Effects of echinomycin on endothelin-2 expression and ovulation in immature rats primed with gonadotropins

Abstract

Echinomycin is a small-molecule inhibitor of hypoxia- inducible factor-1 DNA-binding activity, which plays a crucial role in ovarian ovulation in mammalians. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1α-mediated endothelin (ET)-2 expressions contributed to ovarian ovulation in response to human chorionic gonadotropin (hCG) during gonadotropin-induced superuvulation. By real-time RT-PCR analysis, ET-2 mRNA level was found to significantly decrease in the ovaries after echinomycin treatment, while HIF-1α mRNA and protein expression was not obviously changed. Further analysis also showed that these changes of ET-2 mRNA were consistent with HIF-1 activity in the ovaires, which is similar with HIF-1α and ET-2 expression in the granulosa cells with gonadotropin and echinomycin treatments. The results of HIF-1α and ET-2 expression in the granulosa cells transfected with cis-element oligodeoxynucleotide (dsODN) under gonadotropin treatment further indicated HIF-1α directly mediated the transcriptional activation of ET-2 during gonadotropin- induced superuvulation. Taken together, these results demonstrated that HIF-1α-mediated ET-2 transcriptional activation is one of the important mechanisms regulating gonadotropin-induced mammalian ovulatory precess in vivo.

Figure 1

Effects of enchinomycin on ovarian ovulation in rats. After PMSG priming for 48 h, immature rats were treated with hCG to induce ovulation. (A) In a set of enchinomycin dose dependent experiments, rats were injected with either vehicle (dimethylsulfoxide) or various dose of enchinomycin (0.01, 0.1, 1 or 10 mg/kg body weight) at 6 h after hCG. (B) In another treatment time dependent experiments, rats were given either vehicle or a fixed dose of enchinomycin (1 mg/kg body weight) at 3, 6, 9 or 10 h after hCG. The ovulated eggs were counted at 20 h after hCG, and their numbers were compared between the enchinomycin-treated or untreated groups. Each value represents the means ± SE. One-way analysis of variance (ANOVA) was used to analyze the data. Different superscripts denote significant values (P < 0.05) by Tukey's multiple-range test.

Figure 2

Effects of enchinomycin on ET-2 mRNA and HIF-1α mRNA levels in rat ovaries. After PMSG priming for 48 h, immature rats were treated with hCG to induce ovulation and ovaries were collected from each group at 11 h after hCG. (A) The relative mRNA levels of ET-2 by real-time RT-PCR analysis. (B) The relative mRNA levels of HIF-1α by real-time RT-PCR analysis. Each value represents the mean ± SE. Student's t test was used to evaluate statistical significance of differences between two groups. The asterisk denotes significant values (P < 0.05). Ech, enchinomycin.

Figure 3

Effects of enchinomycin on HIF-1α protein expression in rat ovaries. After PMSG priming for 48 h, immature rats were treated with hCG to induce ovulation and ovaries were collected from each group at 11 h after hCG. (A) Representative ECL gel documents of Western blot analyses depicting the protein level of HIF-1α. (B) Summarized intensities of HIF-1α blot normalized to control. Different superscripts and asterisk denote significant values (P < 0.05) by Student's t test. Ech, enchinomycin.

Figure 4

Effects of enchinomycin on HIF-1 binding activity in rat ovaries. After PMSG priming for 48 h, immature rats were treated with hCG to induce ovulation and ovaries were collected from each group at 11 h after hCG. HIF-1 binding assay in ovarian nuclear extracts from each experiment group, The asterisk denotes significant values (P < 0.05) by Student's t test. Ech, enchinomycin.

Figure 5

Effects of enchinomycin on ET-2 mRNA levels in ovarian granulosa cells. Animals were sacrificed after 48 h PMSG-treatment for collecting ovaries and granulosa cells were isolated for in vitro culture experiments with hCG and Ech. The relative mRNA levels of ET-2 by real-time RT-PCR analysis. Each value represents the mean ± SE. One-way analysis of variance (ANOVA) was used to analyze the data. The asterisk denotes significant values (P < 0.05) by Tukey's multiple-range test. n = 6 batches of cells. Ech, enchinomycin.

Figure 6

Effects of HIF-1α decoy ODNs transfection on ET-2 mRNA, HIF-1α expression and HIF-1 binding activity in response to hCG in granulosa cells. Animals were sacrificed after 48 h PMSG-treatment for collecting ovaries and granulosa cells were isolated for in vitro culture experiments with hCG and dsODN. (A) Representative ECL gel documents of Western blot analyses depicting the protein level of HIF-1α. (B) Summarized intensities of HIF-1α blot normalized to control. (C) The relative mRNA levels of ET-2 by real-time RT-PCR analysis. (D) HIF-1 binding assay in the nuclear extracts from different granulosa cell treatment groups. Each value represents the mean ± SE. One-way analysis of variance (ANOVA) was used to analyze the data. The asterisk denotes significant values (P < 0.05) by Tukey's multiple-range test. n = 6 batches of cells.