【作者】 左兆云；

【导师】 罗海玲；

【作者基本信息】 中国农业大学 ， 动物营养与饲料科学， 2014， 博士

【摘要】 本团队前期研究证明,日粮维生素E可以提高公羔繁殖器官的发育、抗氧化性能、精子发育和精液品质。据此,为进一步探索日粮维生素E对公羊繁殖性能的作用机理,本研究选择健康断奶后20-30天的滩羊公羔35只(16.20±1.65kg),随机分为5个处理组,每组7只。对照组(E0)饲喂基础日粮,不添加维生素E；试验组维生素E的添加剂量分别为20(E20)、100(E100)、200(E200)、2000(E2000) IU羊-1.天-1,试验期为120天。屠宰后取睾丸及各组织,从维生素E转运因子、睾丸差异基因、抗氧化酶基因和生殖激素合成相关基因的角度,研究日粮维生素E对绵羊睾丸繁殖机能影响的分子机制,为揭示维生素E调控繁殖性能提供理论依据。试验一：日粮维生素E对滩羊各组织α-生育酚转移蛋白(α-TTP)基因表达水平的影响。免疫组织化学试验表明：滩羊的心脏、脾脏、肺脏、肾脏组织均检测到α-TTP的阳性信号,并且α-TTP定位在细胞质中；而睾丸、背最长肌、臀肌没有检测到阳性信号。各组织α-TTP基因表达量的结果表明：肾脏、背最长肌和臀肌中,维生素E添加量对α-TTP mRNA表达量没有显著影响(P>0.05)。E200组肺脏α-TTP mRNA表达量显著(P<0.05)高于E2000组；E200组脾脏α-TTP mRNA表达量显著(P<0.05)高于E100组；与此相反,在心脏中,E200组α-TTP mRNA表达量显著(P<0.05)低于E0组。维生素E添加量对睾丸α-TTP mRNA表达量没有显著影响(P>0.05),但随着维生素E添加水平的提高,α-TTP mRNA的表达水平有一定的下降趋势,而E200和E2000组显著提高了睾丸维生素E含量(P<0.05)。本试验表明,日粮维生素E水平对滩羊不同组织α-TTP mRNA的表达量有一定的影响,并且组织间存在差异,影响维生素E在不同组织发挥作用。而日粮维生素E对睾丸α-TTP mRNA表达量和维生素E含量的影响,表明α-TTP水平与维生素E在睾丸发挥功能相关,但是与繁殖性能的直接关联性有待于进一步研究。试验二：构建滩羊睾丸cDNA文库的构建并鉴定质量。结果表明：目前成功得到滩羊睾丸cDNA文库,其初级库容约2.0x106cfu,初级文库甘油菌保存液滴度为5.1×104cfu/1.8mL,插入片段平均长度为1Kb,重组率为93.75%,最终得到文库质粒约3.7mg。本试验表明滩羊睾丸cDNA文库符合建库要求,可作为进一步克隆滩羊睾丸特异表达基因的可靠材料,为研究滩羊睾丸繁殖性能提供基础。试验三：日粮不同维生素E水平下滩羊睾丸差异基因的筛选。结果表明：通过GO生物学功能进行分析,差异基因参与发育过程、免疫系统过程、生物粘连生物学过程、能量脂质代谢过程和氧化应激应答过程。KEGG通路分析结果显示,筛选出的维生素E差异基因涉及PPAR信号通路、MAPK信号通路、细胞粘附、脂质代谢、糖类代谢和氧化相关基因。综合分析表明,维生素E调控睾丸基因表达的作用机制可能包括：调节组织氧化水平；调节信号通路中受体表达或转录因子活性；影响代谢相关基因的表达。试验四：日粮维生素E对睾丸抗氧化酶基因mRNA和蛋白表达的影响。荧光定量结果显示：与对照组相比,E200组显著(P<0.05)提高了睾丸谷胱甘肽过氧化物酶3(GPx3)和谷胱甘肽转移酶A1(GSTA1) mRNA表达量,E20组则显著(P<0.05)提高了GPx3的mRNA表达量。Western blot结果显示：日粮维生素E显著(P<0.05)提高了睾丸GPx3蛋白表达量；与对照组相比,E20组和E100组显著(P<0.05)提高了GSTA1蛋白表达量。本试验表明,日粮添加维生素E能不同程度地提高GPx3和GSTA1mRNA或蛋白表达量,荧光定量结果与基因芯片反映的结果一致。试验五：日粮维生素E对睾丸生殖激素相关基因mRNA和蛋白表达的影响。荧光定量结果显示：随着维生素E添加水平的增高,睾丸GATA结合蛋白4(GATA-4)、类固醇急性调节蛋白(StAR)、细胞色素P450A1(CYP11A1) mRNA的表达水平有先升高后下降的趋势。与对照组相比,E200组显著(P<0.05)提高了GATA-4的mRNA表达量,E20组三种基因rnRNA表达量显著提高(P<0.05)；而E100组三种基因mRNA表达量均有显著(P<0.05)低于E20,但是与对照组差异不显著(P>0.05)。Western blot结果显示：维生素E有提高GATA-4蛋白表达量的趋势；与对照组相比,E20和E2000组均显著(P<0.05)提高了睾丸StAR蛋白表达量。本试验表明,维生素E对生殖激素相关基因mRNA和蛋白表达量有一定的提高作用。结论：日粮维生素E对绵羊睾丸基因表达有一定的调节作用,维生素E可在一定程度上提高抗氧化酶基因和生殖激素合成基因mRNA和蛋白表达量,进而提高抗氧化酶活性,是维生素E提高绵羊睾丸繁殖性能的主要原因。更多还原

