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Endocrinology, doi:10.1210/en.2003-1595
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Endocrinology Vol. 145, No. 5 2157-2164
Copyright © 2004 by The Endocrine Society

Paralogues of Porcine Aromatase Cytochrome P450: A Novel Hydroxylase Activity Is Associated with the Survival of a Duplicated Gene

C. Jo Corbin, S. M. Mapes, J. Marcos, C. H. Shackleton, D. Morrow, S. Safe, T. Wise, J. Joe Ford and A. J. Conley

Department of Population Health and Reproduction, University of California School of Veterinary Medicine (C.J.C., S.M.M., A.J.C.), Davis, California 95616; Children’s Hospital of Oakland Research Institute (J.M., C.H.S.), Oakland, California 94609; Department of Veterinary Physiology and Pharmacology, Texas A&M University (D.M., S.S.), College Station, Texas 77843; and U.S. Department of Agriculture, Agricultural Research Service, Roman L. Hruska U.S. Meat Animal Research Center (T.W., J.J.F.), Clay Center, Nebraska 68933

Address all correspondence and requests for reprints to: Dr. Alan Conley, VM-PHR, School of Veterinary Medicine, 1114 Tupper Hall, University of California, Davis, California 95616. E-mail: ajconley{at}ucdavis.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The gonadal and placental paralogues of porcine aromatase cytochrome P450 (P450arom) were examined for novel catalytic properties to shed light on the evolutionary survival of duplicated copies of an enzyme critical to reproduction. Recombinant gonadal P450arom catalyzed the formation of a novel metabolite from testosterone, identified by gas chromatography/mass spectrometry and biochemical analyses as 1ß-hydroxytestosterone (1ßOH-T), in almost equal proportion to 17ß-estradiol (E2). This activity was absent in reactions with the porcine placental paralogue (or other orthologues) of P450arom and was minimal with androstenedione. Incubations with both porcine enzymes and with bovine and human P450arom demonstrated that 1ßOH-T was not aromatizable, and 1ßOH-T activated the androgen receptor of prostate cancer cells in vitro. Porcine testicular and follicular granulosa tissues synthesized 1ßOH-T, which was also detected in testicular venous plasma. These results constitute the first of identification of a novel, perhaps potent, nonaromatizable metabolite of testosterone, whose synthesis (paradoxically) can be definitively ascribed to the activity of the gonadal paralogue of porcine P450arom. It probably represents an evolutionary gain of function associated with fixation and the survival of the genes after CYP19 duplication. Novel activities and adaptive functions may exist among other duplicated vertebrate aromatases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE EXISTENCE OF functionally distinct paralogues of a vertebrate aromatase cytochrome P450 (P450arom) was first described by Corbin et al. (1) in the domestic pig (Sus scrofa domestica). This discovery was of particular interest because P450arom, the only enzyme capable of synthesizing estrogens from androgen substrates (2), was previously thought to represent a single copy gene (2, 3, 4), consistent with the high degree of sequence identity and functional conservation among the cloned orthologues (5). Subsequent studies identified two paralogues of P450arom in certain species of fish (6) that are encoded by genes located on different chromosomes (7), indicating a relatively long evolutionary history. The domestic pig remains unique among mammals studied in possessing three separate paralogues of P450arom that are encoded by tandem, duplicated genes on the same chromosome (chromosome 1) (5, 8, 9). The porcine paralogues share 87–93% amino acid sequence identity (10), and each is quite tissue-specific in its expression. One is restricted to the gonads (testes, theca, and granulosa) and adrenal gland (11), a second occurs in placenta (12) and endometrium (13), and a third is present in preimplantation blastocysts (14). Thus, the porcine CYP19 cluster provides an interesting case of a gene that has duplicated relatively recently, is tissue-specifically expressed (8), and plays a pivotal role in male and female reproduction and fertility (5).

