Quantitative evaluation of virilizing activity of steroids by measuring morphological changes in uro-genital region of rats.

A new technique to measure the uro-genital parameters such as the lengths of urovaginal septum, corpora cavernosa and anogenital distance on the sagittal sections of the pelvic region of female fetus of rat under microscope equipped with a micrometer was developed. In the examination of 180 normal female fetuses on the 21st day of gestation, relationships were observed between the fetal body weight and the length of urovaginal septum as well as anogenital distance, but not on the length of corpora cavernosa. Following maternal subcutaneous administration of various doses of 17alpha-methyltestosterone between the 17th and 20th day of gestation, dose-dependent abridgment in urovaginal septum length and extensions in corpora cavernosa length and anogenital distance were observed in female fetus on the 21st day's examination. When these three parametars were calculated on the relative value to the fetal body weight, however, linear relationships against log-dose were observed in all parameters. Among these three parameters the abridgment in urovaginal septum was shown to be the most sensitive. A quantitative assay of virilizing activity of steroids in female fetuses was examined on rats treated subcutaneously with 17alpha-methyltestosterone and norethandrolone. Linear regressions against the log-doses of both steroids were demonstrated in urovaginal septum length, and parallelism was noted between both regression lines. The relative potency of norethandrolone to 17alpha-methyltestosterone calculated on urovaginal septum length was 0.354 with fiducial limits of 0.293-0.44l, and it was suggested that the virilizing activities of steroids can be evaluated quantitatively.     

Virilizing activities of various steroids in female rat fetuses

Virilizing activities of nineteen steroids were examined by a method measuring the abridgment of urovaginal septum length of female rat fetuses directly under microscope, following oral administration of the steroids to the mothers for 4 days during the late period of gestation. The characteristics of the virilizing activities of the steroids seemed to depend on their chemical structures. No virilizing activities were observed in progesterone, retroprogesterone, chlormadinone acetate and nandrolone phenpropionate. The introduction of 6-methyl and 17-acetoxy groups into progesterone exhibited a marked virilizing activity. With testosterone and its derivatives, the introduction of 17alpha-ethynyl group into testosterone slightly decreased the virilizing activity compared with the parent compound. On the contrary, the introduction of [3, 2-C] pyrazole ring in addition to the saturation of A-ring strikingly increased the virilizing activity. The most active virilizing steroid in this group was stanozolol followed by oxymetholone, methyl-testosterone and dimethysterone. Testosterone propionate was assumed to be less active than methyltestosterone by this route. With 19-nortestosterone derivatives, the virilizing activities of compounds with 17alpha-ethynyl group were moderate, but the presence of 17alpha-ethyl group caused a marked increase of virilizing activity. While, nandrolone phenpropionate with long side chain at C-17 exhibited no significant virilizing activity. The present results show the possibility of the evaluation on a potential virilizing activity of compounds in the female fetus, and also suggest that a dossociation between the virilizing activity and the androgenic activity may exist among some compounds.     

Influence of diet on plasma steroids and sex hormone-binding globulin levels in adult men

Several experimental studies have suggested that diet can alter the production and metabolism of steroids in men. The purpose of this study was to determine the levels of unconjugated steroids and steroid glucuronides as well as sex hormone-binding globulin (SHBG) among normal adult men who were either omnivorous or vegetarians. The participants were white volunteers ranging from 25-35 years of age and the blood samples were taken between 0900 h and 1000 h and between 1600 h and 1700 h for two consecutive days. No significant statistical change was found in plasma dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, dihydrotestosterone and estradiol levels. Vegetarian group showed a higher levels of sex hormone-binding globulin (SHBG) while the free androgen index (FAI; calculated by the ratio testosterone/SHBG) was lower in this group. Although the concentrations of androsterone glucuronide were higher in vegetarian group, the vegetarians had a 25-50% lower level of androstane-3 alpha, 17 beta-diol glucuronide and androstane-3 beta,17 beta-diol glucuronide. Our data further indicate that both, androstane-3 alpha,17 beta-diol glucuronide and androstane-3 beta,17 beta-diol glucuronide concentrations are significantly correlated with SHBG levels and with the FAI values. The increases in androstane-3 alpha,17 beta-diol glucuronide and androstane-3 beta,17 beta-diol glucuronide levels in the omnivorous group are probably a consequence of the elevation of the FAI. Our data suggest that in a vegetarian group, less testosterone is available for androgenic action.     

