Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (23): 4940-4949.doi: 10.3864/j.issn.0578-1752.2020.23.018

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

Effect of KISS1 Interference on the Function of Porcine Granulosa Cells in Porcine Ovary

LU SiYu1(),HE YingTing1,ZHOU XiaoFeng1,XIN XiaoPing1,ZHANG AiLing2,YUAN XiaoLong1,ZHANG Zhe1,LI JiaQi1()   

  1. 1Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding/National Engineering Research Center for Breeding Swine Industry/College of Animal Science, South China Agricultural University, Guangzhou 510642
    2Guangdong Development Center of Applied Ecology and Ecological Engineering in Universities/Biology and Food Engineering Institute,Guangdong University of Education, Guangzhou 510310
  • Received:2019-11-28 Accepted:2020-10-29 Online:2020-12-01 Published:2020-12-09
  • Contact: JiaQi LI E-mail:siyu_lu19@163.com;jqli@scau.edu.cn

RichHTML

16

PDF

486

Abstract:

【Background】 As the basic structural and functional unit of ovary, the main functions of follicles are ovulation and hormone secretion. Granulosa cells proliferation can promote the development of follicles, while the excessive apoptosis of granulosa cells will inhibit the development of follicles, induce follicular atresia, reduce the estrus frequency and affect the reproductive capacity of female animals. Recent studies have found that the KISS1 plays an important role in ovary. 【Objective】 This study was aimed to explore the effects of KISS1 on apoptosis, cell cycle and estrogen secretion of porcine granulosa cells, through interfering the expression of KISS1, so as to provide some useful information in molecular regulation mechanism of KISS1 on porcine granulosa cells. 【Method】 To investigate the effect of knockdown KISS1 on the transcription of key genes on phosphatidylinositol 3-kinase (PI3K) signaling pathway in porcine granulosa cells, KISS1-siRNA were designed and transfected to porcine granulosa cells in vitro, and then quantitative real time PCR (qRT PCR) was applied to detect the transcription of KISS1. Then, flow cytometry, Annexin V-FITC, and ELISA were utilized to explore the effects of KISS1-siRNA on granulosa cell cycle, apoptosis, and secretion of E2, respectively. Finally, qRT-PCR was applied to explore the effects of KISS1-siRNA on estrogen receptor and estrogen signaling pathway. 【Result】 In porcine granulosa cells, interfering KISS1 gene could significantly decrease the transcription of AKT1(P<0.05), while PIK3CG, PI3CI and PDK1 decreased with no significant difference (P>0.05). The mRNA levels of the genes, involving in activation of PI3K signaling pathway like FOXO3, TSC2, and BAD, were also decreased with no significant difference (P>0.05). Transfecting KISS1-siRNA to porcine granulosa cells could significantly promote cell apoptosis (P<0.01), arrest cell cycle at G0/G1 phase (P<0.01), and decrease the E2 secretion (P<0.01). After knockdown KISS1, the transcription of estrogen receptor ESR1 and ESR2, as well as Star, CYP17, 3B-HSD, 17B-HSD and CYP19A of estrogen pathway were also decreased significantly (P<0.05). 【Conclusion】 This study confirmed that KISS1 gene could participate in PI3K and estrogen signaling pathways. Interfering KISS1 could promote porcine granulosa cells apoptosis, arrest cell cycle at G0/G1 phase (P<0.01), and decrease the E2 secretion. It suggested that KISS1 played an important role in the division, growth and estrogen secretion of porcine granulosa cells.

