Conversion of Androstenedione and Androstadienedione by Sterol-Degrading Bacteria

The composition of steroid metabolites formed during the conversion of androstenedione and androstadienedione, products of the degradation of sterol side chains by soil and mutant strains of the bacterial genera Mycobacterium and Protaminobacter, was studied. Testololactone was absent from the conversion products. This favors the idea of different cleavage pathways of steroid ring D in bacteria and fungi. Very small amounts of two new 14-hydroxy derivatives with a cleaved B ring were isolated after the conversion of androstenedione by soil strains. It was shown that a mutant Mycobacterium smegmatis strain, as well as wild strains, could perform 14-hydroxylation of steroids. It is suggested that cleavage of the steroid nucleus at the side of rings D and C starts with the introduction of a 14-hydroxy group followed by dehydration.     

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.     

Selection of Mycobacterium sp. strains with capacity to biotransform high concentrations of β-sitosterol

In this work, phytosterol-biotransforming strains were selected from Mycobacterium sp., using a high concentration of beta-sitosterol. The selection was made by culturing the strains in a medium enriched with 14 g beta-sitosterol/l as the unique source of carbon. During 2 months, the bacterial cultures were transferred successively. The extraction of the biotransformation products was made with methanol and ethyl acetate. The qualitative and quantitative analysis was made by means of thin-layer chromatography, gas-liquid chromatography (GLC) and GLC-mass spectrometry. Under these conditions, it was observed that after seven transfers, the strains MYcobacterium sp. MB-3683 and the Mycobacterium fortuitum B-11045 increased their biotransformation capacity from 20% to 64% and from 34% to 55%, respectively. The products in the highest proportion identified for each trial were androstenedione and androstadienedione. The results suggest that the high substrate concentration could be a selective mechanism to obtain strains more efficient in the biotransformation of beta-sitosterol into steroidal bases.     

The involvement of cytochrome P-450 in hepatic microsomal steroid hydroxylation reactions supported by sodium periodate, sodium chlorite, and organic hydroperoxides.

The mechanism of steroid hydroxylation in rat liver microsomes has been investigated by employing NaIO4, NaClO2, and various organic hydroperoxides as hydroxylating agents and comparing the reaction rates and steroid products formed with those of the NADPH-dependent reaction. Androstenedione, testosterone, progesterone, and 17beta-estradiol were found to act as good substrates. NaIO4 was by far the most effective hydroxylating agent followed by cumene hydroperoxide, NADPH, NaClO2, pregnenolone 17alpha-hydroperoxide, tert-butyl hydroperoxide, and linoleic acid hydroperoxide. Androstenedione was chosen as the model substrate for inducer and inhibitor studies. The steroid was converted to its respective 6beta-, 7alpha, 15-, and 16alpha-hydroxy derivatives when incubated with microsomal fractions fortified with hydroxylating agent. Evidence for cytochrome P-450 involvement in androstenedione hydroxylation included a marked inhibition by substrates and modifiers of cytochrome P-450 and by reagents which convert cytochrome P-450 to cytochrome P-420. The ratios of the steroid products varied according to the type of hydroxylating agent used and were also modified by in vivo phenobarbital pretreatment. It was suggested that multiple forms of cytochrome P-450 exhibiting different affinities for hydroxylating agent are responsible for these different ratios. Horse-radish peroxidase, catalase, and metmyoglobin could not catalyze androstenedione hydroxylation. Addition of NaIO4, NaClO2, cumene hydroperoxide and other organic hydroperoxides to microsomal suspensions resulted in the appearance of a transient spectral change in the difference spectrum characterized by a peak at about 440 nm and a trough at 420 nm. The efficiency of these oxidizing agents in promoting steroid hydroxylation in microsomes appeared to be related to their effectiveness in eliciting the spectral complex. Electron donors, substrates, and modifiers of cytochrome P-450 greatly diminished the magnitude of the spectral change. It is proposed that NaIO4, NaClO2, and organic hydroperoxides promote steroid hydroxylation by forming a transient ferryl ion (compound I) of cytochrome P-450 which may be the common intermediate hydroxylating species involved in hydroxylations catalyzed by cytochrome P-450.     

