Intrauterine position effects on steroid metabolism and steroid receptors of reproductive organs in male mice.

Mice differ in their adult reproductive characteristics as a function of whether they developed in utero between two male fetuses (2M males), which have higher testosterone levels, or between two female fetuses (0M males), which have higher estradiol levels. The present study was designed to further characterize biochemical parameters of 2M and 0M adult male mice. Activities of testicular steroidogenic enzymes, namely delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase, 17 alpha-hydroxylase, and C17,20-lyase (C21SCC P450), were measured by means of radiometric assays and HPLC fractionation of substrate and products. Activity of 5 alpha-reductase in both seminal vesicle and prostate was measured by similar techniques. Estrogen and androgen receptor concentrations, which indicate capacity to respond to steroid hormones, were also examined in the accessory sex organs. For both seminal vesicle and prostate, 5 alpha-reductase activities were approximately 60% greater in 2M males than in 0M males, indicating greater capacity to form dihydrotestosterone from testosterone in organs from 2M mice. No significant differences were found in testicular steroidogenic enzymes between 2M and 0M animals, whereas the trend for all three activities was higher for 2M males than for 0M males. While no differences were found in estrogen receptor concentrations, 0M prostates had three times the concentration of androgen receptors (occupied receptors) compared to 2M prostates. Our findings suggest that intrauterine fetal position exerts a significant influence on subsequent adult androgen metabolism and androgen responsiveness in reproductive organs of adult male mice.     

Early steroid metabolism in Xenopus laevis, Rana temporaria and Triturus vulgaris embryos.

Embryos of Xenopus laevis, Rana temporaria and Triturus vulgaris exposed to radioactive pregnenolone have been found to convert it to progesterone. Incubations with radioactive progesterone showed that it was actively metabolized by oocytes and embryos. In Xenopus incubations progesterone was converted to 5alpha-pregnane-3,20-dione, 17alpha-hydroxy-4-pregnen-3-one, 4-androstene-3,17-dione and 17alpha-20alpha-dihydroxy-4-pregne-3-one, indicating 5alpha-reductase, 17alpha-hydroxylase, 19-20-desmolase and 20alpha-hydroxylase activities. In oocytes of Triturus and Rana no evidence of 19-20-desmolase was found. In Rana oocytes were also not evidence of 17alpha-hydroxylase activity. All identified activities except 20alpha-hydroxylase were common to embryos of all three species. It is suggested that the steroid enzyme activities present in the embryos are not solely derived from the oocytes but synthentized during early development. Possible meaning of this kind of metabolism during differentiation remains open.     

Progesterone metabolism in T47Dco human breast cancer cells--II. Intracellular metabolic path of progesterone and synthetic progestins

We show here that progesterone added to the medium of proliferating T47Dco human breast cancer cells is metabolized with a half life of 2-4h. The final metabolic product, 5 alpha-pregnan-3 beta,6 alpha-diol-20-one, (P-metabolite) is released into the medium. This structure suggested that the intracellular metabolism of progesterone involves the enzymes 5 alpha-reductase, 3 beta-hydroxysteroid dehydrogenase, and 6 alpha-hydroxylase. To investigate this pathway, the cells were incubated with a variety of potential substrates. In addition to progesterone, only precursors with the 5 alpha-configuration served as substrates for the enzymes leading to P-metabolite formation. Some precursors with a 5 beta-configuration were also metabolized by T47Dco cells. This metabolism reflected activity by either 3 beta-hydroxysteroid dehydrogenase and/or 6 alpha-hydroxylase but, in contrast to progesterone metabolism, the rates were different and the products were often mixtures. In T47Dco and MCF-7 human breast tumor cells, the reduction at C-3 followed by 6 alpha-hydroxylation, appear to be the major, and possibly only, route of progesterone metabolism. In contrast, preliminary data suggest that in normal human breast epithelial cells, this is not an exclusive route. Androgens are partially subject to the same metabolic enzymes, but synthetic progestins are not metabolized by T47Dco during an 18 h incubation.     

