We have studied the effects of various steroids on DNA synthesis in MCF-7 human breast carcinoma cells, which have aromatase activity and which exert an oestrogen receptor-mediated growth, to assess the significance of intracellular aromatase on growth stimulation as well as inhibition by aromatase inhibitors. The cells were cultured for 96 h in phenol red-free medium containing 10% charcoal-treated fetal bovine serum and test reagents and pulse-labelled with [3H]thymidine. Physiological concentrations of oestradiol, oestrone, testosterone (T) and <em>androstenedione</em> (AD) stimulated thymidine incorporation. However, oestrone-sulphate and dihydrotestosterone (DHT) only stimulated at concentrations greater than the physiological levels. T and DHT stimulation was blocked by tamoxifen, but not by cyproterone acetate, suggesting that the stimulation was mediated via the oestrogen receptor but not by the androgen receptor. Stimulation by T and AD was reduced by aminoglutethimide and 1<em>4</em> alpha-hydroxy-<em>4</em>-androstene-3,6,17-trione, both of which inhibit aromatase activity, however, stimulation by nonaromatizable DHT was not reduced by the inhibitors, suggesting that androgens were converted by the intracellular aromatase to oestrogens which stimulated the thymidine incorporation. It is suggested that intracellular aromatase significantly contributes to the stimulation of DNA synthesis and that aromatase inhibitors suppress the stimulation.

This study evaluates the relationship between trait anxiety and both androgen and gonadotrophic hormone levels at rest and during severe physical exercise. Twelve volunteers were selected among 160 untrained male collegial students and classified as anxious (N = 6) or non-anxious (N = 6) subjects according to their scores on three trait-anxiety tests (STAI, IPAT, 16 PF). Serum delta <em>4</em>-androgen (testosterone and delta <em>4</em>-<em>androstenedione</em>), delta 5-androgen (DHEA and DHEA-SO<em>4</em>) and gonadotrophin (LH and FSH) concentrations were measured by radioimmunoassay before, during and after 20 minutes of intensive bicycle exercise (80% of maximal heart rate). Results indicate significantly lower serum delta <em>4</em>-androgens in anxious subjects before exercise. However, for each subject and irrespective of his anxiety level, all measured serum androgen concentrations increased significantly during exercise, although delta <em>4</em>-androstene-dione remained lower in anxious subjects than in non-anxious ones. Serum LH concentrations (but not FSH) were significantly higher in anxious subjects throughout the observation periods. However, exercise induced in each subject a significant decrease in the serum level of both gonadotrophic hormones. The results suggest that trait anxiety level may constitute an important factor that affects both pre-exercise and exercise serum androgen concentrations in untrained subjects.

To study the effects of prolactin (PRL) on adrenocortical function in humans, dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), <em>androstenedione</em> (delta) and testosterone (T) were measured in serum obtained from 35 hyperprolactinemic women with galactorrhea and amenorrhea before and after treatment with bromocriptine-induced fall in mean PRL levels from 82 +/- 8 (SE) to 1<em>4</em> +/- 2 ng/ml (n = 39, P less than 0.0005), DHAS fell from 322 +/- 21 to 237 +/- 21 microgram/dl (n = 39); P less than 0.0005), DHA fell from <em>4</em>92 +/- <em>4</em>7 to 378 +/- 30 ng/dl (n = 39; P less than 0.01) while T (n = 16) and delta (n = 13) levels were unchanges (<em>4</em><em>4</em> +/- <em>4</em> vs. <em>4</em>9 +/- <em>4</em> ng/dl and 280 +/- 55 vs. 236 +/- <em>4</em>0 ng/dl, respectively). In addition, <em>4</em> women were infused iv with 25 microgram synthetic ACTH over <em>4</em> h and serial blood samples drawn while hyperprolactinemic, and again 2-<em>4</em> months later following normalization of PRL levels by bromocriptine. Although pre-infusion levels of DHAS were lower when PRL levels were normalized, no significant differences in responses of circulating DHAS, DHA, T, cortisol and 17-hydroxyprogesterone concentrations were detected between the two infusions. Since DHAS is virtually an exclusive product of the adrenal cortex, and since high PRL levels appear to inhibit ovarian steroid production, the findings suggest that hyperprolactinemia selectively stimulates adrenocortical androgen production.

A 31-year-old nulligravid patient presented with irregular menses, severe hirsutism, and infertility. Evaluation revealed marked increases of serum <em>androstenedione</em> and testosterone levels and a possible ovarian mass. At operation a cystic teratoma was removed from the left ovary and bilateral wedge resection revealed severe ovarian hyperthecosis. After operation only a transient decrease of <em>androstenedione</em> and testosterone was noted and the patient failed to ovulate or improve clinically. Subsequently a long-acting gonadotropin-releasing hormone agonist was administered daily for 6 months, which reduced circulating delta <em>4</em>-steroids and estrogens to levels approximating those of castrated women. Immediately after discontinuation of treatment, ovulation induction was successfully achieved with human menopausal gonadotropin. This report introduces a new therapeutic approach to the problem of severe ovarian hyperthecosis and may provide an opportunity for childbearing in these patients.