Fragments formed by the side chain cleavage of a 20-aryl analog of 20alpha-hydroxycholesterol by adrenal mitochondria.

An analog of 20alpha-hydroxycholesterol, (20R)-20-phenyl-5-pregnene-3beta,20-diol, which is completely substituted at C-22 was prepared with radioisotopes at various positions. The analog labeled with 3H at C-M and 14C at C-4 and C-IU was converted into radioactive pregnenolone by an enzyme preparation derived from adrenal mitochondria. Cleavage of the phenyl analog labeled with 3H in the aromatic ring by the same enzyme preparation led to the formation of [3H]phenol. Using the substrate doubly labeled with 14C at C-4 and 3H in the aromatic ring, it appeared that the products of the reactions, pregnenolone and phenol, were formed in equal amounts. During incubation of the side chain labeled substrate, another labeled fragment was formed. It was identified as acetophenone, a product resulting from cleavage of the C17,20 bond. The steroidal fragment corresponding to this C8 ketone was traced using nuclear label analog. From its nonpolar chromatographic properties it appears to be a C-17-deoxy-C19 steroid.     

Influence of substituents at C11 on hydroxylation of progesterone analogs by Bacillus sp

Transformation of progesterone analogs viz., progesterone, 11 alpha-, 11 beta-hydroxyprogesterones and 11-ketoprogesterone by Bacillus sp. is reported. Both progesterone and 11-ketoprogesterone were hydroxylated while the C(11) epimeric alcohols of progesterone remained unaltered under the conditions used. The major bioconverted products obtained from progesterone and 11-ketoprogesterone were characterized as 6 beta- and 14 alpha-hydroxyprogesterones and 14 alpha-hydroxy-11-ketoprogesterone respectively by mass and NMR spectra. The conversion of 11-ketoprogesterone to its 14 alpha-hydroxy derivative by microbe is unprecedented and novel. Moreover, hydroxylation at 6 beta- and 14 alpha-positions of progesterone by Bacillus sp. is significant. In conclusion, the present data showed that the substituents at 11-position of steroid play important role on hydroxylation by microbe.     

Degradation of pyrene, benz[a]anthracene, and benzo[a]pyrene by Mycobacterium sp. strain RJGII-135, isolated from a former coal gasification site.

The degradation of three polycyclic aromatic hydrocarbons (PAH), pyrene (PYR), benz[a]anthracene (BAA), and benzo[a]pyrene (BaP), by Mycobacterium sp. strain RJGII-135 was studied. The bacterium was isolated from an abandoned coal gasification site soil by analog enrichment techniques and found to mineralize [14C]PYR. Further degradation studies with PYR showed three metabolites formed by Mycobacterium sp. strain RJGII-135, including 4,5-phenanthrene-dicarboxylic acid not previously isolated, 4-phenanthrene-carboxylic acid, and 4,5-pyrene-dihydrodiol. At least two dihydrodiols, 5,6-BAA-dihydrodiol and 10,11-BAA-dihydrodiol, were confirmed by high-resolution mass spectral and fluorescence analyses as products of the biodegradation of BAA by Mycobacterium sp. strain RJGII-135. Additionally, a cleavage product of BAA was also isolated. Mass spectra and fluorescence data support two different routes for the degradation of BaP by Mycobacterium sp. strain RJGII-135. The 7,8-BaP-dihydrodiol and three cleavage products of BaP, including 4,5-chrysene-dicarboxylic acid and a dihydro-pyrene-carboxylic acid metabolite, have been isolated and identified as degradation products formed by Mycobacterium sp. strain RJGII-135. These latter results represent the first example of the isolation of BaP ring fission products formed by a bacterial isolate. We propose that while this bacterium appears to attack only one site of the PYR molecule, it is capable of degrading different sites of the BAA and BaP molecules, and although the sites of attack may be different, the ability of this bacterium to degrade these PAH is well supported. The proposed pathways for biodegradation of these compounds by this Mycobacterium sp. strain RJGII-135 support the dioxygenase enzymatic processes reported previously for other bacteria. Microorganisms like Mycobacterium sp. strain RJGII-135 will be invaluable in attaining the goal of remediation of sites containing mixtures of these PAH.     

