Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (18): 3582-3590.doi: 10.3864/j.issn.0578-1752.2018.18.014

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Effects of FSH Treatment on Steroidogenic Enzymes Expression and Histone H3 Modification in Pig Granulosa Cells

JinBi ZHANG(), Wang YAO, ZengXiang PAN, HongLin LIU()   

  1. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095
  • Received:2017-04-25 Accepted:2018-07-06 Online:2018-09-16 Published:2018-09-16

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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

Table 1

Primer and PCR reaction conditions"

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
R: CATAGGACCTGCCGTGTCTG
CYP11A1 NM_214427.1 F: AGACACTGAGACTCCACCCCA 110
R: GACGGCCACTTGTACCAATGT
HSD3B NM_001004049.1 F: CCCAGTGTTTTCTGGTTCCT 131
R: TTCTCCTCCAGCAACAAGTG
CYP19A NM_214431.1 F: TGCTGGACACCTCTAACAA 264
R: TCAACTCAGTGGCGAAAT
FSHR NM_214386.3 F: GAATTGAAAAGGCCAACAAC 214
R: CTTTCAAAACTTAGTCCCACG
LHR NM_214449.1 F: ACTCCAATGTGCTCCCGAAC 222
R: AGTAGCAGGTAGAGCCCCAT
XIAP NM_001097436.1 F: ACTGGCCAGACTATGCTCAC 107
R: CAGTTTCCCGCCACAACAAA
FasL-2 AY033634 F: CCACCACTCCTGCCATCAA 135
R: CAGCCCCAATCCAACCA
GAPDH AF017079 F: GGACTCATGACCACGGTCCAT 220
R: TCAGATCCACAACCGACACGT
STAR-ChIP chr15:55501189
-55501271		 F: GAGCAACATTCCTCCCTAGACT 83
R: CAGCCCCAATCCAACCA
HSD3B-ChIP chr4:111565035
-111565175		 F: GCCGGCTGTGACTATTTGGA 141
R: TGCTTCAAGGACTGTGTCCC
CYP19A-ChIP chr1:133903003
-133903130		 F: ACCCTTAACTCAAAAGAACCCA 128
R: AATTTTGCAGTGGCGGGATT

Fig. 1

Steroidogenic enzymes expression in response to FSH treatment in granulosa cells *. P<0.05,**. P<0.01 The same as below"

Fig. 2

Pituitary hormone receptors expression in response to FSH treatment in granulosa cells"

Fig. 3

Apoptosis related genes expression in response to FSH treatment in granulosa cells"

Fig. 4

Histone modification on HSD3B gene regulatory region in response to FSH treatment in granulosa cells"

Fig. 5

Histone modification on STAR gene regulatory region in response to FSH treatment in granulosa cells"

Fig. 6

Histone modification on CYP19A gene regulatory region in response to FSH treatment in granulosa cells"

Fig. 7

Biosynthesis of steroid hormones Steroid hormones are in bold, steroidogenic enzymes are encircled"

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