Characterization And Manipulation Of O-Glcnacylation In Granulosa Cells Of Bovine Ovarian Antral Follicles

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 (OGlcNAcylation). O-GlcNAcylation is an evolutionarily-conserved, post-translational process that modifies serine and threonine residues on a variety of proteins. OGlcNAcylation is also considered a nutrient sensor that can regulate cellular processes such as metabolism, signal transduction, and proliferation. In this respect, OGlcNAcylation may be similar to, and possibly mediate, AMP-activated protein kinase (AMPK) signaling and its nutrient-sensing actions. However, the occurrence of OGlcNAcylation 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 OGlcNAcylation 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 OGlcNAcylation 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 OGlcNAcylation (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 nutrientsensing pathway to influence GC function and folliculogenesis in the bovine ovary.