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The activities of the hepatic microsomal 2α-, 2β-, 18- and 7α-hydroxylase systems active on 5α-[4-14C] androstane-3α,17β-diol were studied in male and female rats which had been castrated and spayed at 14, 24, 35 and 45 days of age, treated for 5 days with 500 μg of estradiol benzoate per kg body weight and killed 6 days after gonadectomy. The hepatic microsomal 15β-hydroxylase enzyme system active on 5α-[1,2-3H] androstane-3α, 17β-diol 3,17-disulphate was also measured in these animals as well as in an additional group of animals that were gonadectomized at 56 days of age, treated with estradiol benzoate and killed at 62 days of age. The hydroxylase systems active on the free steroid substrate were all relatively unresponsive towards enstradiol in the developing rat, in contrast to the strong effects on hepatic hydroxylase activities previously noted following treatment of adult male rats with estradiol. On the other hand, the 15β-hydroxylase system active on disulphurylated 5α-androstane-3α, 17β-diol was inducible in both male and female rats of 41 and 51 days of age and in male rats of 61 days of age.

Clinical and biochemical investigation of a virilized woman has shown an adrenal cortical adenoma to be the source of elevated plasma testosterone levels and to be responsive to gonadotropin administration in vivo (Givens et al.) (2). In the present study, the gonadotropin responsiveness and biosynthetic potential of the adenoma were evaluated in vitro . Incubation of minced adrenal tumor with hCG resulted in increased 14C-acetate incorporation into pregnenolone, progesterone, dehydroepiandrosterone (DHA), androstenedione, and testosterone. Androstenedione and testosterone were the major products, accounting for 27% and 20% respectively of the total radio-activity added, when 3H-pregnenolone was incubated with homogenized tissue. Estrogen synthesis was not observed in the tumor. The adenoma contained 9.0 μg/g testosterone and 1.9 μg/g androstenedione as determined by radio immunoassay. 17β-hydroxysteroid oxido-reductase was active in the adenoma. Androstenedione was reduced to testosterone at a rate of 0.6 μg/100mg/hr. Under the same conditions, reduction of estrone to estradiol was undetectable. The reductase activity was present in both the mitochondrial and microsomal fractions. NADPH was the required cofactor. When NADH was substituted, the rate was less than 10% of that with NADPH in both particulate fractions. The experimental results indicate the presence of steroid path-way(s) necessary to synthesize testosterone, and represent the first in vitro demonstration of gonadotropin sensitive steroidogenesis in an adrenal cortical adenoma.

The metabolism of testosterone-4- 14 C by hamster skin and flank organ was studied; flank organ metabolized more testostrone than an equal weight of adjacent skin. Androstenedione, † androsterone, androstanedione, dihydrotestosterone (DHT), and 3α-androstanediol were identified by chromatography and reverse isotopic dilution as metabolites formed by flank organ. The metabolites of flank organ and general body skin showed identical chromatographic mobilities. Flank organ produced 4 times as much DHT as an equal weight of adjacent skin. The ratio of total 5α-metabolites formed by both tissues was the same as the DHT ratio, indicating that the greater amount of DHT formed is due to greater formation of 5α-metabolites, in general. In contrast, both tissues produced the same amount of 17-ketosteroids from testosterone and had similar DNA contents, suggesting that the observed difference in 5α-reduction was not due to differences in cellularity but to a greater capacity of the flank organ to reduce testosterone. No 3β-hydroxysteroids, the formation of which has been reported in human skin were found.

SUMMARY The site of secretion of androstenedione in women was investigated by measuring the concentration of androstenedione in peripheral and ovarian venous plasma by radioimmunoassay at different stages of the menstrual cycle. The concentration in peripheral plasma rose from 1·45 ± 0·11 (s.e.m.) ng/ml on day 1 to a peak on the 7 days around mid-cycle (2·29 ± 0·08) before declining gradually during the luteal phase. The concentration of androstenedione in plasma draining ovaries containing the pre-ovulatory follicle or a corpus luteum (33·2 ± 9·0) was significantly higher than in plasma draining the contralateral ovary (10·1 ± 1·6, P < 0·001). In women in whom ovulation was induced with gonadotrophins the concentration of androstenedione in peripheral plasma rose from 1·42 ± 0·21 before treatment to reach maximum levels (2·5–4·5 ng/ml) in the luteal phase. There was a significant (P < 0·001) correlation between the concentration of androstenedione and oestradiol-17β in peripheral (r = 0·50) and ovarian (r = 0·79) venous plasma. These findings indicate that all three cellular compartments in the ovary secrete significant amounts of androstenedione in women during their reproductive life.

