Using a preparation of highly purified, adult rat Leydig cells and conditions of culture which we found to optimize testosterone production during 2<em>4</em> h, we sought to maintain optimal testosterone production for 3 d. Leydig cells cultured on Cytodex 3 beads at 19% O2 in Dulbecco's modified Eagle's medium-Ham's nutrient mixture F12 (1:1; vol/vol) containing 0.5 mg/ml total bovine lipoproteins (less than 1.222 g/ml) with maximal luteinizing hormone (LH) stimulation failed to maintain a constant amount of testosterone for 3 d. These cells did however secrete a similar amount of total delta <em>4</em>-3-ketosteroids on each of the 3 culture d, indicating that their viability was preserved. The predominance of progesterone and 170H-progesterone relative to the amount of <em>androstenedione</em> found on Days 2 and 3 suggested that the activity of the cytochrome P<em>4</em>50 C17-hydroxylase-C17,20-lyase enzyme in the smooth endoplasmic reticulum was diminished when Leydig cells were maintained in our primary culture for longer than 2<em>4</em> h. Decreasing the oxygen tension of the cultures from 19 to 5%, and decreasing the concentration of LH used to stimulate the Leydig cells from 100 to 0.1 ng/ml, were necessary to achieve maintenance of testosterone secretion without accumulation of other delta <em>4</em>-3-ketosteroids during a 3-d period. Cells cultured in this fashion were still able to respond to maximal LH stimulation during Day 3, producing as much testosterone as if cultured for 2<em>4</em> h on Day 1 at 19% O2 with 100 ng/ml LH stimulation.

BACKGROUND

In women with polycystic ovary syndrome (PCOS), the basis for ovarian androgen overproduction involves an overall increase of steroidogenesis, notably in the delta-<em>4</em> pathway. However, in vitro studies have suggested that excessive androgen production occurs predominantly through the delta-5 pathway.

OBJECTIVE

This study was performed to assess androgen dose-responses after human chorionic gonadotropin (hCG) stimulation in PCOS and normal women.

METHODS

We conducted a prospective study to compare androgen production after iv hCG in PCOS and normal women.

METHODS

The study was conducted in a General Clinical Research Center in an academic medical center.

METHODS

Women with PCOS (age, 18-37 yr; n = 10) and normal ovulatory controls (age, 18-37 yr; n = 11) were recruited.

METHODS

For dose-response studies, blood samples were obtained before and at 0.5, 2<em>4</em>, and <em>4</em>8 h after iv recombinant hCG (1, 10, 25, 100, and 250 μg). A subset of subjects underwent frequent blood sampling over 2<em>4</em> h after iv injection of 25 μg of recombinant hCG.

METHODS

We measured basal and stimulated serum 17-hydroxyprogesterone (17-OHP), androstenedione (A), testosterone (T), dehydroepiandrosterone, estradiol, and progesterone responses after hCG administration.

RESULTS

In PCOS women, maximal A and T production was observed at the lowest doses of hCG, whereas responses were minimal in normal women. Incremental responses of 17-OHP, estradiol, and progesterone were greater in PCOS compared to normal women.

CONCLUSIONS

In PCOS women, maximal A and T responses to hCG relative to those of 17-OHP are consistent with ovarian androgen overproduction via the delta-5 pathway.

The present study was designed to characterize the granulosa cell subpopulations derived from rats in which ovarian growth was induced by diethylstilbestrol (DES) or in which growth and differentiation was induced by pregnant mare's serum gonadotropin (PMSG). In the DES model, immature rats were given two separate injections of 2 mg DES/rat s.c. on 25 and 26 days of age and were killed 2<em>4</em> hr after the second injection. In the PMSG model, rats on day 28 were given 8 IU of PMSG s.c. and were killed 5<em>4</em> hr later. Granulosa cells were isolated from the ovaries, separated on a continuous Percoll density gradient, and divided into 12 fractions. The cells in each fraction were cultured in the presence of <em>androstenedione</em> with or without 20 ng/ml of follicle-stimulating hormone (FSH) and estradiol and progesterone in the incubation medium were measured. In DES-treated rats, granulosa cells in fractions <em>4</em> and 5 and fractions 9 and 10 contained about 30-<em>4</em>0% of total cell yield and showed high steroidogenic potential. Granulosa cells from fractions 6, 7, and 8, which represent 55-60% of total cell yield, produced relatively low amounts of steroids on a per-million-cell basis. FSH was required for the stimulation of steroidogenesis. In granulosa cells from the PMSG treated rats, aromatase activity appeared maximally induced and incubation with FSH in vitro did not bring about any further increase. However, in vitro incubation with FSH was required for progesterone production. Furthermore, the granulosa cells from the PMSG treated rats also showed much more active estradiol and progesterone synthesis in fractions 9-12 as compared with lower density fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies in six Arab individuals from Gaza with familial male pseudohermaphroditism (MPH) due to 17-ketoreductase deficiency revealed several metabolic aberrations associated with the disorder. Plasma LH, FSH, testosterone, and <em>androstenedione</em> concentrations were low in the two prepubertal patients. After hCG administration plasma <em>androstenedione</em> increased markedly. The four postpubertal MPH patients had very high plasma gonadotropin and <em>androstenedione</em> concentrations, the latter increasing further after hCG administration. Plasma testosterone concentrations in all six patients were moderately low or normal for age and increased little after hCG administration. Spermatic venous testosterone concentrations, measured in three adults, were within the normal range in two and low in one, while <em>androstenedione</em> concentrations were markedly elevated (15- to 32-fold) in all three patients. Kinetic analyses of progesterone and <em>androstenedione</em> metabolism were performed in testicular tissue of these patients and compared to the results in two control subjects. While testicular tissue from the two prepubertal patients metabolized progesterone only to <em>androstenedione</em>, and that to a limited extent, the tissue from the four postpubertal patients metabolized progesterone to 16 alpha- and 16 beta-hydroxyprogesterone, 17 alpha-hydroxyprogesterone, <em>androstenedione</em>, and testosterone and metabolized <em>androstenedione</em> to testosterone. The Michaelis constants of these reactions were similar in the tissue from the MPH and the control subjects. The production of 16 alpha- plus 16 beta-hydroxyprogesterone was 5.<em>4</em>- to 10.3-fold greater, and 17-hydroxylase activity was 5.8- to 8.1-fold lower in the testes of the postpubertal MPH patients compared to values in the control subjects. The preference of <em>androstenedione</em> production through the delta <em>4</em>- or delta 5-pathways was examined in the testes of two adult MPH patients using an equimolar concentration of [1<em>4</em>C]progesterone and [3H]pregnenolone as substrates. While the flow of substrates in the control testes was equal or slightly greater through the delta <em>4</em>-pathway, the delta 5-pathway predominated in the testes of the MPH patients. A large amount of dehydroepiandrosterone accumulated when NAD, the cofactor for 3 beta-hydroxysteroid dehydrogenase-isomerase, was omitted, supporting the contention that <em>androstenedione</em> was produced in the testes of the MPH patients mainly through the delta 5-pathway. Additional support for this suggestion was the finding that the 3H/1<em>4</em>C ratio in <em>androstenedione</em> and testosterone produced from both substrates was 8 times higher in the testes from MPH patients than in those from the control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

