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 a slaughterhouse, 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.

Keywords: O-GlcNAcylation; bovine; glucose; granulosa cell; metabolism; ovary.

Figure 1

Schematic of glucose metabolism through the hexosamine biosynthesis pathway (HBP) and the process of O-GlcNAcylation. Approximately 2–5% of all glucose metabolized by the cell enters the HBP. Glutamine fructose-6-phosphate aminotransferase (GFAT) is a rate-limiting enzyme of O-GlcNAcylation that partitions fructose-6-phosphate (an intermediate of glycolysis) to form glucosamine-6-phosphate, and ultimately, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Subsequently, the enzyme, O-GlcNAc transferase (OGT) adds GlcNAc sugars from the donor substrate, UDP-GlcNAc, to serine/threonine residues of proteins. Conversely, the enzyme O-GlcNAcase (OGA), removes these same GlcNAc sugars from proteins. OGT and OGA enzymes are the sole regulators of O-GlcNAcylation and influence the process in a cyclic manner [1].

Figure 2

Relative concentrations of glucose and lactate in follicular fluid of bovine antral follicles according to follicle size. (A) Glucose concentrations and (B) lactate concentrations are depicted for small (SF; 3–5 mm) and large (LF; >10 mm) bovine follicles. Results represent n = 7 ovary pairs, run in duplicate, with the mean ± SEM concentration (mM) of a given metabolite shown. Different letters denote differences between SF and LF at P < 0.05.

Figure 3

Immunoblots and measurement of global O-GlcNAcylation and OGT expression in freshly isolated bovine granulosa cells of small and large antral follicles. (A) Immunoblot of global O-GlcNAcylation and OGT expression (MW:110 kDa) in freshly isolated bovine granulosa cells of small (SF; 3–5 mm) and large (LF; >10 mm) follicles. (B) Bar graphs of the densitometric analysis of O-GlcNAcylated proteins and OGT expression relative to total protein expression. Results represent n = 7 ovary pairs run in triplicate, with the mean ± SEM signal intensity shown. Different letters denote differences between SF and LF at P < 0.05.

Figure 4

Immunoblots, measurement of global O-GlcNAcylation, and quantification of cell proliferation following treatment of cultured bovine granulosa cells from small and large antral follicles with the glutamine fructose-6-phosphate aminotransferase (GFAT) inhibitor, DON. (A–C) Effects of 24-hour exposure to DON (50 μM) on global O-GlcNAcylation (A, B) and granulosa cell proliferation (C) in cells from small antral follicles (3–5 mm). (D–F) Effects of 24-hour exposure to DON (50 μM) on global O-GlcNAcylation (D, E) and granulosa cell proliferation (F) in cells from large antral follicles (>10 mm). Representative immunoblots and corresponding bar graphs of the densitometric analyses of global O-GlcNAcylation relative to total protein for the two follicle sizes (A and B, D and E) are shown. Bar graphs depicting granulosa cell proliferation as measured by MTS assay for the two follicle sizes (C and F) are also shown. Results representative of n = 4–5 independent experiments run in triplicate, with the mean ± SEM signal intensity or absorbance at 490 nm indicated. Different letters denote differences from control at P < 0.05.

Figure 5

Immunoblots, measurement of global O-GlcNAcylation, and quantification of cell proliferation following time-course treatment of cultured bovine granulosa cells from small and large antral follicles with the O-GlcNAc transferase (OGT) inhibitor, OSMI-1. (A–C) Effects of OSMI-1(50 μM) on global O-GlcNAcylation after 4, 8, 12, and 24 h of exposure to the inhibitor (A, B); and on granulosa cell proliferation after 24 h of exposure to the inhibitor (C) in cells from small antral follicles (3–5 mm). (D–F) Effects of OSMI-1 (50 μM) on global O-GlcNAcylation after 4, 8, 12, and 24 h of exposure to the inhibitor (D, E); and on granulosa cell proliferation after 24 h of exposure to the inhibitor (F) in cells from large antral follicles (>10 mm). Representative immunoblots and corresponding bar graphs of the densitometric analyses of global O-GlcNAcylation relative to total protein for the two follicle sizes (A and B, D and E) are shown. Bar graphs depicting granulosa cell proliferation as measured by MTS assay for the two follicle sizes (C and F) are also shown. Results represent n = 3–6 independent experiments run in triplicate, with the mean ± SEM signal intensity or absorbance at 490 nm indicated. Different letters or asterisks denote differences from control at P < 0.05.

Figure 6

Immunoblots, measurement of global O-GlcNAcylation, and quantification of cell proliferation following treatment of cultured bovine granulosa cells from small and large antral follicles with the O-GlcNAcase (OGA) inhibitor, Thiamet-G. (A–C) Effects of 24 h exposure to Thiamet-G (2.5 μM) on global O-GlcNAcylation (A, B) and granulosa cell proliferation (C) in cells from small antral follicles (3–5 mm). (D, E, F) Effects of 24 h exposure to Thiamet-G (2.5 μM) on global O-GlcNAcylation (D, E) and granulosa cell proliferation (F) in cells from large antral follicles (>10 mm). Representative immunoblots and corresponding bar graphs of the densitometric analyses of global O-GlcNAcylation relative to total protein for the two follicle sizes (A and B, D and E) are shown. Bar graphs depicting granulosa cell proliferation as measured by MTS assay for the two follicle sizes (C and F) are also shown. Results represent n = 3–6 independent experiments run in triplicate, with the mean ± SEM signal intensity or absorbance at 490 nm shown indicated. Different letters denote differences from control at P < 0.05.

Figure 7

Immunodetection of granulosa cell proliferation (via Ki-67 staining) following augmentation and inhibition of O-GlcNAcylation with Thiamet-G (2.5 μM) and OSMI-1 (50 μM), respectively. Bar graphs depicting the percentage of Ki-67 positive cells from small antral follicles (SF, 3–5 mm; A) and large antral follicles (LF, >10 mm; B) are shown. (C) Representative photomicrographs (Bar = 150 μm) depicting Ki-67 staining (darkened cells) in cultured bovine granulosa cells of small and large antral follicles (SF and LF, respectively) following 24-hour treatment with DMSO (control), Thiamet-G, and OSMI-1. Results represent n = 5 independent experiments, run in duplicate, with the mean ± SEM of Ki-67 expression shown. Different letters denote differences among treatments at P < 0.05.