The effects of steroid hormones and gonadotropins on in vitro placental conversion of pregnenolone to progesterone

Using human term placental mitochondrial preparations, optimal conversion of [3H]pregnenolone to [3H]progesterone was obtained at 30 min incubation and with a mitochondrial protein content of 2.5-3.5 mg/ml. Estradiol, estrone, progesterone and testosterone in a dose range of 0.03-8.66 mumol inhibited the in vitro conversion of [3H]pregnenolone to [3H]progesterone by placental homogenates. All four steroids inhibited the pregnenolone to progesterone conversion in a dose-dependent manner. The ID50 (dose required to inhibit conversion of pregnenolone to progesterone by 50%) was 0.04 mumol for estradiol, 0.13 mumol for testosterone, 0.3 mumol for progesterone and 1.0 mumol for estriol. Neither gonadotropin releasing hormone (50-1000 ng) nor human chorionic gonadotropin (5-500 IU) affected the placental basal conversion rate of pregnenolone to progesterone in vitro. Our findings indicate that steroid hormones such as estradiol, estrone, testosterone and progesterone can inhibit local placental progesterone biosynthesis through inhibition of the enzyme complex 5-ene-3 beta-hydroxysteroid dehydrogenase.     

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.     

Progesterone attenuates the inhibitory effects of cardiotonic digitalis on pregnenolone production in rat luteal cells

Previous studies have shown that digoxin decreases testosterone secretion in testicular interstitial cells. However, the effect of digoxin on progesterone secretion in luteal cells is unclear. Progesterone is known as an endogenous digoxin-like hormone (EDLH). This study investigates how digitalis affected progesterone production and whether progesterone antagonized the effects of digitalis. Digoxin or digitoxin, but not ouabain, decreased the basal and human chorionic gonadotropin (hCG)-stimulated progesterone secretion as well as the activity of cytochrome P450 side chain cleavage enzyme (P450scc) in luteal cells. 8-Br-cAMP and forskolin did not affect the reduction. Neither the amount of P450scc, the amount of steroidogenic acute regulatory (StAR) protein, nor the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) was affected by digoxin or digitoxin. Moreover, in testicular interstitial and luteal cells, progesterone partially attenuated the reduction of pregnenolone by digoxin or digitoxin and the progesterone antagonist, RU486, blocked this attenuation. These new findings indicated that (1) digoxin or digitoxin inhibited pregnenolone production by decreasing the activity of P450scc enzyme, but not Na(+)-K(+)-ATPase, resulting in a decrease on progesterone secretion in rat luteal cells, and (2) the inhibitory effect on pregnenolone production by digoxin or digitoxin was reversed partially by progesterone. In conclusion, digoxin or digitoxin decreased progesterone production via the inhibition of pregnenolone by decreasing P450scc activity. Progesterone, an EDLH, could antagonize the effects of digoxin or digitoxin in luteal cells.     

Testicular steroidogenesis in the rhesus monkey (Macaca mulatta.

Studies were undertaken to investigate testicular steroidogenesis in the Rhesus monkey Macaca mulatta. Testicular fragments (50 mg) were incubated for 3 hr with pregnenolone-7-3H or with progesterone-7-3H. The major metabolite of pregnenolone was progesterone (70.1%), with a lesser conversion to 17-hydroxyprogesterone (1.6%), androstenedione (3.3%), and testosterone (7.2%). The delta-5 intermediates 17-hydroxypregnenolone (4.6%) and dehydroepiandrosterone (8.6%) were also identified in the pregnenolone incubates. A majority of the progesterone substrate was not metabolized by the testicular fragments (80.1%), while some conversion to 17-hydroxyprogesterone (3.4%), androstenedione (4.8%), and testosterone (11.7%) occurred in the incubates. These results suggest that testicular fragments from the Rhesus monkey may convert pregnenolone to testosterone through both the delta-4 and the delta-5 pathways.     

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.     

Biosynthesis of testicular steroids in the immature, adult and senescent guinea-pig

