Organization of Cytochrome P450 Enzymes Involved in Sex Steroid Synthesis: PROTEIN-PROTEIN INTERACTIONS IN LIPID MEMBRANES*

Mounting evidence underscores the importance of protein-protein interactions in the functional regulation of drug-metabolizing P450s, but few studies have been conducted in membrane environments, and none have examined P450s catalyzing sex steroid synthesis. Here we report specific protein-protein interactions for full-length, human, wild type steroidogenic cytochrome P450 (P450, CYP) enzymes: 17α-hydroxylase/17,20-lyase (P450c17, CYP17) and aromatase (P450arom, CYP19), as well as their electron donor NADPH-cytochrome P450 oxidoreductase (CPR). Fluorescence resonance energy transfer (FRET)³ in live cells, coupled with quartz crystal microbalance (QCM), and atomic force microscopy (AFM) studies on phosphatidyl choline ± cholesterol (mammalian) biomimetic membranes were used to investigate steroidogenic P450 interactions. The FRET results in living cells demonstrated that both P450c17 and P450arom homodimerize but do not heterodimerize, although they each heterodimerize with CPR. The lack of heteroassociation between P450c17 and P450arom was confirmed by QCM, wherein neither enzyme bound a membrane saturated with the other. In contrast, the CPR bound readily to either P450c17- or P450arom-saturated surfaces. Interestingly, N-terminally modified P450arom was stably incorporated and gave similar results to the wild type, although saturation was achieved with much less protein, suggesting that the putative transmembrane domain is not required for membrane association but for orientation. In fact, all of the proteins were remarkably stable in the membrane, such that high resolution AFM images were obtained, further supporting the formation of P450c17, P450arom, and CPR homodimers and oligomers in lipid bilayers. This unique combination of in vivo and in vitro studies has provided strong evidence for homodimerization and perhaps some higher order interactions for both P450c17 and P450arom.     

Preparation and pharmacological profile of 7-(alpha-azolylbenzyl)-1H-indoles and indolines as new aromatase inhibitors

Aromatase (P450 arom) is a target of pharmacological interest for the treatment of breast cancer. New series of 7-(alpha-azolylbenzyl)-1H-indoles and indolines were synthesized as non-steroidal inhibitors of P450 arom. Selectivity was studied towards P450 17alpha enzyme. The most active compound, 1-ethyl-7-[(imidazol-1-yl)(4-chlorophenyl)methyl]-1H-indole 12c exhibited promising relative potency (rp) of 336 (rp of aminoglutethimide=1) and most of the described azoles were active and selective towards P450 arom.     

Secretory cell types and cell proliferation of human bronchial epithelial cells in an organ-culture system

We examined the localization of steroidogenic cells in rainbow trout (Oncorhynchus mykiss) testis during spermatogenesis by using polyclonal antibodies generated against rainbow trout cholesterol side-chain cleavage enzyme cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17α-hydroxylase/C17,21 lyase (P450c17), and aromatase cytochrome P450 (P450arom) as markers of steroid production. Since we had previously produced specific antibodies against 3β-HSD and P450arom, antibodies against oligopeptides corresponding to C-terminal sequences of P450scc and P450c17, predicted from rainbow trout P450scc and P450c17 cDNAs, were produced in this study. These two antibodies recognized 54-kDa (P450scc) and 59-kDa (P450c17) bands specifically in several steroidogenic organs, i.e., testis, ovary, and interrenal tissue (head kidney) in Western blots. Immunohistochemically, immunoreactive P450scc, P450c17, and 3β-HSD, but not P450arom, were found only in interstitial Leydig cells of immature and mature testes. Immunoreactive P450arom was not detected in either testis. This study suggests that Sertoli cells and germ cells of rainbow trout testis do not contain P450scc, P450c17, P450arom, or 3β-HSD.     

