Progestin binding in mammary tissue of prepartum,nonlactating and postpartum,lactating cows

Binding of [³H]R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3, 20-dione) to bovine mammary cytosol indicated the presence of progestin binding sites of high-affinity and low-capacity in tissue from prepartum, nonlactating and from postpartum, lactating cows. To prevent binding of [³H]R5020 to glucocorticoid binding sites, a 200-fold molar excess of nonradioactive cortisol was included during all incubations, thus specific binding was limited to progestin binding sites. Nonradioactive R5020 and progesterone effectively inhibited [³H]R5020 binding to progestin binding sites, while estradiol-17β, dihydrotestosterone (17β-hydroxy-5α-androstan-3-one), dexamethasone (9-fluoro-11β, 17, 21-trihydroxy-16α-methyl-1,4-pregnadiene-3,20-dione) or additional cortisol were ineffective. Dissociation constants for specifically bound [³H]R5020 in cytosol from mammary tissue of nonlactating and lactating cows were nearly identical, averaging 1.9 ( ± 0.3) and 0.8( ± 0.2) × 10?⁹M, respectively. However, binding capacities (fmol/mg cytosolic protein) were greater in cytosol from prepartum, nonlactating (179 ± 53) than postpartum, lactating (41 ± 15) cows. Specific binding components in cytosol from lactating cows sedimented in the 6-7S region on linear sucrose density gradients. When subjected to isoelectric focusing, specific binders with isoelectric points (pI) of approximately 6.1, 7.9 and 8.3 were resolved. The decrease in number of binding sites during lactation was due to the virtual absence of the anionic binding species, suggesting that their presence is necessary for progesterone to inhibit milk secretion.     

C19-steroid 5β-reductase and 3- and 17-oxidoreductases of adult male hamster kidney

Male hamster kidney cytosol exhibited strong 5β-reductase activity. Incubation of cytosol with [4-¹⁴C]-testosterone at pH 6.7 yielded 5β-DHT with minor quantities of 5β-androstane-3α,17β-diol and 5β-androstane-3β,17β-diol. Incubation with [4-¹⁴C]-androstendione yielded 5β-androstanedione and smaller quantities of testosterone, 5β-DHT, 3α-hydroxy-5β-androstan-17-one, 3β-hydroxy-5β-androstan-17-one and 5β-androstane-3α,17β-diol. The two major metabolites were progressively increased with increase in the concentration of the respective substrates but the other metabolites showed very little change. The metabolism of the respective substrates was progressively decreased with changes in pH of the incubation mixture from 6.0–7.5 accompanied by a parallel decrease in the formation of the respective major metabolites. NADPH was much more effective than NADH as coenzyme. The microsomes exhibited a trace of 5β-reductase activity only with NADPH and androstenedione.The kidney homogenate at pH 10.1 effectively converted [4-¹⁴C]-testosterone to [4-¹⁴C]-androstenedione. The dehydrogenase activity was present in the cytosol and microsomes. NAD⁺ was more effective than NADP⁺ in the cytosol and the reverse was indicated for the microsomes. Spectrophotometric assay revealed not only NADP⁺-linked Hβ-dehydrogenase activity but also a lower 3α-dehydrogenase activity but no detectable 3β- or 17α-dehydrogenase activity. NAD⁺-linked activity was not explored because of the interference by the very high endogenous NAD⁺-reduetase activity.     

Bile acid biosynthesis: the metabolism of 7 alpha-hydroxy-4-cholesten-3-one in the bile fistula rat.