The potential biosynthetic capacity of testicular hormones was studied in immature, pubertal and aging guinea-pig. In their sexual development towards puberty, changes in the relationship of the steroids involved in the steroidogenic pathways were observed. The testosterone/androstenedione ratio changes markedly, showing an important increase with pubertal proximity. The testosterone in equilibrium androstenedione sequence, reversibly catalyzed by 17 beta-hydroxysteroid oxidoreductase (17 beta-oxido-reductase), clearly shifted towards androstenedione in immature animals irrespective of the precursor utilized. Post-pubertal animals showed a greater enzymatic activity in the 5-ene and 4-ene testicular synthesis pathways, testosterone production being greatest. In the aging animal, hormonal biosynthetic capacity falls. Reversion of the 17 beta-oxido-reductase activity could be one of the mechanisms responsible for the decrease in testosterone, as in immature guinea-pigs. In order to investigate the in vitro steroidogenic capacity of glands at different ages, minces of testicular tissue were incubated with labelled precursors. The studies were conducted in triplicate at 35 degrees C. For equal quantities of incubated tissue the non-metabolized amount of [3H]pregnenolone and [14C]progesterone, utilized as precursors, was different in post-pubertal and senescent animals: 55.7 +/- 3 vs 59.3 +/- 2.3% (P less than 0.01) for pregnenolone, and 50.1 +/- 3.3 vs 56.3 +/- 2.9% (P less than 0.01) for progesterone, respectively. Testosterone production was 12 +/- 2% in adult and 6.7 +/- 2.7% in senescent animals (P less than 0.01). The testosterone/androstenedione ratio was not significantly different in post-pubertal and senescent animals: 2.8 +/- 0.5 vs 2.4 +/- 0.4, but consistently higher than found in immature animals: 0.3 +/- 0.1. The lesser potential capacity of the aging tissue to synthesize testosterone could be explained by a decline in the glands capacity to metabolize the hormonal precursors.     

Regulation of 3 beta-hydroxysteroid dehydrogenase activity by human chorionic gonadotropin, androgens, and anti-androgens in cultured testicular cells

delta 5-3 beta-Hydroxysteroid dehydrogenase is a key enzyme for testicular androgen biosynthesis and a marker for the Leydig cells. The hormonal regulation of this enzyme was studied in cultured rat testicular cells. Human chorionic gonadotropin (hCG) increased testosterone production in vitro while time course studies indicated a biphasic action of the gonadotropin on 3 beta-hydroxysteroid dehydrogenase activity. An initial stimulation (51%) of the enzyme was detected between 3 and 12 h of culture when medium testosterone was low. This is followed by an inhibition of 3 beta-hydroxysteroid dehydrogenase activity on days 2 and 3 of culture when medium testosterone was elevated. Concomitant treatment with a synthetic androgen (R1881) inhibited 3 beta-hydroxysteroid dehydrogenase activity and testosterone production in hCG-treated cultures while an anti-androgen (cyproterone acetate) increased 3 beta-hydroxysteroid dehydrogenase activity and testosterone biosynthesis. Addition of 10(-5) M spironolactone, an inhibitor of 17 alpha-hydroxylase, blocked the hCG stimulation of testosterone production but increased medium progesterone. In the absence of the secreted androgen, hCG stimulated 3 beta-hydroxysteroid dehydrogenase activity in a time- and dose-related manner. Furthermore, hCG stimulation of 3 beta-hydroxysteroid dehydrogenase activity and progesterone accumulation in spironolactone-supplemented cultures was decreased by concomitant treatment with R1881 but was not affected by cyproterone acetate. The inhibitory effect of R1881 was blocked by the anti-androgen. In the absence of hCG, treatment with testosterone, dihydrotestosterone, or R1881, but not promegestone, alone also inhibited 3 beta-hydroxysteroid dehydrogenase activity while the inhibitory effect of testosterone was blocked by cyproterone acetate. Thus, hCG stimulates 3 beta-hydroxysteroid dehydrogenase activity in cultured testicular cells. The androgenic steroidogenic end products, in turn, inhibit this enzyme. The hormonal regulation of 3 beta-hydroxysteroid dehydrogenase activity may be important in the ultrashort loop autoregulation of androgen biosynthesis.     

Ethanol-induced inhibition of testosterone biosynthesis in rat Leydig cells: role of L-glutamate and pyruvate

The mechanisms by which ethanol (EtOH) inhibits testicular testosterone biosynthesis were studied with isolated rat Leydig cells in vitro comparing the effects of EtOH in six different culture media. The actual sites of inhibition by EtOH, identified by measuring the steroidogenic precursors, varied depending on the medium used. In Krebs-Ringer bicarbonate buffer, EtOH inhibited both the conversion of pregnenolone to progesterone and androstenedione to testosterone. In the pyruvate (Pyr) supplemented Dulbecco's Modified Eagle medium, the decreased progesterone concentrations in the presence of EtOH were reflected to all successive steroids 17-OH-progesterone, androstenedione and testosterone. The presence of L-glutamate (Glu) in the medium elevated testosterone production, but EtOH still inhibited the conversion of pregnenolone to progesterone, and also the androstenedione/testosterone ratio was elevated because of the decreased testosterone concentrations. In the presence of both Glu and Pyr in the medium the EtOH-induced decreases in the steroid concentrations were fully recovered in isolated Leydig cells. These results demonstrate that both Pyr and Glu supplementations are essential for the maintenance of maximal rate of testosterone synthesis in vitro in the presence of EtOH.     

