Anti-anxiety, cognitive, and steroid biosynthetic effects of an isoflavone-based dietary supplement are gonad and sex-dependent in rats.

Isoflavone-rich diets are associated with reduced menopausal symptoms and lowered risk of cancers of reproductive tissues. Isoflavones may mimic some effects of estrogen by binding to estrogen receptors, and/or altering steroid availability. Despite their potential health benefits, neither the effects, nor mechanisms, of isoflavones are well understood. We hypothesized that isoflavones would alter behavior and physiology of rats in sex and/or gonad-dependent manner. An isoflavone-based, commercially-available, dietary supplement was administered via subcutaneous implantation to female and male, intact and gonadectomized Long-Evans rats. Affective (elevated plus-maze), cognitive (water-maze), and reproductive (sexual) behavior was examined. Weights of reproductive structures were measured, as an index of trophic effects. Steroid levels in circulation and brain regions associated with behavioral measures were evaluated by radioimmunoassay. The supplement increased anti-anxiety behavior of intact, but not gonadectomized, rats. The supplement enhanced visual-spatial performance of all rats, but this effect was most evident among proestrous female rats, which had the poorest spatial performance. There were neither effects of the supplement on sexual behavior, mass of reproductive tissues, nor plasma steroid levels. The supplement increased levels of 5α-androstane,17ß-diol-3α-diol (3α-diol) in the hippocampus (but not other brain regions) of gonadectomized females. Thus, the supplement altered anxiety and cognitive behavior and brain production of steroids; however, the anti-anxiety effects were limited to rats with an intact reproductive axis and effects on cognitive performance and neurosteriodogenesis were most evident among intact and gonadectomized, female rats respectively.

Neurosteroids: endogenous role in the human brain and therapeutic potentials.

This chapter provides an overview of neurosteroids, especially their impact on the brain, sex differences and their therapeutic potentials. Neurosteroids are synthesized within the brain and rapidly modulate neuronal excitability. They are classified as pregnane neurosteroids, such as allopregnanolone and allotetrahydrodeoxycorticosterone, androstane neurosteroids, such as androstanediol and etiocholanolone, and sulfated neurosteroids such as pregnenolone sulfate. Neurosteroids such as allopregnanolone are positive allosteric modulators of GABA-A receptors with powerful anti-seizure activity in diverse animal models. Neurosteroids increase both synaptic and tonic inhibition. They are endogenous regulators of seizure susceptibility, anxiety, and stress. Sulfated neurosteroids such as pregnenolone sulfate, which are negative GABA-A receptor modulators, are memory-enhancing agents. Sex differences in susceptibility to brain disorders could be due to neurosteroids and sexual dimorphism in specific structures of the human brain. Synthetic neurosteroids that exhibit better bioavailability and efficacy and drugs that enhance neurosteroid synthesis have therapeutic potential in anxiety, epilepsy, and other brain disorders. Clinical trials with the synthetic neurosteroid analog ganaxolone in the treatment of epilepsy have been encouraging. Neurosteroidogenic agents that lack benzodiazepine-like side effects show promise in the treatment of anxiety and depression.

Development of a cryopreservation protocol for Leydig cells.

BACKGROUND: In the present study, we describe a procedure to cryopreserve the postnatal members of the Leydig cell lineage, including progenitor (PLC), immature (ILC) and adult (ALC) Leydig cells from, respectively 21-, 35- and 90-day-old rats. METHODS: The cells were resuspended in a culture medium supplemented with 1% bovine serum albumin (Dulbecco's Modified Eagle's Medium [DMEM]/F12) to a final concentration of 2 x 10(6)cells/ml and the effects of varying concentrations of dimethylsulfoxide (DMSO) (5, 10, 15 or 20%) were assessed after freezing at -70 degrees C and then storing in liquid nitrogen. After 12 months of frozen storage, these cells were thawed rapidly at 37 degrees C and Trypan Blue exclusion staining and attachment to culture dishes were assessed as measures of viability. RESULTS: The trypan blue exclusion and attachment rates for Leydig cell stages were around 85% in the presence of 15% DMSO. After frozen storage, Leydig cell steroidogenic capacity in response to a range of LH doses, (0.01-100 ng/ml) was unchanged compared with freshly isolated control cells. Furthermore, the steady-state mRNA levels for Leydig cell specific transcripts were maintained. CONCLUSIONS: This study demonstrates that purified rat Leydig cells at a range of developmental stages can be frozen and that the cryopreserved cells retain normal function.

