雌性牦牛主要生殖器官中DBI的表达与定位分析

Expression and Localization of DBI in Main Reproductive Organs of Female Yaks

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。

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.