RESUMO
OBJECTIVE: To explore the mechanism of hyperthermia inducing infertility by observing the expression of glial cell line-derived neurotrophic factor (GDNF) in rat Sertoli cells cultured in vitro at different temperatures. METHODS: Using combination enzyme digestion and selective adhesion, we isolated Sertoli cells from male Wistar rats and cultured them in vitro at different temperatures, followed by observation of the changes in their adhesion and morphology and identification by FasL immunohistochemical staining. We divided the Sertoli cells into a control group (35 degrees C) and four experimental groups (36 degrees C, 37 degrees C, 38 degrees C, and 39 degrees C), measured their proliferation by CCK-8, observed their morphology and structure by HE staining, and determined the expression of GDNF by RT-PCR, immunofluorescence and Western blot. RESULTS: Sertoli cells were successfully isolated and in vitro-cultured, with a purity of (95.30 +/- 2.15)% (n = 10). The CCK-8 assay showed that the proliferation of the Sertoli cells was the highest at 36 degrees C, gradually decreasing with the temperature above 36 degrees C, and significantly inhibited at 39 degrees C (P < 0.01). Immunofluorescence revealed the expression of GDNF in the cytoplasm, with the highest fluorescence intensity at 36 degrees C. RT-PCR and Western blot exhibited a decreasing trend of the GDNF expression with the increasing temperature above 36 degrees C. There were statistically significant differences in the expression of GDNF between the control group and the four experimental groups (P < 0.01). CONCLUSION: The proliferation and GDNF expression of in vitro-cultured Sertoli cells differ significantly at different temperatures. At > 36 degrees C, the higher the temperature is, the lower the Sertoli cell proliferation and GDNF expression are. Our findings suggest that high temperature above 36 degrees C suppresses the function of Sertoli cells and may also damage spermatogenesis.