Fecal steroid metabolites and reproductive monitoring in a female Tsushima leopard cat (Prionailurus bengalensis euptilurus)

Although the Tsushima leopard cat (Prionailurus bengalensis euptilurus) is one of the most endangered mammals in Japan, its reproductive physiology and endocrinology have been not elucidated. The objective was to establish the non-invasive monitoring of reproductive endocrinology in a female Tsushima leopard cat and to identify the types of fecal reproductive steroid metabolites in this species. Fecal concentrations of estrogen and progestin were determined by enzyme immunoassays, from 60 d before to 60 d after the last copulation, during three pregnancies. Fecal estrogen metabolite concentrations were increased before/around the mating period and after mid-pregnancy. Fecal progestin metabolite concentrations increased after the last copulation and remained high during pregnancy. The gestation period was 65.0 ± 0.6 d (mean ± SD). Fecal extracts were separated by high-performance liquid chromatography for identification of fecal metabolites. Fecal estrogens were identified as estradiol-17β and estrone. Fecal progestins during pregnancy contained 5α-reduced pregnanes: 5α-pregnan-3α-ol-20-one, 5α-pregnan-3β-ol-20-one and 5α-pregnan-3,20-dione, and nonmetabolized progesterone was barely detected in feces. In conclusion, measurement of fecal estrogen and progestin metabolites was effective for noninvasive reproductive monitoring in the Tsushima leopard cat. An immunoassay for fecal estradiol-17β concentrations seemed useful to monitor follicular activity, whereas an immunoassay with high cross reactivity for 5α-reduced pregnanes was useful to monitor ovarian luteal activity and pregnancy.     

Biotransformation of progesterone by suspension cultures of Digitalis purpurea cultured cells

It has been shown that the cultured cells of Digitalis purpruea are capable of transforming progesterone (I) to 5α-pregnane-3,20-dione (II), 5α-pregnan-3β-ol-20-one (III), its glucoside (IV), 5α-pregnane-3β,20α-diol (V), its glucoside (VI), 5α-pregnane-3β,20β-diol (VII), its glucoside (VIII), Δ⁴-pregnen-20α-ol-3-one (IX), its glucoside (X), Δ-pregnen-20β-ol-3-one (XI) and its glucoside (XII). 5α-Pregnan-3β-ol-20-one glucoside (IV), 5α-pregnane-3β,20α-diol glucoside (VI), 5α-pregnane-3β,20β-diol glucoside (VIII), Δ⁴-pregnen-20α-ol-3-one glucoside (X) and Δ⁴-pregnen-20β-ol-3-one glucoside (XII) have been found for the first time as new metabolises by plant tissue cultures. A scheme for the biotransformation of progesterone (I) has been proposed, and the reduction and glucosidation activities distinctly have been observed in these cultured cells.     

Metabolism of progesterone in subcellular fractions of rat uterus

The metabolism of progesterone and 5α-pregnane-3,20-dione was studied in subcellular fractions of uterus from untreated and estradiol-17β treated immature rats. The reduction of progesterone to 5α-pregnane-3, 20-dione took place in all the particulate fractions of the uterus. The nuclear 5α-reductase accounted for the greatest fraction of enzymatic activity and was stimulated by estradiol treatment in vivo. The 5α-reductase activity in the mitochondrial and microsomal fractions was not increased after estradiol treatment. The reduction of 5α-pregnane-3,20-dione to 3α-hydroxy-5α-pregnan-20-one occurred mainly in the soluble fraction and was only slightly stimulated by estradiol. It proceeded much more rapidly than the reduction of progesterone to pregnanedione. Progesterone was also reduced to 20α-hydroxy-4-pregnen-3-one by a soluble enzyme whose activity was increased after estradiol-17β treatment.     

