SMAD7对山羊卵泡颗粒细胞增殖、凋亡的影响

doi:10.11843/j.issn.0366-6964.2022.08.013

摘要/Abstract

摘要： 旨在探究SMAD7对山羊卵泡颗粒细胞增殖和凋亡的影响。本试验收集3~4月龄大足黑山羊母羊的卵泡颗粒细胞，通过过表达或siRNA干扰、ELISA、qRT-PCR、Western blot及流式细胞术等技术与方法探究SMAD7对颗粒细胞增殖、凋亡及类固醇激素分泌的影响。结果发现，SMAD7过表达显著下调颗粒细胞增殖活力并促进细胞凋亡，抑制PCNA表达（P<0.05），下调BCL2/BAX的比值（P<0.01）；同时，SMAD7干扰显著上调颗粒细胞增殖活力，显著上调PCNA表达（P<0.05）与BCL2/BAX表达量比值（P<0.05）。SMAD7过表达极显著上调颗粒细胞的孕酮分泌，下调雌二醇表达水平（P<0.01）；同时SMAD7干扰极显著下调孕酮分泌，上调雌二醇分泌（P<0.01）。进一步研究发现，SMAD7过表达显著抑制SMAD2、SMAD3的mRNA和蛋白表达（P<0.05）；SMAD7干扰则显著促进SMAD2、SMAD3的mRNA和蛋白表达（P<0.05）。结果表明，SMAD7抑制山羊卵泡颗粒细胞的增殖和雌二醇分泌，促进凋亡和孕酮的合成，并且抑制SMAD2、SMAD3的表达，进而调节卵泡的发育与闭锁。

关键词: SMAD7, 山羊, 颗粒细胞, 增殖, 凋亡

Abstract: The purpose of this study was to explore the effects of SMAD7 on the proliferation and apoptosis of follicular granulosa cells in goat. In this study, follicular granulosa cells were collected from 3-to 4-month-old Dazu black goats. The overexpression, siRNA interference, ELISA, qRT-PCR, Western blot and flow cytometry were used to investigate the effects of SMAD7 on proliferation, apoptosis and steroid secretion of granulosa cells. The results showed that overexpression of SMAD7 significantly reduced the proliferation activity and promoted the apoptosis of granulosa cells, inhibited the expression of PCNA (P<0.05) and decreased the ratio of BCL2/BAX(P<0.01). Oppositely, SMAD7 interference significantly promoted the proliferation activity of granulosa cells, significantly up-regulated the expression of PCNA and the ratio of BCL2/BAX (P<0.05).SMAD7 overexpression significantly increased progesterone secretion of granulated cells, and reduced estradiol expression levels (P<0.01). Meanwhile,SMAD7 interference significantly down-regulated the secretion of progesterone and up-regulated the secretion of estradiol (P<0.01). Further studies showed that SMAD7 overexpression significantly inhibited SMAD2,SMAD3 mRNA and protein expression (P<0.05), but did not affect SMAD4 expression. SMAD7 interference significantly promoted the mRNA and protein levels of SMAD2,SMAD3 (P<0.05). The results suggested that SMAD7 inhibits the proliferation and estradiol secretion, promotes apoptosis and progesterone synthesis, and inhibits the expression of SMAD2,SMAD3 in follicular granulosa cells, thereby regulating the development and atresia of follicles.

Key words: SMAD7, goat, granulosa cell, proliferation, apoptosis

中图分类号:

S827.3

李碧筠, 黄思艺, 王钰锟, 王磊, 何莉娜, 唐雪, 徐德军, 赵中权. SMAD7对山羊卵泡颗粒细胞增殖、凋亡的影响[J]. 畜牧兽医学报, 2022, 53(8): 2548-2557.

LI Bijun, HUANG Siyi, WANG Yukun, WANG Lei, HE Lina, TANG Xue, XU Dejun, ZHAO Zhongquan. Effects of SMAD7 on Proliferation and Apoptosis of Goat Follicular Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(8): 2548-2557.