We constructed expression plasmids for bovine adrenal cytochrome P<em>4</em>50c17 (P<em>4</em>50c17) by inserting the corresponding cDNA between the yeast alcohol dehydrogenase I promoter and terminator of the expression vector pAAH5. Plasmids pA alpha 1 and pA alpha 2 contained the entire coding region for bovine P<em>4</em>50c17, whereas pAC alpha 1 included the cDNA coding for chimeric P<em>4</em>50c alpha consisting of the amino-terminal <em>4</em>5 amino acid residues of rat P<em>4</em>50c and the carboxy-terminal <em>4</em>82 amino acid residues of bovine P<em>4</em>50c17. The transformed Saccharomyces cerevisiae AH22/pA alpha 1, AH22/pA alpha 2, and AH22/pAC alpha 1 cells produced about 1 x 10(5), 1 x 10(5), and 2 x 10(<em>4</em>) molecules per cell of the corresponding P<em>4</em>50 hemoproteins, respectively. On incubation with the cultures of each of the three strains, progesterone was specifically converted into 17 alpha-hydroxyprogesterone, which was not further converted into <em>androstenedione</em>, indicating that the three strains showed 17 alpha-hydroxylase activity, but almost no C17,20-lyase activity. The microsomal fraction prepared from the AH22/pA alpha 1 cells showed 17 alpha-hydroxylase activity toward progesterone and pregnenolone to higher extents, and exhibited C17,20-lyase activity toward 17 alpha-hydroxypregnenolone to a lesser extent and almost no C17,20-lyase activity toward 17 alpha-hydroxyprogesterone. These results indicated that bovine P<em>4</em>50c17 synthesized in S. cerevisiae cells manifests the 17 alpha-hydroxylase activity, but not the C17,20-lyase activity.

The relationship between physiological variations in female sex and androgenic hormones and calciotropic hormones was investigated during the menstrual cycle. Estradiol, progesterone, total and free testosterone, <em>androstenedione</em>, immunoreactive PTH, calcitonin, osteocalcin (OC), and ionized calcium serum levels were determined throughout the menstrual cycle in a population of healthy eumenorrhoic women (n = 12; age range: 20-29 yr; mean: 2<em>4</em>.2 yr). The women were studied from the first day of a menstrual phase until the first day of the following menstrual phase. Cycle length was standardized on the preovulatory estradiol peak (day 0), and values were given for the first day of a menstrual phase, and days -12, -10, -8, -6, -<em>4</em>, -2, 0, 2, <em>4</em>, 6, 8, 10, 12, and 1<em>4</em> of the menstrual cycle. All subjects had a regular ovulatory cycle, as indicated by the hormonal profile. No significant cycle phase-dependent changes in calciotropic hormones were present. Significant positive correlations between total testosterone (r = 0.32, P < 0.001), free testosterone (r = 0.26, P < 0.001), <em>androstenedione</em> (r = 0.35, P < 0.0001), and OC were observed. The significant relations between these variables were confirmed by a time series analysis. For the first time, these findings indicate a relationship between androgens and OC serum levels during the menstrual cycle. An important regulatory role of endogenous androgens in OC secretion, bone formation, and maintenance of normal bone mineral content in the healthy eumenorrhoic woman is hence suggested.

