荣昌猪BMP15基因通过TGF-β RⅡ激活SMAD4信号分子机制研究

doi:10.11843/j.issn.0366-6964.2020.04.011

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (4): 754-762.doi: 10.11843/j.issn.0366-6964.2020.04.011

荣昌猪BMP15基因通过TGF-β RⅡ激活SMAD4信号分子机制研究

易华山^1,2, 马鲜平^1,2, 赵瑶¹, 朱飘¹, 刘骁¹, 邓博远¹, 吴昕炜¹, 朱丹³, 崔燕^4*

1. 西南大学动物科学学院, 重庆 402460;
2. 重庆市兽医科学工程研究中心, 重庆 402460;
3. 重庆市畜牧科学院, 重庆 402460;
4. 甘肃农业大学动物医学院, 兰州 730070

收稿日期:2019-11-06 出版日期:2020-04-25 发布日期:2020-04-21
通讯作者: 崔燕,主要从事基础兽医学研究,E-mail:cuiyan369@sina.com
作者简介:易华山(1980-),男,甘肃临夏人,讲师,博士,主要从事兽医学研究,E-mail:dyxyihuashan@swu.edu.cn;马鲜平(1981-),女,甘肃文县人,讲师,硕士,主要从事基础兽医学研究,E-mail:xianpingma@163.com
基金资助:
中央高校基本科研业务专项基金（XDJK2018C059；XDJK2018C060）；西南大学荣昌校区青年科学基金（20700949）；重庆市基础研究与前沿探索专项项目基金（cstc2018jcyjAX0615）

Study on the Rongchang Pig BMP15 Gene through TGF-β RⅡ Activation SMAD4 Signal Molecular Mechanism

YI Huashan^1,2, MA Xianping^1,2, ZHAO Yao¹, ZHU Piao¹, LIU Xiao¹, DENG Boyuan¹, WU Xinwei¹, ZHU Dan³, CUI Yan^4*

1. College of Animal Science, Southwest University, Chongqing 402460, China;
2. Chongqing Veterinary Science Engineering Research Center, Chongqing 402460, China;
3. Chongqing Academy of Animal Science, Chongqing 402460, China;
4. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China

Received:2019-11-06 Online:2020-04-25 Published:2020-04-21

摘要/Abstract

摘要： 本研究旨在阐明荣昌猪BMP15基因在荣昌猪性成熟前不同发育期的表达特性及其与SMADs信号相关基因的表达关系。采集1月龄、3月龄及5月龄荣昌猪卵巢组织，利用qRT-PCR及石蜡切片免疫荧光染色法分析荣昌猪不同月龄卵巢组织内BMP15基因表达及细胞定位特征；利用5月龄卵巢活组织添加重组人BMP15蛋白及TGF-β受体抑制剂（LY215799和LY2109761），应用qRT-PCR及Western blot方法分析BMP15及SMADs信号通路相关基因SMAD2、SMAD3、SMAD4、SMAD7、TGF-β1、TGF-β2、TGF-β3、TGF-β RⅠ和TGF-β RⅡ表达特征及BMP15/SMADs信号通路。结果表明：从1月龄至5月龄，随着荣昌猪生长发育，卵巢组织内BMP15基因mRNA表达量呈上调表达（P<0.05）；石蜡切片免疫荧光试验表明，5月龄卵巢组织卵母细胞周围颗粒细胞内存在BMP15蛋白荧光信号；从3月龄至5月龄的卵巢组织内BMP15、SMAD4、TGF-β1及TGF-β RⅡ基因在mRNA水平呈上调表达（P<0.05），而SMAD2、TGF-β2及TGF-β RⅠ呈下调表达（P<0.05）；通过5月龄卵巢活组织添加重组人BMP15蛋白、TGF-β RⅠ/Ⅱ及TGF-β RⅠ受体抑制剂培养，发现TGF-β RⅠ/Ⅱ抑制剂（LY2109761）明显抑制TGF-β RⅡ受体蛋白的表达，TGF-β RⅠ抑制剂（LY2157299）不能抑制TGF-β RⅡ受体蛋白的表达。上述结果表明，荣昌猪BMP15基因在荣昌猪性成熟前的卵巢组织内呈上调表达，SMAD4、TGF-β1及TGF-β RⅡ也呈上调表达。本试验研究表明BMP15基因通过TGF-β RⅡ介导SMAD4信号分子调控下游基因的表达，为进一步研究荣昌猪BMP15基因在卵泡发育中发挥的作用提供理论依据。

