Progesterone 5α-reductase Research Articles

Biotransformation of cardenolide-precursors by Saccharomyces cerevisiae

Digitalis plants are the most important source for the extraction of cardenolides used in the treatment of cardiac insufficiency in humans. In order to study putative biosynthetic genes and enzymes we intend to establish a yeast system were glycosylated enzymes and P450 enzymes involved in cardenolide biosynthesis can be functionally expressed. For example, we were unable to express progesterone 5α-reductase, a glycoprotein, in E. coli. We also aim at the reconstruction of consecutive steps of the putative cardenolide pathway in yeast to produce commercially unavailable pregnanes from bulk precursors such as progesterone. We already cloned three genes encoding enzymes that are supposed to transform pregnanes during cardenolide formation: the Δ5–3β-hydroxysteroid dehydrogenase (3β-HSD, EC 1.1.1.51) from Digitalis lanata, which converts pregnenolone to isoprogesterone [1], the Arabidopsis thalianaΔ4,5-steroid 5β-reductase (At5β-StR, EC 1.1.1.145/1.3.1.23) which reduces progesterone to 5β-pregnane-3,20-dione [2] and an enzyme capable of hydroxylating pregnanes in position 21, the steroid-21-hydroxylase (Cyp21A1, EC 1.14.99.10) from Mus musculus. These genes were cloned into the Gateway® pYES-DEST52 vector system and then transformed into Saccharomyces cerevisiae strain BY4741a. Our data indicate, that the Cyp21A1 gene is functionally expressed in the recombinant yeast strain as it is capable of transforming progesterone into 21-hydroxyprogesterone. Product formation was demonstrated by GC-MS and TLC. Further studies are ongoing to show the functional expression of 3β-HSD and At5β-StR in yeast and to cotransform and coexpress these genes.

Dutasteride affects progesterone metabolizing enzyme activity/expression in human breast cell lines resulting in suppression of cell proliferation and detachment

Recent evidence indicates that progesterone metabolites play important roles in regulating breast cancer. Previous studies have shown that breast carcinoma and tumorigenic breast cell lines have higher 5α-reductase and lower 3α-hydroxysteroid oxidoreductase (3α-HSO) and 20α-HSO activities and mRNA expression levels than normal tissue and non-tumorigenic cell lines. The 5α-reduced progesterone metabolites such as 5α-dihydroprogesterone (5αP) promote both mitogenic and metastatic activity in breast cell lines in culture, whereas the 4-pregnene metabolites, 4-pregnen-3α-ol-20-one (3αHP) and 4-pregnen-20α-ol-3-one (20αHP) have the opposite (anti-cancer-like) effects. The 5α-reductase inhibitor dutasteride has been shown to inhibit 5α-reduction of testosterone to 5α-dihydrotestosterone in prostate tissue, resulting in decreased prostate volume. The aim of this study was to determine if dutasteride is an effective inhibitor of progesterone 5α-reduction in human breast cell lines and if such inhibition reduces mammary cell proliferation and detachment. The effect of dutasteride on progesterone metabolizing enzyme activities and mRNA expression were examined in tumorigenic MCF-7 and non-tumorigenic MCF-10A human breast cell lines. Dutasteride (10 −6 M) inhibited progesterone conversion to 5α-pregnanes by >95% and increased 4-pregnene production. The results indicated that effects of dutasteride on the progesterone metabolizing enzymes are due to direct inhibition of 5α-reductase activity and to altered levels of expression of 5α-reductase and HSO mRNAs. Treatment of cells with progesterone without medium change for 72 h resulted in significant conversion to 5α-pregnanes and increases in cell proliferation and detachment. The increases in proliferation and detachment were blocked by dutasteride and were reinstated by concomitant treatment with 5αP, providing proof-of-principle that the effects were due not to progesterone but to the 5α-reduced metabolites. This study provides the first evidence that dutasteride is a potent progesterone 5α-reductase inhibitor and that such inhibition may be beneficial in breast cancer.

