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*17ALPHA-HYDROXYPROGESTERONE
*PROGESTERONE
The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 8 3762-3766
Copyright © 2003 by The Endocrine Society

The 17, 20-Lyase Activity of Cytochrome P450c17 from Human Fetal Testis Favors the {Delta}5 Steroidogenic Pathway

Christa E. Flück, Walter L. Miller and Richard J. Auchus

Department of Pediatrics and Metabolic Research Unit (C.E.F., W.L.M.), University of California, San Francisco, California 94143-0978; and Division of Endocrinology and Metabolism, Department of Internal Medicine (R.J.A.), University of Texas Southwestern Medical Center, Dallas, Texas 75390-8857

Address all correspondence and requests for reprints to: Richard J. Auchus, M.D., Ph.D., Division of Endocrinology and Metabolism, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8857. E-mail: richard.auchus{at}utsouthwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cytochrome P450c17 catalyzes both 17{alpha}-hydroxylation and 17,20-lyase conversion of 21-carbon steroids to 19-carbon precursors of sex steroids. P450c17 can mediate testosterone biosynthesis via the conversion of pregnenolone to dehydroepiandrosterone (the {Delta}5 pathway) or via conversion of progesterone to androstenedione (the {Delta}4 pathway). In many species, the 17, 20-lyase activity of P450c17 for one pathway dominates, reflecting the preferred steroidogenic pathway of that species. All studies of recombinant human P450c17 and of human adrenal microsomes have found high 17, 20-lyase activity only in the {Delta}5 pathway. Because the 17, 20-lyase activities in both the {Delta}4 and {Delta}5 pathways for testicular P450c17 have not been directly compared, however, it is not known if the {Delta}5 pathway dominates in the human testis. To resolve this issue, we assayed the conversion of 17{alpha}-hydroxypregnenolone to dehydroepiandrosterone ({Delta}5 17, 20-lyase activity) and of 17{alpha}-hydroxyprogesterone to androstenedione ({Delta}4 17, 20-lyase activity) by human fetal testicular microsomes. We obtained apparent Michaelis constant (Km) and maximum velocity (Vmax) values of 1.0 µM and 0.73 pmol·min-1·µg-1 for {Delta}5 17, 20-lyase activity and of 3.5 µM and 0.23 pmol·min-1·µg-1 for {Delta}4 17, 20-lyase activity. Catalytic efficiencies, expressed as the ratio Vmax/Km, were 0.73 and 0.066 for the {Delta}5 and {Delta}4 reactions, respectively, indicating 11-fold higher preference for the {Delta}5 pathway. We conclude that the majority of testosterone biosynthesis in the human testis proceeds through the conversion of pregnenolone to dehydroepiandrosterone via the {Delta}5 pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CYTOCHROME P450c17 IS THE sole enzyme that catalyzes both steroid 17{alpha}-hydroxylation and the 17, 20-lyase reaction. In the human adrenal zona glomerulosa, absence of P450c17 directs steroidogenesis to aldosterone. In the zona fasciculata, the 17{alpha}-hydroxylase activity of P450c17 far exceeds its 17, 20-lyase activity, resulting in cortisol production. In the zona reticularis, 17, 20-lyase activity is abundant, resulting in the synthesis of the C19 precursors of sex steroids, principally dehydroepiandrosterone (DHEA) and its sulfate, as well as androstenedione ({Delta}4A) (1).

P450c17 may drive sex steroid production by converting pregnenolone to 17{alpha}-hydroxypregnenolone (17OHPreg) and then to DHEA along the {Delta}5 pathway. DHEA is then converted in two steps to testosterone. Alternatively, flux may simultaneously occur via the {Delta}4 pathway: progesterone to 17{alpha}-hydroxyprogesterone (17OHP) to {Delta}4A (Fig. 1Go). The dominant pathway to C19 steroids varies for P450c17 from different species, due principally to discrepancies in the catalytic efficiencies for the 17, 20-lyase reactions in the {Delta}5 and {Delta}4 pathways. The catalytic activities of bovine P450c17 (2, 3) resemble those of the human enzyme, but pig (4, 5, 6, 7), frog (8), and trout (9) P450c17 have readily measured {Delta}4 17, 20-lyase activity, whereas the {Delta}4 pathway predominates with the rat (10), hamster (11, 12), and guinea pig (13, 14) enzymes. Consequently, many endocrinology texts have diagrammed both adrenal C19 steroid and testicular testosterone biosynthesis proceeding via both the {Delta}4 and {Delta}5 pathway or predominantly via the {Delta}4 pathway.



