Author ORCID Identifier

https://orcid.org/0000-0003-2401-8750

Semester

Spring

Date of Graduation

2024

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

Steven Hardy

Committee Co-Chair

Robert Goodman

Committee Member

Stanley Hileman

Committee Member

Robert Dailey

Committee Member

Kate Weil

Abstract

In female sheep, the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus triggers luteinizing hormone (LH) to be released from the pituitary. Luteinizing hormone, in turn, acts on the ovaries to elicit estradiol (E2) release. E2 acts in a negative feedback manner to control the amount of LH released. The increase in LH release that drives puberty onset results from a decrease in sensitivity to E2 as the animal ages. However, the neural mechanisms mediating this change in sensitivity are not known, as is where in the hypothalamus E2-negative feedback occurs. In the arcuate nucleus (ARC) of the hypothalamus, a population of neurons coexpressing kisspeptin, neurokinin B (NKB), and dynorphin are critical for reproduction and are thought to be responsible for pulsatile GnRH release.

Data from my initial work showed that there is no change in the number of ARC kisspeptin or NKB immunopositive cells, or percent colocalization of the two, with pubertal development. This, in conjunction with a previous finding that the receptor for estradiol, ERa increases instead of decreases within KNDy cells during development, raised the possibility that E2-negative feedback does not directly take place within these cells and that increased production of kisspeptin or NKB is not a limiting factor to puberty onset. However, in my second study, I found that ablating about 75% of KNDy cells with injections of NK3-saporin led to a significant delay in puberty onset. A subsequent experiment in that study also showed that KNDy cells are necessary for the full amplitude of the LH surge, but in a third experiment, ablation of NK3R-containing cells in the ARC was not needed for a positive response to intravenous injections of the NK3R agonist, senktide. Interestingly, in this last experiment, ewes showed clear evidence of normal pulsatile LH secretion despite the delay in puberty onset and previous work in adult ewes showing that ARC NK3-SAP injections in adult ewes blocked pulsatile LH release for at least 4 weeks. This novel work clearly indicated that KNDy cells are critical for timely puberty onset in female sheep but may not be a point of initiation for the decline in E2-negative feedback. Thus, there may be an upstream population or populations of cells that serve to transduce input regarding E2 and other important cues (such as nutritional information) to the reproductive system via KNDy cells. Indeed, in subsequent an increase in KNDy neuron activity was associated with puberty-related changes in LH secretion, further supporting this idea.

In another set of experiments, two hypothalamic areas were assessed as possible sites for E2-negative feedback via placement of E2 microimplants into the hypothalamus of ovariectomized ewes or the ERa antagonist ICI 182,780 into the hypothalamus of intact ewes: the preoptic area (POA) and mediobasal hypothalamus (MBH). While the results of this study were not completely clear, they pointed to a role of both the POA and MBH in E2-negative feedback. The E2 microimplants within either area were not able to suppress LH to the level that subcutaneous E2 implants did on their own, indicating that the two areas act in concert, perhaps with other regions of the brain, to communicate E2 input. Further work will be needed to identity these regions.

Nutritional state and puberty onset are intimately connected, as animals only undergo puberty onset after reaching a critical energetic state. One pathway through which these metabolic signals are communicated to the reproductive system likely occurs via neuronal populations in the ARC that contain neuropeptide Y (NPY), agouti-related peptide (AgRP), and proopiomelanocortin (POMC). The latter controls satiety and increases energy expenditure, and injection of the POMC analogue a-melanocyte stimulating hormone (aMSH) leads to an increase in LH. AgRP and NPY trigger food intake, decrease energy expenditure, and decrease LH. aMSH and AgRP both act via MC3R and MC4R receptors (commonly referred to as the melanocortin system), which have been observed in KNDy neurons. NPY acts on multiple receptor isoforms (Y1-6), but it is unknown if these receptors are expressed by KNDy neurons. In my final study, POMC and NPY immunopositive cell numbers did not change and AgRP cell numbers decreased significantly with pubertal development. The fiber density for NPY in the ARC through development was also determined, but there was no significant change. The activity of POMC neurons increased with puberty-associated changes in LH secretion, and AgRP neuronal activity decreased. AgRP or NPY contacts onto KNDy did not change with development, but there was an increase in POMC contacts onto KNDy cells. Finally, the percentage of POMC and AgRP that expressed ERa through development did not change. This work adds significant knowledge regarding potential neural mechanisms that regulate ovine puberty. The melanocortin system is involved in receiving and transducing important information on the nutritional state of the individual to KNDy neurons, allowing for a pubertal increase in GnRH and LH secretion that drives puberty onset. KNDy neurons are a critical penultimate input to GnRH neurons and ultimately mediate the decline in sensitivity to E2 that leads to puberty onset. However, where and how E2-negative feedback is harbored in the hypothalamus remains a critical but open question.

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