Role of ethanol metabolism in the inhibition of testosterone biosynthesis in rats in vivo: importance of gonadotropin stimulation

The mechanisms by which ethanol (EtOH, 1.5 g/kg) inhibits testicular testosterone synthesis were studied in nonstimulated and human chorionic gonadotropin (hCG, 50 IU/kg)-treated male rats. To dissociate the effects caused by ethanol metabolism, the alcohol dehydrogenase inhibitor 4-methylpyrazole (4MP, 10 mg/kg) was given to half of the rats 30 min before EtOH. The 4MP had little or no effect in the nonstimulated rats on the EtOH-induced decreases in the concentrations of serum testosterone and of the intratesticular steroids of the testosterone biosynthetic pathway measured, but reduced the EtOH-induced elevation in the intratesticular pregnenolone-to-progesterone ratio. In contrast, 4MP pretreatment markedly reversed the EtOH-induced decrease in serum and intratesticular testosterone and increase in intratesticular pregnenolone concentrations in the hCG-stimulated rats. Simultaneously, the EtOH-induced elevations in the intratesticular pregnenolone/progesterone and androstenedione/testosterone ratios were abolished. In the EtOH-treated rats whose EtOH metabolism was blocked by 4MP pretreatment, the intratesticular testosterone concentrations were negatively correlated with the elevated serum corticosterone levels. It is concluded that: (1) EtOH metabolism is involved in the inhibition of testicular steroidogenesis in vivo. This effect is pronounced during gonadotropin-stimulated conditions. Thus, previously reported "discrepancies" between the in vivo and in vitro results are clarified; (2) corticosterone seems also to be involved in the EtOH-induced inhibition of steroidogenesis. This effect is also pronounced during gonadotropin-stimulated conditions; and (3) without external gonadotropin stimulation other inhibitory mechanisms, such as decreased stimulation by luteinizing hormone, are prevalent.     

Inhibition of testicular androgenesis by urinary antigonadotropins and melatonin in rats.

Comparative in vitro and in vivo studies of the effects of urinary gonadotropin-inhibiting substances and melatonin on the androgenesis in rat testicular homogenates were performed. When the urinary extract containing the inhibiting substances or melatonin was added directly to the incubation medium, and was also injected into rats 24 and 48 hrs prior to sacrifice, either one was effective in suppressing the conversion of pregnenolone to testosterone and/or androstenedione in testicular tissues. The urinary extract exerted the inhibitory effect on the conversion of cholesterol to pregnenolone in vitro and in vivo, whereas melatonin did not have this effect in vitro and in vivo. These findings suggest that the antigonadotropic substances are different from melatonin in their action on androgenesis in the rat testes.     

The interaction of hCG, hydroxysteroids and interstitial fluid on rat Leydig cell steroidogenesis in vitro

Rat testicular interstitial fluid and hydroxycholesterol both stimulated testosterone production by isolated Leydig cells in vitro in a dose-dependent manner, but the dose-response lines were not parallel. The addition of cycloheximide blocked the stimulation by interstitial fluid but not that of hydroxycholesterol. Use of the compounds SU 10603 and cyanoketone (which inhibit 3 beta-hydroxysteroid dehydrogenase and 17 alpha-hydroxylase respectively) or aminoglutethimide (which acts on the cholesterol side-chain cleavage enzyme) showed that the stimulatory factor(s) in interstitial fluid stimulated steroidogenesis at the cholesterol side-chain cleavage enzyme, before the conversion of pregnenolone. This enzyme is rate-limiting in the synthesis of testosterone by Leydig cells and a site of action of LH; therefore, these results support the view that an interstitial fluid factor may be involved in the paracrine regulation of testicular steroidogenesis.     

