Decrease In Steroidogenesis Research Articles

Transcriptomic characterization of interactions between sodium selenite and coenzyme Q10 on preventing cadmium-induced testicular defects

The effect of heavy metal cadmium (Cd) on testicular function is recognized. However, the mechanism involved is not well-established. In the present study, we analyzed the testicular transcriptomic changes induced by acute Cd exposure of adult rats with and without supplementation of antioxidants selenium (Se) and/or coenzyme Q10 (CoQ). Cd significantly decreased serum testosterone and two steroidogenic proteins SCARB1 and STAR. RNA-Seq analyses of testicular RNAs revealed specific activation of oxidative stress-, inflammation-, MAPK- and NF-κB-related signaling molecules. In addition, Cd treatment down-regulated gene for I, III and IV complexes of mitochondrial electron transport chain and up-regulated genes for NADPH-oxidase, major cascade in ROS production. The decrease in steroidogenesis and increase in inflammation may result from oxidative stress since supplementation of Se and CoQ, but not with either alone, almost completely prevented these changes, including overall alterations in transcriptome. Cd exposure induced total of 1192 differentially expressed genes (DEGs), which was reduced to 29 without considering confounding factors associated with Se/CoQ, a 97.6% protection rate. In conclusion, Cd exposure inhibited Leydig cell steroidogenesis by down-regulating SCARB1 and STAR through increasing oxidative stress and inflammation, but Se plus CoQ synergistically prevented all the changes induced by the Cd exposure.

DEHP Decreases Steroidogenesis through the cAMP and ERK1/2 Signaling Pathways in FSH-Stimulated Human Granulosa Cells.

DEHP is an endocrine disruptor that interferes with the function of the female reproductive system. Several studies suggested that DEHP affects steroidogenesis in human and rodent granulosa cells (GC). Some studies have shown that DEHP can also affect the FSH-stimulated steroidogenesis in GC; however, the mechanism by which DEHP affects hormone-challenged steroidogenesis in human GC is not understood. Here, we analyzed the mechanism by which DEHP affects steroidogenesis in the primary culture of human cumulus granulosa cells (hCGC) stimulated with FSH. Cells were exposed to DEHP and FSH for 48 h, and steroidogenesis and the activation of cAMP and ERK1/2 were analyzed. The results show that DEHP decreases FSH-stimulated STAR and CYP19A1 expression, which is accompanied by a decrease in progesterone and estradiol production. DEHP lowers cAMP production and CREB phosphorylation in FSH but not cholera toxin- and forskolin-challenged hCGC. DEHP was not able to decrease steroidogenesis in cholera toxin- and forskolin-stimulated hCGC. Furthermore, DEHP decreases FSH-induced ERK1/2 phosphorylation. The addition of EGF rescued ERK1/2 phosphorylation in FSH- and DEHP-treated hCGC and prevented a decrease in steroidogenesis in the FSH- and DEHP-treated hCGC. These results suggest that DEHP inhibits the cAMP and ERK1/2 signaling pathways, leading to the inhibition of steroidogenesis in the FSH-stimulated hCGC.

Effects of SLCO1B1 Genetic Variant on Metabolite Profile in Participants on Simvastatin Treatment.