Aromatase inhibitor and 17alpha-methyltestosterone cause sex-reversal from genetical females to phenotypic males and suppression of P450 aromatase gene expression in Japanese flounder (Paralichthys olivaceus)

The sex of Japanese flounder (Paralichthys olivaceus) is easily altered by water temperature or sex steroid hormone treatment during the period of sex determination. We have previously shown that rearing the genetically female larvae at high water temperature caused the suppression of P450 aromatase (P450arom) gene expression in the gonad and phenotypic sex-reversal of the individuals to males (Kitano et al. 1999. J Mol Endocrinol 23:167-176). In the present study, we show that treatment of genetically female larvae with fadrozole (aromatase inhibitor) or 17alpha-methyltestosterone induces sex-reversal as well as suppression of P450arom gene expression. The effect of fadrozole was counteracted by co-administration of estradiol-17beta. Effective periods for fadrozole treatment to induce sex-reversal were similar to those for high water temperature treatment. RT-PCR did not detect P450arom mRNA in gonad of the sex-reversed, phenotypic males. These results indicate that sex-reversal of the genetically female larvae by aromatase inhibitor (or 17alpha-methyltestosterone) may be due to the suppression of P450arom gene expression and the resultant decrease in the amount of estrogen.     

Sex differentiation and mRNA expression of P450c17, P450arom and AMH in gonads of the chicken

The present study was conducted to reveal effects of in ovo injection of nonsteroidal aromatase inhibitor (Fadrozole) or estradiol at day 3 of incubation on mRNA levels of P45017alphahydroxylase (P450c17), P450 aromatase (P450arom) and anti-Müllerian hormone (AMH) in the chicken gonads. The mRNA levels in the gonads at days 4-8 of incubation were assessed by in situ hybridization analysis using digoxigenin labeling method. The in situ hybridization data were analyzed by relative expression of specific hybridizable signals of each mRNA corrected by the non-specific background by employing an image analyzer. P450c17 mRNA expression increased rapidly at day 6 of incubation in the male but decreased thereafter. In contrast to the transient expression in the male, the expression was gradually increased in the female. P450arom mRNA was not expressed in the male but was detectable in the female as early as day 6 and increased subsequently with days of incubation. AMH mRNA was expressed as early as day 5 of incubation followed by a sharp increase on day 6, which was maintained in the male thereafter. In contrast, the female showed very little expression. The injection of Fadrozole caused no effect on P450c17 mRNA expression, while it suppressed P450arom mRNA expression but increased AMH mRNA expression in the female. In contrast, the injection of estradiol induced P450arom mRNA expression significantly but suppressed AMH mRNA expression in the male. These results indicate that expression of P450arom and AMH is sexually dimorphic and is reciprocally regulated during early ontogenic life in chicken gonads.     

Effects of luteinizing hormone and follicle-stimulating hormone and insulin-like growth factor-I on aromatase activity and P450 aromatase gene expression in the ovarian follicles of red seabream,Pagrus major

To clarify the mechanism of estradiol-17beta production in the ovarian follicle of red seabream, in vitro effects of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and insulin-like growth factor (IGF-I) on aromatase activity (conversion of testosterone to estradiol-17beta) and cytochrome P450 aromatase (P450arom) mRNA expression in ovarian fragments of red seabream were investigated. Of the growth factors used in the present study, only IGF-I stimulated both aromatase activity and P450arom gene expression in the ovarian fragments of red seabream. LH from red seabream pituitary, but not FSH, stimulated both aromatase activity and P450arom gene expression. IGF-I slightly enhanced the LH-induced aromatase activity and P450arom gene expression. These data and our previous results indicate that LH, but not FSH, stimulates estradiol-17beta production in the ovarian follicle of red seabream through stimulation of aromatase activity and P450arom gene expression and IGF-I enhances the LH-stimulated P450arom gene expression.     

Immunolocalization of steroidogenic enzymes in the fetal, neonatal and adult testis of the shiba goat.

The localizations of steroidogenic enzymes (P450scc, 3betaHSD, P450c17 and P450arom) in testes of Shiba goats were investigated by immunohistochemistry. P450scc, 3betaHSD, P450c17 and P450arom were detected in all Leydig cells of adults. P450scc and P450c17 were observed in most Leydig cells in the fetus (90 days) and neonate (15 days). 3betaHSD and P450arom were found in some Leydig cells of the fetus with weak immunostaining but the numbers of immunopositive Leydig cells and intense immunostaining were increased in Leydig cells of the neonate. These results suggest that Shiba goat testes have the ability to synthesize progestin, androgen and estrogen in the fetus, neonate and adult, and synthesis of these steroid hormones showed an age-related rise.     