Biosynthesis of pregnane derivatives in somatic embryos of Digitalis lanata

Activities of the enzymes cholesterol monooxygenase (side chain cleaving) (SCCE) Δ 5-3β- hydroxysteroiddehydrogenase Δ 5-Δ 4- ketosteroid isomerase, progesterone 5β-reductase, 3β-hydroxysteroid 5β-oxidoreductase, 3α-hydroxysteroid 5β-oxidoreductase, progesterone 5α-reductase and 3β-hydroxysteroid 5α-oxidoreductase which are involved in biosynthesis and transformation of pregnane derivatives were determined in different developmental stages of somatic embryos of Digitalis lanata. All enzymes were found to be present in proembryogenic masses (PEMs) as well as in globular and bipolar embryos. Most SCCE activity was found in the mitochondria fraction. Cholesterol, sitosterol and stigmasterol, precursors of the pregnanes, occurred in somatic embryos in amounts of about 1 μg mg −1 protein. Pregnenolone was found in traces only (about 20 ng mg −1 protein). Feeding of progesterone caused an increase of the contents of 5α-pand 5β-pregnandione, progesterone, 5β-pregnane-3α-ol,20-one and 5α-pregnane-3β-ol,-20-one. In contrast, administration of cholesterol caused a small increase of pregnenolone only. These results indicate that the rate limiting step in pregnane (and probably in cardenolide biosynthesis) is compartmentation of CSSE in the mitochondria of the somatic embryos of D. lanata.

Changes in pituitary, hypothalamic and brain progestin-metabolizing enzyme activities during lactation

Progesterone 5α-reductase activity and 5α-dihydroprogesterone 3α-hydroxysteroid oxidoreductase (3α-HSOR) activities (NADH- and NADPH-linked) were measured in anterior pituitaries, hypothalami and brains from lactating rats (8 and 21 days postpartum) and non-lactating (60-day-old cycling) rats. Tissue levels of these three enyme activities varied significantly among the three groups examined. In terms of pituitary, mean levels of both of its 3α-HSOR activities were 40–140% higher in actively lactating rats (8 days postpartum) relative to mean levels in lactating rats at weaning (21 days postpartum) or in non-lactating rats. There were no differences in pituitary progesterone 5α-reductase activity among the three experimental groups. In the hypothalamus, the NADPH-linked 3α-HSOR was elevated (50%) at 8 days of lactation compared to the group at 21 days. Hypothalamic NADH-linked 3α-HSOR levels did not vary among the 3 groups. Hypothalamic progesterone 5α-reductase levels in the actively lactating and weaning groups were 30% lower than those of the non-lactating group. Brain levels of progesterone 5α-reductase were also lower these two lactating groups (35–55%) as compared to the non-lactating control group. In brain, NADPH 3α-HSOR activity did not vary among the three groups, but levels of NADH 3α-HSOR activity were lower (40–50%) in the weaning group as compared to the actively lactating and control groups. These findings suggest the possibility that tissue changes in these progesterone-metabolizing enzyme activities during lactation and at weaning are influencing the in situ supply of 3α,5α-tetrahydroprogesterone and 5α-dihydroprogesterone and their derivative effects on GABA A receptor activity and prolactin and gonadotropin release. The decreased activity of progesterone 5α-reductase in hypothalamus and brain would presumably reduce in situ 5α-dihydroprogesterone formation while increases in 3α-HSOR activity would suggest higher in situ 3α,5α-tetrahydroprogesterone formation especially in the pituitary.

Enzyme Activities in Cardenolide-Accumulating, Mixotrophic Shoot Cultures of Digitalis purpurea L.

Summary Photomixotrophic, aseptically growing shoot cultures of Digitalis purpurea accumulate cardiac glycosides. All cardenolides are 5β-configurated. The progesterone 5β-reductase and the 3β-hydroxysteroid-5β-oxidoreductase present in these cultures are involved in providing precursors for cardenolide synthesis, whereas a progesterone 5α-reductase also present in shoot cultures is discussed with regard to its role in phytosterol biosynthesis. The 3β-hydroxysteroid-5β-oxidoreductase catalyzed the reduction of 5β-pregnane-3,20-dione to 5β-pregnan-3β-ol-20-one, which is a precursor of cardenolides; the reaction was reversible. The progesterone 5α-reductase had an activity maximum 7 days after the onset of cultivation whereas progesterone 5β-reductase had its maximum on day 11. The cardenolide content of the plantlets was highest after 24 days. The soluble 30-hydroxysteroid-5β-oxidoreductase had a pH-optimum at 6.5; the temperature optimum was 40 °C. The apparent K m value for the substrate 5β-pregnane-3,20-dione was 17.1 μM. The enzyme utilizes NADPH 2 and NADH 2 as co-substrates with an apparent K m -value for NADPH 2 of 0.8 μM.