Studies in this laboratory have focused on catalytic function in an attempt to identify biochemical and potential physiological adaptations that might have accompanied the evolutionary survival of the tissue-specific paralogues of porcine CYP19. For instance, we proposed that an enhanced capacity for testosterone metabolism by P450arom expressed in the porcine placenta protected female pig fetuses from the androgenic influence of littermate males in utero (15). Human female infants suffering severe P450arom deficiency are born virilized (16, 17). Although an equally plausible hypothesis in pigs, this still does not address the persistence in the genome of the gonadal paralogue that is less functionally competent. Specifically, the porcine gonadal P450arom has a lower turnover rate (18) and overall catalytic efficiency of estrogen synthesis (18, 19), particularly with respect to testosterone metabolism. This low efficiency of aromatization is offset in the ovary by increased protein expression compared with that in placenta (11) and, uniquely to the pig, by expression of gonadal P450arom in the theca interna as well as the granulosa of the preovulatory follicle (20, 21, 22). Estrogen production by the ovary and testis is essential for fertility, and the evolutionary survival of a less competent P450arom enzyme expressed in the gonads of any species is counterintuitive. Therefore, we reexamined the products of testosterone metabolism by purified recombinant porcine gonadal P450arom and having identified a novel metabolite, examined its synthesis by tissues, secretion by testis, and its potential for biological activity in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and chemicals
All general chemicals were of highest quality and were purchased from AMRESCO (Solon, OH), Fisher Scientific (Pittsburgh, PA), or Sigma-Aldrich Corp. (St. Louis, MO) unless specified otherwise. Restriction enzymes and DNA-modifying enzymes were obtained from Promega Corp. (Madison, WI) or New England BioLabs (Beverly, MA). Insect cell culture media, antibiotics, and competent cells were obtained from Invitrogen Life Technologies (Grand Island, NY). 19-Hydroxyandrostenedione (4-androsten-19-ol-3,17-dione) and stigmasterol were obtained from Steraloids (Wilton, NH). Sodium borohydride reduction was used to produce 19-hydroxytestosterone (19OH-T) from 19-hydroxyandrostenedione. Pyridine and trimethylsylilimidazole were purchased from Pierce Chemical Co. (Rockford, IL), and methoxyamine hydrochloride, sodium borohydride, and Lipidx (hydroxyalkoxypropyl dextran type IX) were obtained from Sigma-Aldrich Corp.

Baculovirus cloning, overexpression, and quantification
The cDNAs encoding the native porcine placental and gonadal P450arom were overexpressed in insect cells after subcloning into pFastBac1 (Invitrogen, Carlsbad, CA). Transformation and transposition into bacmid DNA were carried out according to the manufacturer (Bac-to-Bac Baculovirus Expression System, Invitrogen). PCR was used to identify recombinant clones, which were subsequently used to transfect midlog phase Sf9 insect cells grown in SF900-II SFM (Invitrogen). Rounds of amplification were repeated until the titers reached at least 1 x 107 plaque-forming units/ml. For P450arom expression, High Five insect cells (Invitrogen), grown in suspension culture in Express Five SFM (Invitrogen), were infected at a multiplicity of infection of at least 5 and grown for 72 h at 26 C in the presence of ferrous ammonium sulfate (0.2 mM) and {gamma}-aminolevulinic acid (0.3 mM). Infected High Five cells were pelleted at 500 x g, resuspended at a concentration factor of 20x in cold 0.1 M potassium phosphate (KPO4; pH 8.0), 20% glycerol, 5 mM 2-mercaptoethanol, 5 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, and 0.5 mM phenylmethylsulfonylfluoride. Lysates were homogenized in a tissue grinder and briefly sonicated. Solublization was achieved by gentle rotation for 1 h at 4 C. The unsolubilized fraction was removed by centrifugation for 1 h at 100,000 x g. The final P450arom content was determined by CO difference spectrum measured on a Shimadzu 2401 spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD) and calculated as (A450 – A490) x 0.091 = nmol/ml (23).

