Reactive oxygen species (ROS) are involved in a variety of pathophysiological conditions of the testis, and oxidative stress is known to inhibit ovarian and testicular steroidogenesis. The site of ROS-mediated inhibition of steroidogenesis in the corpus luteum and MA-10 tumor Leydig cells was shown to be the hormone-sensitive mitochondrial cholesterol transfer step. The purpose of this study was to examine the effects of ROS on steroidogenic acute regulatory (StAR) protein in MA-10 cells and determine the extent to which MA-10 cell mitochondria are sensitive to oxidative stress. cAMP-stimulated progesterone production was inhibited in a dose-dependent manner in MA-10 cells exposed to H(2)O(2). StAR protein, but not mRNA levels, was decreased in parallel to changes in progesterone production. Even at the highest concentrations of H(2)O(2) tested, there was no effect on P450 side-chain cleavage enzyme protein levels. Oxidative stress from exposure to exogenous xanthine oxidase and xanthine resulted in the inhibition of both progesterone production and StAR protein expression. The mature 30- and 32-kDa intramitochondrial forms of StAR were decreased relative to the 37-kDa extramitochondrial precursor form of StAR, indicating that the ROS-mediated inhibition of StAR protein was due, in part, to the inhibition of mitochondrial import and processing. Vital staining with the fluorescent dye tetramethylrhodamine ethyl ester was used to visualize changes in the mitochondrial electrochemical gradient-dependent membrane potential (Deltapsim). ROS caused a significant dissipation of Deltapsi(m) and time-dependent loss of tetramethylrhodamine ethyl ester fluorescence. The inhibitory effects of H(2)O(2) were transient. There was no evidence for ROS-induced cell death, and following H(2)O(2) removal in the presence of continuous treatment with 8-bromo-cAMP, StAR protein levels and progesterone production were restored. In addition, there was no loss of cell viability following treatment with H(2)O(2) or xanthine/xanthine oxidase as determined by trypan blue exclusion. H(2)O(2) did not cause a significant decrease in total cellular ATP levels. These data indicate that oxidative stress-mediated perturbation of the mitochondria and dissipation of Deltapsi(m) results in the inhibition of StAR protein expression and its import, processing, and cholesterol transfer activity. These findings confirm earlier studies demonstrating the requirement for maintenance of an intact Deltapsi(m) for StAR protein function in cholesterol transport. The significant reduction in the 32- to 30-kDa mature forms of StAR, cessation of cholesterol transport, and loss of Deltapsi(m) are consistent with mitochondrial perturbation because of oxidative stress. This mechanism likely contributes to a host of pathophysiological events evident in testicular disorders such as infection, reperfusion injury, aging, cryptorchidism, and varicocele.

The metabolism of cholesterol by cytochrome P450 side chain cleavage enzyme is hormonally regulated in steroidogenic tissues via intramitochondrial cholesterol transport. The mediating steroidogenic acute regulatory protein (StAR) is synthesized as a 37-kDa (p37) precursor that is phosphorylated by protein kinase A and cleaved within the mitochondria to generate 30-kDa forms (p30, pp30). The effectiveness of modified recombinant StAR forms in COS-1 cells without mitochondrial import has led to a prevailing view that cholesterol transport is mediated by p37 StAR via activity on the outer mitochondrial membrane. The present study of the activation of cholesterol metabolism by bromo-cAMP in adrenal cells in relation to 35 S-StAR turnover indicates that targeting of pp30 to the inner membrane provides the dominant cholesterol transport mechanism. We show that 1) only newly synthesized StAR is functional, 2) phosphorylation and processing of p37 to pp30 occurs rapidly and stoichiometrically, 3) both steps are necessary for optimum transport, and 4) newly synthesized pp30 exhibits very high activity (400 molecules of cholesterol/StAR/min). Segregation of cAMP activation and synthesis of StAR from cholesterol metabolism showed that very low levels of newly synthesized StAR (1 fmol/min/10 6 cells) sustained activated cholesterol metabolism (0.4 pmol/min/10 6 cells, t1 ⁄2 ‫؍‬ 70 min) long after complete removal of p37 (t1 ⁄2 ‫؍‬ 5 min). This activity was highly sensitive to inhibition of processing by CCCP only until sufficient pp30 was formed. Maximum activation preceded bromo-cAMP-induced StAR expression, indicating other limiting steps in cholesterol metabolism.The conversion of cholesterol to pregnenolone, the first step in steroid synthesis, is catalyzed by cytochrome P450 side chain cleavage enzyme (P450scc), 1 which is localized on the matrix side of the inner mitochondrial membrane (1, 2). The transfer of cholesterol to P450scc, a limiting step in this conversion (3, 4), requires hormonal activation of cholesterol mobilization to the mitochondria. This mobilization is dependent on the uptake of lipoproteins, lysosomal-cytosolic transfer, and hydrolysis of cholesterol esters as well as on two subsequent transfer steps (5). The first step involves transfer of cholesterol to the mitochondria and is dependent on an intact cytoskeleton but not on protein synthesis (6). The second step involves transfer within the mitochondria from outer to inner membrane. This trophic hormone-dependent transport of cholesterol from mitochondrial outer membrane to inner membrane (4, 7, 8) is blocked in vivo within 10 min by protein synthesis inhibitors, such as cycloheximide (CHX) (9, 10). A series of cAMP-stimulated and CHX-sensitive phosphoproteins (30 -37 kDa), which localize to the mitochondria, have been identified in cultured adrenal and testis cells (11)(12)(13)(14)(15)(16)(17). The gene that encodes these proteins has been cloned, and the active form has been named the steroidogenic acute regulatory protein (StAR) (18). The exp...

