Steroid 5α-reductase Activity Research Articles

Indole Alkaloids from Hosta plantaginea and Inhibition of Steroid 5α-Reductase Activities In Vitro

A new indole alkaloid (1), together with eight known indole alkaloid derivatives (2–9), was isolated from Hosta plantaginea for the first time. The structures of compounds 1–9 were elucidated on the basis of comprehensive spectroscopic analyses and references. Compounds 1–9 were evaluated for their inhibition of steroid 5α-reductase activity in vitro. Among them, compounds 1 and 2 showed important inhibition of steroid 5α-reductase activities.

Quintaquinone, a Merosesquiterpene from the Yellow Sponge Verongula cf. rigida Esper.

A chemical investigation of the sponge Verongula cf. rigida led to the isolation of 13 merosesquiterpenes, among which quintaquinone (2), 5-epi-nakijiquinone L (3), and 3-farnesyl-2-hydroxy-5-methoxyquinone (4) were isolated and reported here for the first time. Particularly, compound 2 is the first member of merosesquiterpenes with a polyketide side chain substituted on C-19. All of the isolated compounds were examined for steroid 5α-reductase inhibitory activity. Cyclospongiaquinone 1 (5) showed a strong activity in the same range as that of standard finasteride.

Synthesis, Biological Evaluation and Molecular Docking of Avicequinone C Analogues as Potential Steroid 5α-Reductase Inhibitors.

Avicequinone C (5a), a furanonaphthoquinone isolated from the Thai mangrove Avicennia marina has been shown previously to have interesting steroid 5α-reductase type 1 inhibitory activity. In this study, a series of avicequinone C analogues containing furanonaphthoquinone with different degrees of saturation and substituents at the furan ring were synthesized. The resulting synthetic avicequinone C and analogues (5a-f) along with some related compounds including 2,5-dihydroxy-1,4-benzoquinone (6) and natural naphthoquinones such as lawsone (7a) and lapachol (7b) were evaluated for their in vitro cell viability and steroid 5α-reductase type 1 inhibitory activities using the cultured cell line of human keratinocytes (HaCaT). This cell-based bioassay was performed based on a direct detection of the enzymatic product dihydrotestosterone (2) by using a non-radioactive high performance thin layer chromatography (HPTLC) method. Among the furanonaphthoquinones in this series, 5e having a propionic substituent at furan ring possessed approximately 22-fold more potent than the original isolated compound 5a. However, the compounds without furan motif such as 6, 7a and b could not inhibit the activity of steroid 5α-reductase. Molecular docking results of the in silico three-dimensional steroid 5α-reductase type 1-reduced nicotinamide adenine dinucleotide phosphate (NADPH) binary complex was performed via AutoDock Vina and it illustrated that the furanonaphthoquinone moiety and the substituent at furan ring might play a key role as pharmacophores for the steroid 5α-reductase inhibitory activity.

Hamster SRD5A3 lacks steroid 5α-reductase activity in vitro

According to current knowledge, two steroid 5α-reductases, designated type 1 (SRD5A1) and type 2 (SRD5A2), are present in all species examined to date. These isozymes play a central role in steroid hormone physiology by catalyzing the reduction of 3-keto-4-ene-steroids into more active 5α-reduced derivatives, including the conversion of testosterone (T) to dihydrotestosterone (DHT). A third 5α-reductase (SRD5A3, -type 3), which is overexpressed in hormone-refractory prostate cancer cells, has been identified; however, its enzymatic characteristics are practically unknown. Here, we isolated a cDNA encoding hamster Srd5a3 (hSrd5a3) and performed functional metabolic assays to investigate its biochemical properties. The cloned cDNA encodes a 330 amino acid protein that is 87% identical to the homologous protein in mice and 78% to that in humans. However, hSrd5a3 exhibits low sequence homology with its counterparts hSrd5a1 (19%) and hSrd5a2 (17%). A fusion protein consisting of hSrd5a3 and green fluorescent protein provided evidence for cytoplasmic localization in transfected mammalian cells. Real-time PCR analysis revealed that, Srd5a3 mRNA was present in nearly all hamster tissues, with high expression in the cerebellum, Harderian gland and testis. Functional assays expressing hSrd5a3 cDNA in HEK-293 cells revealed that this isozyme is unable to reduce T into DHT. Further expression assays confirmed that similar to testosterone, progesterone, androstenedione and corticosterone are not reduced by hSrd5a3 or human SRD5A3. Together, these results indicate that hSrd5a3 lacks the catalytic activity to transform 3-keto-4-ene-compounds; therefore 5α-reductase type 3 may not be involved in 5α-reduction of steroids.

