Rat Ventral Prostate Nuclei Research Articles

Partial purification and differential androgen sensitivity of protein phosphokinases from nuclei of rat ventral prostate.

Total protein kinase activity associated with nuclei of rat ventral prostate was extracted and fractionated with the aid of DEAE-Sephadex column chromatography. The flow-through peak contained kinase activities towards dephosphophosvitin, lysine-rich histone, and nonhistone proteins as phosphoprotein substrates. These activities were not stimulated by addition of cAMP, although the cAMP-dependent protein kinase inhibitor reduced the lysine-rich histone kinase activity by 70% or more, suggesting that it might represent the catalytic subunit of the cAMP-dependent histone kinase. By contrast, the inhibitor produced a stimulation of kinase activities toward dephosphophosvitin and nonhistone proteins by 15% and 50%, respectively. The bound fraction on the DEAE Sephadex column was eluted in two peaks of protein kinase activity, one at 0.15–0.20 M (NH4)2SO4, and the other at 0.25–0.30 M (NH4)2SO4. The first peak contained activity only toward nonhistone proteins, whereas the second had activity toward dephosphophosvitin, lysine-rich histone, and nonhistone proteins. None of these was inhibited by the cAMP-dependent protein kinase inhibitor. The effect of orchiectomy on the activity of the various protein kinase fractions was determined. The kinase activity toward dephosphophosvitin, in the flow-through peak, was reduced by about 20% and 40% at 24 and 48 h post-orchiectomy, respectively, and in the bound peak by 33% and 50%, under the same circumstances. Little change was observed in the kinase activity toward lysine-rich histone in the flow-through peak or in the bound peak at 24 h post-orchiectomy. However, at 48 h post-orchiectomy, it increased by 250% in both fractions. This particular kinase activity toward lysine-rich histone might be localized to nucleoplasm rather than chromatin. The protein kinase active toward nonhistone proteins present in the flow-through peak did not change at 24 h post-orchiectomy, but declined by 50% at 48 h post-orchiectomy. Of the two bound nonhistone protein kinase activities, that eluting at 0.25–0.30 M (NH4)2SO4 was reduced by over 80% within 24 h post-orchiectomy, whereas that eluting at 0.15–0.20 M increased by 100% at 24 h and 200% at 48 h post-orchiectomy. These data demonstrate a differential response of prostatic nuclear-associated protein kinases to androgen deprivation.

Expression of 5α-reductase in bacteria as a trp E fusion protein and its use in the production of antibodies for immunocytochemical localization of 5α-reductase

A cDNA encoding a full-length rat 5α-reductase was isolated using female rat liver mRNA and the polymerase chain reaction, and fused to the Escherichia coli trp E gene in a pATH expression vector. The trp E-5α-reductase fusion protein expressed in bacteria and a synthetic oligopeptide corresponding to the C-terminus of rat 5α-reductase were used as antigens to produce rabbit polyclonal antibodies to 5α-reductase. Antibodies to the 5α-reductase portion of the fusion protein and to the peptide were purified by affinity chromatography. Antibodies against the 5α-reductase fusion protein reacted with a single component of rat liver microsomes with M r 26,000 on Western blots, consistent with the size of 5α-reductase predicted from its cDNA, and with a M r 23,000 component on Western blots of detergent extracts of rat ventral prostate nuclei; other rat ventral prostate cellular fractions (mitochondrial, microsomal, cytosol) bound little or no antibody. Antibody against the synthetic peptide reacted with a M r 26,000 component of rat liver microsomes as well as with several components in various cellular fractions of rat ventral prostate. With anti-5α-reductase fusion protein antibodies, specific immunocytochemical staining was observed in the epithelial cell nuclei of the rat ventral prostate, seminal vesicle, epididymis and other accessory sex glands. This nuclear staining was specific, since antibodies from non-immunized rabbits did not give nuclear staining and preincubation of the anti-5α-reductase fusion protein antibodies with the trp E-5α-reductase fusion protein eliminated nuclear staining. Incubation of antibodies with trp E (without the 5α-reductase fusion) had no effect on nuclear staining. Specific staining was not detected in the cytoplasm of these epithelial cells. Little or no specific staining was observed in stromal cells in these rat tissuess. Human prostate was also immunocytochemically stained with this antibody. Specific staining was found in both epithelial and stromal cell nuclei.