Organic-anion-transporting polypeptide 1B1 (OATP1B1), encoded by the solute carrier organic anion transporter family member 1B1 gene (SLCO1B1), is highly expressed in the liver and transports several endogenous metabolites into the liver, including statins. Previous studies have not investigated the association of SLCO1B1 rs4149056 variant with the risk of type 2 diabetes (T2D) or determined the metabolite signature of the C allele of SLCO1B1 rs4149056 (SLCO1B1 rs4149056-C allele) in a large randomly selected population. SLCO1B1 rs4149056-C inhibits OATP1B1 transporter and is associated with increased levels of blood simvastatin concentrations. Our study is to first to show that SLCO1B1 rs4149056 variant is not significantly associated with the risk of T2D, suggesting that simvastatin has a direct effect on the risk of T2D. Additionally, we investigated the effects of SLCO1B1 rs4149056-C on plasma metabolite concentrations in 1373 participants on simvastatin treatment and in 1368 age- and body-mass index (BMI)-matched participants without any statin treatment. We found 31 novel metabolites significantly associated with SLCO1B1 rs4149056-C in the participants on simvastatin treatment and in the participants without statin treatment. Simvastatin decreased concentrations of dicarboxylic acids, such as docosadioate and dodecanedioate, that may increase beta- and peroxisomal oxidation and increased the turnover of cholesterol into bile acids, resulting in a decrease in steroidogenesis due to limited availability of cholesterol for steroid synthesis. Our findings suggest that simvastatin exerts its effects on the lowering of low-density lipoprotein (LDL) cholesterol concentrations through several distinct pathways in the carriers of SLCO1B1 rs4149056-C, including dicarboxylic acids, bile acids, steroids, and glycerophospholipids.

Oleic acid reduces steroidogenesis by changing the lipid type stored in lipid droplets of ovarian granulosa cells

BackgroundOleic acid is an abundant free fatty acid present in livestock that are in a negative energy-balance state, and it may have detrimental effects on female reproduction and fertility. Oleic acid induces lipid accumulation in bovine granulosa cells, which leads to a foam cell-like morphology and reduced steroidogenesis. However, why oleic acid increases lipid accumulation but decreases steroidogenesis remains unclear. This study focused on oleic acid’s effects on lipid type and steroidogenesis.ResultsOleic acid increased the lipid accumulation in a concentration-dependent manner and mainly increased the triglyceride level and decreased the cholesterol ester level. Oleic acid also led to a decline in estradiol and progesterone production in porcine granulosa cells in vitro. In addition, oleic acid up-regulated the expression of CD36 and diacylglycerol acyltransferase 2, but down-regulated the expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, scavenger receptor class B member 1 and acetyl-Coenzyme A acetyltransferase 2, as well as steroidogenesis-related genes, including cytochrome P450 family 11 subfamily A member 1, cytochrome P450 family 19 subfamily A member 1 and 3 as well as steroidogenic acute regulatory protein at the mRNA and protein levels. An oleic acid-rich diet also enhanced the triglyceride levels and reduced the cholesterol levels in ovarian tissues of female mice, which resulted in lower estradiol levels than in control-fed mice. Compared with the control, decreases in estrus days and the numbers of antral follicles and corpora lutea, as well as an increase in the numbers of the atretic follicles, were found in the oleic acid-fed female mice.ConclusionsOleic acid changed the lipid type stored in lipid droplets of ovarian granulosa cells, and led to a decrease in steroidogenesis. These results improve our understanding of fertility decline in livestock that are in a negative energy-balance state.

GONADOPROTECTIVE PROPERTIES OF ANTIOXIDANT COMPLEX IN CONDITIONS CAUSED BY TOXIC EFFECTS OF HERBICIDES

Nowadays, when the nation’s temporary economic difficulties have led to a decline in the agriculture share of the overall economy, the problem of controlling weeds of agricultural land is especially urgent. Undoubtedly, in our country, with the strengthening of the economy, the use of plant protection products is growing, requiring even closer attention to the problem of the environmental impact of herbicides and the rehabilitation of soils contaminated with residues of poisonous chemicals. The general mechanism of the action affecting herbicides of different groups is the violation of redox processes, and, in particular, the increase in free radical lipid peroxidation, first of all, in gonadal cell membranes that can eventually lead to the destruction throughout the reproductive period. Moreover, they produce gonadotoxic effects leading to a decrease in steroidogenesis, and as a consequence, to impaired spermatogenesis.
 The aim of the study was to investigate the gonadoprotective properties of the antioxidant complex, its effect on the parameters of lipid peroxidation, morphological and functional changes in the testes of rats exposed to the toxic effect of the clopyralid herbicide.
 The introduction of this complex under the conditions mentioned above contributed not only to a significant decrease in free radical lipid perozidation in the blood and tissues of the testes, but also to normalization of the state of spermatogenic epithelium, quantitative sperm indicators, normalization of sperm motility. It has been proven the complex of antioxidants not only affects the level of spermatogenic epithelium, but also improves the quality of sperm, contributing to the restoration of sperm motility. The studies of morphological changes in the testes and the functional maturation of the sperm have shown that the most significant changes were registered during the correction with the antioxidant complex, emphasizing its more pronounced gonadoprotective effect.
 The results obtained indicate the feasibility of using this antioxidant complex as gonadoprotectant in cases of chronic intoxication with herbicides.

