Steroid metabolism by rat nasal mucosa: studies on progesterone and testosterone

The metabolism of [4-14C]progesterone and [4-14C]testosterone by slices of the nasal mucosa from rats was studied. As shown by gas chromatography-mass spectrometry there was a preferential formation of reduced progesterone-metabolites (5 alpha-pregnane-3,20-dione, 3 alpha- and 3 beta-hydroxy-5 alpha-pregnane-20-one, 20 alpha- and 20 beta-hydroxypregn-4-en-3-one, 2 alpha,3 alpha-dihydroxy-5 alpha-pregnane-20-one, 3 alpha,16 alpha-dihydroxy-5 alpha-pregnane-20-one) and reduced testosterone-metabolites (4-androstene-3,17-dione, 5 alpha-dihydrotestosterone, 3 alpha-hydroxy-5 alpha-androstane-17-one, and 5 alpha-androstane-3 alpha, 17 beta-diol, 2 alpha-hydroxy-5 alpha-dihydrotestosterone, 5 alpha-androstane-2 alpha,3 alpha, 17 beta-triol) indicating the presence of 5 alpha-reductase, 3 alpha-, 3 beta-, 17 beta-, 20 alpha- and 20 beta-hydroxysteroid oxidoreductase activities in this tissue. Progesterone-metabolites hydroxylated at positions 2 alpha, 6 alpha, 6 beta, 15 alpha and 16 alpha and testosterone-metabolites hydroxylated at positions 1 beta, 2 alpha, 6 beta, 15 beta and 16 alpha were also identified, indicating the presence of several steroid hydroxylases in the nasal mucosa. Autoradiography of the nasal region of rats injected with [4-14C]progesterone or [4-14C]testosterone showed a selective localization of radioactivity in the mucosa covering the olfactory region of the nasal cavity.     

Studies on an extract of rat testicular microsomal fraction that catalyses the transformation of progesterone into 17-hydroxyprogesterone and androgens

The supernatant obtained by centrifugation of Triton N-101-treated freeze-dried rat testicular microsomal fraction at 105000g(av.) for 2h transformed progesterone into testosterone via 17-hydroxypregn-4-ene-3,20-dione and androst-4-ene-3,17-dione. Hydroxylation at C-17 of 3beta-hydroxypregn-5-en-20-one and deoxycorticosterone was not observed. Non-haem iron protein, cytochrome P-450 and material with NADPH dehydrogenase activity were precipitated by 40% saturation of the supernatant with ammonium sulphate; however, it was not possible to establish the participation of these substances in the 17alpha-hydroxylase and side-chain-cleavage activities also present in the precipitate. The results of gel-filtration chromatography indicated that the Triton N-101 extract consisted primarily of a suspension of small particles of microsomes and that the progesterone 17-hydroxylase and the 17-hydroxypregn-4-ene-3,20-dione side-chain-cleavage enzyme were not in true solution.     

Steroid hydroxylation by Whetzelinia sclerotiorum, Phanerochaete chrysosporium and Mucor plumbeus

The fungi Whetzelinia sclerotiorum ATCC 18687, Phanerochaete chrysosporium ATCC 24725 and Mucor plumbeus ATCC 4740 were examined for their ability to perform steroid biotransformations under single phase, pulse feed conditions. The steroids 3beta-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) (1), 17beta-hydroxyandrost-4-en-3-one (testosterone) (5), 3beta-hydroxypregn-5-en-20-one (pregnenolone) (3), pregn-4-ene-3,20-dione (progesterone) (9), 17alpha,21-dihydroxypregn-4-ene-3,11,20-trione (cortisone) (11), 17alpha,21-dihydroxypregna-1,4-diene-3,11,20-trione (prednisone) (14), and 3-hydroxyestra-1,3,5(10)-trien-17-one (estrone) (15) were fed to each fungus. The production of a number of novel metabolites is reported. Of the fungi investigated W. sclerotiorum performed the most interesting biotransformations and had a clear propensity for 2beta, 6beta/7beta and 15beta/16beta hydroxylations. P. chrysosporium was more prone functionalize steroids in the allylic position. Oxygen insertion at C-14 by M. plumbeus is reported for the first time. All three micro-organisms exhibited redox activity.     