Ovaries from 8-week-old female NMRI mice in different stages of the oestrous cycle, or from females neonatally treated with the synthetic oestrogen diethylstilboestrol (DES; 5-10(-6) micrograms daily for 5 days), were studied histologically and for the ability to synthesize steroids from [3H]pregnenolone in vitro. Daily doses of 10(-<em>4</em>) micrograms DES or higher resulted in absence of corpora lutea. In ovaries lacking corpora lutea, the interstitial tissue dominated and the cells in this compartment were large with a clear cytoplasm. The steroids synthesized in ovarian homogenates were separated with thin-layer chromatography. The homogeneity of the steroids was checked in recrystallization experiments. Daily doses of 5-10(-<em>4</em>) micrograms DES in the neonatal period resulted in pronounced deviations in the pattern of ovarian steroids synthesized as compared with control ovaries. In DES-exposed ovaries, the synthesis of <em>androstenedione</em> and, above all, progesterone was high while the synthesis of 17 alpha-hydroxyprogesterone and testosterone was reduced compared with controls. These results could argue for a difference in activities of 17 alpha-hydroxylase and 17 beta-ol-dehydrogenase in ovaries from DES-treated females compared with controls. After transplantation of DES-exposed ovaries to ovariectomized control females, the steroid pattern changed to that typical for control ovaries. Control ovaries transplanted to DES-treated females had a steroid pattern similar to that of DES-exposed ovaries.

Type 5 17beta-HSD, one of the seven types of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) so far characterized in humans, catalyzes the transformation of <em>4</em>-<em>androstenedione</em> (<em>4</em>-dione) into testosterone (T). This reaction is also catalyzed by type 3 17beta-HSD which is responsible for pseudohermaphroditism in deficient man but is asymptomatic in deficient women. Since type 3 17beta-HSD is not found in the ovary, whereas type 5 is, it is suggested that the latter is involved in the conversion of <em>4</em>-<em>androstenedione</em> to testosterone in the ovary. The comparison of type 5 17beta-HSD of different species shows that the human enzyme shares 95 and 78% identity with the monkey and mouse enzymes respectively. In addition, the human and monkey enzymes are labile and are destroyed upon homogenization of the transfected cells, whereas the mouse enzyme is not. Human type 5 17beta-HSD also possesses a high 20alpha-HSD activity that inactivates progesterone, whereas the monkey and mouse enzymes do not have such high 20alpha-HSD activity. Since the endocrine ovary is composed of two types of cells, one producing androgens (theca cells) and the other producing progesterone and estrogens (granulosa cells), it is tempting to suggest that the role of the high 20alpha-HSD activity of type 5 17beta-HSD is to protect the theca cells against the progesterone produced by the granulosa cells. The double activity of type 5 17beta-HSD in the female reproductive tissues is probably necessary to the control of the optimal level of progesterone and sex steroids.

Dehydroepiandrosterone (DHEA) and its conjugates persist in the rat brain, for up to 1 month after ablation of both adrenals and gonads. Since DHEA synthesis in brain from pregnenolone (PREG) was excluded, we have considered other tissular sources including the digestive tract. In situ hybridization with specific oligonucleotide probes showed that the parietal cells of the gastric mucosa, contrary to other cell types, strongly expressed P<em>4</em>50(17) alpha messenger RNA. Expression of the enzyme in the parietal cells was confirmed by immunocytochemistry with specific antibodies. An intense reaction was observed in the stomach of adult males and of cyclic or pregnant females. Access to food did not influence the intensity of immunostaining. It appeared at postnatal days 16-21, then the number of positive cells increased rapidly and leveled off at adult age. Parietal cells were released by pronase digestion of everted stomachs from adult male and female rats and were purified by density gradient centrifugation on Nycodenz. 5 x 10(<em>4</em>) to 1.6 x 10(6) cells were incubated with either 1 microM 1<em>4</em>C-PREG or 1<em>4</em>C-progesterone (1<em>4</em>C-PROG) at 37 C under 95% O2-5% CO2, for 10-180 min. PREG was converted to 17-OH PREG and to androstenediol, whereas PROG was converted to 17-OH PROG and to testosterone. Only minute amounts of either DHEA or <em>androstenedione</em>, respectively, were detected at any incubation time, indicating their fast conversion to the corresponding 17 beta-hydroxysteroids. 3H-25-OH cholesterol was not metabolized to 3H-PREG, and 1<em>4</em>C-PREG was not converted to 1<em>4</em>C-PROG, in accordance with negative immunocytochemical results with antibodies to cytochrome P<em>4</em>50scc and 3 beta-hydroxysteroid dehydrogenase delta 5->><em>4</em>-isomerase (3 beta-HSD). In conclusion, the parietal cells, which are known as the source of gastric acid secretion, can synthesize testosterone from PROG and androstenediol from PREG. The physiological relevance of such conversions remains to be established.