Expression of human aromatase (CYP19) in Escherichia coli by N-terminal replacement and induction of cold stress response

CYP19 (P450arom) catalyzes the aromatization reaction of C19 steroids leading to estrogens. While readily expressed in insect cells, the human P450arom has been a difficult P450 to express in Escherichia coli at useful levels. In the present study, we replaced the N-terminal sequence in human CYP19 with the corresponding sequences of other microsomal P450s (CYP2C11 and CYP17) that are efficiently expressed in E. coli. Although the N-terminal replacement alone was not sufficient for the expression, human P450arom was successfully expressed up to the level of 240nmol/l culture by the combination of the N-terminal replacement and the induction of cold stress response by 1 microg/ml chloramphenicol. Membrane fractions containing the expressed P450arom catalyzed aromatization of androstenedione with a specific activity of 4.9 nmol/min/nmol P450. Our results are important to provide large quantities of human P450arom as an active form for structure-function studies.     

Correlation between messenger RNA expression of cytochrome P450 aromatase and its enzyme activity during oocyte development in the red seabream (Pagrus major)

In teleosts, estradiol-17beta (E2) is an important hormone responsible for oocyte development. To elucidate the molecular mechanisms underlying E2 biosynthesis, we characterized the structure of red seabream (Pagrus major) cytochrome P450 aromatase (P450(arom)) that is directly involved in E2 biosynthesis and found changes in mRNA levels of P450(arom) during oocyte development induced by implantation of gonadotropin-releasing hormone analogue. A cDNA clone encoding P450(arom) is 1779 base pairs in length and encodes a protein of 519 amino acids in length, with a calculated molecular weight of 58.9 kDa. Northern blot analysis showed that P450(arom) mRNA levels increased gradually from Day 8, when oocytes reached the secondary yolk globule stage, and were maintained at high levels at the day of spawning (Day 15). The P450(arom) mRNA levels increased in association with an increase of the gonadosomatic index (gonad weight/body weight x 100%), serum E2, and P450(arom) enzyme activity (in vitro conversion of testosterone to E2 in the ovarian fragments). Furthermore, an increase in mRNA levels of the LHbeta, but not FSHbeta, correlated with increased P450(arom) mRNA levels during the course of ovarian development. In addition, the levels of P450(arom) mRNA increased in isolated ovarian follicles during the course of vitellogenic oocyte growth and became undetectable in follicles at the migratory nucleus and the mature stages. These findings, together with those of the previous studies, suggest that LH, not FSH, may regulate E2 biosynthesis via increased levels of P450(arom) mRNA during oocyte development of red seabream.     

Tamoxifen induces masculinization of genetic females and regulates P450 aromatase and Müllerian inhibiting substance mRNA expression in Japanese flounder (Paralichthys olivaceus)

Japanese flounder, Paralichthys olivaceus, provides an excellent model to elucidate the roles of sex steroid hormones in gonadal sex differentiation because the sex is easily altered by sex steroid treatments or water temperature control during the sex differentiation. We have previously shown that high water temperature, an aromatase inhibitor (fadrozole), or 17alpha-methyltestosterone treatment causes the sex-reversal from genetic females to phenotypic males and suppression of mRNA expression of ovary-type P450 aromatase (P450arom), which is a steroidogenic enzyme responsible for the conversion of androgens to estrogens, in Japanese flounder. In the present study, we demonstrate that treatment of the genetic females with anti-estrogen (tamoxifen) leads to their masculinization, suppresses P450arom mRNA expression, and induces mRNA expression of Müllerian inhibiting substance (MIS), a member of the transforming growth factor-beta (TGF-beta) superfamily, while it has no effect on mRNAs expression of estrogen receptor-alpha (ERalpha) and ERbeta. In contrast, 17beta-estradiol counteracted masculinization of the genetic females by tamoxifen or high water temperature treatment, up-regulated P450arom mRNA expression, and down-regulated MIS mRNA expression. These results strongly suggest that estrogen signaling through ERs dramatically influences the gonadal sex differentiation by regulating P450arom and MIS mRNA expression.     