Phosphorylation and nuclear processing of the androgen receptor

Although transformed androgen receptor (AR) complexes derived from cytosol and nuclear AR complexes have been shown to bind with high affinity to nuclei and DNA, we have shown that the binding characteristics of the two receptor populations to rat ventral prostate nuclei are different. To account for these differences, we investigated the possibility that the two receptor populations differed in phosphorylation status. Significantly, an anti-phosphotyrosine antibody immunoprecipitated androgen binding from the nuclear AR preparation but not from the transformed cytosolic receptor preparation. These studies suggest that (i) further processing of the AR complex takes place after it has become transformed, and (ii) phosphorylation of the complex is one modification which occurs during the processing of the nuclear receptor.

Chemical demonstration of nuclear androgen receptor following affinity chromatography with immobilized ligands.

With increasing purification of the androgen receptor from nuclei of rat ventral prostate, a receptor-like protein could be demonstrated by chemical staining with silver nitrate. After sonication and digestion of nuclei with micrococcal nuclease, the solubilized receptor was applied to a column of Matrex Gel Green A and eluted with a linear gradient of 0-2 M NaCl. Characterized by specific binding of dihydrotestosterone, this form of the receptor was also androgen dependent and yielded an apparent Mr of 33,000 when analyzed by polyacrylamide gel electrophoresis and silver nitrate staining. To facilitate recovery following chromatography, the receptor was precipitated with 0-40% ammonium sulfate. Analysis of the 15-fold enriched fraction by sucrose density-gradient centrifugation confirmed the presence of a 3S androgen-binding protein. About 200 ng of the precipitated protein was applied to a column of dihydrotestosterone-17 beta-succinyl agarose (ligand concentration, 0.25 mumol/ml). The fractions eluted with 50 microM dihydrotestosterone were electrophoresed and stained as before; again, the presence of a 33,000 Mr protein sensitive to castration was demonstrated. Alternatively, when the precipitated protein was fractionated by fast protein liquid chromatography utilizing a Superose 12 HR 10/30 column, the receptor coeluted with nuclear proteins in the 29,000-36,000 Mr range as determined both by retention time and electrophoresis. In combination, the above methods may be used to obtain a receptor protein purified to near homogeneity with a yield of 5-10%. The amount of receptor afforded by the purification sequence is small but nevertheless sufficient for chemical detection. We anticipate that with modification, the procedures may prove suitable for the recovery of nuclear androgen receptor on a preparative scale.

Interaction of androgen receptors with chromatin and DNA

Controlled digestion of rat ventral prostate nuclei by careful adjustment of conditions of temperature, divalent cation concentration, ionic strength and micrococcal nuclease: DNA ratios yielded oligonucleosome fractions corresponding to less than 10% of the total genome which contain the majority of RNA polymerase B activity and androgen-receptor complexes of the nucleus. These parameters were affected acutely by androgen withdrawal and administration: furthermore, such manipulations affected the susceptibility to micrococcal nuclease release of prostate binding protein gene sequences. This transcriptionally-active androgen-influenced fraction was considered ideal for studies of interaction of chromatin components with androgen receptor protein. Androgen receptor was purified approximately 20 000-fold from rat prostate cytosol. The purified protein retained its ability to stimulate RNA polymerase B activity in prostate nuclei and chromatin fractions, and its properties of binding to chromatin and to DNA. However, although purified receptor protein showed tissue-specific binding to prostate chromatin and enhanced binding to fractions released by low nuclease digestion, no such specificity was indicated by binding to total DNA, DNA from specific fractions or cloned prostatic binding protein cDNA.

Extraction of androgen-receptor complexes from regions of rat ventral prostate nuclei sensitive or resistant to nucleases.

Rat ventral prostate nuclei contain androgen-binding sites which are susceptible or resistant to excision by endonucleolytic action. Those which were susceptible were associated both with oligonucleosomal and subnucleosomal particles. The sedimentation profile characteristic of a nuclear androgen-receptor complex could be obtained by exhaustive nucleolytic digestion or by treatment of fractions with KCl (0.6 mol/l). Androgen-binding sites resistant to DNAase I were also resistant to KCl, whereas those sites resistant to micrococcal nuclease were partially extractable with KCl. Nuclease-resistant sites could be extracted with heparin (10 mg/ml). Androgen-receptor complexes obtained from nuclease-sensitive or nuclease-resistant regions by extraction with KCl or heparin were indistinguishable by routine sedimentation analysis.