Ethanol-induced inhibitions of testicular steroidogenesis in the male rat: Mechanisms of actions

Ethanol markedly inhibits the biosynthesis of testosterone in the male of several species. Since several $\text{in vitro}$ studies have suggested that ethanol $\text{per se}$ is not a gonadal toxin and that it must be metabolized to exert its effects, we examined this possibility under $\text{in vivo}$ conditions in the present studies. We found that the administration of the alcohol dehydrogenase inhibitor, pyrazole, to adult male rats significantly elevated blood ethanol levels. However, rather than resulting in a potentiation of the effects of ethanol on testicular steroidogenesis, pyrazole-induced elevations in blood ethanol concentrations produced a significant attenuation of ethanol's effects. In view of these observations, it is difficult to maintain that ethanol itself is responsible for inhibiting the production of testosterone. On the contrary, our results may provide the first $\text{in vivo}$ support for the hypothesis that ethanol must be metabolized to exert its effects on testicular steroidogenesis.     

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.     

The estimation of alcohol dehydrogenase activity in aerobic and anaerobic “permeabilised” baker’s yeast cells

The in vivo and in vitro activity of alcohol dehydrogenase from baker's yeast maintained under aerobic and anaerobic conditions was measured. In vivo measurements were made in cells "permeabilised" with toluene. Michaelis constants (NAD+ as substrate) were found to be almost identical as those reported for purified preparations. In addition the Km of the enzyme from cells incubated under anaerobic conditions was virtually identical to that from cells from aerobic conditions. The activity of the enzyme was found to be greater (in both "permeabilised" cells and extracts) in cells maintained under nitrogen than air. Cells metabolizing glucose in N2 produced greater levels of ethanol than in air and the rate of NAD+ reduction was also found to be greater in N2 than in air. The results indicate that it was feasible to determine rates of this enzyme in vivo and that the difference in activity of alcohol dehydrogenase under N2 and air may conceivably account for differences in rates of glucose utilisation, ethanol production and NAD+ reduction in air and nitrogen.     

Effects of ethanol and acetaldehyde on the biosynthesis of testosterone in the rodent testes

The effects of ethanol and acetaldehyde on testicular steroidogenesis were examined in enzymatically dispersed cells of the rodent testes. Both drugs significantly inhibited gonadotropin-stimulated steroidogenesis, but acetaldehyde was considerably more potent (>1000 times) than ethanol. To determine the step in testosterone's biosynthetic pathway which was inhibited by the two drugs, cells were incubated in the presence of [³H]pregnenolone and [³H]progesterone, and the amount of label incorporated into testosterone and its precursors was determined. Ethanol and acetaldehyde inhibited only the conversion of androstenedione to testosterone; none of the other precursors of testosterone was affected.     

On the regulation of rat testicular steroidogenesis. Short term effect of luteinizing hormone and cycloheximide in vivo and Ca2+ in vitro on steroid production in cell-free systems.

An attempt has been made to correlate the rapid effect of luteinizing hormone on testicular steroid production in vivo with testicular steroid concentrations and in vitro steroid production rates in testis tissue preparations. Within 20 min after intravenous administration of 25 mug luteinizing hormone, increases were observed in testosterone concentrations in testicular venous plasma and in whole testis tissue and in pregnenlone concentrations isolated testis mitochondrial fractions. Testosterone production by whole testis homogenates and pregnenolone production by isolated mitochondrial fractions were significantly increased within 5 min after in vivo administration of luteinizing hormone. Injection of cycloheximide 10 min prior to luteinizing hormone prevented the stimulating effect of luteinizing hormone to steroid levels in testicular venous plasma and testis tissue and on steroid production rates by preparations of rat testis tissue. Cycloheximide treatment of control animals did not significantly alter testosterone concentrations and testosterone production rates vitro, although mitochondrial pregnenolone concentrations and production rates were decreased. Testosterone production by whole testis homogenates as well as the pregnenolone production by isolated mitochondrial fractions obtained from luteinizing hormone treated testes and control glands showed a biphasic time curve A period (5-10 min) of high steroid production was followed by a period lower steroid production. Addition of 25 mug luteinizing hormone or 10(-8)--10(-5) M adenosine 3':5'-monophosphate (cyclic AMP) to the incubation medium had no effect pregnenolone production by isolated mitochondrial fractions. Administration of leuteinizing hormone in vivo markedly enhance the stimulating effect of Ca2+ on testosterone production by whole testis homogenates and on pregnenolone production by isolated mitochondrial fractions.     