Key words: sows, granulosa cells, KISS1, PI3K, E2

Table 1

Quantitative primer"

引物名称 Primer name Gene ID 引物序列 Primer sequence 产物大小 Size (bp)
qRT-KISS1 100145896 F: AACCAGCATCTTCTCACCAGG 192
R: CTTTCTCTCCGCACAACGC
qRT-PIK3CG 396979 F: AACGGGCTTTGAGATAGTGAA 184
R: AAGTTGCTTGGTTGGTGGATA
qRT-PIK3C1 5290 F: CAAGTGAGAATGGTCCGAATG 152
R: GTGGAAGAGTTTGCCTGTTTT
qRT-PDK1 100286871 F: AAATCACCAGGACAGCCAATA 190
R: CTTCTCGGTCACTCATCTTCAC
qRT-AKT1 100126861 F: CTGCACAAACGAGGCGAGT 89
R: CGCTCCTTGTAGCCGATGAA
qRT-FOXO3 733621 F: ACAAACGGCTCACTCTGTCCCA 85
R: GAACTGTTGCTGTCGCCCTTATC
qRT-TSC2 100505408 F: CGAGGTGGTGTCCTACGAGAT 115
R: GAGCAGGCGTTCAATGATGTT
qRT-BAD 100521065 F: AGTCGCCACTGCTCTTACCC 172
R: TCTTGAAGGAACCCTGGAAATC
qRT-ESR1 10013276 F: GATGCCTTGGTCTGGGTGAT 124
R: AGTGTTCCGTGCCCTTGTTA
qRT-ESR2 396697 F: AAGGGAAAAGGAGGATGGGACA 202
R: CAGATAGGGACTGCGTGGAGGT
qRT-Star 396597 F: GGAAAAGACACAGTCATCACCCAT 121
R: CAGCAAGCACACACACGGAAC
qRT-CYP17 100157047 F: AAGCCAAGACGAACGCAGAAAG 228
R: TAGATGGGGCACGATTGAAACC
qRT-3BHSD 445539 F: GGGGCTTCTGTCTTGATTCCA 284
R: GGTTTTCAGTGCTTCCTTGTGC
qRT-17BHSD 397574 F: CCCAACGCAGGAGACTCAAAAT 149
R: CCAGAGCCCATAACGAAGACAGA
qRT-CYP19A 403332 F: GCTGGACACCTCTAACAACCTCTT 91
R: TTGCCATTCATCAAAATAACCCT

Fig. 1

Interference effect of porcine KISS1 in granulosa cells * P<0.05,** P<0.01。下同 The same as below"

Fig. 2

Interfering KISS1 inhibits PI3K signaling pathway A. PI3K signaling pathway activates expression of related genes. B. PI3K signaling pathway inhibits the expression of related genes"

Fig. 3

Interfering KISS1 promotes granulosa cell apoptosis A. Flow cytometry of KISS1-siRNA transfection. B. Flow cytometry of Scrambled-siRNA transfection. C. Interfering KISS1 promotes granulosa cell apoptosis"

Fig. 4

Interfering KISS1 arrests granulosa cells in interphase"

Fig. 5

Interfering KISS1 inhibits granulosa cell secretion of E2"

Fig. 6

Gene transcription level of estrogen secretion related pathway A. Interfering KISS1 inhibits ESR1 and ESR2 expression; B. Interfering KISS1 inhibits the expression of estrogen pathway genes"

