Mitochondrial function in Leydig cell steroidogenesis

The first and rate-limiting step in the biosynthesis of steroid hormones is the transfer of cholesterol into mitochondria, which is facilitated by the steroidogenic acute regulatory (StAR) protein. Recent studies of Leydig cell function have focused on the molecular events controlling steroidogenesis; however, few studies have examined the importance of the mitochondria. The purpose of this investigation was to determine which aspects of mitochondrial function are necessary for Leydig cell steroidogenesis. MA-10 tumor Leydig cells were treated with 8-bromo-cAMP (cAMP) and site-specific mitochondrial disrupters, pro-oxidants, and their effects on progesterone synthesis, StAR expression, mitochondrial membrane potential (ΔΨ_m) and ATP synthesis were determined. Dissipating ΔΨ_m, with CCCP inhibited progesterone synthesis, even in the presence of newly synthesized StAR protein. The electron transport inhibitor antimycin A significantly reduced cellular ATP, inhibited steroidogenesis, and reduced StAR protein expression. The F₀/F₁ ATPase inhibitor oligomycin reduced cellular ATP and inhibited progesterone synthesis and StAR protein expression, but had no effect on ΔΨ_m) Disruption of pH with nigerian significantly reduced progesterone production and StAR protein, but had minimal effects on ΔΨ_m. Sodium arsenite at low concentrations inhibited StAR protein but not mRNA expression and inhibited progesterone without disrupting ΔΨ_m. The mitochondrial Ca²⁺ inhibitor Ru360 also inhibited StAR protein expression. These results demonstrate that ΔΨ_m, ATP synthesis, ΔpH and [Ca²⁺] _mt are all required for steroid biosynthesis, and that mitochondria are sensitive to oxidative stress. These results suggest that mitochondria must be energized, polarized, and actively respiring to support Leydig cell steroidogenesis and alterations in the state of mitochondria may be involved in regulating steroid biosynthesis.