1. The rates were measured at which a number of different steroids were secreted by the adrenal gland of the eviscerated and nephrectomized young pig and dog.2. In addition to cortisol and corticosterone the following steroids were consistently found to be present in the adrenal venous blood: pregnenolone, progesterone, 11betaOH-progesterone, <em>androstenedione</em>, 11betaOH-<em>androstenedione</em> and adrenosterone.3. The sum of the latter steroids could amount to as much as 30% of the total steroid secretion.<em>4</em>. A severe deficit in the blood volume increased the secretion of pregnenolone and the 17-oxo steroids in the pig.5. alpha-ethyltryptamine failed to inhibit ACTH release if the animals were eviscerated and nephrectomized, or if they were anaesthetized with chloralose instead of pentobarbitone sodium.6. The same steroids as in the adrenal venous blood were found in extracts from the adrenal glands of a number of species.7. The amount of individual steroids present in the adrenal gland of the stressed pig and dog was compared with the rate at which each had been secreted immediately before the excision of the gland.8. There was a positive correlation between the adrenal concentrations and the secretion rates of cortisol, the major glucocorticoid secreted by the pig and the dog.9. In contrast, the rate at which pregnenolone was secreted did not show a consistent relationship to its concentration in the gland.10. A storage mechanism for pregnenolone in the adrenal cortex is proposed.

In vitro egg production bySchistosoma mansoni was examined following a 72-hr incubation period in the presence of various steroids, steroid precursors, or steroid synthesis inhibitors. Mevinolin, an inhibitor of cholesterol biosynthesis, significantly decreased egg production at 10(-6) M. However, neither cholesterol (at 10(-5) M) nor any of its hydroxylated derivatives (at 10(-<em>4</em>) M) affectedS. mansoni fecundity. Dianisidine, an inhibitor of cholesterol metabolism to hormonal products, was also ineffective in influencing egg production. The steroid hormones progesterone, 17α-hydroxyprogesterone and medroxyprogesterone acetate, all significantly decreased egg production; however, parasite muscle tension was also significantly reduced by the concentrations of these steroids needed to produce an effect on egg laying. Various androgenic hormones (androsterone, <em>androstenedione</em>, testosterone) and estrogenic hormones (17β-estradiol and estrone) demonstrated no effect on in vitro egg production. Significant elevation in the rate of parasite oviposition was seen in the presence of the corticosteroid prednisolone (at 10(-5) M), but not by dexamethasone, a corticosteroid analog.

The neural pathway most related with ovarian steroidogenesis has been identified as the superior ovarian nerve (SON). This work constitutes the first study of the effects of early ovarian SON transection, which was performed in rats of <em>4</em> days of age (SON-t rats) to magnify the effects of the denervation. The rats were studied at the prepubertal (30 days), peripubertal (<em>4</em>1 days) and adult cyclic in dioestrus (60 days) reproductive stages. The SON-t rats showed a delay of vaginal opening, a notable disruption of oestrous cyclicity, and a large number of corpora lutea. In all the stages, the circulating levels of FSH, prolactin and growth hormone were lower in SON-t rats than in controls, whereas LH did not vary. Serum <em>androstenedione</em> levels were higher in SON-t rats at 30 days and lower at <em>4</em>1 days, compared with control animals while no difference was observed at 60 days. Serum progesterone levels did not differ between control and SON-t, but serum oestradiol concentrations were higher in SON-t rats in all of the stages. At the peripubertal stage, there were fewer ovarian beta-adrenergic receptors in SON-t ovaries, associated with a rise in the ovarian content of norepinephrine, but no changes were observed in SON-t rats at 30 and 60 days with respect to the controls. The release of progesterone in vitro from luteal cell in SON-t rats at 60 days was reduced in basal condition and under ovine LH or FSH stimulation, when compared with control animals; while no difference was observed in presence of isoproterenol or <em>androstenedione</em> in the culture medium. In corpora lutea of SON-t rats at 60 days, no change was observed in the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), but the activity of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) was reduced, suggesting abnormal luteolysis in spite of the large number of corpora lutea. The interruption of innervation at an early age by SON transection is very important in the regulation of ovarian development in prepubertal and cyclic rats. The functional changes observed in the ovary suggest a possible alteration in the hypothalamic-hypophyseal axis.

Local modulation of follicular and gametogenic functions by ovarian androgens and estrogens in mammalian species has been proposed. This study examined the effects of elevated androgen/estrogen ratios during follicular maturation in vivo by inhibiting aromatase activity in rhesus monkeys. To obviate steroid feedback effects, gonadotropin-treated animals were used. Beginning at menses (day 1), animals received human (h) FSH (60 IU/day, im) on days 1-6, followed by hFSH plus hLH (60 IU/day, im) on days 7-9 to promote the growth of multiple follicles. Ovulatory maturation was induced by hCG (1000 IU, im) on day 10. On days 8-10, four animals received an aromatase inhibitor, 1,<em>4</em>,6-androstatrien-3,17-dione (ATD; 1-1.25 g, orally, twice/day), while five served as controls and received no further treatment. Within 8 h of ATD treatment, a 63% reduction in serum estradiol levels relative to control values was evident, which reached maximal suppression (8<em>4</em>%) by day 10. A marked elevation (17-fold) in serum <em>androstenedione</em> and a lesser increase (2.6-fold) in serum testosterone occurred with aromatase inhibition, yielding <em>androstenedione</em>/estradiol (18.0) and testosterone/estradiol (1.9) ratios greater than those in controls (0.6 and 0.3, respectively). ATD treatment did not alter follicular diameters or the total number of follicles per animal (20 +/- 3) relative to control values (16 +/- 3). Of the total cohort classified, the proportion of oocytes collected at prophase I was greater (P < 0.05) after ATD treatment (31%) than in controls (11%). Completion of oocyte meiosis to metaphase II was retarded (P < 0.05) in ATD-treated (<em>4</em>%) compared to control (26%) animals. Furthermore, the in vitro fertilization rate of metaphase II oocytes from ATD-treated animals (9%) was reduced (P < 0.05) relative to that in controls (25%). While basal progesterone production by luteinizing granulosa cells in vitro was similar between groups, the addition of hCG in vitro enhanced progesterone secretion by cells from ATD-treated animals (3.1 +/- 0.3-fold over basal) to a greater extent (P = 0.05) than in controls (1.5 +/- 0.3-fold). Progesterone receptor was detected by immunocytochemistry in nuclei of luteinizing granulosa cells from ATD-treated animals as well as controls. Serum progesterone profiles and the length of the luteal phase were similar between groups. Thus, acute elevation of serum androgen/estrogen ratios in vivo during follicular maturation was detrimental to the gametogenic functions of the primate follicle, but did not alter follicular growth, events of early luteinization, or subsequent luteal function.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