[1] VANDERHYDEN B C, ARMSTRONG D T . Role of cumulus cells and serum on the in vitro maturation, fertilization, and subsequent development of rat oocytes. Biology of Reproduction, 1989,40(4):720-728.
doi: 10.1095/biolreprod40.4.720 pmid: 2752073
[2] MAALOUF S W, LIU W S, PATE J L . MicroRNA in ovarian function. Cell and Tissue Research, 2016,363(1). DOI: 10.1007/s00441-015-2307-4.
doi: 10.1007/s00441-015-2257-x pmid: 26254045
[3] EPPIG J J . Oocyte control of ovarian follicular development and function in mammals. Reproduction, 2001,122(6):829-838.
doi: 10.1530/rep.0.1220829 pmid: 11732978
[4] UDA M, OTTOLENGHI C, CRISPONI L . Foxl2 disruption causes mouse ovarian failure by pervasive blockage of follicle development. Human Molecular Genetics, 2004,13(11):1171-1181.
doi: 10.1093/hmg/ddh124 pmid: 15056605
[5] WEST A, VOJTA P J, WELCH D R . Chromosome localization and genomic structure of the KiSS-1 metastasis suppressor gene(KISS1). Genomics, 1998,54(1):145-148.
doi: 10.1006/geno.1998.5566 pmid: 9806840
[6] LEE J H, MIELE M E, HICKS D J . KiSS-1, a novel human malignant melanoma metastasis-suppressor gene. Journal of the National Cancer Institute, 1996, 88(23):1731-1737.
doi: 10.1093/jnci/88.23.1731 pmid: 8944003
[7] HU K L, ZHAO H, CHANG H M . Kisspeptin/Kisspeptin receptor system in the ovary. Front Endocrinology (Lausanne), 2017,8:365.
[8] GAYTAN F, GAYTAN M, CASTELLANO J M . KiSS-1 in the mammalian ovary: distribution of kisspeptin in human and marmoset and alterations in KiSS-1 mRNA levels in a rat model of ovulatory dysfunction. American Journal of Physiology Endocrinology and Metabolism, 2009,296(3):E520-531.
doi: 10.1152/ajpendo.90895.2008 pmid: 19141682
[9] 郭美君 . 高泌乳素血症通过Kiss1/GPR54系统影响小鼠卵泡发育及排卵[D]. 南昌: 南昌大学, 2016.
GUO M J . Hyperprolactinemia affects the follicles development and ovulation by Kiss1/GPR54 system in mice[D]. Nanchang: Nanchang University. 2016. (in Chinese)
[10] 赖丽丹 . 高泌乳素血症通过Kiss1/GPR54系统对大鼠卵巢黄体细胞内分泌功能的影响及其机制[D]. 南昌: 南昌大学, 2016.
LAI L D . The effect and mechanism of hyperprolactinemia on endocrine function of rat luteal cell by Kiss1/GPR54 system[D]. Nanchang: Nanchang University, 2016. (in Chinese)
[11] 王笑, 王甄真, 陈雁 . PI3K/AKT信号通路在维持血糖平衡中的作用. 生命科学, 2013,25(2):133-139.
WANG X, WANG Z Z, CHEN Y . The functions of PI3K/AKT signaling pathway in glucose homeostasis. Chinese Bulletin of Life Sciences, 2013, 25(2):133-139. (in Chinese)
[12] CANTLEY L C . The phosphoinositide 3-kinase pathway. Science, 2002,296(5573):1655-1657.
doi: 10.1126/science.296.5573.1655 pmid: 12040186
[13] ENGELMAN J A, LUO J, CANTLEY L C . The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nature Reviews Genetics, 2006,7(8):606-619.
doi: 10.1038/nrg1879 pmid: 16847462
[14] JAGARLAMUDI K, LIU L, ADHIKARI D . Oocyte-specific deletion of Pten in mice reveals a stage-specific function of PTEN/PI3K signaling in oocytes in controlling follicular activation. PLoS ONE, 2009,4(7):e6186.
doi: 10.1371/journal.pone.0006186 pmid: 19587782
[15] REDDY P, LIU L, ADHIKARI D . Oocyte-specific deletion of Pten causes premature activation of the primordial follicle pool. Science, 2008,319(5863):611-613.