The two primary bile acids, cholic acid (3α,7α,12α-tri-hydroxy-5β-cholan-24-oic acid) and chenodeoxycholic acid (3α,7α-dihydroxy-5β-cholan-24-oic acid), are initially synthesized by way of identical precursors, and the point of divergence of this pathway is thought to occur at the intermediate 7α-hydroxy-4-cholesten-3-one. In order to test this hypothesis, bile fistula rats received simultaneous intra-venous infusions of ³H-7α-hydroxy-4-cholesten-3-one and ¹⁴C-cholesterol (5-cholesten-3β-ol). Assays of equal specific activities of the two bile acids from an infusion of ¹⁴C-cholesterol demonstrated the achievement of a steady state, and assays of equal specific activities from an infusion of ³H-7α-hydroxy-4-cholesten-3-one would-validate the above postulate. However, the infusion of ³H-7α-hydroxy-4-cholesten-3-one resulted in unequal specific activities in the bile acids of the rats investigated, with cholic acid always of a lower value. These results suggest that either 7α-hydroxy-4-cholesten-3-one is not the last common intermediate in the production of cholic acid and chenodeoxycholic acid, or that the infused bile acid intermediate was not metabolized in a fashion similar to that formed in the liver from cholesterol.     

Hydrocortisone binding receptor in the rat pituitary GH3 cell line.

A receptor with specificity and high affinity for hydrocortisone (HC) has been found in the cytosol of GH₃ cells, a growth hormone (GH) producing culture. Scatchard analysis indicated that the interaction of [³H]HC with the receptor has an apparent dissociation constant (K_d) of about 6.0 × 10^?9M and a concentration of binding sites of approx. 1 × 10^?13 mol/mg cytosol protein. The second order association rate constant was determined to be 1.5 × 10⁶ M^?1 min^?1. The receptor activity is stable at 2°C for several hours, but is destroyed completely by heating at 37°C for 1 hour, or by treatment with pronase, only partially by RNase, but not by DNase. The binding of [³H]HC to the cytosol receptor is inhibited by unlabeled progesterone (PR) or dexamethasone to the same extent as the inhibition by unlabeled HC. However, it is only partially inhibited by testosterone, 17-methyl-testosterone, 17α and 17β-estradiol, and 4-pregnen-20β-ol-3-one, and is unaffected by 5α-pregnan-3β,20β-diol. The biological role for these receptors in the regulation of GH synthesis is supported by the observations that the HC-stimulated production of GH is antagonized by PR, which competes with the binding of HC to the receptor.     

Characterization of a hormone receptor defect in the androgen-insensitivity mutant

Mouse kidney cytosol contains specific receptors that reversibly bind dihydrotestosterone at a concentration of 43 f moles/mg protein. [Nonstandard abbreviation: DHT, dihydrotestosterone, 17 β-hydroxy-5 α-androstan-3-one.] The equilibrium dissociation constant of the receptor-dihydrotestosterone complex is 1.3 × 10^?9M for females and 1.7 × 10^?9M for castrated males. The complex sediments at 8–9S in glycerol gradients. In males bearing the androgen-insensitivity mutation (analogous to human testicular feminization), the specific dihydrotestosterone receptor activity is decreased about 8 fold. The residual binding activity has wild type affinity (K_D = 1.5 × 10^?9M) for dihydrotestosterone and also sediments at 8–9S. Kidney cytosol from castrated mutant mice displays a new binding component with low affinity and high capacity for dihydrotestosterone.     

Steroid metabolism by purified adult rat leydig cells in primary culture

To characterize Leydig cell steroidogensis, we examined the metabolism of (³H)pregnenolone (3β-hydroxy-5-pregnen-20-one) to androgens in the presence and absence of human chorionic gonadotropin (hCG) as a function of culture duration. Approximately 20–30% of the (³H)pregnenolone was converted to testosterone (17_β-hydroxy-4-androsten-3-one) by purified Leydig cells at 0, 3 and 5 days (d) of culture. Androstenedione (4-androstene-3,17-dione) and dihydrotestosterone (17_β-hydroxy-5_α-androstan-3-one) were also produced while on day 5 of culture, significant amounts of progesterone (4-pregnene-3, 20-dione) were isolated. The Δ⁵ intermediates, 17-hydroxypregnenolone (3β, 17-dihydroxy-5-pregnen-20-one) and dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one), accounted for less than 1% of substrate conversion, indicating a clear preference for Leydig cells to metabolize (³H)pregnenolone via the Δ⁴ pathway. On day 0 of culture, unidentified metabolites consisted of predominately polar steroids while on day 5 of culture, the unidentified metabolites consisted of predominately nonpolar steroids. In the presence of hCG, (³H)pregnenolone metabolism did not differ from basal on day 0 or 3 of culture. HCG increased the conversion of pregnenolone to progesterone and 17-hydroxyprogesterone (17-hydroxy-4-pregnene-3, 20-dione) on 5d. This suggests that Leydig cells cultured for 5d have decreased C_17–20 desmolase activity or that hCG acutely stimulates 3β-hydroxysteroid dehydrogenase and Δ⁴-Δ⁵ isomerase activities.     

