Semester

Fall

Date of Graduation

2013

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

Robert L. Goodman

Committee Co-Chair

Heather J. Billings

Committee Member

Steven L. Hardy

Committee Member

Stanley M. Hileman

Committee Member

Adrienne Salm

Abstract

Hypothalamic input is vital to the regulation of reproductive function. Gonadotropin-releasing hormone (GnRH) is secreted by hypothalamic GnRH neurons into the hypophysial portal circulation. When GnRH reaches the anterior pituitary, it stimulates gonadotropes to secrete luteinizing hormone (LH) into the systemic circulation, ultimately leading to ovulation. GnRH/LH secretion occurs in two different patterns: pulsatile or surge. Pulsatile GnRH release is governed by estradiol (E2) negative feedback while surge release is the result of E2 positive feedback. However, GnRH neurons lack the alpha isoform of the estrogen receptor, indicating that E2 feedback on GnRH release is indirect. Other hypothalamic neurons that express ER&agr; have been identified as possible intermediates in this E2 feedback pathway, including those containing neurokinin B (NKB) and kisspeptin.;Kisspeptin has been identified as a potent stimulator of LH secretion in a variety of species. GnRH neurons express the kisspeptin receptor (Kiss1r), indicating that kisspeptin can directly stimulate GnRH secretion. Two anatomically separate populations have been identified in the hypothalamus: one in the arcuate nucleus (ARC) and the other in the preoptic area (POA). Furthermore, ARC kisspeptin neurons have been found to colocalize two other neuropeptides as well: NKB and dynorphin A (DYN). Thus, these neurons were assigned the acronym KNDy neurons. The specific role of these two kisspeptin-containing populations in GnRH/LH regulation is unclear. Therefore we examined the degree to which each of these populations were activated, using cFos as an index of activation, during positive E2 feedback (surge) or following E2 withdraw (i.e. after ovariectomy). We found that both populations exhibited increased activation during the GnRH/LH surge, indicating that both are involved in E2-positive feedback. Only the ARC kisspeptin neurons showed increased activation after E2 withdrawal, indicating that only the KNDy neurons are playing a role in E2-negative feedback control of GnRH release.;NKB neurons have been shown to consistently stimulate GnRH/LH secretion in non-rodent species. Since KNDy neurons express receptors for NKB (NK3R), the current proposed model suggests that NKB acts in a paracrine or autocrine fashion to stimulate kisspeptin release from KNDy neurons which then acts directly to initiate a pulse of GnRH. Dynorphin from KNDy neurons then acts to terminate kisspeptin release and end the pulse of GnRH release. However, high levels of neurokinin-3-receptor (NK3R) are found outside of KNDy neurons in the ovine POA, ARC, retrochiasmatic area (RCh), and paraventricular nucleus (PVN). We first examined whether NKB or senktide, an NK3R agonist, would alter LH secretion if placed within each of these areas. While NKB, in any of those areas, had no effect on LH secretion, senktide placed in either the RCh or POA stimulated a surge-like secretion of LH, suggesting that NK3R activation in these areas may play a role in the GnRH/LH surge. Thus, we subsequently placed an NK3R antagonist in the RCh or POA during an E2-induced LH surge and found that blocking NK3R in the RCh, but not the POA, significantly suppressed LH surge amplitude. These results suggest that NK3R activation in the RCh is essential for the full LH surge.;As mentioned above, one current model suggests that NKB exerts its actions via kisspeptin output to GnRH neurons; however, this has not been examined in ewes. We first assessed activation of the ARC and POA populations of kisspeptin neurons following senktide treatment in either the RCh or POA. We found that senktide in either area significantly increased ARC kisspeptin neuron activation but did not alter activity of POA kisspeptin neurons. To further test the model, we gave a Kiss1R-antagonist (via intracerebroventricular infusion) during senktide treatment to see if blocking kisspeptin receptors would ameliorate senktide-induced surge-like LH secretion. This experiment yielded inconsistent results, with the increase in LH blocked in some ewes by Kiss1R antagonist infusion but not in others. Nonetheless, based on the activation data, we conclude that NK3R activation stimulates LH secretion via ARC kisspeptin neurons.;In conclusion, we have identified a role for NK3R-positive neurons in the RCh in regulating the LH surge in ewes. These data reveal the importance of the RCh in the LH surge, an area whose involvement in this process was previously unknown.

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