Hypoxia Promotes Progesterone Synthesis During Luteinization in Bovine Granulosa Cells

Abstract

To determine whether hypoxia has an effect on luteinization, we examined the influence of hypoxia on a model of bovine luteinizing and non-luteinizing granulosa cell culture. The granulosa cells were obtained from small antral follicles (≤ 6 mm in diameter). To induce luteinization, the cells were treated for 24 h with insulin (2 µg/ml), forskolin (10 µM) or insulin in combination with forskolin at 20% O2. After 24 h, progesterone (P4) production was higher in the treated cells, which we defined as luteinizing granulosa cells, than in non-treated cells, which we defined as non-luteinizing granulosa cells. P4 production by non-luteinizing granulosa cells was not affected by hypoxia (24 h at 10% and 5% O2), while P4 production by granulosa cells treated with insulin in combination with forskolin was significantly increased under hypoxia (24 h at 10% and 5% O2). Because hypoxia affected P4 production by the luteinizing granulosa cells but not by the non-luteinizing granulosa cells, hypoxia seems to promote P4 production during, rather than before, luteinization. In the cells treated with insulin in combination with forskolin, mRNA and protein expression of steroidogenic acute regulatory protein (StAR) and protein expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) increased under 10% O2, while mRNA and protein expressions of key protein and enzymes in P4 biosynthesis did not increase under 5% O2. The overall results suggest that hypoxia plays a role in progressing and completing the luteinization by enhancing P4 production through StAR as well as 3β-HSD expressions in the early time of establishing the corpus luteum.

HIF1 driven transcriptional activity regulates steroidogenesis and proliferation of bovine granulosa cells

“…However, this stimulatory effect of HIF1 on CYP19A1 expression and estradiol production was not seen under hypoxic conditions upon HIF1A knockdown possibly because of the attenuation of FSH and IGF1 signaling. HIF1 has been shown to be involved in the hypoxic synthesis of progesterone in GC 49 . Our results are in the scope of this finding as inhibition of HIF1 activity decreased the progesterone production at hypoxia by possibly inhibiting STAR and HSD3B expression.…”

Induction of altered gene expression profiles in cultured bovine granulosa cells at high cell density

“…The up-regulation of other hypoxia-related genes ( HBA and EGLN3 ), however, suggest that hypoxic conditions occur in GC cultures under high density conditions, presumably within the observed tight cell clusters described in Baufeld et al [3]. Studies by others revealed that the induction of hypoxic factors is necessary for the ongoing differentiation process in the follicle [35, 36]. However, it is still unclear whether hypoxic conditions, in particular those presumably caused by increasing cell density in the GC layer of dominant follicles, are in fact essential signals during the folliculo-luteal transition in vivo [37].…”

Endocrine targets of hypoxia-inducible factors

“…It has been shown that follicle-stimulating hormone (FSH) enhances HIF-1α transcriptional activity, which augments FSH-induced upregulation of luteinizing hormone receptor, inhibin-α and VEGF to promote follicular differentiation to a preovulatory phenotype (Alam et al 2004). HIF-1α mRNA or protein was absent in pre-antral and antral follicles and become evident in the granulosa cells of preovulatory follicles in monkey , porcine (Boonyaprakob et al 2005) (Fadhillah et al 2014), expression of HIF-1α was also detected. These observations indicate that expression of HIF-1α in granulosa cells is likely stimulated by luteinizing hormone (LH).…”

“…HIF-induced VEGF expression certainly is critical for this process. In addition, hypoxia also promotes progesterone production during luteinization (Fadhillah et al 2014). In corpus luteum, HIF-1α was highly expressed in the early corpus luteum and gradually declined as the corpus luteum aged (Boonyaprakob et al 2005.…”