Androgen metabolites in bovine follicular fluid

The values of C21-steroids, Delta4-androgens, estrogens as well as 5alpha-reduced steroids have been determined in follicular fluid obtained from superovulated and untreated cows. In the three cows treated with a hormone regimen to induce superovulation, the levels of progesterone and estradiol determined in 3 to 6 follicles per cow ranged from 65 to 448 ng/ml and 1.9 to 8.6 ng/ml, respectively while the concentrations of androstenedione and testosterone varied between 1.5 to 2.5 ng/ml. Low levels of dihydrotestosterone and androstane-3alpha, 17beta-diol (approximately 30 to 50% of Delta4-androgens) were found in the bovine follicular fluid. In untreated cows, the follicular steroid concentrations were divided into two groups on the basis of the ratio between estrogen and Delta4-androgen concentrations. In estrogen-rich follicles, the ratio of estrogens Delta4- androgens was higher than 1 and in estrogen-poor follicle, the ratio of estrogens Delta4- androgens was lower than 1. Pregnenolone, dehydroepiandrosterone, androst-5-ene-3beta, 17beta-diol, progesterone, androstenedione and testosterone levels were not significantly different in the two groups while the levels of estradiol and estrone were approximately 100-fold higher in the estrogen-rich group. The concentrations of 5alpha-reduced steroids particularly, dihydrotestosterone, androstane-3alpha, 17beta-diol and androsterone as well as their glucuronides which were found at values extremely low (under 1 ng/ml) were not significantly different in both groups. The results indicate that low levels of 5alpha-reduced steroids and their glucuronides are present in bovine follicular fluid and their concentrations remained fairly stable either in estrogen-rich or estrogen-poor groups.     

Effect of neonatal estrogenization on testosterone metabolism in the prostate and in the epididymis of the rat

The present experiments were performed in order to analyze whether the administration of estrogens (single injection of 500 micrograms of estradiol benzoate s.c.) to neonatal male rats might modify the weight of the ventral prostate and the epididymis as well as the metabolism of testosterone in these two organs. The metabolism of testosterone was evaluated in vitro using 14C-radiolabelled testosterone as the substrate. The metabolites dihydrotestosterone (DHT), 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol), 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol), androstenedione, 5 alpha-androstane-3,17-dione (5-A-dione) and 3 alpha-hydroxy-5 alpha-androstane-17-one (androsterone) were quantified. After neonatal estrogen administration animals were killed on days 22 and 90 of age. The following changes were observed: (1) the body weight, the weight of the testes and of the ventral prostate were lower than in controls on both day 22 and 90; (2) the weight of the epididymides was higher than in controls on day 22 and lower on day 90; (3) in the ventral prostate the in vitro formation of DHT was lower and that of the diols was higher than in control tissue on day 22 of age; (4) the in vitro formation of alpha-reduced metabolites of the 17-keto series (5 alpha-A-dione + androsterone) was higher in ventral prostate of treated animals than in that of controls on day 22; (5) in treated animals, no formation of DHT in the caput epididymis was observed at day 22. On the contrary, at the same age the formation of androstenedione was higher than in controls; on day 90 of age the formation of DHT, androstenedione and the 5 alpha-reduced metabolites of the 17-keto series was identical in caput epididymis of the treated animals and of the controls, while the formation of the diols was higher in the treated than in the controls. The data indicate that neonatal estrogenization may induce important changes in testosterone metabolism in the prostates and in the epididymides of the rat.     

