Date of Award
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Animal Nutrition and Food Science
First Advisor
David H. Townson
Second Advisor
Elizabeth Bonney
Abstract
Subfertility in dairy cows causes substantial financial loss to producers. A significant portion of subfertility can be attributed to ovarian and likely granulosa cell (GC) dysfunction. A disrupted metabolic state of the cow is known to negatively impact reproduction, but the mechanism(s) of its influence on GCs and follicular development is poorly understood. Glucose is the preferred metabolic substrate of GCs, yet its role and utilization within GCs is poorly defined. The hexosamine biosynthesis pathway (HBP) is an alternate route of glucose utilization, providing substrate for O-GlcNAcylation, a post-translational modification that influences cellular metabolism, proliferation, and fate. This process is reliant on nutrient availability and is considered a nutrient sensor. Follicular fluid and GCs were aspirated from small (3-5mm) and large (>10mm) antral follicles from bovine ovary pairs. GCs of small follicles exhibited greater expression of O-GlcNAcylation and O-GlcNAc transferase (OGT) than large follicles (P<0.05). Less glucose and more lactate were detectable in the follicular fluid of small versus large follicles (P<0.05). Inhibition of the HBP via the glutamine fructose-6-phosphate aminotransferase inhibitor, DON (50µM) and via the OGT inhibitor, OSMI-1 (50µM), impaired O-GlcNAcylation and GC proliferation (P<0.05). Next, extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of the GCs were measured. GCs from small antral follicles exhibited overall greater metabolic activity than GCs from large antral follicles as evidenced by increased ECAR (P<0.05) and OCR (P<0.05). The glycolytic stress test indicated that GCs from both types of follicles possessed additional glycolytic capacity (P<0.05). Inhibition of cellular respiration by 2-Deoxy-D-glucose (50 mM) impaired OCR only in GCs from small antral follicles (P<0.05), but exposure to the mitochondrial stress test had no effect (P>0.05). Conversely, in GCs from large antral follicles, oxidative metabolism was impaired by the mitochondrial stress test (P<0.05) and was accompanied by a concomitant increase in glycolytic metabolism (P<0.05). Immunodetection of glycolytic enzymes revealed that phosphofructokinase expression is increased in GCs of small antral follicles compared to large follicles (P<0.05). Inhibition of O-GlcNAcylation via OSMI-1 (50μM) impaired the expression of hexokinase, phosphofructokinase, pyruvate kinase and pyruvate dehydrogenase in GCs (P<0.05). Lastly, groups of cows were separated into two groups (negative energy balance or NEB and positive energy balance or PEB) during the postpartum period. Serum concentrations of BHBA and NEFAs were greater in the NEB group than the PEB group (P<0.05). There was no effect of energy balance on the number of healthy versus atretic follicles aspirated from each cow, or the number of perceived ovulatory versus nonovulatory cows (P>0.05). Follicular fluid concentrations of glucose, insulin, and lactate did not differ between the two groups (P>0.05). Additionally, there was no detectable difference in the expression of O-GlcNAcylation in the GCs from NEB versus PEB cows (P>0.05). Collectively, these results provide the first evidence of O-GlcNAcylation in granulosa cells of bovine antral follicles and demonstrate that relative expression of O-GlcNAcylation is associated with changes in concentrations of glucose and lactate within the follicle. Inhibition of O-GlcNAcylation impairs granulosa cell proliferation and cellular metabolism, which indicates a homeostatic level of O-GlcNAcylation is necessary to support granulosa cell function.
Language
en
Number of Pages
176 p.
Recommended Citation
Maucieri, Abigail M., "The influence of the hexosamine biosynthesis pathway and O-GlcNAcylation on glucose metabolism in ovarian antral follicles" (2024). Graduate College Dissertations and Theses. 1931.
https://scholarworks.uvm.edu/graddis/1931