Photoperiodic regulation of ovarian steroidogenesis in a tropical rodent, Funambulus pennanti: role of melatonin and MT1

ABSTRACTTo elucidate the role of photoperiodically modulated endogenous melatonin and its receptor MT1 in the ovary of a diurnal rodent, adult Indian palm squirrel Funambulus pennanti were exposed to Short Day (SD; 16L:8D) and Long Day (LD; 16L:8D) and compared with natural day controls (NDL; 12L:12D). Plasma 17-β estradiol level was significantly low in SD group while expression of StAR, P450SCC, 17-β HSD, and aromatase showed significant down-regulation in the ovary of squirrels maintained under SD when compared to LD and NDL groups suggesting an overall decrease in steroidogenesis. Expression of ERα in the ovary was significantly lower in SD than both LD and NDL groups, having a positive correlation with plasma estradiol level. Circulatory melatonin level was significantly high in SD-exposed squirrels, while expression of MT1R decreased in the ovary when compared to LD and NDL groups, showing a negative correlation with plasma melatonin levels. Expression of cell proliferation marker Proliferating Cell Nuclear Antigen (PCNA) was positively correlated to MT1R expression in the ovary being significantly low in SD-exposed squirrels. Our results suggest that even in tropical rodents seasonal variation in photoperiod is responsible for initiating female reproductive activities where circulatory melatonin via MT1R plays an important role.

Dual effect of insulin resistance and cadmium on human granulosa cells - In vitro study

Combined exposure of cadmium (Cd) and insulin resistance (IR) might be responsible for subfertility. In the present study, we investigated the effects of Cd in vitro in IR human granulosa cells. Isolated human granulosa cells from control and polycystic ovary syndrome (PCOS) follicular fluid samples were confirmed for IR by decrease in protein expression of insulin receptor-β. Control and IR human granulosa cells were then incubated with or without 32μM Cd. The combined effect of IR with 32μM Cd in granulosa cells demonstrated significant decrease in expression of StAR, CYP11A1, CYP19A1, 17β-HSD, 3β-HSD, FSH-R and LH-R. Decrease was also observed in progesterone and estradiol concentrations as compared to control. Additionally, increase in protein expression of cleaved PARP-F2, active caspase-3 and a positive staining for Annexin V and PI indicated apoptosis as the mode of increased cell death ultimately leading to decreased steroidogenesis, as observed through the combined exposure. Taken together the results suggest decrease in steroidogenesis ultimately leading to abnormal development of the follicle thus compromising fertility at the level of preconception.

Melatonin membrane receptor (MT1R) expression and nitro-oxidative stress in testis of golden hamster, Mesocricetus auratus: An age-dependent study