Zymogen secretion and phospholipid metabolism in the pancreas. Phospholipids of the zymogen granule

1. The metabolism of [4-(14)C]pregnenolone to androst-16-enes has been studied in short-term incubations of boar testis tissue. With fresh tissue androsta-5,16-dien-3beta-ol (8%) and 5alpha-androst-16-en-3beta-ol (2%) were formed. Tissue that had been stored at -20 degrees C was still capable of metabolizing pregnenolone to androsta-5,16-dien-3beta-ol. 2. NADPH was essential for the formation of androsta-5,16-dien-3beta-ol from pregnenolone; NADH had less activity and ATP was not necessary for the reaction. 3. [4-(14)C]Androsta-5,16-dien-3beta-ol, prepared biosynthetically from [4-(14)C]pregnenolone, was shown to be converted by boar testis preparations into androsta-4,16-dien-3-one (31%) if NAD(+) was present or into 5alpha-androst-16-en-3beta-ol (4%) if NADPH was present. 4. 17alpha-Hydroxyandrost-4-en-3-one and 3beta,17alpha-dihydroxypregn-5-en-20-one were considered as possible precursors for androst-16-ene formation, but both were shown to be ineffective. 5. No radioactivity was incorporated into androst-5-en-3beta-ol used to trap any corresponding (14)C-labelled compound formed from [4-(14)C]pregnenolone.     

Synthesis of steroids by echidna (Tachyglossus aculeatus) testis and the effects of gonadotrophin administration.

After administration of hCG and PMSG to male echidnas (in non-breeding state) the testis to body weight ratio increased 3-fold and the diameter of the seminiferous tubules doubled, but spermatogenesis was not induced. The major conversion product of testicular homogenate with [4-14C]progesterone as substrate was 17 alpha-hydroxyprogesterone (4% in untreated and 29% in treated echidnas). Testosterone (5%), androstenedione (16%) and 17 alpha,20 beta-dihydroxypregn-4-en-3-one (7%) were also obtained in gonadotrophin-treated animals. In untreated animals these steroids were present in minor amounts (less than 1%).     

Comparison of plasma and testicular testosterone levels during the active season in the common garter snake, Thamnophis sirtalis (L)

Plasma and testicular testosterone levels were determined during three periods (spring, summer and autumn) of the active season in the common garter snake, Thamnophis sirtalis. Plasma testosterone levels were highest in spring and lowest in summer. Testicular testosterone levels were higher in spring than autumn but not significantly different in spring than summer or in summer than autumn. This study presents further evidence that in reptiles, plasma and testicular androgen cycles are not necessarily parallel.     

An appetite suppressant from Hoodia species

Studies conducted at the Council for Scientific and Industrial Research (CSIR, South Africa) identified extracts from Hoodia species, in particular Hoodia pilifera and Hoodia gordonii, as possessing appetite suppressing properties. Two pregnane glycosides were isolated by fractionation of the dried stems of H. gordonii. Their structures were determined as 3beta-[beta-D-thevetopyranosyl-(1-->4)-beta-D- cymaropyranosyl-(1-->4)-beta-D-cymaropyranosyloxy]-12beta-tigloyloxy-14beta-hydroxypregn-5-en-20-one (1) and 3beta-[beta-D-cymaropyranosyl-(1-->4)-beta-D-6-thevetopyranosyl-(1-->4)-beta-D-cymaropyranosyl-(1-->4)-beta-D-cymaropyranosyloxy]-12beta-tigloyloxy-14beta-hydroxypregn-5-en-20-one (2) on the basis of spectroscopic studies and conversion to known compounds. Compounds 1 and 2 were also isolated from H. pilifera. Compound 1 was tested for its appetite suppressant properties in rats by oral gavage at 6.25-50 mg/kg and the results showed that all doses resulted in a decrease of food consumption over an eight day period and a body mass decrease when compared to the control sample receiving only the vehicle. In a comparative study against a fenfluramine control sample, compound 1 resulted in a reduction in food intake over the study period, with a concomitant overall decrease in body weight while fenfluramine resulted in a small decrease in food intake, but an increase in body weight (though less than control group) over the same period of time.     