Dehydroepiandrosterone (DHA) augments the activity of human chorionic gonadotrophin (HCG) in the rat by increasing endogenous pituitary gonadotrophin secretion. The following experiments were undertaken to investigate the mechanism underlying this effect. Androstenedione (40 μg), dihydrotestosterone (200 μg) and testosterone (200 μg) augmented the rat uterine weight response to 0.5 IU of HCG. At these doses, the steroids did not affect basal uterine weight although this was increased when 1 mg of a steroid was injected. Androsterone (1 mg), 17α-hydroxypregnenolone (1 mg) and progesterone (200 μg) neither augmented HCG activity nor increased basal uterine weight. Ovarian weight differences were not significant in any of the experiments. Androstenedione, DHA, dihydrotestosterone and testosterone (200 μg dose level) did not significantly affect the uterine weight of castrated animals, and responses to 0.04 μg of oestradiol were not potentiated. The results with androstenedione, dihydrotestosterone and testosterone are identical to those obtained with DHA and suggest that these steroids may also increase pituitary gonadotrophin secretion.

This report describes a 17-yr-old female who developed acanthosis nigricans, hirsutism and amenorrhea concurrently at age 15. Plasma androstenedione (A) was extremely elevated ranging from 3500 to 5000 ng/100 ml. Plasma testosterone (T) and urinary 17-ketosteroids (17-KS) were also elevated at 357 ng/100 ml and 32–42 mg/24 hr respectively. Plasma A and T were not reduced by dexamethasone (Dex), but human chorionic gonadotropin (hCG) increased plasma A to 23,500 ng/100 ml, plasma T to 3666 ng/100 ml and the 17-KS to 156 mg/24 hr. Ethinyl estradiol (EE) plus Dex reduced LH to a nondetectable level, A to 72 ng/100 ml and T to 10 ng/100 ml. ACTH did not increase A or T. The patient was treated for 7 months with 0.15 mg/day of EE and there was marked improvement of the acanthosis nigricans associated with correction of the hyperandrogenism. Because of menorrhagia, EE was discontinued and the high levels of A and T returned and the acanthosis nigricans worsened. Exploratory laparotomy was performed and...

In a series of studies involving the determination of the mechanism(s) of estrogen production in women with endometrial neoplasia, it has been ascertained that the estrogenic hormonal milieu of endometrial adenocarcinoma is the near-exclusive production at extraglandular site(s) of the estrogen product hormone, estrone from the aromatization of plasma androstenedione. Further, the results of these studies indicate that those metabolic aberrations which give rise to the capability for increased extraglandular estrone production are precisely the same as those constitutional stigmata and/or ovarian abnormalities that are known to be associated with an increased incidence of endometrial neoplasia. Increased estrone production at extraglandular site(s) occurs as a consequence of one of two events; i.e., increased availability of the plasma precursor, androstenedione, or increased extent of conversion of the plasma precursor, androstenedione, to its product hormone, estrone at extraglandular site(s). Thus, a hypothesis has emerged which relates the estrogenic hormonal milieu of near-exclusive estrone production to the occurrence of endometrial neoplasia and suggests that those constitutional stigmata which are associated with an increased occurrence of endometrial neoplasia are identical with those that give rise to metabolic mechanism(s) for increasing extraglandular estrone production.

