Genetic control of extraglandular aromatase activity in the chicken

Feminization of feathers in the Sebright cock is the result of increase in the activity of skin aromatase. This increased estrogen synthesis is the consequence of an autosomal dominant mutation that causes an increase in the specific androgen-binding cytochrome P450 oxidase involved in the reaction. Since this oxidase appears to be kinetically indistinguishable from the activity in control ovary we believe that the mutation causes an increased steady-state level of normal enzyme. The mechanism by which the mutation acts is unknown, but its presence implies that in normal birds an allele of the mutation limits the activity of the enzyme in all tissues other than ovary.     

Inheritance of the henny feathering trait in the golden Campine chicken: evidence for allelism with the gene that causes henny feathering in the Sebright bantam

The henny feathering mutation causes roosters to develop female feathering morphology as a result of increased conversion of androgen to estrogen (aromatase activity) in extraglandular tissues, including skin. This trait is maintained in two breeds of chickens: the Sebright Bantam and the Golden Campine. To characterize the inheritance of this trait further, we did breeding studies of the Golden Campine and identified the trait by measuring aromatase activity in biopsied skin. As previously established for the Sebright Bantam, the trait is transmitted in the Campine by an autosomal, incomplete dominant mechanism; heterozygous offspring express half the levels of extraglandular aromatase as do homozygous Campines on average. No reversions to wild-type levels were observed in 555 heterozygous offspring of crosses between homozygous Campines and normals. Compound heterozygotes for the trait were obtained by mating homozygous Sebrights and homozygous Campines. When these compound heterozygote birds were crossed to control birds, all 98 offspring had elevated aromatase activity in skin, suggesting that the traits in Sebright and Campine birds are allelic. Furthermore, the restriction fragment length polymorphism pattern performed on genomic DNA was the same in the Sebright and Campine birds. Thus, the phenotypic, endocrine, and genetic features suggest that the traits in Sebright and Campine birds are the same. The trait in the Campine probably was derived from the Sebright.     

Estrogen production by fetal rat gonads

Aromatase activity in fetal rat testes and ovaries was demonstrated by the conversion of tritiated testosterone or 19-hydroxyandrostenedione into estrone and estradiol, which were identified and quantified by double isotopic dilution and recrystallization to constant specific activity. Testes formed mostly estradiol, ovaries mostly estrone. Aromatase activity was stimulated by cAMP in both the testes and ovaries as early as 17 days of fetal life. Stimulation by FSH was noted at this same stage in the testis, but not before 3–4 days after birth in the ovary. LH was without effect on aromatase activity in both kinds of gonads. Basal estrogen secretion was non-existent or undetectable in both the testes and ovaries in fetal stages. In the presence of cAMP and as early as 17 days of fetal life, the testes released estradiol, as early as 14 days the ovaries released estrone. Estrogen secretion was stimulated by LH and FSH at fetal stages in the testis and at infantile stages in the ovary. Responsiveness to gonadotrophins closely followed the appearance of the receptors.     

Inheritance of the henny-feathering trait of the Sebright bantam chicken

The henny -feathering trait of the Sebright bantam chicken is the result of an enhanced rate of estrogen synthesis (aromatase activity) in skin and other peripheral tissues of this bird. To gain insight into the underlying nature of this mutation, we examined the inheritance of this trait using a sensitive isotopic assay for aromatase activity. All birds of the F1 generation obtained by crossing homozygous henny X non- henny chickens expressed increased aromatase in the skin, and the males exhibited henny -feathering plumage. The average rate of aromatase activity in the skin of F1 chicks was approximately half that of homozygous henny chicks. The distribution of increased aromatase activity in the F2 generation and in the backcrosses of F1 to the two types of parental strains suggest that the enzyme activity in extragonadal tissues is regulated predominantly by one autosomal gene. Attempts to demonstrate linkage to several known loci by backcrossing F1 heterozygotes with parental chickens carrying a variety of genetic markers were unsuccessful. We conclude that the enhanced estrogen synthesis in extragonadal tissues in chickens carrying this gene is inherited as an autosomal codominant but that a half maximal level of the enzyme is sufficient to allow full development of female feathering in affected male birds so that henny -feathering is transmitted as a dominant trait.     

Sebright and Campine chickens express aromatase P-450 messenger RNA inappropriately in extraglandular tissues and in skin fibroblasts.

As the result of a single gene mutation, Sebright and Campine chickens have increased activity of cytochrome P-450 aromatase and increased formation of estrogen in extragonadal tissues. Affected roosters develop a feminizing state characterized by a feathering pattern typical of hens. In this paper we demonstrate that the expression of extraglandular aromatase in these chickens is due to the accumulation of aromatase mRNA similar to that expressed in the ovary of Leghorn and Campine hens. Furthermore, in fibroblasts cultured from Sebright skin, but not in normal Leghorn fibroblasts, aromatase mRNA levels are enhanced in response to 5-azacytidine and sodium butyrate, and aromatase mRNA levels in these fibroblasts correlate with enzymatic activity. We conclude that the accumulation of aromatase mRNA is a critical step in the expression of this mutation.     