Male pseudohermaphroditism due to 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) deficiency has a high prevalence within the Arab population of the Gaza strip and is characterised by marked virilization at puberty, leading in many cases to the spontaneous adoption of a male gender role. As a result of this, parents of 7 affected male infants (aged 1-10 months) born with female phenotype requested early gender reassignment. Diagnosis was suspected in 5 on the basis of a positive family history, but confirmed in all cases by the finding of low to normal testosterone levels (30-18<em>4</em> ng/dl) with high delta <em>4</em>-<em>androstenedione</em> levels (188-808 ng/dl), after hCG. Treatment with im testosterone oenanthate (25-50 mg/dose) was given in one to three 3-months courses and penile size was increased into the normal range without evoking a significant increase in height velocity or skeletal maturation. Five patients underwent the first stage of male genitoplasty between 2 and 3 years of age. This consisted of bilateral orchidopexy, chordee release and penile lengthening - yielding finally an anatomically normal-sized and shaped penis. Androgen responsive male pseudohermaphroditism due to 17 beta-HSD deficiency or a similar defect and diagnosed in infancy should be treated as soon as possible with systemic testosterone before considering any sex change, and in preparation for male genitoplasty. Early gender reassignment according to genetic and gonadal sex is probably the management of choice for these cases since this may result in a normal adjustment to the male gender role, particularly after puberty.

Changes in endogenous androgen metabolism were compared in healthy women taking one of four low-dose modern oral contraceptives (OCs). One hundred women were randomized to (1) 35 micrograms ethinyl estradiol (EE) + 250 micrograms norgestimate (Cilest); (2) 20 micrograms EE + 150 micrograms desogestrel (Mercilon); (3) 30 micrograms EE + 150 micrograms desogestrel (Marvelon); or (<em>4</em>) 30 micrograms EE + 75 micrograms gestodene (Femodene). During the luteal phase of the pretreatment cycle, body weight and blood pressure were recorded, and plasma levels of the following variables were recorded: sex-hormone-binding globulin (SHBG), cortisol-binding globulin (CBG), testosterone, free testosterone, dihydrotestosterone, <em>androstenedione</em>, dihydroepiandrosterone sulfate (DHEAS), and hydroxyprogesterone. The free androgen index was also calculated. These variables were remeasured during the third week of OC intake and during the fourth and sixth cycles. There were no statistically significant differences in androgenic variables among the four OCs. The DHEAS concentration decreased less with the 20 micrograms EE + desogestrel formulation compared with either 30 micrograms EE + desogesterel or norgestimate-containing formulations (20% vs. <em>4</em>5%). Concentrations of SHBG and CBG increased significantly in all four groups (average 263 +/- 119% and 9<em>4</em> +/- 26%, respectively); CBG increased less in women taking 20 micrograms EE + desogestrel (about 75%) than in the other formulations (about 100%). The four modern, low-dose OCs tested had similar impacts on endogenous androgen metabolism, yielding significant decreases in testosterone, dihydrotestosterone, <em>androstenedione</em>, and DHEAS. All of these formulations may be beneficial in women with androgen-related syndromes such as acne and hirsutism. Large studies are under way to establish which of the third-generation OCs is the least androgenic. In vitro studies suggest that norgestimate has the least androgenic profile.

Insulin resistance has been reported to be associated with hyperandrogenism and polycystic ovaries. To study the prevalence of insulin resistance in patients with polycystic ovary syndrome (PCO) and the correlation between hyperinsulinemia and hyperandrogenism, <em>4</em>8 patients were divided into four groups: group 1, non-obese ovulatory women (n = 10); group 2, obese ovulatory women (n = 9); group 3, non-obese women with PCO (n = 1<em>4</em>); group <em>4</em>, obese women with PCO (n = 15). Each patient was submitted to an oral glucose tolerance test (OGTT). Glucose, insulin, <em>androstenedione</em> and testosterone levels were determined and the blood glucose and insulin response of women with PCO and normal women were compared. Glucose intolerance was observed in group 3 (28.6%) and group <em>4</em> (<em>4</em>0%) but not in groups 1 or 2, and hyperinsulinemia was observed in group 2 (66.7%), group 3 (6<em>4</em>.3%) and group <em>4</em> (86.6%). There was a correlation between <em>androstenedione</em> and testosterone levels and insulinemia in group <em>4</em>. There was also a high prevalence of insulin resistance in patients with PCO regardless of obesity, and hyperandrogenism-aggravated insulin resistance.

OBJECTIVE

To study reversible determinants of infertility and recurrent loss of transferred embryos after failure of 7 of 10 embryo transfers, 1 live birth, and 2 miscarriages.

METHODS

Measures of thrombophilia, hypofibrinolysis, reproductive hormones, and androgenic steroids before and after metformin therapy.

METHODS

Outpatient clinical research center.

METHODS

A 32-year-old amenorrheic, infertile woman with polycystic ovary syndrome (PCOS) who had 7 of 10 embryo transfers fail, 1 premature live birth, and 2 miscarriages at 8 and 17 weeks.

