Department of Animal Science
First Advisor
Brett R. White
Date of this Version
12-2022
Citation
Elsken, D.H. Molecular Mechanisms Underlying Aberrant Circulating Steroid Hormones In GnRHR-II Knockdown Boars and Their Control Littermates. MS Thesis. University of Nebraska-Lincoln. 2022.
Abstract
Swine are the only livestock species that produce a functional protein for both GnRH-II and its receptor (GnRHR-II). Our laboratory reported that the interaction between GnRH-II and GnRHR-II facilitates testosterone production, independent of LH stimulation. To further examine GnRHR-II function, we developed a swine line with 70% reduced GnRHR-II mRNA levels (GnRHR-II KD) compared to control littermates. Mass spectrometry analysis of serum revealed that circulating levels of gonadal steroids were reduced in mature transgenic vs. control boars. Paradoxically, evaluation of testicular tissue indicated no difference between lines for gonadal steroid hormone levels. To address a potential cause for this discrepancy, we examined sulfoconjugation, which plays an important role in steroid homeostasis. Moreover, swine produce high concentrations of sulfoconjugates and testicular proteins associated with this pathway. Therefore, we designed primers of two sulfotransferases (SULT2A1 and SULT1E1), steroid sulfatase (STS) and the rate-limiting enzyme, 3'-phosphoadenosine 5'-phosphosulfate synthase (PAPSS), as well as a sulfate exporter (ABCC1) and importer (OSCP1) to compare mRNA levels in GnRHR-II KD and control testes using digital droplet PCR. Results suggested no expression differences between testicular tissue of GnRHR-II KD (n = 13) and control littermates (n = 11; P > 0.05) for any of these genes. Although amounts of mRNA for these target genes did not differ between lines, the role of this important pathway in the GnRHR-II KD phenotype requires more investigation. Next, we evaluated the testicular transcriptome of mature GnRHR-II KD (n = 10) and control (n = 7) boars via RNA sequencing. Read mapping with a false discovery rate adjusted analysis (P≤ 0.05) described 81,209 transcripts, of which we determined the presence of 24 differentially expressed genes (DEGs). Of these, 13 DEGs were upregulated and 11 DEGs were downregulated in GnRHR-II KD boars. Putative functions of DEGs include spermatogenesis (LOC106504199), cell signaling (GUCY1A3), mitochondrial function (LOC100524239), transcriptional regulation (PUF, ZNF33B), ubiquitination (UBE2W, USP42) and differentiation (EGR1, FIBIN, MSI2). These informative DEGs provide potential targets for improvement of boar fertility.
Advisor: Brett R. White
Comments
A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Animal Science, Under the Supervision of Professor Brett White. Lincoln, Nebraska: November 2022
Copyright © 2022 Dorothy H. Elsken