FSH处理对猪颗粒细胞中类固醇合成酶基因的表达 及其调控区组蛋白H3修饰的影响

doi:10.3864/j.issn.0578-1752.2018.18.014

摘要/Abstract

摘要：

【目的】研究FSH处理对猪卵巢颗粒细胞类固醇合成酶、垂体激素受体、凋亡相关等基因表达的影响及此过程中组蛋白H3修饰的变化情况。【方法】首先,采集猪卵巢组织并用注射器抽取方法收集卵泡颗粒细胞,用含血清体系体外培养颗粒细胞至贴壁,血清饥饿16h后用终浓度5IU?mL^-1的FSH处理24h,并收集细胞。其次,提取细胞mRNA,采用qRT-PCR方法检测类固醇合成酶（STAR、CYP11A1、HSD3B和CYP19A1）、垂体激素受体（FSHR和LHR）、凋亡相关基因（XIAP和FasL）mRNA的表达变化,最后,相同处理后固定细胞,采用染色质免疫沉淀结合qPCR（ChIP-qPCR）方法检测类固醇合成酶基因STAR、CYP19A1和HSD3B上游转录调控区组蛋白H3修饰（H3K4me2、H3K4me3、H3K9ac和H3K14ac）状况。【结果】5IU?mL^-1的FSH处理引起类固醇合成酶基因STAR、CYP19A1和HSD3B分别为2倍（P<0.01）、2.8倍（P <0.01）和3.6倍（P<0.05）的显著上调,而CYP11A1表达水平没有显著变化;FSH处理对垂体激素受体FSHR、LHR和凋亡相关基因XIAP、FasL影响不显著。在上调的三个类固醇合成酶基因中,HSD3B调控区组蛋白H3修饰变化最为显著,H3K4me2、H3K4me3、H3K9ac和H3K14ac结合分别有14.7倍（P<0.01）、 13.6倍（P<0.01）、19.7（P<0.01）倍和2.5倍（P<0.05）的显著上调;STAR基因调控区的H3K9ac在处理后有11.1倍的显著下降（P<0.05）;CYP19A基因调控区的H3K4me3和H3K9ac分别有0.5倍的上调（P<0.01）和10.4倍（P<0.01）的下降,其余组蛋白修饰在处理前后没有显著变化。【结论】FSH处理24h对颗粒细胞类固醇合成酶基因转录有显著上调作用,对其转录过程有H3组蛋白修饰参与,组蛋白修饰模式具有基因特异性。垂体激素受体和凋亡相关基因的应答可能需要FSH和其他因素的联合作用。

关键词: 猪, FSH, 颗粒细胞, 类固醇合成酶, 组蛋白修饰

Abstract:

【Objective】The objective of this study was to explore whether FSH treatment affect expressions of genes including steroidogenic enzymes, pituitary hormone receptors and apoptosis related genes in porcine granulosa cells, and to detect the histone H3 modification on specific gene regulation regions involved in this process. 【Method】 Firstly, ovary granulosa cells were collected using syringe extraction method from porcine ovaries and cultured in media with serum until the cells attached. After 16 h of non-serum culture, granulosa cells were treated by 5 IU?mL^-1 FSH for another 24 h culture and harvested for following experiment. Secondly, transcriptional expression changes of steroidogenic enzymes (STAR, CYP11A1, HSD3B and CYP19A1), pituitary hormone receptors (FSHR and LHR) and apoptosis related genes (XIAP and FasL) were detected using qRT-PCR method. Finally, histone H3 modification (H3K4me2, H3K4me3, H3K9ac and H3K14ac) status on regulatory regions of STAR, CYP19A1 and HSD3B genes were detected by ChIP-qPCR.【Result】 Treatment of 5 IU?mL^-1 FSH induced a significant upregulation of STAR, CYP19A1 and HSD3B genes with fold changes of 2 (P<0.01), 2.8 (P<0.0), and 3.6 (P<0.05), respectively, but had no significant effect on CYP11A1, pituitary hormone receptors FSHR, LHR and apoptosis related genes XIAP, FasL. Among the three steroidogenic genes, the histone modifications of HSD3B regulatory region were the most significant. The fold change of H3K4me2, H3K4me3, H3K9ac and H3K14ac was 14.7 (P<0.01), 13.6 (P<0.01), 19.7 (P<0.01) and 2.5 (P<0.05), respectively. H3K9ac on STAR gene regulation region decreased 11.1 (P<0.01) times. H3K4me3 on CYP19A regulation region increased 0.5 (P<0.01) times while H3K9ac decreased 10.4 (P<0.01) times. Other histone modification changes were not significant. 【Conclusion】24 h of FSH treatment enhanced the transcription levels of steroidogenic enzymes in pig granulosa cells. The up-regulation process involved H3 histone modifications in a gene-specific manner. Independent FSH treatment was not capable to induce significant effect on candidate pituitary hormone receptor and apoptosis related genes.