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.     

Utilization and metabolic effects of acetaldehyde and ethanol in the perfused rat liver

1. The formation of the two 16-unsaturated alcohols 5alpha-androst-16-en-3alpha-ol and 5alpha-androst-16-en-3beta-ol from [5alpha-(3)H]5alpha-androst-16-en-3-one has been demonstrated in boar testis homogenates. 2. The optimum yield (23%) of the 3alpha-alcohol was obtained in the presence of NADPH, whereas that for the 3beta-alcohol (74%) was obtained when NADH was the added cofactor. 3. The two alcohols were not interconvertible. 4. Prolonged storage of boar testis tissue at -20 degrees C abolished the ability to form all androst-16-enes except androsta-4,16-dien-3-one from [4-(14)C]progesterone. 5. The production of 5alpha-androst-16-en-3-one and the two alcohols from [7alpha-(3)H]androsta-4,16-dien-3-one only occurred when fresh tissue was used, whereas reduction of [5alpha-(3)H]5alpha-androst-16-en-3-one was unaffected by storage of testis at -20 degrees C. 6. NADPH was the preferred cofactor for the reduction of androsta-4,16-dien-3-one. 7. The previously established conversion of androsta-5,16-dien-3beta-ol into androsta-4,16-dien-3-one was shown to be reversible, NADH and NADPH being equally effective cofactors. 8. Pathways of biosynthesis of 5alpha-androst-16-en-3alpha- and 3beta-ols, with the C(19) 3-oxo steroids as intermediates, are presented.     

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.     

Nucleosteroids: carbocyclic nucleoside analogs of androst-4-en-17 beta-ol

Steroidal nucleoside analogs were synthesized starting from testosterone. By reduction of the oxime of 17 beta-hydroxy-androst-4-en-3-one (testosterone), a mixture of the two amino epimers of C-3 were obtained. The 3 alpha-amino-androst-4-en-17 beta-ol was crystallized in 73% yield and coupled with 5-amino-4,6-dichloropyrimidine to give 3 alpha-(5'-amino-4'-chloro-pyrimidin-6'-yl)amino-androst-4-en-17 beta-ol. This compound was treated with triethyl orthoformate in acid media to give the corresponding purinyl steroid adduct 3 alpha-(6'-chloro-purin-9'-yl)-androst-4-en-17 beta-ol in 98% yield. This substance, in turn, was converted with good yield into the 6'-thio, 6'-methylamino, and 6'-diethyl aminopurinyl derivatives through nucleophilic reactions at C-6 of the purine nucleus.     

Seasonal variation in the activity of the Leydig cells in Egyptian Nubian goat (Zaraibi) bucks

Ten Egyptian Nubian goat bucks were used to evaluate the effect of season on testicular hormonal activity and ultrastructure. Parameters were recorded for 7 consecutive weeks in the middle of the four seasons, with blood samples being collected weekly. At the end of each of these seasons, testicular biopsies were obtained surgically for histological and cytological studies. Season had a significant effect on plasma testosterone concentration, being at its lowest level (P < 0.01) during winter and spring (1.2 and 2.6 ng/ml, respectively), while at its highest during summer (10 ng/ml). The effect of season on plasma LH concentration was higher (P < 0.01) in autumn (2.9 mIU/ml) and less in spring and summer (0.4 mIU/ml). Season of the year influenced the percentage of sectional tissue area occupied by the seminiferous tubules and interstitial tissue. Seminiferous tubules occupied the majority of the testicular tissue during winter (76.6%), with the least being occupied during spring (49.8%). The thickness of the seminiferous tubules was maximal during autumn, followed by summer (53 and 36 μm, respectively). In summer the Leydig cells contained abundant smooth endoplasmic reticulum (sER), while some areas of the cytoplasm were occupied exclusively by tubular sER, arranged in parallel—indicating the highest activity of these cells. A characteristic multivesicular structure with numerous large lipid droplets and vacuoles was recorded in the Leydig cells during spring and winter, denoting low or even arrested activity of the cells. It could be concluded that season influences the activity of the Leydig cells of Egyptian Nubian bucks, and this is reflected by their ultrastructure and secretive activity.     