Reconstituted P450arom activity and thin layer chromatographic (TLC) metabolite analysis
Stereospecific loss was measured by incubating 2 pmol purified P450arom with a mixture of either [1ß-3H]- and [4-14C]androstenedione (770 nM) or [1ß,2ß-3H]- and [4-14C]testosterone (770 nM; all from NEN Life Science Products, Boston, MA) with 20 pmol recombinant P450 reductase at 37 C for 2–6 h in the presence of an NADPH-generating system (1 mM NADPH, 2 mM NADP, 17 mM glucose-6-phosphate, and 1 U glucose-6-phosphate dehydrogenase). Metabolite formation was determined by incubation of recombinant enzyme (2 pmol) or tissue microsomal fraction (100 µg) with either [4-14C]androstenedione or [4-14C]testosterone as described above. After incubation, the samples were extracted with 10 vol methylene chloride, and the organic phase was evaporated to dryness and resuspended in 25 µl ethyl acetate. Samples were applied to silica gel plates (Whatman, Maidstone, UK) along with unlabeled standards and developed once in chloroform/methanol (98:2, vol/vol) for separation of androstenedione or ethyl acetate (100%) for separation of testosterone metabolites. Plates were placed on storage phosphor screens (Amersham Pharmacia Biotech, Piscataway, NJ) and scanned the following day on a Molecular Dynamics Typhoon 8600. The appropriate products were scraped from the TLC plate, eluted with ethyl acetate, evaporated, and quantified by liquid scintillation counting. 1-Hydroxytestosterone was generated by incubating recombinant porcine gonadal P450arom with testosterone, scaling up the previously described conditions as needed.

Tissues, cells, and plasma
Procedures for handling all animals in this study were approved by the MARC animal care committee and the animal use and care administrative advisory committee of University of California-Davis. Porcine granulosa cells were dissected from dominant, preovulatory follicles from the ovaries of animals in standing estrus, and testicular tissue was obtained by castration of 2- to 3-wk-old neonatal boars. Aromatase activities in these tissues, determined using androstenedione as substrate, have been previously reported (22, 24). Briefly, tissues were homogenized in 0.1 M KPO4 (pH 7.4), 20% glycerol, 5 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonylfluoride, and 1 µg/ml aprotinin. After brief sonication, the homogenate was centrifuged at 15,000 x g for 10 min. The supernatant was subjected to a 100,000 x g spin for 1 h, and the resultant microsomal pellet was resuspended in buffer containing 1 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. Protein concentrations were determined using the bicinchoninic acid protein reagent (Pierce Chemical Co.). Testicular venous plasma was also obtained from three sexually mature boars while under anesthesia before surgical castration, extracted twice with methylene chloride, and subjected to analysis by gas chromatography/mass spectrometry (GC-MS) as described below. No attempt was made to capture or investigate sulfo-conjugated steroids or those synthesized by cytosolic or mitochondrial fractions.

Sample preparation and analysis by GC-MS
After adding 1 µg stigmasterol, used as internal standard, samples were evaporated. The dried extract was dissolved in 100 ml 2% methoxyamine hydrochloride in pyridine. After 1 h at 55 C, pyridine was evaporated, and 50 µl trimethylsylilimidazole were added. This was left at 100 C overnight. The derivative was purified by Lipidx 5000 chromatography (25), dissolved in 20 µl cyclohexane, and transferred to an autosampler vial. GC-MS was carried out on a 5890 gas chromatograph coupled with a 5970 MSD (Hewlett-Packard, Palo Alto, CA). The steroids were separated on a DB 1 cross-linked methyl-silicone column (inside diamter, 15 m x 0.25 mm; film thickness, 0.25 µm; J&W Scientific, Folsom, CA). Helium was used as the carrier gas at a constant pressure of 5 {psi}. A 2-µl aliquot of the final derivatized extract was injected into the system operated in splitless mode (valve opened at 2 min). The GC temperature was ramped as follows: initially 50 C, held for 3 min, increased to 230 C at 30 C min–1, thereafter increased to 285 C at 2 C min–1. The injector and transfer line were kept at 260 and 280 C, respectively. The mass range scanned was from 90–500 atomic mass units.

