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| The Characteristic Analysis of Yak (Bos grunniens) CLDN3 Gene and the Effects of FSH on Its Expression in Sertoli Cell |
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Abstract Claudin3 (CLDN3) regulates the movement of small molecules across the sertoli cell (SC) tight junctions (TJ) and creates physical barrier for preleptotene / leptotene spermatocytes. In order to explore biological effects of CLDN3 on yak SC, this study used reverse transcription polymerase chain reaction (RT-PCR) to clone CLDN3 cDNA of yak (Bos grunniens) and used bioinformatics method to analyze it's biological characteristics. The different concentrations of follicle-stimulating hormone (FSH) (0, 10, 25, 50, 75, 100 ng/mL) were added into culture medium during yak sertoli cells (SCs) cultured in vitro, and the levels of CLDN3 mRNA and protein were evaluated by quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting (WB). What's more, the location of CLDN3 protein on SC was detected by immunofluorescence. The results showed that yak SC exhibited bipolar corpuscula in nucleus after Feulgen staining and had a positive reaction of Fasl by the immunocytochemical identification. The CLDN3 cDNA of yak was successfully cloned, and it contained ORF of 1 074 bp length (GenBank No. YK411920) and 660 bp CDS, encoding 219 amino acids. The sequence alignment showed that the homology between yak and other species were relatively high (≥90%). CLDN3 protein could be detected in nucleus, cytoplasm and plasma membrane of SC from immunofluorescence analysis. qRT-PCR detection showed that the 10, 25, 50, 75 and 100 ng/mL FSH treatment groups could improve relative expression of SC CLDN3 mRNA. The CLDN3 mRNA expression level both 25 and 50 ng/mL groups were significantly higher than other groups (P＜0.01). The CLDN3 mRNA of 25 ng/mL group was the highest expression level (P＜0.01). The difference between 10 ng/mL and control group was not significant (P＞0.05). 75 and 100 ng/mL were significantly different from the control group (P＜0.05). 25 ng/mL FSH group of CLDN3 protein was the highest of the groups (P＜0.01), and the expression of CLDN3 protein in 100 ng/mL FSH was lower than that in the control group (P＞0.05). In summary, CLDN3 amino acids sequence was conserved in the different genus. FSH may be as an important hormone during the formation of TJ in testis of yak. The expression both of CLDN3 mRNA and CLDN3 protein relied on concentration of FSH in which the optimum FSH concentration was 25 ng/mL (P＜0.01). The research could provide the basis for revealing the biological effect of yak CLDN3 during sperm migration.
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Received: 18 July 2017
Published: 14 February 2018
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