参考文献 37

[1]	KIM I Y, KIM M M, KIM S J.Transforming growth factor-β:biology and clinical relevance[J].J Biochem Mol Biol, 2005, 38(1):1-8.
[2]	BATLLE E, MASSAGUÉ J.Transforming growth factor-β signaling in immunity and cancer[J].Immunity, 2019, 50(4):924-940.
[3]	CHEN W J, TEN DIJKE P.Immunoregulation by members of the TGF β superfamily[J].Nat Rev Immunol, 2016, 16(12):723-740.
[4]	AI K, ZHU X, KANG Y, et al.miR-130a-3p inhibition protects against renal fibrosis in vitro via the TGF-β1/Smad pathway by targeting SnoN[J].Exp Mol Pathol, 2020, 112:104358.
[5]	GWON M G, AN H J, KIM J Y, et al.Anti-fibrotic effects of synthetic TGF-β1 and Smad oligodeoxynucleotide on kidney fibrosis in vivo and in vitro through inhibition of both epithelial dedifferentiation and endothelial-mesenchymal transitions[J].FASEB J, 2020, 34(1):333-349.
[6]	LI R J, QI C L, FENG Q, et al.Alprostadil alleviates myocardial fibrosis in rats with diabetes mellitus through TGF-β1/Smad signaling pathway[J].Minerva Endocrinol, 2020, 45(3):270-273.
[7]	MENG G H, LAURIA A, MALDOTTI M, et al.Genome-wide analysis of smad7-mediated transcription in mouse embryonic stem cells[J].Int J Mol Sci, 2021, 22(24):13598.
[8]	XIAO H P, LI B D, YANG X M, et al.Interference of TGF-β1/Smad7 signal pathway affects myocardial fibrosis in hypertension[J].Pak J Pharm Sci, 2020, 33(6):2625-2631.
[9]	HE D, RUAN Z B, SONG G X, et al.miR-15a-5p regulates myocardial fibrosis in atrial fibrillation by targeting Smad7[J].Peer J, 2021, 9:e12686.
[10]	GAO Y, WEN H X, WANG C, et al.SMAD7 antagonizes key TGFβ superfamily signaling in mouse granulosa cells in vitro[J].Reproduction, 2013, 146(1):1-11.
[11]	孙玉英, 陈淑萍, 谈勇.滋阴补阳序贯法联合西药对卵巢储备功能下降排卵障碍性不孕大鼠TGF-β1/Smads信号通路的影响[J].中南大学学报:医学版, 2018, 43(10):1068-1074.SUN Y Y, CHEN S P, TAN Y.Influence of nourishing yin and tonifying yang sequential therapy combined with Western medicine on TGF-β1/SMADs signaling pathway in anovulatory infertility rats with diminished ovarian reserve[J].Journal of Central South University:Medical Science, 2018, 43(10):1068-1074.(in Chinese)
[12]	YU X Y, SUN Q, ZHANG Y M, et al.TGF-β/SMAD signaling pathway in tubulointerstitial fibrosis[J]. Front Pharmacol, 2022, 13:860588.
[13]	LI Q L, PANGAS S A, JORGEZ C J, et al.Redundant roles of SMAD2 and SMAD3 in ovarian granulosa cells in vivo[J].Mol Cell Biol, 2008, 28(23):7001-7011.
[14]	常迪, 董晓晨, 郭勇, 等.TGF-βRⅠ及SMAD2表达情况对绵羊腔前卵泡发育的影响[J].中国农学通报, 2016, 32(32):7-14.CHANG D, DONG X C, GUO Y, et al.Expression of TGF-βRⅠ and smad2 affecting the development of sheep preantral follicles[J].Chinese Agricultural Science Bulletin, 2016, 32(32):7-14.(in Chinese)
[15]	NOMURA M, SAKAMOTO R, MORINAGA H, et al.Activin stimulates CYP19A gene expression in human ovarian granulosa cell-like KGN cells via the SMAD2 signaling pathway[J].Biochem Biophys Res Commun, 2013, 436(3):443-448.
[16]	徐瑛蕾.SF-1和SMAD3相互作用促进小鼠颗粒细胞中Cyp19a1的表达[D].合肥:中国科学技术大学, 2011:69.XU Y L.The interaction between SF-1 and SMAD3 enhance Cyp19a1 expression in mouse ovariangranulosacells[D].Hefei:University of Science and Technology of China, 2011:69.(in Chinese)
[17]	RICHARDS J S, ASCOLI M.Endocrine, paracrine, andautocrine signaling pathways that regulate ovulation[J].Trends Endocrinol Metab, 2018, 29(5):313-325.
[18]	KISHI H, KITAHARA Y, IMAI F, et al.Expression of the gonadotropin receptors during follicular development[J].Reprod Med Biol, 2018, 17(1):11-19.
[19]	TESFAYE D, HAILAY T, SALILEW-WONDIM D, et al.Extracellular vesicle mediated molecular signaling in ovarian follicle:implicationfor oocyte developmental competence[J].Theriogenology, 2020, 150:70-74.