Androgen responsiveness of the liver of the developing rat

The activities of the hepatic microsomal 2alpha-, 2beta-, 7alpha- and 18-hydroxylase systems active on 5alpha-[4-(14)C]androstane-3alpha,17beta-diol were studied in male and female rats which had been castrated at birth and at the age of 7, 13, 21, 27, 34, 43 and 55 days, treated for 5 days with 2mg of testosterone propionate/kg body weight and killed 6 days after castration. The 7alpha-hydroxylase system was affected very little by androgen treatment at all stages during development. On the other hand it was found that the rat liver passed through three phases during development with respect to androgen responsiveness as judged by changes in the activities of the 2alpha, 2beta- and 18-hydroxylase systems: a first phase (from the neonatal period up to about 19 days of age) with a relative androgen unresponsiveness in both male and female rats, a second phase (from about 27 to about 33 days of age) when male and female rats responded equally well to androgens and a final phase (from about 40 days of age) with a successively decreasing androgen responsiveness in female rats but with a retained responsiveness in male rats. The hypothesis is presented that neonatal imprinting of the liver by testicular androgen(s) determines the development and degree of androgen responsiveness of liver tissue in the rat.     

HPLC-RIA analysis of steroid hormone profile in a virilizing stromal tumor of the ovary

The pathological steroid biosynthesis of a virilizing ovarian tumor was examined via high performance liquid chromatography-radioimmunoassay (HPLC-RIA) determination of the intratissular steroid concentrations. Sex cord-stromal tumor of the ovary was obtained surgically from an 18-year-old female patient with extremely high androst-4-ene-3,17-dione (4-en-dione) and testosterone (Test) blood serum levels. The tissue specimen was extracted with ethyl acetate and the extract was then purified on a C18 mini-column with methanol-water eluents. Steroids were isolated by reversed-phase HPLC on a C18 silica gel column with 51%, 55% and 64% v/v methanol-water eluents. Steroids in the collected eluent fractions were detected by the radioactivity of tritiated internal standards and then quantified by specific RIAs. In the tumor specimen, very high 17alpha-hydroxyprogesterone (17-OH-Prog; 6300 fmol/g), dehydro-epiandrosterone (2870 fmol/g), androst-4-ene-3,17-dione (3000 fmol/g), testosterone (5700 fmol/g) concentrations, and less progesterone (PROG; 320 fmol/g) and androst-5-ene-3beta,17beta-diol (5-en-diol; 320 fmol/g), were determined. Tissue levels of 5alpha-dihydrotestosterone (DHT), 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), 5alpha-androstane-3beta,17beta-diol (3beta-diol), and 17beta-estradiol were found to be 71, 20, 28, and 12 fmol/g, respectively. Steroid profile analysis verified a pathological steroid biosynthesis in the ovarian tumor and suggested that the 17alpha-hydroxylase (17alpha-H), 17,20-lyase (17,20-L), and 3beta-hydroxysteroid dehydrogenase/Delta5-4-isomerase (Delta5-3beta-HSD) activities were particularly elevated in this tumorous tissue. Present data demonstrate that the analysis of intratissular steroid profile by a HPLC-RIA method may valuably contribute to the steroidal pathophysiology of endocrine tumors.     

Virilizing effect of methyltestosterone on female descendants in the rat

Pregnant rats were given daily a subcutaneous injection of methyltestosterone for 4 days from the 17th to the 20th day of gestation, and were allowed to be delivered to their offsprings (F1) which were used for the examination of later reproductive functioning. When observed for 21 weeks after birth, the growth rate of F1 from methyltestosterone-treated groups was higher than that of F1 from the control group. The anogenital distance in 50-microgram-treated F1 females started to become significantly longer on the 14th day and in 5-microgram-treated F1 females on the 28th day after birth than that in F1 from the control. The day on which vaginal opening took place in 50% of females was 34.4 days of age in both the control and the 5 microgram groups, but it delayed until 40.7 days in the 50 microgram group. Furthermore, persistent estrus was observed after about 90 days of age in the 50 microgram group. This persistent estrus disappeared by placing these females with males, resulting no pregnancy. In the 5 microgram group females could be pregnant, but their female fetuses (F2), when examined on the 21st day of gestation, had significantly shortened the length of the urovaginal septum. The observations show that virilization can be induced in the third generation.     