Inhibition of rat testicular androgenesis by a polychlorinated biphenyl mixture aroclor 1248

Polychlorinated biphenyls (PCBs) are complex mixtures of congeners that exhibit carcinogenic and toxicant activities in a variety of mammalian tissues. Here, we studied the acute in vivo and in vitro effects of a commercially used PCB product, Aroclor 1248 (A1248), a mixture of tri-, tetra-, and pentachloro congeners. Single intraperitoneal (i.p.) or bilateral intratesticular (i.t.) injections of A1248 decreased serum androgen levels in both groups 24 h after injection. Chorionic gonadotropin-stimulated androgen production by acute testicular cultures from both groups was also reduced, and progesterone production was attenuated in cultures from i.t.-treated animals. The capacity of the postmitochondrial fractions from testes of i.t.-treated animals to convert pregnenolone to progesterone and progesterone to testosterone was reduced as well. In vitro studies revealed that a 10- to 15-min exposure of postmitochondrial testicular fractions and intact interstitial cells from normal animals to A1248 in a subnanomolar concentration range was sufficient to attenuate the conversion of pregnenolone to progesterone and progesterone to testosterone. At micromolar concentrations, A1248 added in vitro also inhibited the conversion of Delta(4)-androstendione to testosterone without affecting the viability of interstitial cells. These results indicate that A1248 down-regulates the testicular androgenesis by an acute inhibition of 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase/lyase, and 17beta-hydroxysteroid dehydrogenase activities.     

Steroid production in toads

In Bufo arenarum, androgen biosynthesis occurs through a complete 5-ene pathway, including 5-androstane-3β,17β-diol as the immediate precursor of testosterone. Besides, steroidogenesis changes during the breeding period, turning from androgens to C₂₁-steroids such as 5-pregnan-3,20-diol, 3-hydroxy-5-pregnan-20-one and 5-pregnan-3,20-dione. In B. arenarum, steroid hormones are not involved in hCG-induced spermiation, suggesting that the steroidogenic shift to C₂₁-steroids during the breeding be not related to spermiation. The activity of 17-hydroxylase-C_17–20 lyase (CypP450_c17) decreases during the reproductive season, suggesting that this enzyme would represent a key enzyme in the regulation of seasonal changes. However, the increase in the affinity for pregnenolone of 3β-hydroxysteroid dehydrogenase (3HSD)/isomerase could also be involved. Moreover, the reduction in CypP450_c17 leading to a reduction in C₁₉-steroids, among them dehydroepiandrosterone (DHE), would contribute to the conversion of pregnenolone into progesterone, avoiding the non-competitive inhibition exerted by DHE on this transformation. Additionally, CypP450_c17 possesses a higher affinity for pregnenolone than for progesterone, explaining the predominance of the 5-ene pathway for testosterone biosynthesis. Animals in reproductive condition showed a significant reduction in circulating androgens, enhancing the physiological relevance of all the in vitro results. The in vitro effects of mGnRH and hrFSH on testicular steroidogenesis revealed that both hormones inhibited CypP450_c17 activity. In summary, these results demonstrate that, in B. arenarum, the change in testicular steroidogenesis during the reproductive period could be partially due to an FSH and GnRH-induced decrease in CypP450_c17 activity.     

