| MicroRNAs(miRNAs) are a class of widely distributive and highly conservative non-coding RNAs with 22 nucleotides(nt). They negatively regulated the expression of target gene by combining with the 3’-UTR. There are some of mi RNAs in the testis of mammals. Previous studies had shown that some mi RNAs were related with the function of germ cells and sertoli cells. In this study, we tried to reveal function of mi R-10 a during spermatogensis through the mice which micro RNA-10 a were over-expressed in germ cells(Mvh-Cre;floxp mi R-10alsl-tetoff;mi R-10a+/-).1. The mi R-10a+/-mice showed the obstacle in spermatogenesis.The mice which micro RNA-10 a was over-expressed in germ cells(mi R-10a+/-) were obtained by the Cre-Loxp system.The testis of mi R-10a+/-mice were smaller than the control groups from postnatal 10 days. Mi R-10a+/-adult mice displayed male sterility by mating test. The seminiferous tubule of adult mi R-10a+/-mice was empty and there were not sperm by H.E.staining. While there were not significant difference in histology at 7-day-old mice.2. Meiosis of germ cells of mi R-10a+/- mice was blocked by mi R-10 a inhibiting Rad51Location of SYCP3 and SYCP1 were not influenced in the testis of mi R-10a+/- mice whileγ-H2 AX appeared not only in sex chromosome but also in autosome by immunofluorescence.It showed that the DNA damage repair of germ cells was prevented during the meiotic.Furthermore, the expression level of Rad51 was down regulated in the testis of mi R-10a+/-mouse. Rad51 was a potential target gene of micro RNA-10 a by double fluorescence analysis report system(DLR)assays and prediction of targeted sites. And then and both m RNA and protein of Rad51 is down regulated by RT-q PCR and Western-blot when mi R-10 a mimic was transferred into 3T3, GC-1, Hela, and 293 T cells, respectively. Rad51 was confirmed a target gene of mi R-10 a. Because Rad51 had the most significant change and play an important role in meiosis. Western-blot Analysis protein level of the Rad51 was dramaticlly down-regulated in testis of mi R-10a+/-mouse and wide type mouse.3. mi R-10 a inhibited SSCs differentiation in testis of mi R-10a+/- mouse.We found that Mvh and Stra8 positive cells were significantly reduced but the PLZFand GATA4 positive cells were no change by immunofluorescence staining. The results displayed that the number of SSCs had no change but the number of the differentiated germ cells were significantly low.4. Differentiation of germ cells was inhibited in testes of mi R-10a+/- mice by the disorder polarity of germ cell in early spermatogenesis.To further verify the germ cell polarity, the of cdc42 and γ-actin were detected by immunohistochemisty staining in testis of 6-day-old, 10-day-old, 14-day-old, and 60-day-old mi R-10a+/-mice and wild-type mice, respectively. These results showed that there were no difference between 6-day-old mi R-10a+/- mice and wild-type mice. Location of γ-actin in the germ cell nucleus around of 10-day-old mi R-10a+/- mice significantly reduced comparing to wild-type mice. Cdc42, as a sign protein of cell polarity, was analyzed in seminiferous tubules epithelium of mouse testis. We found that cdc42 was higher expression in of the germ cells10-day-old mi R-10a+/- mice than that of wild-type. These results showed that the cdc42 andγ-actin expression may be suppressed. It was inferred that differentiation of the germ cells was inhibited in early spermatogenesis by polarity disorder of germ cells. |
PDF Full Text Request