Pituitary progestin-metabolizing enzyme activities in the aged female rat

Progesterone 5α-reductase activity and 5α-dihydroprogesterone 3α-hydroxysteroid oxidoreductase (3α-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 durng these stages of reproductive aging, we also determined enzyme levels from ovariectomized rats from each stage treated with estrogen or vehicle. Progesterone 5α-reductase and NADH-linked 3α-HSOR activities were 2-fold higher in pituitaries of CE rats as compared to those of PSP and AN rats. NADPH-linked 3α-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), 5α-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 5α-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 (10μg/day) restored 5α-reductase levels. Ovariectomy had no e ffect on the NADPH-linked 3α-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 3α-HSOR levels except for the CE group. The overiectomized CE rats exhibited reduced activity pituitary NADH-linked 3α-HSOR levels (30%). In contrast, young rats exhibit elevated pituitary NADH-linked 3α-HSOR levels after ovariectomy (4- to 5-fold). These changes suggest the possibility that altered processing of progesterone and its 5α- and 3α-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.

Progesterone metabolism in the microsomal fraction of the testis, head kidney, and trunk kidney from the rainbow trout

In the present study on male rainbow trout, well-defined microsomal fractions from gonad, trunk kidney, and head kidney were used to study enzymes active on progesterone. The metabolites produced were identified by mass spectrometric analysis. In the testis the main metabolite was 17α-hydroxyprogesterone which is an intermediate in the steroid biosynthetic pathway. 17α-Hydroxyprogesterone was also identified in incubations from head and trunk kidney. The 17α-hydroxylase activity was higher in the head kidney than in the trunk kidney which probably reflects the presence of steroid producing interrenal cells in this part of the kidney. The conversion of progesterone to 17α-hydroxylated products in the testis and head kidney was NADPH dependent and inhibited by carbon monoxide, indicating the participation of cytochrome P450 monooxygenase in this reaction. NADH supported the reaction to some extent (27% of the NADPH-dependent activity) in the testis but not in the head kidney. In addition to 17α-hydroxyprogesterone the head and trunk kidney microsomes gave rise to 16α- and 6β-hydroxyprogesterone. These activities were low or absent in testis microsomes. Progesterone 5α-reductase activity was only detected in trunk kidney microsomes.

Characterization and Solubilization of Progesterone 5α-Reductase from Cell Cultures ofDigitalis lanata

Characterization and Solubilization of Progesterone 5α-Reductase from Cell Cultures of<i>Digitalis lanata</i>

The kinetic mechanism of the hypothalamic progesterone 5α-reductase

The kinetic mechanism of the hypothalamic NADPH-linked progesterone 5α-reductase from female rats was determined to be equilibrium ordered sequential by initial velocity, product inhibition and dead-end inhibition studies. Analysis of the initial velocity data resulted in intersecting double reciprocal plots indicating a sequential mechanism (apparent K m (progesterone) = 95.4 ± 4.5 nM; apparent K ia (NADPH) = 9.9 ± 0.7±M). The plot of l/v vs l/progesterone intersected on the ordinale which is consistent with an equilibrium ordered mechanism. Ordered addition of the substrates was also supported by product inhibition studies with NADP versus NADPH and NADP versus progesterone. NADP is a competitive inhibitor versus NADPH (apparent K is = 4.3 ± 1.3μM) and a noncompetitive inhibitor versus progesterone (apparent K is = 31.9 ± 1.4μM and apparent K ii = 145.4 ± 15.5μM). These inhibition patterns show that NADPH binds prior to progesterone. Taken together, these analyses indicate that the cofactor, NADPH, binds to the enzyme in rapid equilibrium and preferentially precedes the binding of progesterone.