Age-dependent decline in melatonin level induces nitro-oxidative stress that compromises physiological homeostasis including reproduction. However, less information exist regarding the age-dependent variation in local melatonin (lMel) concentration and MT1R expression in testis and its interaction with testicular steroidogenesis and nitro-oxidative stress in golden hamster, Mesocricetus auratus. Therefore, we evaluated lMel level along with MT1R expression and its possible interaction with steroidogenesis and nitro-oxidative stress in testes of young (6weeks), adult (15weeks) and old (2years) aged hamsters. Further, we injected the old hamsters with melatonin to address whether age-related decline in lMel and MT1R is responsible for the reduction in testicular steroidogenesis and antioxidant status. Increased expression of steroidogenic markers suggests increased testicular steroidogenesis in adult hamsters that declined in old hamsters. An age-dependent elevation in the level of NOX, TBARS, corticosterone and the expression of iNOS and GR with a concomitant decrease in enzyme activities for SOD, CAT, GSH-PX indicate increased nitro-oxidative stress in testes. Data suggest that reproductive senescence in male hamsters might be a consequence of declined lMel concentration with MT1R expression inducing nitro-oxidative stress resulting in diminished testicular steroidogenesis. However, administration of Mel in old-aged hamsters significantly increased steroidogenesis and antioxidant status without a significant variation in lMel concentration and MT1R expression in testes. Therefore, decreased lMel and MT1R might not be the causative factor underlying the age-associated decrease in antioxidant defence and steroidogenesis in testes. In conclusion, Mel induced amelioration of testicular oxidative insult and elevation of steroidogenic activity suggests a potential role of increased nitro-oxidative stress underlying the age-dependent decrease in steroidogenesis.

Adrenal and Ovarian Phenotype of a Tissue-Specific Urocortin 2-Overexpressing Mouse Model.

Urocortin 2 (UCN2) is a neuropeptide of the CRH family, involved in homeostatic mechanisms, the stress response, and control of anxiety. To elucidate the effects of UCN2 on steroidogenesis, we developed a mouse model that allows a Cre recombinase-determined conditional overexpression of UCN2 (UCN2-COE). In these mice SF1-Cre-driven overexpression of UCN2 was restricted to the adrenal glands, gonads, and parts of the hypothalamus. UCN2-COE animals of both sexes revealed significantly higher plasma UCN2 levels and significantly higher UCN2 expression levels in the adrenals and ovaries. In contrast, the baseline expression of UCN2 was already high in the testes of control mice with no further increase achievable in UCN2-COE animals. Adrenal steroidogenesis of UCN2-COE animals was investigated under baseline conditions, upon an ACTH stimulation test, and following a restraint stress test. A tendency toward lower expression of steroidogenic enzymes was detectable in UCN2-COE animals of both sexes with slight differences between males and females. A similar reduction in the expression levels of the final steps of ovarian steroidogenesis, accompanied by reduced plasma estradiol levels, was observed in female UCN2-COE animals. Thus, adrenal UCN2 overexpression resulted in down-regulation of adrenal steroidogenesis, suggesting a reduction in the stress response in the mouse (stress coping behavior). Similarly, UCN2 overexpression in the ovaries caused a decrease in steroidogenesis and reduction of follicles that had undergone ovulation. Nevertheless, this finding was not associated with reduced fertility.

The Opposing Roles of Nitric Oxide and cGMP in the Age-Associated Decline in Rat Testicular Steroidogenesis

The molecular mechanism of the aging-associated dysfunction of Leydig cells (LCs) is complex and poorly understood. In this study, we analyzed the contribution of nitric oxide (NO) and cGMP signaling to the age-dependent decline in LC function. Significant (>50%) decreases in serum, intratesticular, and LC androgens in aging rats (15-24 months) were accompanied by a proportional increase in NO production, an up-regulation of cGMP levels, and the expression of soluble guanylyl cyclase-1B and protein kinase G1 in LCs. In contrast, LC cAMP levels decreased with age, most likely reflecting the up-regulation of cAMP-specific phosphodiesterase expression. Moreover, the expression of genes encoding enzymes responsible for cholesterol transport and its conversion to T were reduced. Exposing LCs from aged animals to NO further increased cGMP levels and decreased cAMP and androgen production, whereas the addition of cell-permeable 8-bromoguanosine-cGMP alone had the opposite effect. In vivo inhibition of cGMP-specific phosphodiesterase-5 for 3 and 6 months in aged rats led to a partial restoration of androgens, NO, and cyclic nucleotide levels, as well as the expression of steroidogenic and NO/cGMP signaling genes. These results indicate that a progressive increase in NO production contributes to the age-dependent decrease in steroidogenesis in a cGMP-independent manner, whereas the sustained elevation in cGMP levels significantly slows the decline in LC function.