The actions and the mechanisms of action of epidermal growth factor (EGF) in testicular steroidogenesis were investigated using a model of primary culture of purified porcine Leydig cells from immature intact animals. EGF decreased (1.7-fold) human CG (hCG)-induced dehydroepiandrosterone (DHEA) accumulation in the medium whereas it enhanced (2.5-fold) that of testosterone. The maximal and half-maximal effects on both DHEA and testosterone secretions were observed at similar concentrations which were, respectively, 3 (5 x 10(-10) M) and 0.7 (11 x 10(-11) M) ng/ml EGF, after 72-h treatment. EGF effect on DHEA and testosterone secretion was similarly observed whether the cells were acutely (3 h) stimulated with hCG (1 ng/ml) or with 8-bromo-cAMP (10(-3) M). To further localize the steroidogenic biochemical steps affected by EGF, the growth factor action on steroidogenic enzyme activities was investigated. EGF increased delta 5 steroid intermediate (i.e. pregnenolone and DHEA) formation [evaluated in the presence of 10(-5) M of WIN 2<em>4</em>5<em>4</em>0, an inhibitor of 3 beta-hydroxysteroid dehydrogenase/iosomerase (3 beta-HSDI) activity]. However, this stimulation was observed in cells when acutely (3 h) stimulated with hCG (0.01-1 ng/ml) but not when incubated with 22R-hydroxycholesterol (0.01-10 micrograms/ml). Such findings indicate that EGF did not affect cholesterol side chain cleavage cytochrome P<em>4</em>50 activity but probably increased cholesterol substrate availability for this enzyme in the inner mitochondria. Moreover, EGF 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 EGF enhances 3 beta-HSDI activity. Such effects of EGF are directly exerted on Leydig cells since EGF receptors (Kd = 16 x 10(-11) M) are present in primary cultures of purified porcine Leydig cells. Together, the present findings show that in Leydig cells from intact animals, EGF enhances the gonadotropin action on testosterone formation through an increase in the availability of cholesterol substrate in the mitochondria as well as an increase in the activity of 3 beta-HSDI.

A technique of monolayer tissue culture of human fetal adrenal cells was developed in order to study steroidogenic responses to factors such as ACTH. The daily production of 12 steroids [pregnenolone, 17-hydroxy pregnenolone, dehydroepiandrosterone (DHA), DHA sulfate, progesterone, 17-hydroxyprogesterone, <em>androstenedione</em>, testosterone, corticosterone, 11-desoxycortisol, cortisol, and aldosterone) was measured by RIA. Initially, fresh fetal adrenal cells produced DHA, DHA sulfate, 17-hydroxypregnenolone, and small amounts of cortisol, but in the absence of ACTH, the production of all steroids declined during culture to low levels. The addition of physiological amounts (1-10(<em>4</em>) pg/ml) of either alpha ACTH-1(1-2<em>4</em>) or alpha ACTH-(1-39) or coculture with fetal pituitary cells elicited a progressive rise in steroid production during the first <em>4</em>-6 days of incubation. The lowest ACTH doses elicited a proportionately greater adrenal androgen response (as reflected in the DHA to cortisol ratio), but with increasing ACTH dosage, there was greater stimulation of cortisol production, which equalled or exceeded that of DHA. The data demonstrate that fetal adrenal cells may be maintained in short term culture and can respond to physiological amounts of ACTH. The progressive increase in the production of cortisol and other delta <em>4</em>, 3-ketosteroids in vitro suggests that the characteristic fetal pattern of steroidogenesis may result from the interaction of ACTH with some circulating inhibitor of adrenal 3 beta-hydroxysteroid dehydrogenase.

It has been well known that reactive oxygen species (ROS) are produced in the steroidogenic pathway and spermatozoa. H2O2, one of ROS produced by spermatozoa, appears to be a primary toxic agent. In the present study, we examined the effects of H2O2 on the basal and evoked-testosterone release from primary Leydig cells, the protein expressions of cytochrome P<em>4</em>50 side chain cleavage enzyme (P<em>4</em>50scc) and steroidogenic acute regulatory (StAR) protein were also investigated. Our preparation was found to contain approximately 87% Leydig cells and very few macrophages. The results demonstrated that H2O2 (>1 x 10(-<em>4</em>) M) significantly inhibited the basal and hCG-stimulated testosterone release. H2O2 abolished forskolin- or 8-Br-cAMP-evoked testosterone release. In the presence of pregnenolone, progesterone, or <em>androstenedione</em>, the inhibitory effect of H2O2 on testosterone release was prevented. H2O2 also inhibited pregnenolone production in the presence of trilostane (an inhibitor of 3beta-hydroxysteroid dehydrogenase), therefore diminished the activity of P<em>4</em>50scc in Leydig cells. In addition to the inhibition of hormone secretion, H2O2 also regulated steroidogenesis by diminishing protein expression of StAR. These results suggest that H2O2 acts directly on rat Leydig cells to diminish testosterone production by inhibiting P<em>4</em>50scc activity and StAR protein expression.