A high-performance liquid chromatography-tandem mass spectrometry assay of the androgenic neurosteroid 3alpha-androstanediol (5alpha-androstane-3alpha,17beta-diol) in plasma.

The testosterone metabolite 3alpha-androstanediol (5alpha-androstane-3alpha,17-diol) is a potential GABA(A) receptor-modulating neurosteroid with anticonvulsant properties and hence could act as a key neuromodulator in the central nervous system. However, there is no specific and sensitive assay for quantitative determination of the androgenic neurosteroid 3alpha-androstanediol in biological samples. We have established a liquid chromatography-tandem mass spectrometry (LC-MS-MS) assay to measure 3alpha-androstanediol in rat plasma. Standard 3alpha-androstanediol added to rat plasma has been successfully analysed with excellent linearity, specificity, sensitivity, and reproducibility. The sensitivity of the method was < 10 ng/ml with a detection limit of 2 ng/ml (6.8 nmol/l) and a linear range of 10-2000 ng/ml. The method was used for the analysis of testosterone-induced increase in plasma 3alpha-androstanediol levels in rats. Testosterone produced a dose-dependent elevation in plasma 3alpha-androstanediol, which was almost completely prevented by pretreatment with the 5alpha-reductase inhibitor finasteride, indicating that 3alpha-androstanediol is synthesized from testosterone via a 5alpha-reductase pathway. This LC-MS-MS method allows accurate, high-throughput analysis of 3alpha-androstanediol in small amounts (200 microl) of plasma and possibly other biological samples.

Unsolved problems in male physiology: studies in a marsupial.

Testicular androgens induce formation of the male urogenital tract in all mammals. In marsupials male development occurs after birth and over a prolonged period. For example, in the tammar wallaby virilization of the Wolffian ducts begins by day 20, prostate formation begins about day 25, and phallic development starts after day 80 of pouch life. Between days 20 and 40 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol) is formed in tammar testes and secreted into plasma. Administration of 5alpha-adiol to pouch young females induces urogenital sinus virilization by day 40 and formation of a mature male prostate and phallus by day 150. 5alpha-Adiol is synthesized in pouch young testes by two pathways, one involving testosterone and dihydrotestosterone and the other 5alpha-pregnane-3alpha,17alpha-diol-20-one and androsterone as intermediates, both utilizing steroid 5alpha-reductase. In target tissues 5alpha-adiol acts via the androgen receptor after conversion to dihydrotestosterone but may have other actions as well. Whether 5alpha-adiol plays a role in male development in placental mammals is uncertain.

5alpha-androstane-3alpha,17beta-diol is formed in tammar wallaby pouch young testes by a pathway involving 5alpha-pregnane-3alpha,17alpha-diol-20-one as a key intermediate.

The synthetic pathway by which 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol) is formed in the testes of tammar wallaby pouch young was investigated by incubating testes from d 20-40 males with various radioactive precursors and analyzing the metabolites by thin-layer chromatography and HPLC. [(3)H]Progesterone was converted to 17-hydroxyprogesterone, which was converted to 5alpha-adiol by two pathways: One involves the formation of testosterone and dihydrotestosterone as intermediates, and the other involves formation of 5alpha-pregnane-3alpha,17alpha-diol-20-one (5alpha-pdiol) and androsterone as intermediates. Formation of 5alpha-adiol from both [(3)H]testosterone and [(3)H]progesterone was blocked by the 5alpha-reductase inhibitor 4MA. The addition of nonradioactive 5alpha-pdiol blocked the conversion of [(3)H]progesterone to 5alpha-adiol, and [(3)H]5alpha-pdiol was efficiently converted to androsterone and 5alpha-adiol. We conclude that expression of steroid 5alpha-reductase in the developing wallaby testes allows formation of 5alpha-reduced androgens by a pathway that does not involve testosterone as an intermediate.

Altered balance between the 5 alpha-reductase and aromatase pathways of androgen metabolism during controlled ovarian hyperstimulation with human menopausal gonadotropins.