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  		FIG. 1. Potential pathways of testosterone synthesis in the human testis. Cholesterol is converted to pregnenolone by P450scc; this is the rate-limiting step. Conversion of pregnenolone to testosterone might take place by either of two pathways. In the {Delta}5 pathway, pregnenolone undergoes 17{alpha}-hydroxylation to 17{alpha}-hydroxypregnenolone (17OHPREG) and scission of the C17-C20 bond to yield DHEA, both catalyzed by P450c17. DHEA is converted to androstenediol by 17ßHSD3, and testosterone biosynthesis is completed by 3ßHSD2. Testicular 3ßHSD2 can also convert each {Delta}5 steroid to its corresponding {Delta}4 steroid, including the conversion of nascent pregnenolone to progesterone. For progesterone to be converted to testosterone, it must follow a {Delta}4 pathway through 17{alpha}-hydroxyprogesterone (17OHPROG) and {Delta}4A to testosterone. The relative efficiency of the human testicular P450c17 in converting 17-hydroxysteroid intermediates to the C19 steroids DHEA and {Delta}4A with its 17, 20-lyase activities is the subject of this study.

 
Both clinical observations of human adrenal physiology and direct biochemical assays of recombinant human P450c17 demonstrate that the 17, 20-lyase activity of the human enzyme strongly prefers the {Delta}5 pathway. Thus, in newborns with 21-hydroxylase deficiency, serum concentrations of 17OHP are generally elevated 100-fold, but concentrations of {Delta}4A are elevated only 5- to 10-fold (15), indicating poor conversion of 17OHP to {Delta}4A by adrenal {Delta}4 17, 20-lyase activity. When human P450c17 is expressed in transfected COS-1 cells (16, 17) or in microsomes from genetically engineered yeast (18), it catalyzes the conversion of 17OHPreg to DHEA approximately 20 times more efficiently than the conversion of 17OHP to {Delta}4A. The same dominance of the {Delta}5 17, 20-lyase activity is observed when purified, modified human P450c17 is studied in reconstituted assay systems (19), and the {Delta}5 pathway also dominates in P450c17 from several other primate species (20).

The pathway leading to C19 testosterone precursors in the human testis, however, has been the subject of some controversy. Early studies using [3H]-pregnenolone and -progesterone precursors suggested that the human testis makes C19 steroids through both the {Delta}4 and the {Delta}5 pathways (21). Some data even suggested that the adrenal glands and gonads used different isozymes of P450c17 (22), but the cloning of the human adrenal and testicular P450c17 cDNAs showed that a single gene encodes the same protein in both tissues (23). Nevertheless, it is conceivable that the dominant pathways to C19 steroids might differ in the human adrenal and gonads, and that such a testicular pathway was not seen in previous studies of P450c17 in the adrenal or in vitro.

P450c17 functions as part of a catalytic system comprised of P450c17 itself, cytochrome P450 oxidoreductase, and cytochrome b5 (b5). The 17, 20-lyase activities of the P450c17 system can be dramatically modulated by at least three posttranslational events. First, a high molar ratio of the reductase to P450c17 favors 17, 20-lyase activity (7, 17). Second, b5 acts allostericly to favor 17, 20-lyase activity (18, 24, 25), and b5 is preferentially expressed in the zona reticularis of the adrenal (26, 27, 28), which produces large amounts of C19-steroids. Finally, the 17, 20-lyase activity of P450c17 is strongly influenced by serine/threonine phosphorylation (29, 30). Thus, it is conceivable that other, as yet unidentified, posttranslational events might favor {Delta}4 17, 20-lyase activity in the Leydig cell but not occur in the adrenal or in in vitro systems. To address this question directly, we examined the 17, 20-lyase activity of P450c17 in human fetal testis microsomes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Fetal testis microsome preparation