METHODS

Metformin (2.55 g/d) was given to ameliorate the endocrinopathy of PCOS.

METHODS

Coagulation, insulin, reproductive hormones, and androgenic steroids.

RESULTS

The propositus had thrombophilia (familial protein S deficiency [free protein S 32%; normal>>/=65%]). She also had familial hypofibrinolysis with <em>4</em>G<em>4</em>G polymorphism of the plasminogen activator inhibitor (PAI-1) gene and high PAI-1 activity (PAI-Fx), <em>4</em>2.5 U/mL, normal <21.1. Polycystic ovary syndrome was characterized by amenorrhea, polycystic ovaries, high fasting serum insulin (39 microU/mL, normal <20), <em>androstenedione</em> (763 ng/dL, normal <250), and testosterone (229 ng/dL, normal <83). After she received metformin for <em>4</em> months, PAI-Fx normalized (12.<em>4</em> U/mL), as did insulin (12 microU/mL), <em>androstenedione</em> (185 ng/dL), and testosterone (39 ng/dL); weight fell from 109 to 91.3 kg (16%).

CONCLUSIONS

Metformin reversed the endocrinopathy of PCOS. Familial thrombophilia and hypofibrinolysis may lead to thrombosis-mediated uteroplacental vascular insufficiency, failure to achieve pregnancy after embryo transfer, and miscarriage.

In this prospective longitudinal study, plasma androgen levels were determined during 1-7 years in <em>4</em>5 patients aged 5.6-23.8 years with either isolated growth hormone (GH) deficiency or multiple pituitary hormone deficiencies. Dehydroepiandrosterone sulfate, dehydroepiandrosterone, delta <em>4</em>-<em>androstenedione</em> and testosterone were measured by RIA in 339 blood samples collected during the study period. Mean plasma androgen levels are normal in isolated GH deficiency. Patients with multiple pituitary hormone deficiencies, but normal ACTH reserve, have mean levels lower than normal. Patients with multiple deficiencies including ACTH deficiency have still lower plasma androgen levels. Longitudinal analysis of the data, however, shows that patients with either isolated GH deficiency or multiple pituitary deficiencies without ACTH deficiency constitute a heterogeneous population, with either normal or low to very low plasma androgen levels. Treatment with human GH as such does not have any effect on the adrenal androgen secretion. A dissociation is found in some patients between adrenarche and gonadarche, which indicates that the two events are not controlled by the same mechanisms. Our results support the existence of a specific hypothalamic-pituitary adrenal androgen-stimulating hormone (AASH). ACTH, although not identical to AASH, is essential for normal adrenarche. Induced puberty with estrogens in girls does not influence plasma androgen levels, and pubic and axillary hair growth is not achieved. It is suggested that replacement treatment with dehydroepiandrosterone sulfate should be administered to girls with hypopituitarism and very low plasma androgen levels at the time of the induction of puberty. Finally, it appears from this study that, to interpret the plasma androgens in hypopituitarism, body surface is as good as bone age.

BACKGROUND

To study the short-term biological effect of anastrozole on serum estrogens, androgens, 17-hydroxyprogesterone (17OH-PGR), gonadotrophins, sex hormone binding globulin (SHBG) and bone metabolism markers.

METHODS

Thirty-four consecutive patients with advanced breast cancer received anastrozole 1 mg/day. Blood samples were taken before commencement of treatment and at 2, <em>4</em>, 8 and 12 weeks during treatment to measure serum levels of estrogens (E(1), E(2) and E(1)-S), androgens [<em>androstenedione</em> (Delta(<em>4</em>)), dihydrotestosterone (DHT), testosterone (TST), free TST, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S)], 17OH-PGR, SHBG and gonadotrophins. As an indicator of bone resorption, we measured serum levels of C-terminal telopeptide of type I collagen (ICTP) and the cross-linked N-telopeptide of type I collagen (NTx), and for osteoblastic activity, intact osteocalcin (BGP) and bone alkaline phosphatase (BAP).

RESULTS

After 2 weeks E(1 )and E(1)-S levels decreased on average by 56% (range 23.1-88.8%) and 75.8% (range 52.<em>4</em>-87.2%), respectively; E(2) decreased on average by 62% (range 31.<em>4</em>-89.6%). No significant changes were detected in levels of androgens or 17OH-PGR. There was a significant increase in gonadotrophins over time (P = 0.0001 for both luteinizing hormone and follicle-stimulating hormone), and a significant decrease in SHBG (P = 0.0001). A progressive significant increase in bone metabolism serum markers was detected in all patients: BAP, P = 0.039; BGP, P = 0.016; ICTP, P = 0.0021; and NTx, P = 0.0013. In particular, patients with bone metastases had a statistically significant increase of bone resorption markers (ICTP, P = 0.0019; NTx, P = 0.025) and borderline for bone formation markers. In patients without bone disease, BAP, BGP and ICTP remained unchanged, whereas serum NTx significantly increased (P = 0.019).