Sexual dimorphism of growth plate prehypertrophic and hypertrophic chondrocytes in response to testosterone requires metabolism to dihydrotestosterone (DHT) by steroid 5-alpha reductase type 1

Rat costochondral growth plate chondrocytes exhibit sex-specific and cell maturation dependent responses to testosterone. Only male cells respond to testosterone, although testosterone receptors are present in both male and female cells, suggesting other mechanisms are involved. We examined the hypothesis that the sex-specific response of rat costochondral cartilage cells to testosterone requires further metabolism of the hormone to dihydrotestosterone (DHT). Resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) chondrocytes from male and female Sabra strain rats exhibited sex-specific responses to testosterone and DHT: only male cells were responsive. Testosterone and DHT treatment for 24 h caused a comparable dose-dependent increase in [3H]-thymidine incorporation in quiescent preconfluent cultures of male GC cells, and a comparable increase in alkaline phosphatase specific activity in confluent cultures. RC cells responded in a differential manner to testosterone and DHT. Testosterone decreased DNA synthesis in male RC cells but DHT had no effect and alkaline phosphatase specific activity of male RC cells was unaffected by either hormone. Inhibition of steroid 5alpha-reductase activity with finasteride (1, 5, or 10 microg/ml), reduced the response of male GC cells to testosterone in a dose-dependent manner, indicating that metabolism to DHT was required. RT-PCR showed that both male and female cells expressed mRNAs for steroid 5alpha-reductase type 1 but lacked mRNAs for the type 2 form of the enzyme. Male cells also exhibited 5alpha-reductase activity but activity of this enzyme was undetectable in female cells. These observations show that sex-specific responses of rat growth zone chondrocytes to testosterone requires the further metabolism of the hormone to DHT and that the effect of DHT in the male growth plate is maturation-state dependent. Failure of female chondrocytes to respond to testosterone may reflect differences in testosterone metabolism, since these cells possess greater ability to aromatize the hormone to estradiol.     

3H]Oestradiol metabolism by 18-day rat interstitial cells in culture and the effect of FSH: presence of 16 alpha-hydroxylase

The metabolism of [3H]oestradiol by interstitial cells in culture prepared from 18-day old rat testes was investigated. Interstitial cells were able to convert [3H]oestradiol to [3H]oestriol as confirmed by recrystallization of oestriol to constant specific activity from samples containing cells and not from controls. This demonstrated for the first time the presence of 16 alpha-hydroxylase in rat testicular interstitial cells. The effect of in vitro FSH treatment on the cells in culture was also investigated. FSH failed to affect 16 alpha-hydroxylase activity since we could not demonstrate a significant difference between treated and untreated preparations. The 16 alpha-hydroxylation of phenolic steroids is widely regarded as the major pathway of oestrogen metabolism in mammals. This metabolic step significantly reduces the biological activity of oestradiol. The presence of 16 alpha-hydroxylase in interstitial cells suggests that it may play a role in inactivating the oestradiol that is produced in the Leydig cells and thus prevent its intracellular accumulation. Such activity may conceivably play a role in the overall local "fine tuning" of androgen biosynthesis.     

Steroid hormone production in testis, ovary, and adrenal gland of immature rats irradiated in utero with 60Co