Synthesis and reduction of steroid C-20 alkylidene cyanoacetates.

The geometry of the condensation products between ethyl cyanoacetate and 20-ketosteroids (5 alpha-pregnane-20-one, 3 beta-hydroxypregn-5-en-20-one and 3 beta-acetoxypregn-5-en-20-one) was established by NMR spectra. Reduction of these steroid C-20 alkylidene cyanoacetates was shown to afford one of the two possible 20-C epimers, which seen to correspond to the 20 beta-methyl configuration.     

Subcellular distribution and properties of progesterone (delta4-steroid) 5alpha-reductase in rat medial basal hypothalamus.

The subcellular distribution and properties of rat hypothalamic progesterone 5 alpha-reductase, which accelerates the conversion of progesterone to 5 alpha-pregnane-3,20-dione, have been investigated by utilizing 3H-labeled substrate and a reverse isotopic dilution assay system. The enxymic activity was associated primarily with a cell debris-membranes fraction deribed from the 100 x g pellet. This fraction contained mainly membrane-like particulates and was free of nuclei. Little or no activity was associated with the purified nuclei. The hypothalamic 5 alpha-reductase was stimulated by NADPH but not by NADH. The reaction proceeded optimally over a pH range of 6.0 to 7.2 and at a temperaturhe substrate specificity of the enzyme for other delta 4-3-ketosteroids and the ability of these steroids to inhibit the 5 alpha reduction of [1,2-3H]progesterone as well as the effect of 17 beta-estradiol were also studied. 20 alpha-hydroxypregn-4-en-3-one was more reactive that progesterone, while testosterone was the least reactive. The estimated Km for 20 alpha-hydroxypregn-4-en-3-one was 8.6 +/- 1.9 x 10(-7) M, and for testosterone, 1.6 +/- 1.4 x 10(-5) M. The inhibition studies indicate that 20 alpha-hydroxypregn-4-en-3-one and 17 beta-estradiol are competitive and noncompetitive inhibitors, respectively, of the 5 alpha reduction of progesterone with Ki of 6.0 +/- 3.0 x 10(-8) M for 20 alpha-hydroxypregn-4-en-3-one and Kii (intercept inhibition constant) of 2.6 +/- 0.7 x 10(-5) M and Kis (slope inhibition constant) of 3.6 +/- 0.6 x 10(-5) M for 17 beta-estradiol. Testosterone is a poor competitive inhibitor of the reaction.     

Base promoted air oxidation of 13beta-ethyl-11-methylenegon-4-en-17-one.

The structure of 13-ethyl-11-methylene-18,19-dinor-17alpha-pregn-4-en-20-yn-16beta,17-diol (3, 16beta-OH desogestrel), a by-product obtained in the last step of the synthesis of desogestrel (1) by reaction of monolithium acetylide-ethylenediamine complex with 13beta-ethyl-11-methylenegon-4-en-17-one (2), is here reported. The structural assignments were supported by NMR 1H-, 13C-, 1H-1H COSY, 1H-13C HSQC, COLOC) and mass spectroscopy, and the configuration at the C-16 and C-17 stereocentres was established by X-ray crystallography. When the same 17-ketoderivative 2 was treated with a non-alkylating base, such as potassium tert-butoxide, instead of the expected 16-hydroxylated ketone, a dimeric product, 13beta-ethyl-16-[2'-(des-D-13"-carboxy-13"beta-ethyl-11"-methylenegon-4"-en-14"-yl)-ethyliden]-11-methylenegon-4-en-17-one (4), was isolated in good yield; it was characterized by NMR, mass, ultraviolet spectroscopy, and chemical transformations. Compounds 3 and 4 originate from the high reactivity of the 16-methylenic position of the 17-keto substrate (2) toward molecular oxygen under basic conditions.     

