The Molecular Mechanism Of Promoting The Testis Reproduction Performance By Dietary Vitamin E In Sheep

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.