Different areas from brains of 7- day-old female rats were incubated with testosterone- 7-3H (T) or androstenedione- 1-23H (A). Hypothalami from 120-day-old females were also incubated with T. The conversion of these precursors to estradiol (E2) and estrone (E1) was studied using double isotope dilution technique. In addition, the conversion of E2 to E1 and E1 to E2 by brain of the immature females was studied. T was converted predominantly to E2 and A to E1. The yield of E2from T was greatest in incubations of hypothalamus and amygdala, less for the combined septal and preoptic areas and least for the cortex. Conversion of T to E2 by hypothalami of adult rats was less than by that from the immatures. The yield of E1 from A was also greater in hypothalamic than in cortical incubations. There was only minimal conversion less than 3%) of E2 to E1 and of E1, to E2.(Endocrinology 94: 616, 1974)

Summary Seminal vesicles of anuran amphibian Discoglossus pictus were incubated in vitro with testosterone-4-14C. The following metabolites were isolated and identified: 5α-androstane-17β-ol-3-one (5α-DHT), 5α-androstane-3α,17β-diol,5α-androstane-3β,17β-diol and androstenedione. The intracellular distribution of 5α-reductase activity has been studied; aΔ4-3-ketosteroid 5α-oxydoreductase has been found in the nuclear fraction, in the microsomal fraction and in the cytosol fraction (200.000 × g supernatant during 60 minutes). No purification of the 5α-reductase could be achieved in any fraction. Approximatively one half of the total 5α-reductase activity was associated with the nuclei and about 30 p. cent with the supernatant fraction. The enzyme was shown to have a pH optimum of around 6,6 and a temperature optimum about 35°C. The 5α-reductase activity exhibited an absolute requirement for NADPH. The apparent Km values obtained were 1.4 × 10−5 M for NADPH and 0.4 × 10−6 M for testosterone.

The annual reproductive cycle has been investigated in the male pipistrelle bat Pipistrellus pipistrellus. Spermatogenesis occurs during summer and spermatozoa are produced in August and September. The seminiferous tubules then regress rapidly but sperm are stored throughout winter in the cauda epididymidis. Little change is apparent in the morphology of the Leydig cells throughout the year, and histochemical studies have revealed no seasonal differences in the occurrence of enzymes involved in steroidogenesis. The percentage yields of testosterone and androstenedione after incubation of testis and adrenal tissues in vitro with tritiated pregnenolone have been determined and measurements made of the concentration of these hormones in testes and whole blood. Although the highest levels of these androgens are synchronous with spermatogenesis, both continue to be produced by the testes during winter. Studies in castrated bats have confirmed these observations and indicate that the activity of the accessory reproductive organs during winter is dependent on testicular androgens. The testicular cycle of the pipistrelle is not thought to be complicated by adrenal androgenesis, or storage of androgens in the brown fat. The interrelationships between reproduction and hibernation in bats are discussed.

Specific antiserum has been developed for use in measuring 11β-hydroxyandrost-4-ene-3, 17-dione by radioimmunoassay (RIA). Rabbit antiserum was generated by employing the conjugate prepared by coupling 6β,11β-dihydroxyandrost-4-ene-3,17-dione 6-hemisuccinate with bovine serum albumin. The antiserum bound 68% of 50 picograms of 11β-hydroxyandrost-4-ene-3,17-dione-[1,2,6,7-3H] during characterization at a dilution of 1:12,500. Among the numerous steroids tested for cross-reactivity, 5α-androstane-3,17-dione, androst-4-ene-3,17-dione, and 11β-hydroxy-5α-androstane-3, 17-dione showed 2%, 5%, and 30% cross-reactivity respectively. The Rivanol-treated antiserum was coupled to Enzacryl AA, in order to study the feasibility of a solid-phase RIA, and this complex showed 50% binding with the labeled antigen at a dilution of 1:3000. The complex retained high specificity and should prove useful in a simple solid-phase RIA.

Female rats were implanted on the day of birth with Silastic capsules containing nonesterified testosterone, androstenedione, or dihydrotestosterone. The date of vaginal opening was assessed until sacrifice. The animals were ovariectomized, treated with estradiol benzoate and progesterone, and tested for the display of lordosis. The animals were then administered testosterone propionate and the size of the phallus was taken. Testosterone and dihydrotestosterone completely inhibited vaginal opening; androstenedione was partially effective. Testosterone almost completely inhibited lordosis behavior; androstenedione was partially effective and dihydrotestosterone was ineffective. All three androgens facilitated phallic development.