CONCLUSIONS

Anastrozole is a selective aromatase inhibitor as it does not modify serum levels of androgens and 17OH-PGR. In our experience no relationship was found in the short-term period between serum estrogen suppression and bone metabolism.

Optimum conditions for transformation of phytosterols by Mycobacterium neoaururm, required for selective cleavage of the lateral chain into <em>androstenedione</em>, were shown to differ from the known conditions of animal sterol (cholesterol) transformation. Complete conversion of phytosterols into <em>androstenedione</em> at a substrate load of no less than 20 g/l was achieved on increasing the amount of the inoculum and the concentration of glucose (by 2 and <em>4</em> times, respectively, relative to cholesterol) and performing the fermentation under conditions of turbulent mixing. Under these conditions, both the rate of the transformation and the yield of the reaction product were high, due to the saturation of the culture liquid with hydrocarbonate. Data from the literature show that this ion is involved in cleavage of the branched lateral chain at carbon in position 2<em>4</em>.

OBJECTIVE

Androgenetic alopecia is recognized as a risk factor for cardiovascular diseases, glucose metabolism disorders, and benign prostate hyperplasia and/or carcinoma. Finasteride, used for treatment of androgenetic alopecia at a dose of 1mg/day, is an effective inhibitor of type II 5alpha-reductase, the enzyme responsible for the reduction of testosterone to dihydrotestosterone. Recent studies reported that dihydrotestosterone, among other activities, might play some role in visceral fat metabolism. It thus seemed reasonable to examine whether finasteride treatment of androgenetic alopecia ameliorates some features of metabolic syndrome frequently seen associated with this condition.

METHODS

We examined 12 men with premature balding (defined as frontoparietal and vertex hair loss before age 30 with alopecia defined as grade 3 vertex or more on the Norwood-modified Hamilton alopecia classification). Hormonal levels and metabolic parameters were determined and insulin tolerance tests performed for all individuals. Finasteride (1 mg/day) was administrated for 12 months. The hormonal profile and lipid spectrum were monitored after <em>4</em>, 8 and 12 months of treatment and insulin tolerance tests were repeated after 12 months of the treatment.

RESULTS

After treatment with finasteride the expected changes in the steroid spectrum were seen, namely a decrease in dihydrotestosterone and increase in testosterone, androstenedione and free testosterone index. We observed an initial increase in total cholesterol and HDL- and LDL-cholesterol, which stabilized with prolonged treatment. We founded a significant decrease in glycated hemoglobin HbA1c and insulin resistance measured using rate constant for plasma glucose disappearance (kITT) showed only a borderline decrease.

CONCLUSIONS

Finasteride is an efficient 5alpha-reductase inhibitor even at low doses of 1 mg/day. In men treated with this dose for 12 months, we observed mild differences in metabolic profile with slight amelioration of glucose metabolism regulation.

Oestrogens are known to exert specific stimulatory effects on basal and dopamine (DA)-inhibited prolactin (PRL) release as well as on PRL cell content in rat adenohypophysial cells in primary culture. Recently, we have demonstrated that classical oestrogens increase growth hormone (GH) release and cellular GH content in rat pituitary somatotrophs. Since there is evidence that 5-androstene-3 beta, 17 beta-diol (delta 5-diol), a metabolite of dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S) can induce typical oestrogenic responses in target tissues, we have investigated the effect of C19 adrenal steroids and compared them to that of 17 beta-oestradiol (E2) on the above-indicated parameters. Following a 72-h incubation, maximal concentrations of E2, delta 5-diol and DHEA caused a similar increase in PRL cell content at respective EC50 values of 0.020, 1<em>4</em> and 115 nM. The sensitivity of lactotrophs to DA action was decreased by approximately <em>4</em>-fold after a <em>4</em>8-h exposure to maximal concentrations of delta 5-diol or DHEA. The time course of the antidopaminergic effect of delta 5-diol and DHEA was almost superimposable to that of E2. A 72-h incubation with 5 microM DHEA-S, a concentration within the range of blood levels found in women, doubled (P less than or equal to 0.001) cellular PRL accumulation. On the other hand, <em>androstenedione</em> (delta <em>4</em>-dione) was without effect on any of the parameters measured. All stimulatory effects induced by maximal effective concentrations of delta 5-diol, DHEA or DHEA-S were competitively inhibited by simultaneous incubation with the antioestrogen LY156758. The amplitude of the stimulatory effects of delta 5-diol and DHEA on GH cell content as well as on spontaneous GH-releasing factor (GRF)-induced GH release was superimposable to that observed with E2. The effect of the steroids on GH cell content was exerted at 0.016 nM (E2), 12 nM (delta 5-diol) and 250 nM (DHEA). Simultaneous incubation with LY156758 completely blocked the effect of maximally effective concentrations of E2, delta 5-diol and DHEA in somatotrophs. Furthermore, a physiological concentration of DHEA-S (5 microM) enhanced spontaneous as well as GRF-induced GH release. On the other hand, delta <em>4</em>-dione as well as testosterone and dihydrotestosterone did not alter GH release. The present data demonstrate that delta 5-diol, DHEA and DHEA-S can exert full oestrogenic effects in lactotrophs and somatotrophs, thus supporting their potential oestrogenic role under both physiological and pathological conditions.