Human testis tissue from a fetus 21.5 wk of gestation was obtained from the American Association of Tissue Banks (McLean, VA; aatb@aatb.org). The fetal testis tissue was disrupted with 30 strokes of a glass homogenizer on ice with 0.25 ml of 0.25 M sucrose/5 mM EDTA/10 mM Tris·HCl, pH 7.4. Crude cellular debris was removed, and a premicrosomal pellet was obtained after centrifugation at 10,000 x g for 10 min. The supernatant was centrifuged at 100,000 x g for 45 min, and the pellet, containing enriched microsomes, was resuspended in 0.1 ml of TEG (50 mM Tris·HCl/1 mM EDTA, pH 8.0/20% (vol/vol) glycerol). The microsome preparation was kept frozen at -70 C until needed. Microsomal protein concentration was determined colorimetrically (Protein Assay Dye Reagent, Bio-Rad, Hercules, CA) using BSA as a standard. Microsomal P450 content was estimated from type I difference spectra with progesterone, recorded on a Shimadzu UV-160U spectrophotometer as described (20).

P450c17 enzyme assays

Enzyme kinetic studies were performed using a constant amount of microsomal protein (2.3 µg for testing the {Delta}5 pathway, 9.2 µg for testing the {Delta}4 pathway) and four different substrate concentrations. Fetal testis microsomes were preincubated in 50 mM potassium phosphate buffer (pH 7.4) with 0.5–4.0 µM steroids (added in 4 µl of ethanol) in a total volume of 200 µl at 37 C for 2 min. Each reaction also contained either 100,000 cpm of [3H]-17OHPreg (21.2 Ci/mmol) or 40,000 cpm of [3H]-17OHP (66 Ci/mmol) (Perkin-Elmer Life Sciences, Boston, MA). To start the reaction, 1 mM reduced nicotinamide-adenine dinucleotide phosphate (Sigma, St. Louis, MO) was added, and the reaction was returned to 37 C for 50 min (17OHPreg) or 60 min (17OHP). Steroids were extracted from the reaction mixture with 400 µl of ethyl acetate/isooctane (1:1), concentrated by evaporation under continuous nitrogen flow, and separated by thin-layer chromatography on phosPE SIL G/UV silica gel plates (Whatman, Maidstone, Kent, UK) using 3:1 chloroform/ethyl acetate as the solvent system (18). Incubations with [14C]-progesterone or -pregnenolone at 1 µM steroid, which approximates intratesticular concentrations of many steroids (31), were conducted in 100 µl of phosphate buffer as described above for the 17-hydroxysteroids at 37 C for 60 min with 2.3 µg microsomal protein. The radiolabeled steroids were quantified by phosphorimaging analysis on a Storm 860 PhosphorImager using ImageQuant software version 1.2 for Macintosh (Molecular Dynamics, Sunnyvale, CA).

Data analysis

All assays were performed three times, with each data point in duplicate or triplicate, and data are presented as means ± SEM. Kinetic behavior was approximated as a Michaelis-Menten system, and data are plotted as described by Lineweaver and Burk and by Dixon and Webb (32). Curve fitting for calculation of maximum velocity (Vmax) and apparent Michaelis constant (Km) values was performed with PRISM 3.02 (GraphPad Software, Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To study the P450c17 catalytic system from human testicular Leydig cells, we prepared microsomes from human mid-term fetal testes, which are about 50% Leydig cells by mass (33, 34). The microsomes prepared from this tissue contained 66 pmol cytochrome P450/mg of protein. Human testis lacks most other microsomal cytochromes P450 such as steroid 21-hydroxylase (35) and has low aromatase content (36), and as cytochrome P450scc is a mitochondrial enzyme, this spectroscopic assay primarily measures P450c17 content.