Examination of the role of follicle stimulating hormone in estrogen biosynthesis in vivo and in vitro in the ovary of the cyclic hamster

The effect of neutralizing endogenous follicle stimulating hormone (FSH) or luteinizing hormone (LH) with specific antisera on the in vivo and in vitro synthesis of estrogen in the ovary of cycling hamster was studied. Neutralization of FSH or LH on proestrus resulted in a reduction in the estradiol concentration of the ovary on diestrus-2 and next proestrus, suggesting an impairment in follicular development. Injection of FSH antiserum at 0900 h of diestrus-2 significantly reduced the ovarian estradiol concentration within 6--7 h. Further, these ovaries on incubation with testosterone (T) in vitro at 1600 h of the same day or the next day synthesized significantly lower amounts of estradiol, compared to corresponding control ovaries. Although testosterone itself, in the absence of endogenous FSH, could stimulate estrogen synthesis to some extent, FSH had to be supplemented with T to restore estrogen synthesis to the level seen in control ovaries incubated with T. Lack of FSH thus appeared to affect the aromatization step in the estrogen biosynthetic pathway in the ovary of hamster on diestrus-2. In contrast to this, FSH antiserum given on the morning of proestrus had no effect on the in vivo and in vitro synthesis of estrogen, when examined 6--7 h later. The results suggest that there could be a difference in the need for FSH at different times of the cycle. Neutralization of LH either on diestrus-2 or proestrus resulted in a drastic reduction in estradiol concentration of the ovary. This block was at the level of androgen synthesis, since supplementing testerone alone in vitro could stimulate estrogen synthesis to a more or less similar extent as in the ovaries of control hamsters.     

The synthesis of 19-norandrostenedione from dehydroepiandrosterone in equine placenta is inhibited by aromatase inhibitors 4-hydroxyandrostenedione and fadrozole

19-Norandrostenedione was synthesized in vitro from dehydroepiandrosterone by explants of equine full-term placenta. The synthesis of 19-norandrostenedione was inhibited by two specific aromatase inhibitors, 4-hydroxyandrostenedione and fadrozole.     

Effect of 4-hydroxyandrostenedione on 19-hydroxylation of deoxycorticosterone in the golden Syrian hamster

4-Hydroxyandrostenedione, a known inhibitor of ovarian and peripheral aromatization of androgen (testosterone and androstenedione) to form estrogen, was studied for its inhibitory effect on the 19-hydroxylating enzyme system of the adrenal for the conversion of deoxycorticosterone to 19-hydroxydeoxycorticosterone. In vitro incubation of Golden Syrian hamster adrenal homogenates with tritiated deoxycorticosterone demonstrated (80-85%) reduction in label incorporated into 19-hydroxydeoxycorticosterone in the presence of 4-hydroxyandrostenedione.     

The aromatase inhibitor 4-hydroxyandrostenedione inhibits the formation of 19-norandrostenedione by porcine grakulosa cells in vitro {Poster 32}

Porcine granulosa cells convert androstenedione (A) to 19-norandrostenedione (19-norA) and 19-hydroxyandrostenedione (19-OHA) in the presence of FSH and 10% porcine serum; 19-norA is also formed from independent incubations with 19-OHA. 19-NorA and 19-OHA formation from A, and 19-norA from 19-OHA, is blocked by 4-hydroxyandrostenedione, an irreversible inhibitor of aromatase.     

Aromatase--key enzyme of estrogen biosynthesis

Estrogens control a large range pivotal life functions as reproductive development and fertility, bone growth and sexual behavior. Aromatase is a key enzyme of estrogen biosynthesis. The property, structure and reaction mechanism of aromatase as well as detailed structure of human aromatase cytochrome P450 gene (CYP19) was discussed in this article. It was pointed that unique human CYP19 gene expression results from presence of many tissue specific promoters and alternative splicing. The molecular mechanism of control aromatase cytochrome P450 gene expression in various species ovaries, testes and human adipose tissue and placenta was discussed in details. Because of a very important role of estrogen in breast cancer a molecular base of aberrant expression CYP19 gene in breast tumor and adipose tissue proximal to breast tumor and potential possibility of pharmacological silencing of this gene expression was discussed in the article.     

17-Hydroxylase/C17,20-lyase (CYP17) is not the enzyme responsible for side-chain cleavage of cortisol and its metabolites

The question addressed in this study was the nature of the enzyme required to remove the side-chain of 17-hydroxycorticosteroids, leading in the case of cortisol to the excretion of 11β-hydroxyandrosterone, 11-oxo-androsterone and the corresponding etiocholanolones. We questioned whether it could be CYP17, the 17-hydroxylase/17,20-lyase utilized in androgen synthesis. The conversion of exogenous cortisol to C₁₉ steroids in patients with complete 17-hydroxylase deficiency (17HD) was studied rationalizing that if CYP17 was involved no C₁₉ steroids would be formed. The urinary excretion of the four 11-oxy-C₁₉ steroids as well as many of the major C₂₁ cortisol metabolites were measured by GC/MS. Our results showed that the conversion of cortisol to C₁₉ steroids was normal in 17HD indicating that a currently unidentified enzyme must be responsible for this transformation.