Specific stimulation of steroid 5α-reductase solubilized from rat liver microsomes by endogenous phosphatidylserine

Dilauroylphosphatidylcholine caused a marked increase in progesterone 5α-reductase activity solubilized from rat liver microsomes, whereas naturally occurring phosphatidylcholines from biological sources as well as dioleoylphosphatidylcholine had not effect on the activity. Therefore, the stimulatory effect of phospholipids normally found in rat liver microsomes was examined. The lipid extracts were prepared from the fraction which was freed from 5α-reductase activity by DEAE-cellulose chromatography, and found to exhibit a strong stimulatory effect. The lipid extracts were then separated into phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine by chromatography on silicic acid column and preparative thin-layer plate. Among these endogenous phospholipids, only phosphatidylserine stimulated the 5α-reductase, suggesting that the lipid requirement is specific for phosphatidylserine in steroid 5α-reductase from liver microsomes.

Distribution and ovarian control of progestin-metabolizing enzymes in various rat hypothalamic regions

The three principal hypothalamic progesterone metabolizing enzyme activities, namely the progesterone 5α-reductase and 5α-dihydroprogesterone NADH- and NADPH-linked 3α-hydroxysteroid oxidoreductase (3α-HSOR) activities, were examined in discrete rat hypothalamic subsections throughout the estrous cycle and from ovariectomized rats treated with estradiol benzoate or vehicle. The regions studied included the median eminence, the medial preoptic area and the ventromedial and arcuate nuclei. The enzyme assays were performed using radiolabeled steroid substrates and reverse isotopic dilution analysis. While all four hypothalamic regions obtained from intact cycling animals possessed substantial amounts of these three enzyme activities, the median eminence generally had the highest activity levels (2- to 4-fold greater) except during estrus. The other three regions usually had comparable levels. No significant fluctuations were observed in any enzyme activity over the estrous cycle. After ovariectomy, there was a significant decrease (~ 35%) in the level of the NADPH-linked 3α-HSOR activity in the median eminence compared to the level observed in intact cycling animals, suggesting ovarian control. Estrogen treatment for 3 days did not restore this enzyme level to that observed in intact animals. The NADPH-linked 3α-HSOR activity from the other three hypothalamic regions, as well as the NADH-linked 3α-HSOR and the 5α-reductase activities from all four brain regions, did not change significantly after ovariectomy. These results indicate that the median eminence possesses an increased capacity for progesterone metabolism relative to the other hypothalamic regions tested, and that the NADPH-linked 3α-HSOR activity in this region may be under ovarian control.

The kinetic mechanism of the anterior pituitary progesterone 5α-reductase

An analysis of the kinetic mechanism of the microsomal NADPH-linked progesterone 5α-reductase obtained from female rat anterior pituitaries was performed. Initial velocity, product inhibition and dead-end inhibition studies indicate that the kinetic mechanism for the progesterone 5α-reductase is equilibrium ordered sequential. Analysis of the initial velocity data resulted in intersecting double reciprocal plots suggesting a sequential mechanism [apparent K m(progesterone) = 88.2 ± 8.2 nM; apparent K ia(NADPH) = 7.7 ± 1.1 μM]. Furthermore, the plot of 1/v vs 1/progesterone intersected on the ordinale which is indicative of an equilibrium ordered mechanism. Additional support for ordered substrate binding was provided by the product inhibition studies with NADPH versus NADP and progesterone versus NADP. NADP is a competitive inhibitor versus NADPH (apparent K is = 7.8 ± 1.0 μM) and a noncompetitive inhibitor versus progesterone (apparent K is = 9.85 ± 2.1 μM and apparent K ii = 63.2 ± 12.5 μM). These inhibition patterns suggest that NADPH binds prior to progesterone. In sum, these kinetic studies indicate that NADPH binds to the microsomal enzyme in rapid equilibrium and preferentially precedes the binding of progesterone.