The central biochemical mechanisms involved in primate parturition are still unclear. Studies in both humans and nonhuman primates such as the baboon and rhesus monkey indicate that many factors play a part in the cascade of interactive positive feedforward loops that progressively promote parturition: changes in maternal endocrinology, a nocturnal switch in myometrial activity from low amplitude, infrequent contractures to high amplitude, high frequency contractions (see Fig. 1), dilation of the cervix and biochemical changes in the fetal membranes that lead to rupture. Here we demonstrate that infusion of the aromatase inhibitor <em>4</em>-hydroxy<em>androstenedione</em> (<em>4</em>OHA) inhibits conversion of androgen to estrogen and prevents premature delivery caused by administration of androgen to pregnant rhesus monkeys at 0.8 of pregnancy term. <em>4</em>OHA also inhibited the <em>androstenedione</em> induced maternal endocrine and fetal membrane biochemical changes, and alteration of myometrial activity patterns. Secondly, peripheral estrogen infusions increased myometrial activity but did not produce preterm delivery or fetal membrane changes. We conclude that paracrine functions of estrogen at its site of production play critical and central roles in delivery in the non-human primate.

A number of inhibitors of estrogen synthesis are now becoming available which could be of value in the treatment of breast cancer. <em>4</em>-Hydroxy<em>androstenedione</em> (<em>4</em>-OHA), the first of these compounds to enter the clinic has been found to be effective in postmenopausal patients who have relapsed from tamoxifen. Thus, in studies of 2<em>4</em>0 patients, 26% patients experienced partial or complete response to treatment. An additional 25% patients had disease stabilization. <em>4</em>-OHA is a potent selective, steroidal inhibitor which causes inactivation of aromatase in vitro. It is effective in reducing concentrations of ovarian estrogens in rats and of ovarian and peripheral estrogens in non-human primate species. The compound has been shown to lower serum estrogen levels in postmenopausal breast cancer patients. However, not all of these patients experienced disease remission, suggesting that their tumors were hormone insensitive rather than that the dose of <em>4</em>-OHA was suboptimal. In trials of patients who had not received prior tamoxifen treatment, <em>4</em>-OHA (250 mg i.m. every 2 weeks) was found to induce complete or partial tumor regression in 33% of patients. The response of patients was not significantly different from that observed in patients treated with tamoxifen (30 mg o.d) of 37%. No significant difference between treatments was observed for disease stabilization, the duration of response or median survival. Several other steroidal aromatase inhibitors have been studied, such as 7 alpha-substituted <em>androstenedione</em> derivatives. MDL 18962 [10-(2-propynyl)estr-<em>4</em>-ene-3,17-dione] and FCE 2<em>4</em>30<em>4</em> (6-methylen-androsta-1,<em>4</em>-diene-3,17-dione) are currently in clinical trials. Non-steroidal inhibitors of cytochrome P-<em>4</em>50 enzymes, such as imidazole and triazole derivatives have been developed which are highly selective for aromatase. Three triazoles which are very potent and selective inhibitors are vorazole (6-[(<em>4</em>-chlorophenyl)(1H-1,2,<em>4</em>-triazol-1-yl)-methyl]1-methyl-1H- benzotriazole R 76713, arimidex 2,2'[5-(1H-1,2,<em>4</em>-triazol-1-yl methyl)-1,3-phenylene]bis(2-methylpropiononitrile) (ZD1033) and letrozole <em>4</em>-[1-(cyanophenyl)-1-(1,2,<em>4</em>-triazolyl)methyl]benzonitril (CGS 20267). These compounds reduce serum estradiol concentration to undetectable levels in breast cancer patients. These highly potent inhibitors provide the opportunity to determine whether a further degree of estrogen suppression will be important in producing greater clinical response.(ABSTRACT TRUNCATED AT <em>4</em>00 WORDS)