In vitro androgen receptor activation
LNCaP and 22RV1 human prostate cancer cells (American Type Culture Collection, Manassas, VA) were seeded into 12-well plates at 275,000 cells/well in DMEM (D2902, Sigma-Aldrich Corp.) containing 2.5% dextran/charcoal-stripped serum. Cells were allowed to gain substrate attachment for at least 12 h, then were cotransfected with 500 ng/well pXP2-PB-luc reporter plasmid (gift from Robert J. Matusik, Vanderbilt University Medical Center, Nashville, TN) (26) and 250 ng pcDNA3.1-cytomegalovirus-lacZ control plasmid (Invitrogen) using Lipofectamine Plus reagents according to the manufacturer’s instructions (Invitrogen). After 24 h, transfection cocktail-containing medium was removed, and identical medium containing dihydrotestosterone (A8380, Sigma-Aldrich Corp.), testosterone propionate (T1875, Sigma-Aldrich Corp.), or test compound was added at 0.1, 1.0, and 10 nM. After 36-h treatment, cells were assayed for luciferase (Promega) and ß-galactosidase (Tropix, Bedford, MA) activities in a Packard luminometer according to the manufacturer’s instructions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The analysis of testosterone metabolism by the recombinant gonadal paralogue was initially conducted by TLC. It was immediately clear that there was an additional steroid product quite distinct from the expected intermediates and from 17ß-estradiol (E2). This metabolite migrated between 19-hydroxy- and 19-oxo(-al)-testosterone (Fig. 1AGo), but was only seen when testosterone was used as a substrate for the gonadal paralogue. Incubation of the porcine placental P450arom and bovine and human P450arom enzymes yielded 19-hydroxy- and 19-oxotestosterone along with E2, but no other metabolite. Trace amounts of another unknown metabolite were seen along with 19-hydroxy and 19-oxo intermediates (in addition to estrone) in incubations of the porcine gonadal paralogue with androstenedione, but none with the porcine placental paralogue, bovine or human P450arom (data not shown). Further incubations were conducted with porcine recombinant gonadal P450arom using testosterone as substrate, and the products were pooled and submitted for analysis by GC-MS. These analyses confirmed the identities of the 19-hydroxy- and 19-oxotestosterone intermediates in addition to generating a unique spectrum for the unknown product (Fig. 1BGo), which proved to be identical to 1OH-T (Fig. 1CGo) as reported by Lisboa and Gustafsson (27). In the absence of available authentic standard, the epimeric form of the steroid was determined by examining stereo-specific loss of 3H (Table 1Go), which is known to be predominantly from the ß position at C-1 and C-2 of the steroidal A ring during estrogen formation (28). For reactions with the gonadal paralogue, the rate of 3H loss was similar during the synthesis of 1OH-T and E2 (57% and 58%, respectively), indicating that it was, in fact, 1ßOH-T. The rate of tritium loss was higher than this during the aromatization of androstenedione (82%), but was similar among the porcine gonadal and placental paralogues and the human and bovine orthologues (Table 1Go).



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  		FIG. 1. Identification of 1ßOH-T4) synthesis from testosterone by recombinant porcine gonadal P450arom. A, TLC of products of metabolism of [4-14C]testosterone (T4; final concentration, 1000 nM) by 2 pmol recombinant gonadal P450arom. Intermediates [19OH-T (19-OH) and 19-oxotestosterone (19-oxo)] and E2 are also shown. B, Analysis of derivatized (MO-TMS) extracts analyzed by GC-MS. Peaks at 15.9 and 16.4 min (representing syn and anti forms of the same compound; not shown) gave unambiguous and unique spectra corresponding to those previously published for 1OH-T, as shown below. Identities of the other 19OH and 19-oxo (19-al) metabolites shown were similarly confirmed. C, Original mass spectrum for 1ßOH-T4 as reported by Lisboa and Gustafsson (27 ). The 479 (parent) and 389 ion species are 2 U bigger than those reported by Lisboa and Gustafsson (477 and 387) because of the use of 4-14C-labeled substrate. The 133 µ ion accounts for the major species in the spectra.