Production of testosterone from progesterone by rat testicular microsomes without release of the intermediates 17 alpha-hydroxyprogesterone and androstenedione

It has been shown that during the in vitro conversion of progesterone to androstenedione, 17 alpha-hydroxyprogesterone is not an obligatory intermediate which equilibrates with freely diffusible steroids in the incubation medium. Recently a cytochrome P-450 was purified that catalyzed, in addition to hydroxylase/lyase activities, reduction of androstenedione to testosterone. In order to determine whether progesterone could be transformed to testosterone without both intermediates (17 alpha-hydroxyprogesterone and androstenedione) being equilibrated with steroids in the medium, several double-label double-substrate experiments were performed. When rat microsomes were incubated with an equimolar mixture of [14C]progesterone and 17 alpha-hydroxy[3H]progesterone, androstenedione was isolated with a 11-fold higher 14C/3H ratio than 17 alpha-hydroxyprogesterone, indicating that androstenedione could not be produced from free, diffusible 17 alpha-hydroxyprogesterone. Incubation of an equimolar mixture of 17 alpha-hydroxy[3H]progesterone and [14C]androstenedione with testicular microsomes resulted in the incorporation of 3-4-fold more 17 alpha-hydroxyprogesterone into testosterone than of androstenedione, although the latter is the immediate precursor of testosterone. In an experiment in which equimolar concentrations of [3H]progesterone and [14C]androstenedione were incubated with testicular microsomes, the large pool of progesterone inhibited competitively lyase activity, but still the label of progesterone was incorporated into testosterone to the same extent as that of androstenedione. These results indicate that testosterone can be produced by immature rat testicular microsomes from added progesterone on an organized unit without the intermediates equilibrating with the incubation medium.     

The facile bioconversion of testosterone by alginate-immobilised filamentous fungi

The potential for biotransformation of the substrate 17β-hydroxyandrost-4-en-3-one (testosterone) by six filamentous fungi, namely, Rhizopus oryzae ATCC 11145, Mucor plumbeus ATCC 4740, Cunninghamella echinulata var. elegans ATCC 8688a, Aspergillus niger ATCC 9142, Phanerochaete chrysosporium ATCC 24725 and Whetzelinia sclerotiorum ATCC 18687, was investigated. In this study both free cells and macerated mycelia immobilised in calcium alginate were utilised and the results (products, % yields, % transformation) were compared. In general the encapsulated cells of the microorganisms effectively generated products similar to those found using free cells. However, with immobilised macerated mycelia, isolation of the transformation products was expedited by the simple work up procedure, and their purification was facilitated by the absence of fungal secondary metabolites. Twenty seven analogues of testosterone were generated, wherein the androstane skeleton was functionalised at C-1β, -2β, -6β, -7α, -11α, -14, -15α, -15β and -16β by the moulds. Redox chemistry was also observed. Seven of the analogues, 6β,11α,17β-trihydroxyandrost-4-en-3-one, 6β,14α,17β-trihydroxyandrost-4-en-3-one, 2,6β-dihydroxyandrosta-1,4-diene-3,17-dione, 2β,16β-dihydroxyandrost-4-ene-3,17-dione, 2β,6β-dihydroxyandrost-4-ene-3,17-dione, 2β,15β,17β-trihydroxyandrost-4-en-3-one and 2β,3α,17β-trihydroxyandrost-4-ene, were novel compounds. Five others, namely, 7α,17β-dihydroxyandrost-4-en-3-one, 6β,14α-dihydroxyandrost-4-ene-3,17-dione, 15α,17β-dihydroxyandrost-4-en-3-one, 16β,17α-dihydroxyandrost-4-en-3-one and 2β,16β,17β-trihydroxyandrost-4-en-3-one, were fully characterised for the first time.     