Kinetic analysis of 3 beta-hydroxysteroid dehydrogenase activity in microsomes from complete hydatidiform mole

Microsomes isolated from complete hydatidiform moles (CHM) were able to convert [3H]pregnenolone to [3H]progesterone which indicates the presence of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD) activity. The kinetic parameters found (Km = 0.63 microM and Vmax = 1-3.05 nmol/min/mg of protein) were like those observed in microsomes from normal early placenta (NEP) of similar gestational age (herein) and term placenta suggesting that the enzymes from the three sources are kinetically similar. Testosterone, progesterone and estradiol in a dose range of 0.05-5 mumol/l inhibited differently the in vitro conversion of [3H]pregnenolone to [3H]progesterone in a dose-dependent manner. The steroid concentrations necessary to inhibit the conversion of pregnenolone to progesterone by 50% (ID50) in CHM were 0.1 microM for testosterone, 0.6 microM for progesterone and 3 microM for estradiol, whereas in NEP they were 2.5, 1 and 5 microM respectively. The Ki values calculated from these ID50 in CHM together with the reported levels of endogenous steroids indicate that the accumulation of testosterone and progesterone inside the molar vesicle could physiologically regulate the rate of further conversion of pregnenolone to progesterone. The present findings could provide an explanation for the low level of progesterone in patients with CHM in the second trimester of pregnancy which in turn may directly or indirectly affect the spontaneous expulsion of this aberrant tissue.     

Direct biphasic modulation of gonadotropin-stimulated testicular androgen biosynthesis by prolactin

Prolactin (PRL) exerts both stimulatory and inhibitory effects upon testicular steroidogenesis in vivo. The direct effects of PRL on biosynthesis of testicular androgen were studied in primary cultures of testicular cells obtained from adult, hypophysectomized or neonatal, intact rats. In cells from adult animals, treatment with human chorionic gonadotropin (hCG) (10 ng/ml) significantly increased testosterone and progesterone production relative to their respective controls. In contrast, neither steroid was increased by treatment with rat PRL (rPRL) or ovine PRL (oPRL) alone. Upon addition of 0.1-3 ng/ml of either rPRL or oPRL to the hCG-treated cultures, testosterone production was progressively increased up to a maximum of 70% greater than with hCG alone. However, when PRL exceeded 3 ng/ml, the testosterone response began to decline and was 39 or 24% less than from cells treated with hCG alone at 300 ng/ml of rPRL or oPRL, respectively. A similar biphasic response pattern was observed in cells from neonatal animals. In contrast to the biphasic effect of PRL on production of androgen, PRL treatment enhanced hCG-stimulated production of progesterone in a dose-related manner without exerting an inhibitory effect. At 3 and 300 ng/ml, rPRL augmented hCG action by 2.5- and 8-fold, respectively. Similarly, in the presence of inhibitors of pregnenolone metabolism, rPRL also enhanced hCG-stimulated production of pregnenolone. Quantitation of steroid intermediates in the testosterone biosynthetic pathway revealed that the stimulatory effect of 3 ng/ml rPRL on testosterone production was associated with 1.3- and 2.8-fold increases in accumulation of androstenedione and 17 alpha-hydroxyprogesterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endogenous steroid production in cellular and subcellular fractions of rat testis after prolonged treatment with gonadotropins.