Spectrophotometric Method for the Assay of Steroid 5α-Reductase Activity of Rat Liver and Prostate Microsomes

A simple spectrophotometric method for the assay of steroid 5α-reductase (5α-SR) was developed in which 5α-dihydrotestosterone (5α-DHT) and 5α-androstane-3α,17β-diol (5α-diol), metabolites formed in the NADPH-dependent reduction of testosterone with enzyme sources of 5α-SR, were measured by enzymatic cycling using 3α-hydroxysteroid dehydrogenase in the presence of excess thionicotinamide-adenine dinucleotide (thio-NAD) and NADH. It was found that 5α-SR activity was proportional to the accumulated thio-NADH having an absorption maximum at 400 nm. Because of the high cycling rate (> 600 cycle per min) and no interference from testosterone, enzymatic cycling can determine the sum of 5α-DHT and 5α-diol at the picomole level without separation from excess testosterone. The present method was readily applicable to the assay of 5α-SR activity of rat liver and prostate microsomes as well as to the assay of inhibitory activity of finasteride, a synthetic inhibitor of 5α-SR.

Gas chromatography–mass spectrometry profiling of steroids in urine of patients with acute intermittent porphyria

ObjectiveAcute intermittent porphyria (AIP) is an autosomal dominant disease that results from a deficiency of hydroxymethylbilane synthase, the third enzyme of the heme biosynthetic pathway. AIP carriers may present acute neurovisceral attacks with hepatic overproduction of heme-precursors. In some patients, remission of the acute symptoms leads to long-term hepatic metabolic abnormalities. In this study, gas chromatography–mass spectrometry (GC/MS) was used to investigate urinary steroid metabolome of AIP patients. Design and methodsSteroid profiling in urine was performed in a group of AIP patients with biochemically active disease (n=22) and healthy controls (n=20). Five asymptomatic AIP family carriers were also studied. Commonly used ratios for the evaluation of disturbances in the steroid metabolism were calculated. ResultsWe found that etiocholanolone/androsterone and tetrahydrocortisol/5α-tetrahydrocortisol (THF/5α-THF) metabolic ratios were significantly increased in the urine of AIP patients compared to controls (2.3±0.3 vs 0.8±0.1; p<0.001 and 2.9±0.7 vs 0.9±0.1; p<0.01). The (THF+5α-THF)/tetrahydrocortisone ratio was reduced among the AIP patients (p<0.01). Quantification of the steroid absolute concentrations showed that these variations were due to a decrease of the 5α metabolites. Other ratios, like cortisol/cortisone and 6β-hydroxycortisol/cortisol in the free steroid fraction did not show differences between patients and controls. All ratios were normal among the family carriers. ConclusionA significant number of AIP patients present a basal decrease of steroid 5α-reductase activity in the liver. The deficiency may be related to malnutrition and hepatic energy misbalance associated with active AIP. Urinary steroid profiling by GC/MS may be a valuable tool to assess hepatic metabolome in AIP.

Human castration resistant prostate cancer rather prefer to decreased 5α-reductase activity

Physiologically relevant steroid 5α-reductase (SRD5A) activity that is essential for dihydrotestosterone (DHT) biosynthesis in human castration-resistant prostate cancer (CRPC) has not been fully characterized yet. In this study to ascertain the potential SRD5A activity, we cultured two human CRPC cell lines, C4-2 and C4-2AT6, with the steroid precursor: 13C-[2,3,4]-androstenedione (13C-Adione), and analyzed the sequential biosynthesis of 13C-[2,3,4]-testosterone (13C-T) and 13C-[2,3,4]-DHT (13C-DHT) by liquid chromatography/mass spectrometry (LC/MS/MS). The 13C-DHT/13C-T concentration ratio detected by LC/MS/MS in C4-2AT6 cells appeared to reflect the SRD5A activity. The ratio in C4-2AT6 was significantly lower than that in C4-2. An increased concentration of DHT did not have a positive effect on cell proliferation, rather it exhibited inhibitory effects. 5α-reductase inhibitors did not have any inhibitory effect at clinically achievable concentrations. These results indicate that CRPC cells may have an unknown regulation system to protect themselves from an androgenic suppressive effect mediated by SRD5A activity.