Synthesis of [19- 2H3]-analogs of dehydroepiandrosterone and pregnenolone and their sulfates

Deuterated analogs of pregnenolone and pregnenolone sulfate with three atoms of deuterium in position 19 were prepared. The synthetic approach was developed on derivatives of dehydroepiandrosterone, where initial intermediates were well characterized, and then applied to the pregnenolone series. Starting 19-hydroxy compounds were transformed into 3alpha,5-cycloderivatives to simplify the Jones oxidation into the corresponding 19-oic acids. After oxidation, rearrangement to 3-hydroxy-5-enes, and suitable protection, two deuterium atoms were introduced by lithium aluminum deuteride reduction. Mesylate exchange by iodide in the presence of zinc and deuterium oxide added third deuterium atom. Deprotection gave title analogs with about 93-95% content of d3-derivative, the rest was mainly not fully deuterated d2-analogue as followed from the mass spectra analysis. Thus, 3beta-hydroxy[19-2H3]androst-5-en-17-one was prepared in 14 steps from 19-hydroxy-17-oxoandrost-5-en-3beta-yl acetate in 8.9% yield, the analogous sequence in the pregnenolone series gave 3beta-hydroxy[19-2H3]pregn-5-en-20-one in 7.3% yield. Corresponding sulfates were prepared via pyridinium salts in 53 and 57% yields, respectively. Fully assigned NMR data of selected pregnenolone derivatives were given.     

The compartmentation of non-esterified and esterified cholesterol in the superovulated rat ovary

1. The specific radioactivities of non-esterified and esterified cholesterol, progesterone and 20alpha-hydroxypregn-4-en-3-one were determined in slices of superovulated rat ovary after incubation with [1-(14)C]acetate in vitro for various times. The specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal, and (during the fourth hour of incubation) exceeded those of the non-esterified cholesterol and the esterified cholesterol by factors of 2.8 and 7.6 respectively. 2. After separation of homogenates of superovulated rat ovary slices previously incubated with [(14)C]acetate into subcellular fractions by differential centrifugation, the specific radioactivities of non-esterified cholesterol in the cytosol, mitochondria, lipid-containing storage granules and microsomal fraction were 1220, 1510, 1420 and 4020d.p.m./mumol respectively; the corresponding values for the specific radioactivity of the esterified cholesterol were 600, 700, 730 and 760d.p.m./mumol. The specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal in all fractions; the corresponding mean specific radioactivity of progesterone+20alpha-hydroxypregn-4-en-3-one was 6150d.p.m./mumol. 3. By using glutamate dehydrogenase and cytochrome (a+a(3)) as mitochondrial markers, the presence of cholesterol side-chain cleavage enzyme was demonstrated in microsomal fraction free of mitochondrial contamination. 4. The specific radioactivities of ovarian non-esterified and esterified cholesterol, progesterone and 20alpha-hydroxypregn-4-en-3-one were determined up to 8h after the intravenous injection of [4-(14)C]cholesterol into superovulated rats. At all times the specific radioactivities of progesterone and 20alpha-hydroxypregn-4-en-3-one were equal to the specific radioactivity of non-esterified cholesterol and exceeded, by up to 3.3-fold, that of the esterified cholesterol. 5. It is concluded that non-esterified cholesterol formed from [(14)C]acetate in the endoplasmic reticulum equilibrates slowly with non-esterified cholesterol in other subcellular fractions, and is preferentially converted into steroids. Such a mechanism presupposes the operation of a microsomal cholesterol side-chain cleavage enzyme using non-esterified cholesterol as its substrate. Unrelated evidence is presented in support of the existence of such an enzyme. The results are discussed in the light of other biochemical and electron-microscopic findings relating to the compartmentation of cholesterol in steroidogenic tissues.