OBJECTIVETo investigate the effect of hypoxia on the proliferation and occludin expression of primary rat Sertoli

METHODSWe constructed a primary Sertoli cell system by two-step enzymatic digestion in 18 -22 days old Wistar rats and identified it by oil red O and immunofluorescence methods. We randomly divided the Sertoli cells into five groups to be cultured in oxygen at the concentrations of 20%, 15%, 10%, 5% and 1%, respectively, for 6, 12, 24, 48 and 72 hours. We detected the proliferation of the Sertoli cells by CCK-8 assay, determined the expression of occludin by Western blot, and analyzed the differences among the five groups.

RESULTSOil red O staining revealed red lipid droplets in the cytoplasm of the Sertoli cells, and immunofluorescence showed the positive expression of the FasL protein, with the purity of Sertoli cells over 95% in vitro. Compared with the 20% normoxic group, the proliferation of the Sertoli cells was gradually reduced in the 15% and 10% hypoxia groups, and significantly declined in the 5% and 1% groups (P < 0.01). At 12 hours, the expression of occludin began to decrease with the prolonging of time and reduction of oxygen concentration (P < 0.01).

CONCLUSIONHypoxia suppresses the proliferation of Sertoli cells and reduces the expression of occludin. It could be inferred that hypoxia could damage the integrity of blood-testis barrier and spermatogenesis of the testis.