不同浓度GnIH对鸭颗粒细胞周期和增殖的影响

doi:10.11843/j.issn.0366-6964.2021.012

Abstract

Abstract: The study aimed to explore the effects of different concentrations of GnIH on cell cycle, proliferation and related gene expression of duck granulosa cells. Primary duck granulosa cells were cultured in vitro and treated with different concentrations of GnIH (0, 0.1, 1, 10 and 100 ng·mL^-1) for 24 h (n=3). The cell growth status was observed, and the cell cycle and proliferation were detected by flow cytometry and EdU method, and the expression of proliferation-related genes CDK6, CyclinD1, IGF-2, IGFBP-2, p27^kip1 were detected by qRT-PCR. The results showed that the growth status of the cells in the GnIH treatment group at each concentration was good, the morphology was normal, the cell outline was clear, and the number of dead cells were not significantly different between groups (P>0.05). In the 0.1 and 1 ng·mL^-1 GnIH treatment groups, the proportion of cells in the G2 phase increased significantly (P<0.05). With the increase of the concentration of GnIH, the percentage of EdU-positive cells were decreased. In the 0.1 and 1 ng·mL^-1 GnIH treatment groups, the relative expression of CDK6, CyclinD1, IGF-2, IGFBP-2, p27^kip1 genes were downregulated in granulosa cells. The relative expression of these genes were increased in the 10 and 100 ng·mL^-1 GnIH treatment groups. Studies have shown that in the duck granulosa cells cultured in vitro, GnIH could hinder the cell cycle in G2 phase, meanwhile the percentage of EdU-positive cells were decreased and the expre-ssion of proliferation-related genes were downregulated, which could inhibit the proliferation of granulosa cells to affect the reproductive performance of animals.

Key words: GnIH, granulosa cells, cell cycle, cell proliferation, duck

CLC Number:

S834.3

CHEN Shijian, LIU Wenjun, YANG Chen, JIANG Danli, OUYANG Hongjia, HUANG Yunmao, TIAN Yunbo. Effects of Different Concentrations of GnIH on Cell Cycle and Proliferation of Duck Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(1): 116-125.

