The mechanism of manganese-induced inhibition of steroidogenesis in rat primary Leydig cells

In previous studies in cultured primary rat Leydig cells, manganese was shown to inhibit hCG-stimulated steroidogenesis of Leydig cells, and the data showed that while the inhibition of StAR protein expression and/or function and mitochondrial dysfunction contribute to the acute reduction of steroidogenesis (2 and 4 h manganese treatment), the enzyme activities of P450scc and 3β-HSD were only reduced after 24 h manganese treatment, we hypothesize that there were different mechanisms for its effect at later stage (24 and 48 h manganese treatment). We further our study by examining StAR mRNA level in cultured primary rat Leydig cells to understand if inhibition of StAR protein expression occurs at the level of transcription of StAR mRNA. The cellular ATP content was measured to determine the extent that manganese altered mitochondrial function. Since mitochondria are regulators of Ca2+ homeostasis, and there are indications that manganese affects intracellular Ca2+ levels, [Ca2+]i was also tested. The effects of manganese on Leydig cell apoptosis and cell cycle distribution were studied to see whether these effects contribute to the reduction of steroidogenesis by manganese at later stage of manganese treatment. In the present study, we demonstrated that manganese could increase [Ca2+]i and reduced ATP contents in primary Leydig cells after 4 h treatment, while the effects on StAR mRNA level appeared later (24 h). Manganese could also induce arrest at the G0/G1 phase cell cycle after 24 h manganese treatment and subsequently increased in the sub-G1 phase DNA contents, indicating induction of apoptosis. Combined with our previous studies, the results indicate that inhibition of StAR protein expression and/or function, mitochondrial dysfunction and disturbance of calcium homeostasis contribute to the adverse effects of manganese on the Leydig cells at the early/immediate stage after treatment (2 and 4 h). However, at later stages (24 and 48 h) manganese could arrest the cell cycle and induce apoptosis of primary Leydig cells, StAR mRNA and enzyme activities of P450scc and 3β-HSD were also reduced, leading to reduced level of steroidogenesis in cultured primary Leydig cells.