Steroid 5α-reductase inhibitory activity of condensed tannins from woody plants

In humans, steroid 5α-reductase is involved in the development of benign prostatic hyperplasia (BPH). We tested the steroid 5α-reductase inhibitory activity of the 70% acetone extract of woody plants. The tannin polymer prepared from the 70% acetone extract showed the highest steroid 5α-reductase inhibitory activity. Furthermore, the steroid 5α-reductase inhibitory activity of tannins was not affected by the addition of superoxide dismutase or catalase. It was concluded that the steroid 5α-reductase inhibitory activity of condensed tannin was caused by binding to the steroid 5α-reductase, rather than from the peroxide/superoxide produced by tannins. The tannins, prepared from tree barks, with potential for steroid 5α-reductase inhibitory activity might be advantageous in therapy for steroid 5α-reductase diseases such as BPH or prostate cancer.

In vitro effect of D-004, a lipid extract of the fruit of the cuban royal palm ( Roystonea regia), on prostate steroid 5α-reductase activity

Background: D-004, a lipid extract of the fruit of the Cuban royal palm ( Roystonea regia), has been found to reduce prostatic hyperplasia (PH) induced with testosterone (T), but not PH induced with dihydrotestosterone (DHT), in rodents, suggesting the inhibition of prostate 5α-reductase activity. Objectives: The aims of this study were to assess whether D-004 inhibits prostate 5α-reductase activity in vitro and to examine the effects of D-004 on enzyme kinetics. Methods: This experimental study was conducted at the Pharmacology Department, Center of Natural Products, National Center for Scientific Research, Havana, Cuba. Soluble rat prostate preparations were used as the source of 5α-reductase, and ( 3H)-DHT production was measured to determine prostate 5α-reductase activity. Cell-free rat prostate homogenates were pre-incubated with carboxymethyl cellulose 2% alone (control tubes) or D-004 (0.24–125 μg/mL) suspended in the vehicle (treated tubes) for 10 minutes prior to adding the labeled substrate ( 3H)-T Once the reaction was stopped, sterols were extracted with chloroform and aliquots were applied on silica gel plates developed in benzene-acetone (4:1, v/v). Areas containing DHT were scraped and radioactivity was counted. The median inhibitory concentration (IC 50) was determined by measuring the conversion of T to DHT The apparent Michaelis-Menten constant (K m) and V max values before and after adding D-004 were determined in kinetic studies using labeled T (0.5-25 μmol/L). Results: Compared with controls, D-004 significantly and dose-dependently inhibited the enzymatic reaction at doses of 1.95 to 125.0 μg/mL) (all, P < 0.05). The IC 50 of D-004 required to inhibit 5a-reductase activity was 2.25 μg/mL. Enzyme inhibition was noncompetitive, since D-004 lowered the V max from 15.3 to 10.0 nmol DHT/min · mg −1 protein, while the K m (4.54 μmol/L) was almost unaffected. Conclusions: D-004 dose-dependently and noncompetitively inhibited in vitro 5α-reductase activity in soluble fractions of rat prostate. Although the extent of the maximal inhibition was high and the value of IC 50 was low, the relevance of such inhibition requires further study in vivo.