To determine the catalytic efficiencies of the 17, 20-lyase reactions for both the {Delta}4 and the {Delta}5 pathways in human testis microsomes, we measured the conversion of [3H]-17OHPreg to DHEA and of [3H]-17OHP to {Delta}4A. By using microsomal protein preparations and radiolabeled 17-hydroxysteroid intermediates, we directly compared the kinetics of the two 17, 20-lyase reactions, avoiding the assumptions, side reactions, and pitfalls that have led to incorrect assessments of steroid metabolism by other methods (37). Calculation of the apparent Michaelis constants (Km) and maximum velocities (Vmax) (38) for 17OHPreg and 17OHP showed a large preference for the {Delta}5 pathway (Fig. 2Go). The Km for {Delta}5 17OHPreg was 1.0 µM, whereas the Km for {Delta}4 17OHP was 3.5 µM, favoring the {Delta}5 substrate by 3.5-fold. The Vmax for {Delta}5 17OHPreg was 0.73 pmol·min-1·µg-1, whereas the Vmax for {Delta}4 17OHP was 0.23 pmol·min-1·µg-1, demonstrating that the 17, 20-lyase reaction proceeded over three times faster for the {Delta}5 pathway at saturating substrate concentrations. The ratio Vmax/Km, an index of overall catalytic efficiency, was 11-fold higher for the {Delta}5 pathway than the {Delta}4 pathway (Table 1Go). These data demonstrate that, by all kinetic measures, the P450c17 catalytic system of the human testis favors the {Delta}5 pathway for the 17, 20-lyase reaction.



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  		FIG. 2. The 17, 20-lyase activities of human fetal testicular microsomes. A, Representative phosphorimage of thin-layer chromatograms showing the conversion of the indicated concentrations of [3H]-{Delta}5 17OHPreg to DHEA and of [3H]-{Delta}4 17OHP to {Delta}4A. Each assay was performed three times, with each data point assayed in duplicate or triplicate. To measure the conversion of 17OHP to {Delta}4A, the amount of microsomal protein per incubation was increased by a factor of four to achieve sufficient product to measure reliably. B, Lineweaver-Burk plot of 17, 20-lyase activities. Data from phosphorimage analyses were plotted, and lines were generated by least-squares fits to data points ({Delta}5, open squares, r2 = 0.91; {Delta}4, filled squares, r2 = 0.99). The apparent Km and Vmax values obtained from these data are shown in Table 1Go.

 

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  		TABLE 1. Apparent kinetic constants for the 17, 20-lyase activities of human P450c17

 
To confirm that metabolic flux to C19 steroids in the fetal testis is greater for the {Delta}5 pathway than for the {Delta}4 pathway, we incubated fetal testis microsomes with 1 µM [14C]-pregnenolone and -progesterone. After 60 min, comparable amounts of 17-hydroxysteroids had accumulated in both incubations, yet little {Delta}4A was produced from progesterone during this time (Fig. 3Go). In contrast, substantial amounts of DHEA were formed at a time when about 1/3 of the pregnenolone substrate still remained (Fig. 3Go). This result confirms that net conversion of C21 precursors to C19 steroids through both steps of the human testicular P450c17 reaction sequence (17{alpha}-hydroxylase reaction followed by 17, 20-lyase reaction) is more efficient for the {Delta}5 pathway than for the {Delta}4 pathway.



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  		FIG. 3. Metabolism of progesterone and pregnenolone by human fetal testicular microsomes. Phosphorimage of thin-layer chromatogram obtained after incubating microsomes with [14C]-progesterone (PROG) or -pregnenolone (PREG). The amount of 17, 20-lyase product for the {Delta}5 pathway, DHEA, greatly exceeds the amount of {Delta}4A.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Human gonadal differentiation and testicular sex steroid production begin at about wk 6 of gestation. The testicular content of mRNA for P450scc and P450c17 are maximal at about 14 wk (39), and plasma testosterone concentrations peak between 14 and 18 wk of gestation and remain near adult levels until late gestation (21, 34). Thus, our use of 21-wk gestation human testicular tissue corresponds to a time when both testicular P450c17 expression and testosterone production are high. Because Leydig cells comprise roughly half the mass of human fetal testis (33, 34), microsomes from fetal testis are a rich source of the testicular P450c17 catalytic system. Enzymatic studies of these microsomes, which are minimally confounded by the coexistence of other microsomal cytochromes P450 or by other mitochondrial or soluble steroidogenic enzymes, therefore, closely approximate testicular P450c17 activities in vivo.