【Abstract】 The previous research conducted in our laboratory showed that dietary supplementation of vitamin E improved the development of reproductive organs, antioxidant properties, sperm development and semen quality in male kids. Under these results, this study was carried out to investigate the mechanism of dietary vitamin E promoting the ram reproduction performance. Thirty-five local male kids of Tan sheep (20to30days after weaning) with similar body weight (16.20±1.65kg) were randomly allocated into five groups (with7animals in each group) and fed with diets supplemented with0(control group),20,100,200and2,000IU sheep-1day-1vitamin E (treatments denoted as E0, E20, E100, E200, and E2000, respectively) for120days. Then the sheep were slaughtered and the samples of testis, heart, spleen, lung, kidney, longissimus dorsi and gluteus muscle were collected. In the present study, experiments those from the perspective of a-tocopherol transfer protein, differential genes, antioxidant enzyme, and reproductive hormone synthesis related genes, were conducted to investigate the molecular mechanism of promoting the testis reproduction performance by dietary vitamin E in Tan sheep.Exp.1:Dietary vitamin E affects a-TTP mRNA levels in different tissues of the Tan sheep. The results showed that in heart, spleen, lung, and kidney tissues, a-TTP was located mainly in the cytoplasm, while no a-TTP immunoreactivity was detected in the cytoplasm of both the testis, longissimus dorsi and gluteus muscle samples. In the present study, the results showed that dietary vitamin E did not affect the a-TTP mRNA levels in the kidney, the longissimus dorsi muscle and the gluteus muscle. However, compared with the E100group, the E200group had higher (P<0.05) α-TTP mRNA levels in spleen. Similarly, a-TTP mRNA levels in the lung in E200group were higher (P<0.05) than those in the group of E2000. In contrast, compared with the control, the E200group had lower (P<0.05) a-TTP mRNA levels in heart tissues. Dietary vitamin E did not affect the testis a-TTP mRNA levels, but there has a trend of decrease in response to increased dosage. While compared with the control, the E200and E2000group had significantly (P<0.05) higher vitamin E concentration in testis. In conclusion, dietary vitamin E levels had effects on the expression levels of a-TTP in some tissues of Tan sheep and the effect was clearly tissue-specific. And the relevance between the a-TTP mRNA levels and vitamin E concentration in testis warrant further investigation.Exp.2:cDNA library of testis from Tan sheep was constructed with plasmid vector and the quality of the cDNA libraries was identified. The results showed that the titer of testis cDNA library was5.1×104cfu/1.8mL, the total number of colony-forming units (cfu) was2.0×106cfu, the recombinant rate was93.75%and the average insert size was about1.0Kb. The library titer, recombinant rate and the average insert size all matched quality requirements for cDNA library and this indicated that cDNA libraries of ovine testis was successfully constructed, which will be valuable resource for reproduction performance of the Tan sheep.Exp.3:Tan sheep testis samples from different dietary vitamin E were collected to research the differential expression genes in our experiment by gene chip. The differential expression genes by GO analysis can find notations on development, response to reactive oxygen species, DNA metabolic process, cell adhesion, and lipid transport. The differential expression genes by KEGG pathway analysis had founded in the pathway of PPAR、 MAPK, Toll-like receptor signaling pathway, focal adhesion, glutathione metabolism and starch and sucrose metabolism. Based on the analysis of these genes, it was speculated that vitamin E might affect gene expression of testis by modulating the oxidation level, by affecting the expression of receptor and transcription factors in pathway, and regulating the expression of metabolism-associated genes.Exp.4:Dietary vitamin E affects the expression of antioxidant enzyme genes in testis of the Tan sheep. The results showed that the E200group had higher (P<0.05) GPx3and GSTA1mRNA levels and E20group had higher (P<0.05) GPx3mRNA levels compared with the control. Dietary vitamin E could increase (P<0.05) the GPx3protein expression of testis. While compared with the control, E20and E100group had higher (P<0.05) GSTA1protein levels. In conclusion, dietary vitamin E could increase (P<0.05) the mRNA and protein expression of GPx3and GSTA1at different levels. The results of qRT-PCR for antioxidant enzyme genes were consistent with those from gene chip.Exp.5:Dietary vitamin E affects the expression of reproductive hormone synthesis related genes in testis of the Tan sheep. The results showed that:As the levels of vitamin E increasing from0to2000IU sheep-1day-1diet, mRNA expression of GATA-4, StAR and CYP11A1first increased, then decreased. Compared with the control, the E20group increased (P<0.05) these three genes mRNA levels and the E200group increased (P<0.05) the expression of GATA-4mRNA. In contrast, these three genes mRNA levels in E100group decreased (P<0.05) compared with the E20group. And the E20and E2000group had the highest (P<0.05) protein expression of StAR. In conclusion, dietary vitamin E could increase (P<0.05) the mRNA and protein expression of GATA-4, StAR and CYP11A1at different levels.It was concluded that the supplementation of vitamin E could regulate the gene expression in testis. Dietary vitamin E could increase the mRNA and protein expression of reproductive hormone synthesis related genes and antioxidant enzyme genes, and then increase the activity of antioxidant enzyme, were the main reasons for promoting the testis reproduction performance by dietary vitamin E.更多还原