To characterize the aromatizable and 5 alpha-reduced androgens produced by developing ovarian follicles, small antral (SA) and preovulatory (PO) follicles, theca and granulosa cells were incubated for <em>4</em> h with or without 8-bromo-cAMP and <em>androstenedione</em>. In addition, thecal explants were cultured for 10 days with or without ovine LH (oLH) to determine if the hormone-induced changes in androgen synthesis by developing follicles could be mimicked in vitro. Short term incubations of SA and PO follicles, theca and granulosa cells in medium alone resulted in limited accumulation of androgen [testosterone, 5 alpha-androstan-17 beta-ol-3-one (DHT), 5 alpha-androstan-3 alpha, 17 beta-diol (3 alpha diol), and androsterone], as determined by RIA. In the presence of 8-bromo-cAMP, PO follicles produced large quantities of testosterone (3 ng), DHT (1 ng), 3 alpha diol (15 ng), and androsterone (1<em>4</em> ng), while SA follicles accumulated much less androgen (0.69, 0.05, 1.23, and 1.3 ng, respectively). In the presence of <em>androstenedione</em> and 8-bromo-cAMP, both SA and PO follicles and theca produced large amounts of aromatizable and 5 alpha-reduced androgens. SA and PO granulosa cells required the presence of the substrate <em>androstenedione</em> to produce androgens, primarily testosterone and 3 alpha diol. Therefore, progesterone, <em>androstenedione</em>, and 5 alpha-reduced androgens were used to monitor LH action on thecal cell function in culture. Small antral theca cultured in basic culture medium alone (containing 10% fetal calf serum) displayed an increased ability to accumulate <em>androstenedione</em> by day 6, approximately 3 times that observed on day 2. However, a 5-fold further increase in <em>androstenedione</em> accumulation was observed by day 6 for SA theca cultured in the presence of oLH. Maintenance of progesterone accumulation by SA theca throughout the culture period also was dependent on the presence of LH. In contrast, <em>androstenedione</em> accumulation by PO theca required the presence of LH in the culture medium, while progesterone accumulation in these cultures did not. Little or no 5 alpha-reduced androgen accumulated in the media of SA and PO theca cultured in basic culture medium alone. However, SA and PO theca cultured with oLH accumulated approximately 1 ng androsterone by day 10. We conclude that 1) SA and PO follicles, theca and granulosa cells possess the enzymes required to produce large amounts of 3 alpha diol and androsterone; 2) low concentrations of oLH are required to stimulate SA thecal steroidogenesis and to maintain PO thecal <em>androstenedione</em> accumulation in culture.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

The ability of Sertoli cells to metabolize progesterone to testosterone (T) and 5alpha-dihydrotestosterone (DHT) was investigated in vitro. Cultures of Sertoli cells isolated from testes of 80-day-old rats were incubated with [7(n)-3H]progesterone (10 muCi/3.0 nmol) for 0.5, 1, 2, and 3 h. The amount of progesterone converted to T, <em>androstenedione</em> (A), DHT, 17alpha-hydroxyprogesterone, and 20alpha-dihydroprogesterone was calculated on the basis of crystallization data. The amount of substrate converted to the various steroids increased between 0.5-3 h of incubation. During this period, T increased 6-fold; A, 2-fold; DHT, <em>4</em>-fold; 17alpha-hydroxyprogesterone, 3-fold; and 20alpha-dihydroprogesterone, 12-fold. The amount of substrate converted to C-19 steroids (T, A, and DHT) increased linearly with time; 19.<em>4</em> ng androgen/1 x 10(6) cells were formed within 3 h, the largest amount (11.83 ng) being DHT. Although the amount of 3H-labeled C-19 steroids formed from [3H]-progesterone by Sertoli cells is relatively small (1.8% conversion) compared to T formation by whole testicular tissue or by isolated interstitial cells, the ability of Sertoli cells to form T and DHT from progesterone may be physiologically important in the local regulation of Sertoli cell function and spermatogenesis.

Intact ovarian follicles were dissected from a single ovary and groups of follicles incubated in balanced saline at 6 degrees with and without a pacific salmon gonadotrophin (GTH) preparation. Aliquots of the incubation media were withdrawn at intervals, the steroids were extracted with solvent, and 17 alpha, 20 beta-dihydroxy-<em>4</em>-pregnen-3-one (17 alpha 20 beta P), 17 alpha-hydroxyprogesterone, progesterone, <em>androstenedione</em>, testosterone, and oestradiol were separated chromatographically and measured by radioimmunoassay. The main findings were (1) germinal vesicle breakdown commenced on Day 8 under the influence of GTH, (2) after an induction period of about 2 days there was a dramatic and continuing release of 17 alpha 20 beta P in the presence of GTH, (3) GTH promoted a moderate release of 17 alpha-hydroxyprogesterone, (<em>4</em>) GTH stimulated testosterone release up to Day 6 but thereafter levels fell, reflecting either absorption or catabolism of the released steroid, (5) GTH induced a precipitous release of <em>androstenedione</em> during the first few hours of incubation, and (6) progesterone and oestradiol release were little affected by GTH. Steroid concentrations were also measured in ova stripped from naturally ovulating wild fish and the relative amounts were shown to bear some similarity to those released into the incubation medium in the presence of GTH. The routes of biosynthesis and functions of these steroids are considered in light of the results.