Initial studies of testosterone synthesis in human fetal testis found comparable conversion rates of pregnenolone and progesterone to testosterone (21). Other investigators found that homogenates from whole testes of adult men also converted pregnenolone rapidly through 17OHPreg to DHEA (40). The adult testis homogenates, in contrast, produced little progesterone and {Delta}4A from pregnenolone, and 17OHP accumulated slowly, suggesting poor {Delta}4 17, 20-lyase activity relative to the {Delta}5 pathway (40, 41). The enzyme sources used by these two groups are not equivalent because, unlike the fetal testis, over 90% of the mass of the adult testis is comprised of seminferous tubules (42). Furthermore, neither study performed incubations with 17-hydroxysteroid intermediates, and 17, 20-lyase activity was inferred from multistep transformations to testosterone in complex mixtures. Because pregnenolone is the first committed intermediate in sex steroid biosynthesis from cholesterol, studies starting with progesterone might overestimate testosterone production rates by omitting the 3ß-hydroxysteroid dehydrogenase-{Delta}5/4-isomerase (3ß-HSD) step. With pregnenolone as the substrate, the Km of 3ß-HSD is more than five times higher than that of P450c17 (43), suggesting that 3ß-HSD may be the rate-limiting step in the conversion of pregnenolone to testosterone in the testis. This explanation would reconcile previous studies with our data. We infer that our results also apply to adult Leydig cells, but adult testis tissue was not available for our study.

Our results demonstrate that the catalytic efficiency for the 17, 20-lyase reaction of P450c17 in human fetal testis is 11-fold higher for the {Delta}5 pathway than for the {Delta}4 pathway. This {Delta}5 preference is consistent with results obtained with human P450c17 obtained from adrenal microsomes (18) or expressed in heterologous systems (16, 18, 19). We calculate a Vmax for the conversion of 17OHPreg to DHEA in fetal testis microsomes (the {Delta}5 17, 20-lyase reaction) of more than 11 pmol·min-1·pmol P450-1, which is 4–10 times faster than the highest rates measured in optimized assays with recombinant enzyme (18, 19). Consequently, the rates of the 17, 20-lyase reactions in the fetal testis, even for the minor {Delta}4 pathway, are higher than in other systems, but the preference of the {Delta}5 pathway is lower (18) (Table 1Go).

Thus, the 17, 20-lyase activity of the P450c17 catalytic system in the human fetal testis is relatively high in both the {Delta}5 and {Delta}4 pathways, exceeding rates measured with recombinant P450c17 in heterologous expression systems. Reasons for the rapid turnover rates might include optimal abundance of P450 oxidoreductase (7) and b5 (18, 25), favorable composition of phospholipids, low amounts of competing enzymes, and proper phosphorylation state (29, 30). Because the catalytic efficiency of the 17, 20-lyase reaction for the testicular P450c17 system favors the {Delta}5 pathway by 11-fold, the majority of testosterone biosynthesis in the human testis derives from the conversion of pregnenolone to DHEA via the {Delta}5 pathway.


   Footnotes
 
This work was supported by a grant from Swiss Foundation for Medical-Biological Grants (to C.E.F.) and by NIH Grants HD41958 (to W.L.M.) and K08DK02387 (to R.J.A.).

Abbreviations: {Delta}4A, Androstenedione; DHEA, dehydroepiandrosterone; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase-{Delta}5/4-isomerase; Km, apparent Michaelis constant; 17OHP, 17{alpha}-hydroxyprogesterone; 17OHPreg, 17{alpha}-hydroxypregnenolone; Vmax, maximum velocity.

Received January 28, 2003.

Accepted April 10, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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