Costs and consequences of cellular compartmentalization and substrate competition among human enzymes involved in androgen and estrogen synthesis

The impact of compartmental expression of steroidogenic enzymes and of changes in flux through delta5 and delta4 metabolism on sex steroid synthesis was investigated by rebuilding pathways using recombinant enzyme expression by infection of insect cells with recombinant baculovirus constructs. Human cytochromes 17alpha-hydroxylase/17,20-lyase (P450c17) and aromatase (P450arom), always coexpressed with their redox partner NADPH-P450 oxidoreductase (CPR) and 3beta-hydroxysteroid dehydrogenase/delta5-4 isomerase (3betaHSD; types 1 or 2), were compartmentally expressed in different cell populations or coexpressed together with pregnenolone (100 nM) as substrate. Estrone was compared among cell compartments expressing different enzyme combinations or in cells coexpressing all enzymes (experiment 1). Additionally, P450c17, 3betaHSD, and CPR were all coexpressed, and androstenedione was measured in cells with different 3betaHSD expression levels or activity using an inhibitor, trilostane (experiment 2). Steroids were measured by immunoassay and mass spectrometry. In experiment 1, partitioning of P450c17, P450arom, and 3betaHSD markedly decreased estrone synthesis in comparison to cells coexpressing enzymes in different combinations. However, partitioning P450arom with 3betaHSD from P450c17 in different cell populations resulted in more estrone than either of the other two-cell compartment models. In experiment 2 (cells coexpressing P450c17, 3betaHSD, and CPR), androstenedione secretion was (paradoxically) higher at lower levels of 3betaHSD, and partial inhibition of 3betaHSD by trilostane also increased androstenedione when 3betaHSD expression was high. We conclude 1) that tissue or cell-specific, partitioned expression of sex steroid synthesizing enzymes limits rather than maximizes estrogen synthesis and 2) that limiting metabolism by 3betaHSD can paradoxically promote androgen synthesis when 3betaHSD expression is high by promoting delta5-steroid flux.     

P450 aromatase expression in the temperature-sensitive sexual differentiation of salamander (Hynobius retardatus) gonads

Sex differentiation of gonads in amphibians is believed to be controlled genetically, but altered epigenetically or environmentally. When larvae of the salamander Hynobius retardatus were reared at defined temperatures from hatching to metamorphic stages, a high temperature (28 degrees C) induced exclusively female gonads (ovaries), whereas intermediate (20 and 23 degrees C) or lower (16 degrees C) temperatures produced a 1:1 sex ratio of the morphological gonads. The thermosensitive period was determined to be restricted from 15 to 30 days after hatching, just before or when sexual differentiation occurred. Hynobius P450 aromatase (P450arom) cDNA was isolated from adult gonads and the partial nucleotide or deduced amino acid sequences were determined, showing a high level of identity with various vertebrate species. The P450arom gene was expressed predominantly in the adult ovary and brain, weakly in testis, but not in other somatic organs. A typical sexual dimorphism in P450arom expression was detected in normally developing larvae by a quantitative competitive RT-PCR; strong expression in the female gonads but very weak in male gonads. The dimorphism was detected much earlier than the morphological sexual differentiation of the gonads. When larvae were reared at the female-producing temperature (28 degrees C), strong expression was detected in all the temperature-treated larvae, suggesting that P450arom was up-regulated, even in genetic males. Our results confirm the importance of the P450arom regulation in the sexual differentiation of gonads and demonstrate that an up-regulation of P450arom is involved in the process of temperature-sensitive sex reversal in this species.     

Expression of 3beta-hydroxysteroid dehydrogenase, cytochrome p450 17alpha-hydroxylase/17,20-lyase and cytochrome p450 aromatase enzymes in corpora lutea of diestrous and early pregnant mares

In the pregnant mare, luteal estrogen production increases at the onset of equine chorionic gonadotropin (eCG) secretion by endometrial cups. In previous studies, we have demonstrated that eCG stimulates luteal androgen and estrogen production in pregnant mares. To further elucidate the regulation of steroidogenesis within the equine corpus luteum (CL) of pregnancy, we examined the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20 lyase (P450(17alpha)) and cytochrome P450 aromatase (P450(arom)) in luteal tissue samples collected during diestrus (Days 7 to 10) and pregnancy before (Days 29 to 35) and after (Days 42 to 45) the onset of eCG secretion. Immunoblot analyses revealed a single protein per enzyme with molecular weights of 48 kDa (3beta-HSD), 58 kDa (P450(17alpha)) and 56 kDa (P450(arom)). Steady-state levels of 3beta-HSD were lower in luteal tissue of diestrus than pregnancy, but expression did not change during pregnancy. Steady-state expression of P450(17alpha) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, P450(17alpha) expression was significantly higher after the onset of eCG secretion. Steady-state expression of P450(arom) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, luteal expression of P450(arom) was significantly lower after the onset of eCG secretion. These data support the hypotheses that eCG has a differential effect on the expression of luteal steroidogenic enzymes, that the eCG-induced increase in luteal estrogen production is the result of an increase in available aromatizable androgen due to an increase in P450(17alpha) expression and activity, and that increased luteal estrogen production is not due to an increase in aromatase expression.     