Key words: pig, FSH, granulosa cells, steroidogenic enzymes, histone modification

张金璧, 姚望, 潘增祥, 刘红林. FSH处理对猪颗粒细胞中类固醇合成酶基因的表达及其调控区组蛋白H3修饰的影响[J]. 中国农业科学, 2018, 51(18): 3582-3590.

JinBi ZHANG, Wang YAO, ZengXiang PAN, HongLin LIU. Effects of FSH Treatment on Steroidogenic Enzymes Expression and Histone H3 Modification in Pig Granulosa Cells[J]. Scientia Agricultura Sinica, 2018, 51(18): 3582-3590.

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基因Gene	登陆号Acc. No.	引物(5'- 3')Primer (5'- 3')	产物长度Product length（bp）
STAR	XM_005671764.2	F: GCTGAGCCCTTTCGTGTCTA	140
STAR	XM_005671764.2	R: CATAGGACCTGCCGTGTCTG	140
CYP11A1	NM_214427.1	F: AGACACTGAGACTCCACCCCA	110
CYP11A1	NM_214427.1	R: GACGGCCACTTGTACCAATGT	110
HSD3B	NM_001004049.1	F: CCCAGTGTTTTCTGGTTCCT	131
HSD3B	NM_001004049.1	R: TTCTCCTCCAGCAACAAGTG	131
CYP19A	NM_214431.1	F: TGCTGGACACCTCTAACAA	264
CYP19A	NM_214431.1	R: TCAACTCAGTGGCGAAAT	264
FSHR	NM_214386.3	F: GAATTGAAAAGGCCAACAAC	214
FSHR	NM_214386.3	R: CTTTCAAAACTTAGTCCCACG	214
LHR	NM_214449.1	F: ACTCCAATGTGCTCCCGAAC	222
LHR	NM_214449.1	R: AGTAGCAGGTAGAGCCCCAT	222
XIAP	NM_001097436.1	F: ACTGGCCAGACTATGCTCAC	107
XIAP	NM_001097436.1	R: CAGTTTCCCGCCACAACAAA	107
FasL-2	AY033634	F: CCACCACTCCTGCCATCAA	135
FasL-2	AY033634	R: CAGCCCCAATCCAACCA	135
GAPDH	AF017079	F: GGACTCATGACCACGGTCCAT	220
GAPDH	AF017079	R: TCAGATCCACAACCGACACGT	220
STAR-ChIP	chr15:55501189 -55501271	F: GAGCAACATTCCTCCCTAGACT	83
STAR-ChIP	chr15:55501189 -55501271	R: CAGCCCCAATCCAACCA	83
HSD3B-ChIP	chr4:111565035 -111565175	F: GCCGGCTGTGACTATTTGGA	141
HSD3B-ChIP	chr4:111565035 -111565175	R: TGCTTCAAGGACTGTGTCCC	141
CYP19A-ChIP	chr1:133903003 -133903130	F: ACCCTTAACTCAAAAGAACCCA	128
CYP19A-ChIP	chr1:133903003 -133903130	R: AATTTTGCAGTGGCGGGATT	128