The HMG-CoA reductase inhibitor simvastatin suppresses human testicular testosterone synthesis in vitro by a selective inhibitory effect on 17-ketosteroid-oxidoreductase enzyme activity

In concentrations probably exceeding those achieved in vivo, the cholesterol lowering compound simvastatin was found to suppress the synthesis of the androgens androstenediol and testosterone in vitro by human testicular homogenates. It was demonstrated that simvastatin in addition to its known inhibitory effect on HMG-CoA reductase activity, also affects the later steps of testicular steroidogenesis by selectively inhibiting the 17-ketosteroid-oxidoreductase catalyzed conversion of dehydroepiandrosterone and androstenedione to androstenediol and testosterone respectively. There was no effect of simvastatin on the Cytochrome P-450-dependent microsomal enzymes. Although in doses conventionally used in the treatment of hypercholesterolemia, simvastatin does not affect testicular steroidogenesis, at higher doses--especially when inadvertently administered during early pregnancy--adverse effects on normal testosterone biosynthesis and thereby fetal development should be considered.     

Inhibition of testosterone biosynthesis by ethanol. Relation to hepatic and testicular acetaldehyde, ketone bodies and cytosolic redox state in rats.

In experiments in which liver and testis freeze-stops were performed on pentobarbital-anaesthetized rats, ethanol (1.5 g/kg body wt.) reduced plasma testosterone concentration from 13.1 to 3.2 nmol/litre. 4-Methylpyrazole abolished the ethanol-induced hepatic and testicular increase in the lactate/pyruvate ratio, and the testicular acetaldehyde level, but did not diminish the reduction in plasma testosterone concentration. In testes, but not in liver, ethanol decreased the 3-hydroxybutyrate/acetoacetate ratio, and 4-methylpyrazole did not prevent this effect. In experiments in which freeze-stop was performed after cervical dislocation, ethanol decreased the testis testosterone concentration from 590 to 220 pmol per g wet wt. The effects of ethanol and 4-methylpyrazole on testis acetaldehyde, lactate/pyruvate and 3-hydroxybutyrate/acetoacetate ratios were the same as found during anaesthesia. The NAD+-dependent ethanol oxidation capacity in testis ranged from 0.1 to 0.2 mumol/min per g wet wt. and seemed to be inhibited by 4-methylpyrazole both in vivo and in vitro. In additional experiments, ethanol doses between 0.3 and 0.9 g/kg body wt. did not alter the plasma testosterone concentration in rats treated, or not treated, with cyanamide, which induced elevated acetaldehyde levels in blood and testes. The results suggest that ethanol-induced inhibition of testosterone biosynthesis was not caused by extratesticular redox increases, or by extra- or intra-testicular acetaldehyde per se. The inhibition is accompanied by changes in testicular ketone-body metabolism.     

Ethanol inhibits the naloxone-induced release of luteinizing hormone-releasing hormone from the hypothalamus of the male rat

It has been inferred that ethanol suppresses the secretion of luteinizing hormone (LH) in the male by depressing the release of LH-releasing hormone (LH-RH) from the hypothalamus. Direct support for this inference has been difficult to obtain, however, because of significant technical difficulties in measuring LH-RH release under in vivo conditions. To circumvent these problems, we made use of the opiate antagonist naloxone, as a neuroendocrine probe, to elicit the release of LH-RH under in vivo conditions. We found that ethanol was a potent suppressor of the increase in serum LH levels evoked by naloxone at extremely low blood ethanol concentrations ( less than 60 mg/dl). Furthermore, we observed that the antagonism between ethanol and naloxone appeared to be competitive in nature since a fixed dose of ethanol (1 g/kg, blood ethanol concentration 60 mg/dl) shifted the naloxone dose-response curve significantly to the right and high doses of the antagonist overcame ethanol's effects. Finally, we found that the interaction between ethanol and naloxone took place at the level of the hypothalamus. Our results, therefore, seem to provide the first in vivo evidence supporting the widely-held hypothesis that ethanol reduces serum LH levels by depressing the hypothalamically-medicated release of LH-RH. The mechanisms underlying ethanol's depression of naloxone-induced increases in the release of LH-RH are not fully understood at this time, but one prominent possibility is that ethanol enhances the synthesis or release of endogenous opioids which in turn override naloxone's effects.     

