SMAD2、SMAD3和SMAD4 基因在小尾寒羊卵巢活动中的功能分析

doi:10.13304/j.nykjdb.2022.1118

摘要/Abstract

摘要：

为探究SMAD基因家族重要成员SMAD2、SMAD3和SMAD4基因在小尾寒羊繁殖活动中的生物学功能，利用生物信息学技术对SMAD2、SMAD3和SMAD4 基因的蛋白理化性质、亲疏水性和亚细胞定位进行分析，并对其二级结构、蛋白互作进行预测及GO（gene ontology）和KEGG（kyoto encyclopedia of genes and genomes）的富集分析；然后以小尾寒羊为研究对象，通过免疫组织化学染色（immunohistochemistry，IHC）确定SMAD2、SMAD3和SMAD4蛋白在卵巢组织中的表达，利用实时荧光定量PCR（real-time quantitative PCR，RT-qPCR）和蛋白免疫印迹（western blot，WB）方法测定SMAD2、SMAD3和SMAD4基因mRNA及其编码蛋白在不同生理阶段卵巢组织和不同直径卵泡中表达水平。结果显示，SMAD2、SMAD3和SMAD4蛋白为亲水性蛋白，理论等电点分别为6.67、6.73和6.50，在不同生理阶段卵巢组织中的阳性表达主要位于卵母细胞、卵泡膜细胞和颗粒细胞，且二级结构中α螺旋和无规则卷曲的占比较高，其蛋白与FOXH1、ACVR1B、SMAD1、TGFβR1、TGFβR2、ZFYVE9、SKI、SKIL、TGIF1和ENSOARP0000001869蛋白存在互作关系。SMAD2、SMAD3和SMAD4基因在TGF-β受体信号通路、TGF-β受体结合、TGF-β激活受体活性中发挥生物学功能，且在TGF-β信号通路富集。SMAD2和SMAD4蛋白在撤栓后42 h卵巢组织中的表达量显著高于撤栓后18 h，其mRNA表达趋势及其编码蛋白表达趋势总体一致；而SMAD3基因mRNA及其编码蛋白的表达量在不同生理阶段卵巢组织中差异不显著。SMAD3基因mRNA及其编码蛋白在中卵泡中的表达量显著高于大卵泡；SMAD2和SMAD4基因表达量在不同直径卵泡中差异不显著。综上所述，SMAD2、SMAD3和SMAD4 基因在小尾寒羊卵巢周期性活动中的生物学功能存在差异，SMAD2和SMAD4 基因参与小尾寒羊发情初期至排卵前的卵巢生理变化，但不参与卵泡发育；而SMAD3参与卵泡发育。以上研究结果为进一步探索SMAD基因家族在小尾寒羊卵巢活动中的分子机理提供理论参考。

关键词: 小尾寒羊, 卵巢, 卵泡, SMAD2, SMAD3, SMAD4

Abstract:

To investigate the biological functions of SMAD2， SMAD3 and SMAD4 genes in the reproductive activity of small-tailed cold sheep， which are important members of the SMAD gene family， the bioinformatics techniques were used to analyze the physicochemical properties， hydrophilicity and subcellular localisation of the SMAD2， SMAD3 and SMAD4 genes， as well as secondary structures， protein interaction prediction and enrichment analysis of gene ontology （GO） and kyoto encyclopedia of genes and genomes （KEGG）. The small-tailed sheep was as material， positive expression of SMAD2， SMAD3 and SMAD4 proteins in ovarian tissue was determined by immunohistochemistry staining （IHC）， and the real-time quantitative PCR （RT-qPCR） and western blot （WB） were used to determine the expression levels of SMAD2， SMAD3 and SMAD4 mRNAs and their encoded proteins in ovarian tissues and follicles of different diameters at different physiological stages. The results showed that SMAD2， SMAD3 and SMAD4 proteins were hydrophilic proteins with theoretical isoelectric points of 6.67， 6.73 and 6.50. The positive expressions in ovarian tissues at different physiological stages were mainly located in oocytes， follicular membrane cells and granulosa cells， and the highest proportion of α-helices and random nematic clusters in the secondary structure， which were associated with FOXH1， ACVR1B， SMAD1， TGFβR1， TGFβR2， ZFYVE9， SKI， SKIL， TGIF1 and ENSOARP0000001869 proteins. SMAD2， SMAD3 and SMAD4 genes played biological functions in the TGF-β receptor signalling pathway， TGF-β receptor binding， TGF-β activated receptor activity and were enriched in TGF-β signalling pathway. The expressions of SMAD2 and SMAD4 proteins were significantly higher in ovarian tissues at 42 h after bolus withdrawal than those at 18 h after bolus withdrawal， and their mRNA expression showed the consistent trends with proteins. However， the expressions of SMAD3 gene mRNA and its encoded protein were not significant in ovarian tissues between different physiological stages. The expressions of SMAD3 and its encoded protein were significantly higher in medium follicles than those in large follicles， while the expressions of SMAD2 and SMAD4 were no significant differences between different follicle diameters. In conclusion， SMAD2， SMAD3 and SMAD4 genes had different biological functions in ovarian cycling of small-tailed cold sheep， i.e. SMAD2 and SMAD4 genes were involved in ovarian physiological changes from early oestrus to preovulation in small-tailed cold sheep， but not in follicle development； SMAD3 was involved in follicle development. Above results provided a theoretical reference to further explore the molecular mechanism of the SMAD gene family in ovarian activity in small-tailed cold sheep.

