雌性牦牛主要生殖器官中DBI的表达与定位分析

doi:10.11843/j.issn.0366-6964.2022.01.017

摘要/Abstract

摘要： 为加深对DBI基因功能的探究，揭示其在牦牛生殖生理过程中的作用。本试验采集健康母牦牛（3～6岁）繁殖周期不同阶段（卵泡期、黄体期和妊娠期）的卵巢、输卵管和子宫组织，共分为9组，每组设置3个生物学重复。采用实时荧光定量PCR （qRT-PCR）和蛋白免疫印迹技术（Western blotting，WB）检测牦牛DBI在繁殖周期不同阶段雌性牦牛生殖器官中的相对表达量；通过免疫组织化学方法（immunohistochemistry，IHC）定位DBI蛋白在雌性牦牛卵巢、输卵管和子宫中的分布情况。结果表明：1） qRT-PCR结果显示，在卵巢中，DBI在黄体期和妊娠期的表达量显著高于卵泡期（P<0.05）；在输卵管中，DBI在黄体期表达量显著高于卵泡期和妊娠期（P<0.05）；在子宫中，DBI在妊娠期表达量最高，黄体期次之，卵泡期最低。2） Western blotting结果表明，在卵巢中，DBI在妊娠期的水平显著高于卵泡期和黄体期（P<0.05）；在输卵管中，DBI在黄体期水平显著高于卵泡期和妊娠期（P<0.05）；在子宫中，DBI在妊娠期水平明显高于黄体期和卵泡期（P<0.05）。3） IHC结果显示，DBI主要位于生殖上皮细胞、颗粒细胞、卵泡膜细胞、黄体细胞、输卵管上皮细胞、子宫基质细胞和子宫腺中。DBI在牦牛繁殖周期不同阶段的卵巢、输卵管和子宫组织中的表达量存在显著差异，说明其参与牦牛生殖生理调控，为进一步挖掘DBI基因在高原哺乳动物生殖方面的研究奠定相应的理论基础。

关键词: 牦牛, DBI, 卵巢, 输卵管, 子宫, 表达

Abstract: The study aimed to investigate the function of DBI gene and reveal its role in reproductive physiology of yak. The ovaries, fallopian tubes and uterine tissues of healthy female yaks (3-6 years old) at different stages of reproductive cycle (follicular stage, luteal stage and gestation stage) were collected and divided into 9 groups with 3 biological replicates in each group. Real-time quantitative PCR (qRT-PCR) and Western blotting (WB) assays were used to detect the rela-tive expression of DBI in reproductive organs of female yaks at different stages of reproductive cycle. Immunohistochemistry (IHC) was used to determine the distribution of DBI protein in the ovaries, fallopian tubes and uterus of female yaks. The results showed as follows: 1) qRT-PCR result showed that,in ovary,the expression of DBI was significantly higher in luteal stage and pregnancy stage than in follicular stage (P<0.05); In fallopian tube, the expression of DBI in luteal stage was significantly higher than that in follicular and gestation stages (P<0.05). In utero, the expression level of DBI was highest during gestation stage, followed by luteal phase, and the lowest during follicular phase. 2) Western blotting results showed that,in ovary, the level of DBI was significantly higher in pregnancy stage than in follicular and luteal stages (P<0.05); In fallopian tube, the level of DBI in luteal stage was significantly higher than that in follicular stage and gestation stage (P<0.05); In uterus, the expression level of DBI in pregnancy stage was significantly higher than that in luteal phase and follicular phase (P<0.05). 3) IHC results showed that DBI was mainly located in reproductive epithelial cells, granulosa cells, follicular membrane cells, luteal cells, tubal epithelial cells, uterine stromal cells and uterine glands. The expression levels of DBI in the ovaries, fallopian tubes and uterus tissues of yaks at different stages of reproductive cycle are significantly different, indicating that DBI is involved in the reproductive physiological regulation of yaks, which lays a theoretical foundation for further exploring the role of DBI gene in the reproductive aspects of plateau mammals.