Androgen metabolism in rat skeletal muscle in vitro

Androgen metabolism by the cytosol fraction of rat skeletal muscle was investigated. Testosterone metabolism was low, the main metabolite being 4-androstene-3α, 17β-diol. In addition, small amounts of 5α-androstane-3a,17β-diol were formed, but no 17β-hydroxy-5α-androstane-3-one could be detected. 4-Androstene-3α,17β-diol was metabolized only to testosterone in this system of incubation. When 17β-hydroxy-5α-androstane-3-one was incubated with muscle cytosol, considerable metabolism to 5α-androstane-3α,17β-diol and to 5α-androstane-3β,17β-diol could be detected. Low 5α-reduction of testosterone and rapid conversion of formed 17α-hydroxy-5α-androstane-3-one to 5α-androstane-3α, 17β-diol and 5α-androstane-3β,17β-diol gave limited ability of the muscle preparation employed to accumulate 17β-hydroxy-5α-androstane-3-one.     

Synthesis of 5α-Androstane-3α,16α,17β-triol from 3β-hydroxy-5-androsten-17-one

5α-Androstane-3α, 16α 17β-triol was synthesized from 3β-hy-droxy-5-androsten-17-one. The procedure Involved catalytic hydrogenation of 3β-hydroxy-5-androsten-17-one to 3β-hydroxy-5α-androstan-17-one. This was followed by conversion of the 3β-hydroxy group to 3α-benzoyloxy group by the Mitsunobu reaction. Further treatment with isopropenyl acetate yielded 5α-androsten-16-ene-3α, 17-diol 3-benzoate 17-acetate. This was then converted to 3α, 17-dihydroxy-5α-androstan-16-one 3-benzoate 17-acetate via the unstable epoxide intermediate after treatment with $\text{m}$-cloroperoxybenzoic acid. LiAlH₄ reduction of this compound formed 5α-androstane-3α, 16α, 17β-trlol. ¹H and ¹³C NMR of various steroids are presented to confirm the structure of this compound.     

3 -Hydroxy- 5 -C 19 -and C 21 -steroid oxidoreductase activity in rat liver

The presence of small amounts of 3β-hydroxy-Δ⁵-C₁₉- and C₂₁-steroid oxidoreductase in the microsomal fraction of rat liver is shown. NAD was the preferred cofactor. K_m for the oxidation of dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one) into androstenedione (4-androstene-3,17-dione) was 3 × 10⁻⁶ M. In similarity to the adrenal and gonadal 3β-hydroxy-Δ⁵-C₁₉-steroid oxidoreductase, but in contrast to the hepatic 3β-hydroxy-Δ⁵-C₂₇-steroid oxidoreductase involved in the biosynthesis of bile acids, the hepatic 3β-hydroxy-Δ⁵-C₁₉-steroid oxidoreductase was inhibited by the 3β-hydroxy-Δ⁵-steroid oxidoreductase inhibitor, 2α-cyano-4,4,17-trimethyl-17β-hydroxy-5-androsten-3-one, and the activity was greatly reduced with microsomes from immature rats.     