关键词: 荣昌猪, BMP15, SMAD, 卵母细胞, TGF-β

Abstract: In order to clarify BMP15 gene expression characteristics in Rongchang pigs at different developmental stages in relation to their sexual maturity, as well as the relationship between the expression of the BMP15 gene and SMADs signaling pathways genes, ovarian tissues from 1 month, 3 months, and 5 months old Rongchang pigs were collected to analyze different BMP15 gene expression and cellular localization characteristics in ovarian tissues of the Rongchang pigs at different developmental stages using qRT-PCR and paraffin sectioning methods. BMP15 and SMADs signaling pathways related genes SMAD2, SMAD3, SMAD4, SMAD7, TGF-β1, TGF-β2, TGF-β3, TGF-β RⅠ and TGF-β RⅡ expression characteristics were also revealed using qRT-PCR and Western blot analysis by adding recombinant human BMP15 protein and TGF-β receptor inhibitor (LY215799 and LY2109761) to 5-month-old ovarian biopsy. The results showed that from the age of 1 month to 5 months, the mRNA expression of BMP15 gene in ovarian tissues were up-regulated (P<0.05)with the growth and development of Rongchang pigs. Immunofluorescence and paraffin sectioning experiments showed that BMP15 protein fluorescent signals existd in granulosa cells surrounding oocyte in the 5-month-old ovarian tissues. Within the ovarian tissues, from 3-5 months old, BMP15, SMAD4, TGF-β1 and TGF-β RⅡ genes mRNA expressions were raised and SMAD2, TGF-β2 and TGF-β RⅠ expressions reduced (P<0.05). By adding the re combinant human BMP15 protein, TGF-β RⅠ/ Ⅱ and TGF-β RⅠ receptor inhibitor in the age of 5 months old ovarian biopsy, the results showed that TGF-β RⅠ/ Ⅱ receptor inhibitor (LY2109761) significantly inhibited the expression of TGF-β RⅡ proteins and TGF-β RⅠ receptor inhibitor (LY2157299) could not inhibit the expression of TGF-β RⅡ proteins. In conclusion, BMP15 gene expression before sexual maturity in Rongchang pig ovarian tissues were raised and SMAD4, TGF-β1 and TGF-β RⅡ genes expression were also up-regulated. The study illuminated that BMP15 gene mediated SMAD4 signaling molecules and regulated the downstream genes expression through TGF-β R Ⅱ, which provided theoretical basis for the further study of Rongchang pig BMP15 gene roles in the follicles development.

Key words: Rongchang pig, BMP15 gene, SMAD, oocyte, TGF-β

中图分类号:

S828.8

易华山, 马鲜平, 赵瑶, 朱飘, 刘骁, 邓博远, 吴昕炜, 朱丹, 崔燕. 荣昌猪BMP15基因通过TGF-β RⅡ激活SMAD4信号分子机制研究[J]. 畜牧兽医学报, 2020, 51(4): 754-762.

YI Huashan, MA Xianping, ZHAO Yao, ZHU Piao, LIU Xiao, DENG Boyuan, WU Xinwei, ZHU Dan, CUI Yan. Study on the Rongchang Pig BMP15 Gene through TGF-β RⅡ Activation SMAD4 Signal Molecular Mechanism[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(4): 754-762.