 

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  		TABLE 1. Verification that hydroxylation of testosterone by porcine gonadal P450arom is at the 1ß position of the steroid A ring using dual tracer metabolism

 
Experiments were conducted with recombinant porcine gonadal P450arom to determine the time course of appearance and the relative abundance of 1ßOH-T, its proximate substrate, and subsequent fate. Incubations with recombinant gonadal P450arom demonstrated that 1ßOH-T synthesis, although faster in the early stages of reactions, almost paralleled that of E2 after the first 15 min, particularly so as testosterone became depleted (Fig. 2Go). Identical time-course studies conducted with the placental P450arom (and bovine and human enzymes; data not shown) demonstrated much more rapid accumulation of E2 from testosterone, but no detectable 1ßOH-T was produced (Fig. 3Go). Similar incubations with the gonadal paralogue with 19-hydroxy- and 19-oxotestosterone demonstrated that 19-oxo- was formed from 19OH-T, and that 19-oxotestosterone was converted to E2, but that 1ßOH-T was formed only from testosterone (Fig. 4Go). Moreover, incubation of 1ßOH-T with the porcine gonadal and placental paralogues or with bovine or human P450arom (data not shown) failed to provide evidence of any further metabolism regardless of reaction time.



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  		FIG. 2. Time course of testosterone metabolism by porcine gonadal and placental (inset) aromatase cytochromes P450 (P450arom). Pu-rified recombinant P450arom enzymes (2 pmol) were reconstituted with purified NADPH-cytochrome P450 oxidoreductase (20 pmol) and an NADPH-generating system and incubated with [4-14C]testosterone. Reactions were stopped at the times indicated, extracted, and analyzed by TLC. Plates were subjected to phosphorimage analysis from which metabolism was quantified. The mean ± SD (n = 3) are shown. Note that the time course of incubations with placental P450arom (inset) extended to 40 min only.

 


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  		FIG. 3. Synthesis of 1ßOH-T4 from testosterone by porcine gonadal microsomes. Ovarian granulosa (lane 1) and testicular (lane 2) microsomes (100 µg each), and recombinant gonadal P450arom (2 pmol; lane 3) were incubated with [4-14C]testosterone (1 µM) and extracted. Products were separated by TLC (100% ethyl acetate) and subjected to phosphorimage analysis. Incubations extended over 5, 10, and 20 min with recombinant placental P450arom as indicated above each lane and over 15 min for the recombinant gonadal P450arom. Tissue microsome incubations were performed for 2 h. The migration of available standards is shown at the left.

 


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  		FIG. 4. Schematic representation of alternative pathways of testosterone metabolism by porcine gonadal aromatase cytochrome P450 leading to 1ß-hydroxylation or 19-hydroxylation at the methyl also projecting from the ß-face of the steroid.

 
Synthesis of 1ßOH-T ex vivo was investigated using porcine testes and granulosa microsomes. In vivo evidence of synthesis was sought by GC-MS analysis of testicular venous plasma, and its potential bioactivity was examined in vitro. Microsomes were enriched from porcine preovulatory ovarian granulosa cells (22) and from neonatal testicular tissues (24) and were used in incubations conducted in parallel with recombinant porcine gonadal P450arom. All reactions clearly demonstrated 1ßOH-T as a product along with 19-hydroxy- and 19-oxotestosterone intermediates (Fig. 4Go). Synthesis in vivo was confirmed further by identification of 1ßOH-T in testicular venous plasma from three adult boars, among a variety of other steroids, including 1{alpha}OH-T, as well as 19OH-, 11OH-, 11-keto-, and 19-nor-testosterone, 19-nor-androstenedione, epiandrosterone, and dehydroepiandrosterone. The ratio of 1-hydroxylated products to testosterone itself in testicular venous plasma was estimated to be perhaps as high as 0.36, an approximate value as these peaks coeluted with interferents. Potential androgenic activity was also investigated in vitro. Reactions were again scaled up and pooled, and 1ßOH-T was isolated after separation by TLC. The quantity of purified steroid was estimated by optical absorbance at 240 nm using testosterone as a standard and used to compare the ability of 1ßOH-T and 5{alpha}-dihydrotestosterone (DHT) to drive expression of luciferase from reporter constructs containing one or more copies of the androgen response region of the probasin gene in LNCaP and 22RV1 cell lines. Each androgen was added to cultures in equal concentrations (0.1, 1, and 10 nM) after transfection with reporter plasmids and corrected for transfection efficiency. Luciferase activity was stimulated by both DHT and 1ßOH-T with comparable potencies (Fig. 5Go). Similar experiments were conducted using reporter constructs driven by estrogen receptor {alpha}, but no evidence for estrogenic activity was observed (data not shown).