References 42

[1]	TSUTSUI K,SAIGOH E,UKENA K,et al.A novel avian hypothalamic peptide inhibiting gonadotropin release[J].Biochem Biophys Res Commun,2000,275(2):661-667.
[2]	TSUTSUI K.A new key neurohormone controlling reproduction,gonadotropin-inhibitory hormone (GnIH):Biosynthesis,mode of action and functional significance[J].Prog Neurobiol,2009,88(1):76-88.
[3]	WANG X Y,LI X,HU C H.Distribution of gonadotropin-inhibitory hormone (GnIH) in male Luchuan piglets[J].Gene Expr Patterns,2018,28:42-53.
[4]	TSUTSUI K,UBUKA T.How to contribute to the progress of neuroendocrinology:Discovery of GnIH and progress of GnIH research[J].Front Endocrinol (Lausanne),2018,9:662.
[5]	SON Y L,UBUKA T,TSUTSUI K.Molecular mechanisms of gonadotropin-inhibitory hormone (GnIH) actions in target cells and regulation of GnIH expression[J].Front Endocrinol (Lausanne),2019,10:110.
[6]	JAIN A,JAIN T,KUMAR P,et al.Follicle-stimulating hormone-induced rescue of cumulus cell apoptosis and enhanced development ability of buffalo oocytes[J].Domest Anim Endocrinol,2016,55:74-82.
[7]	BRAMBILLASCA F,GUGLIELMO M C,COTICCHIO G,et al.The current challenges to efficient immature oocyte cryopreservation[J].J Assist Reprod Genet,2013,30(12):1531-1539.
[8]	李鹏飞,岳文斌,庞钰莹,等.FSH和胰岛素对绵羊卵巢卵泡颗粒细胞体外培养的影响[J].畜牧兽医学报,2013,44(9):1386-1391.LI P F,YUE W B,PANG Y Y,et al.Effects of FSH and insulin on sheep ovarian follicular granulosa cells in vitro culture[J].Acta Veterinaria et Zootechnica Sinica,2013,44(9):1386-1391.(in Chinese)
[9]	UBUKA T,SON Y L,BENTLEY G E,et al.Gonadotropin-inhibitory hormone (GnIH),GnIH receptor and cell signaling[J].Gen Comp Endocrinol,2013,190:10-17.
[10]	SINGH P,KRISHNA A,TSUTSUI K.Effects of gonadotropin-inhibitory hormone on folliculogenesis and steroidogenesis of cyclic mice[J].Fertil Steril,2011,95(4):1397-1404.
[11]	MADDINENI S R,OCÓN-GROVE O M,KRZYSIK-WALKER S M,et al.Gonadotropin-inhibitory hormone (GnIH) receptor gene is expressed in the chicken ovary:potential role of GnIH in follicular maturation[J].Reproduction,2008,135(2):267-274.
[12]	SINGH P,KRISHNA A,SRIDARAN R,et al.Immunohistochemical localization of GnRH and RFamide-related peptide-3 in the ovaries of mice during the estrous cycle[J].J Mol Histol,2011,42(5):371-381.
[13]	董霞.鹅等级前卵泡发育研究及GnIH在等级前卵泡中的表达分析[D].雅安:四川农业大学,2013.DONG X.The development study of geese pre-hierarchical follicles and the analysis of GnlH expression in pre-hierarchical follicles[D].Ya'an:Sichuan Agricultural University,2013.(in Chinese)
[14]	LI X,SU J,FANG R,et al.The effects of RFRP-3,the mammalian ortholog of GnIH,on the female pig reproductive axis in vitro[J].Mol Cell Endocrinol,2013,372(1-2):65-72.
[15]	甘超.鹅GnIH及其受体基因的克隆、组织表达以及在颗粒细胞中的研究[D].雅安:四川农业大学,2014.GAN C.Cloning of goose GnIH and its receptor,the gene expression and research in granulosa cells[D].Ya'an:Sichuan Agricultural University,2014.(in Chinese)
[16]	BENTLEY G E,TSUTSUI K,KRIEGSFELD L J.Recent studies of gonadotropin-inhibitory hormone (GnIH) in the mammalian hypothalamus,pituitary and gonads[J].Brain Res,2010,1364:62-71.
[17]	ULLAH R,SHEN Y,ZHOU Y D,et al.Expression and actions of GnIH and its orthologs in vertebrates:Current status and advanced knowledge[J].Neuropeptides,2016,59:9-20.
[18]	TSUTSUI K,UBUKA T,SON Y L,et al.Contribution of GnIH research to the progress of reproductive neuroendocrinology[J]. Front Endocrinol (Lausanne),2015,6:179.
[19]	ANJUM S,KRISHNA A,TSUTSUI K.Possible role of GnIH as a mediator between adiposity and impaired testicular function[J].Front Endocrinol (Lausanne),2016,7:6.
[20]	RIZWAN M Z,POLING M C,CORR M,et al.RFamide-related peptide-3 receptor gene expression in GnRH and kisspeptin neurons and GnRH-dependent mechanism of action[J].Endocrinology,2012,153(8):3770-3779.
[21]	KHAN A R,KAUFFMAN A S.The role of kisspeptin and RFamide-related peptide-3 neurones in the circadian-timed preovulatory luteinising hormone surge[J].J Neuroendocrinol,2012,24(1):131-143.
[22]	UBUKA T,TSUTSUI K.Gonadotropin-inhibitory hormone inhibits aggressive behavior of male quail by increasing neuroestrogen synthesis in the brain beyond its optimum concentration[J].Gen Comp Endocrinol,2014,205:49-54.
