Evidence and manipulation of O-GlcNAcylation in granulosa cells of bovine antral follicles†

Abstract

Glucose is a preferred energy substrate for metabolism by bovine granulosa cells (GCs). O-linked N-acetylglucosaminylation (O-GlcNAcylation), is a product of glucose metabolism that occurs as the hexosamine biosynthesis pathway (HBP) shunts O-GlcNAc sugars to serine and threonine residues of proteins. O-GlcNAcylation through the HBP is considered a nutrient sensing mechanism that regulates many cellular processes. Yet little is known of its importance in GCs. Here, O-GlcNAcylation in GCs and its effects on GC proliferation were determined. Bovine ovaries from an abattoir, staged to the mid-to-late estrous period were used. Follicular fluid and GCs were aspirated from small (3-5 mm) and large (>10 mm) antral follicles. Freshly isolated GCs of small follicles exhibited greater expression of O-GlcNAcylation and O-GlcNAc transferase (OGT) than large follicles. Less glucose and more lactate was detectable in the follicular fluid of small versus large follicles. Culture of GCs revealed that inhibition of the HBP via the glutamine fructose-6-phosphate aminotransferase inhibitor, DON (50 μM), impaired O-GlcNAcylation and GC proliferation, regardless of follicle size. Direct inhibition of O-GlcNAcylation via the OGT inhibitor, OSMI-1 (50 μM), also prevented proliferation, but only in GCs of small follicles. Augmentation of O-GlcNAcylation via the O-GlcNAcase inhibitor, Thiamet-G (2.5 μM), had no effect on GC proliferation, regardless of follicle size. The results indicate GCs of bovine antral follicles undergo O-GlcNAcylation, and O-GlcNAcylation is associated with alterations of glucose and lactate in follicular fluid. Disruption of O-GlcNAcylation impairs GC proliferation. Thus, the HBP via O-GlcNAcylation constitutes a plausible nutrient-sensing pathway influencing bovine GC function and follicular growth.

Disruption of O-GlcNAcylation Homeostasis Induced Ovarian Granulosa Cell Injury in Bovine

“…In the current study, we observed that altered O-GlcNAc cycling in response to OGT or OGA pharmacological inhibition reduced cell viability, decreased the expression levels of the proliferation-associated genes CDC42 and PCNA , induced apoptosis, and caused the upregulated BAX/BCL-2 mRNA ratio and CASPASE-3 transcripts, which corroborates the findings of a previous study showing that the accumulation of O-GlcNAc in mouse embryonic neural precursor cells induced by treatment with the OGA inhibitor, PUGNAc, activated Caspase-3 activity and severely damaged the cell viability [ 38 ]. However, this is slightly different from a recent study conducted on GCs of bovine antral follicles, which revealed that the manipulation of O-GlcNAc by inhibiting the HBP or via the OGT inhibitor, OSMI-1, impairs GC proliferation, but perturbation of O-GlcNAc via the OGA inhibitor, Thiamet-G, has no obvious effect on GC proliferation [ 24 ]. This discrepancy may be due to the different OGA inhibitor used.…”

“…Glucose is an important metabolic substrate and preferably utilized by GCs to provide the oocytes with energy production [ 23 ]. A recent study demonstrated that O-GlcNAcylation varies in different sizes of antral follicles and influences GC proliferation in bovines [ 24 ]. However, the detailed molecular regulation of O-GlcNAcylation in GC function is just beginning to be clarified.…”

Protein O‐GlcNAcylation: The sweet hub in liver metabolic flexibility from a (patho)physiological perspective

“…104 The cellular O-GlcNAcylation cycle is believed to be strictly controlled by HBP flow and UDP-GlcNAc concentration. 105 The HBP and UDP-GlcNAc link altered metabolism and O-GlcNAcylation, providing an important mechanism for cells to perceive and respond to nutrient availability. 106 For example, hyperglycaemia causes an increase in intracellular UDP-GlcNAc…”

Saturated fatty acids inhibit unsaturated fatty acid induced glucose uptake involving GLUT10 and aerobic glycolysis in bovine granulosa cells

“…Also, in sheep, glycolytic metabolism was upregulated in gonadotropin-stimulated antral follicles, as reflected by increased glucose uptake and lactate production 31 . Abigail et al found that GCs of small follicles exhibit enhanced glucose uptake and glycolytic metabolism in bovine 32 . In vitro studies in pigs showed that increased proliferation of GCs in small follicles undergoing development is accompanied by a metabolic shift toward aerobic glycolysis 33 , indicating that the glycolytic pathway is a major player of GCs function in different species.…”