OPN5对鸭颗粒细胞凋亡、增殖及类固醇激素生成的影响

doi:10.11843/j.issn.0366-6964.2021.012.015

摘要/Abstract

摘要： 旨在研究视神经蛋白（neuropsin，OPN5）对鸭颗粒细胞凋亡、增殖及类固醇激素生成的影响。本研究分别对鸭颗粒细胞进行OPN5过表达质粒和OPN5 siRNA处理72 h （n=6），应用EdU细胞增殖检测技术、流式细胞技术、Annexin V-FITC、RT-PCR、Western blot及ELISA技术系统地检测颗粒细胞的增殖、凋亡情况及生殖相关基因的mRNA、蛋白水平及激素水平的变化规律。结果显示，OPN5过表达能促进鸭卵泡颗粒细胞增殖，抑制鸭卵泡颗粒细胞凋亡；能极显著地促进GnRHR、FSHR、LHR的表达，并抑制GnIH、GnIHR的表达（P<0.01）；能显著或极显著上调StAR、CYP11A1、3β-HSD、CYP17A1、CYP19A1的mRNA水平（P<0.05或P<0.01）；显著升高OPN5、3β-HSD及CYP19A1的蛋白表达水平（P<0.05）和极显著升高E2、P4分泌水平（P<0.01），并极显著降低INHβ的水平（P<0.01）。OPN5 siRNA能够显著降低OPN5的表达水平（P<0.01），抑制卵泡颗粒细胞增殖并促进凋亡，极显著下调GnRH、GnRHR、FSHR、LHR（P<0.01），促进GnIH、GnIHR的表达（P<0.01）；并极显著抑制StAR、CYP11A1、CYP17A1的表达（P<0.01）；显著降低OPN5、3β-HSD及CYP19A1蛋白表达水平（P<0.05）及极显著降低E2、P4的分泌水平（P<0.01），并能极显著升高INHβ的水平（P<0.01）。研究表明，OPN5能促进鸭卵泡颗粒细胞的增殖，抑制其凋亡，促进颗粒细胞中类固醇激素的生成和分泌。

关键词: OPN5, 鸭, 颗粒细胞, 凋亡, 增殖, 类固醇激素

Abstract: This study aimed to characterize the effects of neuropsin (OPN5) on apoptosis, prolife-ration and steroid hormone production of granulosa cells of duck. The duck granulosa cells were isolated and transfected with OPN5 overexpression plasmid and OPN5 siRNA, respectively. After continuous culture for 72 h (n=6), both proliferation and apoptosis of granulosa cell were detected using EdU cell proliferation assay, flow cytometry, Annexin V-FITC. The mRNA and protein expression levels, as well as hormone secretion were investigated by RT-PCR, Western blot and ELISA, respectively. The results showed that overexpression of OPN5 promoted prolife-ration and inhibited apoptosis of follicular granulosa cells in ducks. Moreover, overexpression of OPN5 significantly promoted the expression of GnRHR, FSHR, LHR, inhibited the expression of GnIH and GnIHR (P<0.01), significantly or highly significantly upregulated the mRNA levels of StAR, CYP11A1, 3β-HSD, CYP17A1 and CYP19A1 (P<0.05 or P<0.01), significantly increased the protein expression levels of OPN5, 3β-HSD and CYP19A1 (P<0.05), highly significantly increased the secretion levels of E2 and P4 (P<0.01). However, the levels of INHβ was highly significantly decreased (P<0.01). OPN5 knockdown with siRNA could effectively downregulate OPN5 expression in transfected cells (P<0.01), inhibit proliferation and promote apoptosis of follicular granulosa cells. siRNA transfection significantly downregulated the expression levels of GnRH, GnRHR, FSHR, LHR (P<0.01), promoted the expression of GnIH and GnIHR (P<0.01), significantly inhibited the expression of StAR, CYP11A1, CYP17A1 (P<0.01). OPN5 knockdown significantly decreased the protein expression levels of OPN5, 3β-HSD and CYP19A1 (P<0.05), highly significantly decreased the secretion levels of E2 and P4 (P<0.01), and highly significantly increased the level of INHβ (P<0.01). In conclusion, OPN5 promotes the proliferation and inhibits the apoptosis of duck follicular granulosa cells, and promotes the production and secretion of steroid hormones in granulosa cells.

Key words: OPN5, duck, granulosa cell, apoptosis, proliferation, steroid hormone

中图分类号:

S834.83

刘付穗, 潘建秋, 江丹莉, 沈栩, 许丹宁, 田允波, 黄运茂. OPN5对鸭颗粒细胞凋亡、增殖及类固醇激素生成的影响[J]. 畜牧兽医学报, 2021, 52(12): 3480-3491.

