Abstract
Objective
The objective of this study were to determine whether the inhibition of clusterin expression in rat Sertoli cells enhances heat stress-induced apoptosis.
Methods
Adult male Sprague-Dawley rats were used in this study. The scrotum of rats were completely immersed in a water bath and exposed to either 43 or 37 °C as a control for 15 min. Rats were then sacrificed on days 1, 4, 7, 14 and 21 after heat treatment. Expression levels of clusterin mRNA and protein were evaluated respectively, and immunohistochemical staining of clusterin and TUNEL assay were also performed. We then purified Sertoli cells from rat testes, and expression vector containing short hairpin RNA (shRNA) targeting rat clusterin gene or control vector alone was transiently transfected into Sertoli cells.
Results
Clusterin mRNA and protein expression levels were significantly up-regulated and peaked on day 21. The apoptotic index was markedly increased 1 day. In purified Sertoli cells, following exposure to heat stress at 41 °C for 12 h, clusterin mRNA was markedly up-regulated after transfection with the control vector; however, the transfection of siRNA targeting clusterin resulted in <70% reduction in the expression of clusterin mRNA. Furthermore, the apoptotic index in these Sertoli cells was significantly higher after the treatment with siRNA targeting clusterin than control, and the most prominent difference was observed within 24 h after the heat treatment.
Conclusions
These results suggest that an increase in the secretion of clusterin by Sertoli cells protects the testes from heat stress-induced injury.
Keywords: Clusterin, heat stress, Sertoli cell, apoptosis