Pathway of testosterone biosynthesis in the testis of the marmoset Saguinus oedipus

These studies were undertaken to determine the principal pathway of androgen biosynthesis by the testis of the marmoset $\text{Saguinus oedipus}$. Testicular fragments (25 mg) were incubated at 37°C in Krebs-Ringer bicarbonate buffer, pH 7.4, containing pregnenolone-7-³H (3β-hydroxy-5-pregnen-20-one) or progesterone-7-³H. Duplicate fragments were incubated with each substrate for 30 min, one hr, three hr, or five hr, for a total of 16 separate incubations. Metabolites were separated by paper and thin-layer chromatography, with identity established by recrystallization to constant specific activities and ³H/¹⁴C ratios. Pregnenolone was readily metabolized to progesterone, 17α-hydroxyprogesterone, androstenedione (4-androstene-3, 17-dione) and testosterone. Progesterone was converted to 17α-hydroxyprogesterone, androstenedione and testosterone. 17α-hydroxyprogesterone was the predominant metabolite obtained from both substrates at one, three and five hrs of incubation. Neither 17α-hydroxypregnenolone (3β-17-dihydroxy-5-pregnen-20-one) nor dehydroepiandrosterone (3β-hydroxy-5-androsten17-one) was detected in the incubates. These data suggest a predominant Δ⁴ pathway with accumulation of 17α-hydroxyprogesterone in the testis of this primate specie.     

Comparative studies of 5α-reductase inhibitors within MCF-7 human breast cancer cells

We have compared the inhibitory effects of six synthetic steroid analogs (17β-carboxy-4-androsten-3-one benzylanilide (VP-1), 17α -acetoxy-6-methylene-4-pregnene-3, 20-dione (VP-2), 6-methylene-4-pregnene-3, 20-dione (VP-3), 17β-acetoxy-6-methylene-4-androsten-3-one (VP-4), 17β -acetoxy-16, 16-dimethyl-6-methylene-4-androsten-3-one (VP-5), and 3β-hydroxy-16-methylene-5-androsten-17-one (VP-6)) upon 5α-reductase activity within MCF-7 human breast cancer cells and rat prostate. Enzyme assays were performed by quantifying the amounts of [³H]5α-androstan-3α-17β-diol and/or [³H]dihydrotestosterone formed from 40 nM [³H]testosterone within each system. Five μM concentrations of VP-2 and VP-3 inhibited prostatic 5α-reductase by 55 and 65%, respectively, whereas the other analogs showed little activity. In contrast, each of the six analogs was active against MCF-7 homogenate 5α reductase activity. VP-2 and VP-4 demonstrated approx 65 and 70% inhibitions, respectively, whereas the other four compounds inhibited enzyme activity by 40–55% in this system. These results suggest that rat prostate and MCP-7 cells contain different 5α-reductase isozymes. When these agents were examined for 5α-reductase inhibitory activity following 1 h preincubations with intact MCF-7 cultures, VP-1 and 3 demonstrated potencies similar to those in MCF-7 homogenate. The other compounds, however, were far less active under these conditions. Longer culture preincubations (16 h) were associated with substantially increased VP-6 potency, moderate increases for VP-4 and 5, but no change in VP-2 activity. Additional studies examining the abilities of these agents to bind to MCF-7 androgen receptor (AR) and progesterone receptor (PR) revealed moderate AR binding activities of VP-2, 3, and 4, and substantial PR binding for VP-2 and 3. Finally, VP-4 failed to inhibit estrogen-dependent MCF-7 PR synthesis, suggesting that it has no androgenic activity despite its ability to interact with MCF-7 AR.     

Specific cytosol binding of diethylstilbestrol in rat skeletal muscle

Rat skeletal muscle cytosol proteins bound ³H-diethylstilbestrol (³H-DES). More than 90% of this binding was high capacity and low affinity. Serum albumin accounted for roughly 50–60% of the binding, as evidenced by its precipitation with anti-rat albumin IgG. About half of the binding was distinguishable from albumin and other serum proteins by its precipitation in 40% saturated ammonium sulfate. This material sedimented at 4–5S in high-salt sucrose gradients, and resolved into two components (8S and 4–5S) in low-salt. Following incubation at 23–27°C for one hour, 2% of the bound ³H-DES in whole cytosol (approximately 2 fmole/mg cytosol protein) was retained by DNA-cellulose, and was eluted with 0.6 M KCl. This small fraction of the total binding was inhibited by estrogens and DES analogues: estradiol-17β, DES, dienestrol, and hexestrol were strong inhibitors; isodienestrol, dimethylstilbestrol, estradiol-17α, estrone, tamoxifen, MER-25, CI-628, and nafoxidine were weak inhibitors; dihydrotestosterone, testosterone, and prednisone did not compete. These observations indicate that specific estrogen-binding sites exist in rat skeletal muscle.     