The direct effect of LH on estrogen secretion by rat granulosa cells was investigated. Ovarian granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were primed with FSH for 2 days in vitro to induce LH receptors. After the FSH priming, the granulosa cells were washed, and recultured for <em>4</em> additional days in media containing aromatase substrate (10(-7) M <em>androstenedione</em>) and purified FSH or LH. After the incubations, estrogen (E), progesterone (P) and 20 alpha-dihydroprogesterone (20 alpha-OH-P) in the media were measured by RIA. When granulosa cells from hypophysectomized DES-treated rats were cultured for 6 days with FSH and <em>androstenedione</em>, the production of E, P and 20 alpha-OH-P was stimulated to a maximum of 100-, 200- and 270-fold, respectively, above that of control levels. In contrast, LH did not increase steroidogenesis in these cells. Following 2 days of FSH priming in vitro, however, the cultured granulosa cells exhibited marked increases (<em>4</em>00-600%) in E, P and 20 alpha-OH-P production in response to LH treatment over a <em>4</em>-day incubation period. This stimulatory effect of LH on estrogen and progestin production was dose-related; the minimum and maximum effective doses of LH for steroid production were 3 and 30 ng/ml, respectively, and the ED50 was calculated to be 6 ng/ml of LH. As with LH, FSH also stimulated steroidogenesis in a dose-related manner and the apparent ED50 of FSH on steroidogenesis was <em>4</em>5 ng/ml. To investigate whether LH can also stimulate aromatase activity in granulosa cells primed with FSH in vivo, immature hypophysectomized DES-treated rats were injected for 2 days with FSH after which the granulosa cells were isolated and cultured for <em>4</em> days in medium containing 10(-7) M <em>androstenedione</em> and LH or FSH. Both LH and FSH stimulated E, P and 20 alpha-OH-P production, and the maximum steroidogenic responses of LH and FSH were similar to those observed in cultured granulosa cells primed with FSH in vitro. THese results have demonstrated that LH is effective in stimulating both estrogen and progestin secretion in rat granulosa cells pretreated with FSH. This suggests an important role of LH in the direct control of both aromatization and luteinization in the granulosa cell.

The objectives were to compare expression of mRNA for cytochrome P<em>4</em>50 cholesterol side-chain cleavage (P<em>4</em>50scc), cytochrome P<em>4</em>50 17alpha-hydroxylase (P<em>4</em>50c17), cytochrome P<em>4</em>50 aromatase (P<em>4</em>50arom), 3beta-hydroxysteroid dehydrogenase Delta(<em>4</em>), Delta(5) isomerase (3beta-HSD), FSH receptor (FSHr) and LH receptor (LHr) in bovine ovarian follicles of the first and second waves of the bovine oestrous cycle and to determine if LH infusion changes growth, steroidogenesis and gene expression in second wave follicles. Transrectal ultrasonography was used to examine follicular size changes during the oestrous cycle in non-lactating Holstein cows (n=31). Saline or purified bovine LH was infused intravenously into cows at emergence of follicular waves for 2 or <em>4</em> days using a computer-controlled syringe pump (n=5-6 per treatment). Treatments were: wave 1, saline (W1S); wave 2, saline (W2S) or LH (25 microg/h; W2LH). During infusion, blood samples were collected at 12min intervals for 8h via i.v. catheters for measurement of serum LH concentrations. Ovaries were removed from cows on days 2 or <em>4</em> after emergence of follicular waves. Follicles were frozen and stored at -80 degrees C. Follicular fluid (FF, 50 microl) was collected for determination of progesterone (P<em>4</em>), oestradiol-17beta (E2) and <em>androstenedione</em> (A<em>4</em>) concentrations. Frozen sections (1<em>4</em> microm) were used for in situ hybridization to measure expression of mRNA (% pixel intensity) for P<em>4</em>50scc, P<em>4</em>50c17, P<em>4</em>50arom, 3beta-HSD, FSHr, and LHr. LH infusion resulted in a serum LH pattern (high frequency) similar to the early luteal phase. There were no significant differences in size of follicles among the three treatment groups. Follicular fluid concentrations of E2 and A<em>4</em> in W2S were lower than those of W1S on day 2 of a follicular wave. LH infusion into cows during the midluteal phase increased follicular fluid E2 and A<em>4</em> concentrations in second wave follicles on day 2 of a follicular wave (W2LH) compared to those of W2S. The increase in follicular fluid E2 on day 2 in wave 2 follicles after LH infusion occurred possibly through an increase in mRNA expression of P<em>4</em>50c17 and 3beta-HSD. In conclusion, follicular fluid concentrations of E2 and A<em>4</em> were lower in W2S than in W1S and E2 and A<em>4</em> concentrations were restored by infusion of LH in W2LH with an increase in mRNA expression of P<em>4</em>50c17 and 3beta-HSD.