Pregnant rats received whole-body irradiation at 20 days of gestation with 2.6 Gy lambda rays from a 60Co source. Endocrinological effects before maturation were studied using testes and adrenal glands obtained from male offspring and ovaries from female offspring irradiated in utero. Seminiferous tubules of the irradiated male offspring were remarkably atrophied with free germinal epithelium and containing only Sertoli cells. Female offspring also had atrophied ovaries. Testicular tissue obtained from intact and 60Co-irradiated rats was incubated with 14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, and androstenedione as a substrate. Intermediates for androgen production and catabolic metabolites were isolated after the incubation. The amounts of these metabolites produced by the irradiated testes were low in comparison with the control. The activities of delta 5-3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, C17,20-lyase, and delta 4-5 alpha-reductase in the irradiated testes were 30-40% of those in nonirradiated testes. Also, the activities of 17 beta- and 20 alpha-hydroxysteroid dehydrogenases were 72 and 52% of the control, respectively. In adrenal glands, the 21-hydroxylase activity of the irradiated animals was 38% of the control, but the delta 5-3 beta-hydroxysteroid dehydrogenase activity was comparable to that of the control. On the other hand, the activity of delta 5-3 beta-hydroxysteroid dehydrogenase of the irradiated ovary was only 19% of the control. These results suggest that 60Co irradiation of the fetus in utero markedly affects the production of steroid hormones in testes, ovaries, and adrenal glands after birth.     

Some properties of progesterone 5 alpha-reductase solubilized from rat liver microsomes

The experiments described in this paper were undertaken to examine the requirements of NADPH-cytochrome c reductase and phosphatidylcholine for hepatic steroid 5 alpha-reduction, previously proposed by Golf and Graef (Golf, S. W. and Graef, V. (1978) J. Steroid Biochem. 9, 369-371). To determine how NADPH-cytochrome c reductase participates in hepatic 5 alpha-reductase activity, antibodies against the purified NADPH-cytochrome c reductase were added to 5 alpha-reductase preparation solubilized from rat liver microsomes. Whereas both NADPH-cytochrome c reductase and progesterone 16 alpha-hydroxylase in the preparation were inhibited by the antiserum, the inhibitory effect on 5 alpha-reductase activity was not observed. In addition, chromatography of the polyethylene glycol fraction active in 5 alpha-reduction on DEAE-cellulose resulted in the complete separation of 5 alpha-reductase activity from NADPH-cytochrome c reductase activity. Unlike NADPH-cytochrome c reductase, phosphatidylcholine increased the activity of the partially purified 5 alpha-reductase about 2.5 fold. Phosphatidylserine also enhanced the activity to an extent identical for phosphatidylcholine. Phosphatidic acid and lysophosphatidylcholine were stimulatory to lesser extents.     

Adipose tissue intracrinology: potential importance of local androgen/estrogen metabolism in the regulation of adiposity.

The present article summarizes some of the studies available on steroid hormone conversion through the specific expression of steroidogenic enzymes in adipose tissue (adipose tissue intracrinology) and discusses the potential impact of local adipose tissue steroid metabolism on the regulation of adipocyte function and other metabolic parameters. Several studies have demonstrated significant steroid hormone uptake and conversion by adipose tissues from various body sites and in various cell fractions. Activities and/or mRNAs of aromatase, 3beta-hydroxysteroid dehydrogenase (HSD), 3alpha-HSD, 11beta-HSD, 17beta-HSD, 7alpha-hydroxylase, 17alpha-hydroxylase, 5alpha-reductase and UDP-glucuronosyltransferase 2B15 have been detected in adipose tissue or adipose cells. These studies have demonstrated potentially important roles for these enzymes in obesity, central fat accumulation, and the metabolic syndrome. Future studies on adipose tissue intracrinology will contribute further to our understanding of steroid action in adipocytes.     

4-pregnene-3-one-20 beta-carboxaldehyde: a potent inhibitor of 17 alpha-hydroxylase/C17,20-lyase and of 5 alpha-reductase.