Androgen regulation of progestin biosynthetic enzymes in FSH-treated rat granulosa cells in vitro

The influence of androgens on the FSH modulation of progestin biosynthetic enzymes was studied $\text{in vitro}$. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 3 days in a serum-free medium containing FSH (20 ng/ml) with or without increasing concentrations (10^?9?10^?6 M) of 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone; DHT), 5α-androstane-3α, 17β-diol (3α-diol), or the synthetic androgen 17β-hydroxy-17-methyl-4,9,11-estratrien-3-one (methyltrienolone; R1881). FSH treatment increased progesterone and 20α-hydroxy-4-pregnen-3-one(20α-OH-P) production by 10.2- and 11-fold, respectively. Concurrent androgen treatment augmented FSH-stimulated progesterone and 20α-OH-P production in a dose-related manner (R1881 > 3α-diol > DHT). In the presence of an inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), the FSH-stimulated pregnenolone (3β-hydroxy-5-pregnen-20-one) production (a 20-fold increase) was further enhanced by co-treatment with R1881, 3α-diol or DHT. Furthermore, FSH treatment increased 4.4-fold the activity of 3β-HSD, which converts pregnenolone to progesterone. This stimulatory action of FSH was further augmented by concurrent androgen treatment. In contrast, androgen treatment did not affect FSH-stimulated activity of a progesterone breakdown enzyme, 20α-hydroxysteroid dehydrogenase(20α-HSD). These results demonstrate that the augmenting effect of androgens upon FSH-stimulated progesterone biosynthesis is not due to changes in the conversion of progesterone to 20α-OH-P, but involves an enhancing action upon 3β-HSDΔ⁵, Δ⁴-isomerase complexes and additional enzymes prior to pregnenolone biosynthesis.     

Chemical constituents from the roots of Acanthus ilicifolius var. xiamenensis

Chemical investigation of Acanthus ilicifolius var. xiamenensis led to the isolation of eleven compounds, and their structures were identified to be 2-benzoxazolinone (1), 2-hydroxy-2H-1,4-benzoxazin-3(4H)-one (2), (2R)-2-O-β-d-glucopyranosyl-2H-1,4-benzox azin-3(4H)-one (3), (2R)-2-O-β-d-glucopyranosyl-4-hydroxy-2H-1,4-benzoxazin-3(4H)-one (4), (2R)-2-O-β-d-glucopyranosyl-7-hydroxy-2H-1,4-benzoxazin-3(4H)-one (5), lyoniside (6), 3′-methoxy-luteolin-7-O-β-d-glucopyranoside (7), β-sitosterol-3-O-β-d-glucopyranoside (8), stigmasterol octadecanoate (9), β-sitosterol octadecanoate (10), stigmasterol-3-O-β-d-glucopyranoside (11) on the basis of mass and NMR spectra. This is the first report on the occurrence of compound 6 and 7 in Acanthaceae. This work also represents the first phytochemical work on the roots of A. ilicifolius var. xiamenensis.     

Androgen metabolism by rat epididymis. 1. Metabolic conversion of 3 H-testosterone in vivo

The epididymis of adult rats metabolize ³H-testosterone by experiments $\text{in vivo}$. Thirty minutes after the injection of 100 μCi ³H-testosterone, some 10 per cent of the total radioactivity of the epididymis was found in the water-soluble fraction, whereas 90 per cent was found in the ether soluble fraction (free steroids). The free steroids were examined further and the following androgenic metabolites identified: $\text{testosterone}$ (17β-hydroxy-4-androsten-3-one) 8, 9%, $\text{androstendipne}$ (4-androstene-3, 17-dione, 2,7%,$\text{5α-A-dione}$ (5α-androstane-3, 17-dione) 6,5%, $\text{DHT}$ (17β-hydroxy-5α-androstan-3-one) 47, 2%, $\text{3β-diol}$ (5α-androstane-3β, 17β-diol) 4, 4%, $\text{3α-diol}$ (5α-androstane-3α,17β-diol) 20, 8% and $\text{androsterone}$ (3α-hydroxy-5α-androstan-3-one) 3,4%. The relative amount of each metabolite is given in per cent of total radioactivity in the ether soluble fraction.     