The brain is a steroidogenic organ and is thus dependent on estrogen for many aspects of its development and maintenance in both males and females. The purpose of this study was to develop a model to investigate the effect of estrogen on zebrafish sensory-motor (S-M) maturation through mechanisms found in the central nervous (CNS) and peripheral nervous (PNS) systems. In these experiments the aromatase inhibitor (AI), <em>4</em>-hydroxy <em>androstenedione</em> (<em>4</em>-OH-A), which blocks estrogen synthesis, was used to diminish estrogen's effects on zebrafish CNS and PNS development. During these various treatments the zebrafish were analyzed for neurological deficits, including tactile response, swimming movements, vestibular behavior, pectoral fin and eye movements. Over a three to five day time period (<em>4</em>8-168 h post fertilization), in response to AI treatment, none of these S-M behaviors were developmentally expressed creating a "listless" or non-responding condition. Furthermore, when the AI was removed from the treatment medium the S-M behaviors were fully expressed over a two to three day time period. Most importantly, when estrogen was added at the same time as the AI in a co-treatment paradigm, normal developmental appearance of S-M behaviors was rescued in all neurological parameters measured. Furthermore, the addition of estrogen alone after AI washout accelerated the recovery of the tactile response during the first 2<em>4</em> h of treatment. Treatment of developing zebrafish with the selective estrogen receptor blocker ICI 182,780 mimicked the deficit in S-M behaviors caused by AI treatment. This deficit was overcome by low concentrations of estrogen in a co-treatment paradigm with high ICI levels indicating the possibility of a non-genomic mechanism for estrogen's actions on the developmental expression of these S-M behaviors. Eventually, AI exposed fish died of cardiac arrest <em>4</em> to 5 days after the start of treatment; however, AI/estrogen co-treatment allowed for 90-100% survival and the maintenance of normal heart rates during this time period. In conclusion, these studies have demonstrated that the presence of estrogen in the early developing zebrafish embryo is necessary for the proper developmental expression of critical nervous system S-M behaviors necessary for survival, as well as the health of the cardiovascular system. These studies also establish a unique "listless" model for further analysis of estrogen's role in the development of brain, brainstem, and spinal cord circuitry related to the maturation of these behavioral and cardiovascular phenomena.

The main purpose of the study was to identify the principal gonadal steroids synthesized by male and female sea lampreys, Petromyzon marinus. To achieve this, we used high performance liquid chromatography to separate the steroids in the serum of sexually mature animals, and to separate the steroids produced by gonadal tissue incubated in the presence of radiolabelled precursor steroids, as a means of identifying the major steroidogenic pathways. We were unable to detect evidence of the 'classical' steroids, such as 17beta-estradiol (E(2)) or testosterone (T) in the serum of either male or female lampreys. Instead, the principal chromatographic peaks contained very polar compounds that had elution times consistent with 15alpha-hydroxylated estrogens and androgens, and there were sex-specific differences in the chemical nature and the quantity of these compounds. Testis fragments or ovarian follicles co-incubated with tritium-labelled pregnenolone ([3H]P(5)), 17-hydroxyprogesterone ([3H]17OHP(<em>4</em>)), or <em>androstenedione</em> ([3H]A(<em>4</em>)), provided additional confirmation that the gonads synthesize a range of very polar steroids, and the metabolites found were consistent with the presence of a 15alpha-hydroxylated (15alphaOH) metabolic pathway common to testis and ovary. For ovarian tissue, the major 'end product' metabolites from all three precursors were 15alphaOH-estrogens, and for testis tissue 15alpha-hydroxyprogesterone (15alphaOHP(<em>4</em>)) and 15alpha-hydroxytestosterone (15alphaOHT) and small amounts of 15alphaOH estrogen. Small amounts of E(2) were also produced by both ovarian (all substrates) and testicular tissue (some substrates). Although it was assumed that the E(2) was synthesized via the aromatization of T, [3H]T was not found as an intermediate metabolite. The study suggests that the principal gonadal steroids in sea lamprey are 15alpha-OH compounds, and that only small amounts of E(2) or T are synthesized by the gonads at this stage of reproductive development. There was no direct evidence of progesterone (P(<em>4</em>)) synthesis from [3H]P(5), although the metabolites synthesized by both testis and ovary were indicative of a metabolic pathway that involved P(<em>4</em>) as an intermediate.

We investigated whether Sertoli cell spent media (SCM) contain a factor (or factors) which influences steroidogenesis in Leydig cells. Freshly prepared prepubertal interstitial cells or Percoll-purified Leydig cells and similar cells cultured in the presence or absence of LH were incubated for 2<em>4</em> h in the presence of a 5 alpha-reductase inhibitor and in the presence or absence of SCM. The accumulation of C19-steroids (testosterone and <em>androstenedione</em>), C21-steroids (progesterone, 17 alpha-hydroxyprogesterone and 20 alpha-hydroxypregn-<em>4</em>-en-3-one) and cyclic AMP was measured by radioimmunoassay. It could be demonstrated that SCM contains a factor that stimulates an early step in the steroidogenic pathway but at the same time hampers the conversion of C21-precursors into androgens. In freshly prepared Leydig cells the final effect is a stimulation of the androgen output. In Leydig cells cultured in the absence of LH, mainly C21-steroid output is increased. These biological effects resemble those observed with LHRH and its agonists. The activity of the Sertoli cell factor is not affected by an LHRH antagonist, however, and maximally effective concentrations of the factor and LHRH have additive effects, suggesting that they act by distinct receptor systems. Preliminary characterization shows that the factor in SCM is a thermolabile protein with a MW greater than 10 000. The production of the factor decreases during prolonged culture in serum-free medium. Addition of fetal calf serum causes a marked and dose-dependent increase in the production or activity of the factor. Several permanent cell lines (B16, Bowes, BHK, Ratec, RK13, Vero) produce a factor with comparable biological effects on Leydig cells. Nonetheless, the observation that the production of this factor by Sertoli cell cultures is stimulated by FSH and dbcAMP suggests that, in the testis, it may play a role in the paracrine control of Leydig cell function.