参考文献 25

[1]	PANGAS S A,LI X H,UMANS L,et al.Conditional deletion of Smad1 and Smad5 in somatic cells of male and female gonads leads to metastatic tumor development in mice[J].Mol Cell Biol,2008,28(1):248-257.
[2]	ZHAI B,LIU H Y,LI X,et al.BMP15 prevents cumulus cell apoptosis through CCL2 and FBN1 in porcine ovaries[J].Cell Physiol Biochem,2013,32(2):264-278.
[3]	GASPERIN B G,FERREIRA R,ROVAN M T,et al.Expression of receptors for BMP15 is differentially regulated in dominant and subordinate follicles during follicle deviation in cattle[J].Anim Reprod Sci,2014,144(3-4):72-78.
[4]	MOORE R K,SHIMASAKI S.Molecular biology and physiological role of the oocyte factor,BMP-15[J].Mol Cell Endocrinol,2005,234(1-2):67-73.
[5]	MIYAZAWA K,SHINOZAKI M,HARA T,et al.Two major Smad pathways in TGF-β superfamily signalling[J].Genes Cells,2002,7(12):1191-1204.
[6]	CUI X R,JING X,WU X Q,et al.Abnormal expression levels of BMP15/Smad1 are associated with granulosa cell apoptosis in patients with polycystic ovary syndrome[J].Mol Med Rep,2017,16(6):8231-8236.
[7]	林鹏飞.猪有腔卵泡发育与闭锁的内分泌及分子细胞学变化研究[D].南京:南京农业大学,2010.LIN P F.Study on endocrine and molecular cytological alterations of porcine antral follicles during development and atresia[D].Nanjing:Nanjing Agricultural University,2010.(in Chinese)
[8]	马鲜平,毕俊萱,龙安格,等.哺乳动物卵母细胞凋亡机制研究进展[J].中国畜牧兽医,2016,43(12):3263-3267.MA X P,BI J X,LONG A G,et al.Advance of apoptotic mechanism in mammalian oocyte[J].China Animal Husbandry & Veterinary Medicine,2016,43(12):3263-3267.(in Chinese)
[9]	CHEN H Y,LIU C,JIANG H,et al.Regulatory role of miRNA-375 in expression of BMP15/GDF9 receptors and its effect on proliferation and apoptosis of bovine cumulus cells[J].Cell Physiol Biochem,2017,41(2):439-450.
[10]	翟博.BMP15调节猪卵丘细胞凋亡的信号通路研究[D].长春:吉林大学,2013.ZHAI B.Study on BMP15 signal pathway to regulate the apoptosis of porcine cumulus cells[D].Changchun:Jilin University,2013.(in Chinese)
[11]	马鲜平,朱涛,龙安格,等.荣昌猪骨形态发生蛋白15基因克隆与表达谱分析[J].华北农学报,2016,31(S1):458-462.MA X P,ZHU T,LONG A G,et al.Sequence and transcripts analysis of the Bone Morphogenetic Protein 15 in Porcine of Rongchang[J].Acta Agriculturae Boreali-Sinica,2016,31(S1):458-462.(in Chinese)
[12]	翟向玮.小鼠和家兔卵母细胞体外发生的培养体系与发育特征的研究[D].青岛:青岛农业大学,2008.ZHAI X W.The culture system and developmental characteristic of oogenesis in vitro of mouse and rabbit[D].Qingdao:Qingdao Agricultural University,2008.(in Chinese)
[13]	冯怀亮,秦鹏春,杨庆章,等.貉卵巢卵母细胞体外培养的研究[J].东北农学院学报,1992,23(3):306-308.FENG H L,QIN P C,YANG Q Z,et al.Maturation of Racoon dog ovarian oocytes cultured in vitro[J].Journal of Northeast Agricultural College,1992,23(3):306-308.(in Chinese)
[14]	李琴.microRNA对哺乳动物卵泡发育的影响[J].畜牧兽医学报,2018,49(12):2558-2566.LI Q.Roles of microRNA on mammalian follicle development[J].Acta Veterinaria et Zootechnica Sinica,2018,49(12):2558-2566.(in Chinese)
[15]	FILATOV M,KHRAMOVA Y,PARSHINA E,et al. Influence of gonadotropins on ovarian follicle growth and development in vivo and in vitro[J].Zygote,2017,25(3):235-243.
[16]	ROY S,GANDRA D,SEGER C,et al.Oocyte-derived factors (GDF9 and BMP15) and FSH regulate AMH expression via modulation of H3K27AC in granulosa cells[J].Endocrinology,2018,159(9):3433-3445.
[17]	SHIMIZU K,NAKAMURA T,BAYASULA B,et al.Molecular mechanism of FSHR expression induced by BMP15 in human granulosa cells[J].J Assist Reprod Genet,2019,36(6):1185-1194.
[18]	KAMATO D,TA H,AFROZ R,et al.Mechanisms of PAR-1 mediated kinase receptor transactivation:smad linker region phosphorylation[J].J Cell Commun Signal,2019,13(4):539-548.
[19]	LI X H,TRIPURANI S K,JAMES R,et al.Minimal fertility defects in mice deficient in oocyte-expressed Smad4[J].Biol Reprod,2012,86(1):1-6.
[20]	BELLI M,SHIMASAKI S.Molecular aspects and clinical relevance of GDF9 and BMP15 in ovarian function[J].Vitam Horm,2018,107:317-348.
[21]	ALAM M H,LEE J,MIYANO T.GDF9 and BMP15 induce development of antrum-like structures by bovine granulosa cells without oocytes[J].J Reprod Dev,2018,64(5):423-431.
[22]	DU X,PAN Z X,LI Q Q,et al.SMAD4 feedback regulates the canonical TGF-β signaling pathway to control granulosa cell apoptosis[J].Cell Death Dis,2018,9(2):151.
[23]	CRAIG J,ORISAKA M,WANG H M,et al.Gonadotropin and intra-ovarian signals regulating follicle development and atresia:the delicate balance between life and death[J].Front Biosci,2007,12:3628-3639.
[24]	PRESSING A,DIAL G D,ESBENSHADE K L,et al.Hourly administration of GnRH to prepubertal gilts:endocrine and ovulatory responses from 70 to 190 days of age[J].J Anim Sci,1992,70(1):232-242.
[25]	梁学超,蒋明,罗玉茹,等.猪卵巢发育的组织学变化及卵泡闭锁规律研究[J].畜牧兽医学报,2017,48(10):1863-1870.LIANG X C,JIANG M,LUO Y R,et al.Study on histology and patterns of follicular atresia during ovarian development in pig[J].Acta Veterinaria et Zootechnica Sinica,2017,48(10):1863-1870.(in Chinese)

荣昌猪BMP15基因通过TGF-β RⅡ激活SMAD4信号分子机制研究

Study on the Rongchang Pig BMP15 Gene through TGF-β RⅡ Activation SMAD4 Signal Molecular Mechanism

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