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  		FIG. 5. Androgen receptor activation by DHT and 1ßOH-T. 1ßOH-T4 was isolated by TLC from the products of testosterone metabolism by recombinant porcine gonadal P450arom and estimated by optical absorbance at 240 nm using testosterone as a reference. Prostate cancer cells (upper panel, LNCaP; lower panel, 22RV1) were cultured in charcoal-stripped serum and transfected with a reporter plasmid (AAR3TK-luciferase) encoding luciferase driven by the androgen-response region (AAR) of the probasin gene and a LacZ control plasmid to correct for transfection efficiency. Cells were treated with increasing concentrations (nanomolar) of DHT and 1ßOH-T4 for 36 h, then assayed for luciferase activity. Results are expressed (mean ± SD; n = 3) as fold induction over vehicle (dimethylsulfoxide) control after correction for transfection efficiency. Similar experiments were conducted using reporter constructs driven by estrogen receptor {alpha}, but no evidence for estrogenic activity was detected (data not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of the current studies identify 1ßOH-T as a novel product (from an unusual P450arom activity) of the gonadal paralogue of P450arom in domestic pigs that possibly provides insight into the survival of the gene after duplication of the CYP19 gene locus. Logically, genes that influence reproductive fitness directly might be expected to become fixed or to degenerate more rapidly than others (29). Thus, survival of duplicate genes encoding P450arom would be expected to be under heavy selection pressure, because this is the sole enzyme system responsible for the synthesis of estrogens and thus is central to the coordination of vertebrate reproduction (5). A low biosynthetic efficiency of estrogen synthesis, such as that exhibited by the gonadal relative to the placental paralogue (23), necessitating increased expression in both thecal and granulosa compartments of the follicle, for instance (11), might present a barrier to both efficient reproduction and ultimate survival of the gene in functional form. It is critical that the product, 1ßOH-T, was identified in reactions using purified P450arom protein, that it was not synthesized from either 19OH-T or 19-oxotestosterone (intermediates in aromatization of testosterone) or in cell-based systems, and that it was identified definitively by GC-MS. Several previous studies have reported that 19-nor-androgens are novel products of porcine aromatases (30, 31, 32, 33) and even the equine enzyme (34, 35). Nor-androgens have not been seen in our studies using transiently transfected cells (1), with stable expressing cell lines (15, 19) or in reconstituted assays with the purified gonadal and placental paralogues (18, 23), systems in which redox partner support varied greatly. The observed inhibition of 19-nor-androgen synthesis by aromatase inhibitors (30) suggests that these steroids arise from 19OH or 19-oxo intermediates, but are not direct products of P450arom activity itself. Subsequent studies with purified equine P450arom did not report 19-nor-androgen as a product (36) and are consistent with this interpretation. Others have suggested that they arise as an artifact of isolation (37). Collectively, identification of 1ßOH-T as a novel, and quantitatively significant, product of the gonadal enzyme of the domestic pig suggests a physiological role for this steroid in gonadal function and reproduction in this species.