A physiological dose of estradiol with progesterone affects striatum biogenic amines

The acute effect of estradiol and progesterone on dopamine and serotonin metabolism in rat striatum was studied. One subcutaneous injection of 17 beta-estradiol (300 ng) and progesterone (150 micrograms) into intact male rats increased plasma levels of these steroids, while testosterone, corticosterone, and estrone remained unchanged. Dehydroepiandrosterone, androstane-3 beta, 17 beta-diol and dihydrotestosterone remained undetectably low. Prolactin decreased and androstane-3 alpha, 17 beta-diol, and 17-OH progesterone increased, but less than estradiol and progesterone. Peak levels of striatal dopamine, dihydroxyphenylacetic acid, and homovanillic acid were observed 15-45 min after steroid injection with a return to control values after 45-60 min, while serotonin and 5-hydroxyindoleacetic acid levels were slightly decreased. An injection of estradiol (70 ng) with progesterone (70 micrograms) to ovariectomized female rats left plasma prolactin levels unchanged, while striatum dopamine and serotonin as well as their metabolite concentrations peaked 15-60 min after steroid injection and returned to control values after 45-75 min. To allow for a better comparison of the action of these steroids, the effect of estradiol or progesterone alone and in combination on the brain of ovariectomized rats was compared in the same experiment. A similar increase in metabolites of dopamine levels was observed after these steroids alone or in combination, while dopamine levels were increased only after progesterone alone or in combination with estradiol. An injection of estradiol or progesterone to ovariectomized rats led to peak steroid concentrations at approximately the same time in the brain and plasma. In addition, plasma and brain steroid levels were significantly correlated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Onset of the hormone-sensitive perinatal period for sexual differentiation of the sexually dimorphic nucleus of the preoptic area in female rats

The volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) of the rat brain is severalfold larger in males than in females. The volume of the SDN-POA can be influenced significantly by the hormonal milieu during the perinatal "critical period" of sexual differentiation of the brain. The purpose of the present study was to determine the onset of this period of sexual differentiation of the SDN-POA. Pregnant rats received no treatment or were injected subcutaneously with oil on day 17, 18, or 20, or testosterone (T;5 mg) on days 16-22 of gestation. On postnatal day 15, unilateral SDN-POA volumes from female offspring prenatally exposed to testosterone on day 16 or 17 were not different from values of control (untreated or oil-injected) offspring. Female offspring from mothers treated with testosterone on day 18, 19, or 20 of gestation showed a significant and similar increase in SDN-POA volume over values from control animals. SDN-POA volumes from female offspring exposed to testosterone on day 21 or 22, although larger than those of controls, were not different statistically. We conclude that with the specific paradigm used in this study SDN-POA development is insensitive prior to day 18 of gestation, the day on which the onset of the hormone-sensitive period occurs.     

In vitro metabolism of 4-androstene-3,17-dione and testosterone by rat submaxillary gland.

Tritiated 4-androstene-3,17-dione and testosterone were incubated with submaxillary gland homogenates of male and female rats. The metabolism was predominately reductive. In 15 and 180 min incubations submaxillary tissue converted 4-androstene-3,17-dione chiefly to androsterone. Less testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one, 5 alpha-androstane-3,17-dione, 5 alpha-androstane-3 alpha, 17 beta-diol, and 4-androstene-3 alpha, 17 beta-diol were also identified. Testosterone was converted to the same products plus 4-androstene-3,17-dione. 5 alpha-Androstane-3 alpha, 17 beta-diol was the major testosterone metabolite. Qualitatively the metabolism by male and female submaxillary gland was similar.     

Effect of 3-week treatment with [D-Trp6, des-Gly-NH10(2)]LHRH ethylamide, aminoglutethimide, ketoconazole or flutamide alone or in combination on testicular, serum, adrenal and prostatic steroid levels in the dog

Adult male mongrel dogs were treated with the LHRH agonist [D-Trp6, des-Gly-NH10(2)]LHRH ethylamide, aminoglutethimide, ketoconazole or flutamide alone or in combination for 21 days before measurement of steroid levels in the testes, prostate, adrenals and serum. Ketoconazole alone caused a marked stimulation of the intra-testicular concentration of pregnenolone, 17OH-pregnenolone, progesterone and 17OH-progesterone with no or little change of androstenedione, testosterone and dihydrotestosterone. Aminoglutethimide caused a 30-95% inhibition in the concentration of all steroids in the tests while treatment with the LHRH agonist caused a near complete inhibition of all testicular steroids. When administered concomitantly with the LHRH agonist, ketoconazole partly prevented the inhibitory effect of the LHRH agonist on testicular steroid levels. Serum levels of dehydroepiandrosterone, androst-5-ene-3 beta,17 beta-diol, androstenedione and androstane-3 alpha, 17 beta-diol were 75 to 95% inhibited by the LHRH agonist while serum testosterone and dihydrotestosterone concentrations were reduced below detection limits by the same treatment. Moreover, treatment with the LHRH agonist caused a 70-95% reduction in the intraprostatic concentration of testosterone and dihydrotestosterone in all the groups although maximal effect was observed when the LHRH agonist was combined with any of the three other agents. The present data show that while treatment with ketoconazole, aminoglutethimide or Flutamide alone has only partial inhibitory effects on androgen levels, combination with an LHRH agonist provides maximal inhibition. In addition to its direct blockade of the androgen receptor, some of the effect of Flutamide could be related to its blockade of testicular 3 beta-hydroxy-steroid dehydrogenase activity.     

