Date of Award

2022

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Animal Science

First Advisor

David H. Townson

Abstract

Glucose is widely recognized as the preferred energy substrate for metabolism by granulosa cells (GCs). Yet in most cells, 2-5% of glucose is shunted through the hexosamine biosynthesis pathway (HBP) for O-linked N-acetylglucosaminylation (O-GlcNAcylation). O-GlcNAcylation is an evolutionarily-conserved, post-translational process that modifies serine and threonine residues on a variety of proteins. O-GlcNAcylation is also considered a nutrient sensor that can regulate cellular processes such as metabolism, signal transduction, and proliferation. In this respect, O-GlcNAcylation may be similar to, and possibly mediate, AMP-activated protein kinase (AMPK) signaling and its nutrient-sensing actions. However, the occurrence of O-GlcNAcylation and its relative importance to GC function has not been determined. Here, we characterized relative O-GlcNAcylation in bovine GCs from small and large antral follicles and determined its effects on GC proliferation.

Bovine ovary pairs morphologically staged to the mid-to-late estrous period were used. Granulosa cells and follicular fluid were aspirated from small (3-5mm) and large (>10mm) follicles. Freshly isolated GCs of small follicles exhibited greater immunodetectable expression of O-GlcNAcylation and the O-GlcNAcylation enzyme, O-GlcNAc transferase (OGT) expression than large follicles (P<0.05, n=7 ovary pairs). Less glucose (0.4mM vs 2.2 mM, P<0.05) and more lactate (33.3mM vs 9.6mM, P<0.05) was present in the follicular fluid of small follicles compared to large follicles (P<0.05, n=7). Steroid profiles revealed a progesterone to estradiol ratio >10 in all small follicle pools, indicative of highly atretic follicles. Similarly, 5 of the 7 large follicles pools were highly atretic and 2 were intermediately atretic (>1<10). Culture of GCs in serum free conditions revealed that inhibition of the HBP via the glutamine fructose-6-phosphate aminotransferase (GFAT) inhibitor, DON (50µM), impaired O-GlcNAcylation for both follicle sizes (P<0.05, n=5 independent expts.). The inhibitor DON also prevented GC proliferation regardless of follicle size (P<0.05, n=4). Direct inhibition of O-GlcNAcylation via the OGT inhibitor, OSMI-1 (50µM), prevented proliferation of GCs from small follicles (P<0.05, n=3). Augmentation of O-GlcNAcylation via the O-GlcNAcase (OGA) inhibitor, Thiamet-G (2.5µM), enhanced O-GlcNAcylation in GCs from both follicle sizes (P<0.05, n=3) but had no effect (P>0.05, n=3) on GC proliferation for either follicle size. Lastly, the use of the AMPK activator, Metformin (10mM), revealed that while AMPK activation inhibited GC proliferation from small follicles (P<0.05, n=4) as anticipated, it had no effect on O-GlcNAcylation (P>0.05 n=5).

The results indicate that: 1) O-GlcNAcylation occurs in GCs of bovine antral follicles, 2) Relative expression of O-GlcNAcylation is associated with alterations of glucose and lactate within the follicle, 3) Disruption of O-GlcNAcylation impairs GC proliferation, and 4) AMPK activation does not affect O-GlcNAcylation. In conclusion, the HBP and O-GlcNAcylation in GCs constitute an alternative, potential nutrient-sensing pathway to influence GC function and folliculogenesis in the bovine ovary.

Language

en

Number of Pages

95 p.

Available for download on Thursday, August 12, 2021

Share

COinS