LIUFU Sui, PAN Jianqiu, JIANG Danli, SHEN Xu, XU Danning, TIAN Yunbo, HUANG Yunmao. Effects of OPN5 on Apoptosis, Proliferation and Steroid Hormone Synthesis of Duck Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(12): 3480-3491.

参考文献 35

[1]	TARTTELIN E E, BELLINGHAM J, HANKINS M W, et al.Neuropsin (Opn5):a novel opsin identified in mammalian neural tissue[J].FEBS Lett, 2003, 554(3):410-416.
[2]	NAKANE Y, YOSHIMURA T.Photoperiodic regulation of reproduction in vertebrates[J].Annu Rev Anim Biosci, 2019, 7:173-194.
[3]	KOJIMA D, MORI S, TORII M, et al.UV-sensitive photoreceptor protein OPN5 in humans and mice[J].PLoS One, 2011, 6(10):e26388.
[4]	STEVENSON T J, BALL G F.Disruption of neuropsin mRNA expression via RNA interference facilitates the photoinduced increase in thyrotropin-stimulating subunit β in birds[J].Eur J Neurosci, 2012, 36(6):2859-2865.
[5]	张磊, 康波, 姜冬梅, 等.神经视蛋白的结构及其生物学功能研究进展[J].西北农林科技大学学报:自然科学版, 2015, 43(8):46-50.ZHANG L, KANG B, JIANG D M, et al.Progress in structure and biological functions of neuropsin[J].Journal of Northwest A & F University:Natural Science Edition, 2015, 43(8):46-50.(in Chinese)
[6]	KANG S W, KUENZEL W J.Deep-brain photoreceptors (DBPs) involved in the photoperiodic gonadal response in an avian species, Gallus gallus[J].Gen Comp Endocrinol, 2015, 211:106-113.
[7]	BEAUDRY F E G, IWANICKI T W, MARILUZ B R Z, et al.The non-visual opsins:eighteen in the ancestor of vertebrates, astonishing increase in ray-finned fish, and loss in amniotes[J].J Exp Zoo B Mol Dev Evol, 2017, 328(7):685-696.
[8]	ZHU H X, LIU X Q, HU M D, et al.Endocrine and molecular regulation mechanisms of the reproductive system of Hungarian White geese investigated under two artificial photoperiodic programs[J].Theriogenology, 2019, 123:167-176.
[9]	ZHU H X, HU M D, GUO B B, et al.Effect and molecular regulatory mechanism of monochromatic light colors on the egg-laying performance of Yangzhou geese[J].Anim Reprod Sci, 2019, 204:131-139.
[10]	ZHU G Q, FANG C, LI J, et al.Transcriptomic diversification of granulosa cells during follicular development in chicken[J].Sci Rep, 2019, 9(1):5462.
[11]	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)
[12]	JIN L, REN L, LU J, et al.CXCL12 and its receptors regulate granulosa cell apoptosis in PCOS rats and human KGN tumor cells[J].Reproduction, 2021, 161(2):145-157.
[13]	KUROWSKA P, MLYCZYŃ SKA E, DAWID M, et al.In vitro effects of Vaspin on porcine granulosa cell proliferation, cell cycle progression, and apoptosis by activation of GRP78 receptor and several kinase signaling pathways including MAP3/1, AKT, and STAT3[J].Int J Mol Sci, 2019, 20(22):5816.
[14]	CHEN S X, WANG F G, LIU Z, et al.Brain-derived neurotrophic factor promotes proliferation and progesterone synthesis in bovine granulosa cells[J].J Cell Physiol, 2019, 234(6):8776-8787.
[15]	陈世健, 刘文俊, 杨晨, 等.不同浓度GnIH对鸭颗粒细胞周期和增殖的影响[J].畜牧兽医学报, 2021, 52(1):116-125.CHEN S J, LIU W J, YANG C, et al.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.(in Chinese)
[16]	ZENG C, SHANG J, JIN A M, et al.The effect of luteal GnRH antagonist on moderate and severe early ovarian hyperstimulation syndrome during in vitro fertilization treatment:a prospective cohort study[J].Arch Gynecol Obstet, 2019, 300(1):223-233.
[17]	MISHRA I, AGARWAL N, RANI S, et al.Scotostimulation of reproductive neural pathways and gonadal maturation are not correlated with hypothalamic expression of deiodinases in subtropical spotted munia[J].J Neuroendocrinol, 2018, 30(9):e12627.