Induction of lordosis behavior in female rats by intravenous administration of progestins

Progesterone is rapidly metabolized by neural cells in the rat. Progesterone could, therefore, act as a “prohormone,” stimulating lordosis behavior in estrogen-primed rats only after metabolic conversion. Were such the case, one might expect one or more of the naturally occurring metabolites of progesterone to be more potent than the parent compound. Estradiol benzoate-primed rats were therefore administered intravenously 200 μg of progesterone or one of five immediate metabolites of progesterone. The steroid 20α-dihydroprogesterone was found to be more potent than progesterone. Both 20α-hydroxy-5α-pregnan-3-one and 3α-hydroxy-5α-pregnan-20-one were less potent than progesterone, but more potent than the vehicle propylene glycol. Neither 5α-pregnane-3α, 20α-diol nor 5α-pregnane-3,20-dione (dihydroprogesterone, DHP) differed from the vehicle in potency. The data suggest that 20α-dihydroprogesterone, which is secreted at high levels during the estrous cycle, could play a role in the regulation of sexual receptivity. The data also suggest that 5α-reduction is probably not crucial for progesterone's action.     

Metabolism of progesterone by chorionic cells of the early sheep conceptus invitro

Progesterone-4-¹⁴C was extensively metabolized during incubation with dispersed trophoblast prepared from chorionic membranes of the 21-day sheep conceptus. Of the metabolites formed, 17,20α-dihydroxypregn-4-en-3-one, 20α-hydroxypregn-4-en-3-one, 20(β-hydroxypregn-4-en-3-one, 5α-pregnane-3α,17,20α-triol, 5β-pregnane-3ga, 17,20α-triol, 5β-pregnane-3g,20α-diol, 3β-hydroxy-5α-pregnan-20-one, 3α-hydroxy-5β-pregnan-20-one, 20β-hydroxy-5β-pregnan-3-one, 5α-pregnane-3,20-dione and 5β-pregnane-3,20-dione were identified. These findings indicate that the sheep conceptus acquires extensive steroid metabolizing capability very early in pregnancy.     

Metabolism of progesterone by Dioscorea deltoidea suspension cultures

Dioscorea deltoidea plant tissue suspension cultures are capable of metabolizing progesterone to 5α-pregnan-3-β-ol-20-one and 5α-pregnan-3β,20β-diol. The latter product has not previously been reported as a metabolic product of progesterone by plant systems. Both transformation products are present as conjugates in this plant tissue culture.     

Stereospecific reduction of progesterone by Pisum sativum

[4 -¹⁴C]-Progesterone was applied to the leaves of growing pea plants, Pisum sativum. After 3 weeks, about 50% of the administered steroid was reduced, about 20% being reduced to 5α-pregnane-3α,20β-diol as the major metabolite. The radioactivities of 5α-pregnane-3α,20α-diol and 5α-pregnane-3α,20β-diol after 3 weeks were more than twice those after one week. The following radioactive metabolises were also isolated: 5α-pregnane-3,20-dione; 20α-hydroxy-4- pregnen-3-one; 20β-hydroxy-4-pregnen-3-one; 3α-hydroxy-5α-pregnan-20-one; 3α-hydroxy-5β-pregnan-20-one; 3β-hydroxy- 5α-pregnan-20-one; 20β-hydroxy-5α-pregnan-3-one; 5α-pregnane-3β,20β-diol; and 5β-pregnane-3α,20β-diol. The radioactivities of the 5α-pregnane derivatives were considerably higher than those of the corresponding 5β-pregnane derivatives.     

The in vivo metabolites of [14C] progesterone in bovine muscle and adipose tissue

Muscle and adipose tissue were obtained from steers and dairy cows following subcutaneous administration of [¹⁴C] progesterone. Following extraction, purification and separation by column, thin layer and gas-liquid chromatography, various radioactive residues from these tissues were identified by their Chromatographic mobility, crystallization to constant specific activity and mass spectra. Progesterone constituted 54% of free radioactivity extracted from muscle and 69 and 73% of radioactivity in the free and conjugated portions of extracts, respectively, from fat. Metabolites identified were: 5α-pregnane-3,20-dione, 9%, 0%, 0%, 20β-hydroxy-4-pregnen-3-one, 8%, 11%, 3%; 3α-hydroxy-5β-pregnan-20-one, 13%, 2%, 2%; 3α-hydroxy-5α-pregnan-20-one, 3%, 3%, 6%; 20α-hydroxy-5α-pregnan-3-one, 0%, 2%, 3%; of radioactivity in muscle (free) and fat (free and conjugated fractions), respectively. Tentatively identified in fat extracts by chromatographic mobility were: 20α-hydroxy-4-pregnen-3-one, 1%, 1% and 3β-hydroxy-5β-pregnan-20-one, 0%, 2% of radioactivity in free and conjugated fractions, respectively. The average concentration of steroid in these animals due solely to treatment, calculated from the specific activity of the [¹⁴C] progesterone administered, was 3.4 and 18.1 ng/g in muscle and subcutaneous fat, respectively.     

