the granulosa cell (GC) layer from the largest the pre-recruitment follicle to pituitary-derived follicle stimulating hormone (FSH) and the initiation of receptor-mediated cyclic adenosine monophosphate production. However, the exact mechanism that initiates this sensitization is still unknown. The first objective of the present work was to determine if pre-recruitment follicles are differentially responsive to FSH according to size, in vivo. Results indicated that in vivo injection of exogenous FSH induced recruitment of multiple pre-recruitment follicles within the duration of an ovulatory cycle, in a dose-dependent manner. These results indicate that pre-recruitment follicles are differentially responsive to FSH, in vivo, and the GC layer can respond to FSH to initiate differentiation when adequately stimulated. Moreover, under normal circumstances there is an inhibitory mechanism that prevents follicles from responding to normal circulating concentrations of FSH, but that there is an FSH concentration threshold above which pre-recruitment follicles respond and the threshold increases as the follicle size decreases. To determine potential markers in pre-recruitment follicles that confer an increased ability to respond to FSH as they increase in size, the second objective of the present work was to characterize changes in protein expression in the four largest pre-recruitment and the most recently recruited follicles. Results from a shotgun proteomics study indicated that there were no significant changes detected within the theca from the largest pre-recruitment follicle compared to the most recently recruited follicle. Within GC layers, a comparison between the most recently recruited follicle and the 4 largest pre-recruitment follicles determined that very few proteins (≤ 6) were differentially expressed, all of which were involved in cholesterol or yolk transport. These proteins are synthesized in the liver and are then transported in the plasma to the ovary. The protein profile of the largest pre-recruitment follicle was more similar to that of the most recently recruited follicle than those from the 2nd, 3rd and 4th largest pre-recruitment follicles. Pathway analysis indicated that, in GC from the most recently recruited follicle, transforming growth factor beta 1 and microRNA-21 pathways were predicted to be activated, whereas the pro-apoptotic cytokine oncostatin M pathway was predicted to be inhibited. We also report for the first time, expression of G-protein coupled receptor ligand RELAXIN-3 protein and mRNA in GC but not theca from pre-recruitment follicles, with highest expression detected in the most recently recruited follicle. The results suggest that the few changes in protein expression detected in pre-recruitment follicles are in GC and not TH. Accordingly, it is proposed that cyclic recruitment is primarily driven by rapid changes in signal transduction, and RELAXIN-3 signaling may play an important role in mediating these changes. Determining if cyclic recruitment and ovulation are functionally linked would greatly facilitate determining the exact time of cyclic recruitment and inform investigations on the most proximal event that leads to cyclic recruitment. Accordingly, the third objective of the present work was to determine if failure of ovulation leads to failure of cyclic recruitment. Results determined that cyclic recruitment can occur even when ovulation fails to occur. In addition, cyclic recruitment and ovulation did not appear to occur at the same time of the day. These results indicate that the mechanisms regulating time of cyclic recruitment are independent of the ovulatory cycle. We conclude that cyclic recruitment is a continuous process whereby a pre-recruitment follicle gains responsiveness to FSH as it increases in size. Although cyclic recruitment is mediated at least in part by changes in protein expression in GC, it is most likely driven by rapid changes in cell signaling due to sensitization of G-protein coupled receptors that stimulate cAMP production. Finally, time and occurrence of cyclic recruitment are not linked to time and occurrence of ovulation. We propose instead that time of cyclic recruitment is pre-determined by the position the follicle will assume in the ovulatory sequence which, in turn is most likely determined during early stages of pre-recruitment follicle development.
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