Abstract
During follicle maturation, oxygen levels continuously decrease in the follicular fluid and reach lowest levels in the preovulatory follicle. The current study was designed to comprehensively understand effects of low oxygen levels on bovine granulosa cells (GC) using our established estrogen active GC culture model. As evident from flow cytometry analysis the viability of GC was not found to be affected at severely low oxygen condition (1% O2) compared to normal (atmospheric) oxygen condition (21% O2). Estimations of hormone concentrations using competitive radioimmunoassay revealed that the production of estradiol and progesterone was significantly reduced at low oxygen condition. To understand the genome-wide changes of gene expression, mRNA microarray analysis was performed using Affymetrix’s Bovine Gene 1.0 ST Arrays. This resulted in the identification of 1104 differentially regulated genes of which 505 were up- and 599 down-regulated under low oxygen conditions. Pathway analysis using Ingenuity pathway analyzer (IPA) identified 36 significantly affected (p < 0.05) canonical pathways. Importantly, pathways like “Estrogen-mediated S-phase Entry” and “Cyclins and Cell Cycle Regulation” were found to be greatly down-regulated at low oxygen levels. This was experimentally validated using flow cytometry based cell cycle analysis. Up-regulation of critical genes associated with angiogenesis, inflammation, and glucose metabolism, and down-regulation of FSH signaling, steroidogenesis and cell proliferation indicated that low oxygen levels induced early luteinization associated changes in granulosa cells. Identification of unmethylated CpG sites in the CYP19A1 promoter region suggests that granulosa cells were not completely transformed into luteal cells under the present low oxygen in vitro condition. In addition, the comparison with earlier published in vivo microarray data indicated that 1107 genes showed a similar expression pattern in granulosa cells at low oxygen levels (in vitro) as found in preovulatory follicles after the LH surge (in vivo). Overall, our findings demonstrate for the first time that low oxygen levels in preovulatory follicles may play an important role in supporting early events of luteinization in granulosa cells.
Highlights
Ovaries are the female’s primary reproductive organs, which contain a large pool of primordial follicles
After subjecting granulosa cells (GC) to low and normal oxygen levels (Figure 1 and Supplementary Figure S1), the percentage of live, apoptotic and dead cells was determined using flow cytometric analysis by adding propidium iodide (PI) and annexin-V reagents to the detached cells. This revealed that GC did not show significant variation in healthy viable (PI−, Annexin−), apoptotic (PI−, Annexin+) and dead (PI+, Annexin+) cell counts at low oxygen levels compared to cells grown at normal oxygen levels (Figure 1C)
Unlike the viability status of the cells, levels of estradiol and progesterone were significantly reduced at low oxygen levels (Figure 1D)
Summary
Ovaries are the female’s primary reproductive organs, which contain a large pool of primordial follicles. Lower amounts of oxygen will diffuse through the basement membrane to reach the multiple layers of mural followed by antral GC and to the cumulus – oocyte complex (COC). This clearly suggests that antral GC along with the COC are exposed to relatively low oxygen levels in large antral follicles. Several other studies agree with these estimates and reported that the dissolved oxygen levels in follicular fluid is between 1 and 5% (Van Blerkom et al, 1997; Huey et al, 1999), which is far less than the atmospheric oxygen concentration (21%) that is generally used for culturing of follicular granulosa cells
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