Effects of Electro-Acupuncture on Ovarian P450arom,P450c17α and mRNA Expression Induced by Letrozole in PCOS Rats

Hyperandrogenism is a core factor in the series of reproductive and endocrine metabolic disorders involved in polycystic ovary syndrome (PCOS). Abnormalities in enzymatic activity and the expression of ovarian granular cell layer P450arom and theca cell P450c17α can lead to an atypical environment of local ovarian hormones, including excessive androgen levels. Rat models prepared with letrozole exhibit similar endocrine and histological changes to those that occur in human PCOS. We used such a model to study the role of electro-acupuncture (EA) in regulating ovarian P450arom and P450c17α enzymatic activity and mRNA expression in PCOS rats. Female Sprague Dawley (SD) rats aged 42 days were randomly divided into 3 groups (control, PCOS, and PCOS EA) consisting of 10 rats each. The PCOS and PCOS EA groups were administered a gavage of 1.0 mg/kg⁻¹ of letrozole solution once daily for 21 consecutive days. Beginning in the ninth week, the PCOS EA group was administered low-frequency EA treatment daily for 14 consecutive days. After the treatment, we obtained the following results. The estrous cycles were restored in 8 of the 10 rats in the PCOS EA group, and their ovarian morphologies and ultrastructures normalized. The peripheral blood measurements (with ELISA) showed significantly decreased androgens (i.e., androstenedione and testosterone) with significantly increased estrogens (i.e., estrone, estradiol) and increased P450arom with decreased P450C17α. Immunohistochemistry and Western blotting methods showed enhanced expression of ovarian granular cell layer P450arom as well as decreased expression of theca cell layer P450C17α. Fluorescence quantitative PCR methods showed enhanced expression of ovarian granular cell layer P450arom mRNA as well as decreased expression of theca cell layer P450C17α mRNA. These results may help explain the effects of electro-acupuncture in changing the local ovarian hyperandrogenic environment and improving reproductive and endocrine metabolic disorders in PCOS.     

Hormonal regulation of estradiol biosynthesis, aromatase activity, and aromatase mRNA in rat ovarian follicles and corpora lutea

To determine the molecular basis for changes in aromatase (P450arom) activity in rat ovarian follicles and corpora lutea, seven clones for rat P450arom cDNA have been identified and isolated from a rat granulosa cell λgtll cDNA expression library using a 62 mer deoxyoligonucleotide probe (derived from an amino acid sequence of purified human placental aromatase) and a human placental P450arom cDNA probe. One of the rat P450arom cDNA clones contained an insert 1.2 kb in size. Both the human 1.8 kb cDNA and the rat 1.2 kb cDNA probes hybridized to a single species of P450arom mRNA that was 2.6 kb in size. Northern blot analysis revealed that corpora lutea isolated on day 15 of pregnancy contained high amounts of P450arom mRNA, whereas granulosa cells of antral follicles of hormonally primed, hypophysectomized rats (i.e., those from which mRNA was isolated to construct the cDNA library) contained only low amounts of P450arom mRNA. The lower amounts of P450arom in granulosa cells of preovulatory follicles in the estradiol-follicle-stimulating hormone primed hypophysectomized rats were unexpected because follicles incubated in medium containing testosterone substrate produce more estradiol than do corpora lutea isolated on day 15 of pregnancy and incubated under similar conditions. Additional studies will determine the hormonal events responsible for the elevated amounts and constitutive maintenance of P450arom mRNA and aromatase activity in luteal cells in vivo and in vitro.     