Intratesticular unconjugated steroids in elderly men

As an extension of our studies on the influence of age on testicular function and with the aim of detecting whether the decline in testosterone production by aged testes is accompanied by a block in the biosynthetic chain leading from cholesterol to testosterone, we determined in the testis of young and elderly men, who died suddenly either from a cardiac incident or from accident, intratesticular steroids: pregnenolone, 17 hydroxypregnenolone (3 beta, 17 alpha-dihydroxy-5-pregnen-20-one), dehydroepiandrosterone, androstenediol, (5-androsten-3 beta, 17 beta-diol), progesterone, 17 hydroxyprogesterone, androstenedione, 17 beta-estradiol as well as testosterone, dihydrotestosterone (5 alpha-androstan-17 beta-ol-3-one) and androstanediol (5 alpha androstane-3 alpha, 17 beta-diol). The intratesticular steroid pattern in elderly men was essentially characterized by a decrease of the 5-ene steroid concentration, whereas we did not observe a decrease in the 4-ene steroids, progesterone concentration being even significantly higher in the aged testes. There was no evidence for a decrease in either lyase or 17-hydroxylase activity. It is suggested that the steroid pattern as observed in the aged testes is the consequence of a decreased oxygen supply, due to a decreased testicular perfusion.     

Experimental study of umbilical cord length as a marker of fetal alcohol syndrome

Umbilical cord length has long been investigated as a potential marker of intrauterine events that may place the neonate at risk for future adverse developmental sequelae. Experimentally, significantly shortened cords have been reported in association with prenatal exposure to common drugs of abuse. This study in rats reports the time course of effects on umbilical cord length of a daily maternal ethanol gavage (3,200 mg/kg) from gestational day 6 through termination of pregnancy at either day 17, 18, 19, or 20. A total of 786 fetuses derived from 60 litters were examined. Control fetuses demonstrated a linear increase in umbilical cord length and body weight gain during late gestation, findings that support previous studies. The body weights of the ethanol-exposed fetuses were reduced significantly on all gestational days examined, indicating intrauterine growth retardation, a characteristic of fetal alcohol syndrome. Similarly, acute fetal akinesia as well as long-term sequelae stemming from impaired neurological development would result from the elevated blood ethanol levels achieved in this study. The umbilical cords of ethanol-exposed fetuses were significantly shorter on gestational days 19 and 20 in comparison to their controls, while cord lengths on days 17 and 18 were not shortened significantly. A stretch hypothesis has been proposed suggesting that the degree of fetal activity is the main determinant of umbilical cord length. In rats, there is a physiologic diminution of the volume of amniotic fluid (oligohydramnios) in late gestation (day 19 to term), which restricts fetal movements but does not appear to alter the linear relationships between gestational age and cord length in controls, thus arguing against the stretch hypothesis. However, cord lengths in the ethanol-exposed fetuses plateaued in late gestation, suggesting possible adherence to a stretch hypothesis. This dichotomy is discussed emphasizing fetal growth and activity as well as intrauterine space.     

Metabolism of testosterone sulfate in the rat: analysis of biliary metabolites.