Key words: yak, DBI, ovary, oviduct, uterus, expression

中图分类号:

S823.85

王强龙, 潘阳阳, 闫翠霞, 张同享, 何翃闳, 崔燕, 徐庚全, 王立斌, 樊江峰, 余四九. 雌性牦牛主要生殖器官中DBI的表达与定位分析[J]. 畜牧兽医学报, 2022, 53(1): 169-178.

WANG Qianglong, PAN Yangyang, YAN Cuixia, ZHANG Tongxiang, HE Honghong, CUI Yan, XU Gengquan, WANG Libin, FAN Jiangfeng, YU Sijiu. Expression and Localization of DBI in Main Reproductive Organs of Female Yaks[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 169-178.

参考文献 40

[1]	ROSENDAL J,ERTBJERG P,KNUDSEN J.Characterization of ligand binding to acyl-CoA-binding protein[J].Biochem J,1993,290(2):321-326.
[2]	ALQUIER T,CHRISTIAN C A,ALFONSO J,et al.From benzodiazepines to fatty acids and beyond:revisiting the role of ACBP/DBI[J].Trends Endocrinol Metab,2021,32(11):890-903.
[3]	GUIDOTTI A,FORCHETTI C M,CORDA M G,et al.Isolation,characterization,and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors[J].Proc Natl Acad Sci U S A,1983,80(11):3531-3535.
[4]	PAPADOPOULOS V,BERKOVICH A,KRUEGER K E,et al.Diazepam binding inhibitor and its processing products stimulate mitochondrial steroid biosynthesis via an interaction with mitochondrial benzodiazepine receptors[J].Endocrinology, 1991, 129(3):1481-1488.
[5]	GUIDOTTI A,CORDA M G,COSTA E.Strategies for the isolation and characterization of an endogenous effector of the benzodiazepine recognition sites[J].Adv Biochem Psychopharmacol,1983,38:95-103.
[6]	AUGOFF K,KOLONDRA A,CHORZALSKA A,et al.Expression,purification and functional characterization of recombinant human acyl-CoA-binding protein (ACBP) from erythroid cells[J].Acta Biochim Pol,2010,57(4):533-540.
[7]	NEESS D,BEK S,ENGELSBY H,et al.Long-chain acyl-CoA esters in metabolism and signaling:role of acyl-CoA binding proteins[J].Prog Lipid Res,2015,59:1-25.
[8]	李林春,王家庆,何振峰,等.青鳉鱼DBI基因全长cDNA的克隆与蛋白结构分析[J].基因组学与应用生物学,2010, 29(5):885-889.LI L C,WANG J Q,HE Z F,et al.The full-length cDNA cloning of DBI gene and its protein structure analysis derived from medaka (Oryzias latipes)[J].Genomics and Applied Biology,2010,29(5):885-889.(in Chinese)
[9]	杨珊珊,何翃闳,潘阳阳,等.整合素αv在牦牛繁殖周期输卵管不同部位中的表达与定位[J].畜牧兽医学报,2020, 51(11):2710-2719.YANG S S,HE H H,PAN Y Y,et al.Expression and localization of integrin αv in different parts of the fallopian tube in reproduction cycle of yak[J].Acta Veterinaria et Zootechnica Sinica,2020,51(11):2710-2719.(in Chinese)
[10]	YU S J,LI F D.Profiles of plasma progesterone before and at the onset of puberty in yak heifers[J].Anim Reprod Sci,2001,65(1-2):67-73.
[11]	潘阳阳,王萌,芮弦,等.IGF-1调控RBM3表达抑制低温应激诱导牦牛卵丘细胞凋亡[J].中国农业科学,2020, 53(11):2285-2296.PAN Y Y,WANG M,RUI X,et al.RNA-binding motif protein 3(RBM3) expression is regulated by insulin-like growth factor(IGF-1) for protecting yak(Bos grunniens) cumulus cells from apoptosis during hypothermia stress[J].Scientia Agricultura Sinica, 2020,53(11):2285-2296.(in Chinese)
