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. 2016 Mar 15:9:15.
doi: 10.1186/s13048-016-0225-z.

Changes in gene expression of histone modification enzymes in rat granulosa cells undergoing luteinization during ovulation

Ryo Maekawa  1 , Lifa Lee  1 , Maki Okada  1 , Hiromi Asada  1 , Masahiro Shinagawa  1 , Isao Tamura  1 , Shun Sato  1 , Hiroshi Tamura  1 , Norihiro Sugino  2
Affiliations

Changes in gene expression of histone modification enzymes in rat granulosa cells undergoing luteinization during ovulation

Ryo Maekawa et al. J Ovarian Res. .

Abstract

Background: The ovulatory LH surge rapidly alters the expression of steroidogenesis-related genes such as steroidogenic acute regulatory protein (StAR) in granulosa cells (GCs) undergoing luteinization. We recently reported that histone modifications contribute to these changes. Histone modifications are regulated by a variety of histone modification enzymes. This study investigated the changes in gene expression of histone modification enzymes in rat GCs undergoing luteinization after the induction of ovulation. The extracellular regulated kinase (ERK)-1/2 is a mediator in the intracellular signaling pathway stimulated by the ovulatory LH surge and regulates the expression of a number of genes in GCs. We further investigated whether ERK-1/2 is involved in the regulation of the histone modification at the StAR promoter region in GCs undergoing luteinization.

Results: GCs were obtained from rats treated with equine chorionic gonadotropin (CG) before (0 h) and after human (h) CG injection. The expressions of 84 genes regulating histone modifications or DNA methylation were measured using a PCR array. Five genes (HDAC4, HDAC10, EZH2, SETDB2, and CIITA) were identified as histone acetylation- or histone methylation-related genes, and were significantly altered after hCG injection. None of the genes were related to DNA methylation. mRNA levels of EZH2, SETDB2, HDAC4, and HDAC10 decreased and CIITA mRNA levels increased 4 or 12 h after hCG injection. GCs isolated after eCG injection were incubated with hCG for 4 h to induce luteinization. StAR mRNA levels were significantly increased by hCG accompanied by the increase in H3K4me3 of the StAR promoter region. StAR mRNA expression was inhibited by the ERK inhibitor with the significant decrease of H3K4me3. These results suggest that hCG increases StAR gene expression through the ERK-1/2-mediated signaling which is also associated with histone modification of the promoter region.

Conclusions: Gene expressions of histone modification enzymes change in GCs undergoing luteinization after ovulation induction. This change may play important roles in regulating the expression of various genes during the early stage of luteinization, which may be critical for the subsequent corpus luteum formation.

Keywords: Granulosa cells; Histone modification; Luteinization; Ovulation.

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Figures

Fig. 1
Fig. 1
mRNA expression of EZH2, SETDB2, HDAC4, HDAC10, and CIITA in rat granulosa cells undergoing luteinization after ovulation induction. Granulosa cells were obtained from rats treated with eCG before (0 h), 4, and 12 h after hCG injection. EZH2, SETDB2, HDAC4, HDAC10, and CIITA were identified as genes related to histone acetylation or histone methylation, and validated with additional samples (n = 5 in each time point) by real-time RT-PCR. The value of mRNA of each gene was normalized to that of an internal control (GADPH). Data were expressed as a ratio of mRNA of each gene to GADPH. Each bar represents the mean +/− SEM of five animals. a; p < 0.01 vs. 0 h, b; p < 0.05 vs. 0 h
Fig. 2
Fig. 2
mRNA expression of NCOA7 and HP1BP3 in granulosa cells undergoing luteinization after ovulation induction. NCOA7 and HP1BP3 are related to histone modification or chromatin remodeling and act downstream of the ERK1/2 signal after ovulation induction. mRNA expressions of both genes were analyzed in the same model as described in Figure 1. The value of mRNA of each gene was normalized to that of the internal control (GADPH). Each bar represents the mean +/− SEM of five animals. a; p < 0.01 vs. 0 h
Fig. 3
Fig. 3
Recruitment of EZH2 to the promoter regions of StAR and cyp19a1. Granulosa cells were obtained from three rats treated with eCG before (0 h), 4, and 12 h after hCG injection. Binding activities of EZH2 to the promoter region were analyzed by a ChIP assay. Each bar shows the IP/INPUT ratio. Data are representatives of four independent experiments
Fig. 4
Fig. 4
Effects of the inhibitor of ERK-1/2 on H3K4me3 of the StAR promoter region and StAR mRNA expression in granulosa cells undergoing luteinization. Granulosa cells were isolated 48 h after eCG injection, and incubated with or without hCG for 4 h to induce luteinization (a, b). Granulosa cells were also incubated with or without the inhibitor of ERK (U0126, 10 M) for 2 h prior to hCG stimulation (c, d). StAR mRNA expression was measured by real-time RT-PCR. The value was normalized to an internal control (GADPH). Data were expressed as a percentage of the group without hCG. H3K4me3 of the StAR promoter region was analyzed by a ChIP assay. The IP/INPUT ratio was calculated and data were expressed as a percentage of the group without U0126. Each bar represents the mean +/− SEM of three independent experiments. *; p < 0.05 vs. hCG (−) or U0126 (−)
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