The pregnene derivative, 4-pregnene-3-one-20 beta-carboxaldehyde (22-A) was evaluated as an inhibitor of 17 alpha-hydroxylase/C17,20-lyase in rat testicular microsomes and of 5 alpha-reductase in human prostatic homogenates. The effect of the compound in vivo was studied in adult male rats. The 22-A demonstrated potent and competitive inhibition of 17 alpha-hydroxylase and C17,20-lyase with Ki values 8.48 and 0.41 microM, respectively, significantly below the Km values for these two enzymes (33.75 and 4.55 microM). This compound also showed potent inhibition of 5 alpha-reductase with a Ki value of 15.6 nM (Km for this enzyme is 50 nM). By comparison, ketoconazole, a currently studied 17 alpha-hydroxylase/C17,20-lyase inhibitor for the treatment of prostatic cancer, showed less potent inhibition of 17 alpha-hydroxylase (Ki 39.5 microM) and C17,20-lyase (Ki 3.6 microM) and did not inhibit 5 alpha-reductase. Progesterone which has been reported to inhibit the 17 alpha-hydroxylase/C17,20-lyase, did not significantly reduce the production of testosterone by rat testes in vitro in comparison to controls, while the same concentration of 22-A demonstrated a 42% reduction of testosterone biosynthesis. When the adult male rats were injected s.c. with 22-A at 50 mg/day/kg for a 2 week period, the testosterone concentrations in the rat sera were significantly lower than control values (P less than 0.05), whereas serum corticosterone levels did not change. These results suggest that 22-A is a selective potent inhibitor for 17 alpha-hydroxylase and C17,20-lyase, but is more potent for the C17,20-lyase. The compound also inhibits 5 alpha-reductase, and therefore may reduce biosynthesis of testosterone and dihydrotestosterone effectively. Thus, 22-A may be useful in the treatment of problems associated with the androgen excess and prostatic cancer.     

Properties of a 4-ene-3-ketosteroid-5 alpha-reductase in cell extracts of the intestinal anaerobe, Eubacterium sp. strain 144

When Eubacterium sp. 144 was grown in the presence of progesterone, extracts of these cells contained a 4-ene-3-ketosteroid-5 alpha-reductase (5 alpha-reductase). No evidence for the presence of a 5 beta-steroid-reductase or a 5 alpha to 5 beta-steroid-isomerase was found. 5 alpha-Reductase activity was dependent on reduced methyl viologen as the electron donor and this could be generated biologically by adding pyruvate or H2 to cell extracts or chemically by adding sodium dithionite. NADH or NADPH with or without flavin nucleotides were not electron donors for 5 alpha-reductase. Most of the 5 alpha-reductase activity (60-65%) of crude extracts was located in the membrane fraction and the enzyme was solubilized by treatment with 1% Triton X-100. Optimum 5 alpha-reductase activity occurred at pH 7.0-7.5 in potassium phosphate buffer but was stimulated by Tris-HCl buffer (pH 8.0-9.0). 5 alpha-Reductase activity was highest at 10% (v/v) methanol and was progressively inhibited by higher methanol concentrations. Sulfhydryl reagents strongly inhibited 5 alpha-reductase but the enzyme was not affected by other metabolic inhibitors. Extracts prepared from cells induced with 16-dehydroprogesterone and grown without hemin contained 5 alpha-reductase and 16-dehydroprogesterone-reductase activities equivalent to those found in extracts of induced cells grown with hemin. This indicates that hemin is not required for the synthesis of active steroid double bond-reductases in strain 144.     

Inhibition of steroid 5 alpha-reductase and its effects on testosterone hydroxylation by rat liver microsomal cytochrome P-450