总状毛霉对4-烯-3-酮甾体的生物转化研究

从土样中筛选到一株能转化甾体的菌株,经形态观察,鉴定为总状毛霉(Mucor racemosus)。首次利用该菌株对4-烯-3-酮类甾体衍生物进行生物转化,目的是合成具有潜在活性的羟基类4-烯-3-酮衍生物。转化条件为27℃,220r/min振荡培养4d。转化产物经乙酸乙酯萃取,用硅胶柱层析法分离,通过红外、质谱和核磁分析确定了甾体转化产物的化学结构。黄体酮生物转化得到的产物是14α-羟基-4-孕甾烯-3,20-二酮和7α,14α-二羟基-4-孕甾烯-3,20-二酮;4-雄烯二酮的转化产物是14α-羟基-雄甾-4-烯-3,17-二酮1、4α,17β-二羟基-雄甾-4-烯-3-酮和6α,17β-二羟基-雄甾-4-烯-3-酮。研究结果表明总状毛霉具有转化甾体的能力,对4-烯-3-酮类甾体进行生物转化的主要产物是14α-羟基甾体衍生物。     

A patchwork pathway for oxygenase‐independent degradation of side chain containing steroids

The denitrifying betaproteobacterium Sterolibacterium denitrificans serves as model organism for studying the oxygen‐independent degradation of cholesterol. Here, we demonstrate its capability of degrading various globally abundant side chain containing zoo‐, phyto‐ and mycosterols. We provide the complete genome that empowered an integrated genomics/proteomics/metabolomics approach, accompanied by the characterization of a characteristic enzyme of steroid side chain degradation. The results indicate that individual molybdopterin‐containing steroid dehydrogenases are involved in C25‐hydroxylations of steroids with different isoprenoid side chains, followed by the unusual conversion to C26‐oic acids. Side chain degradation to androsta‐1,4‐diene‐3,17‐dione (ADD) via aldolytic C–C bond cleavages involves acyl‐CoA synthetases/dehydrogenases specific for the respective 26‐, 24‐ and 22‐oic acids/‐oyl‐CoAs and promiscuous MaoC‐like enoyl‐CoA hydratases, aldolases and aldehyde dehydrogenases. Degradation of rings A and B depends on gene products uniquely found in anaerobic steroid degraders, which after hydrolytic cleavage of ring A, again involves CoA‐ester intermediates. The degradation of the remaining CD rings via hydrolytic cleavage appears to be highly similar in aerobic and anaerobic bacteria. Anaerobic cholesterol degradation employs a composite repertoire of more than 40 genes partially known from aerobic degradation in gammaproteobacteria/actinobacteria, supplemented by unique genes that are required to circumvent oxygenase‐dependent reactions.     

Initial steps in the anaerobic degradation of 3,4,5-trihydroxybenzoate by Eubacterium oxidoreducens: characterization of mutants and role of 1,2,3,5-tetrahydroxybenzene.

Chemical mutagenesis and antibiotic enrichment techniques were used to isolate five mutant strains of the obligate anaerobe Eubacterium oxidoreducens that were unable to grow on 3,4,5-trihydroxybenzoate (gallate). Two strains could not transform gallate and showed no detectable gallate decarboxylase activity. Two other strains transformed gallate to pyrogallol and dihydrophloroglucinol but lacked the hydrolase activity responsible for ring cleavage. A fifth strain accumulated pyrogallol, although it contained adequate levels of the enzymes proposed for the complete transformation of gallate to the ring cleavage product. The conversion of pyrogallol to phloroglucinol by cell extract of the wild-type strain was dependent on the addition of 1,2,3,5-tetrahydroxybenzene or dimethyl sulfoxide. This activity was induced by growth on gallate, while the other enzymes involved in the initial reactions of gallate catabolism were constitutively expressed during growth on crotonate. The results confirm the initial steps in the pathway previously proposed for the metabolism of gallate by E. oxidoreducens, except for the conversion of pyrogallol to phloroglucinol.     