Key words: small tail cold sheep, ovary, follicles, SMAD2, SMAD3, SMAD4

中图分类号:

S826

王龙斌, 李明娜, 罗玉柱, 王继卿, 郝志云, 沈继源, 甄慧敏, 张玉庭, 杨舒童. SMAD2、SMAD3和SMAD4 基因在小尾寒羊卵巢活动中的功能分析[J]. 中国农业科技导报, 2024, 26(7): 69-79.

Longbin WANG, Mingna LI, Yuzhu LUO, Jiqing WANG, Zhiyun HAO, Jiyuan SHEN, Huimin ZHEN, Yuting ZHANG, Shutong YANG. Functional Analysis of SMAD2， SMAD3 and SMAD4 Genes in Ovarian Activity in Lesser Caecilian Sheep[J]. Journal of Agricultural Science and Technology, 2024, 26(7): 69-79.

图/表 9

参考文献 37

1	国家畜禽遗传资源委员会.中国畜禽遗传资源志:羊志[M].北京:中国农业出版社,2011.
2	JUENGEL J L, FRENCH M C, QUIRKE L D, et al.. Reproduction differential expression of cart in ewes with differing ovulation rates [J]. Soc. Study Fert., 2017, 153(4):471-479.
3	SEKELSKY J J, NEWFELD S J, RAFTERY L A, et al.. Genetic characterization and cloning of mothers against dpp, a gene required for decapentaplegic function in Drosophila melanogaster [J/OL]. Genetics, 1995, 139(3): 1347 [2022-11-20]. .
4	严晓华,章隽宇,陈晔光.抑制性Smad蛋白对TGF-β超家族信号转导的调控及其生理意义[J].细胞生物学杂志,2009,31(2):4-13.
	YAN X H, ZHANG J Y, CHEN Y G. Regulation of TGF-β superfamily signaling by inhibitory Smad [J]. Chin. J. Cell Biol., 2009,31(2):4-13.
5	MULSANT P, LECERF F, FABRE S, et al.. Mutation in bone morp-hogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Merino ewes [J]. Proc. Natl. Acad. Sci. USA, 2001, 98:5104-5109.
6	ZHANG Y, MUSCI T, DERYNCK R. The tumor suppressor Smad4/DPC 4 as a central mediator of Smad function [J]. Current Biol. Cb, 1997, 7(4):270-276.
7	MING Z, LOPA M, CHU X D. The role of TGF-β/SMAD4 signaling in cancer [J]. Int. J. Biol. Sci., 2018, 14(2):111-123.
8	廖火城,刘勇,周彬,等.四逆汤对异丙肾上腺素诱导的大鼠心肌纤维化Smad2和Smad7的影响[J].中国中西医结合杂志,2012,32(7):934-938.
	LIAO H C, LIU Y, ZHOU B, et al.. Effects of Sin decoction on the expressions of Smad2 and Smad7 in isoproterenol induced myocardial fibrosis rats [J]. Chin. J. Integrative Med., 2012, 32(7):934-938.
9	LIU C, LI W, ZHANG X, et al.. The critical role of myostatin in differentiation of sheep myoblasts [J]. Bioch. Biophy. Res. Comm., 2012, 422(3):381-386.
10	张秉芬,周胜红,王哲.延龄草皂苷通过抑制TGF-β/Smad3与Wnt/β-catenin信号通路改善大鼠肺纤维化[J].山东大学学报(医学版),2022,60(8):23-29.
	ZHANG B F, ZHOU S H, WANG Z. Trillium saponins ameliorates pulmonary fibrosis in rats by inhibiting TGF-β/Smad3 and Wnt/β-catenin signaling pathways [J]. J. Shandong Univ. (Health Sci.), 2022, 60(8):23-29.
11	范李静,张苗,郑瑛红,等.miR-34a-5p靶向Smad4抑制子宫内膜纤维化的机制研究[J].现代妇产科进展,2022,31(9):678-682.
	FAN L J, ZHANG M, ZHENG Y H, et al.. Inhibitory effect of miR-34a-5p on fibrosis of endometrial by down-regulating Smad4 [J]. Prog. Obstet. Gynecol., 2022, 31(9):678-682.
12	王雪,张景萍,郭丽丽,等.TGF-β/SMAD信号通路在哺乳动物卵巢发育调控中的作用研究进展[J].中国畜牧杂志,2023,59(3):61-69.