A novel nuclear protein in rat ventral prostate androgen-dependent and age-related change

A novel protein was found in the nuclei of rat ventral prostate. This protein has a molecular weight of about 21 kDa as measured by SDS-polyacrylamide gel electrophoresis. It showed a characteristic change between 3 and 84 weeks after birth in close association with the level of testosterone in the blood. After castration, the level of the 21-kDa protein decreased to 1 60 of normal in 7 days, but on daily injection of testosterone the level was restored to normal in 8 days and to twice the normal level in 14 days. Unlike H 1 and H 1 0 histone and high mobility group proteins, the 21-kDa protein was not extracted with 5% HClO 4, but was partially extracted with 0.35 M NaCl. The 21-kDa protein was not found in kidney, liver, or brain, suggesting that it is specific to the ventral prostate.

Androgen-dependent regulation of androgen nuclear receptor in the rat ventral prostate.

Methods have been developed for the measurement of nonradioactive or radioactive dihydrotestosterone (DHT) bound to androgen receptor in rat ventral prostate nuclei (DHTRn). Using 1-day-castrated adult Sprague-Dawley rats (300--320 g body weight), the effects of testosterone (T) on nuclear androgen receptor formation were investigated in the absence and presence of cycloheximide or emetine. DHTRn was measured 4 h after injection of increasing amounts of T, and a maximal value of 14.6--17.8 pmol DHTRn/prostate was reached with doses of T greater than 37.5 micrograms. The amount of DHTRn 30 min after administration of 75 micrograms T was already 3 times greater than the concentration of cytosol receptor initially present in untreated castrated rats (2.0 +/- 0.3 pmol/prostate). In rats that received 2 mg cycloheximide ip 30 min before T, DHTRn did not exceed 5 pmol/prostate; in rats pretreated with 10 mg emetine administered at 1 h and 15 min before T, DHTRn did not exceed 8 pmol/prostate. These results suggest a two-component mechanism: 1) the translocation of preexisting cytosolic-receptor-hormone complexes to the nucleus; and 2) de novo formation of androgen receptor dependent on protein synthesis, which upon interaction with exogenous steroid is likewise accumulated in the nucleus. Progesterone (1 mg) and estradiol (0.6 mg) were also injected in amounts calculated to produce the same degree of occupancy of androgen receptor sites as did T. With both of these steroids, nuclear accumulation of androgen receptor was observed, but neither progesterone nor estradiol induced de novo synthesis of androgen receptor.

Responses to androgens of rat ventral prostate nuclear androgen-binding sites sensitive and resistant to micrococcal nuclease.

Rat ventral prostate nuclei were separated into three major fractions by mild digestion with micrococcal nuclease and two fractions by extensive digestion. All fractions contained androgen-binding sites. Almost 50% of nuclear binding sites were resistant to enzymic digestion when only 5-15% of total DNA was resistant. Under milder digestion conditions, 21% of nuclear binding sites were associated with an intermediate fraction, representing 16% of total nuclear DNA, which was enriched in specific androgen-regulated gene sequences. This fraction was rapidly degraded by more extensive digestion. The nuclease sensitivity of these particular genes was markedly influenced by castration and the administration of dihydrotestosterone to castrated animals. The nuclear content of both nuclease-resistant and -sensitive androgen-binding sites was decreased by castration. Whereas the administration of androgen to animals castrated 1 day previously preferentially replenished nuclease-resistant sites, nuclease-sensitive sites, including those associated with transcriptionally active regions, had apparent priority when androgen was supplied to animals castrated 7 days previously. The significance of these observations to the regulation of nuclear processes and the possible interrelationships of nuclease-sensitive and -insensitive sites are discussed.

Enzymic properties and effects of androgen on nuclear histone phosphatase activity of rat ventral prostate.