The likelihood that 1ßOH-T is physiologically active is supported by several lines of evidence. 1ßOH-T activated the androgen receptor in two different cell lines. Definitive data on potency await the availability of sufficient quantities of authentic steroid to conduct animal- and/or tissue-based studies, as the androgen receptor in LNCaP (and probably 22RV1) cells is mutated and somewhat promiscuous (38). However, it is significant that, like DHT, 1ßOH-T is nonaromatizable, a competitive inhibitor of P450arom (39) and, as shown here, apparently resistant to metabolism by testis microsomes in general. Moreover, testosterone was readily metabolized by gonadal microsomes to 1ßOH-T without detectable DHT synthesis, suggesting that 1ßOH-T may be the more abundant of the two, nonaromatizable testicular androgens. Testicular microsomal 5{alpha}-reductase activity was very low in 3-wk-old neonatal pigs compared with peripubertal animals (40), consistent with the general decline in androgens during this period (41). Clearly a great deal more work is also required to document the relative levels of these androgens in testicular tissue and venous plasma and thereby define the physiological activity and significance of 1ßOH-T4 production in the gonads. Levels no doubt will change developmentally and with reproductive state, and 1ßOH-T4 might be further metabolized to more or less active steroids locally within tissues. However, 1ßOH-T4 synthesis by the gonadal P450arom may simply balance the extraordinarily high rates of estrogen production by the boar testes (42, 43), a seemingly paradoxical phenomena still not understood. There is also support for a possible adaptive function in the female from studies in gilts demonstrating that administration of a nonaromatizable androgen increases granulosa cell FSH receptor expression and ovulation rate (44). Thus, we believe that 1ßOH-T4 synthesis represents a gain of function, especially because it occurs at the direct expense of efficient E2 production.

Distinct catalytic differences have yet to be clearly defined for any of the identified paralogues of P450arom in fish (45), and no hypothesis has been offered to explain their fixation in the vertebrate genome. We propose that the evolution of a novel androgen-hydroxylating activity is likely to have contributed to the survival of the gonadal paralogue after duplication of porcine CYP19. Evidence of at least two CYP19 genes in the related Suide, the collared peccary (Corbin, C. J., and A. J. Conley, unpublished observations), and a transcribed pseudogene in domestic cattle (46) is consistent with the likelihood that duplication was present in a common ancestor. Thus, the porcine paralogues of P450arom have survived perhaps 50 million yr, based on estimates of the time of divergence of pigs from other artiodactyla (47). The different enzymatic properties of the gonadal and placental forms of porcine P450arom also provide evidence of functional divergence existing among even the most conserved proteins. Functional changes in enzymes arising from duplicated genes as subtle as those involving fractional shifts in pH optima have been proposed to drive survival in evolutionary time (29). The fish CYP19 genes have survived longer than those of the pig, based on their segregation onto different chromosomes, and their functional divergence may be even greater, but this still requires investigation, and novel catalytic activities have yet to be reported.

Many theories have been advanced on how metabolic pathways might have arisen. Retro-evolution from the synthesis of an essential metabolite (48) is a particularly controversial hypothesis (49), suggesting that limitations in substrate availability drive development of a means to produce it (50). This presupposes that the terminal enzyme in a cascade is the most ancestral of those required to generate substrates. CYP19 is one of the most ancient of all microsomal P450s based on phylogenetic analyses, certainly one of the oldest of the steroid hydroxylases (51, 52). The only other example of functional paralogues among the P450s involved in steroid synthesis is CYP11B, the gene that encodes 11ß-hydroxylase cytochrome P450 (P450c11). A single mitochondrial enzyme (P450c11) catalyzes cortisol and aldosterone in some species (53), and this is probably the ancestral state. However, humans, rats, mice, and guinea pigs have two functional CYP11B genes (54, 55), duplicated, but still tandemly arranged, that have given rise to P450 enzymes adapted specifically for cortisol (P450c11) and aldosterone (P450aldo, aldosynthase) synthesis. There are other intriguing observations linking P450arom and P450c11. The porcine gonadal P450arom is inhibited by etomidate (18), better known as an inhibitor of P450c11, and bovine P450c11 can aromatize androgen (56). Although porcine testes exhibit 11ß-hydroxylase activity (57, 58), our data confirm that this is not due to P450arom. Whether retrograde or not, the above data suggest the possibility that steroidogenesis may indeed be evolving from the terminal enzymes in the synthetic cascade by the duplication of genes encoding them and divergence of catalytic function among surviving copies.