It has been shown previously that liver microsomal steroid 5 alpha-reductase activity increases with age in female but not male rats, which coincides with a female-specific, age-dependent decline in the cytochrome P-450-dependent oxidation of testosterone to 1 beta-, 2 alpha-, 2 beta-, 6 alpha-, 6 beta-, 7 alpha-, 15 beta-, 16 alpha-, 16 beta-, and 18-hydroxytestosterone and androstenedione. To determine whether the increase in steroid 5 alpha-reductase activity is responsible for the decrease in testosterone oxidation, we have examined the effects of the steroid 5 alpha-reductase inhibitor, 4-MA (17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one), on the pathways of testosterone oxidation catalyzed by rat liver microsomes. We have also determined which hydroxytestosterone metabolites are substrates for steroid 5 alpha-reductase. At concentrations of 0.1 to 10 microM, 4-MA completely inhibited steroid 5 alpha-reductase activity without inhibiting the pathways of testosterone oxidation catalyzed by liver microsomes from rats of different age and sex, and from rats induced with phenobarbital or pregnenolone-16 alpha-carbonitrile. 4-MA (10 microM) had little or no effect on the oxidation of testosterone catalyzed by liver microsomes from mature male rats (which have low steroid 5 alpha-reductase activity). In contrast, the hydroxylated testosterone metabolites formed by liver microsomes from mature female rats (which have high steroid 5 alpha-reductase activity) accumulated to a much greater extent in the presence of 4-MA. Evidence is presented that 4-MA increases the accumulation of hydroxytestosterones by two mechanisms. First, 4-MA inhibited the 5 alpha-reduction of those metabolites (such as 6 beta-hydroxytestosterone) that were found to be excellent substrates for steroid 5 alpha-reductase. In the absence of 4-MA, these metabolites eventually disappeared from incubations containing liver microsomes from mature female rats. Second, 4-MA inhibited the formation of 5 alpha-dihydrotestosterone, which otherwise competed with testosterone for oxidation by cytochrome P-450. This second mechanism explains why 4-MA increased the accumulation of metabolites (such as 7 alpha-hydroxytestosterone) that were found to be poor substrates for steroid 5 alpha-reductase. Despite its marked effect on the accumulation of hydroxylated testosterone metabolites, 4-MA had no effect on their initial rate of formation by liver microsomes from either male or female rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of flutamide on 5 alpha-reductases, 5 beta-reductases, and 3-hydroxysteroid dehydrogenases in rat liver

Flutamide (0.5 mM) decreased in vitro the activity of NADH-5 alpha-reductase (substrate testosterone) in liver homogenate of male and female rats, whereas no change of activity of NADPH-5 alpha-reductase was observed. NADH- and NADPH-5 beta-reductase activity increased only in liver of female, but not of male rats. NAD+-3 beta-hydroxysteroid dehydrogenase and NAD+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 alpha-dihydro-testosterone) in liver homogenate from female rats were inhibited by flutamide (0.5 mM), whereas the activity of NADP+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 alpha-dihydrotestosterone) and of NAD+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 beta-dihydrotestosterone) increased in presence of flutamide. The activity of NADH- and NADPH-5 alpha-reductase decreased after flutamide administration to female rats at a dose of 5 mg per day for 7 days.     

Steroidogenesis in the testes and the adrenals of adult male rats after gamma-irradiation in utero at late pregnancy

Pregnant rats were irradiated with 2.1 Gy gamma-ray of 60Co at day 20 of gestation. Seventy days after birth, the body weight of the fetally irradiated male pups was significantly lower than the control. The testes, ventral prostates and seminal vesicles were atrophied by irradiation, whereas no decreased weight of the adrenals was observed. Histological examination of the testes of the irradiated rats revealed a complete disappearance of germinal cells. Sertoli cells and Leydig cells appeared normal, and no apparent histological difference was observed in the adrenals between the control and the irradiated rats. Activities of microsomal delta 5-3 beta-hydroxysteroid dehydrogenase (HSD) + isomerase, 17 alpha-hydroxylase/C17,20-lyase, 17 beta-HSD and 7 alpha-hydroxylase per pair of testes were decreased in the irradiated rats (36-86% of the control). In contrast, no decreased activity of 20 alpha-HSD in the cytosol fraction was observed by irradiation. No decreased activity of adrenocortical enzymes, such as delta 5-3 beta-HSD + isomerase, 21-hydroxylase, 11 beta-/18-hydroxylase and 5 alpha-reductase, was also observed in the irradiated group. Concentrations of LH, FSH, TSH, prolactin, testosterone, progesterone and aldosterone in serum were measured by radioimmunoassay. Only the FSH concentration was significantly increased by the irradiation, while no difference was found in the concentration of other hormones. It was concluded that irreversible damage was induced in spermatogenesis and androgen production by the fetal irradiation, whereas corticoidogenesis was not affected.     