Abstract Nuclei of rat ventral prostate have been demonstrated to possess a protein phosphatase activity utilizing 32 P-labelled, lysine-rich histone (calf thymus) as the phosphoprotein substrate. This phosphatase has a pH optimum of 7.1 and was stimulated by the sulfhydryl protective agents dithiothreitol and 2-mercaptoethanol. This nuclear protein phosphatase did not appear to require divalent cations; rather, small inhibitions of activity were found in the presence of 2.4 mM Mg 2+ , Mn 2+ , and Ca 2+ . Divalent cations such as Zn 2+ or Cu 2+ were found to be much stronger inhibitors, giving about 80% inhibition at 1 mM. Monovalent cations were also found to inhibit the histone phosphatase, e.g., 43% at 200 mM NaCl. Ammonium molybdate did not influence the enzyme activity whereas ADP and ATP reduced it by 72 and 82% respectively at 1 mM. There was no change in activity of the histone phosphatase up to 96 h post-orchiectomy when specific activity was based per unit of nuclear protein. However, a small decrease is noted if specific activity is expressed per unit of nuclear DNA (19% at 48 h and 36% at 96 h orchiectomy). This difference reflects the decreased nuclear protein content of the prostate observed following castration. Our data suggest that the decline in prostatic nuclear histone phosphorylation observed following orchiectomy is not due to increased phosphatase activity.

Acidic-phosphoprotein phosphatase activity of rat ventral prostate nuclei apparent lack of effect of androgens

A protein phosphatase activity has been demonstrated in nuclei of rat ventral prostate utilizing 32P-labelled phosvitin as a model acidic phosphoprotein substrate. This phosphoprotein phosphatase has a pH optimum of 6.7, is unaffected by the sulphydryl protecting agent 2-mercaptoethanol, and requires a divalent cation for maximal activity. Of the various divalent cations tested, Mg2+ is the most effective in reactivating the EDTA-inhibited enzyme. The phosphatase is inhibited by sodium flouride, sodium oxalate, N-ethylmaleimide, ATP and ADP but is relatively insensitive to ammonium molybdate. Increased ionic strength of the reaction medium also causes a reduction in the enzyme activity, e.g., by 48% at 200 mM sodium chloride. The activity of the acidic phosphoprotein phosphatase did not change significantly at 48 h or 96 h post-orchiectomy when expressed per unit of nuclear protein. However, it is reduced by approx. 30% at these times after castration if based on DNA content. The decline in activity per nucleus reflects the decrease in the realtive nuclear protein content observed at 48 h or 96 h post-orchiectomy. This suggests that the decline in the phosphorylation of prostatic nuclear acidic proteins which occurs upon androgen withdrawal is not due to increased nuclear phosphatase activity.

Inhibition of the nuclear dihydrotestosterone receptor complex from rat ventral prostate by antiandrogens and stilboestrol

The inhibitory effects of stilboestrol and representatives from three main classes of antiandrogens on the dihydrotestosterone receptor complex isolated from the nuclei of rat ventral prostate were compared. After labelling of the tissue in vivo or in vitro with [ 3H]testosterone in the presence or absence of test drugs, the nuclear extracts were prepared and the inhibitory effect on the steroid-receptor complex was evaluated. Equimolar doses (1 μmol/100 g body weight) of cyproterone acetate, chlormadinone acetate, SC 9022 and flutamide inhibited the binding of dihydrotestosterone to the receptor complex by 48–54%, whereas in the in vitro studies an equimolar concentration (0.1 μM) inhibited the binding by 45–58% with the exception of flutamide which had no effect. Under these experimental conditions stilboestrol had no effect on the binding of dihydrotestosterone to the nuclear receptor.

Enzymic characteristics and effects of testosterone treatment on nucleolar and chromatin-associated histone phosphokinase activity of rat ventral prostate

Histone phosphokinase activity is present in nucleoli and chromatin isolated from nuclei of rat ventral prostate. The activity (utilizing lysine-rich histone as substrate) in each of these subnuclear fractions is maximal at pH 8.0–8.2, is optimally stimulated in the presence of dithiothreitol and NaCl, and requires Mg 2+. Enzymic properties described for the histone phosphokinase activities are similar for nucleolus and chromatin, but are different from those demonstrated by kinases which readily phosphorylate phosvitin. Histone phosphokinase activity of the nucleolus declines about 18% at 24 h and 45% at 48 h post orchiectomy. This decrease in activity is prevented by testosterone replacement therapy. Chromatin-associated histone phosphokinase activity also declines following orchiectomy (21% at 48 h and 66% by 96 h). Thus, the rates of decline of nueleolar and chromatin-associated histone phosphokinase activity in orchiectomized rats described here are slower compared to the enzymic activity toward acidic protein substrates. This further suggests that changes in androgenic status results in differential effects on the protein phosphokinase reactions associated with the prostatic nucleus.