	WANG X, ZHANG J P, GUO L L, et al.. Research progress on the regulation of TGF-‍β/Smad signaling pathway in mammalian ovarian development [J]. Chin. J. Anim. Sci., 2023, 59(3):61-69.
13	LIU Y, DU S Y, DING M, et al.. The BMP4-Smad signaling pathway regulates hyperandrogenism development in a female mouse model [J]. J. Biol. Chem., 2017, 292(28):11740-11750.
14	OSTERLUND C. TGFbeta receptor types Ⅰ and Ⅱ and the substrate proteins Smad 2 and 3 are present in human oocytes [J/OL]. Mol. Human Reprod., 2000, 6(6):498 [2022-11-20]. .
15	ZHANG K, RAJPUT S K, LEE K B, et al.. Evidence supporting a role for smad 2/3 in bovine early embryonic development: potential implications for embryotropic actions of follistatin [J/OL]. Biol. Reprod., 2015, 86:130278 [2022-11-20]. .
16	GUERIPEL X, BENAHMED M, GOUGEON A. Sequential gonadotropin treatment of immature mice leads to amplification of transforming growth factor β action, via upregulation of receptor-type1, Smad2 and 4, and downregulation of Smad6 [J]. Biol. Reprod., 2004, 70(3):640-648.
17	PANGAS S A, LI X, ROBERTSON E J, et al.. Prematureluteinization and cumulus cell defects in ovar-ian-specific Smad4 knockout mice [J]. Mol. Endocrinol., 2006, 20(6):1406-1422, .
18	FINDIAY J K, DRUMMOND A E, DYSON M, et al.. Produc-tion and actions of inhibin and activin during folliculogenesis in the rat [J]. Mol. Cell Endocr-inol., 2001, 180(1-2):139-144.
19	JING J, JIANG X, CHEN J, et al.. Notch signaling pathway promotes the development of ovine ovarian follicular granulosa cells [J]. Anim. Reprod. Sci., 2017, 181(1):69-78.
20	CHOU K C, SHEN H B. A new method for predicting the subcellular localization of eukaryotic proteins with both single and multiple sites: Euk-mPLoc 2.0 [J/OL]. PloS One, 2010, 5(4): e9931[2022-11-20]..
21	梁维炜,李明娜,罗玉柱,等.性成熟期辽宁绒山羊与子午岭黑山羊睾丸发育比较[J].畜牧兽医学报,2021,52(12):3461-3470.
	LIANG W W, LI M N, LUO Y Z, et al.. Comparation of the testes development between Liaoning cashmere goat and Ziwuling blackgoat at swxual matreity [J]. J. Anim. Husb. Vet. Med., 2021, 52(12):3461-3470.
22	汤继顺.利用转录组测序和蛋白质组学分析筛选绵羊多羔候选基因的研究[D].北京:中国农业科学院,2019.
	TANG J S. Study on screening polytocous candidate genes in sheep based on transcriptome sequencing and proteomics analysis [D]. Beijing: Chinese Academy of Agricultural Sciences, 2019.
23	盖玉强,杜宝霞,马龙,等.不同发情时期Smad4基因在绵羊卵巢中表达规律的研究[J].吉林畜牧兽医,2020,41(10):1-2, 4.
24	HE Z, DENG F, XIONG S, et al.. Expression and regulation of Smad2 by gonadotropins in the protogynous hermaphroditic ricefield eel (Monopterus albus) [J/OL]. Fish Physiol. Biochem., 2020, 4:9 [2022-11-20]. .
25	苗竹林,王自能,章韵,等.Smad4蛋白及mRNA在卵巢不同发育阶段的表达[J].中国病理生理杂志,2005,21(5):1009-1013.