PURPOSE: To evaluate androgen production and metabolism during controlled ovarian hyperstimulation. METHODS: Five women, aged 33-42, were studied. All participants were undergoing controlled ovarian hyperstimulation with gonadotropin-releasing hormone agonist and human menopausal gonadotropins. Serum estradiol, estrone, androstenedione, testosterone, 3 alpha-androstanediol glucuronide, and sex hormone-binding globulin levels were measured at 6 time points during the cycle. RESULTS: The levels of all steroids increased significantly from baseline during controlled ovarian hyperstimulation. Mean total testosterone levels increased from 0.29 +/- 0.05 ng/mL to 0.58 +/- 0.07 ng/mL after gonadotropin stimulation. Sex hormone-binding gonadotropin levels increased from 50 +/- 16 nM to 73 +/- 12 nM after gonadotropin stimulation. Estrone/androstenedione and estradiol/testosterone ratios, reflecting the aromatase pathway, increased whereas 3 alpha-androstanediol glucuronide/androstenedione and 3 alpha-androstanediol glucuronide/testosterone ratios, reflecting 5 alpha-reductase activity, decreased. CONCLUSIONS: Controlled ovarian hyperstimulation with human menopausal gonadotropins results in increased serum testosterone and androstenedione levels. Whereas there is an enhancement in androgen metabolism by aromatase, 5 alpha-reductase activity with regard to androgen metabolism is diminished.

Evidence for significant differences in microsomal drug glucuronidation by canine and human liver and kidney.

The in vitro glucuronidation of a range of structurally diverse chemicals has been studied in hepatic and renal microsomes from human donors and the beagle dog. These studies were undertaken to improve on the limited knowledge of glucuronidation by the dog and to assess its suitability as a model species for pharmacokinetic studies. In general, the compounds studied were glucuronidated severalfold more rapidly (based on intrinsic clearance estimates) by DLM than by HLM. Intrinsic clearance values for human UGT1A1 and UGT2B7 substrates were an order of magnitude higher in DLM than in HLM (e.g., gemfibrozil: 31 microl/min/mg versus 3.0 microl/min/mg; ketoprofen: 2.4 microl/min/mg versus 0.2 microl/min/mg). There were also drug-specific differences. HLM readily glucuronidated propofol (2.4 microl/min/mg) whereas DLM appeared unable to glucuronidate this drug directly. Regioselective differences in morphine glucuronidation were also apparent. Human kidney microsomes catalyzed the glucuronidation of many xenobiotics, although glucuronidation of the endobiotic bilirubin was not detectable in this tissue. In direct contrast, dog kidney microsomes glucuronidated bilirubin only (no glucuronidation of all other xenobiotics was detected). These preliminary studies indicated significant differences in the glucuronidation of xenobiotics by microsomes from the livers and kidneys of human and dog and should be confirmed using a larger panel of tissues from individual dogs. Early knowledge of the relative rates of in vitro glucuronidation, the UGTs responsible for drug glucuronidation, and their tissue distribution in different species could assist the design and analysis of preclinical pharmacokinetic and safety evaluation studies.

Developmental response by Leydig cells to acidic and basic fibroblast growth factor.

The present study examines the effects of acidic (FGF-1) and basic (FGF-2) fibroblast growth factors on Leydig cell steroidogenesis by cells from 5-, 21- and 90-day-old rats. These ages represent three distinct time points in Leydig cell development: fetal Leydig cells (day 5), immature Leydig cells (day 21) and adult Leydig cells (day 90). The results demonstrate that the actions of the two growth factors on steroidogenesis are developmentally regulated, and require the presence of heparan sulphate proteoglycans (HSPG). FGF-1 and FGF-2 both had stimulatory effects on basal, but not maximally LH-stimulated, testosterone production by fetal Leydig cells, and both growth factors stimulated basal 5 alpha-androstane-3 alpha, 17 beta-diol production by immature Leydig cells. These effects were mediated by heparan sulphate-proteoglycans (HSPG), as they were blocked by the addition of protamine sulphate and sodium chlorate. FGF-1 and FGF-2 had no effect on basal testosterone production by adult Leydig cells, however, FGF-1 alone inhibited LH-stimulated testosterone production by adult Leydig cells in a dose-dependent manner. These data demonstrate that the effects of FGF-1 and FGF-2 are dependent on the specific stage of Leydig cell differentiation and development and may vary accordingly. Furthermore, although FGF-1 and FGF-2 are closely related structurally, a different effect of these two growth factors can be observed on the same type of Leydig cells. The data therefore suggest that these growth factors may have different but specific roles in the regulation of Leydig cell steroidogenesis, at different stages of development.