Steroid production and enzyme activities were examined in preparations of whole testis tissue, isolated interstitial tissue and seminiferous tubules obtained from adult rats with intact pituitaries receiving daily subcutaneous injections of 100 I.U. human chorionic gonadotropin for 5 days and from control animals. After human chorionic gonadotropin administration testosterone concentrations were increased in total homogenates of whole testis tissue, interstitial tissue and seminiferous tubules. The testosterone production from endogenous precursors was enhanced only in total homogenates of whole testis tissue and interstitial tissue obtained from testes of human chorionic gonadotropin-treated rats. The production of testosterone in the corresponding homogenates of isolated seminiferous tubules was very low. The specific activity of 3 beta-hydroxysteroid dehydrogenase was increased in total homogenates of whole testis tissue, isolated interstitial tissue and seminiferous tubules. No effect was observed on the specific activities of marker enzymes such as cytochrome c oxidase, monoamine oxidase, steroid sulfatase and lactate dehydrogenase, whereas the specific activities of carboxyl esterase were decreased in homogenates of whole testis tissue and interstitial tissue. Total activity of monoamine oxidase was increased in homogenates of interstitial tissue of tests from human chorionic gonadotropin treated rats. After the same prolonged human chorionic gonadotropin treatment the concentration of pregnenolone was increased in mitochondrial fractions of whole testis tissue, interstitial tissue and seminiferous tubules, and the amount of protein isolated in the mitochondrial fraction of interstitial tissue increased by 40%. Steroid production (estimated as pregnenolone) from endogenous precusors by mitochondrial fractions of whole testis tissue and interstitial tissue were increased after human chorionic gonadotropin treatment, for whole testis from 580 pmol/mg mitochondrial protein per h to 1420 pmol/mg per h; and for interstitial tissue from 2665 pmol/mg per h to 7050 pmol/mg per h. The production of pregnenolone in mitochondrial fractions obtaine from isolated seminiferous tubules was very low and contributed hardly at all to the total pregnenolone production in mitochondrial fractions of whole testis tissue from normal rats as well as from human chorionic gonadotropin-treated rats.     

Decreased luteinizing hormone-stimulated progesterone secretion by preovulatory follicles isolated from cyclic rats treated with the progesterone antagonist RU486.

Since administration of the antiprogesterone RU486 to cyclic female rats at metestrus and diestrus results in increased serum levels of LH, estradiol, and testosterone at proestrus, we investigated whether RU486 affects follicular steroidogenesis. Female rats with a 4-day estrous cycle, induced experimentally by a single injection of bromocriptine on the morning of estrus, were given RU486 (2 mg) twice daily (0900 and 1700 h) on metestrus and diestrus. At proestrus the preovulatory follicles were isolated and incubated for 4 h in the absence and presence of LH. In the absence of LH, accumulation of estradiol, testosterone, and progesterone in the medium was not different for RU486-treated rats and oil-treated controls. In contrast, LH-stimulated estradiol, testosterone, and progesterone secretions were significantly lower in RU486-treated rats compared with controls. Addition of pregnenolone to the incubation medium resulted in a significantly lower increase of progesterone in follicles from RU486-treated rats compared with those from oil-treated controls. This suggests that 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity is decreased by administration of RU486 in vivo. Aromatase and 17 alpha-hydroxylase/C17-20 lyase activities were not affected: addition of substrate (androstenedione and progesterone respectively) did not affect differently the amount of product formed (estradiol and testosterone) in RU486- and oil-treated rats. However, LH-stimulated pregnenolone secretion was lower in follicles from RU486-treated rats compared with follicles from oil-treated controls, suggesting that either cholesterol side-chain cleavage activity or LH responsiveness is decreased. At proestrus the preovulatory follicles from RU486- and oil-treated rats were not morphologically different.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of A23187 and dibutyryl cyclic AMP on progestagen production by rat granulosa cells in vitro

The gonadotrophic regulation of progesterone production by rat granulosa cells was examined in a chemically-defined medium containing FSH, dibutyryl cyclic AMP [Bu)2cAMP) and the calcium ionophore, A23187. FSH and A23187 alone significantly enhanced the production of pregnenolone, progesterone and its metabolite, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) from endogenous substrate(s). Stimulation of progesterone production by A23187 was accompanied by an increase in 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) but not 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity, as attested by enhancement of the metabolism of exogenous pregnenolone to progesterone but not of progesterone to 20 alpha-OH-P. In contrast, although (Bu)2cAMP increased pregnenolone and progesterone production and the metabolism of exogenous progesterone to 20 alpha-OH-P, it failed to stimulate the conversion of exogenous pregnenolone to progesterone. The increase in progesterone production and in the conversion of exogenous pregnenolone to progesterone by FSH and A23187 was concentration- and time-dependent. Whereas maximal stimulation of de-novo progesterone synthesis by FSH was evident by 6 h (earliest time examined), a significant increase in the conversion of exogenous pregnenolone to progesterone in the presence of FSH or the ionophore was not noted until 12 h of incubation. Although a small but significant increase in progesterone production was also noted as early as 6 h of incubation in the presence of the calcium ionophore, this was markedly smaller than that elicited by FSH. We conclude that the calcium ionophore A23187 and (Bu)2cAMP have similar as well as distinct effects on progesterone production in rat granulosa cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monodansylcadaverine inhibition of testicular 17-ketosteroid reductase

Dispersed mouse testicular interstitial cells were treated with the transglutaminase inhibitor monodansylcadaverine (500 microM) for 30 min. Subsequent incubation of the cells with [3H]pregnenolone increased formation of steroidogenic intermediates, tentatively identified as progesterone, 17 alpha-hydroxyprogesterone, and androstenedione, but decreased testosterone formation by monodansylcadaverine-treated cells. Measurement of 17-ketosteroid reductase activity (the enzyme that converts androstenedione to testosterone) demonstrated that monodansylcadaverine treatment caused a reversible, noncompetitive inhibition of this enzyme. These results suggest that transglutaminase catalyzed protein cross-links may influence the activity of 17-ketosteroid reductase.     