Presence and androgen control of an alkaline phosphatase in the nucleus of rat ventral prostate

The presence of alkaline phosphatase (EC 3.1.3.1) activity has been demonstrated in nuclei of rat ventral prostate. This enzyme activity remained after washing of isolated nuclei with 0.5% Triton X-100; an acid phosphatase initially present with the nuclear fraction was removed by this treatment. The nuclear alkaline phosphatase, examined by utilizing p- nitrophenyl phosphate as substrate, had a pH optimum of 9.5–10.3, and a broad substrate specificity: p- nitrophenyl phosphate > phosphothreonine > β-glycerophosphate > phosphoserine. The nuclear phosphatase was sensitive to denaturation by heat or urea treatments and was also inhibited by P i, l-phenylalanine, homoarginine, dithiothreitol, and EDTA. The EDTA-inhibited enzyme was maximally reactivated by Zn 2+, although Mg 2+, or Ca 2+ were also effective at somewhat higher concentrations. Orchiectomy of adult rats resulted in an increase in the nuclear alkaline phosphatase activity (2–3-fold at 24 or 48 h postorchiectomy). A decline in the protein: DNA ratio also occurred following orchiectomy, but the increase in phosphatase specific activity was evident whether expressed per unit of protein or per unit of DNA. Testosterone replacement following orchiectomy abolished the increase in nuclear phosphatase activity. The results suggest that the prostatic nuclear alkaline phosphatase may be involved in events related to inactivation of the prostate nucleus following androgen deprivation.

The differential response of prostatic nucleolar and extra-nucleolar protein phosphokinase activities following androgen deprivation.

Protein phosphokinase activities of nucleolar and extra-nucleolar compartments of rat ventral prostate nuclei were measured using the model acidic phosphoprotein, dephosphophosvitin, as substrate. Following orchiectomy, the activity in both of these fractions declined; however, the kinase activity of the nucleolus decreased at a much greater rate than that in the extra-nucleolar portion of the nucleus. Testosterone maintenance of castrated animals prevented this decline in activity. The regulation of protein phosphokinases which phosphorylate prostatic nucleolar acidic proteins may be an important mechanism in the androgen mediated activation of the nucleolus in this target tissue.

Localization of protein phosphokinase activities in the nucleolus distinct from extra-nucleolar regions in rat ventral prostate nuclei

Rat ventral prostate nucleoli contain protein phosphokinase(s) which have distinctly different characteristics from protein phosphokinase(s) localized in the extra-nucleolar regions of the nucleus. The differences pertain to pH vs activity profiles and activation by divalent cations, utilizing dephospho-phosvitin as substrate. Lysine-rich and arginine-rich F3 histones are also phosphorylated by nucleolar protein phosphokinase(s). Phosphorylation of histones by either nucleolar or extra-nucleolar fractions was not stimulated by cAMP, whereas that of phosvitin was slightly inhibited.

Direct Evidence on Incorporation of the Receptor into the Nuclei of Rat Ventral Prostate in the Form of the Complex with Dihydrotestosterone

Cytosol 9S receptor was prepared from the supernatant fluid at 105,000 X g of rat prostate homogenates by a Sephadex G-100 gel chromatography, and was labeled with 131I. The 131I-labeled 9S receptor retained the activity of forming a complex with 3H-dihydrotestosterone, similarly to the intact cytosol receptor, when examined by a sucrose density gradient centrifugation. When the 131I-labeled cytosol 9S receptor was incubated with isolated prostatic nuclei in the presence of 3H-dihydrotestosterone, it was found that the 131I-labeled receptor was directly incorporated into the nuclei in a form of the complex bound to 3H-dihydrotestosterone. 131I-Labeled receptor-3H-dihydrotestosterone complex which was incorporated into the nuclei was extracted with 0.5 M KCl solution, and the nuclear complex sedimented with a velocity of 5S. The incorporation of 131I-labeled receptor-3H-dihydrotestosterone complex into the nuclei increased along with the raised temperature of incubation, whereas association of the complex with the chromatin reached maximum at 35 degrees C and then decreased gradually beyond this temperature. In the time course study, either the incorporation of the complex into the nuclei or the association with the chromatin reached the maximal levels within 10 min and leveled off up to 60 min. On the other hand, 131I-labeled serum protein was far less efficiently incorporated into the nuclei than the radioiodinated 9S receptor, and association of the serum protein with the chromatin was limited to a very little extent. The 131I-labeled 9S receptor-dihydrotestosterone complex associated with the chromatin was found to be preferably distributed into the non-histone protein as well as the DNA itself of the chromatin. The radioactivity lost by dehistonization of the chromatin was almost negligible. Furthermore, the cytosol 9S receptor fraction bound to 3H-dihydrotestosterone was purified about 100 fold by the two consecutive column chromatographies. The partially purified receptor fraction which was labeled with radioiodine incorporated mostly into non-histone protein and DNA fractions.

