Date of Award
2024
Degree Type
Thesis
Degree Name
Master of Science in Biological and Environmental Sciences (MSBES)
First Advisor
David Townson
Abstract
The global population is on the rise putting pressure on the agricultural industry to increase production and increase the global food supply. The dairy industry has responded to this pressure by genetically selecting for higher milk production. This increase in production has coincided with a decline in fertility among dairy cows, due in part to a loss of reproductive cyclicity. Ovarian cyclicity and, in turn, reproductive cyclicity, is dependent upon proper development of ovarian follicles and the function of the cells that comprise the follicle and support the oocyte. One of these cell types is the granulosa cell. Granulosa cells support the growth and maturation of the oocyte of the follicle while producing estradiol to regulate reproductive cyclicity and behavior of the dairy cow (bovine). The functional capacity of bovine granulosa cells has been linked to a post-translational protein modification called O-linked-N-acetylglucosaminylation, or O-GlcANcylation. O-GlcNAcylation influences the proliferation of bovine granulosa cells, and possibly their metabolic activity. However, a connection between O-GlcNAcylation and estradiol production has yet to be explored. Additionally, considering the lack of a vascular supply within the ovarian follicle during its development, it is unclear whether the optimal metabolic environment for granulosa cells is low oxygen tension conditions (hypoxia). In this study granulosa cells from bovine small antral follicles were cultured to evaluate the effects of O-GlcNAcylation and hypoxia on estradiol production. Briefly, the cells were cultured in the absence or presence of small molecule inhibitors (OSMI-1; 50μM and Thiamet-G; 2.5μM) to manipulate O-GlcNAcylation, and in a separate set of experiments, cultured under normoxic (20% O2). or hypoxic (2% O2) conditions. The conditioned medium was collected at the end of the 144hr culture period to evaluate estradiol production. Conversely, the cells were collected and lysed for qPCR analysis of transcripts of the estradiol synthesis pathway (i.e., FSHR, STAR, and CYP19A1) and for immunodetection of hypoxia (i.e., HIF1α). Hormonal stimulation of the granulosa cells stimulated estradiol production (P<0.05) as expected. However, manipulation of O-GlcNAcylation under these circumstances had no effect (P>0.05). The only detectable effect of O-GlcNAcylation occurred with transcript expression of STAR, which decreased (P>0.05) as O-GlcNAcylation was inhibited. Hypoxia increased estradiol production (P<0.05) by the granulosa cells, but only under basal culture conditions, and there was no effect of hypoxia on any of the evaluated transcripts of estradiol synthesis (P>0.05). Surprisingly, HIF1α was undetectable in the cell lysates, so induction of hypoxia remained unverifiable. The results indicate O-GlcNAcylation has minimal effects on hormone-stimulated estradiol synthesis and secretion by bovine granulosa cells, and that hypoxia may be the optimal environment for granulosa cell metabolism.
Recommended Citation
DeCastro, Lauren, "EVALUATION OF THE EFFECTS OF O-GLCNACYLATION AND HYPOXIA ON ESTRADIOL PRODUCTION BY BOVINE GRANULOSA CELLS" (2024). Open Access Master's Theses. Paper 2505.
https://digitalcommons.uri.edu/theses/2505