[23]	LI X,SU J,LEI Z H,et al.Gonadotropin-inhibitory hormone (GnIH) and its receptor in the female pig:cDNA cloning,expression in tissues and expression pattern in the reproductive axis during the estrous cycle[J].Peptides,2012,36(2):176-185.
[24]	BENTLEY G E,WILSTERMAN K,ERNST D K,et al.Neural versus gonadal GnIH:Are they independent systems? A mini-review[J].Integr Comp Biol,2017,57(6):1194-1203.
[25]	TSUTSUI K,BENTLEY G E,BEDECARRATS G,et al.Gonadotropin-inhibitory hormone (GnIH) and its control of central and peripheral reproductive function[J].Front Neuroendocrinol,2010,31(3):284-295.
[26]	KING K L,CIDLOWSKI J A.Cell cycle regulation and apoptosis[J].Annu Rev Physiol,1998,60:601-617.
[27]	HOLLEY S L,HEIGHWAY J,HOBAN P R.Induced expression of human CCND1 alternative transcripts in mouse Cyl-1 knockout fibroblasts highlights functional differences[J].Int J Cancer,2005,114(3):364-370.
[28]	BACHS O,GALLASTEGUI E,ORLANDO S,et al.Role of p27Kip1 as a transcriptional regulator[J]. Oncotarget, 2018,9(40):26259-26278.
[29]	WANG X Y,LI X,HU C H.RFRP-3,the mammalian ortholog of GnIH,induces cell cycle arrest at G2/M in porcine ovarian granulosa cells[J].Peptides,2018,101:106-111.
[30]	李昀.GnIH对猪卵巢颗粒细胞增殖和MAPKs信号通路活化的影响[D].南宁:广西大学,2017.LI Y.Effects of GnIH on proliferation of porcine ovarian granulosa cells and activation of MAPKs signaling pathway[D]. Nanning:Guangxi University,2017.(in Chinese)
[31]	汪瑶,李珣,李敏婕,等.GnIH对母猪生殖调控的研究[J].中国农业科学,2014,47(18):3716-3724.WANG Y,LI X,LI M J,et al.Regulation of GnIH on reproduction of female pig[J].Scientia Agricultura Sinica, 2014,47(18):3716-3724.(in Chinese)
[32]	赵秀华.胰岛素样生长因子结合蛋白-2基因研究进展[J].中国家禽,2014,36(5):38-39.ZHAO X H.The research progress of IGFBP-2 gene[J].China Poultry,2014,36(5):38-39.(in Chinese)
[33]	果双双,李辉,施振旦,等.脂多糖对体外培养猪颗粒细胞增殖、凋亡和雌二醇分泌的影响[J].畜牧兽医学报, 2016, 47(4):693-699.GUO S S,LI H,SHI Z D,et al.Effects of lipopolysaccharide on proliferation,apoptosis and estradiol secretion of in vitro cultured porcine granulosa cells[J].Acta Veterinaria et Zootechnica Sinica,2016,47(4):693-699.(in Chinese)
[34]	BENTLEY G E,UBUKA T,MCGUIRE N L,et al.Gonadotropin-inhibitory hormone and its receptor in the avian reproductive system[J].Gen Comp Endocrinol,2008,156(1):34-43.
[35]	UBUKA T,UKENA K,SHARP P J,et al.Gonadotropin-inhibitory hormone inhibits gonadal development and maintenance by decreasing gonadotropin synthesis and release in male quail[J].Endocrinology,2006,147(3):1187-1194.
[36]	MCGUIRE N L,KANGAS K,BENTLEY G E.Effects of melatonin on peripheral reproductive function:Regulation of testicular GnIH and testosterone[J].Endocrinology,2011,152(9):3461-3470.
[37]	KUO Y M,SHIUE Y L,CHEN C F,et al.Proteomic analysis of hypothalamic proteins of high and low egg production strains of chickens[J].Theriogenology,2005,64(7):1490-1502.
[38]	张克山,胡彦竞科,韩笑哲,等.鹅不同繁殖时期GnRH和GnIH基因表达和激素浓度变化分析[J].畜牧兽医学报,2016,47(8):1720-1726.ZHANG K S,HU Y J K,HAN X Z,et al.Analysis of the serum concentrations and mRNA expression levels of GnRH and GnIH in geese during different reproductive periods[J].Acta Veterinaria et Zootechnica Sinica,2016,47(8):1720-1726.(in Chinese)
[39]	KADOKAWA H,SHIBATA M,TANAKA Y,et al.Bovine C-terminal octapeptide of RFamide-related peptide-3 suppresses luteinizing hormone (LH) secretion from the pituitary as well as pulsatile LH secretion in bovines[J].Domest Anim Endocrinol, 2009,36(4):219-224.
[40]	UBUKA T,SON Y L,TSUTSUI K.Molecular,cellular,morphological,physiological and behavioral aspects of gonadotropin-inhibitory hormone[J]. Gen Comp Endocrinol,2016,227:27-50.
[41]	ANJUM S,KRISHNA A,SRIDARAN R,et al.Localization of gonadotropin-releasing hormone (GnRH),gonadotropin-inhibitory hormone (GnIH),kisspeptin and GnRH receptor and their possible roles in testicular activities from birth to senescence in mice[J].J Exp Zool A Ecol Genet Physiol,2012,317(10):630-644.
[42]	ANJUM S,KRISHNA A,TSUTSUI K.Inhibitory roles of the mammalian GnIH ortholog RFRP3 in testicular activities in adult mice[J].J Endocrinol,2014,223(1):79-91.