Inflammatory disease has been established to affect male reproductive function and fertility. Relevant inflammatory diseases include general and chronic infectious diseases as well as localized acute or chronic infections of the male genitourinary tract. Male accessory gland infections account for almost 15% of all cases of male infertility seen in infertility clinics while fertility usually is not a clinical objective among patients with acute systemic infections such as Gram-negative sepsis. Infections of the male accessory glands frequently are associated with increased counts of white blood cells in semen and elevated levels of proinflammatory cytokines in semen and the testis. There is a mounting body of evidence that demonstrates the importance of cytokines and chemokines in the regulation of testicular and glandular function during pathophysiological states as well as under normal physiological conditions when cytokines act as growth and differentiation factors. The purpose of this review is to examine the role of cytokines in the regulation of steroidogenesis and spermatogenesis in the testis under physiological and pathophysiological conditions and considers clinical investigations that help to improve the evaluation and treatment of male infertility.

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 study of Leydig cell function has focused on the mechanisms regulating steroidogenesis; however, few investigations have examined the importance of mitochondria in this process. The purpose of this investigation was to determine which aspects of mitochondrial function are necessary for acute cAMP-stimulated Leydig cell steroidogenesis. MA-10 cells were treated with 8-bromoadenosine 3',5'-cyclic monophosphate (cAMP) and different site-specific agents that disrupt mitochondrial function, and the effects on acute cAMP-stimulated progesterone synthesis, StAR mRNA and protein, mitochondrial membrane potential (Deltapsim), and ATP synthesis were determined. cAMP treatment of MA-10 cells resulted in significant increases in both cellular respiration and Deltapsim. Dissipating Deltapsim with carbonyl cyanide m-chlorophenyl hydrazone resulted in a profound reduction in progesterone synthesis, even in the presence of newly synthesized StAR protein. Preventing electron transport in mitochondria with antimycin A significantly reduced cellular ATP, potently inhibited steroidogenesis, and reduced StAR protein levels. Inhibiting mitochondrial ATP synthesis with oligomycin reduced cellular ATP, inhibited progesterone synthesis and StAR protein, but had no effect on Deltapsim. Disruption of intramitochondrial pH with nigericin significantly reduced progesterone production and StAR protein but had minimal effects on Deltapsim. 22(R)-hydroxycholesterol-stimulated progesterone synthesis was not inhibited by any of the mitochondrial reagents, indicating that neither P450 side-chain cleavage nor 3beta-hydroxysteroid dehydrogenase activity was inhibited. These results indicate that Deltapsim, mitochondrial ATP synthesis, and mitochondrial pH are all required for acute steroid biosynthesis. 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.

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 (delta psi(m)) and ATP synthesis were determined. Dissipating delta psi(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 delta psi(m). Disruption of pH with nigericin significantly reduced progesterone production and StAR protein, but had minimal effects on delta psi(m). Sodium arsenite at low concentrations inhibited StAR protein but not mRNA expression and inhibited progesterone without disrupting delta psi(m). The mitochondrial Ca2+ inhibitor Ru360 also inhibited StAR protein expression. These results demonstrate that delta psi(m), ATP synthesis, delta pH 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.