Resveratrol potentiates effects of simvastatin on inhibition of rat ovarian theca-interstitial cell steroidogenesis

We have previously reported that simvastatin (SIM), a competitive inhibitor of HMG-CoA reductase, a rate-limiting step of the mevalonate pathway, inhibits rat theca-interstitial cell steroidogenesis by decreasing CYP17A1 gene expression, the key enzyme of the androgen biosynthesis pathway. Recently, we demonstrated that resveratrol (RES), a bioflavonoid abundant in red grapes, decreases HMG-CoA reductase gene expression in rat theca-interstitial cells and may enhance the inhibitory effect of simvastatin on the mevalonate pathway. The present study evaluated the effect of combining RES and SIM treatments on rat theca-interstitial cell steroidogenesis. In-vitro study. Rat theca-interstitial cells isolated from 30 day-old female rats were cultured for up to 48 hrs in the absence (control) or in the presence of SIM (1 μM) and/or RES (10-30 μM). Gene expression of steroidogenic enzymes (CYP11A1, CYP17A1) was evaluated by qrt-PCR. Androstenedione and androsterone levels in culture media were determined using liquid chromatography-mass spectrometry. Comparisons between groups were performed using ANOVA and Tukey test. SIM decreased androstenedione levels in culture media by 92% (P<0.001) and androsterone production by 60% (P<0.001), whereas the addition of RES to SIM-treated cultures decreased androstenedione levels by 79-88% (P<0.001) and androsterone production by 82-91% (P<0.001), respectively. SIM inhibited both CYP11A1 and CYP17A1 gene expression, respectively, by 24% (P<0.001) and 87% (P<0.001). However, RES in combination with SIM decreased both CYP11A1 and CYP17A1 gene expression, respectively, by 55-72% (P<0.001) and 95-99% (P<0.001). SIM inhibits androgen production in theca-interstitial cells, and this SIM-induced decrease in steroidogenesis is enhanced by the addition of RES. The present findings may lead to the development of novel treatments of PCOS involving a combination of SIM and RES.

Protein kinase G-mediated stimulation of basal Leydig cell steroidogenesis

The androgen-secreting Leydig cells produce cGMP, but the pathways responsible for generation and actions of this intracellular messenger have been incompletely characterized in these cells. Here, we show the presence of mRNA transcripts for the membrane-bound and soluble guanylyl cyclases (sGC), the cGMP-specific phosphodiesterase 5, and the cGMP-dependent protein kinase I (PKG I) and PKG II in purified rat Leydig cells from adult animals. Stimulation of both guanylyl cyclases and inhibition of phosphodiesterase 5 in vitro were accompanied by elevations in cGMP and androgen production, whereas inhibition of sGC and PKG led to a decrease in steroidogenesis. The stimulatory action of cGMP on steroidogenesis was preserved in cells with inhibited cAMP-dependent protein kinases. Experiments with exogenously added substrates revealed the dependence of cGMP-induced progesterone and androgen synthesis on cholesterol but not on 22-OH cholesterol, pregnenolone, progesterone, and Delta(4)-androstenedione. Treatment with nitric oxide donor increased phosphorylation of the steroidogenic acute regulatory protein (StAR). In contrast, inhibition of sGC and PKG, but not protein kinase A, significantly reduced StAR phosphorylation. These results suggest that cGMP contributes to the control of basal steroidogenesis in Leydig cells through the PKG-dependent modification of the StAR protein.

Caspase-3 is a pivotal mediator of apoptosis during regression of the ovarian corpus luteum.