Evaluation of progesterone and 20-oxo-progestagens in the plasma of Asian (Elephas maximus) and African (Loxodonta africana) elephants

The corpus luteum of African elephants produces high amounts of 5α-reduced progesterone metabolites (5α-pregnane-3,20-dione and 5-α-pregnane-3α-ol-20-one), whereas progesterone itself is quantitatively less important, and plasma levels of progesterone during the estrous cycle in elephants are considerably lower than those of other mammals. The objective of this study was to compare the concentration of progesterone in plasma of Asian and African elephants as determined by specific progesterone assays with those of total immunoreactive progestagens containing a 20-oxo-group (20-oxo-P). These metabolites were determined by an enzyme immunoassay using an antibody against 5-α-pregnane-3β-ol-20-one, 3HS:BSA. Plasma of non-pregnant Asian (n = 4) and African (n = 4) elephants was collected at weekly intervals for periods of 8–15 months and at random intervals during pregnancy in one Asian elephant. High-performance liquid chromatography separation of plasma samples of both species demonstrated that in the 20-oxo-P assay, 5α-pregnane-3,20-dione makes up ˜60% of the total immunoreactive material. The progesterone and 20-oxo-P values during the estrous cycle showed a parallel pattern and were significantly correlated (P < 0.001; Asian: r = 0.80; y = 3.76 × –0.10; African: r = 0.75; y = 2.66 × –0.08). Progesterone and 20-oxo-P values in Asian and African elephants were <0.15 ng/ml during the follicular phase (weeks –4 to 0) of the estrous cycle; progesterone values during the luteal phase (weeks 2–9) were 0.60 ± 0.03 and 0.53 ± 0.03 ng/ml, and the 20-oxo-P values were 2.19 ± 0.16 and 1.48 ± 0.12 ng/ml, respectively. The 20-oxo-P values of the pregnant animal, although slightly higher, were comparable to those of non-pregnant elephants during the luteal phase. Total immunoreactive 20-oxo-P values are about three times higher than those of progesterone during the luteal phase, and 5α-pregnane-3,20-dione is the major immunoreactive 20-oxo-P in the plasma of Asian and African elephants. Zoo Biol 16:403–413, 1997. © 1997 Wiley-Liss, Inc.     

Comparative facilitation and inhibition of lordosis in the guinea pig with progesterone, 5-alpha-pregnane-3,2-dione, or 3-alpha-hydroxy-5-alpha-pregnan-20-one

The major 5α-reduced metabolites of progesterone tentatively identified in neural tissue of the guinea pig were evaluated in this species for their ability to facilitate and inhibit lordosis responses of spayed females after estradiol benzoate (EB) pretreatment. 5α-Dihydroprogesterone was found to be an effective facilitative agent, but at doses of 0.05-0.3 mg administered at time intervals from 12–60 hr after estradiol, it was not as potent as progesterone. The steroids 3α-hydroxy-5α-pregnan-20-one and 5β-pregnane-3,20-dione, evaluated at only one dose level (0.18 mg) and at one time interval after estradiol (36 hr), were found to have moderate facilitative effects, but they were not as effective as 5α-dihydroprogesterone.The inhibitory influences of the metabolites studied were found to be weak relative to progesterone when given at doses of 0.6 mg 1 hr after EB. However, when 5α-dihydroprogesterone was given at a higher dose (3.6 mg) it was then found to be an effective inhibitor of the lordosis response. The results indicate that this metabolite has behavioral influences similar to those of progesterone for both facilitation and inhibition of estrus. It was suggested that the superior potency of injected progesterone may be due to mechanisms of bioavailability, including relative solubility differences of the two steroids when administered subcutaneously.     