Pituitary progesterone 5α-reductase: Solubilization and partial characterization

The microsomal progesterone 5α-reductase activity from female rat anterior pituitary has been solubilized and partially characterized with regard to some of its kinetic and physical properties. The solubilization of progesterone 5α-reductase has been achieved through the use of either an n-octyl glucoside (OG)-KC1- or a digitonin-KCl-extraction. The total yield and specific activity of solubilized enzyme activity is greater using the OG-KC1 method. Kinetic analyses of microsomal and OG-KCl-solubilized progesterone 5α-reductase have indicated that both of these preparations exhibit a similar apparent K m for progesterone (microsomal K m = 117 ± 12 nM; solubilized K m = 123 ± 11 nM), suggesting that the solubilization procedure does not appreciably alter the kinetic behavior of this enzyme activity. The OG-KCl-extracted progesterone 5α-reductase activity also appears quite stable, since essentially no enzyme activity is lost following dialysis at 4°C for 22 h. In addition, the activity of the solubilized-dialyzed enzyme preparation can be slightly stimulated via the addition of phospholipids. Studies on the properties of the microsomal enzyme activity have indicated that this preparation is unaffected by metal chelators (EDTA or EGTA) but can be completely inhibited by the powerful sulfhydryl blocking agent p-chloromercuribenzoic acid. An evaluation of the possible role of flavins (as a hydride carrier between NADPH and the steroid) has shown that progesterone 5α-reduction is inhibited by high levels of flavins and flavin analogs.

Partial characterization of the microsomal and solubilized hypothalamic progesterone 5α-reductase

Microsomal progesterone 5α-reductase activity from female rat hypothalamus has been solubilized and partially characterized in terms of kinetic and physical properties. The solubilization of progesterone 5α-reductase has been accomplished through the use of a digitonin/KCL-extraction. Both the microsomal and solubilized enzyme activities exhibit similar kinetic and physical characteristics. These include their apparent K m for progesterone (Microsomal K m = 113 ± 11 nM; solubilized K m = 144 ± 20 nM) and their affinity (~7nM) for the 5α-dihydroprogesterone analog, 4-aza-4-methyl-5α-pregnane-3,20-dione, which is a potent inhibitor of progesterone 5α-reduction. Both activities are inhibited by divalent cations (Zn 2+ and Cu 2+) and the sulfhydryl-blocking agent p-chloromercuribenzoic acid. Studies aimed at optimizing isolation and assay conditions for the hypothalamic progesterone 5α-reductase indicate that the microsomal activity is enhanced in the presence of monovalent cations (particularly K + and Li +) and the metal chelator EDTA, but is unaffected by the sulfhydryl reducing agent dithiothreitol. The activity of the solubilized enzyme is also enhanced by EDTA but slightly stimulated by dithiothreitol. Analysis of hypothalamic progesterone 5α-reduction for possible flavin involvement (as a hydride carrier between NADPH and the steroid) indicates that the enzyme activity is decreased by high levels of flavins, flavin analogs and riboflavin deficiency.

Progesterone 5α-reductase in mouse brain

We have examined the distribution of progesterone 5α-reductase activity in minces of discrete brain areas from two strains of adult ovariectomized mice known to be differentially responsive to the behavioral effects of exogenously administered 5α-pregnane-3,20-dione (5α-DHP). Minces of freshly dissected midbrain tegmentum (TEG), hypothalamus-preoptic area (HPOA), hippocampus (HIP), and parietal cortex (CTX) were incubated with [4- 14C]progesterone in a Krebs-Henseleit bicarbonate buffer solution containing 10 mM glucose for 2 h at 37°C. The [ 14C]5α-DHP produced was identified by comparison with known standards in paper and thin-layer chromatography systems, derivative formation, and recrystallization to a constant 14C/ 3H ratio. We found a 6-fold difference in the extent to which various brain areas formed 5α-DHP. The amount of conversion was similar for both strains of mice. When expressed as picomoles of 5α-DHP formed per 2 h per 20 mg of tissue, the following order of activity was observed: TEG > HPOA > HIP > CTX.

The effects of neurotransmitters and other cellular modulators and factors on hypothalamic and anterior pituitary Δ 4 -steroid (progesterone) 5α-reductase activity