Metabolism of (3H) 5 alpha-androstane-3 alpha, 17 beta-diol by cultures of isolated rat sertoli cells and the effect of LH and FSH

Sertoli cells from immature rats metabolized (3H) 5 alpha-androstane-3 alpha, 17 beta-diol to (3H) 5 alpha-androstane-3 alpha, 16 alpha, 17 beta-triol and (3H) 3 alpha-hydroxy-5 alpha-androstan-17-one. This is the first report of 16 alpha-hydroxylation of 5 alpha-reduced androgens in the testis. FSH significantly stimulated 16 alpha-hydroxylation while LH significantly decreased this activity. 3 alpha-Hydroxy-5 alpha-androstan-17-one was the major metabolite formed and its production was significantly increased in the presence of both LH and FSH, although FSH stimulation was significantly more than LH. The possible role of 16 alpha-hydroxylase in androgen metabolism by immature rat Sertoli cells is discussed.     

Cholesterol 7 alpha-hydroxylase in isolated rat liver cells.

1. The activity of cholesterol 7 alpha-hydroxylase found in the 10000 x g supernatant prepared from isolated rat liver cells was comparable to that found with microsomal fractions from whole liver. 2. The activity of cholesterol 7 alpha-hydroxylase from cells prepared from livers of rats fed the bile salt sequestering agent cholestyramine was 2--3 fold higher than the activity of this enzyme found in cells isolated from animals on a control diet. 3. On incubation of hepatocytes in a suitable medium at 37 degrees C, cholesterol 7 alpha-hydroxylase activity declined to about 50% of its original value after three hours despite the fact that the cells retained a high level of viability over 5--6 h as measured by various sensitive criteria. 4. The decrease in cholesterol 7 alpha-hydroxylase activity was observed whether cholestyramine was included in the diet or excluded from the diet of the animals used as sources of the liver cells. 5. The change in cholesterol 7 alpha-hydroxylase activity seen on incubation of the cells was not affected by including in the incubation medium additional nutrients such as amino acids, the glucocorticoid cortisol, phospholipid dispersions, or sodium taurocholate. 6. Changing the incubation medium in which the cells were suspended at regular intervals during the three-hour experiments failed to prevent this decline in the cholesterol 7 alpha-hydroxylase activity during the incubation of these cells. 7. Although isolated liver cells have been shown to lose glutathione on incubation, addition of physiological levels of this compound did not prevent the decline in cholesterol 7 alpha-hydroxylase activity. 8. Cycloheximide addition to the incubation medium accelerated the decrease in cholesterol 7 alpha-hydroxylase activity. This suggests that some protein synthesis associated with cholesterol 7 alpha-hydroxylase activity occurs during the incubation and inhibition of such protein synthesis accelerated the decrease in this enzyme activity. 9. The cytochrome P-450 content of the 10000 x g supernatant prepared from hepatocytes declined slowly to about 65% of its original value after four hours of incubation at 37 degrees C. This decline in the 10000 x g supernatant cytochrome P-450 content may partly explain the observed loss of cholesterol 7 alpha-hydroxylase activity during incubations in vitro. 10. Isolated hepatocytes rapidly take up radioactively labelled sodium cholate. Subsequent excretion of the radioactivity was also very rapid even in the presence of large amounts of this bile salt in the medium.     

Assessment of porcine and human 16-ene-synthase, a third activity of P450c17, in the formation of an androstenol precursor. Role of recombinant cytochrome b5 and P450 reductase.

