| The synthesis of testosterone in mammals is regulated by many genes,some of which are missing or reduced in expression will affect the level of testosterone synthesis,testosterone deficiency will have an adverse effect on the spermatogenesis process.Studies have found that STAR,CYP11A1,HSD3B1,CYP17A1,HSD17B3 and GLOD4 are expressed in the testes of male animals,which are closely related to the maintenance of testicular function and the process of testosterone synthesis in male animals,but their expression patterns and functions are different in different species.Therefore,the study used q RT-PCR and Western Blot methods to detect the expression characteristics of STAR,CYP11A1,HSD3B1,CYP17A1,HSD17B3 and GLOD4 in the testes and epididymides tissues of Tibetan sheep at three key developmental stages: 3 months old(before sexual maturity),1 year old(sexual maturity),and 3 years old(adult),and used immunofluorescence technology to detect the positive signal distribution characteristics of its protein.In addition,q RT-PCR technology and dual luciferase reporter assay were used to verify the expression characteristics of oar-miR-190-3p and oar-miR-363-3p and targeting relationship with HSD17B3.The main findings are as follows:1.Compared with 3 months old,the expression levels of STAR,CYP11A1,HSD3B1,CYP17A1 and HSD17B3 mRNA in 1 and 3 years old Tibetan sheep testes were significantly down-regulated(P<0.01),while the expression of STAR,HSD3B1,CYP17A1 and HSD17B3 protein were significantly up-regulated(P<0.01),the expressions of CYP11A1 and GLOD4 mRNA and protein in the testes of 1 and 3 years old were all significantly up-regulated(P<0.01).The results of immunofluorescence staining showed that STAR,CYP11A1,HSD3B1,CYP17A1,HSD17B3 and GLOD4 protein predominantly existed in Leydig cells from postpubertal(1 year old and 3 years old)testes,and small amounts were present in germ cells of the throughout development stages.The results of the study indicate that testosterone synthesisrelated genes may participate in spermatogenesis by maintaining the function of testicular stromal cells and regulating the secretion of testosterone in Tibetan sheep stromal cells.2.STAR,CYP11A1,HSD3B1,CYP17A1 and HSD17B3 mRNA and protein are expressed in the epididymal caput,corpus and cauda of Tibetan sheep at different developmental stages.STAR,CYP11A1,HSD3B1,CYP17A1 and HSD17B3 mRNA are differentially expressed in the developing epididymides(caput,corpus and cauda),while their protein expressions were all significantly up-regulated(P<0.01).Immunofluorescence results showed that STAR,CYP11A1,HSD3B1,CYP17A1 and HSD17B3 proteins showed strong positive signal distribution in epididymal epithelial cells at different developmental stages of Tibetan sheep,and the signal intensity gradually increased with age.In addition,there is also a moderately strong positive signal were distributed in the sperm of the epididymal lumen after sexual maturity.The results demonstrated that STAR,CYP11A1,HSD3B1,CYP17A1 and HSD17B3 participate in epididymal development and sperm maturation by regulating the secretion and absorption function of epididymal epithelial cells in Tibetan sheep epididymis at different developmental stages.3.With the increase of age,the expression of oar-miR-190-3p and oar-miR-363-3p were significantly up-regulated in the testis at different developmental stages(P<0.01),and they were differentially expressed in the epididymal caput,corpus and cauda.The results of dual luciferase reporter gene detection found that oar-miR-190-3p and oar-miR-363-3p mimics significantly decreased the luciferase activity of HSD17B3 3’UTR wild-type(P<0.05).These results indicate that oar-miR-190-3p and oar-miR-363-3p can target and regulate the expression of sheep HSD17B3 gene. |
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