Derivatization of progesterone to a neurally active steroid by pituitary neurointermediate lobe

The melanotrophs of the neurointermediate lobe and peptidergic terminals of the neural lobe are regulated by gamma-aminobutyric acid (GABA) via GABA-A receptors and therefore, may be important sites for the modulatory actions of neurally active steroids. These steroid compounds might be produced peripherally, synthesized de novo in the pituitary, or derivatized from circulating steroids, each pathway having different physiological implications. In the present study, we show that neurointermediate lobe tissue can derivatize progesterone to the neurally active steroid 3α-hydroxy-5α-pregnan-20-one. The neurointermediate lobe was found to be four times as active as anterior pituitary and mediobasal hypothalamus in conversion of progesterone to 3α-hydroxy-5α-pregnan-20-one; mediobasal hypothalamus was relatively more active in the production of the intermediate 5α-pregnan-3,20-dione. The identity of the compounds was confirmed by the method of serial isotopic dilution. We observed rates of synthesis in the neurointermediate lobe consistent with the production of physiologically relevant quantities of 3α-hydroxy-5α-pregnan-20-one from concentrations of progesterone which can occur naturally. In support of these findings, we demonstrate the presence of 3α-hydroxysteroid oxidoreductase in neurointermediate lobe by immunocytochemistry.     

Identification of 2 alpha-hydroxy-4-pregnene-3,20-dione in human pregnancy urine.

2 alpha-Hydroxyprogesterone (2 alpha-hydroxy-4-pregnene-3,20-dione) was identified in human late pregnancy urine by liquid-gel chromatography, GLC and GC-MS. In addition, the following 2-hydroxylated C21 steroids were found and identified as 2 zeta-hydroxy-5 zeta-pregnane-3,20-dione, 2 zeta,20 zeta-dihydroxy-4-pregnen-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha- (and 5 beta)-pregnan-20-one, two isomers of pregnane-2,3,20-triol and 2 zeta,3 zeta,16 zeta-trihydroxy-5 zeta-pregnan-20-one.     

Evidence for steroid metabolism during the in vitro induction of maturation in oocytes of Rana pipiens

Oocytes of Rana pipiens exposed to exogenous progesterone in order to induce maturation have been observed to extensively metabolize this hormone. When progesterone was injected directly into the oocytes, they did not mature, but similar metabolism of progesterone occurred. The metabolites have been tentatively identified as the 5α-reduced derivatives, 5α-pregnanedione, 5α-pregnan-20α-ol-3-one, and 5α-pregnan-3β, 20α-diol, and the pathway of conversion has been examined. Samples of these steroids obtained from commercial sources and those extracted from progesterone-treated oocytes were effective in inducing maturation when added to the medium. Evidence is presented which suggests that steroid metabolism is not a prerequisite for maturation and that the metabolites like progesterone must interact with the oocyte surface to be effective.     

Metabolism of 5β-pregnane-3,20-dione and 3β-hydroxy-5β-pregnan-20-one by Digitalis suspension cultures

Digitalis purpurea normal callus suspension culture is capable of metabolizing 5β-pregnane-3,20-dione (1) to 3β-hydroxy-5β-pregnan-20-one (2), 3α-hydroxy-5β-pregnan-20-one (3), 3β-hydroxy-5β-pregnan-20-one glucoside (7) and 3α-hydroxy-5β-pregnan-20-one glucoside (8). Digitalis purpurea habituated callus suspension culture is also capable of metabolizing 1 to 2, 3, 5β-pregnane-3β,20β-diol (5), (7), (8), 5β-pregnane-3β,20α-diol monoglucoside (9) and 5β-pregnane-3α,20α-diol monoglucoside (11). Furthermore, it was observed that 3β-hydroxy-5β-pregnan-20-one (2) is converted to 7, 9 and 11 by both suspension cultures. At the same time, 1, 3, 5 and 8 were detected in normal callus, while 5β-pregnane-3β,20α-diol (4) and 5β-pregnane-3β,20β-diol monoglucoside (10) were present in the habituated callus culture.     