Recently, we have shown that the biosynthesis of androstenol, a potential endogenous ligand for the orphan receptors constitutive androstane receptor and pregnane-X-receptor, requires the presence of enzymes of the steroidogenic pathway, such as 3 beta-hydroxysteroid dehydrogenase, 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase. In this report, we examine at the molecular level whether the enzyme 17 alpha-hydroxylase/17,20-lyase (P450c17), which possesses dual 17 alpha-hydroxylase and 17,20-lyase activities and catalyzes the production of precursors for glucocorticoids and sex steroids, is also able to catalyze the formation of a third class of active steroids, 16-ene steroids (including androstenol). The role of components of the P450 complex is also assessed. We transfected human embryonic kidney (HEK-293) cells with various amounts of vectors expressing P450c17, NADPH-cytochrome P450 reductase, and cytochrome b5. Our results showed that P450c17 possesses a 16-ene-synthase activity able to transform pregnenolone into 5,16-androstadien-3 beta-ol, without the formation of the precursor 17-hydroxypregnenolone. Cytochrome b5 has a much stronger effect on the 16-ene-synthase activity than on the 17 alpha-hydroxylase/17,20-lyase activities. On the other hand, P450reductase has a drastic effect on the latter, but a negligible one on 5,16-androstadien-3 beta-ol synthesis. Our results therefore demonstrate that human P450c17, as other enzymes of the classical steroidogenic pathway, is involved in the biosynthetic pathway leading to the formation of androstenol.     

5 alpha-reductase activity in rat adipose tissue

We measured the 5 alpha-reductase activity in isolated cell preparations of rat adipose tissue using the formation of [3H]dihydrotestosterone from [3H]testosterone as an endpoint. Stromal cells were prepared from the epididymal fat pad, perinephric fat, and subcutaneous fat of male rats and from perinephric fat of female rats. Adipocytes were prepared from the epididymal fat pad and perinephric fat of male rats. Stromal cells from the epididymal fat pad and perinephric fat contained greater 5 alpha-reductase activity than did the adipocytes from these depots. Stromal cells from the epididymal fat pad contained greater activity than those from perinephric and subcutaneous depots. Perinephric stromal cells from female rats were slightly more active than those from male rats. Estradiol (10(-8) M), when added to the medium, caused a 90% decrease in 5 alpha-reductase activity. Aromatase activity was minimal, several orders of magnitude less than 5 alpha-reductase activity in each tissue studied.     

Hormonal regulation of activities of 4-ene-5 beta- and 5 alpha-reductases and 17 beta-hydroxy-dehydrogenase in immature golden hamster testis

We have reported [1-3] in immature golden hamster testis that 5 beta-reductase is localized in the tubular nongerm cells, while 5 alpha-reductase is present in the interstitial tissue and that the 17 beta-hydroxy-dehydrogenase activity is found predominantly in the tubular nongerm cells. Hormonal regulation of these enzyme activities was examined in the present study. Male golden hamsters were hypophysectomized on day 22 after birth. The hypophysectomized hamsters in groups of 3-8 were injected daily with 10 micrograms NIH-LH-S19, 50 micrograms NIAMD-Rat-FSH-B-1, 8 or 16 micrograms NIAMD-oFSH-13, 8 micrograms NIAMD-oFSH-13 plus 5 or 10 micrograms NIH-LH-S19, 1 mg testosterone propionate or saline for 5 days starting from day 23. Testicular homogenates of the treated hamsters and intact hamsters on day 28 were incubated with [14C]4-androstene-3,17-dione and NADPH, and enzyme activity (nmol/testes/h) was estimated. The activities of 5 beta- and 5 alpha-reductases and 17 beta-hydroxy-dehydrogenase decreased significantly 6 days after hypophysectomy. In the hypophysectomized hamster testis, a distinct response to FSH but not to LH in the activities of 5 beta-reductase and 17 beta-hydroxy-dehydrogenase was found. The injection of LH in addition to FSH showed no significant additive effects on these enzyme activities. The 5 alpha-reductase activity was stimulated significantly by LH plus FSH but not by LH alone, FSH alone or androgen. These results show that 5 beta-reduction of 4-ene-3-ketosteroids takes place in the Sertoli cells under the influence of FSH while 5 alpha-reduction occurs in the interstitial cells under the influence of LH and FSH in immature hamster testis.     