Site-directed mutagenesis studies of rat steroid 5α-reductase (isozyme-1): mutation of residues in the cofactor binding and C-terminal regions

We have investigated the roles of highly conserved glycine (G175, G185), negatively charged (E188, D165) and histidine residues (H233, H237) in rat steroid 5α-reductase (isozyme-1), on NADPH, testosterone (T) binding and enzyme activity. The mutations G175R and G175S result in a two- to threefold increase in K m NADPH and an approximately fourfold decrease in the V max with no change in K m T. The mutation G185W resulted in a fivefold decrease in K m NADPH and an eightfold decrease in V max, with no change in K m T, whereas the mutations E188Q and D165N both resulted in inactive enzyme. Steady-state kinetic measurements showed that the mutation H233R resulted in an approximately 40-fold decrease in V max, an approximately 20-fold increase in K m T and no alteration in K m NADPH, whereas the mutation H237R resulted in virtually inactive enzyme. The results suggest that the conserved glycines are not essential for cofactor binding and activity, and that the negatively charged residues may contribute to enzyme stability, whereas the C-terminal histidines appear to be involved in substrate binding and catalytic activity.

In vitro modulation of steroid 5α-reductase activity by phospholipases in epithelium and stroma of human benign prostatic hyperplasia

Membrane components, such as phospholipids, play an important role in the regulation of prostatic 5α-reductase activity. To describe in more detail the impact of such regulation on 5α-reductase activity, epithelial and stromal cell homogenates of human BPH were treated with phospholipases to specifically alter the structure of cellular phospholipid components. Phospholipase A 2 (PLA 2) was used to alter the structure of the nonpolar, hydrophobic region of the membrane bilayer. Various types of phospholipase C (PLC) affect the polar, hydrophilic region of phospholipids. In epithelium and stroma, 5α-reductase activity was dose-dependently inhibited by PLA 2 and PLC type III. In epithelium and stroma, the mean IC 50 values of PLA 2 were 9.4 ± 1.1 and 13.9 ± 2.6 [U/mg protein ± SEM], respectively. The mean IC 50 values of PLC type III in epithelium and stroma were 4.5 ± 1.2 and 1.7 ± 0.2 [U/mg protein ± SEM], respectively. In epithelium as well as in stroma, 5α-reductase activity was more greatly inhibited by PLC type III than by PLA 2. Both in epithelium and stroma, PLA 2 significantly decreased the V max of 5α-reductase whereas its K m remained unaffected. A similar decrease in V max was found with PLC type III in epithelium and stroma. Furthermore, the K m of epithelial 5α-reductase increased significantly following the addition of PLC type III. The two phospholipases, with their specific substrate affinities and sites of hydrolysis, exhibited significantly different effects on 5α-reductase, indicating that 5α-reductase activity is not unspecifically affected by modification of the hydrophilic milieu. Rather, 5α-reductase activity is specifically modulated by various phospholipids and/or phospholipolysis mediated degradation products. These findings suggest that the structural composition of the lipid environment plays a fundamental role in the post-translational regulation of 5α-reductase activity in the epithelium and stroma of human BPH. Thus, changes in membrane phospholipid content seem to be instrumental in the expression of DHT-dependent processes.

Biochemical and pharmacogenetic dissection of human steroid 5 alpha-reductase type II.

Human prostatic steroid 5alpha-reductase, encoded by the SRD5A2 gene on chromosome band 2p23, catalyses the irreversible conversion of testosterone to dihydrotestosterone (DHT), the most active androgen in the prostate, with NADPH as its cofactor. This enzyme has never been purified but a number of competitive inhibitors have been developed for this enzyme since increased steroid 5alpha-reductase activity may cause benign prostatic hypertrophy and prostate cancer. We report here the detailed biochemical and pharmacogenetic dissection of the human enzyme by analysing 10 missense substitutions and three double mutants which are all naturally found in humans. Nine of these 13 mutants reduce activity (measured as Vmax) by 20% or more, three increase steroid 5alpha-reductase by more than 15% and one results in essentially unaltered kinetic properties suggesting that it is a truly neutral ('polymorphic') amino acid substitution. Substantial pharmacogenetic variation among the mutants was also observed when three competitive inhibitors, finasteride, GG745 (dutasteride) and PNU157706, were investigated. Our studies not only define the substrate and cofactor binding sites of human steroid 5alpha-reductase, but also have significant consequences for the pharmacological usage of steroid 5alpha-reductase inhibitors in human patients treated for prostatic conditions.

