Changes in Testosterone Metabolism Associated with the Evolution of Placental and Gonadal Isozymes of Porcine Aromatase Cytochrome P450 1

0013-7227/99/$03.00/0 Endocrinology Copyright © 1999 by The Endocrine Society Vol. 140, No. 11 Printed in U.S.A. Changes in Testosterone Metabolism Associated with the Evolution of Placental and Gonadal Isozymes of Porcine Aromatase Cytochrome P450* C. J. CORBIN, J. M. TRANT, K. W. WALTERS, AND A. J. CONLEY Department of Population Health and Reproduction, School of Veterinary Medicine, University of California (C.J.C., K.W.W., A.J.C.), Davis, California 95616; and the Center of Marine Biotechnology, University of Maryland Biotechnology Institute (J.M.T.), Baltimore, Maryland 21202 ABSTRACT Differences in the catalytic activity of the placental and gonadal isozymes of porcine aromatase cytochrome P450 (P450arom) were examined in cell lines exhibiting stable expression of recombinant enzyme. Cell lines were selected that expressed high, but similar, immunodetectable levels of each isozyme based on Western analysis. Aromatase activity varied with growth in culture, decreasing at confluence from a peak reached between 50 – 80% cell density. Cells expressing the placental isozyme had 3–5 times higher catalytic activity (per mg protein) than those expressing the gonadal isozyme. The P450arom inhibitor fadrazole (1 mM) inhibited more than 97% of this activity, whereas another imidazole, etomidate (1 mM), selectively inhibited gonadal P450arom activity by 92%. Estrogen synthesis from androstenedione and testosterone was determined by RIA and confirmed by HPLC analysis, which also identified the accumulation of the 19-hydroxy and 19-oxo intermediates of the respective substrates. A ROMATASE cytochrome P450 (P450arom) is the catalytic component of the aromatase enzyme complex responsible for the synthesis of estrogens from androgens. As such, this enzyme complex plays a unique role in maintaining a physiological balance between androgens and estrogen, which is critical for reproductive development and function in vertebrates. Furthermore, estrogen synthesis, through aromatase expression, contributes significantly to the normal growth and well-being of both males and females (1, 2). Consistent with a fundamental contribution to reproduction and fertility, P450arom is highly conserved, demonstrating 50 –90% peptide sequence identity among mammalian, avian, and fish enzymes (3–14). Despite the recent explosion of sequence information on the vertebrate aromatases, most of what is understood concerning the catalytic properties of this important enzyme has been derived from studies in human tissues, principally the placenta (15), which expresses unusually high levels of activity compared with placentas of other primates or domestic animal species (16). Much less is known concerning the functional biochemistry of P450arom expressed in other tissues or other species. Aromatase cytochrome P450 is encoded by a single gene Received June 8, 1999. Address all correspondence and requests for reprints to: Dr. A. J. Conley, VM-PHR, School of Veterinary Medicine, University of California, Davis, California 95616. E-mail: . * This work was supported in part by USDA 98 –35203-6439 and HD-36913– 01 (to A.J.C.) and USDA 94 –37203-0761 (to J.M.T.). There was no evidence of other steroid metabolites accumulating in the media of cell lines expressing either isozyme. Tritiated water formed during aromatization of substrates 3H labeled at the C1 and C2 positions was stereo-selective for the b orientation, but less so for testosterone than androstenedione during metabolism by the porcine placental (and human) isozyme than the gonadal isozyme. Testosterone showed a higher affinity for the porcine placental P450arom than the gonadal P450arom, but both isozymes had similar affinities for androstenedione. Testosterone was also aromatized more slowly than androstenedione by the porcine gonadal P450arom. These data suggest that catalytic differences have arisen in the substrate binding pocket during the evolution of isozymes of porcine P450arom that affect androgen metabolism, particularly the aromatization of testosterone. The physiological significance of these differences to the reproductive biology of the pig remains to be determined. (Endocrinology 140: 5202–5210, 1999) in humans, even though it is expressed in a broad array of tissues, including many that are steroidogenic in the classical sense as well as others generally considered nonsteroidogenic (15). For instance, in addition to the placenta and the gonads of both men (17) and women (18), P450arom is expressed in brain, liver (19), adipose tissue (20), and a number of tissues in the fetus (21). Additionally, tumors from numerous sites, such as breast (22, 23), gonads (24), prostate (19), and even myeloid leukemia cells (25), have been shown to express P450arom. An elaborate mechanism involving alternative splicing of untranslated first exons and associated promoter elements differentially regulates tissue-specific expression in these sites (15). The ovary is notable in this regard because it appears to be the only tissue expressing P450arom in which splicing is not invoked. Instead, the first untranslated exon is contiguous with exon II (26). Hinshelwood et al. (27) showed that this characteristic of human ovarian P450arom expression is apparently shared in equine and porcine species, even at the nucleotide level. Moreover, this contrasts the lack of sequence conservation in the 59-untranslated region among placental P450arom transcripts from these species (27) and even P450arom transcripts expressed in the porcine testis (28). Thus, this feature of the basic structure of the CYP19 gene regulating P450arom expression appears conserved only in the female gonad. These observations are consistent with the suggestion that P450arom may have first evolved in the vertebrate ovary (15) and that expression in the placenta is a more recent event among eu- 5202 FUNCTIONAL EVOLUTION OF PORCINE AROMATASES therian mammals. How the functional properties of P450arom might have changed to accommodate the physiological needs of the developing fetus in utero for both androgen metabolism and/or estrogen synthesis is an interesting, but unanswered, question. As noted above, and in contrast to the wealth of information on human P450arom, many fewer studies have been conducted on the aromatase enzyme system of other mammals. Recent investigations in this laboratory and others have determined that there are essential differences in the biology of P450arom in the pig. Unlike any other mammal investigated to date, the pig expresses functionally distinct isozymes in a tissue-specific manner (12). Evidence continues to accumulate that these are encoded by completely duplicated genes clustered on chromosome 1 (29 –31). Our studies have shown that, unlike other mammals, a gonadal isozyme is expressed in the theca interna as well as the stratum granulosum of the ovarian follicle (32), the Leydig cell of the testes, and, interestingly, the zona reticularis of the adr (...truncated)