[18]	NAKANE Y, YOSHIMURA T.Universality and diversity in the signal transduction pathway that regulates seasonal reproduction in vertebrates[J].Front Neurosci, 2014, 8:115.
[19]	RZASA J, SECHMAN A, PACZOSKA-ELIASIEWICZ H, et al.Effect of tamoxifen on sex steroid concentrations in chicken ovarian follicles[J].Acta Vet Hung, 2009, 57(1):85-97.
[20]	LI Y, WANG H X, ZHOU D X, et al.Up-Regulation of long noncoding RNA SRA promotes cell growth, inhibits Cell apoptosis, and induces secretion of estradiol and progesterone in ovarian granular cells of mice[J].Med Sci Monit, 2018, 24:2384-2390.
[21]	LI L, WU J, LUO M, et al.The effect of heat stress on gene expression, synthesis of steroids, and apoptosis in bovine granulosa cells[J]. Cell Stress Chaperones, 2016, 21(3):467-475.
[22]	PETRO E M L, LEROY J L M R, VAN CRUCHTEN S J M, et al.Endocrine disruptors and female fertility:Focus on (bovine) ovarian follicular physiology[J].Theriogenology, 2012, 78(9):1887-1900.
[23]	BURGER H G, HEE J P C, MAMERS P, et al.Serum inhibin during lactation:relation to the gonadotrophins and gonadal steroids[J]. Clin Endocrinol, 1994, 41(6):771-777.
[24]	M'BAYE M, HUA G H, KHAN H A, et al.RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells[J].J Reprod Dev, 2015, 61(5):391-397.
[25]	TAN T Q, GE C T, MI Y L, et al.Ginsenosides promote proliferation of granulosa cells from chicken prehierarchical follicles through PKC activation and up-regulated cyclin gene expression[J].Cell Biol Int, 2010, 34(7):769-775.
[26]	夏露.胆固醇转运相关基因在鹅卵泡类固醇激素合成中的作用研究[D].雅安:四川农业大学, 2013.XIA L.Study on the role of cholesterol trafficking genes related to steroid hormones synthesis in goose follicles[D].Ya'an:Sichuan Agricultural University, 2013.(in Chinese)
[27]	FANG L L, LI Y R, WANG S J, et al.Melatonin induces progesterone production in human granulosa-lutein cells through upregulation of StAR expression[J].Aging (Albany NY), 2019, 11(20):9013-9024.
[28]	CHAKRAVARTHI V P, RATRI A, MASUMI S, et al.Granulosa cell genes that regulate ovarian follicle development beyond the antral stage:the role of estrogen receptor β[J].Mol Cell Endocrinol, 2021, 528:111212.
[29]	FISHER C R, GRAVES K H, PARLOW A F, et al.Characterization of mice deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene[J].Proc Natl Acad Sci U S A, 1998, 95(12):6965-6970.
[30]	张美莉, 王剑平.家禽生殖激素的研究进展[J].畜牧兽医杂志, 1999, 18(1):11-14.ZHANG M L, WANG J P.Research progress of reproductive hormone in poultry[J].Journal of Animal Science and Veterinary Medicine, 1999, 18(1):11-14.(in Chinese)
[31]	王琬, 何永鑫, 陈君, 等.大鼠去势后肾上腺与前列腺中CYP17a1基因的表达[J].高等学校化学学报, 2008, 29(7):1349-1351.WANG W, HE Y X, CHEN J, et al.CYP17a1 gene expression in rat's adrenal and prostate after orchiectomy[J].Chemical Journal of Chinese Universities, 2008, 29(7):1349-1351.(in Chinese)
[32]	YAN H N, LI C C, ZOU C, et al.Perfluoroundecanoic acid inhibits Leydig cell development in pubertal male rats via inducing oxidative stress and autophagy[J].Toxicol Appl Pharmacol, 2021, 415:115440.
[33]	MADDINENI S, OCÓN-GROVE O M, KRZYSIK-WALKER S M, et al.Gonadotrophin-inhibitory hormone receptor expression in the chicken pituitary gland:potential influence of sexual maturation and ovarian steroids[J].J Neuroendocrinol, 2008, 20(9):1078-1088.
[34]	NAKANE Y, SHIMMURA T, ABE H, et al.Intrinsic photosensitivity of a deep brain photoreceptor[J].Curr Biol, 2014, 24(13):R596-R597.
[35]	杨博.不同光照时间对蛋鸭产蛋性能、免疫力及肠道微生物的影响[D].广州:仲恺农业工程学院, 2018.YANG B.Effects of different photo period on laying performance, immunity and intestinal microbes in duck[D].Guangzhou:Zhongkai University of Agriculture and Engineering, 2018.(in Chinese)