The pinneal organs of orchiectomized rats incubated in vitro with 3H-testosterone took up and retained 3H-radioactivity up to a tissue concentration 18-fold that of the incubation media. Maxima in tissue/medium ratios (T/M) were observed after 2-hr incubations at 37 C. Pineal organs of castrated rats exhibited higher T/M ratios than sham-operated controls. Pineal T/M ratio was 8- and 6-fold that of the cerebral cortex and tibial muscle, respectively, and about half that of the prostate. Testosterone, β-hydroxy-5α-androstan-3-one, 5α-androstane-3α, 17β-diol, androstenedione, and 5α-androstane-3, 17-dione were identified in the incubated rat pineals by thin-layer chromatography and recrystallization to constant specific activity. At low concentrations of testosterone (up to 2 × 10−11 M), T/M ratios for 3H-radioactivity depended upon the concentration of the hormone in the incubation medium. At higher concentrations pineal T/M ratio remained constant regardless of the concentration of testosterone in the med...

Cumene hydroperoxide is capable of supporting the aromatic hydroxylation of a variety of compounds in the presence of hepatic microsomes. NADPH and molecular oxygen are not required. Cytochrome P-450 acts as the catalyst and could not be replaced by other hemoproteins. One mole of hydroperoxide is consumed for every mole of substrate hydroxylated. It is suggested that the oxenoid species of cytochrome P-450 involved in microsomal aromatic hydroxylation is present in a form equivalent to the ferryl from.

Two 46 XY siblings with familial incomplete male pseudohermaphroditism, Type 2, inherited as an apparent autosomal recessive trait, were investigated. The phenotype was distinctive in that male wolffian-duct structures (epididymis, vas deferens and seminal vesicles) were present whereas tissues derived from the urogenital sinus and from the anlage of the external genitalia were female in character. Studies of estrogen and androgen dynamics revealed normal male blood testosterone and estrogen levels, production rates and interconversions. The normal blood luteinizing hormone level and normal estrogen production rate suggest that androgen resistance in this syndrome is different from that in other androgen-resistant states. The demonstration of markedly deficient dihydrotestosterone formation in slices of perineal skin, epididymis and phallus, considered with the fact that dihydrotestosterone is the fetal hormone responsible for male differentiation of the external genitalia, is compatible with the...

By judicious choice of the site of attachment of steroid haptens to a protein carrier it is possible to elicit the formation of antibodies with enhanced specificity towards selected parts of the steroid molecule. Coupling the steroid through a position remote from the functional groups responsible for its hormonal specificity preserves these substituents as antigenic determinants. This has been effected in gonadal steroids by inserting “chemical handles” (e.g. carboxymethyl oxime, carboxyethyl thioether or hemisuccinate groups) into the steroid skeleton at positions 1,6,7 or 11. Thus, immunization with oestrone, oestradiol-17β or oestriol coupled to protein at position 6 gave rise to antibodies discriminating between the three major oestrogenic hormones and closely related metabolites that are not distinguished by antibodies to oestrogens conjugated to protein at C-17. Likewise, antibodies to progesterone-6-, 7- or 11-conjugates recognize changes in the 17-sidechain more efficiently than antibodies to the corresponding 20-conjugate. However, the antiserum to progesterone-6—protein was least specific towards changes in the A-ring. This approach has been extended to key progesterone metabolites and to androgens. Thyroglobulin or BSA conjugates of 1-carboxyethyl thioethers of testosterone, 5α-dihydrotestosterone (DHT), androstenedione (Ad) and progesterone have been prepared. Antisera reacting preferentially with DHT, and antisera to testosterone showing minimal cross-reaction with DHT (8 %) and none with Ad (

SUMMARYThree experiments were carried out to investigate the anabolic effects of androstenedione administered at various levels to female and castrated male lambs in one experiment and to entire and castrated male calves in two other experiments. Significant increases in growth were not effected in normally fed animals but a sex × dose level interaction was detected in female lambs. Under normal husbandry conditions androstenedione appears to have little application in meat production.