Conversion of phytosterols into androstenedione by Mycobacterium neoaurum

Optimum conditions for transformation of phytosterols by Mycobacterium neoaururm, required for selective cleavage of the lateral chain into androstenedione, were shown to differ from the known conditions of animal sterol (cholesterol) transformation. Complete conversion of phytosterols into androstenedione at a substrate load of no less than 20 g/l was achieved on increasing the amount of the inoculum and the concentration of glucose (by 2 and 4 times, respectively, relative to cholesterol) and performing the fermentation under conditions of turbulent mixing. Under these conditions, both the rate of the transformation and the yield of the reaction product were high, due to the saturation of the culture liquid with hydrocarbonate. Data from the literature show that this ion is involved in cleavage of the branched lateral chain at carbon in position 24.     

Conversion of phytosterols into androstenedione by <Emphasis Type="Italic">Mycobacterium neoaurum</Emphasis>

Optimum conditions for transformation of phytosterols by Mycobacterium neoaurum, required for selective cleavage of the lateral chain into androstenedione, were shown to differ from the known conditions of animal sterol (cholesterol) transformation. Complete conversion of phytosterols into androstenedione at a substrate load of no less than 20 g/l was achieved on increasing the amount of the inoculum and the concentration of glucose (by 2 and 4 times, respectively, relative to cholesterol) and performing the fermentation under conditions of turbulent mixing. Under these conditions, both the rate of the transformation and the yield of the reaction product were high, due to the saturation of the culture liquid with hydrocarbonate. Data from the literature show that this ion is involved in cleavage of the branched lateral chain at carbon in position 24.     

Photoperiod-induced changes in the testicular metabolism of [4-14C]17 alpha-hydroxyprogesterone in the bank vole (Clethrionomys glareolus)

Minces of the testes of bank voles, born and reared in a long (18L:6D) photoperiod until weaning (18-22 days of age) and subjected thereafter to a short (6L:18D, Group S) or a long (18L:6D, Group L) photoperiod for 6-9 weeks, were incubated with [4-14C]17 alpha-hydroxyprogesterone in the presence of cofactors (NADP/NADPH, 1.3 mmol/1) for 1 h at 37 degrees C. The radioactive metabolites were characterized and identified by thin-layer chromatography with derivative formation and chromatography to constant specific activity and isotope ratio. In Group L virtually all of the substrate was utilized and it was readily converted to androgens (48% of the radioactivity recovered) such as androstenedione and testosterone. The only pregnane metabolite identified was 17 alpha-hydroxy,20 alpha-dihydroxyprogesterone (43.3%). In Group S there was a decreased production of 17 alpha-hydroxy,20 alpha-dihydroprogesterone and androgens (25.4% and 10.4% respectively) and a substantial portion of the substrate was not metabolized (38.8%). The main androgen metabolites identified, androst-4-ene-3 beta,17 beta-diol and 5 alpha-androstane-3,17-dione are hormonally quite inert steroids. No androstenedione or testosterone was found. The results indicate that exposure to short photoperiod induces a decrease in the testicular C17-C20 lyase and 20 alpha-hydroxysteroid dehydrogenase.     

Studies on steroid monooxygenase from Cylindrocarpon radicicola ATCC 11011. Oxygenative lactonization of androstenedione to testololactone

A steroid monooxygenase of Cylindrocarpon radicicola was found to catalyze oxygenative lactonization of 17-ketosteroid, androstenedione, to yield D-homo-17 alpha-oxasteroid, testololactone, i.e., the androstenedione monooxygenase reaction, in addition to catalyzing the progesterone monooxygenase reaction. The reaction product was identified by TLC, GLC, and mass spectrometry. The oxygenation proceeded with unitary stoichiometry for 17-ketosteroid, NADPH, and molecular oxygen, indicating that it is a typical monooxygenase reaction of the external electron donor type. The enzyme catalyzed successively the side chain cleavage reaction of 17 alpha-hydroxy-20-ketosteroid to produce its 17-keto derivative and the lactonization of the product. The effects of pH and of the concentration of substrate steroids on the androstenedione monooxygenase reaction were different from those on the progesterone monooxygenase reaction. Progesterone is a strong and competitive inhibitor of the lactonization of 17-ketosteroids. The steroid monooxygenase is concluded to have the activities of both oxygenative esterification of 20-ketosteroids and oxygenative lactonization of 17-ketosteroids.     