	MIAO Z L, WANG Z N, ZHANG Y, et al.. Expression of Smad4 protein and mRNA in different developmental stages of the rat ovary [J]. Chin. J. Pathophysiol., 2005, 21(5):1009-1013.
26	刘利杰.山羊卵母细胞减数分裂机制的初步研究[D].杨凌:西北农林科技大学,2008.
	LIU L J. The study on the regulation of meiotic of goat oocyte [D]. Yangling: Northwest A & F University, 2008.
27	郑健.高低繁湖羊垂体差异表达lncRNA/mRNA筛选及候选基因SMAD2的相关研究[D].南京:南京农业大学,2019.
	ZHENG J. Pituitary transcriptomic study reveals the differential regulation of incRNAs/mRNAs related to prolificacy in sheep and study on the candidate gene SMAD2 [D]. Nanjing: Nanjing Agricultural University, 2019.
28	李碧筠,黄思艺,王钰锟,等. 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 B J, HUANG S Y, WANG Y K, et al.. Effect of SMAD7 on proliferation and apoptosis of goat follicular granulosa cells [J]. J. Anim. Husb. Vet. Med., 2022, 53(8):2548-2557.
29	NOMURA M, SAKAMOTO R, MORINAGAH, et al.. Activin stimulates CYP19A gene expression in human ovarian granulosa cell-like KGN cells via the SMAD2 signaling pathway [J]. Biochem. Biophys. Res. Comm., 2013, 436(3):443-448.
30	YU C, ZHANG Y L, FAN H Y. Selective Smad4 knockout in ovarian preovulatory follicles results in multiple defects in ovulation [J]. Mol. Endocrinol., 2013, 27(6):966-978.
31	张娟.Smad2、Smad3及Smad7蛋白在绝经过渡期大鼠卵巢中的表达及意义[D].张家口:河北北方学院,2017.
	ZHANG J. The expression and significance of Smad protein in the ovary of the rat during menopausal transition [D]. Zhangjiakou: Hebei North College, 2017.
32	袁丽娟,任君旭,姬宏宇,等.转化生长因子-β和Smad4在绝经过渡期大鼠卵巢颗粒细胞中的表达[J].解剖学报, 2018, 49(1):108-112.
	YUAN L J, REN J X, JI H Y, et al.. Expression of transforming growth factor-β and Smad4 in ovarian granulosa cells in menopausal transitional rat [J]. J. Anatomy, 2018, 49(1):108-112.
33	LI Y, SCHANG G, BOEHM U, et al.. SMAD3 regulates follicle-stimulating hormone synthesis by pituitary gonadotrope cells in vivo [J]. J. Biol. Chem., 2017, 292(6):2301-2314.
34	LI Y, JIN Y, LIU Y, et al.. SMAD3 regulates the diverse functions of rat granulosa cells relating to the FSHR/PKA signaling pathway [J/OL]. Reproduction, 2013, 146(2):0325 [2022-11-20]. .
35	GRY K S, KASPER A, ALEXANDRO C C, et al.. Expression of TGF-beta superfamily growth factors, their receptors, the associated SMADs and antagonists in five isolated size-matched populations of pre-antral follicles from normal human ovaries [J]. Mol. Human Reprod., 2014, 20(4):293-308.
36	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.
37	ZHOU J L, LIU J Y, PAN Z X, et al.. The let-7g microRNA promotes follicular granulosa cell apoptosis by targeting transforming growth factor-β type 1 receptor [J]. Mol. Cellular Endocrinol., 2015, 409(7):103-112.