Effects of Different Concentrations of GnIH on Cell Cycle and Proliferation of Duck Granulosa Cells

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 42

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	SHI Shengjie, WANG Liguang, GAO Lei, CAI Chuanjiang, HE Weixian, CHU Guiyan. Effect of miR-24-3p on Estradiol Synthesis in Porcine Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 169-178.
[2]	DUAN Xiangru, KANG Jia, YANG Ruochen, SHAN Xinyu, LI Taichun, ZHAO Wen, ZHANG Yingjie, LIU Yueqin. Effect of L-cysteine on Proliferation, Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 179-191.
[3]	LIN Yan, HUANG Min, LI Xiujin, ZHANG Xumeng, HUANG Yunmao, TIAN Yunbo, WU Zhongping. Uncovering Genome-wide Copy Number Variations in 8 Duck Breeds Using Whole Genome Resequencing Data [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3700-3709.
[4]	ZHANG Wanfeng, ZHAO Tianzhi, LI Jiao, YOU Ziwei, YANG Yang, CAI Chunbo, GAO Pengfei, CAO Guoqing, GUO Xiaohong, LI Bugao. Study on NR2F2 Gene Regulating Proliferation and Apoptosis of Porcine PK15 Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3242-3251.
[5]	HE Mingyang, MA Yujing, WANG Yong, YANG Ruochen, LIU Yueqin, ZHANG Yingjie, DUAN Chunhui. Effects of Melatonin on Proliferation, Apoptosis of Ovarian Granulosa Cells, and Its Secretion of Steroid Hormones of Sheep [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3313-3324.
[6]	WANG Wanjie, CHEN Nanzhu, ZOU Huiying, ZHOU Xinyi, HAO Haisheng, PANG Yunwei, ZHU Huabin, ZHAO Xueming, YU Dawei, DU Weihua. Effects of Histone Methyltransferase ASH1L Overexpression on Proliferation and Apoptosis of Bovine Cumulus Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3358-3368.
[7]	ZHANG Peng, WANG Mingxiu, JING Kemin, PENG Wei, TIAN Yuan, LI Yuqian, FU Changqi, SHU Shi, ZHONG Jincheng, CAI Xin. Abnormal Expression of FGFs/FGFRs and Their Mediated Signaling Pathway Genes Affect the Proliferative Activity of Undifferentiated Spermatogonia in Cattleyak [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(7): 2886-2897.
[8]	LIU Jie, XU Xiangping, DENG Ming, ZOU Xian, JIANG Shengwei, LIU Dewu, LIU Guangbin, SUN Baoli, GUO Yongqing, LI Yaokun. Effect of miR-144-5p Targeting WNT5a on the Proliferation and Apoptosis of Goat Ovarian Granulosa Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2421-2435.
[9]	ZENG Chengrong, WANG Na, BI Wenwen, MEI Shihui, HE Guangxia, ZHANG Junjie, CHEN Ze, WEN Ming, ZHOU Bijun. Metabonomics Analysis of Duck Ileum Infected by Clostridium perfringens Type A [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2555-2569.
[10]	XU Tiantian, ZHANG Tongtong, WANG Meng, WANG Xin. Transcription Factor Foxq1 Affects the Proliferation of Hair Follicle Stem Cells in Cashmere Goats via WNT/β-catenin Signaling Pathway [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2653-2661.
[11]	SUN Junfeng, PAN Jianqiu, ZHANG Zhuoshen, JIANG Danli, SHENG Xu, CHEN Rong, XU Danning, TIAN Yunbo, HUANG Yunmao. The Expression Patterns of OPN5-TSH-DIO2/DIO3 and VIP-PRL in the Process of Repressing the Testicular Function of Male Shanma Duck under Short-day Photoperiod [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2001-2012.
[12]	CAO Xiuyun, LIU Jiwen, TANG Zhihui, ZHENG Ziyi, YAN Liping, SONG Suquan. Isolation, Identification and Pathogenicity Analysis of a Duck Adenovirus Type 3 [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2050-2061.
[13]	DAN Yixin, YANG Lu, XIANG Hua, ZHANG Huanrong, REN Yupeng, YANG Falong, HE Honghong, ZHU Jiangjiang. Effects of BIRC5 on the Cycle and Apoptosis of Goat Testis Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1511-1524.
[14]	REN Xiaoli, FAN Yuying, HUANGFU Heping, LIU Yun, SHI Dongmei. Effect of GSK126 on Epithelial-mesenchymal Transition of Canine Mammary Tumor Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1721-1729.
[15]	YANG Guang, XU Jing, LI Xin, HU Debao, GUO Yiwen, DING Xiangbin, GUO Hong, ZHANG Linlin. Effect of Interfering lncbMD on Proliferation and Differentiation of Bovine Skeletal Muscle Satellite Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1015-1025.