The present study examines how the hormonal action of gonadotropin is modulated by transforming growth factor-beta 1 (TGF beta 1) and epidermal growth factor (EGF) in primary cultures of purified porcine Leydig cells. Although TGF beta 1 (1 ng/ml; <em>4</em>8 h) and EGF (10 ng/ml; 72 h) individually enhanced hCG-stimulated testosterone formation, the effects of EGF were more pronounced than those of TGF beta 1. When studied in combination, the effects of maximal concentrations of TGF beta 1 and EGF were additive on gonadotropin hormonal action. In the present study we demonstrate that their additive effects resulted from a complex interaction occurring at the levels of cholesterol substrate availability in the mitochondria and of 3 beta-hydroxysteroid dehydrogenase/isomerase activity (3 beta HSDI). First, TGF beta 1 (1 ng/ml; <em>4</em>8 h) and EGF (10 ng/ml; 72 h) were, respectively, shown to reduce and enhance dehydroepiandrosterone (DHEA) formation (evaluated in the presence of 10(-5) M WIN 2<em>4</em>5<em>4</em>0, an inhibitor of 3 beta HSDI) in Leydig cells when acutely (3 h) stimulated with hCG (0.01-1 ng/ml), but not when incubated with 22R-hydroxycholesterol (3 micrograms/ml). Such findings indicate that TGF beta 1 and EGF did not affect cholesterol side-chain cleavage cytochrome P<em>4</em>50 activity, but, respectively, decreased and increased cholesterol substrate availability for this enzyme in the mitochondria. Furthermore, when Leydig cells were treated with the combined factors, the formation of delta 5-steroid intermediates (such as DHEA) in untreated (control) and EGF-plus TGF beta 1-treated cells was not significantly different whether the cells were acutely stimulated with the gonadotropin or incubated with 22R-hydroxycholesterol. Such findings indicate that the effects of EGF and TGF beta 1 on cholesterol substrate availability in the mitochondria are antagonistic. Second, EGF, TGF beta 1, and EGF plus TGF beta 1 significantly (P less than 0.001) increased delta 5-steroid intermediate (i.e. pregnenolone and DHEA), but not delta <em>4</em>-steroid intermediate (i.e. progesterone and <em>androstenedione</em>), conversion into testosterone, indicating that the growth factors increased, individually or in combination in an additive manner, 3 beta HSDI activity (respectively, 90.7 +/- 0.6%, 80.6 +/- 2.6%, and 16<em>4</em> +/- <em>4</em>.5% increase in the presence of EGF, TGF beta 1, and EGF plus TGF beta 1). Together, the reciprocal suppression of the effects of TGF beta 1 and EGF on the mitochondrial cholesterol substrate availability coupled to their stimulatory additive actions on 3 beta HSDI activity provide an explanation of the additive actions of the two growth factors on gonadotropin-induced testicular androgen formation.

We studied the effects of shakuyaku-kanzo-toh (a Chinese herbal medicine) and its components on testosterone production by rat gonads. We used paeoniflorin as a main component of shakuyaku (paeoniae radix), glycyrrhizin as a main component of kanzo (glycyrrhizae radix) and glycyrrhetinic acid as a main metabolite of glycyrrhizin. Oral administration of shakuyaku-kanzo-toh, glycyrrhizin, and glycyrrhetinic acid decreased in vitro basal testosterone production in Leydig cells by LH stimulation. Glycyrrhizin and glycyrrhetinic acid caused a significant decrease in testosterone production with an accumulation of 17 alpha-hydroxyprogesterone when incubated with isolated Leydig cells, while paeoniflorin showed no such effect. The inhibitory effect of glycyrrhetinic acid was far more potent than that of glycyrrhizin, causing about 90% inhibition at 10 micrograms/ml. Glycyrrhizin and glycyrrhetinic acid did not change the cyclic AMP or progesterone level in the Leydig cells. When 1<em>4</em>C-labeled <em>androstenedione</em> was incubated with microsomal fraction of testicular or ovarian tissue, glycyrrhizin and glycyrrhetinic acid inhibited the conversion of <em>androstenedione</em> to testosterone, indicating that these compounds inhibit the activity of 17 beta-hydroxysteroid dehydrogenase (EC. 1.1.1.6<em>4</em>). The ED50 of glycyrrhetinic acid was about <em>4</em> microM.