Pretranslational up-regulation of the hepatic microsomal δ 4-3-oxosteroid 5α-oxidoreductase in male rat liver by all- trans-retinoic acid

Administration of all- trans-retinoic acid (ATRA; 60 mg/kg daily for 3 days) to male rats increased the rate of 5α-dihydrotestosterone (5α-DHT) formation from testosterone in microsomal fractions in vitro. The formation of androstane-3α,17β-diol from testosterone was also increased because of the higher concentration of 5α-DHT produced in microsomal incubations. Northern analysis confirmed that the increased rate of 5α-DHT formation was due to the pretranslational up-regulation in Δ 4-3-oxosteroid 5α-oxidoreductase (EC 1.3.99.5) mRNA expression in ATRA-treated male rat liver. Thus, ATRA elicited in male rat liver a partial feminization of the expression of this enzyme, which normally exhibits a female-selective distribution in the rat. Subsequent experiments evaluated whether the administration of human chorionic gonadotropin or thyroxine to ATRA-treated male rats decreases 5α-reductase activity to that observed in untreated male rat liver. Although these treatments did not decrease 5α-reductase to untreated male levels, it was found that administration of ATRA to gonadectomized male rats produced complete feminization of the enzyme. Again, up-regulation was confirmed at the mRNA level. The activity of the male-specific cytochrome P450 2C11 (as reflected by microsomal testosterone 16α-hydroxylation activity) was correspondingly decreased by treatments that increased steroid 5α-reductase activity. Thus, gonadectomy in combination with ATRA administration effected a more pronounced decrease in 16α-hydroxylation activity than either treatment alone. These findings suggest that ATRA is a novel positive regulator of the 5α-reductase that in combination with the removal of circulating androgen, which normally suppresses 5α-reductase levels, feminizes the expression of this enzyme in rat liver.

Synthesis of benzanilide derivatives as dual acting agents with α 1-adrenoceptor antagonistic action and steroid 5-α reductase inhibitory activity

Synthesis of benzanilide derivatives which have dual α 1-adrenoceptor antagonistic action and steroid 5α-reductase inhibitory activity and their structure-activity relationships is described.

Insulin-like growth factor 1 (IGF-I) effects on sex-specific cytochrome P450 enzymes in normal and hypophysectomised male rats

The role of growth hormone (GH) in the regulation of the sex-differentiated rat cytochrome P450 (CYP) enzymes has been extensively studied. However, little is known about the involvement of insulin-like growth factor I (IGF-I) as a mediator in this regulation. We wanted to study if IGF-I had effects on sex-differentiated CYP enzymes and to compare the effects of IGF-I to the effects of GH. IGF-I, GH or saline was administered continuously via osmotic minipumps to normal and hypophysectomised rats for seven days. After treatment, the expression of several sex-differentiated liver enzymes (CYP2C11, CYP2C12), the female-dominant steroid 5α-reductase, and the male-dominant CYP3A2 enzyme was studied at mRNA, protein and/or functional levels. Our results demonstrate that IGF-I has marked effects on the sex-specific expression of CYP2C11 and CYP2C12. The effects of IGF-I were similar to those of GH. In contrast, in hypophysectomised rats IGF-I gave effects opposite to those observed after GH treatment to normal rats on the CYP3A-associated cortisol 6β-hydroxylation. No effects of IGF-I on the steroid 5α-reductase activity were observed.

Impact of tamoxifen on peripubertal Androgen imprinting of rat hepatic cytochrome P450 2C11, cytochrome P450 3A2, and steroid 5α-reductase