Incorporation of [1-14C]-acetate into testosterone,androstenediol, dehydroepiandrosterone and progestogens by ram testis following human chorionic gonadotropin administration

Testicular steroidogenesis in rams was examined by constant infusion (3 hr) of [1-¹⁴C]-acetate into the testicular artery of four conscious standing animals.The following steroids (in order of decreasing levels of [¹⁴C] labeling) were secreted by the testis and found in testicular tissue: testosterone, dehydroepiandrosterone, 3β-hydroxy-5-androsten-17-one, androstenediol, 5-androsten-3β,17β-diol and 17-hydroxy-4-pregnene-3,20-dione. In addition, [¹⁴C] labeling of 17,20α-dihydroxy-4-pregnen-3-one occurred in testicular tissue but not in blood. This $\text{in vivo}$ system with the conscious standing ram demonstrated an operative Δ⁵ steroidal pathway to testosterone. The physiological significance of 17,20α-dihydroxy-4-pregnen-3-one is not yet explained in this species.     

Testicular 3 alpha-hydroxysteroid oxidoreductase activity in the developing male rat.

In the testes, 17β-hydroxy-5α-androstan-3-one (dihydrotestosterone, DHT) is converted to 5α-androstane-3α,17β-diol (3α-diol) by the enzyme 3α-hydroxysteroid oxidoreductase (3α-HSO). This steroid has been shown to possess biological activity in the male rat. The secretion of 3α-diol is much greater in the prepubertal animal than in the adult. This study is designed to quantitate the activity of 3α-HSO in the cytosol fraction of testes from male rats throughout sexual development. Following homogenizatlon of whole testes, the 105,000 × g supernatant or cytosol fraction was incubated with ³H DHT and varying concentrations of unlabelled DHT in the presence of 0.25μm NADPH. The incubation was carried out at 34°C for 10 min at a pH of 7.4. The K_m of 3α-HSO in testicular cytosol was calculated to be 1.25μM. The specific activity of testicular cytosol 3α-HSO, expressed as pmoles of 3α-diol converted from DHT per min per mg testicular cytosol protein, was high in young rats from 10 to 22 days of age, and was followed by a decline between day 22 and 37, with activity remaining low throughout adulthood. Total testicular cytosol activity of 3α-HSO, expressed as nmoles of 3α-diol converted from DHT per min per pair of testes, gradually increased from day 10 to day 60 and remained high in the adult rat. In the post-pubertal period, a possible lack of available substrate, DHT, or possible endogenous testicular regulatory mechanisms acting on 3α-HSO activity might account for the actual decrease in 3α-diol concentration in the blood and testes of mature rats.     

In vitro conversion of 5alpha-reduced metabolites of testosterone by the seminal vesicles, ventral prostate glands and testes of adult rats

In order to ascertain the ability of rat seminal vesicles, testes and ventral prostate glands to interconvert 5α-reduced androgens, these three organs were incubated with either tritiated 17β-hydroxy-5αandrostan-3-one (5α-dihydrotestosterone,DHT), 5α-androstane-3α, 17βdiol (3α-diol) or 5α-androstane-3β, 17β-diol (3β-diol). The incubation environment utilized (Krebs-Ringer bicarbonate glucose buffer) was selected because the histologic appearance of the tissue at the conclusion of the incubation was indistinguishable from tissue fixed immediately after sacrifice of the animal, thereby approximating the physiologic conditions as closely as possible. In incubations of rat seminal vesicles, ³H.-3β-diol was not metabolized while 26.7 ± 3.8% of ³H-3α-diol appeared as DHT and 17.2 ± 1.5% of ³H-DHT was metabolized to 3α-diol. A small amount (7.5 ± 0.8%) of ³H-DHT was, however, converted to 3β-diol. In incubations of rat testes, the major metabolite, regardless of substrate, was 3α-diol. The conversion of 75.7 ± 2.1% of ³H-3β-diol to 3α-diol has demonstrated, for the first time, that this steroid can be metabolized by the rat testis. Rat ventral prostate glands metabolized $\text{18.5 ± 2.5\%}\text{of}{}^3\text{H-3β-}\text{diol}$ to DHT and 61± 2.9% of ³H-3α-diol to DHT. When ³H-DHT served as the substrate, 83.2 ± 1.5% remained unmetabolized. The prostate glands are, therefore, capable of metabolizing 3β-diol to DHT.     