Progesterone fate in rabbit cornea

Excised cornea from adult New Zealand rabbits were incubated with progesterone-4-14C in Eagle's media for 96 hr. Samples were inactivated at intervals of 24 hr incubation periods. The following metabolites of progesterone were isolated: 20 alpha-Hydroxy-4-pregnen-3-one, 20-hydroxy-4-pregnen-3-one, 5 alpha-pregnane-3,20-dione; 5 beta-pregnane-3,20-dione and 6 beta-hydroxy-4-pregnen-3,20-dione. 20 alpha-Hydroxy-pregnen-3-one was the predominant metabolite of progesterone-4-14C. A linear increase was observed throughout 96 hr. The opposite was found for 5 alpha and 5 beta pregnane-3,20-dione. Compounds remaining at the origin of the paper chromatograms contained 6 beta-hydroxy-4-pregnen-3,20-dione and other still unidentified metabolites of progesterone-4-14C. Presence of 20 alpha and 20 beta-reductase; 5 alpha and 5 beta-reductase and 6 beta-hydroxylase enzyme systems are involved in corneal progesterone metabolism. No fungal neither bacterial enzymatic biotransformation occurred in the culture media.     

Metabolism of progesterone in mouse vaginal tissue

The $\text{in vitro}$ and $\text{in vivo}$ metabolism of 1,2- ³H-progesterone was studied in estrogen-stimulated and control vaginae of ovariectomized mice. Employing two-dimensional thin-layer chromatography, gas-liquid chromatography and metabolite “trapping” techniques, the major and minor pathways for progesterone metabolism were determined $\text{in vitro}$ and shown to involve saturation of the Δ⁴-double bond to yield 5α-pregnane compounds and reduction of the C20 and C3 ketone groups to form 20α- and 3α- and 3β-hydroxy derivatives, respectively. The quantities of 20β-hydroxy metabolites and 5β-epimers that were detected were considered not to be significant. The major metabolites formed by untreated tissues following $\text{in vitro}$ incubation in the presence of both high (10^?6M) and low (10^?8M) progesterone concentrations were 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione. Although these two derivatives were also found in sizable quantities in estrogen-treated tissues, a marked increase (5-fold) in the rate of C20 ketone reduction at high progesterone concentrations (10^?6M) to yield 20α-hydroxy-4-pregnen-3-one was demonstrated. Following intravaginal administration of ³H-progesterone $\text{in vivo}$, only progesterone and 3α-hydroxy-5α-pregnan-20-one were retained in appreciable quantities through 2 hr, suggesting rapid loss of 20α-hydroxy-4-pregnen-3-one and the 5α-pregnanediols from this tissue under $\text{in vivo}$ conditions.     

Interaction of progestins with steroid receptors in human uterus

Norethindrone (17β-hydroxy-19-nor-17α-pregn-4-en-20-yn-3-one) and norethindrone acetate (17β-acetoxy-19-nor-17α-pregn-4-en-20-yn-3-one) interfered to a varying degree, by competitive inhibition, with the binding of progesterone and oestradiol to respective cytoplasmic receptors in the human uterus. Progesterone binding to 4S macromolecule was saturable and co-specific for progestins. Competitors like norgestrel (17β-hydroxy-18-methyl-19-nor-17α-pregn-4-en-20-yn-3-one), 19-norprogesterone, medroxyprogesterone acetate (17α-acetoxy-6α-methylpregn-4-ene-3,20-dione) and compound R₅₀₂₀ (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) possessed higher binding affinities for the progestin receptor. The dissociation constant (K_d) for the progesterone–receptor interaction was 0.6–1.6nm and the receptor concentration ranged between 6600 and 8200 sites/cell. Norethindrone and norethindrone acetate competed for the progesterone receptor with inhibition constants (K_i) of 6.8 and 72nm respectively. Gradient displacement and competitive-receptor assays indicated that norethindrone acetate-binding affinity for progestin receptor was approximately one-tenth that of norethindrone and progesterone. The progestins also inhibited oestradiol binding to 4.6S oestrogenic receptor by 8–12%, involving interaction at the oestradiol-binding site with a calculated K_i value of 0.5–0.8μm. The competitive interaction of progestins with steroid receptors may be of putative importance in explaining the progestin action at the target site.     