doi: 10.1126/science.1152257 pmid: 18239123
[16] FAN H Y, LIU Z, CAHILL N . Targeted disruption of Pten in ovarian granulosa cells enhances ovulation and extends the life span of luteal cells. Molecular Endocrinology, 2008,22(9):2128-2140.
doi: 10.1210/me.2008-0095 pmid: 18606860
[17] 蔡竞 . 基于PI3K/AKT信号通路研究新加归肾丸促卵泡发育的分子机制[D]. 成都: 成都中医药大学. 2013.
CAI J . Based on PI3K/AKT signaling pathway to study the molecular mechanism of Xinjiaguishen pill promoting follicular development[D]. Chengdu: Chengdu University of TCM. 2013. (in Chinese)
[18] XIN X, LI Z, ZHONG Y . KISS1 Suppresses apoptosis and stimulates the synthesis of E2 in porcine ovarian granulosa cells. Animals (Basel), 2019,9(2). DOI: 10. 3390/ani9020054.
[19] TOMIKAWA J, HOMMA T, TAJIMA S . Molecular characterization and estrogen regulation of hypothalamic KISS1 gene in the pig. Biology of Reproduction, 2010,82(2):313-319.
doi: 10.1095/biolreprod.109.079863 pmid: 19828777
[20] 臧猛, 晁利刚, 张志强, 台玉磊, 王静, 武宇晓, 杨国宇 . 猪KISS-1基因克隆、序列分析. 江西农业学报, 2008,20(11):5-7.
ZANG M, CHAO L G, ZHANG Z Q, TAI Y L, WANG J, WU Y X, YANG G Y . Molecular cloning and sequence analysis of porcine gene KISS-1. Acta Agriculturae Jiangxi, 2008,20(11):5-7. (in Chinese)
[21] OHTAKI T, SHINTANI Y, HONDA S . Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature, 2001,411(6837):613-617.
doi: 10.1038/35079135 pmid: 11385580
[22] IRWIG M S, FRALEY G S, SMITH J T . Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology, 2004,80(4):264-272.
doi: 10.1159/000083140 pmid: 15665556
[23] FUNES S, HEDRICK J A, VASSILEVA G . The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system. Biochemical and Biophysical Research Communications, 2003,312(4):1357-1363.
doi: 10.1016/j.bbrc.2003.11.066 pmid: 14652023
[24] PAPAOICONOMOU E, MSAOUEL P, MAKRI A . The role of kisspeptin/GPR54 in the reproductive system. In Vivo, 2011,25(3):343-354.
pmid: 21576407
[25] HUNTER M G . Oocyte maturation and ovum quality in pigs. Reviews of Reproduction, 2000,5(2):122-130.
doi: 10.1530/ror.0.0050122 pmid: 10864857
[26] MCGEE E A, HSUEH A J . Initial and cyclic recruitment of ovarian follicles. Endocrine Reviews, 2000,21(2):200-214.
doi: 10.1210/edrv.21.2.0394 pmid: 10782364
[27] SIMPSON E R, CLYNE C, RUBIN G . Aromatase-A brief overview. Annual Review of Physiology, 2002,64:93-127.
doi: 10.1146/annurev.physiol.64.081601.142703 pmid: 11826265
[28] 魏泽辉, 贾存灵 . 家禽卵泡选择过程中颗粒细胞的分子调控机制. 中国家禽, 2017,39(21):1-5.
WEI Z H, JIA C L . The molecular regulation mechanism of granulosa cells in the process of poultry follicle selection. Chinese Poultry, 2017,39(21):1-5. (in Chinese)
[29] MISHIMA T, TODA S, ANDO Y . Rapid proliferation of activated lymph node CD4(+) T cells is achieved by greatly curtailing the duration of gap phases in cell cycle progression. Cellular & Molecular Biology Letters, 2014,19(4):638-648.
doi: 10.2478/s11658-014-0219-z pmid: 25424911
[30] CHEN H, XU X, WANG G . CDK4 protein is degraded by anaphase-promoting complex/cyclosome in mitosis and reaccumulates in early G1 phase to initiate a new cell cycle in HeLa cells. Journal of Biological Chemistry, 2017,292(24):10131-10141.