The effect of alcohol stress on nicotinamide adenine dinucleotide (NAD+) levels in Drosophila

Previous studies carried out in mammalian systems indicated that an organism's NAD+/NADH balance is carefully regulated but can be destabilized by dietary stresses. Since Drosophila alcohol dehydrogenase (ADH) uses NAD+ to remove a hydrogen from ethanol in the first step of alcohol catabolism, it is possible that under alcohol stress conditions the in vivo NAD+ levels in Drosophila may decrease. In this study genetically homozygous flies were stressed with maximally sublethal concentrations of ethanol (10%) for periods of up to 24 hr. The results indicate that NAD+ levels do in fact drop by at least 20% in response to ethanol stress. Evidence is presented that suggests that this decrease is the direct result of ADH-mediated catabolism.     

Effect of Ca2+, ruthenium red and ageing on pregnenolone production by mitochondrial fractions from normal and luteinizing hormone treated rat testes

The effect of Ca²⁺ in vitro on pregnenolone production rates under various incubation conditions by mitochondrial fractions fractions isolated from testes of normal rats and of rats after in vivo treatment with luteinizing hormone has been investigated. Concentrations of Ca²⁺ in the range of 0.1–0.5 mM stimulated succinate supported pregnenolone production in mitochondrial fractions from both control and luteinizing hormone treated testes. When mitochondrial fractions were isolated in 0.25 M sucrose without additions, Ca²⁺ in vitro increased succinate supported pregnenolone production rates in mitochondrial fractions isolated from control testes to a greater extent than in mitochondrial fractions, from luteinizing hormone treated testes. Production rates in control mitochondrial fractions, incubated in the presence of initial Ca²⁺ concentrations of 0.7 mM and higher were almost similar to production rates in relevant luteinizing hormone treated mitochondria.Pregnenolone production from endogenous substrates in mitochondrial fractions isolated in 0.25 M sucrose from control and luteinizing hormone treated testes incubated in the absence of added succinate and Ca²⁺, was maintained during 10–20 min.After longer incubation times no further steroid synthesis took place. Addition of 0.5 mM Ca²⁺ to the incubation medium at time zero slightly stimulated initial pregnenolone production rates in control mitochondrial fractions, but had no effect during prolonged incubations. Addition of 0.5 mM Ca²⁺ to mitochondrial fractions isolated from luteinizing hormone treated glands showed no effect either on initial production rate or during prolonged incubations.Pregnenolone production rates were maintained during 90 min in the presence of 20 mM succinate in the incubation medium. Under such conditions production rates during the first 20 min in mitochondrial fractions obtained from luteinizing hormone treated glands were approx. 3 times higher than in relevant control samples. Addition of 0.5 mM Ca²⁺ to the incubation medium containing 20 mM succinate markedly stimulated initial pregnenolone production rates in control mitochondrial fractions, but gave only a small stimulation of succinate-supported production rates in luteinizing hormone treated testicular mitochondrial fractions. These results indicate that Ca²⁺ in vitro can mimic the trophic effect of luteinizing hormone in vivo on mitochondrial pregnenolone production.Ageing of mitochondrial protein for 60 min at 33°C resulted in a marked increase in pregnenolone production rates in mitochondrial fractions obtained from control testes. The same treatement hardly influenced production rates in mitochondrial fractions isolated from luteinizing hormone treated testes. Ageing may have an effect on the ultrastructure of freshly prepared mitochondria, causing a change in the amount of cholesterol readily available for the enzyme complex.The gluco- and mucoprotein specific agent Ruthenium red (50–2000 ng/ml) did not inhibit pregnenolone production in either control or hormone treated testicular mitochondrial fractions, incubated in the absence of added Ca²⁺. the presence of 200–2000 ng Ruthenium red per ml incubation mixture.The present results have been discussed in relation to the possible involvement of Ca²⁺ in the molecular mechanism of short-term action of luteinizing hormone on testicular androgen production.     