Pituitary progestin-metabolizing enzyme activities in the aged female rat.

Progesterone 5 alpha-reductase activity and 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) enzymic activities (NADH-linked and NADPH-linked) were measured in anterior pituitaries (AP) from aged female rats during three stages of reproductive senescence (constant estrus: CE; repeated pseudopregnancies: PSP; and anestrus: AN). To assess ovarian influence on these enzymes during these stages of reproductive aging, we also determined enzyme levels from ovariectomized rats from each stage treated with estrogen or vehicle. Progesterone 5 alpha-reductase and NADH-linked 3 alpha-HSOR activities were 2-fold higher in pituitaries of CE rats as compared to those of PSP and AN rats. NADPH-linked 3 alpha-HSOR levels did not differ among the three stages. All three enzyme levels were elevated 2- to 5-fold as compared to the corresponding enzyme levels from young cycling rats. After ovariectomy (10 days), 5 alpha-reductase activity in PSP and AN rats was elevated 3- to 4-fold relative to mean levels in intact PSP and AN rats. Ovariectomy had no effect on 5 alpha-reductase levels in CE rats. Under similar conditions, young cycling rats exhibit a 10-12-fold increase. Treatment of ovariectomized PSP and AN rats for 3 days with estradiol benzoate (10 micrograms/day) restored 5 alpha-reductase levels. Ovariectomy had no effect on the NADPH-linked 3 alpha-HSOR levels in CE, PSP or AN animals which is similar to that observed with young rats. Ovariectomy also had no effect on the NADH-linked 3 alpha-HSOR levels except for the CE group. The ovariectomized CE rats exhibited reduced pituitary NADH-linked 3 alpha-HSOR levels (30%). In contrast, young rats exhibit elevated pituitary NADH-linked 3 alpha-HSOR levels after ovariectomy (4- to 5-fold). These changes suggest the possibility that altered processing of progesterone and its 5 alpha- and 3 alpha-reduced products may be one means by which the effectiveness of progesterone is reduced during aging. The results also suggest an altered ovarian role in the regulation of these enzymes.     

Steroid 5 alpha-reductase activity in endothelial cells from human umbilical cord vessels

The metabolism of radiolabeled progesterone and androstenedione was evaluated in endothelial cells from human umbilical cord vein and arteries maintained in culture. The predominant metabolite of progesterone was 5 alpha-pregnane-3,20-dione and that of androstenedione was 5 alpha-androstane-3,17-dione. Thus, the major pathway of progesterone and androstenedione metabolism within these cells is via steroid 5 alpha-reductase. The rate of formation of 5 alpha-pregnane-3,20-dione from progesterone by venous endothelial cells was linear with incubation time up to 4 h and with cell number up to 1.6 X 10(6) cells/ml. The apparent Km of 5 alpha-reductase for progesterone was 0.4 microM; and, the Vmax was 55 pmol 5 alpha-pregnane-3,20-dione formed/mg protein X h. The rate of 5 alpha-androstane-3,17-dione formation from androstenedione also was linear with incubation time up to 4 h. In addition to 5 alpha-androstane-3,17-dione, the metabolism of androstenedione by either venous or arterial cells resulted in the formation of various minor metabolites, including testosterone and 5 alpha-reduced steroids, viz. 5 alpha-dihydrotestosterone, androsterone, isoandrosterone, 5 alpha-androstane-3 alpha, 17 beta-diol, and 5 alpha-androstane-3 beta, 17 beta-diol. Estrogens (i.e. estradiol-17 beta and estrone) were not detected as products of androstenedione metabolism. The formation of these metabolites are indicative that the steroid-metabolizing enzymes present in endothelial cells are: 5 alpha-reductase, 17 beta-hydroxysteroid oxidoreductase, 3 alpha-hydroxysteroid oxidoreductase, and 3 beta-hydroxysteroid oxidoreductase.