[20]	ZHANG J B, XU Y X, LIU H L, et al.MicroRNAs in ovarian follicular atresia and granulosa cell apoptosis[J].Reprod Biol Endocrinol, 2019, 17(1):9.
[21]	BRITT K L, DRUMMOND A E, COX V A, et al.An age-related ovarian phenotype in mice with targeted disruption of the Cyp 19(aromatase) gene[J].Endocrinology, 2000, 141(7):2614-2623.
[22]	SOLANO M E, ARCK P C.Steroids, pregnancy and fetal development[J].FrontImmunol, 2020, 10:3017.
[23]	NILSSON E E, SKINNER M K.Progesterone regulation of primordial follicle assembly in bovine fetal ovaries[J]. Mol Cell Endocrinol, 2009, 313(1-2):9-16.
[24]	USLU S, YÖRVK M, MIS L, et al.Localisation of estrogen receptor alpha and progesterone receptor b in goat ovaries during breeding and non-breeding season[J].Kafkas Univ Vet Fak Derg, 2016, 22(6):877-882.
[25]	ZHAO Z Q, GUO F Y, SUN X W, et al.BMP15 regulates AMH expression via the p38 MAPK pathway in granulosa cells from goat[J].Theriogenology, 2018, 118:72-79.
[26]	TANG J M, LI X, CHENG T L, et al.miR-21-5p/SMAD7 axis promotes the progress of lung cancer[J].Thorac Cancer, 2021, 12(17):2307-2313.
[27]	SOBRAL L M, ASEREDO F, AGOSTINI M, et al.Molecular events associated with ciclosporinA-induced gingival overgrowth are attenuated by Smad7 overexpression in fibroblasts[J].J Periodont Res, 2012, 47(2):149-158.
[28]	LALLEMAND F, MAZARS A, PRUNIER C, et al.Smad7 inhibits the survival nuclear factor κB and potentiates apoptosis in epithelial cells[J].Oncogene, 2001, 20(7):879-884.
[29]	YAO W, PAN Z X, DU X, et al.miR-181b-induced SMAD7 downregulation controls granulosa cell apoptosis through TGF-β signaling by interacting with the TGFBR1 promoter[J].J Cell Physiol, 2018, 233(9):6807-6821.
[30]	KACZOROWSKI M, BIECEK P, DONIZY P, et al.SMAD7 is a novel independent predictor of survival in patients with cutaneous melanoma[J].Transl Res, 2019, 204:72-81.
[31]	DI H S, WANG L G, WANG G L, et al.Thesignaling mechanism of TGF-β1induced bovine mammary epithelial cell apoptosis[J].Asian Australas J Anim Sci, 2012, 25(3):304-310.
[32]	NAN W X, LI G Y, SI H Z, et al.All-trans-retinoic acid inhibits mink hair follicle growth via inhibiting proliferation and inducing apoptosis of dermal papilla cells through TGF-β2/Smad2/3 pathway[J].Acta Histochem, 2020, 122(7):151603.
[33]	陈炜.脑源性神经营养因子对人黄素化卵巢颗粒细胞合成雌二醇和孕酮功能调控的研究[D].广州:第一军医大学, 2004:49.CHEN W.Effect and mechanism of brain-derived neurotrophinfactor on secretion of estradiol and progesterone of human granulose-luteal cells[D].Guangzhou:First Military Medical University, 2004:49.(in Chinese)
[34]	王亮.发情周期中青年母牛卵巢、雌二醇和孕酮的变化规律[D].扬州:扬州大学, 2009:52.WANG L.The changes of ovarian, Estradiol and progesterone concentration of the young cows in the estrus cycle[D]. Yangzhou:Yangzhou University, 2009:52.(in Chinese)
[35]	张金璧, 林飞, 潘增祥, 等.猪不同程度闭锁卵泡判定方法的比较研究[J].南京农业大学学报, 2013, 36(1):115-119.ZHANG J B, LIN F, PAN Z X, et al.Comparative study of methods to determine the follicular atresia extent in pigs[J].Journal of Nanjing Agricultural University, 2013, 36(1):115-119.(in Chinese)
[36]	NAGYOVA E, CAMAIONI A, SCSUKOVA S, et al.Activation of cumulus cell SMAD2/3 and epidermal growth factor receptor pathways are involved in porcine oocyte-cumulus cell expansion and steroidogenesis[J].Mol Reprod Dev, 2011, 78(6):391-402.
[37]	FANG L L, CHANG H M, CHENG J C, et al.Growth differentiation factor-8 decreases StARexpression through ALK5-mediated Smad3 and ERK1/2 signaling pathways in luteinized human granulosa cells[J].Endocrinology, 2015, 156(12):468.