The novel 1ß-hydroxylase activity of the gonadal paralogue of porcine P450arom also provides an unusual opportunity to define structure-function relationships between epitopes in the active site of P450arom and different ligands, particularly testosterone. Although the placental and gonadal paralogues exhibit similar, very high affinities for androstenedione, and these are much higher than those for testosterone, the placental isozyme has a higher affinity for testosterone than does the gonadal enzyme (15). Previous studies showed that stereo-specific loss of 3H from the A ring during aromatization of [1ß,2ß-3H]testosterone was also different for the gonadal and placental P450arom enzymes (15), based on estimates of E2 synthesis measured by immunoassay. The current results provide an explanation, at least with respect to the discrepancy between tritium release and E2 synthesis when measured by immunoassay, because 1ßOH-T synthesis is associated with as much tritium release such as occurs during E2 formation, but is unlikely to be detected by immunoassays for estrogen. However, microsomes isolated from human placenta and rat ovary also differed in the rates of 3H loss from labeled testosterone during aromatization (59). This emphasizes the additional importance of rates of metabolism and thereby possible isotope effects as well as essential differences in substrate interactions with active site residues in the stereo-specific loss of hydrogen during androgen metabolism by P450arom (28). These early studies also demonstrated that human placental microsomes were capable of 1ß-hydroxylating 19-norandrostenedione (60, 61), which, like 1ßOH-T, was not aromatizable. Osawa et al. (62) identified 1ßOH-T as a catalytic product of 19-[3H3]testosterone metabolism by purified human placental P450arom, but considered it to result from metabolic switching caused by a large isotope effect at C19. Our data demonstrate that 1ß-hydroxylation is relatively substrate- and certainly paralogue-specific and therefore results from an interplay between the orientation of testosterone and uniquely positioned residues in the active site of the porcine gonadal paralogue, somehow favored in the case of the 17-hydroxy substrate. The spatial proximity of hydrogens at the 1ß-, 11ß-, and C19 positions no doubt facilitates different activities arising from relatively subtle changes in substrate orientation. Future experiments to determine the amino acid residues mediating this unusual activity and orientation of steroids in the active site will probably lead to a better understanding of substrate binding to P450 enzymes.

In conclusion, the present results document for the first time the existence of a novel activity of a mammalian P450arom that leads to the paradoxical synthesis of a nonaromatizable metabolite of testosterone, 1ßOH-T, by the gonadal paralogue of porcine P450arom. The observed synthesis of 1ßOH-T in the porcine gonad provides a plausible explanation of what may have contributed to the survival of this catalytically inefficient paralogue of P450arom after du-plication of the CYP19 gene locus. A functional analysis of proteins is necessary to fully appreciate the pressures of purifying and/or diversifying selection on duplicated genes, and the biological and biochemical adaptations accompanying their evolution and survival. These data also provide evidence that steroidogenesis may still be evolving in some species at least, especially from the terminal enzymes in the metabolic cascade.


   Acknowledgments
 
We express our gratitude to Drs. Bill Lasley and Kala Natarajan for conducting in vitro analyses of estrogen receptor activation by 1ßOH-T. Additional thanks to Drs. Valentine Lance, Bill Lasley, Charles Langley, and Michael Turelli for helpful discussions.


   Footnotes
 
This work was supported by USDA Grant CREES 98-35203-6439 and National Institutes of Health Grant 1R01-HD-36913.

Abbreviations: DHT, 5{alpha}-Dihydrotestosterone; E2, 17ß-estradiol; GC-MS, gas chromatography/mass spectrometry; OH-T, hydroxytestosterone; P450arom, aromatase cytochrome P450; TLC, thin layer chromatography.

Received November 24, 2003.

Accepted for publication January 27, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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