Effect of testosterone on testicular steroidogenesis in the hypogonadal (hpg) mouse

Previous studies have shown that androgens have direct inhibitory effects on steroidogenesis in active Leydig cells. It is not clear what effect androgens have on inactive Leydig cell either through direct action on the cell itself or indirectly through stimulation of Sertoli cell activity. The hpg mouse has undetectable levels of circulating gonadotrophins and the gonads fail to develop post-natally. The effect of androgen treatment on testicular steroidogenesis and morphology was examined in these animals. Treatment with testosterone propionate for two weeks significantly increased testicular and seminal vesicle weight. Seminiferous tubules showed marked development in androgen-treated animals, indicating increased Sertoli cell activity, but the abnormal Leydig cell morphology of the hpg testis was unchanged. Androgen production per testis in vitro was low in control hpg animals and remained unaffected by treatment with androgen. Similarly, the pattern of [3H]pregnenolone metabolism was not significantly affected by androgen treatment. The androgen content of the testis was higher in androgen-treated animals but this could be accounted for by uptake of administered steroid from the circulation. It is concluded that androgens have no direct trophic effect on Leydig cells and that stimulation of Sertoli cell activity is not, in itself, sufficient to affect Leydig cell function.     

In vivo and in vitro adenosine stimulation of ethanol oxidation by hepatocytes, and the role of the malate-aspartate shuttle

In this study, a pronounced increase of ethanol oxidation was found in hepatocytes obtained from adenosine-treated rats, or after in vitro additional of the nucleoside; this finding was accompanied by a maintenance of the normal cytoplasmic redox state. These results suggest a higher availability of cytoplasmic NAD in these cells. Therefore, the metabolic pathways which carry out the reoxidation of cytosolic reducing equivalents, namely, malate-aspartate and alpha-glycerophosphate shuttles, were examined. Isolated mitochondria from adenosine-treated rats had an increased NADH oxidation by the malate-aspartate shuttle; furthermore, in vivo and in vitro addition of adenosine to the hepatocytes induced changes in the equilibrium of the malate-aspartate shuttle, as evidenced by the subcellular distribution of the intermediates of this pathway. Acetaldehyde removal was also increased by adenosine and this fact was related to an elevated NAD/NADH ratio in the mitochondria. Thus, under these conditions, an increased ethanol uptake was accompanied by enhanced acetaldehyde removal in the animal. In conclusion, adenosine administration stimulates the transport of cytoplasmic reducing equivalents to the mitochondria, mainly through the malate-aspartate shuttle. This action, which may be located at the level of the mitochondrial membrane, is reflected by an enhancement of ethanol and acetaldehyde oxidations.     

Leuprolide acetate reduces both in vivo and in vitro ovarian steroidogenesis in infertile women undergoing assisted reproduction

Despite the probable inhibitory effects of GnRH analogues on ovarian steroidogenesis in vitro, their association with assisted reproduction protocols shows favorable results. This suggests that there are important differences in the behaviors of these drugs when administered in vivo versus in vitro. To clarify these differences, this study was designed to analyze the effect of leuprolide acetate (LA) on ovarian steroidogenesis in women undergoing In Vitro Fertilization (IVF). A prospective, randomized open label study was conducted on 14 women (26-35 years): seven receiving only gonadotrophins (Group 1) and seven receiving gonadotrophin plus LA at 1mg/day (Group 2). The LA in vivo effect was determined with serum and follicular fluid (FF) samples and via luteinized granulosa cell cultivation (GCC), where cells were obtained during oocyte retrieval after ovarian hyperstimulation. In vitro analysis was performed via addition of LA to GCC only for Group 1 (without LA) at progressively higher concentrations (0, 10(-12), 10(-9) and 10(-6)M). In vivo, the main observation was a reduction in androgen production in Group 2, represented by lower androstenedione production in FF (G1=6479+/-3458; G2=3021+/-1119 ng/ml; p=0.04) and a lower testosterone peak in GC at 96h (G1=0.64+/-0.12 ng/ml; G2=0.50+/-0.19 ng/ml; P=0.02), but a higher fertilization rate (G1=67%; G2=83%; p=0.009). In vitro, testosterone, estradiol and progesterone were also reduced by LA, even though this reduction occurred for progesterone only at the highest LA dosage (10(-6)M; 606.0+/-114.3 ng/ml versus 1524.0+/-246.5 ng/ml; p=0.02). Results show that LA reduces ovarian steroidogenesis in vivo by essentially inhibiting androgen synthesis; whereas, in vitro, ovarian steroidogenesis is reduced overall.     

