Doctoral Dissertations

Orcid ID

0000-0003-0606-522X

Date of Award

5-2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Brian K. Whitlock

Committee Members

Madhu S. Dhar, Rebecca A. Prosser, Cheryl J. Kojima, Travis Mulliniks

Abstract

Stressors have a negative impact on reproductive efficiency in humans and other animals, which has an economic cost due to infertility treatments for humans and reduced income for food producers. We wished to determine the molecular pathways by which metabolic disturbances and low-level inflammation impact the hypothalamic pituitary gonadal (HPG) axis using an in vitro model. To this end, we used the GT1-7 cell line, an immortalized line expressing the Kiss1R receptor that responds to kisspeptin stimulation with the release of GnRH to assess the impact of stressors on the GnRH-releasing cells. Additionally, we used the KTaV-3 and KTaR-1[A1] cell lines, immortalize lines derived from the rodent anteroventral periventricular and arcuate nuclei, respectively, to assess the impact of stressors on kisspeptin-producing cells. We modeled metabolic disease and negative energy balance by exposing cells to beta-hydroxybutyric acid (BHB) and inflammation by exposure to tumor necrosis factor alpha (TNFα [alpha]). We determined that exposure to BHB significantly increased the intrinsic production of GnRH by GT1-7 cells, but that there were no additional changes in the expression of GnRH mRNA or release of GnRH protein. Additionally, there were no significant changes in calcium signaling or in the phosphorylation of ERK1/2, suggesting that the direct effect of stressors does not impact the GnRH-producing cells of the hypothalamus. Exposure to stressors in culture did not change the expression of kiss1 mRNA in KTaV-3 cells upon exposure to estradiol. However, KTaV-1 cells exhibited a significantly decreased expression of kiss1 mRNA and kiss1 protein release upon exposure to both BHB and tumor necrosis factor alpha. There was also a reduction in the expression of neurokinin B (nkb) mRNA under these conditions, suggesting that the impact of stressors on the HPG axis may be acting through the suppression of signaling in the arcuate nucleus. Disruption of neurokinin B and kisspeptin expression in the arcuate nucleus could impact the production of tonic levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH), resulting in a deficiency in follicle production and oocyte maturation.

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