Androstenedione production by biotransformation of phytosterols

Androstenedione is a key intermediate of microbial steroid metabolism. It belongs to the 17-keto steroid family and is used as starting material for the preparation of different steroids. Androstenedione can be produced by microbial side chain cleavage of phytosterol, which is an alternative to multi-step chemical synthesis. In this review, various methods of biotransformation of sterols to androstenedione are surveyed. It begins with the history and current research status in this field. The existing methods of chemical and biochemical synthesis are examined. Various issues related to these methods and how researchers have addressed them is presented. Among these, the low solubility of sterols in aqueous systems is a critical problem since it limits the product yield. The main content of this review focuses on new methods of biotransformation that are being investigated. Recent biotechnological advances in this field are presented. The review ends with a note on future perspectives.     

Cholesterol binding to cytochrome P450 7A1, a key enzyme in bile acid biosynthesis

The conversion of cholesterol to 7alpha-hydroxycholesterol catalyzed by cytochrome P450 7A1 (CYP7A1) initiates the major pathway for cholesterol elimination in mammals. In the present work we focused on identification of determinants of the CYP7A1 substrate specificity inside the active site using a homology model with a novel P450-fold, site-directed mutagenesis, and substrate-binding and kinetic studies. Forty-one mutants, encompassing twenty-six amino acid residues, were generated and characterized, and of these, seven residues appear to determine cholesterol binding in the active site. In addition, four cholesterol derivatives were used as active site probes in the wild type and the seven mutant enzymes, and the spectral binding constants and products were analyzed. It was concluded that Asn288 in the I helix plays a key role in the P450-cholesterol contacts by hydrogen bonding to the steroid 3beta-hydroxyl, while Val280 and Ala284 are beside and the Trp283 is above the steroid nucleus orienting the cholesterol molecule. Leu360 and Ala358 between the K helix and the beta1-4 strand and Leu485 in the beta4 sheet-turn appear to define the size of the active site over the heme pyrrole ring A, thus limiting the orientation and size of the substrate at the steroid A ring. Additionally, the A358V mutant was found to form two new products, one being 7beta-hydroxycholesterol. Our data indicate that a tight fit of cholesterol in the enzyme active site is in part responsible for the high efficiency of cholesterol turnover by CYP7A1.     

Cholesterol oxidase ChoD is not a critical enzyme accounting for oxidation of sterols to 3-keto-4-ene steroids in fast-growing Mycobacterium sp. VKM Ac-1815D

Fast-growing strain of Mycobacterium sp. VKM Ac-1815D is capable of effective oxidizing of sterols (phytosterol, cholesterol, ergosterol) to androstenedione and other valuable 3-oxo-steroids. To elucidate the role of cholesterol oxidase in sterol catabolism by the strain, the choD gene has been cloned and sequenced. The deduced gene product (M(r) 63.5kDa) showed homologies over its entire length to a large number of proteins belonging to the InterPro-family EPR006076, which includes various FAD dependent oxidoreductases. The expression of choD in Escherichia coli was shown to result in the synthesis of membrane associated cholesterol oxidase. In addition to cholesterol, the enzyme oxidized β-sitosterol, dehydroepiandrosterone, ergosterol, pregnenolone, and lithocholic acid. Knock-out of choD in Mycobacterium sp. VKM Ac-1815D strain was obtained by the gene replacement technique. The mutant strain transformed sitosterol forming exclusively 3-keto-4-ene steroids with androstenedione as a major product, thus evidencing that choD knock out did not abrogate sterol A-ring oxidation. The results indicated that ChoD is not a critical enzyme responsible for modification of 3β-hydroxy-5-ene- to 3-keto-4-ene steroids in Mycobacterium sp. VKM Ac-1815D. Article from a special issue on steroids and microorganisms.     