Expression of sex-dependent rat hepatic cytochromes P450 and steroid 5α-reductase is regulated mainly by the sex-specific pattern of growth hormone (GH) secretion and is subject to androgen imprinting. Since tamoxifen suppresses GH pulse amplitude and nadir levels, we investigated the effect of tamoxifen on peripubertal testosterone imprinting of hepatic CYP2C11, CYP3A2, CYP2A1, and steroid 5α-reductase. Prepubertal tamoxifen administration (5 mg once daily s.c. on days 28 and 29 of age) to non-ovariectomized female Sprague-Dawley rats did not affect hepatic microsomal CYP2C11-dependent testosterone 2α-hydroxylase, CYP3A-mediated testosterone 6β-hydroxylase, CYP2A1-dependent testosterone 7α-hydroxylase, or steroid 5αreductase activity in adult rats. Testosterone treatment (5 μmol/kg, s.c., once daily) of intact female rats during either puberty (days 35–49 of age) or adult life (days 69–77 of age) had no effect on these enzyme activities in adult (78-day-old) female rats, but the same treatment given during both of these periods induced the male-specific testosterone 2α- and 6β-hydroxylase activities and suppressed the female-predominant testosterone 7α-hydroxylase and steroid 5α-reductase activities, indicating that peripubertal testosterone administration imprints the adult androgen responsiveness but not the basal levels of these enzyme activities in non-ovariectomized female rats. However, peripubertal androgen imprinting of the basal levels of testosterone 2α-hydroxylase and steroid 5α-reductase activities was observed in female rats administered tamoxifen prepubertally. Tamoxifen pretreatment also enhanced testosterone imprinting of the adult androgen responsiveness of testosterone 2α- and 6β-hydroxylase and steroid 5α-reductase activities. The enhanced testosterone hydroxylase activities were, however, not associated with an increase in microsomal NADPH-cytochrome P450 reductase activity, but were accompanied by elevated hepatic CYP2C11 and CYP3A2 protein levels. Overall, the present study indicates that prepubertal tamoxifen administration does not interfere with the normal sex differentiation of the gender-dependent hepatic cytochromes P450 and steroid 5α-reductase, but this drug modulates peripubertal androgen imprinting of CYP2C11, CYP3A2, and steroid 5α-reductase in adult female rats.

Kinetic analysis of rat steroid 5α-reductase activity in prostate and epididymis homogenates at neutral pH: Evidence for type I activity in epididymis

Immunocytochemical studies and mRNA measurements have shown that the rat epididymis—like the rat prostate—expresses both rat steroid 5α-reductase isozymes, i.e. type I and II. So far, enzyme activity measurements in rat epididymis homogenates, however, do not support the presence of type I 5α-reductase activity. Incubating homogenates of both tissues with a wide range of substrate concentrations, we were able to detect activity of both isozymes in rat prostate and epididymis tissues at neutral pH. In rat prostate the amount of type I activity, as measured by the V max at pH 7.0, exceeds that of type II 5α-reductase 50-fold. The efficiency ratio, V max K m , of the type I isozyme accounts for 25% of the total in vivo potential activity. A possible anabolic role for the type I isozyme in rat prostate was thus surmised. In rat epididymis the V max of type I and type II 5α-reductase at pH 7.0 were similar. Comparison of the efficiency ratio V max K m of either isozyme in the rat epididymis, however, suggested that the type II isozyme would play the major role in the 5α-reduction of testosterone at physiological concentrations and at neutral pH. The specific localization of the isozymes should be considered to allow for correct quantification of their in vivo contribution to dihydrotestosterone formation.

Kinetic analysis of steroid 5α-reductase activity at neutral pH in benign prostatic hyperplastic tissue: Evidence for type I isozyme activity in the human prostate

In human benign prostatic hyperplastic (BPH) tissue homogenates 5α-reduction of testosterone was examined at neutral pH. As Lineweaver-Burk and Eadie-Scatchard plots of estimated initial velocities against a wide range of substrate concentrations of 2nM to 3.2 μM were non-linear, the existence of two 5α-reductase isozymes in this tissue was surmised. Indeed, enzyme parameters at pH 7.0 suggested the presence of two isozymes with affinity constants of 1995 and 11.8 nM, characteristic of the well established human steroid 5α-reductase isozymes type I and II, respectively. The physiological roles of these isozyme activities remain puzzling. The specific activities, V max, of these subtypes indicated an approx. 6-fold higher maximum velocity of type I than of type II 5α-reductase in the human hyperplastic prostate at this pH. In contrast, the efficiency ratios, V max K m , demonstrated that the type II isozyme had a nearly 27 times higher potential in vivo activity than the type I isozyme, and is therefore most probably quantitatively responsible for dihydrotestosterone formation at physiological testosterone levels in this tissue at neutral pH. This is the first full paper on type I 5α-reductase activity in human BPH tissue.