Separation of multiple dihydrotestosterone receptors in rat ventral prostate by a novel micromethod of electro-focusing Blocking action of cyproterone acetate and uptake by nuclear chromatin

Six dihydrotestosterone-binding proteins in rat ventral prostate nuclei and cytosol have been separated and identified by a microelectrofocusing procedure based on their isoelectric points: pI 3.7, α; 4.6, β; 5.6, γ; 6.7, δ; 7.8, ε and 8.5, ζ. Further resolution of these peaks accomplished by expansion of the pI range of electrofocusing separates one or more labelled proteins from these peaks: pI 3.6, α 1; 4.1, α 2; 4.7, β 1; 5.1, β 2; 5.4, γ 1; 5.8, γ 2; 6.2, δ 1; 6.5, δ 2; 6.8, δ 3; 7.1, ε 1; 7.4, ε 2; 7.8, ε 3; 8.3, ζ 1 and 8.7, ζ 2. Components at pI 3.6, α 1; 4.1, α 2 and 5.8, γ 2 are specifically bound by dihydrotestosterone but of these only γ 2 is taken up by nuclear chromatin and the binding of dihydrotestosterone to γ 2 is blocked by cyproterone acetate. Three other components, pI 6.8, δ 3; 7.8, ε 3; and 8.3, ζ are also taken up by chromatin and blocked by the anti-androgen but also bind testosterone or progesterone ( δ 3, ε 3 and ζ 1) or estradiol-17β ( δ 3). These four components can be extracted from the nuclei ( δ 3, ε 3 and ζ 1 by 0.5 M KCL and γ 2 by 1 M KCL) and all these nuclear extracts except ζ 1 retain their capacity for absorption as dihydrotestosterone complexes by nuclear chromatin. Thus, there are four dihydrotestosterone-binding proteins in rat prostate cytosol which fulfill the criteria for receptor proteins, 5.8, γ 2; 6.8, δ 3; 7.8, ε 3 and 8.3, ζ 1, and it is possible that androgen action may have to be explained by multiple receptor proteins.

Action of a Nonsteroidal Antiandrogen, Flutamide, on the Receptor Binding and Nuclear Retention of 5α-Dihydrotestosterone in Rat Ventral Prostate1

The nonsteroidal anntiandrogen, flutamide (α-α-α-trifluoro-2-methyl-4′-nitro-m-propionotoluidide or SCH 13521), suppresses the retention of a specific 5α-dihydrotestosteronereceptor complex by cell nuclei of rat ventral prostate in vivo. At 1–10 µ-M concentrations, flutamide also inhibits the binding of 5α-dihydrotestosterone in vitro by a receptor protein in the prostate cytosol, and the nuclear retention of the androgen-receptor complex during the incubation of minced prostate. The nonantiandrogenic analogue, acetanilide, at 100 µM, does not show such an inhibitory effect. While flutamide and cyproterone are equipotent as antiandrogens in vivo, cyproterone is about 10 times more active than flutamide in inhibiting the receptor binding of 5 αdihydrotestosterone. This discrepancy may be due to a metabolic activation in the whole animal and' or inactivation of flutamide in the prostate. The suppressive action of flutamide (or its active metabolite) may be related to its geometrical structure, which is simi...

Inhibition of nuclear testosterone 5 alpha-reductase in rat ventral prostate by estrogens and anti-androgens.

The activity of testosterone 5α-reductase in purified nuclei of rat ventral prostate was inhibited by addition of estradiol-17β, estrone or estriol in vitro. Rate of the inhibition of the enzyme activity by these estrogens was of similar degree. Therefore, this inhibitory effect does not seem to be correlated to their estrogenic potency.Effect of three anti-androgens (Ro 2-7239, SK & F 7690 and TSAA 291) on the activity of testosterone 5α-reductase was also examined and it was revealed that SK & F 7690 and TSAA 291 showed the inhibitory effect on the enzyme activity.