Following intraperitoneal injection of a mixture of testosterone-7-3-H-17-sulfate and testosterone-4-14-C into male and female rats with bile fistulas, biliary metabolites were separated and purified by a combination of column chromatography, enzymic hydrolysis or solvolysis of the conjugate fractions and identification of the liberated aglycones. The injected steroids were extensively metabolized and excreted predominantly in the bile. The major portion of the 3H was excreted in the disulfate fraction in both sexes. Solvolysis of the disulfate revealed the sex-specific aglycone pattern: 5alpha-Androstane-3beta,17beta-diol was the major metabolite in the male rat, whereas 5alpha-androstane-3alpha,17beta-diol and polar steroids were found in the female. In marked contrast, testosterone was metabolized in a different way than testosterone sulfate. 14-C radioactivity was distributed in monoglucosiduronate, monosulfate, and diconjugate fractions. Analysis of the aglycones showed that polar steroids were the main metabolites in the male. In the female, testosterone was metabolized to polar steroids, androsterone, and 5alpha-androstane-3alpha,17beta-diol.     

Activation of phospholipases during masculine differentiation of embryonic genitalia

We have investigated whether the androgen-induced masculine differentiation of the sex organs involves an induction of phospholipases. We have measured phosphatidylinositol-specific phospholipase C and phosphatidylcholine-specific phospholipase A2 in the reproductive tract of male and female mouse (CD-I) fetuses at the 18th day of gestation. We report here that (1) the activity of these two enzymes is higher in the male genitalia than in the female genitalia; (2) exogenous testosterone at the 13th to 17th day of pregnancy induces both phospholipase A2 and phospholipase C in the female fetal genitalia; and (3) prenatal administration of cyproterone acetate, an antiandrogen, known to produce feminized males, completely prevents the stimulation of phospholipase A2 and C by testosterone in the female fetuses. In the male fetuses, however, cyproterone acetate inhibits the PLC activity but is unable to alter phospholipase A2 activity. These findings provide evidence that the mechanism by which testosterone organizes the genitalia may involve a modification of phospholipases A2 and C.     

Effects of testosterone metabolites on serum gonadotropin concentrations in immature male rats.

The ability of testosterone, androsterone, 5alpha-androstane-3alpha,17beta-diol, and 5alpha-androstane-3beta,17beta-diol to prevent the castration-induced rise in serum gonadotropin levels was investigated in immature male rats. Rats castrated at 30 days of age were treated once per day by subcutaneous injection of 12.5-100 mug of the steroid per 100 g body weight per day for 3 days, beginning on the day of castration. The animals were sacrificed 24 h after the last injection. Testosterone propionate, androsterone propionate, and 5alpha-androstane-3alpha,17beta-diol dipropionate were also tested at the approximate molar equivalent of 100 mug of the free alcohol form per 100 g body weight per day. Testosterone propionate and 5alpha-androstane-3alpha,17beta-diol were the only compounds tested that prevented the castration induced rise in luteinizing hormone (LH) concentrations. Testosterone propionate also inhibited the rise in follicle stimulating hormone (FSH) concentrations whereas 5alpha-androstane-3alpha,17beta-diol inhibited the rise in FSH in one but not in another experiment. These were the only compounds tested that affected serum FSH concentrations. The lower doses of testosterone tested significantly increased serum LH, but not FSH concentrations compared to castrate control animals. The highest dose tested partially inhibited the rise in serum LH concentrations. Both androsterone and androsterone propionate maintained ventral prostate weights. Although neither compound prevented the castration induced rise in serum LH, two groups receiving androsterone had serum LH concentrations significantly lower than the castrate control group. 5alpha-Androstane-3beta,17beta-diol and 5alpha-androstane-3alpha,17beta-diol dipropionate failed to maintain ventral prostate weights or prevent the rise in serum gonadotropin levels. These results indicate that 5alpha-androstane-3alpha,17beta-diol is capable of preventing the castration induced rise in serum LH concentrations in the immature male rat and thus may participate in the regulation of LH secretion in these animals.     