Effect of gossypol on 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activities in adult rat testes.

It has been reported that little to no 5 alpha-reductase can be detected in adult rat testes when progesterone is used as substrate. The 5 alpha-reductase activity in 4-month-old rats and the inhibitory action of gossypol on steroidobiosynthesis were studied. Testicular sections (10 microns thickness) were incubated at 30.5 degrees C in the presence of NADPH with 3H-testosterone and cold testosterone as substrates (9 microM total), and with or without gossypol as the test sample and control, respectively. Endogenous testosterone level was evaluated by radioimmunoassay. Reverse phase high performance liquid chromatography (HPLC) was used to separate the substrate and products. Components of interest were collected and their recovery monitored. At 200 microM concentration, gossypol significantly decreased dihydrotestosterone (DHT) formation by 21% when compared to that of control (0.6 pm/mg protein/min), and decreased 5 alpha-androstane-3 alpha,17 beta-diol formation by 35% vs control (2 pm/mg protein/min). In the current study, gossypol was found to have inhibitory effects of noncompetitive nature on 5 alpha-reductase, which catalyzes the conversion of testosterone to DHT, and on 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), which interconverts DHT and dihydroandrostanediol.

Androstanediol glucuronide production in human liver, prostate, and skin. Evidence for the importance of the liver in 5 alpha-reduced androgen metabolism.

Androstanediol glucuronide (Adiol G) has been reported to be a marker of peripheral androgen metabolism and action. It consists of two isomers, Adiol 3-G and Adiol 17-G. Adiol G is formed from unconjugated precursors by the enzyme glucuronyl transferase. To determine the likely source of Adiol G formation in man, we developed a glucuronyl transferase assay and measured the activity of this enzyme in human liver, abdominal and scalp skin, and prostate. In human liver, glucuronyl transferase activity was linear with respect to time (up to 120 min) and tissue concentration (up to 1 mg/ml). Apparent Michaelis-Menten constant Km (micromolar) and maximum velocity (Vmax) (picomoles per mg/30 min) were 5.6 and 140 for dihydrotestosterone, 8.9 and 1300 for androstanediol, and 3.1 and 46 for androsterone, respectively. Conversion of androstanediol to Adiol G (/0.5 mg tissue.30 min) was 5.8-13.2%. Over 80% of the Adiol G formed in human liver was Adiol 17-G, similar to what has been previously found in human serum. Glucuronyl transferase activity was present at low levels in human prostate (conversion of androstanediol to Adiol G was 0.04-4.6%/50 mg tissue.120 min). Analogous conversion rates (/50 mg tissue.120 min) for human scalp skin were 0.2-0.4% and for human abdominal skin were 0.07-0.14%. Although dihydrotestosterone may be converted to androstanediol in peripheral tissues such as skin and prostate, our results suggest that the principal site of androgen conjugation to glucuronic acid is the liver. The present results cast doubt upon the role of androstanediol glucuronide as a specific marker of cutaneous androgen metabolism.

Ageing of the neuroendocrine system in the brain of male rats: receptor mechanisms and steroid metabolism.

The work described in this article gives information on the effects of ageing on the hypothalamo-pituitary-testicular axis in rats. The hypothalami of young and old male rats contain similar amounts of luteinizing-hormone-releasing hormone (LHRH); when perifused in vitro they release comparable amounts of LHRH under basal conditions and in response to K+. The addition of an LHRH analogue to the perifusion medium blocks the release of LHRH induced by K+ from the hypothalami of young and old male rats, indicating that the ultrashort feedback mechanism controlling LHRH release functions normally in aged male rats. Ageing also exerts important effects on the density of mu- and kappa-opioid receptors in the brain. The number of hypothalamic mu-opioid receptors was significantly decreased in aged animals; a replacement treatment with testosterone does not reverse this decrease, indicating that the decline of hypothalamic mu receptors and of serum titres of testosterone in old rats are independent phenomena. The number of kappa-opioid receptors in the brain increases in the amygdala and in the thalamus with ageing. Apparently ageing does not influence the number of delta receptors in any of the brain areas investigated. The number of pituitary LHRH receptors decreases in old animals, which might explain the low serum concentration of gonadotrophins in aged rats caused by an inadequate response of the pituitary to hypothalamic LHRH. The impaired secretion of testosterone in aged male rats is accompanied by an increase in the number of testicular LHRH receptors, indicating that the intratesticular mechanisms controlling testosterone release also undergo significant alterations during ageing. The rate of conversion of testosterone to dihydrotestosterone (DHT) and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) is the same in the hypothalami of young and old rats. However, the yields of DHT obtained from the pituitaries of aged male rats are significantly lower than those recorded in the pituitaries of young animals. These results show that the enzymes necessary for metabolizing testosterone via the 5 alpha-reductase pathway are maintained both in the hypothalamus and in the anterior pituitary of aged male rats. However, the 5 alpha-reductase activity of the anterior pituitary of senescent animals appears to be lower than that in the younger controls.