Activin and inhibin have opposite effects on steroid 5α-reductase activity in genital skin fibroblasts

The transforming growth factor β (TGF-β) superfamily includes several closely related peptides including the activins and inhibins. Since we recently reported that TGF-β1 and β2 are potent inducers of steroid 5α-reductase (5αR), we have now studied the effects of these other peptides using primary cultures of human scrotal skin fibroblasts. Recombinant human activin A or inhibin A were added to cultured cells (2 × 10 5 cells) for 2 days in a serum free media and 5αR activity was measured by the %-conversion of tracer [ 3H]-testosterone to dihydrotestosterone (DHT) over a 4-h period. Activin significantly stimulated 5αR activity in a dose related manner (control 3.0 ± 0.4%, activin (1.2 × 10 −9 M) 6 ± 0.7%, P < 0.01, (2.4 × 10 −9 M) 8.5 ± 0.6%, P < 0.001). In comparison, androgen (DHT 10 −7 M) induction of 5αR was 4.7 ± 0.2%, P < 0.05. Combined exposure of fibroblasts to activin (1.2 × 10 −9 M) and androgen (10 −7 M) did not result in additive or synergistic effect on 5αR activity. In contrast, exposure of cells to an androgen (10 −7 M) and TGF-β (2 × 10 −10 M) led to synergistic effects on 5αR activity (control 1.5 ± 0.1%, DHT 2.6 ± 0.2% TGF-β1 4.8 ± 0.5, TGF-β1+DHT 9.2 ± 1.2%). Finasteride, a 4-aza steroid inhibitor of 5αR (10 −8 M) inhibited both activin and TGF-β-induced 5αR activity suggesting that the type II isoenzyme is induced by these peptides. Activin mediated 5αR activity was abolished by the addition of cycloheximide, consistent with the proposition that enzyme induction requires new protein synthesis. Recombinant human inhibin alone did not alter basal 5αR activity but dose dependently inhibited DHT (10 −7 M)-induced 5αR activity (control 4.1 ± 0.4%, DHT 7.5 ± 0.7%, DHT + inhibin (0.6 × 10 −9 M) 5.7 ± 0.5%, P < 0.05 DHT + inhibin (1.2 × 10 −9 M] 4.3 ± 0.2%, P < 0.001). The effects of activin or inhibin were not associated with changes in cell number or thymidine uptake. These studies indicate that activin is 100 times more potent on a molar basis than androgen in induction of 5αR activity. Although both activin and TGF-β1 induce 5αR activity, the actions of the two peptides differ in the presence of an androgen. In contrast, inhibin significantly inhibits androgen induction of 5αR. Activin and inhibin, two closely related molecules, potentially play opposite roles in DHT formation in sexual tissue.

Steroid 5α-Reductase Activity in the Harderian Glands of Male and Female Syrian Hamster (Mesocricetus auratus)

The activity of the enzyme steroid 5α-reductase in Harderian glands of adult syrian hamsters was assessed by measuring the conversion of testosterone (T) to 5αdibydrotestosterone (DHT). The optimal conditions for this reaction were determined in vitro using whole gland homogenates and [3H]testosterone as substrate. Enzyme activity was maximal at pH 5.5. The Michaelis-Menten constant of the Harderian enzyme for T was 4.6 ± 1.2 × 10-6M in females and 4.2 ± 0.39 × 10-6M in males, estimated by Eadie-Hofstee plots. On the basis of relative maximum velocity values, there was 9 or 10 times more 5α-reductase in females (2.8 ± 0.67 nmol/mg protein/hr) than in males (0.289 ± 0.029 nmol/mg protein/hr). Consistently, glands of intact male hamsters had lower 5α-reductase activities than those of females. Castration of males significantly increased the enzymatic activity, which within 4 weeks reached female-like values. The levels of 5α-reductase mRNA also increased with castration. There was a direct correlation between activity and mRNA levels of the enzyme in castrated male glands. Further, the administration of T or DHT to ovariectomized hamsters led to intact male values in the enzymatic activity of the gland. The sex differences in 5α-reductase activity may be of relevance to the differential regulation exerted by androgen upon the physiology of male and female glands. The results are consistent with the view that 5α-dihydrotestosterone is the active androgen in the Harderian gland.