[12]	潘阳阳,王萌,余四九,等.EGF对牦牛冻精运动性能、细胞凋亡和IVF胚胎发育潜力的影响[J].畜牧兽医学报,2017, 48(5):854-862.PAN Y Y,WANG M,YU S J,et al.The effects of EGF on yak frozen sperm motility,apoptosis and the development competence of embryos after IVF[J].Acta Veterinaria et Zootechnica Sinica,2017,48(5):854-862.(in Chinese)
[13]	黄成渝.DBI对胎盘滋养细胞类固醇激素合成的影响以及在自然流产发生中的意义[D].重庆:重庆医科大学,2020.HUANG C Y. DBI is involved in regulating the expression of anabolic hormone genes in trophoblast cells of placenta and its association with spontaneous abortion[D].Chongqing:Chongqing Medical University,2020.(in Chinese)
[14]	BÜRGI B,LICHTENSTEIGER W,LAUBER M E,et al.Ontogeny of diazepam binding inhibitor/acyl-CoA binding protein mRNA and peripheral benzodiazepine receptor mRNA expression in the rat[J].J Neuroendocrinol,1999,11(2):85-100.
[15]	盛丹,刘琴,滕晓红,等.槟榔江水牛ACBP基因克隆及分子特征、组织表达谱分析[J].华北农学报,2018,33(6):72-79.SHENG D,LIU Q,TENG X H,et al.Cloning,molecular characteristics of ACBP gene and its expression profile analysis in Binglangjiang buffalo[J].Acta Agriculturae Boreali-Sinica,2018,33(6):72-79.(in Chinese)
[16]	CHAO H,ZHOU M L,MCINTOSH A,et al.ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT[J].J Lipid Res,2003,44(1):72-83.
[17]	SMITH G V S,SMITH O W.The urinary excretion of estrogenic and gonadotropic hormones during menstrual cycles,the period of conception and early pregnancy[J].N Engl J Med,1936,215(20):908-914.
[18]	GALLEGO S F,SPRENGER R R,NEESS D,et al.Quantitative lipidomics reveals age-dependent perturbations of whole-body lipid metabolism in ACBP deficient mice[J].Biochim Biophys Acta,2017,1862(2):145-155.
[19]	高泽川,赵凌,潘阳阳,等.HAS2和PTGS2在牦牛不同时期卵巢中的表达与定位[J].西北农林科技大学学报:自然科学版,2021,49(9):1-8.GAO Z C,ZHAO L,PAN Y Y,et al.Expression and localization of HAS2 and PTGS2 in yak ovary at different stages[J].Journal of Northwest A&F University:Natural Science Edition,2021,49(9):1-8.(in Chinese)
[20]	YU S J,YONG Y H,CUI Y.Oocyte morphology from primordial to early tertiary follicles of yak[J].Reprod Domest Anim,2010,45(5):779-785.
[21]	何翃闳,潘阳阳,张慧珠,等.FSH对牦牛卵母细胞EGF、EGFR表达及其细胞凋亡的影响[J].畜牧兽医学报,2018,49(9):1899-1907.HE H H,PAN Y Y,ZHANG H Z,et al.The effects of follicle-stimulating hormone (FSH) on the expression of EGF and EGFR in yak oocytes and apoptosis[J].Acta Veterinaria et Zootechnica Sinica,2018,49(9):1899-1907.(in Chinese)
[22]	YAMAMOTO Y,KUWAHARA A,TANIGUCHI Y,et al.Tumor necrosis factor alpha inhibits ovulation and induces granulosa cell death in rat ovaries[J].Reprod Med Biol,2015,14(3):107-115.
[23]	BAUMAN D E,CURRIE W B.Partitioning of nutrients during pregnancy and lactation:a review of mechanisms involving homeostasis and homeorhesis[J].J Dairy Sci,1980,63(9):1514-1529.
[24]	PEÑA-ZANONI M,GARCÍA D C,ROLDÁN-OLARTE M,et al.A long-term in vitro culture of bovine epithelial cells on collagen rafts[J].Reprod Domest Anim,2021,56(4):684-687.
[25]	王静瑜,王萌,潘阳阳,等.牦牛FAF1基因的分子特征及其在不同阶段卵巢、输卵管、子宫中的表达[J].生物技术通报,2020, 36(7):80-89.WANG J Y,WANG M,PAN Y Y,et al.Molecular characteristics of bosgrunniens FAF1 gene and its expression in ovaries,fallopian tubes and uterus at different stages[J].Biotechnology Bulletin,2020,36(7):80-89.(in Chinese)
