Effects of local testicular heat treatment on Leydig cell hyperplasia and testosterone biosynthesis in rat testes

Abstract

Cryptorchidism or local testicular heat treatment induces reversible oligospermia or azoospermia in rodents and humans via increased germ cell apoptosis. Research in this field has concentrated on the impact of heat on spermatogenesis, with rather little attention paid to the molecular effects of heat treatment on Leydig cell function. In the present study, we investigated the effects of exposure to heat stress on the proliferative activity and testosterone biosynthesis of Leydig cells. We subjected adult rats to a single local testicular heat treatment of water at 43°C for 30 min. The expression of Leydig cell-specific markers, such as cholesterol side-chain cleavage (P450SCC) and 3β-hydroxysteroid dehydrogenase, was evaluated by immunohistochemistry and western blot analysis. The proliferative activity of Leydig cells was detected by immunostaining with proliferation-associated markers, including Ki67, bromodeoxyuridine and phosphohistone-H3 (pHH3). The mRNA and protein levels of cell cycle proteins and testosterone synthesis-related enzymes were measured by real-time polymerase chain reaction and western blot analysis, respectively. The testes of heat-treated rats contained 50% more Leydig cells than those of control rats, indicating induction of Leydig cell hyperplasia by testicular heat treatment. Increased proliferative activity in Leydig cells, evidenced by enhanced expression of cell cycle proteins, was the main cause of Leydig cell hyperplasia. In addition, heat treatment reduced serum and testicular testosterone concentrations. Consistent with this finding, heat stress downregulated two enzymes required for testosterone biosynthesis, namely cytochrome P450, family 17 (CYP17) and steroidogenic acute regulatory protein, in Leydig cells. Together, the results suggest that testicular heat leads to Leydig cell hyperplasia and a reduction in testosterone biosynthesis in adult rat testes.