Whereas it has long been known that normal mammalian reproduction depends upon the pulsatile secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, it is yet unclear what cellular and molecular mechanisms lie at the heart of this exceptionally timed pulse release. Additionally, while previous studies have implicated the 24 hour biological clock in the control of reproductive hormone secretion, it remains unknown how the circadian clock might modulate the amplitude or frequency of synchronous GnRH secretory release in order to regulate reproduction. Preliminary work in this laboratory, recently submitted for publication, indicates that not only are all molecular clock components, such as mPer1, mPer2, and clock present in the GnRH-secreting GT1-7 cell line, but that transcripts of mPer1 and mPer2 oscillate in these cultured cells with a circadian period. Strikingly, perturbation of the clock in perifused GT1-7 cells, via transient transfection of a dominant negative clock, disrupts normal secretory pulse patterns, suggesting that an intracellular circadian clock within GnRH neurons may function to modulate secretion. To investigate this further, the following proposal will attempt to 1) determine the extent of regulation of GnRH by the molecular clock, by examining both transcriptional and secretory effects; 2) investigate how cell-specific disruption of clock function only in GnRH neurons in vivo impacts fertility in transgenic mice; 3) dissect inter- and intracellular mechanisms linking clock oscillation to timed GnRH pulsatile secretion, by examining real-time changes in both clock gene oscillations and membrane kinetics. Results from this proposal have the potential to answer many fundamental questions regarding the nature of the GnRH """"""""pulse generator,"""""""" provide insight into broad mechanisms of endocrine neurosecretion, and allow for the candidate to fully develop into a principal investigator capable of eventually becoming a leader in reproductive neurobiology. Additionally, these studies could also advance the field of circadian biology, by ultimately demonstrating how oscillation of transcripts at the molecular level can control synchronous events at the multi-cellular and tissue level, in order to regulate numerous biological processes, from cellular metabolism to exocytosis, and even to orchestrate complex series of behaviors. Potential applications could lead to new directions in treating a range of physiological disorders that result from malfunction of hypothalamic neurosecretion, such as polycystic ovarian syndrome and primary ideopathic hypogonadism.
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