SUMMARY The concentrations of luteinizing hormone (LH), progesterone, testosterone, androstenedione, oestrone and oestradiol in mixed umbilical cord blood taken from foetal calves during months 3 to 9 of gestation have been measured by radioimmunoassay. The levels of progesterone were low (compared with those in maternal plasma) and constant during pregnancy. There were large variations in the concentrations of androstenedione, oestrone, oestradiol and LH, with lower values found during months 7 to 8 of gestation. Significant differences (P < 0·05–< 0·001) in hormone levels between male and female foetuses were found for testosterone, LH, and the oestrogens during months 3 to 7, 3 and 4, and 7 and 8, respectively. No significant differences (P > 0·05) between sexes were found at any time between the concentrations of progesterone and androstenedione.

The addition of androstenedione and NADPH to human placental microsomes results in a marked increase in the rate of O2 and NADPH utilization concomitant with estrogen formation. Initially, 5 to 12 moles of O2 and an equivalent amount of NADPH are consumed per mole of estrogen formed, the ratio varying from one microsomal preparation to another. In the presence, however, of 10 to 20 mm KCN, 3 moles of O2, and 3 moles of NADPH are consumed per mole of estrogen formed. This ratio was invariant even when a concentration of KCN sufficiently high to inhibit aromatization was employed. Both NADPH and O2 are required for the conversion of 19-hydroxyandrostenedione to 19-oxoandrostenedione, suggesting that a second hydroxylation is responsible for this conversion rather than oxidoreduction of the 19-alcohol. These results are consistent with previous data suggesting three successive hydroxylations catalyzed by a mixed function oxidase(s) are required during C19 steroid aromatization. The rate of appearance of estrogen intermediates was examined in an effort to determine to what extent the accumulation of these compounds might account for the high rate of O2 consumption observed in the absence of KCN. It was determined that essentially all of the O2 consumed could be accounted for by accumulation of 19-hydroxyandrostenedione and 19-oxoandrostenedione and estrogens. In the presence of KCN, the rates of formation of 19-hydroxyandrostenedione and 19-oxoandrostenedione were greatly inhibited under steady state conditions while the rate of aromatization was only slightly decreased. Moreover, under all conditions, the rate of formation of 19-hydroxyandrostenedione was greater than its rate of conversion to estrogen. Also, the rate of formation of 19-oxoandrostenedione was greater than its rate of disappearance in the absence of KCN. These observations suggest that the final hydroxylation is the rate-limiting step in the aromatization of androstenedione.

Adult male hamsters were castrated, and four weeks later, after a single mating test, began receiving daily 100 μg injections of testosterone (T), androstenedione (AD), or testosterone propionate (TP). Mating tests were conducted at regular intervals during the eight week treatment period. Results indicated the superiority of TP in reinstating intromissions and ejaculations. The T and AD groups did not differ on any of the measures, a result which agrees with previous similar experiments using rats.

To investigate the mechanism of the increased plasma estradiol levels in spontaneous hyperthyroidism, the contribution, by peripheral conversion, of androstenedione and testosterone to the circulating estrogens was determined. The conversion ratio of androstenedione to estrone was significantly increased in both males and females (0.0396 and 0.0256, respectively). The conversion ratio of androstenedione to estradiol was increased in the male (0.0094) but not in the female (0.0051) patients. The conversion ratio of testosterone to estrone was increased in both males (0.0072) and females (0.0070). The conversion of testosterone to estradiol, however, was not increased in either sex. The plasma concentration of androstenedione was significantly increased (0.34 μg/100 ml) in the male patients with hyperthyroidism. However, the production rate of androstenedione was increased in both sexes (8.59 and 6.72 mg/day). The contribution of testosterone and androstenedione to plasma estrone and estradiol (pro...

