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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.

SUBMITTER: Maucieri AM

PROVIDER: S-EPMC8023420 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAt later stages of folliculogenesis, the mammalian ovarian follicle contains layers of epithelial granulosa cells surrounding an antral cavity. During follicle development granulosa cells replicate, secrete hormones and support the growth of the oocyte. In cattle, the follicle needs to grow > 10 mm in diameter to allow an oocyte to ovulate, following which the granulosa cells cease dividing and differentiate into the specialised cells of the corpus luteum. To better understand the molecular basis of follicular growth and granulosa cell maturation, we undertook transcriptome profiling of granulosa cells from small (< 5 mm; n = 10) and large (> 10 mm, n = 4) healthy bovine follicles using Affymetrix microarrays (24,128 probe sets).ResultsPrincipal component analysis for the first two components and hierarchical clustering showed clustering into two groups, small and large, with the former being more heterogeneous. Size-frequency distributions of the coefficient of variation of the signal intensities of each probe set also revealed that small follicles were more heterogeneous than the large. IPA and GO enrichment analyses revealed that processes of axonal guidance, immune signalling and cell rearrangement were most affected in large follicles. The most important networks were associated with: (A) Notch, SLIT/ROBO and PI3K signalling, and (B) ITGB5 and extracellular matrix signalling through extracellular signal related kinases (ERKs). Upstream regulator genes which were predicted to be active in large follicles included STAT and XBP1. By comparison, developmental processes such as those stimulated by KIT, IHH and MEST were most active in small follicles. MGEA5 was identified as an upstream regulator in small follicles. It encodes an enzyme that modifies the activity of many target proteins, including those involved in energy sensing, by removal of N-acetylglucosamine from serine and threonine residues.ConclusionsOur data suggest that as follicles enlarge more genes and/or pathways are activated than are inactivated, and gene expression becomes more uniform. These findings could be interpreted that either the cells in large follicles are more uniform in their gene expression, or that follicles are more uniform or a combination of both and that additional factors, such as LH, are additionally controlling the granulosa cells.

Project description:BackgroundThe major function of the ovary is to produce oocytes for fertilisation. Oocytes mature in follicles surrounded by nurturing granulosa cells and all are enclosed by a basal lamina. During growth, granulosa cells replicate and a large fluid-filled cavity (the antrum) develops in the centre. Only follicles that have enlarged to over 10 mm can ovulate in cows. In mammals, the number of primordial follicles far exceeds the numbers that ever ovulate and atresia or regression of follicles is a mechanism to regulate the number of oocytes ovulated and to contribute to the timing of ovulation. To better understand the molecular basis of follicular atresia, we undertook transcriptome profiling of granulosa cells from healthy (n = 10) and atretic (n = 5) bovine follicles at early antral stages (< 5 mm).ResultsPrincipal Component Analysis (PCA) and hierarchical classification of the signal intensity plots for the arrays showed primary clustering into two groups, healthy and atretic. These analyses and size-frequency plots of coefficients of variation of signal intensities revealed that the healthy follicles were more heterogeneous. Examining the differentially-expressed genes the most significantly affected functions in atretic follicles were cell death, organ development, tissue development and embryonic development. The overall processes influenced by transcription factor gene TP53 were predicted to be activated, whereas those of MYC were inhibited on the basis of known interactions with the genes in our dataset. The top ranked canonical pathway contained signalling molecules common to various inflammatory/fibrotic pathways such as the transforming growth factor-β and tumour necrosis factor-α pathways. The two most significant networks also reflect this pattern of tissue remodelling/fibrosis gene expression. These networks also contain molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor-β signalling and were up regulated.ConclusionsSmall healthy antral follicles, which have a number of growth outcomes, exhibit greater variability in gene expression, particularly in genes associated with cell division and other growth-related functions. Atresia, on the other hand, not only involves cell death but clearly is an active process similar to wound healing.

Project description:Antral follicle size might be a valuable additive predictive marker for IVF outcome. However, while some studies show positive relations between follicle size and reproductive outcome, others have not been successful in establishing this relation. To better understand consequences of antral follicle size for reproductive outcome, we aimed to obtain insight in follicle size-related granulosa cell processes, as granulosa cells play an essential role in follicular development via the production of growth factors, steroids and metabolic intermediates, needed for follicular growth and oocyte development. Using pigs as a model, we compared gene and protein expression in granulosa cells of smaller and larger follicles in the healthy antral follicle pool at the start of the follicular phase of the estrous cycle. In sows, the early antral follicle pool is very heterogeneous when e.g. size and steroid content of the follicular fluid are considered. To which extent this variety contributes to the developmental competence of the follicles is not clear. Therefore, sows with high variation in antral follicle size (HighVAR) as well as sows with low variation in antral follicle size (LowVAR) were used. Granulosa cells of smaller antral follicles in the healthy antral follicle pool show increased cell proliferation, which was accompanied by a metabolic shift towards aerobic glycolysis (i.e. the Warburg effect), similar to other highly proliferating cells. High granulosa cell proliferation rates in smaller follicles might be regulated via increased granulosa cell expression of AR and EGFR which are activated in response to locally produced mitogens. While granulosa cells of smaller follicles in the pool were more proliferative, indicative of higher follicular growth, granulosa cells of larger follicles in the pool showed less proliferation and were more differentiated, as they showed a higher expression of follicular maturation marker IGF1 and ANGPT1. Our results imply that the inclusion of strict criteria of antral follicle size in IVF protocols might improve reproductive outcome. In addition, we have granulosa cell gene expression of healthy follicles to unravel underlying mechanisms of differences in COC morphology. We compared gene expression in sows with low vs. high-COC-health and found a decreased expression of genes involved in ovarian steroidogenesis (e.g. CYP19A1, ADM, SPP1) and higher expression of genes involved in follicular atresia (e.g. GADD45A, INHBB) in sows with low-COC-health. Thereby we have identified several genes which may serve as markers for follicle developmental competence.

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

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