Estrogen-producing steroidogenic pathways in parietal cells of the rat gastric mucosa

Recently we demonstrated the presence of aromatase (P450(arom)), estrogen synthetase, and the active production of estrogen in parietal cells of the rat stomach. We therefore investigated the steroidogenic pathways of estrogen and also other steroid metabolites in the gastric mucosa of male rats, by showing the mRNA expression of steroidogenic enzymes using RT-PCR and in situ hybridization histochemistry, and by measuring the blood concentration of steroids in the artery and the portal vein. RT-PCR analysis showed the strong mRNA expression of 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), 17beta-hydroxysteroid dehydrogenase (HSD) type III and P450(arom), and the weak mRNA expression of 17beta-HSD type II, 5alpha-reductase type I and 3alpha-HSD. The other mRNAs of steroidogenic enzymes examined were not detected. In situ hybridization histochemistry demonstrated the localization of mRNAs for P450(17alpha), 17beta-HSD type III and P450(arom) in the parietal cells. Higher levels of progesterone and testosterone were found in the artery compared with the portal vein. Higher amounts of estrone and 17beta-estradiol, by contrast, were present in the portal vein compared with the artery. These results indicate that parietal cells of rat stomach convert circulating progesterone and/through androstenedione and testosterone to synthesize both estrone and 17beta-estradiol, which then enter the liver via the portal vein.     

Expression of P450 aromatase and 17beta-hydroxysteroid dehydrogenase type 1 at fetal-maternal interface during tubal pregnancy

Steroidogenesis in the placenta has been studied widely, but little is known about steroid metabolism in ectopic pregnancy. Previous studies have indicated that trophoblast invasion and placentation in the uterus and the fallopian tube may be controlled by similar mechanisms. As far as 17β-estradiol (E₂) production is concerned, it has been well demonstrated that its biosynthesis in the placenta involves the action of P450 aromatase (P450arom) and 17β-hydroxysteroid dehydrogenase type 1 (17HSD1). The purpose of this study was to characterize the expression pattern of P450arom and 17HSD1 at the fetal–maternal interface, particularly in various trophoblast cells, in tubal pregnancy. Using in situ hybridization, P450arom mRNA was localized in syncytiotrophoblast (ST) cells, which are mainly responsible for hormone production during pregnancy, whereas no signal was detected in villous cytotrophoblast (VCT), column CT and extravillous CT (EVCT) cells. Immunohistochemical assays revealed that 17HSD1 is present in ST cells, a large portion of EVCT cells and 20% of column CT cells. On the other hand, no expression of 17HSD1 was detected in VCT cells. In addition, 17HSD1 was found in epithelial cells of the fallopian tube. Interestingly, the expression level of 17HSD1 in fallopian tube epithelium during tubal pregnancy was significantly higher than that during normal cycle. Our data provide the first evidence that normal and tubal pregnancies possess identical expression of P450arom and 17HSD1 in ST cells and therefore, similar E₂ production in the placenta. Further, the association of 17HSD1 with EVCT cells indicates that 17HSD1 perhaps play a role in trophoblast invasion. Finally, increased expression of 17HSD1 in epithelial cells of fallopian tube may lead to a local E₂ supply sufficient for the maintenance of tubal pregnancy.     

黄鳝P-450芳香化酶基因的克隆及序列分析

P-450芳香化酶(P450arom)是催化雄激素生物合成雌激素的关键酶。本文采用RT-PCR和RACE(Rapid amplifi- cation of cDNA ends)法,首次分离和克隆了雌雄同体鱼黄鳝卵巢中P450 arom基因。该基因cDNA全长1802bp(不包 括poly(A)),5'端非翻译区有49bp,3'端202bp(不包含poly(A)),阅读框(Open reading frame,ORF)1551bp,翻译成517 个氨基酸,计算的蛋白质分子量58．2kDa。同源性分析显示,黄鳝卵巢P450arom的氨基酸序列与其他鱼卵巢 P450arom具有63％-80％同源性,与其他鱼脑P450arom为58％-60％同源,与人胚盘和鸡卵巢P450arom则为 50％-52％同源;但在芳香化酶高保守区(包括1-螺旋区,芳香化酶特异保守区和血红素结合区)的同源性高达 76％-92％。系统发育分析表明芳香化酶基因是单起源,黄鳝卵巢芳香化酶基因与鳉鱼卵巢的关系最近,与鱼类卵 巢P450arom属于同一分支的,与鱼类脑及鸡和人的属于不同分支。