Because caspase-3 is considered a primary executioner of apoptosis and has been implicated as a mediator of luteal regression, we hypothesized that corpora lutea (CL) derived from caspase-3 null mice would exhibit a delayed onset of apoptosis during luteal regression, when compared with CL derived from wild-type (WT) mice. To test this hypothesis, ovulation was synchronized in immature (postpartum d 24-27) WT and caspase-3-deficient female littermates by exogenous gonadotropins. Individual CL were isolated by manual dissection, 30 h after ovulation, and placed in organ culture dishes in the absence of serum and growth factors. At the time of isolation (0 h) and after 24, 48, and 72 h in culture, the CL were removed and assessed for the presence of processed (active) caspase-3 enzyme and for apoptosis by multiple criteria. There was no evidence of active caspase-3 enzyme or apoptosis in either WT or caspase-3-deficient CL before culture. However, CL derived from the WT mice exhibited a time-dependent increase in the level of active caspase-3 and apoptosis during culture. By comparison, CL derived from caspase-3-deficient mice, cultured in parallel, failed to exhibit any detectable active caspase-3 and showed attenuated rates of apoptosis. To extend these findings derived from ex vivo culture experiments, ovaries were collected from WT and caspase-3 null female littermates at 2, 4, or 6 d post ovulation, and the occurrence of apoptosis within the CL was analyzed. Whereas ovaries of WT mice had only residual luteal tissue at d 6 post ovulation, ovaries collected from caspase-3-deficient mice retained many CL, at d 6 post ovulation, that were similar in size to those observed in the early luteal phase of WT mice. Importantly, there was no dramatic increase in apoptosis in CL of caspase-3-deficient mice at any time point examined post ovulation, indicating that the involution process had indeed been delayed. In contrast, the levels of progesterone declined regardless of genotype. These data provide the first direct evidence that caspase-3 is functionally required for apoptosis to proceed normally during luteal regression. However, caspase-3 is not a direct mediator of the decrease in steroidogenesis associated with luteolysis.

Ethanol-induced inhibition of testosterone biosynthesis in rat leydig cells: Central role of mitochondrial NADH redox state

The mechanisms by which ethanol (EtOH) inhibits the human chorionic gonadotropin (hCG)-stimulated testosterone synthesis was studied in isolated rat Leydig cells in vitro. EtOH inhibited steroidogenesis, but this inhibition was reversed by L-glutamate (Glu) and an uncoupler of the oxidative phosphorylation, 2,4-dinitrophenol (DNP). The mechanism of EtOH-induced inhibition was studied by measuring steroidogenic precursors and comparing them with the cytosolic and mitochondrial NADH redox states during uncoupling or in the presence of Glu. DNP had a dual effect. Low concentrations abolished the EtOH-induced inhibition of progesterone to testosterone formation suggesting that the inhibitory step was at or before progesterone formation. A large concentration led to an overall decrease in steroidogenesis indicating toxic effects on steroidogenesis. The mitochondrial NADH/NAD+ ratio, measured as the 3-hydroxybutyrate/acetoacetate ratio, decreased simultaneously when steroidogenesis was stimulated, either during uncoupling or in the presence of Glu, whereas cytosolic NADH/NAD+ ratio, measured as lactate/pyruvate ratio showed no response. These results demonstrate that the rise in the mitochondrial NADH/NAD+ ratio rather than in the cytosolic one is connected with the inhibition of testosterone synthesis by EtOH in isolated Leydig cells. The EtOH-induced high mitochondrial NADH/NAD+ ratio may deplete mitochondrial oxalacetate concentrations. This can decrease the activity of several transport shuttles and interrupt the flow of mitochondrial citrate into the smooth endoplasmic reticulum, which then reflects to decreased rate of steroidogenesis in the presence of ethanol.

Action of ACTH, cAMP and cytochalasin B on steroid production by Y-1 mouse adrenal tumor cells in culture.

Continued incubation of Y-1 mouse adrenal tumor cells with adrenocorticotrophic hormone (ACTH) or with dibutyryl-3′,5′-cyclic adenosine monophosphate (dbcAMP) resulted in an initial increase and a subsequent decrease in steroidogenesis. ACTH-or dbcAMP-stimulated steroidogenesis was inhibited by cytochalasin B (CB) to approximately the same extent during the entire period of incubation; CB inhibition of the ACTH response was reversible.