[26]	TEIJEIRO J M,MARINI P E.Hormone-regulated PKA activity in porcine oviductal epithelial cells[J].Cell Tissue Res,2020, 380(3):657-667.
[27]	李晓丹,曹贵方,杨宏新,等.雌激素作用下输卵管上皮细胞S100A8和S100A9的表达及其作用探讨[J].中国药理学通报,2020,36(4):562-568.LI X D,CAO G F,YANG H X,et al.Expression of S100A8 and S100A9 in oviduct epithelial cells induced by estrogen and their possible role in oviduct[J].Chinese Pharmacological Bulletin,2020,36(4):562-568.(in Chinese)
[28]	LI S,WINUTHAYANON W.Oviduct:roles in fertilization and early embryo development[J].J Endocrinol,2017,232(1):R1-R26.
[29]	谭娟.牦牛生殖周期中输卵管的组织结构观察[D].兰州:甘肃农业大学,2008.TAN J.Observation on the histological structure of the yak (Bos grunniens) oviduct during reproductive cycle[D].Lanzhou:Gansu Agricultural University,2008.(in Chinese)
[30]	包图雅.17β-雌二醇对绵羊输卵管上皮细胞β-防御素基因表达的影响及可能的信号通路[D].呼和浩特:内蒙古农业大学,2010.BAO T Y.The effect of 17β-estradiol on B-defensin gene expression in the ovine oviduct epithelial cells and its signal transduction pathways[D].Hohhot:Inner Mongolia Agricultural University,2010.(in Chinese)
[31]	BRENNER R M.Oviduct physiology and sperm/oviduct interactions:an introduction[J].Biol Reprod,1998,58(5):1097.
[32]	VERHAGE H G,MAVROGIANIS P A,O’DAY-BOWMAN M B,et al.Characteristics of an oviductal glycoprotein and its potential role in the fertilization process[J].Biol Reprod,1998,58(5):1098-1101.
[33]	谭秀文,马所峰,刘新勇,等.不同物种输卵管上皮细胞培养、纯度检测及支持小鼠胚胎发育的能力[J].畜牧兽医学报,2006,37(9):878-882.TAN X W,MA S F,LIU X Y,et al.Culture,purification and ability to support mouse embryo development of oviductal epithelial cells from different species[J].Acta Veterinaria et Zootechnica Sinica,2006,37(9):878-882.(in Chinese)
[34]	ABUDUREYIMU A,CAI Y,HUO S D,et al.Expression and localization of follicle-stimulating hormone receptor in the yak uterus during different stages of the oestrous cycle[J].Reprod Domest Anim,2018,53(6):1539-1545.
[35]	FUKUI Y,HIROTA Y,MATSUO M,et al.Uterine receptivity,embryo attachment,and embryo invasion:multistep processes in embryo implantation[J].Reprod Med Biol,2019,18(3):234-240.
[36]	DEY S K,LIM H,DAS S K,et al.Molecular cues to implantation[J].Endocr Rev,2004,25(3):341-373.
[37]	TRANGUCH S,DAIKOKU T,GUO Y,et al.Molecular complexity in establishing uterine receptivity and implantation[J].Cell Mol Life Sci,2005,62(17):1964-1973.
[38]	WANG H B,DEY S K.Roadmap to embryo implantation:clues from mouse models[J].Nat Rev Genet,2006,7(3):185-199.
[39]	德尔曼,布朗.兽医组织学[M].秦鹏春,聂其灼,译.北京:农业出版社,1989:290-295.DELLMANN H D,BROWN E M.Textbook of vetcriuary histoiogy[M].QIN P C,NIE Q Z,trans.Beijing:Agricultural Press,1989: 290-295.(in Chinese)
[40]	阿依木古丽.发情周期不同阶段牦牛子宫中生殖激素受体表达的研究[D].兰州:甘肃农业大学,2011.AYIMUGULI.Different expression of reproductive hormone receptors in yak uterus during estrous cycle[D].Lanzhou:Gansu Agricultural University,2011.(in Chinese)