Hydrogen exchange during oxidoreduction and epimerization at C-3 of C19 steroid sulphates in the rat

Mixtures of 17 beta-hydroxy-5 alpha-[16,16,17 alpha-2H3]androstan-3-one 17-sulphate and 5 alpha-[3 beta (or 3 alpha)-2H]androstane-3 alpha (or 3 beta), 17 beta-diol 17-sulphate were incubated with isolated hepatocytes from female rats or infused intravenously in female rats with bile fistulas. The androstanediols formed were analyzed by gas chromatography-mass spectrometry. Metabolism of 3H-labelled steroids was also studied in corresponding experiments. Isolated hepatocytes rapidly reduced the 3-oxosteroid to the corresponding 3 alpha-hydroxysteroid, which was more rapidly sulphated than the incubated 3 alpha-androstanediol. The 3 alpha-hydroxysteroid was extensively oxidoreduced both in vivo and in isolated hepatocytes. The intermediate formed during oxidoreduction in vivo was incompletely mixed with the infused 3-oxosteroid indicating extrahepatic uptake of the latter. The 3 beta-hydroxysteroid was sulphated without significant oxidoreduction and a minor fraction was converted to 3 alpha-hydroxysteroid both in vivo and in isolated hepatocytes. The incubated 3 beta-hydroxysteroid contributed more to the disulphate of the isolated 3 alpha-hydroxysteroid than to the monosulphate, indicating that the incubated 3-oxosteroid and the intermediate in the inversion were not completely mixed. Deuterium from the 3 beta- or 3 alpha-positions of the incubated [3-2H]androstanediols was not incorporated in androstanediol molecules derived from the 3-oxosteroid. However, both in vivo and in isolated hepatocytes the 5 alpha-[3 alpha-2H]androstane-3 beta,17 beta-diol 17-sulphate molecules which underwent inversion at C-3 retained 50-80% of the deuterium. This indicates that the inversion was not caused by two separate oxidoreductases.     

Androgen metabolism and actions in rat ventral prostate epithelial and stromal cell cultures

The rat ventral prostate requires androgens for normal development, growth, and function. To investigate the relationship between androgen metabolism and its effects in the prostate and to examine differences between the epithelial and stromal cells, we have established a system of primary cell cultures of immature rat ventral prostate cells. Cultures of both cell types after reaching confluency (6-7 days) actively metabolized 3H-labelled testosterone (T), 5 alpha-dihydrotestosterone (5 alpha-DHT), 5 alpha-androstane-3 alpha,17 beta-diol, and 5 alpha-androstane-3 beta,17 beta-diol. The epithelial cells actively reduced T to 5 alpha-DHT and formed significant amounts of 5 alpha-androstane-3,17-dione from T, 5 alpha-DHT, and 5 alpha-androstane-3 alpha,17 beta-diol. All substrates were converted to significant amounts of C19O3 metabolites. The stromal cells also metabolized all substrates, but very little 5 alpha-androstane-3,17-dione was formed. The metabolism studies indicate that both cell types have delta 4-5 alpha-reductase, 3 alpha- and 3 beta-hydroxysteroid oxidoreductase and hydroxylase activities. The epithelial cells have significant 17 beta-hydroxysteroid oxidoreductase activity. The epithelial cells cultures grown in the presence of T have higher acid phosphatase (AP) contents (demonstrated histochemically and by biochemical assay). Tartrate inhibition studies indicate that the epithelial cells grown in the presence of T are making secretory AP. Stromal cell AP is not influenced by T. The results indicate that the cultured cells maintain differentiated prostatic functions: ability to metabolize androgens and, in the case of the epithelial cells, synthesize secretory AP.     

Sex difference in whole-body androgen content in rats on fetal days 18 and 19 without evidence that androgen passes from males to females

The whole-body content of androgen (testosterone + 5 alpha-dihydrotestosterone) was significantly higher on Fetal Days 18 and 19 in male than in female rats; androgen content was equivalent in the two sexes at other fetal ages, including Days 16, 17, 20, and 21, and prior to parturition on Fetal Day 22. These results partially corroborate previous data of Weisz and Ward (Endocrinology 1980; 106:306-316), who measured testosterone in pooled plasma from rat fetuses and suggest that androgens contribute to masculine brain sexual differentiation only briefly during fetal life. No significant differences in whole-body androgen content were observed among groups of females situated in utero between 0, 1, or 2 males on each side (contiguous male model) or among groups of females with 0, 1, or 2 or more males located caudally (on the cervical side) in the same uterine horn, regardless of whether combined data from Fetal Days 17-22 or only Fetal Days 18 and 19 were considered. These results provide no evidence that androgens from males reach female fetuses in the same uterine horn.