Rational design of novel mutants of fungal 17beta-hydroxysteroid dehydrogenase

Reduction of 17-ketosteroids is a biocatalytic process of economic significance for the production of steroid drugs. This reaction can be catalyzed by different microbial 17beta-hydroxysteroid dehydrogenases (17beta-HSD), like the 17beta-HSD activity of Saccharomyces cerevisiae, Pichia faranosa and Mycobacterium sp., and by purified 3beta,17beta-HSD from Pseudomonas testosteroni. In addition to the bacterial 3beta,17beta-HSD the 17beta-HSD of the filamentous fungus Cochliobolus lunatus is the only microbial 17beta-HSD that has been expressed as a recombinant protein and fully characterized. On the basis of its modeled 3D structure, we selected several positions for the replacement of amino acids by site-directed mutagenesis to change substrate specificity, alter coenzyme requirements, and improve overall catalytic activity. Replacement of Val161 and Tyr212 in the substrate-binding region by Gly and Ala, respectively, increased the initial rates for the conversion of androstenedione to testosterone. Replacement of Tyr49 within the coenzyme binding site by Asp changed the coenzyme specificity of the enzyme. This latter mutant can convert the steroids not only in the presence of NADP(+) and NADPH, but also in the presence of NADH and NAD(+). The replacement of His164, located in the non-flexible part of the 'lid' covering the active center resulted in a conformation of the enzyme that possessed a higher catalytic activity.     

Influence of hydroxypropyl-β-cyclodextrin on phytosterol biotransformation by different strains of Mycobacterium neoaurum

Cyclodextrins (CDs) can improve productivity in the biotransformation of steroids by increasing conversion rate, conversion ratio, or substrate concentration. However, little is known of the proportion of products formed by multi-catabolic enzymes, e.g., via sterol side chain cleavage. Using three strains with different androst-1,4-diene-3,17-dione (ADD) to androst-4-ene-3,17-dione (AD) ratios, Mycobacterium neoaurum TCCC 11028 (MNR), M. neoaurum TCCC 11028 M1 (MNR M1), and M. neoaurum TCCC 11028 M3 (MNR M3), we found that hydroxypropyl-β-cyclodextrin (HP-β-CD) can appreciably increase the ratio of ADD to AD, the reaction rate, and the molar conversion. In the presence of HP-β-CD, conversion of 0.5?g/L of phytosterol (PS) was 2.4, 2.4, and 2.3 times higher in the MNR, MNR M1, and MNR M3 systems, respectively, than in the controls. The ADD proportion increased by 38.4, 61.5, and 5.9?% compared with the control experiment, which resulted in a strong shift in the ADD/AD ratio in the ADD direction. Our results imply that the three PS-biotransforming strains cause efficient side chain degradation of PS, and the increased conversion of PS when using HP-β-CD may be associated with the higher PS concentration in each case. A similar solubilizing effect may not induce a prominent influence on the ADD/AD ratio. However, the different activities of the Δ(1)-dehydrogenase of PS-biotransforming strains result in different incremental percentage yields of ADD and ADD/AD ratio in the presence of HP-β-CD.     

Steroid metabolism by monkey and human spermatozoa

Freshly ejaculated spermatozoa from monkey and human were washed and incubated with tritium labelled androgens or estradiol to study the pattern of spermatozoa steroid metabolism. When equal concentrations of steroid substrates were used for incubation, monkey and human spermatozoa showed very similar pattern of steroid conversion. Spermatozoa from both species converted testosterone mainly to androstenedione, but reverse conversion of androstenedione to testosterone was negligible. Estradiol-17 beta was converted mainly to estrone. The close similarity between the spermatozoa of monkey and men in their steroid metabolic pattern indicates that the rhesus monkey could be an useful animal model to study the effect of drugs on the metabolic pattern of human spermatozoa.