Author ORCID Identifier

https://orcid.org/0000-0002-4569-1664

Semester

Summer

Date of Graduation

2025

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

Eric Kelley

Committee Member

Stanley Hileman

Committee Member

Robert Goodman

Committee Member

Elizabeth Bowdridge

Committee Member

Werner Geldenhuys

Abstract

Gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), and follicle stimulating hormone (FSH) are essential for the reproduction of all mammalian species, including both sheep and humans. GnRH is secreted by hypothalamic neurons into the hypophyseal portal vasculature in a pulsatile manner, which then acts within the anterior pituitary to elicit the release of LH and FSH into wider systemic circulation. In females LH and FSH act on the ovaries to promote estradiol (E2) production, follicular development, and under certain conditions in regard to LH, ovulation. Furthermore, while E2 inhibits GnRH and subsequent LH secretion under most conditions, at high concentrations E2 acts as a positive regulator in the hypothalamus to generate an elevated and prolonged release of GnRH, and thus LH, termed the GnRH/LH surge, which culminates in ovulation. However, hypothalamic GnRH neurons lack estrogen receptors, thus E2-mediated feedback must occur upstream of GnRH neurons.

Arcuate nucleus (ARC) located kisspeptin-, neurokinin B-, and dynorphin A-containing neurons, known as KNDy neurons, are thought to mediate E2-negative feedback, but their possible role in E2-postive feedback was not completely understood. Within this dissertation we demonstrate that the ablation of KNDy neurons significantly blunted the amplitude of the GnRH/LH surge, suggesting a role in E2-positive feedback as well. Additional hypothalamic neuronal populations are thought to contribute to the GnRH/LH surge, such as retrochiasmatic area neurokinin 3 receptor- (NK3R) and galanin-containing neurons, and ARC kisspeptin receptor-containing (Kiss1R) neurons. By blocking the action of galanin within the ARC and on the KNDy neurons located there, we subtly but significantly reduced the amplitude of the GnRH/LH surge, while ablating ARC Kiss1R-containing neurons prevented the occurrence of the surge out right.

In females, puberty relies on both increased pulsatile GnRH/LH secretion as well as the emergence of the E2 induced GnRH/LH surge. While KNDy neuron number does not change during ovine puberty, other neurons contribute to this physiological process as well. ARC Kiss1R-containing neurons, which are crucial for the GnRH/LH surge, were ablated in prepubertal female lambs and significantly delayed puberty onset. The mechanism for this is not clear, but GnRH/LH surge amplitude was significantly reduced, but not blocked as seen in adults, suggesting a possible disruption to the generation of the preovulatory surge. Additionally, preoptic area NK3R-containing neurons, which contribute to the GnRH/LH surge in adult ewes, were ablated in prepubertal female lambs as well. This significantly accelerated puberty onset but had no effect on the timing or amplitude of the GnRH/LH surge and, surprisingly, no reduction in cell number was observed. Further studies are needed in characterizing preoptic NK3R-containing neurons and to determine whether they are truly inhibitory to puberty onset in sheep.

Body weight and metabolism are tightly linked to reproduction and fertility, as well as to puberty onset. The melanocortin system, consisting of pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons located in the ARC, act to integrate metabolic cues into the reproductive axis. POMC, through its cleavage product alpha-melanocyte stimulating hormone, acts as an excitatory stimulus, while AgRP plays an inhibitory role. We examined both the number of POMC and AgRP neurons and their activation throughout pubertal development in female lambs. POMC cell numbers did not change, but their activation, as measured by c-Fos, was elevated at the postpubertal time point. In contrast, AgRP cell numbers obviously and significantly decreased from the pre- to postpubertal time point and their activation significantly decreased as well. Thus, puberty onset in sheep may in part be mediated through the withdrawal of inhibitory AgRP signaling and an increase in stimulation via POMC neurons.

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