Study of estramustine binding protein (EMBP): purification of rat EMBP and establishment of RIA

Estramustine binding protein (EMBP) was purified from the ventral prostate of the rat using DEAE-cellulose chromatography, concanavalin-A affinity chromatography and DEAE-sepharose chromatography. At the final step of the purification, two different peaks (Peaks A and B) of A280 nm were obtained. Peak A had a high binding activity to [3H] estramustine. On the other hand, Peak B had a low binding activity. On the analysis of polyacrylamide gel electrophoresis, Peak A gave two protein bands, whereas Peak B gave a single band. The molecular weight of the markedly stained band of Peak A was approximately 27,000, whereas that of Peak B was 18,000, as estimated by analysis of Fargusson's plot. The antibody against Peak B was used for establishing a radioimmunoassay (RIA) of EMBP. The sensitivity of this assay system was sufficient to measure of 1 ng of EMBP. The dilution curve of rat prostatic cytosol was paralleled with the standard curve. As a result obtained from this RIA, the mean concentration of immunoreactive EMBP was 8.01 ng/mg cytosol protein in human benign hyperplastic prostate (BPH) and 4.28 ng/mg protein in human prostatic carcinoma (PC), respectively. These results here obtained indicate that human prostate has an immunoreactive protein to the purified EMBP obtained from the ventral lobe of rat prostate.     

An analysis of the metabolites of progesterone produced by isolated sertoli cells at the onset of gametogenesis

Sertoli cells isolated from 17 day old rats were maintained in culture and incubated with [¹⁴C]-progesterone for 20 h. The cells and media were extracted with ether/chloroform and the extracts chromatographed two-dimensionally on TLC and the radioactive metabolites visualized by autoradiography. Nine of the metabolites (constituting about 88% of total metabolite radioactivity) were identified by relative mobilities of the compounds and their derivatives in TLC and GC systems and by recrystallizations with authentic steroids as the following: 20α-hydroxypregn-4-en-3-one, 3α-hydroxy-5α-pregnan-20-one, 5α-pregnane3α,20α-diol, 17β-hydroxy-5α-androstan-3-one, 5α-pregnane-3,20-dione, 17-hydroxypregn-4-ene-3,20-dione, testosterone, 5α-androstane-3α,17β-diol and androst-4-ene-3,17-dione. Over 71% of the metabolite radioactivity was due to 20α-hydroxypregn-4-en-3-one, the major metabolite. 5α-reduced pregnanes constituted about 12% and C₁₉ steroids comprised about 2.9% of the radioactivity of the metabolites. Calculation of relative steroidogenic enzyme activities from initial reaction rates suggested the following activities in μunits/mg Sertoli cell protein: 20α-hydroxysteroid oxidoreductase (20α-HS0; 7.71), 5α-reductase (4.77), 3α-HS0 (3.57), 17α-hydroxylase (0.93), 17β-HS0 (0.34) and C₁₇-C₂₀ lyase (0.34). The relatively high rate of steroidogenic enzyme activities in the Sertoli cells of young rats may indicate that Sertoli cells are less dependent on Leydig cell steroidogenesis than has been assumed. Since nearly all the metabolites of progesterone and testosterone are now identified, it is possible to construct a picture of Sertoli cell steroidogenic activity.