15β-hydroxysteroids (Part IV). Steroids of the human perinatal period: The synthesis of 3α,15β,17α-trihydroxy-5α-pregnan-20-one and its A/B-ring configurational isomers

In recent years several 15β-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3α,15β,17α-trihydroxy-5β-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3ξ,5ξ-isomers, namely 3α,15β,17α-trihydroxy-5α-pregnan-20-one (3), 3β,15β,17α-trihydroxy-5α-pregnan-20-one (7) and 3β,15β,17α-trihydroxy-5β-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3β,15β-Diacetoxy-17α-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15β,17α-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15β-acetoxy-3β,17α-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15β-acetoxy-3β,17α-dihydroxy-5α-pregnan-20-one (13) a common intermediate for the synthesis of the 3β(and α),5α-isomers. Hydrolysis of the 15β-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15β-acetoxy-17α-hydroxy-5α-pregnan-3,20-dione (14) which on reduction with -Selectride and hydrolysis of the 15β-acetate gave 3. Finally, hydrogenation of 4 gave 15β,17α-dihydroxy-5β-pregnan-3,20-dione (10) which on reduction with -Selectride gave 8.     

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.     

Human placental 3beta-hydroxysteroid dehydrogenase: delta5-isomerase. Demonstration of an intermediate in the conversion of 3beta-hydroxypregn-5-en-20-one to pregn-4-ene-3,20-dione.

3beta-Hydroxypregn-5-en-20-one (pregnenolone) and NAD+ were incubated with a solubilized preparation of the coupled enzyme 3beta-hydroxysteroid:NAD(P) oxidoreductase-3-ketosteroid delta4,delta5-isomerase (3beta-hydroxysteroid dehydrogenase: delta5-isomerase) from the mitochondrial fraction of human placenta. Unconverted pregnenolone, pregn-4-ene-3,20-dione (rogesterone), and a small but detectable amount of pregn-5-ene-3,20-dione were isolated from the medium by Sephadex LH-20 chromomatography. The identification of pregn-5-ene-3,20-dione, confirmed by mass fragmentography, has provided the first direct evidence for the formation of the hypothetical delta5,3-ketone intermediate in the conversion of pregnenolone to progesterone. When tritium-labeled pregnenolone and [4-14C]pregnenolone were incubated simultaneously the 3H:14C ratio in isolated pregn-5-ene-3,20-dione was 4.6 times greater than in isolated progesterone and pregnenolone, indicating a kinetic isotope effect in the enzymatic isomerization of tritium-labeled pregn-5-ene-3,20-dione. Exposure of the enzyme to two steroids which inhibit the overall enzyme reaction, 2alpha-cyano-17beta-hydroxy-4,4,17alpha-trimethylandrost-5-en-3-one (cyanoketone) and 3-hydroxyestra-1,3,5(10),6,8-pentaen-17-one (equilenin), increased the relative yield of labeled pregn-5-ene-3,20-dione as well as the recovery of radioactivity remaining as unconverted pregnenolone, suggesting that both the dehydrogenase and isomerase activities were inhibited. Exposure of the enzyme to equilenin increased the ratio of isolated pregn-5-ene-3,20-dione radioactivity to progesterone radioactivity as progesterone synthesis was inhibited. Equilenin also diminished the tritium isotope effect on the isomerase reaction. Both findings suggest that it is possible to inhibit the isomerase to a greater extent than the dehydrogenase. In order to measure the rate of progesterone produced by the coupled enzymes, we have modified a radiochemical method which involves precipitation of pregnenolone by digitonin. Digitonin precipitation proved to be effective in separating unconverted pregnenolone from the steroid products of both enzyme reactions, progesterone and pregn-5-ene-3,20-dione. Neither the steroidal inhibitors nor the kinetic isotope effect altered the accuracy of the method for routine measurement of the overall rate of conversion of delta5,3beta-hydroxysteroid to delta4,3-ketosteroid.