During rat pregnancy the placenta may provide androgens as a source of precursor for estradiol (E2) formation by the ovary. However, the relative importance of testosterone (T) and delta <em>4</em>-<em>androstenedione</em> (delta <em>4</em> A) for ovarian E2 production is unknown. The present study therefore determined the ability of the rat placenta to convert [3H] pregnenolone (P5) substrate to [3H] delta <em>4</em> A and [3H] T, and to [3H] progesterone (P<em>4</em>) in vitro on Days 12, 1<em>4</em>, 16 and 18 of gestation. The placental formation of delta <em>4</em> A and T was correlated with the uterine vein and peripheral sera concentrations of both androgens, and with their ability to be aromatized to E2 in vitro by the ovary. Placental androgen formation from P5 increased and formation of P<em>4</em> decreased with advancing gestation, with the formation of delta <em>4</em> A being approximately 2- to <em>4</em>-fold greater (P less than 0.01) than the formation of T on Days 12 to 16 of gestation. The conversion of P5 to delta <em>4</em> A increased (P less than 0.001) from 18 +/- 0.9 (mean percent conversion +/- SEM) on Day 12 to 53 +/- 3 and 57 +/- <em>4</em> on Days 1<em>4</em> and 16, respectively, then decreased (P less than 0.05) to <em>4</em>2 +/- 2 on Day 18. The uterine vein and peripheral sera concentrations of delta <em>4</em> A were 2- and 3-fold greater (P less than 0.05-0.001) than T, respectively, on Days 12 to 16.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate the role of adrenal androgens in 3 alpha-androstanediol glucuronide (3AG) production in childhood, we compared serum 3AG and androgen levels [dehydroepiandrosterone (DHEA), DHEA sulfate (DS), <em>androstenedione</em> (delta <em>4</em>-A), and testosterone (T)] in 32 children with premature pubarche due to idiopathic premature adrenarche (IPA; n = 26), partial 21-hydroxylase deficiency (n = 2), or 3 beta-hydroxysteroid dehydrogenase deficiency (n = <em>4</em>) with those in 36 normal prepubertal (18 males and 18 females) and 22 normal pubertal Tanner II-III subjects (10 males and 12 females). Serum 3AG (2.7 +/- 2.0 nmol/L) and all androgen concentrations in children with IPA were significantly higher (P less than 0.05-0.001) than those in normal prepubertal children (3AG, 0.8 +/- 0.5 nmol/L). Serum 3AG and androgen levels, except T, in all children with premature pubarche due to 21-hydroxylase deficiency or 3 beta-hydroxysteroid dehydrogenase deficiency were higher than those in the normal prepubertal children. Serum 3AG and all androgen levels in normal Tanner II-III male (3AG, 3.8 +/- 1.7 nmol/L) or female (3AG, 1.7<em>4</em> +/- 0.52 nmol/L) subjects were also significantly higher (P less than 0.05-0.001) than those in prepubertal children. Serum 3AG, DHEA, DS, and delta <em>4</em>-A levels in children with IPA were similar to those in normal Tanner II-III females or males, but serum T in children with IPA (0.37 +/- 0.2 nmol/L) was significantly lower (P less than 0.05-0.001) than that in normal pubertal females (0.71 +/- 0.37 nmol/L) or males (<em>4</em>.5 +/- 2.6 nmol/L). In the combined group (n = 88), 3AG levels correlated better with serum DS (r = 0.7), DHEA (r = 0.6), and delta <em>4</em>-A (r = 0.52), than with T (r = 0.31) levels. These data suggest that the weak adrenal androgens DS, DHEA, and delta <em>4</em>-A contribute substantially to 3AG production in premature and normal pubarche.

The biological activity of certain estrogens and androgens is modulated by enzymes called 17 beta-hydroxysteroid dehydrogenases (17 beta-HSDs), which catalyze the interconversion between less active 17-oxosteroid and more active 17 beta-hydroxysteroid forms. In the present report, we describe cloning of mouse 17 beta-HSD type-1 cDNA from an ovarian library generated from <em>4</em>,<em>4</em>'-(1,2-diethyl-1,2-ethenediyl)bisphenol-(diethylstilbestrol)-tr eated mice, and characterization of the corresponding enzyme. The open reading frame of the mouse 17 beta-HSD type-1 cDNA encodes a peptide of 3<em>4</em><em>4</em> amino acid residues with a predicted molecular mass of 36785 Da. The mouse 17 beta-HSD type-1 enzyme shares 63% and 93% overall identity with human and rat 17 beta-HSD type-1 enzymes, respectively, and the most striking differences between the mouse and human type-1 enzymes are between the amino acid residues 197 and 230 and in the carboxy terminus of the enzymes. Similarly to the human 17 beta-HSD type-1 enzyme, the mouse type-1 enzyme primarily catalyzes reductive reactions from 17-oxo forms to 17 beta-hydroxy forms in intact cultured cells, but unlike the human type-1 enzyme, the mouse enzyme does not prefer phenolic over neutral substrates. Thus, mouse 17 beta-HSD type 1 catalyzes reduction of androst-<em>4</em>-ene-3,17-dione (<em>androstenedione</em>) to 17 beta-hydroxyandrost-<em>4</em>-en-3-one (testosterone) as efficiently as 3 beta-hydroxyestra-1,3,5(10)-trien-17-one (estrone) to estra-1,3,5(10)-triene-3 beta, 17 beta-diol (estradiol). 17 beta-HSD type 1 is predominantly expressed in mouse ovaries, in which it is located in granulosa cells.