doi: 10.1074/jbc.M116.773226
[31] CROSSEN P E, PATHAK S, ARRIGHI F E . A high resolution study of the DNA replication patterns of Chinese hamster chromosomes using sister chromatid differential staining technique. Chromosoma, 1975,52(4):339-347.
doi: 10.1007/BF00364018 pmid: 53130
[32] GOTTSCH M L, CUNNINGHAM M J, SMITH J T . A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. Endocrinology, 2004,145(9):4073-4077.
doi: 10.1210/en.2004-0431 pmid: 15217982
[33] FINDLAY J K, BRITT K, KERR J B . The road to ovulation: The role of oestrogens. Journal of Reproduction and Fertility, 2001,13(7/8):543-547.
[34] TORAN-ALLERAND C D . Minireview: A plethora of estrogen receptors in the brain: Where will it end? Endocrinology, 2004,145(3):1069-1074.
doi: 10.1210/en.2003-1462 pmid: 14670986
[35] BAKER L, MELDRUM K K, WANG M . The role of estrogen in cardiovascular disease. Journal of Surgical Research, 2003,115(2):325-344.
doi: 10.1016/s0022-4804(03)00215-4 pmid: 14697301
[36] 杜彦博 . 雌激素及其相关炎症因子对卵泡发育的影响[D]. 上海: 复旦大学, 2012.
DU Y B . The effects of estrogen and related inflammatory factors on the development of follicle[D]. Shanghai: Fudan University, 2012. (in Chinese).
[37] 屈超, 刘中洋, 袭荣刚, 王晓波 . 雌激素信号通路概述. 生物技术通讯, 2014,25(3):448-450.
QU C, LIU Z Y, XI R G, WANG X B . Estrogen signaling review. Letters in Biotechnology, 2014,25(3):448-450. (in Chinese)
[38] 岳占碰, 张学明, 马琳 . 雌激素对哺乳动物卵泡发育的影响研究进展. 中国畜牧杂志, 2007(1):41-43.
YUE Z P, ZHANG X M, MA L . The production of estrogens in mammalian follicle and its effect on follicular development. Chinese Journal of Animal Science, 2007(1):41-43. (in Chinese)
[39] 罗峰, 谢琪璇, 秋山泰身, 秦俊文 . 调节卵子发育成熟的信号转导通路. 生殖与避孕, 2011,31(7):495-501.
LUO F, XIE Q X, QIUSHAN T S, QIN J W . Signal transduction pathways that regulate egg development and maturation. Reproduction and Contraception, 2011,31(7):495-501. (in Chinese)
[40] 詹晓庆, 王鲜忠, 张家骅 . 雌激素受体和雌性生殖研究进展. 动物医学进展, 2005(12):35-39.
ZHAN X Q, WANG X Z, ZHANG J H . Progress on estrogen receptor and female reproduction. Progress in Veterinary Medicine, 2005(12):35-39. (in Chinese)
[41] 艾芳, 丘彦 . 雌激素受体亚型与多囊卵巢综合征. 国际生殖健康/计划生育杂志, 2009,28(3):190-193.
AI F, QIU Y . Estrogen receptor subtypes and polycystic ovarian syndrome. Journal of International Reproductive Health/Family Planning, 2009,28(3):190-193. (in Chinese)
[1] YUE XiaoYu, ZHAO ShiChen, WANG Qin. The miR-362-3p Regulates the Proliferation and Steroid Hormone Synthesis of Mare Follicular Granulosa Cells by Targeting BMPR2 [J]. Scientia Agricultura Sinica, 2026, 59(7): 1564-1575.
[2] CHEN YaRu, WANG Lei, FU Ming, HUANG Tao, ZHANG Hao, LIANG ZhenHua, PI JinSong, WU Yan. Molecular Mechanism of USP18 Facilitating Ferroptosis by Suppressing GPX4 Ubiquitination and Degradation in Duck Granulosa Cells [J]. Scientia Agricultura Sinica, 2026, 59(5): 1128-1140.
[3] WANG LinYuan, XUAN JingYan, DU Yu, CHEN Tong, NIU RuiYan. Co-Exposure of DON and PRRSV Induces Autophagy in PAM-KNU Cells Through the PI3K/AKT/mTOR Signaling Pathway [J]. Scientia Agricultura Sinica, 2025, 58(13): 2693-2706.
