Dimethoate is a widely used organophosphate insecticide that has been shown to disrupt reproductive function in animals. Although the pathogenesis of Dimethoate-induced reproductive toxicity remains to be determined, a reduction in serum testosterone levels is thought to play an important role in the development of Dimethoate-induced infertility. Since Leydig cells play a crucial role in male reproductive function by producing testosterone, the mouse MA-10 Leydig tumor cell line was used to determine if Dimethoate can directly block steroid hormone biosynthesis and to identify the site of steroidogenic inhibition. Dimethoate inhibited steroidogenesis in both a dose- and time-dependent manner without affecting total protein synthesis or protein kinase A activity. While it decreased the activity of the P450 side chain cleavage (P450 scc) enzyme, a reduction in the activity of this enzyme alone could not account for the level of Bu(2)cAMP-inhibited progesterone production. Instead, our results suggest that Dimethoate inhibited steroidogenesis primarily by blocking transcription of the steroidogenic acute regulatory (StAR) gene. This finding is significant since StAR protein mediates the rate-limiting and acutely-regulated step in steroidogenesis, the transfer of cholesterol from the outer to the inner mitochondrial membrane. This study indicates that StAR may be an important target for environmental pollutants which disrupt steroidogenesis and impair reproductive function.
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LP Walsh, DR Webster, and DM Stocco
C Paz, F Cornejo Maciel, P Maloberti, LP Walsh, DM Stocco, and EJ Podesta
The LH signal transduction pathway features the activation of protein tyrosine phosphatases (PTPs) as one of the components of a cascade that includes other well characterized events such as cAMP-dependent protein kinase A (PKA) activation. Moreover, the action of PTPs is required to increase the rate-limiting step in steroid biosynthesis, namely the cAMP-regulated transfer of cholesterol to the inner mitochondrial membrane. Since both PKA activity and steroidogenic acute regulatory (StAR) protein induction are obligatory steps in this transfer of cholesterol, the present study was performed to investigate the role of PTPs in the regulation of PKA activity and StAR expression in response to LH/chorionic gonadotropin (CG) and 8Br-cAMP in MA-10 cells. While the exposure of MA-10 cells to the PTP inhibitor, phenylarsine oxide (PAO), did not modify PKA activity, it partially inhibited the effect of human CG and cAMP analog on StAR protein levels. Time-course studies demonstrated that PAO inhibited cAMP induction of StAR protein and mRNA. At 30 min, the effect on cAMP-stimulated StAR protein levels was a 35% inhibition, progressing to up to 90% inhibition at 120 min of stimulation. The maximal inhibitory effect on cAMP-induced StAR mRNA level was obtained at 60 min (85%). In summary, these results demonstrate that inhibition of PTP activity affected both StAR protein and mRNA synthesis and suggest that the activity of hormone-regulated PTPs is a requirement in the LH signaling cascade that results in the up-regulation of StAR protein and, subsequently, increased steroid synthesis.