Pituitary and gonadal function during physical exercise in the male rat

The effects of training and acute exercise on serum testosterone, luteinizing hormone (LH) and corticosterone levels and on testicular endocrine function in male rats were studied. In the first part of the study, the rats were trained progressively on a treadmill, over 8 weeks. Training did not change the basal levels of serum testosterone, LH and corticosterone, or the testicular concentrations of testosterone and its precursors progesterone and androstenedione. The levels of testicular LH (30.3 +/- 2.6 ng/g wet wt, mean +/- SEM) and lactogen (150 +/- 14 pg/g) receptors were unchanged after training. However, the capacity of testicular interstitial cell suspensions to produce cAMP and testosterone increased by 20-30% during in vitro gonadotropin stimulation. In the second part, the trained and untrained control animals underwent acute exhaustive exercise. Serum testosterone levels decreased by 74 and 42% in trained and untrained rats, respectively (P less than 0.02), and corticosterone rose by 182% in trained and 146% in untrained rats (P less than 0.01), whereas the LH level was unchanged. Testicular levels of testosterone and its precursors decreased, with the exception of unchanged androstenedione, in trained rats; the cAMP concentration was unchanged. In both trained and untrained rats, acute exercise decreased the capacity of interstitial cell suspensions to produce cAMP, whereas there were no consistent effects on testosterone production. Acute exercise had no effect on LH or lactogen receptors in testis tissue. In conclusion, training had no effect on serum or testicular androgen concentrations, but increased Leydig cell capacity to produce testosterone and cAMP. Acute exercise decreased serum and testicular testosterone concentrations without affecting serum LH. A direct inhibitory effect of the increased serum corticosterone level on the hypothalamic-pituitary level and/or testis may be the explanation for this finding.     

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.     

Effects of chronic ethanol ingestion on steroid profiles in the rat testis

Testosterone, seven of its potential precursors, three of its metabolites and estradiol were analyzed in testes from rats given ethanol for 23 days in a nutritionally adequate liquid diet. The results were compared to those obtained with pair-fed control rats. The concentrations of pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione and testosterone were markedly lowered in four of the five rats given ethanol. The concentrations of the other 3 beta-hydroxy-delta 5 steroids and estradiol were unchanged, resulting in significantly increased ratios between 17-hydroxypregnenolone and 17-hydroxyprogesterone (P less than 0.025) and between androstenediol and testosterone (P less than 0.025) in the ethanol-treated rats. The results indicate that chronic ethanol administration reduces formation of testosterone by affecting a step prior to pregnenolone. There may also be an effect on the conversion of some 3 beta-hydroxy-delta 5 to the corresponding 3-oxo-delta 4 steroids. The levels of testosterone and three other steroids in testes of rats given the liquid diet were significantly lower than those in testes of animals fed a standard rat chow. This indicates a dietary influence on testicular steroid concentrations.     

R1881 regulation of steroidogenesis in cultured testicular cells

The influence of a synthetic androgen R1881 upon hCG stimulated steroidogenesis in cultured rat testicular cells was investigated. Testicular cells were cultured for 8 days in medium alone and thereafter reincubated for 48 h with appropriate treatments before the collection of media for steroid RIA. Addition of R1881 (10(-6) M) resulted in an overall decrease of hCG (0.3-10 ng/ml) stimulated pregnenolone and progesterone production by cultured cells. The conversion of exogenous steroids of the delta 4 pathway (progesterone,17 alpha-OH-P and delta 4-A) was also studied in cultures supplemented with cyanoketone (10(-5) M) and/or spironolactone (10(-5) M) to prevent endogenous testosterone production. R1881 inhibited progesterone and 17 alpha-OH-P conversion to testosterone (T) and was ineffective when delta 4-A served as precursor for T biosynthesis. The inhibitory effect of R1881 upon Testosterone production was prevented by concomitant treatment with CPA. These observations suggest that R1881 decreases the hCG stimulated testosterone production via inhibition of CSCCE,3 beta-HSD,C17-20 Lyase and likely 17 alpha-Hydroxylase, whereas no effect on 17 beta-HSD could be observed.     

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.