RFamide Peptides and Ovulation: Circadian Control and Environmental Disruptors
Wilbur Putney Williams, III
Doctor of Philosophy in Psychology
University of California, Berkeley
Professor Lance J. Kriegsfeld, Chair
Successful reproduction depends upon a highly orchestrated cascade of events during optimal environmental conditions in order to appropriately time ovulation. In spontaneously ovulating rodents, neurons in the hypothalamus secreting gonadotropin-releasing hormone (GnRH) are triggered by a timed, stimulatory signal originating from the circadian clock in the suprachiasmatic nucleus (SCN), initiating a cascade of events that ultimately leads to the release of a mature egg from it's follicle. Reproductive viability is maintained in part by the temporal precision of GnRH signaling, and ageing-related declines in ovulatory function are a direct result of the dysregulation of circadian control of GnRH. The transition to reproductive senescence is characterized by irregular estrous cycles prior to any decline in ovarian reserves, suggesting the central mechanisms controlling GnRH are initially responsible for determining reproductive quiescence.
In addition to the circadian control of the HPG axis, most mammals are markedly affected by the availability of energy with regard to reproductive function. In most natural habitats, food availability and energetic status fluctuate markedly, requiring individuals within a species to prioritize survival via foraging and ingestive behaviors over behaviors that perpetuate the individual during periods of low available energy. Consequently, the mechanisms monitoring available energy also regulate the reproductive axis, primarily through inhibiting or permitting the release of GnRH under specific energetic conditions.
The specific mechanisms connecting the circadian clock and energy balance to the reproductive axis remain to be fully characterized. Two related RFamide (Arg-PHe-NH2), kisspeptin and RFamide-related peptide-3 (RFRP-3) stimulate and inhibit GnRH, respectively, and may bridge the gap between the circadian system, energy balance and reproduction. All studies utilized female Syrian hamsters, which exhibit precise 4-day estrous cycles and are considered to be ideal model systems for understanding reproductive cyclicity. In the first series of experiments, the role of kisspeptin in the circadian control of ovulation was examined. Kisspeptin neurons exhibit a circadian pattern of activation and are regulated by the SCN via vasopressin (AVP) efferents. AVP administration upregulates kisspeptin indiscriminately, but the GnRH response is restricted to the afternoon. Furthermore, GnRH neurons exhibited a timed sensitivity to kisspeptin administration, suggesting these neurons act as a gating site for upstream signals in order to properly time ovulation.
The circadian control of RFamide peptides may be critical to maintain ovulatory function in aging animals. The second set of experiments investigated whether or not the transition to reproductive acyclicity is characterized by disruptions in the timing of RFamide activation. Middle-aged, female Syrian hamsters exhibiting irregular estrous cycles displayed alterations in the timing of RFRP-3 activation, compared to young and middle-aged, regularly cycling hamsters. Furthermore, middle-aged hamsters exhibit abnormally high levels of kisspeptin immunoreactivity around the time of the LH surge, which may be indicative of improper kisspeptin release during the periovulatory period.
The final set of experiments aimed to determine whether or not the neural circuitry detecting energy status communicates to RFRP-3, and if metabolic challenge, such as food deprivation, alters the activational state of this inhibitory peptide. RFRP-3 activation increases following 48 hours of food deprivation, and may downregulate the HPG axis during periods of negative energy balance. Furthermore, immense innervation of RFRP-3 by the orexigenic peptide, NPY, provides a mechanism of control between these two systems. Finally, mild food restriction over 4-12 days increases RFRP-3 activation, as well as motivation to engage in food hoarding over sexual behaviors. Interestingly, the food restriction paradigm does not alter food intake or estrous cyclicity, suggesting a novel role of RFRP-3 in motivated behaviors.