基因 Gene	GenBank 登录号 GenBank ID	引物序列 Primer sequence（5’-3’）	产物长度 Product length/bp
SMAD2	XM_027960887.2	F：AGTTTTTGCCGAGTGCCTAA R：CTGCCTTCGGTATTCTGCTC	256
SMAD3	XM_042252073.1	F：TGGCTCAGTCTGTCAACCAG R：CATCTGGGTGAGGACCTTGT	188
SMAD4	NM_001267886.1	F：TACAGCACCCCAGCTCTCTT R：GCCCTGAAGCTATCTGCAAC	148
GAPDH	NM_001190390.1	F：CACAGTCAAGGCAGAGAACG R：CAGCCTTCTCCATGGTAGTG	152

基因 Gene	GenBank 登录号 GenBank ID	引物序列 Primer sequence（5’-3’）	产物长度 Product length/bp
SMAD2	XM_027960887.2	F：AGTTTTTGCCGAGTGCCTAA R：CTGCCTTCGGTATTCTGCTC	256
SMAD3	XM_042252073.1	F：TGGCTCAGTCTGTCAACCAG R：CATCTGGGTGAGGACCTTGT	188
SMAD4	NM_001267886.1	F：TACAGCACCCCAGCTCTCTT R：GCCCTGAAGCTATCTGCAAC	148
GAPDH	NM_001190390.1	F：CACAGTCAAGGCAGAGAACG R：CAGCCTTCTCCATGGTAGTG	152

KEGG编号 KEGG ID	KEGG描述 KEGG description	富集基因 Gene enrichment
oas04110	细胞周期 Cell cycle	SMAD2， SMAD4， SMAD3
oas04350	TGF-β信号通路 TGF-β signalling pathway	SMAD2， SMAD4， SMAD3
oas04371	Apelin信号通路 Apelin signalling pathway	SMAD2， SMAD4， SMAD3
oas04390	Hippo信号通路 Hippo signalling pathway	SMAD2， SMAD4， SMAD3
oas04550	调控干细胞多能性的信号通路 Signalling pathways regulating stem cell pluripotency	SMAD2， SMAD4， SMAD3
oas04659	Th17细胞分化 Th17 cell differentiation	SMAD2， SMAD4， SMAD3
oas04933	AGE-RAGE信号通路在糖尿病并发症中的作用 AGE-RAGE signalling pathway in diabetic complications	SMAD2， SMAD4， SMAD3
oas05200	癌症中的路径 Pathways in cancer	SMAD2， SMAD4， SMAD3
oas04068	FoxO 信号通路 FoxO signalling pathway	SMAD4， SMAD3
oas04310	Wnt 信号通路 Wnt signalling pathway	SMAD4， SMAD3
oas04218	细胞衰老 Cell senescence	SMAD2， SMAD3
oas04144	内吞作用 Endocytosis	SMAD2， SMAD3

KEGG编号 KEGG ID	KEGG描述 KEGG description	富集基因 Gene enrichment
oas04110	细胞周期 Cell cycle	SMAD2， SMAD4， SMAD3
oas04350	TGF-β信号通路 TGF-β signalling pathway	SMAD2， SMAD4， SMAD3
oas04371	Apelin信号通路 Apelin signalling pathway	SMAD2， SMAD4， SMAD3
oas04390	Hippo信号通路 Hippo signalling pathway	SMAD2， SMAD4， SMAD3
oas04550	调控干细胞多能性的信号通路 Signalling pathways regulating stem cell pluripotency	SMAD2， SMAD4， SMAD3
oas04659	Th17细胞分化 Th17 cell differentiation	SMAD2， SMAD4， SMAD3
oas04933	AGE-RAGE信号通路在糖尿病并发症中的作用 AGE-RAGE signalling pathway in diabetic complications	SMAD2， SMAD4， SMAD3
oas05200	癌症中的路径 Pathways in cancer	SMAD2， SMAD4， SMAD3
oas04068	FoxO 信号通路 FoxO signalling pathway	SMAD4， SMAD3
oas04310	Wnt 信号通路 Wnt signalling pathway	SMAD4， SMAD3
oas04218	细胞衰老 Cell senescence	SMAD2， SMAD3
oas04144	内吞作用 Endocytosis	SMAD2， SMAD3

[1]	唐靓婷, 黄世会, 牛熙, 李升, 王嘉福, 冉雪琴. 发情周期对香猪卵巢生物钟相关基因表达的影响[J]. 中国农业科技导报, 2023, 25(12): 67-84.
[2]	魏伍川[1] 许尚忠[2] 等. 中国小尾寒羊FSHR基因部分序列的克隆与分析[J]. , 2001, 3(6): 61-65.