Sources of calcium and the involvement of calmodulin during steroidogenesis and oocyte maturation in follicles of Rana pipiens

In the amphibian ovarian follicle, progesterone production is thought to induce maturation of the enclosed oocyte. Intracellular mechanisms regulating these events in the somatic and germ cells are incompletely understood. However, calcium appears to play a role in the production and action of progesterone. Experiments using calcium antagonists were carried out to delineate the role of extra- and intracellular calcium during in vitro stimulation of follicular steroidogenesis and oocyte maturation. Calcium-free medium, verapamil, and La3+ were used to block Ca2+ influx and inhibited follicular progesterone accumulation in response to frog pituitary homogenate (FPH) or exogenous cAMP + IBMX. Progesterone accumulation was not impaired under identical conditions when pregnenolone was added to cultured follicles. TMB-8, an inhibitor of intracellular Ca2+ mobilization, partially inhibited progesterone levels stimulated by FPH at low doses but not higher doses of the inhibitor. However, TMB-8 inhibited FPH-induced oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner, as well as maturation due to exogenous progesterone or La3+. Calmodulin antagonists, W-7, R24571, and trifluoperazine, were used to assess the involvement of calmodulin in the responses of these two cell types. All three antagonists inhibited progesterone accumulation induced by FPH with the apparent order of potency being R24571 greater than W-7 greater than TFP. W-7 inhibited cAMP-induced progesterone elevation, but had no effect on conversion of pregnenolone to progesterone. Of these three calmodulin antagonists, only R24571 exhibited a dramatic ability to inhibit GVBD induced by exogenous progesterone and was associated with morphologic alterations in the oocytes. These data suggest that Ca2+, acting through calmodulin at some specific step(s) distal to cAMP elevation and prior to pregnenolone formation, is involved in FPH-induced progesterone accumulation, apparently with the participation of both extracellular and intracellular pools of Ca2+. In the oocyte, mobilization of Ca2+ from intracellular stores appears to be of primary importance to maturation while extracellular Ca2+ is not. These data provide further evidence that Ca2+ mediates the hormonally provoked responses in both cell types in the intact follicle, but that the source of Ca2+ may differ. Using intact follicles it seems apparent that exploiting this difference with selective inhibitors provides a means for differential modulation and functional uncoupling of these cells with regard to steroidogenesis and steroid action.     

Differential metabolism of pregnenolone by testicular homogenates of humans and two species of macaques. Lack of synthesis of the human sex pheromone precursor 5,16-androstadien-3 beta-ol in nonhuman primates.

In previous reports we described the early time sequence in in vitro [4-14C] pregnenolone metabolism in human and rat testicular homogenates and, apart from a difference in the preferred route of the conversion of pregnenolone to testosterone, we demonstrated the presence of delta 16-synthetase activity in human but not in rat testes. In the study of testicular function higher monkeys are increasingly used as a model for human reproduction. The availability of testes from 2 different species of macaques (rhesus and crab eating monkeys) enabled us to compare the in vitro metabolism of pregnenolone in these testes with human testes. The pattern obtained in both monkey species were very similar, but completely different from those found in man. The delta 4 pathway was the preferred route for the conversion of pregnenolone to testosterone in the monkeys tested, the delta 5 pathway in the humans. delta 16-Synthetase activity, a prerequisite for the synthesis of the sex pheromone precursors 5,16-androstadien-3 beta-ol and 4,16-androstadien-3-one, was clearly measurable in the human but not in the monkey testicular homogenates. So far, man and boar are the only species harbouring delta 16-synthetase activity in their testes. These in vitro data indicate that the nonhuman primates studied are not suitable models for the study of human testicular function.