Effect of temperature and the role of testicular descent on post-natal testicular androgen production in the mouse.

Testes from mice aged 3, 15, 25, 30 or 60 days were incubated under basal conditions or in the presence of hCG. One testis from each animal was incubated at 37 degrees C while the contralateral testis was incubated at 32 or 34 degrees C. During development total androgen production in response to hCG (at 32 degrees C) showed a marked increase between 15 and 30 days. The major androgens secreted at this time were testosterone and 5 alpha-androstane-3 alpha,17 beta-diol. There was little change in total androgen production between 30 and 60 days but by 60 days testosterone was the dominant androgen. Both basal and hCG-stimulated androgen production were temperature sensitive. These effects were most pronounced at 30 and 60 days with androgen production significantly inhibited at 37 degrees C. To examine the role of testicular descent in regulating steroidogenesis animals were rendered unilaterally cryptorchid at 19 days of age. At 25 days, when descent is normally completed in the mouse, there was no significant difference in steroidogenesis between scrotal and abdominal testes. By 30 days, however, the steroidogenic potential of the abdominal testis was significantly lower than that of the scrotal testis. These results show that testicular steroidogenesis is sensitive to temperature changes around the time of testicular descent, although descent itself is not required to achieve an adult level of steroidogenesis. The results also show, however, that testicular descent is required to maintain the adult level of steroidogenesis.

Production and secretion of C-19 steroids by rat and guinea pig adrenals.

The concentrations of C-19 steroids were measured in guinea pig and rat adrenals before and after castration as well as after stimulation with adrenocorticotropin hormone (ACTH). Characterization of adrenal C-19 steroids was also carried out by isolation with high-performance liquid chromatography and gas chromatography/mass spectrometry (GC/MS). From radioimmunoassay (RIA) data, androstenedione (4-DIONE) and 11 beta hydroxyandrostenedione (11 beta-DIONE) were the major C-19 steroids found in guinea pig adrenals, and castration induced a decrease of 4-DIONE levels only while all other C-19 steroids remained unchanged. In rat adrenals, the major C-19 steroids were 4-DIONE and testosterone, and they were also markedly inhibited after castration. With the exception of 11 beta-DIONE, all other C-19 steroids in circulation were eliminated after castration in both animals species. After ACTH administration in the guinea pig, adrenal 4-DIONE and 11 beta-DIONE levels were markedly stimulated, while an increase of only 11 beta-DIONE was observed in plasma. In the rat, ACTH had a small stimulatory effect on adrenal 52-androstane-3 alpha, 17 beta-diol (3 alpha-DIOL) and plasma 11 beta-DIONE levels. Analysis of guinea pig adrenal steroids by GC/MS confirmed the presence of C-19 steroids in adrenals (namely, 4-DIONE and 11 beta-DIONE) while, in the rat, this could not be confirmed. Our data indicate that production of C-19 steroids occurs in guinea pig adrenals, and 11 beta-DIONE is the major C-19 steroid as well as the only C-19 steroid secreted into the circulation. In the rat, the production of C-19 steroids detected by RIA is not supported by GC/MS data.

Formation of androstanediol from 13C-labeled testosterone in humans.

The determination of urinary 5 alpha-androstane-3 alpha,17 beta-diol (3a-Diol) by gas chromatography/mass spectometry during and after the infusion of stable-labeled testosterone (T) represents an alternative to the use of radioactive label for turnover studies in vivo. Using this methodology to assess the urinary excretion rates of T and 3a-Diol in healthy men (n = 6) and women (n = 5) during and after the intravenous infusion (t = 4 hours) of 20 mg (men) or 5 mg (women) [13C]testosterone, the cumulative renal excretion of 13C-labeled T was found to be 15.6 +/- 9.6 micrograms/24 hours (men) and 1.1 +/- 1.6 micrograms/24 hours (women), equivalent to 0.08% +/- 0.05% and 0.02% +/- 0.03% of the infused amount of 13C-T, respectively. The cumulative excretion of 13C-3a-Diol was 67.7 +/- 19.9 micrograms/24 hours (men) and 10.0 +/- 6.0 micrograms/24 hours (women), equivalent to 0.3 +/- 0.1% and 0.2 +/- 0.1% of the infused dose of 13C-labeled testosterone, respectively.