In vitro steroid biosynthesis in the male and female gonad of Mytilus edulis was investigated. Using tritium and 14C-labeled precursors, four enzyme systems were demonstrated: a 3β-hydroxysteroid dehydrogenase-Δ5,4-isomerase comples (pregnenolone→progesterone; 17α-hydroxypregnenolone→17α-hydroxyprogesterone; dehydroepiandrosterone→androstenedione), a C17,20-lyase (17α-hydroxypregnenolone→dehydroepiandrosterone; 17α-hydroxyprogesterone→androstenedione), a 17β-hydroxysteroid dehydrogenase (dehydroepiandrosterone→androstenediol; androstenedioneαtestosterone; estradiol-17βαestrone) and a 5α-reductase (testosterone→5α-dihydrotestosterone). The yields were very low (0.2–1%) except when estrogens were used (10–50%). Radioimmunoassay techniques indicate the presence of testosterone and possibly estrogens in the gonad of the mussel at two different stages in its gametogenic cycle.

When [7α-3H] dehydroepiandrosterone was incubated with the adrenal homogenates of human fetus at 22 to 26 weeks gestational age, 16α-hydroxydehydroepiandrosterone and/or its sulfate was formed as the only detectable metabolite. The 16α-hydroxylase activity was concentrated in the microsomal fraction of the adrenal homogenate. [1,2-3H]Androstenedione, [4-14C] pregnenolone and [7α-3H] progesterone were also 16α-hydroxylated by incubation with the microsomal fraction. Amoung these substrates, progesterone gave the highest yield of 16α-hydroxylated products. By incubation with the microsomal fraction, formation of following steroids were also established: 6β-hydroxyandrostenedione from androstenedione; 17-hydroxypregnenolone, 17,21-dihydroxypregnenolone and dehydroepiandrosterone from pregnenolone; 17-hydroxy-progesterone, deoxycorticosterone, 11-deoxycortisol and androstenedione from progesterone.

Incubation of hypothalamic and limbic system homogenates from 21-day rat fetuses and 1, 5, 10-day-old or adult rats with E4- androstene-3-17-dione 7-3H resulted in the formation of 3H-estrone by all tissues with the exception of the limbic system from the female fetuses. In each paired experiment the activity in male tissues exceeded that of the females. In general, the activities of perinatal tissues either equaled or exceeded those of comparable adult tissues. These results support the hypothesis that neuroendocrine effects of androgens on fetal, neonatal, prepubertal and adult rats could be mediated by estrogen metabolites. (Endocrinology 94: 117, 197.4)

A family with incomplete male pseudohermaphroditism inherited as an apparent X-linked recessive trait was investigated. The phenotype in 11 affected family members ranged from a minimal de...

SUMMARY Human skin from forehead, cheek and axilla was incubated in vitro with [7α-3H]dehydroepiandrosterone (DHA), [7α-3H]DHA sulphate, [7α-3H]-androstenedione and [7α-3H]testosterone. The following enzyme activities were detected: 3β-hydroxysteroid dehydrogenase Δ4-5 isomerase, 17β-hydroxysteroid dehydrogenase, 3β-hydroxysteroid dehydrogenase, 3α-hydroxysteroid dehydrogenase, 5α-reductase, 5β-reductase, sulphotransferase, sulphatase, steroid hydroxylase. 5α-Reduced steroids were the major metabolites. All four substrates were converted to 5α-dihydrotestosterone and 5α-androstane-3α,17β-diol. In axillary skin, conversion of 17-oxosteroids to 17β-hydroxysteroids was favoured, 5α-dihydrotestosterone and 5α-androstane-3α,17β-diol being major metabolites. In facial skin, formation of 17-oxosteroids predominated with little accumulation of 5α-dihydrotestosterone or 5α-androstane-3α,17β-diol. 5α-Androstane-3β,17β-diol was a metabolite of DHA, androstenedione and testosterone but was found in lower amounts than 5α-androstane-3α,17β-diol. Similarly conversions to epiandrosterone were much lower than to androsterone in all the skin specimens. It was concluded that the differences in accumulation of 5α-dihydrotestosterone were determined by the differences in 17β-oxidoreduction rather than differences in 5α-reductase, the activity of which was high in all skin specimens.