| Objective:To explore the mechanism of infertility caused by hyperthermia by investigating the expression of GDNF of in vitro-cultured sertoli cells at different temperature conditions.Methods:Using the combination enzyme digestion and selective adhesion method to isolate the sertoli cells from the male Wistar rat, the isolated Sertoli cells were in vitro-cultured separately in different temperatures, observing the adherence and morphology and identifying the changes by FasL immunohistochemistry staining.These Sertoli cells were divided into two groups:control group (35℃),experimental groups(36℃、37℃、38℃、39℃). The proliferation of Sertoli cells was measured by CCK-8. Morphology and structure of Sertoli cells were observed by hematoxylin-eosin staining under microscope. The expression of GDNF were respectively detected by RT-PCR, Immunofluorescence and Western blotting.Results:Sertoli cells were isolated and in vitro-cultured successfully, and the purity was (95.30±2.15)%(n=10).The CCK-8assay showed that, the proliferation of Sertoli reach a peak at36℃, with the temperature higher than36℃,the proliferation become lower, until39℃, the proliferation was obviously inhibited(P<0.01). The fluorescence intensity in cytoplasm was the highest at36℃. The results of RT-PCR and Western blotting showed, when the temperature exceed36℃, the higher the temperature is, the lower expression of GDNF. Compared all the results with the control group, the differences of GDNF in four groups(36℃、37℃、38℃/39℃) are statistically significant (P<0.01).Conclusion:The proliferation and GDNF expression of in vtro-cultured Sertoli cells were significantly different at different temperatures,and when the temperature is higher than36℃, the higher the temperature, the lower expression of GDNF and capacity of multiplication. These results suggested that the functions of SC were prohibited and damaged at high temperatures which may also lead to the destruction of nicke for spermatogenesis. |
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