Testosterone and androstanediol production by regenerating Leydig cells in the ethylene dimethane sulphonate-treated mature rat.

Mature (60-65 day old) male Sprague-Dawley rats received a single intraperitoneal injection of ethylene dimethane sulphonate (EDS; 100 mg/kg) and were subsequently killed at various times from day 2 to day 40 post-treatment. Testes were removed from these animals and age-matched controls and utilized either for light and electron microscopical analyses or for in-vitro assessment of Leydig cell function. Interstitial cells were prepared by collagenase digestion and used to measure 125I-labelled human chorionic gonadotrophin (hCG) binding capacity and androgen production in the presence or absence of hCG or dibutyryl cyclic AMP (dbcAMP). At day 2 after EDS treatment, 125I-labelled hCG binding capacity was reduced to 10% of control values, while the production of testosterone and 5 alpha-androstane-3 alpha, 17 beta-diol (adiol) were non-detectable. Histological observations confirmed the lack of identifiable Leydig cells at day 2-16 after EDS treatment. Between days 24 and 40 post-treatment, Leydig cell regeneration occurred, as indicated by a rise in 125I-labelled hCG binding capacity, increased androgen production and the presence of histologically identifiable Leydig cells. A pattern of adiol production similar to that seen in the immature rat during Leydig cell development was observed with peak synthesis occurring at day 30 post-treatment. Adiol production fell to barely detectable levels by day 36 and remained low at day 40. It is concluded that the steroidogenic pattern of regenerating Leydig cells in the EDS-treated animal is similar to that of developing Leydig cells in the immature animal.

Separation of interstitial and tubular cells of human testis by means of an anti-substance P-antiserum. Androstane-3 beta, 17 beta-diol is exclusively formed by seminiferous tubules.

Leydig cells and seminiferous tubules of human testicular tissue were successfully separated by means of an anti-substance P-antiserum. On incubation with (14C)-testosterone or (14C)-dihydrotestosterone (DHT) it could be shown that besides DHT and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) also 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) originated in the tubular compartment. The ratio 3 alpha/3 beta-diol was found to be higher (0.7) here compared to incubation with whole testicular tissue (0.2), indicating that the testicular interstitium influences the metabolism of DHT by the seminiferous tubules.

Effect of injection of gonadotrophin-releasing hormone on testicular steroidogenesis in the hypogonadal (hpg) mouse.

Hypogonadal (hpg) mice were injected once daily with 10 ng, 50 ng or 1 microgram GnRH for 5, 10 or 20 days or 12 times daily with 4.2 ng GnRH for 5 days. Basal and hCG-stimulated production in vitro of androstenedione, testosterone and 5 alpha-androstane-3 alpha,17 beta-diol (androstanediol) were measured by radioimmunoassay. All doses of GnRH increased testicular weight and in-vitro androgen production although seminal vesicle weights were unchanged and serum testosterone concentrations remained undetectable. After 5 days' treatment androstenedione and androstanediol were the dominant androgens produced, the latter indicating the presence of high levels of 5 alpha-reductase. By 20 days testosterone production was predominant after treatment with higher doses of GnRH. Total androgen production (androstenedione + testosterone + androstanediol) after 5 and 10 days was similar at all concentrations of GnRH used. After 20 days' treatment total androgen production was significantly greater with 50 ng GnRH/day than with 10 or 1000 ng/day. Multiple daily injections of 4.2 ng GnRH (total dose 50 ng/day) had no greater effect on androgen production in vitro compared to single daily injections of 50 ng. This suggests that under the conditions used in this study the testis does not require pulsatile release of the gonadotrophins. The pattern of [3H]pregnenolone metabolism was measured after 5 days injection of 50 ng GnRH/day. Compared to control hpg animals there was a significant increase in formation of C19 steroids, synthesis being solely through the 4-ene pathway. These results show that GnRH treatment of hpg mice will induce testicular steroidogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)