A number of diverse biological compounds involved in the regulation of the hypothalamo-hypophyseal-ovarian axis have been examined for effects on the conversion of 3H-progesterone to 3H-5α-dihydro-progesterone and 3H-3α-hydroxy-5α-pregnan-20-one by female rat hypothalamus and/or anterior pituitary. Broken cell preparations were incubated with 3H-progesterone and NADPH, and product 5α-reduced progestins were quantitated by reverse isotopic dilution analysis. Progesterone 5α-reductase activity was reduced up to 50% in the presence of 10 −2 to 10 −3 M serotonin in both preparations. At 10 −3 M, various indoles including n-acetylserotonin, melatonin, 5-methoxytryptamine, 5-methoxytryptophol, and 5-hydroxyindole acetic acid decreased by 10 to 30% 5α-reduced product formation. At 10 −2 M , carbamylcholine and norepinephrine were without effect, while 10 −2 M dopamine reduced by 20% the 5α-reduction of progesterone only in pituitary homogenates. The LHRH protease inhibitor bacitracin (2 × 10 −3 M ) decreased by 10 to 40% progesterone 5α-reductase activity in both tissues. By itself, LHRH did not affect the 5α-reduction of progesterone nor did it potentiate the bacitracin effect. In the presence of 1 n M ATP, 100 μ M cAMP and 100 μ M cGMP increased 5α-reduced product formation in the hypothalamus by 19 and 14%. The gonadotropins LH and FSH and the prostaglandins E 1, E 2, F 1α, and F 2α were without effect. Thus, these results and others indicate that a number of cellular components and other factors can affect the in vitro 5α-reduction of progesterone in broken cell preparations.

Subcellular location of hypothalamic progesterone metabolizing enzymes and evidence for distinct NADH- and NADHP-linked 3α-hydroxysteroid oxidoreductase activities

The subcellular location of adult female rat hypothalamic steroid 5α-reductase and 3α-hydroxysteroid oxidoreductase (3α-HSD) activities, which catalyze the conversion of progesterone to 3α-hydroxy-5α-pregnan-20-one via 5α-pregnane-3,20-dione, have been investigated using 3H-labeled substrates and an isotopic dilution assay system. In crude homogenates, appreciable conversion of 5α-pregnane-3,20-dione to 3α-hydroxy-5α-pregnan-20-one is observed with either NADH or NADPH as cofactor. The NADH-and NADPH-linked 3α-HSD activities can be partially separated and enriched using subcellular fractionation by differential centrifugation. The subcellular location of these 3α-HSD activities as well as progesterone 5α-reductase activity was determined with the aid of subcellular markers and electron microscopic techniques. When NADPH is the added cofactor, 3α-HSD activity is present primarily in the cytosol and cofractionates with lactate dehydrogenase activity. When NADH is the added cofactor, 3α-HSD activity is enriched in the 1000 g pellet and is further localized to a cell debris-membranes fraction derived from this pellet. Using the muscarinic cholinergic receptor as a plasma membrane marker, the NADH-linked 3α-HSD activity appears to cofractionate with plasma membranes in subcellular fractions. Progesterone 5α-reductase activity is enriched in the 105,000 g pellet and cofractionates with NADPH cytochrome c reductase activity, suggesting a microsomal location. On the basis of these results, there appear to be at least two hypothalamic 3α-HSD's, a cytosolic NADPH-linked activity and a plasma membrane NADH-linked 3α-HSD activity.

The effect of progesterone analogues, naturally occurring steroids, and contraceptive progestins on hypothalamic and anterior pituitary Δ 4-steroid (progesterone) 5α-reductase

The effects of a number of steroids on the conversion of progesterone to 5α-dihydroprogesterone by hypothalamic and pituitary progesterone 5α-reductase have been investigated. Using enzyme preparations from female rats and 3H-progesterone as substrate, 5α-reduced products (5α-dihydroprogesterone and 3α-hydroxy-5α-pregnan-20-one) were analyzed by reverse isotopic dilution analysis. The amount of total 5α-reduced products formed was compared in the presence and absence of the test steroid. Derivatives lacking the Δ 4 and/or the 3-keto moiety were without effect. Corticosterone had no effect. 16β-Methylprogesterone inhibited progesterone 5α-reduction in both tissues by at least 65%, while the 2α-, 6α-, and 7α-methylated derivatives had lesser effects. 3-Oxo-4-pregnene-20β-carboxaldehyde and 21-fluoroprogesterone were potent inhibitors. 17-Hydroxyprogesterone was a competitive inhibitor (substrate) with Ki's of 0.27 μ M (pituitary) and 0.29 μ M (hypothalamus). Medroxyprogesterone exerted little inhibitory effect. Of the 19-norsteroids examined, only norethindrone appreciably inhibited the 5α-reduction. These results suggest that some natural Δ 4-3-ketosteroids can modify enzymatic activity. Also, inhibitory analogues may be useful for studies on the role of this 5α-reduction of progesterone.