[4] XU YuanYuan, HUANG LiQing, LU XingRong, FENG Chao, SHANG JiangHua. Effects of β-mercaptoethanol on Rapamycin Induced Autophagy, Apoptosis and Steroid Hormone Synthesis in Buffalo Granulosa Cells [J]. Scientia Agricultura Sinica, 2025, 58(11): 2265-2274.
[5] ZHANG HuaPeng, ZHANG QingZe, HE Fan, QI MengFan, FU BinBin, LI QingChun, LI MengXun, MA LiPeng, LIU Yi, HUANG Tao. Cloning and Identification of Differentially Expressed lncRNAs in Follicles of Meishan Pigs and Duroc Pigs with Their Correlation Analysis with miRNAs [J]. Scientia Agricultura Sinica, 2024, 57(9): 1807-1819.
[6] MENG ZhaoYi, WANG YunLu, YAO YiLong, XI GuangYin, NIU JiaQiang, SOLANGSIZHU, GUO Min, XU YeFen. lncRNA-MSTRG.7889.1 Competitively Binds to bta-miR-146a Targeting Smad4 to Regulate Apoptosis of Yak Granulosa Cells [J]. Scientia Agricultura Sinica, 2024, 57(4): 797-809.
[7] GUO ZeYuan, DU ZhangSheng, ZHANG YaQi, CHEN ChunLu, MA XiaoYan, CHENG Ying, WANG Kai, LÜ LiHua. Effects of Smad7-Mediated TGF-β Signaling Pathway on Proliferation of Sheep Granulosa Cells [J]. Scientia Agricultura Sinica, 2023, 56(13): 2597-2608.
[8] LIU YuFang,CHEN YuLin,ZHOU ZuYang,CHU MingXing. miR-221-3p Regulates Ovarian Granulosa Cells Apoptosis by Targeting BCL2L11 in Small-Tail Han Sheep [J]. Scientia Agricultura Sinica, 2022, 55(9): 1868-1876.
[9] WANG JiaMin,SHI JiaChen,MA FangFang,CAI Yong,QIAO ZiLin. Effects of Soy Isoflavones on the Proliferation and Apoptosis of Yak Ovarian Granulosa Cells [J]. Scientia Agricultura Sinica, 2022, 55(8): 1667-1675.
[10] WU Yan,ZHANG Hao,LIANG ZhenHua,PAN AiLuan,SHEN Jie,PU YueJin,HUANG Tao,PI JinSong,DU JinPing. circ-13267 Regulates Egg Duck Granulosa Cells Apoptosis Through Let-7-19/ERBB4 Pathway [J]. Scientia Agricultura Sinica, 2022, 55(8): 1657-1666.
[11] LI LiYing,HE YingTing,ZHONG YuYi,ZHOU XiaoFeng,ZHANG Hao,YUAN XiaoLong,LI JiaQi,CHEN ZanMou. CTNNB1 Regulates the Function of Porcine Ovarian Granulosa Cells [J]. Scientia Agricultura Sinica, 2022, 55(15): 3050-3061.
[12] ZHANG Jing,ZHANG JiYue,YUE YongQi,ZHAO Dan,FAN YiLing,MA Yan,XIONG Yan,XIONG XianRong,ZI XiangDong,LI Jian,YANG LiXue. LKB1 Regulates Steroids Synthesis Related Genes Expression in Bovine Granulosa Cells [J]. Scientia Agricultura Sinica, 2022, 55(10): 2057-2066.
[13] SHU JingTing,JI GaiGe,SHAN YanJu,ZHANG Ming,JU XiaoJun,LIU YiFan,TU YunJie,SHENG ZhongWei,TANG YanFei,JIANG HuaLian,ZOU JianMin. Expression Analysis of IGF1-PI3K-Akt-Dependent Pathway Genes in Skeletal Muscle and Liver Tissue of Yellow Feather Broilers [J]. Scientia Agricultura Sinica, 2021, 54(9): 2027-2038.
[14] DU Xing,ZENG Qiang,LIU Lu,LI QiQi,YANG Liu,PAN ZengXiang,LI QiFa. Identification of the Core Promoter of Linc-NORFA and Its Transcriptional Regulation in Erhualian Pig [J]. Scientia Agricultura Sinica, 2021, 54(15): 3331-3342.
[15] Xin ZHANG,KongLin HUO,XingXing SONG,DuoNi ZHANG,Wen HU,ChuanHuo HU,Xun LI. Effects of GnIH on Autophagy and Apoptosis of Porcine Ovarian Granulosa Cells via p38MAPK Signaling Pathway [J]. Scientia Agricultura Sinica, 2020, 53(9): 1904-1912.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd