Regulatory Effect of Hypoxia-Inducible Factor-1α on hCG-Stimulated Endothelin-2 Expression in Granulosa Cells from the PMSG-Treated Rat Ovary

Abstract

Abstract. Endothelin (ET)-2 plays a crucial role in ovarian ovulation in mammals. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1α-mediated transcriptional activation contributes to the increased expression of ET-2 gene in response to hCG in rat ovarian granulosa cells (GCs) during gonadotropin-induced superovulation. By realtime RT-PCR analysis, ET-2 mRNA expression was found to significantly increase in cultured ovarian GCs after treatment with hCG, or even N-carbobenzoxyl-L-leucinyl-L-leucinyl-L-norvalinal , while this increased ET-2 mRNA expression could also be blocked by ferrous ammonium sulfate (FAS) under human chorionic gonadotropin (hCG) treatment. Further analysis also found that these changes of ET-2 mRNA were consistent with HIF-1α expression or HIF-1 activity, and HIF-1α inhibitor echinomycin inhibited ovulation in rats. Taken together, these results indicate that ET-2 is transcriptionally activated by hCG through HIF-1α-mediated mechanism in GCs. This HIF-1α-induced transcriptional activation may be one of the important mechanisms mediating the increase of ET-2 expression in GCs during the gonadotropin-induced mammalian ovulatory process in vivo. Key words: Endothelin-2, Granulosa cells, Hypoxia-inducible factor-1α, Rat (J. Reprod. Dev. 58: [678][679][680][681][682][683][684] 2012) I n female rats, two pituitary gonadotropin hormones, FSH and LH, control reproductive cyclical events, including ovarian follicle maturation, rupture and release of fertilizable oocytes during a 4-day reproductive cycle in the initial stage of puberty. During each cycle, FSH stimulates a pool of preantral follicles to develop into mature follicles, while the ensuing surge of LH induces ovulation, through LH-activated downstream signaling pathways inthe preovulatory follicles and eventually triggers the release ofoocytes from these follicles [1,2]. At present, our understanding of the regulatory mechanisms controlling the growth and final differentiation of a mammalian follicle has advanced exponentially, but our understanding of even the most fundamental pathways is still not complete during these dynamic processes. Endothelin-2 (ET-2) was recently proposed as a granulosa cell-derived contractile signal that facilitates ovulation [3][4][5].Endothelin-2 (ET-2) is a small, 21 amino acid peptide that is produced by granulosa cells at the time of ovulation [3]. Expression of mRNA for ET-2, but not ET-1 or ET-3, is dramatically increased in the periovulatory follicle for only a very brief period of time (1-2 h) around ovulation, and blockade of endothelin receptor binding will delay/inhibit the process of follicular rupture [3]. This rather unique spatial-and temporal-specific pattern of mRNA expression suggests the involvement of direct signaling pathways to accurately coordinate controlled production of this peptide. Therefore, understanding of the regulatory mechanism controlling production of endothelin-2 (ET-2) becomes more and more important for further knowledge of the p...

HIF1 Activity in Granulosa Cells Is Required for FSH-Regulated Vegfa Expression and Follicle Survival in Mice1

“…In agreement with previous studies [23,25,26], we found that HIF1 is required for the regulation of Vegfa by LH in cultured granulosa cells. Unexpectedly however, granulosa cells isolated from Vegfa d/d ovaries 12 h post-hCG showed normal levels of Vegfa expression, indicating a normal response of the Vegfa gene to LH in vivo.…”

“…1B). Together, these results confirm the previously reported [21][22][23][24][25][26] requirement of HIF1 for gonadotropin-regulated Vegfa expression in granulosa cells and pinpoint a single HRE in the Vegfa promoter that is required for HIF1-mediated regulation of Vegfa transcription and gonadotropin responsiveness in granulosa cells in vitro.…”

“…Among the main transcriptional targets of HIF1 identified in granulosa cells to date is Vegfa. Indeed, HIF1 has been shown to be required for LH-regulated Vegfa expression in vitro and in vivo [23,25,26] as well as for FSH-regulated Vegfa expression in vitro [21,22]. The mechanisms whereby LH regulates HIF activity in this context have not been reported, but regulation of HIF1 by FSH would appear to require both PI3K/AKT/mTOR and ERK signaling [22].…”

Expression of hypoxia-inducible factor-1α during ovarian follicular growth and development in Sprague-Dawley rats

“…3) Granulosa cell culture experiment (Zhang et al, 2012a): granulosa cells were isolated from immature rat ovaries and cultured with HIF-1α small interfering RNA (siRNA) and scrambled siRNA transfection.…”

“…When cell fusion was approximately 85%, siRNA transfection was performed using the siLentFect Lipid Reagent (Bio-Rad) according to manufacturer instructions (Zhang et al, 2012a). For a 10-cm dish, 200 pmol siRNA was used.…”