1. A computerized technique is described for the quantitative determination of radiometabolites from incubation studies. 2. Seven steroid substrates have been incubated with human endometrial tissue. The principal radiometabolites were identified and determined after 2hr. incubation without the addition of cofactors and after <em>4</em>hr. incubation with cofactors. 3. The main products from progesterone were 20alpha-dihydroprogesterone and 5alpha-pregnanedione with lower yields of 5beta-pregnanedione and 20beta-dihydroprogesterone. There was no evidence for 17alpha-hydroxylase activity. <em>4</em>. 17alpha-Hydroxyprogesterone was transformed into small yields of 17alpha,20alpha- and 17alpha,20beta-dihydroxypregn-<em>4</em>-en-3-one. In one incubation there was evidence for conversion into <em>androstenedione</em>. 5. Dehydroepiandrosterone was transformed into small amounts of <em>androstenedione</em>, 5alpha-androstanedione and androsterone. 6. <em>Androstenedione</em> and testosterone were interconvertible, the reaction favouring the formation of <em>androstenedione</em>. 5alpha-Androstanedione and androsterone were formed from both substrates. There was no evidence for the formation of phenolic steroids. 7. Oestrone and oestradiol-17beta were interconvertible, the reaction favouring the formation of oestrone.

In adult male Wistar rats, serum and testicular concentration of testosterone (T), estradiol (E2), dihydrotestosterone (DHT), 5 alpha-androstane 3 alpha, 17 beta-diol (Adiol), and their precursors were measured under basal conditions as well as <em>4</em> and 8 h respectively after i.m. injection of 100 I.U. of hCG. Under basal conditions T (203 +/- 30 (SE) ng/dl) was quantitatively by far the most important serum steroid, followed by progesterone (P) (76.5 +/- 12 ng/dl), 17-hydroxyprogesterone (170HP) (37.3 +/- <em>4</em>.1 ng/dl), <em>androstenedione</em> (A) (2<em>4</em>.6 +/- 2.5 ng/dl) and pregnenolone (P5) (22.9 +/- <em>4</em> ng/dl). Estradiol (E2) was present in a low concentration (1.06 +/- 0.26 ng/dl). In the testes, T was quantitatively the most important steroid (89 +/- 7.2 ng/g), followed by 5 alpha-androstane-3 alpha-17 beta-diol (Adiol) (26.5 +/- 2.8 ng/g) whereas 170HP (11.8 +/- 1.0 ng/g), P (11.5 +/- 1.0 ng/g) and P5 (16.6 +/- 1.8 ng/g) were present in roughly the same concentration, concentrations of A, DHT, dehydroepiandrosterone (DHEA), 17 hydroxy-pregnenolone (170HP5) and androst-5-ene-3 beta-17 beta-diol (D5diol) being much lower; E2 (0.06 +/- 0.01 ng/g) was hardly detectable. Within <em>4</em>-8 h after hCG stimulation, serum androgen concentrations increased by a factor of <em>4</em>-12, except for DHEA and D5diol (X2), and E2 (X 1.5). Intratesticular androgens and delta <em>4</em> steroid precursors increased by a factor of 5-10, delta 5 precursors by a factor of 2-<em>4</em> and E2 by a factor of 2, the data tending to confirm that the delta <em>4</em> pathway is preferred over the delta 5 pathway. After hCG the relative increase of T precursors was the most important for P, suggesting that 17 hydroxylation might be the rate limiting step in T biosynthesis.

Mevinolin, putatively a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme-A reductase, was used to assess the contribution of de novo synthesized cholesterol to androgen production by ovarian thecal cells in vitro. Enzymatically dispersed thecal cells from 3- to 6-mm follicles of prepubertal gilts were incubated at 150,000 cells/ml with a maximally effective dose of LH (250 ng/ml) for 2<em>4</em> h. Mevinolin (3-50 microM) caused dose-dependent inhibition of <em>androstenedione</em> production. Addition of 25-hydroxycholesterol (0.025-25 microM) failed to restore <em>androstenedione</em> production to levels seen in the absence of mevinolin, suggesting an additional site of action of mevinolin beyond 3-hydroxy-3-methylglutaryl coenzyme reductase. The site of this inhibitory effect was determined by measuring steroid products formed in the presence of relevant steroid precursors. Mevinolin (12 microM) inhibited the production of 17 alpha-hydroxyprogesterone from progesterone and that of <em>androstenedione</em> from 17 alpha-hydroxyprogesterone, while 25-hydroxycholesterol to progesterone and pregnenolone to progesterone conversions were unimpaired. That mevinolin did not affect 3 beta-hydroxysteroid dehydrogenase:delta 5-delta <em>4</em>-isomerase reactions was confirmed by demonstrating that conversions of pregnenolone, 17 alpha-hydroxypregnenolone, and dehydroepiandrosterone to progesterone, 17 alpha-hydroxyprogesterone, and <em>androstenedione</em>, respectively, were not affected by 12 microM mevinolin. These results indicate that mevinolin has an additional inhibitory action at the level of the 17 alpha-hydroxylase:C-17,20-lyase complex. The degree of inhibition of <em>androstenedione</em> production was not decreased with increased concentrations of progesterone or 17 alpha-hydroxyprogesterone substrate, suggesting that the inhibition was not competitive in nature. As the dose of mevinolin was increased up to 50 microM, progesterone accumulation was unaffected, but pregnenolone concentrations in medium greatly increased. While the mechanism of this effect is unclear, this finding suggests that preformed intracellular cholesterol, rather than that synthesized de novo, is supplying steroidogenic substrate in these cells.