BIOGENESIS OF CORTICOSTEROIDS IN MONOLAYER CULTURES OF HUMAN FOETAL ADRENAL CELLS

Human foetal adrenal cells were grown in monolayer culture and their steroidogenic capacity observed for up to a month. The cells produced a complex array of steroids and some of their ester sulphates from endogenous as well as from [14C] and [3H] precursors. ACTH stimulated corticoidogenesis, particularly cortisol secretion, and markedly enhanced the incorporation of progesterone and pregnenolone into cortisol. Following incubation with the same precursors, large amounts of radioactivity remained water soluble. From the butanol extractable material of this fraction, dehydroepiandrosterone sulphate was characterized as the main metabolite of pregnenolone and corticosterone and 11-deoxycorticosterone sulphates as the main metabolites of progesterone. With time in culture there was a decrease in steroidogenesis as well as a steady decline in responsiveness to ACTH, mainly manifested by cortisol secretion. The medium from homologous foetal pituitary cultures stimulated cortisol production by the human adrenal cell monolayer.

EFFECT OF OP'DDD ON SERUM ANDROGENS IN A GIRL WITH METASTATIC VIRILIZING ADRENAL CARCINOMA

In metastatic adrenal carcinoma, op'DDD has been reported to decrease urinary 17-KS without causing regression of the tumor. This dissociation between the oncolytic and hormonal effects may be explained by our steroidal data. In a 5 y/o girl with metastatic adrenal carcinoma, serum T, Δ4, DHEA, DS and urinary 17-KS were extremely elevated. With op'DDD treatment, the DS and 17-KS fell rapidly while there was a marked delay in the fall of free androgens. The persistence of free steroid secretion with decreased conjugate formation suggests that op'DDD specifically altered sulfatase activity before causing tumor necrosis and total decrease in steroidogenesis. A fall in urinary 17-KS may not indicate tumor regression and serum androgens seem to be a better indicator for the steroidogenic activity of a virilizing adrenal tumor. These data provide some new insight into a drug-steroid interaction.

Control of citrate and acetoacetate synthesis in rat liver

The mechanisms by which ethanol (EtOH) inhibits the human chorionic gonado-tropin (hCG)-stimulated testosterone synthesis was studied in isolated rat Leydig cells in vitro. EtOH inhibited steroidogenesis, but this inhibition was reversed by l-glutamate (Glu) and an uncoupler of the oxidative phosphorylation, 2,4-dinitrophenol (DNP). The mechanism of EtOH-induced inhibition was studied by measuring steroidogenic precursors and comparing them with the cytosolic and mitochondrial NADH redox states during uncoupling or in the presence of Glu. DNP had a dual effect. Low concentrations abolished the EtOH-induced inhibition of progesterone to testosterone formation suggesting that the inhibitory step was at or before progesterone formation. A large concentration led to an overall decrease in steroidogenesis indicating toxic effects on steroidogenesis. The mitochondrial NADH/NAD+ ratio, measured as the 3-hydroxybutyrate/acetoacetate ratio, decreased simultaneously when steroidogenesis was stimulated, either during uncoupling or in the presence of Glu, whereas cytosolic NADH/NAD+ ratio, measured as lactate/pyruvate ratio showed no response. These results demonstrate that the rise in the mitochondrial NADH/NAD+ ratio rather than in the cytosolic one is connected with the inhibition of testosterone synthesis by EtOH in isolated Leydig cells. The EtOH-induced high mitochondrial NADH/NAD+ ratio may deplete mitochondrial oxalacetate concentrations. This can decrease the activity of several transport shuttles and interrupt the flow of mitochondrial citrate into the smooth endoplasmic reticulum, which then reflects to decreased rate of steroidogenesis in the presence of ethanol.