Regulation of testosterone synthesis in Leydig cells by ClC-2 chloride channel

Testicular Leydig cells are the main source of testosterone, which is essential for spermatogenesis and male fertility. Leydig cell steroidogenesis is regulated by chloride channels, the molecular composition of which remains obscure. ClC-2 is a hyperpolarization-activated chloride channel present in virtually all tissues. ClC-2 deficiency is associated with the loss of male germ cells in mice and male azoospermia in humans. However, the functional significance of ClC-2 in Leydig cells is still unclear. Herein, we aimed to test the hypothesis that the ClC-2 chloride channel may play a regulatory role in Leydig cell steroidogenesis. ClC-2 expression was validated in testicular Leydig cells in adult mice. Lentivirus-delivered shRNA knockdown of endogenous ClC-2 expression in the mouse Leydig tumor cell line MA-10 significantly reduced basal mRNA expression of steroidogenic genes involved in testosterone synthesis, resulting in a dramatic decline in basal testosterone secretion. Application of 8-bromo-cAMP effectively increased the expression of steroidogenic genes and production of testosterone in MA-10 cells. These 8-bromo-cAMP-induced effects were notably attenuated following ClC-2 knockdown, as well as inhibition of the T-type voltage-gated calcium channel. Conversely, enhanced ClC-2 protein level promoted steroidogenic gene expression and testosterone synthesis in MA-10 cells. Importantly, in vivo intratesticular injection of a shClC-2 lentivirus potently diminished intratesticular testosterone levels in adult mice. Taken together, our data highlight a critical role of ClC-2 in regulating testosterone production in Leydig cells and a mechanistic link between testosterone deficiency and loss-of-function mutation in the ClC-2 chloride channel.