A secondary goal was to determine to what extent 11-oxy-C₁₉ steroids were metabolites of cortisol or adrenal synthesized 11β-hydroxyandrostenedione. Since cortisol-treated 17HD patients cannot produce androstenedione, all C₁₉ 11-oxy-metabolites excreted must be derived from exogenous cortisol. The extent to which 17HD patients have lower relative excretion of C₁₉ steroids should reflect the absence of 11β-hydroxyandrostenedione metabolites. Our results showed almost all of 11-oxo-etiocholanolone and 11β-hydroxyetiocholanolone were cortisol metabolites, but in contrast the excretion of 11β-hydroxyandrosterone was less than 10% that of normal individuals, indicating that in excess of 90% must be a metabolite of 11β-hydroxyandrostenedione.     

Androgen synthesis and aromatization by equine corpus luteum microsomes

Whereas mare corpus luteum does not produce androgens or estrogens in vivo, the incubation of mare corpus luteum microsomes with progesterone and NADPH resulted in 17 alpha-hydroxyprogesterone and estrogen production with a small yield of androstenedione. In the presence of an aromatase inhibitor (4-hydroxyandrostenedione), 17 alpha-hydroxyprogesterone and androstenedione were accumulated. Aromatization of testosterone and androstenedione occurred via stereospecific loss of the 1 beta, 2 beta hydrogen atoms and was inhibited by MgCl2, KCl, and EDTA. The Km of estrogen synthetase from equine corpus luteum for testosterone was 18.5 +/- 2.7 nM and for androstenedione was 11.5 +/- 1.5 nM. 19-Norandrogens were aromatized with a slightly higher efficiency than were androgens, but the affinity of the aromatase was lower for 19-norandrogens than for androgens. Our results suggest that aromatases from equine testis and corpus luteum are closely related enzymes. On the other hand, the question arises as to the relationship among the cell origin, the synthetizing abilities, and in vivo production of the corpus luteum in different mammalian species.     

3-Methylene-substituted androgens as novel aromatization inhibitors. Evidence of a requirement for C-3 oxygen in C-19 hydroxylations

Substitution of a methylene group for the C-3 oxygen in androstenedione, testosterone, and the corresponding 19-hydroxy and 19-oxo derivatives results in a new category of inhibitors of estrogen biosynthesis by human placental microsomes. The inhibition is of the competitive type with the most effective inhibitors being the 17-ketonic compounds, 3-methyleneandrost-4-en-17-one, 19-hydroxy-3-methyleneandrost-4-en-17-one, and 3-methylene-19-oxoandrost-4-en-17-one with apparent Ki values of 4.7, 13, and 24 nM, respectively. The 3-methylene derivatives of androstenedione and 19-hydroxyandrostenedione were effective substrates for the placental microsomal 17 beta-hydroxy-steroid oxidoreductase but were only marginally hydroxylated at the C-19 position to the respective 19-hydroxy and 19-oxo derivatives. The 3-methylene analogs are thus competitive inhibitors of aromatization but are not substrates for this enzyme complex. Time-dependent inhibition of aromatization by 10 beta-difluoromethylestr-4-ene-3,17-dione and 10 beta-(2-propynyl)estr-4-ene,3,17-dione was abolished by substitution of a methylene function for the C-3 oxygen, suggesting that the presence of an oxygen at C-3 is required for an oxidative transformation at C-19, an initial step in aromatization. The essential role of the C-19 hydroxylation in aromatization is supported by the observation that the 3-methylene derivatives of 19-hydroxy- and 19-oxoandrostenedione showed time-dependent inhibition, but the corresponding 19-methyl compound did not. The 3-methylene androgens are potent inhibitors of placental aromatization but are themselves only marginal substrates for the enzyme. Their high affinity for and inertness to the placental aromatase complex makes them valuable probes of the aromatization process.     

Action of LH, FSH and (Bu)2 cAMP on the conversion of [3H]19-hydroxyandrostenedione into oestrogens by foetal and infantile rat ovaries in organ culture

Ovaries from 18-21-day-old foetal as well as from 2-10-day-old infantile rats were cultured in vitro in the presence of [3H]19-hydroxyandrostenedione and in the presence or absence of LH, FSH or (Bu)2 cAMP, and oestrone and oestradiol formed were determined by double isotopic dilution and recrystallization to constant specific activity. In foetal ovaries, the stimulation factor with FSH was 0.9-1.3, which was considered insignificant in comparison with the 8-13-fold stimulation obtained with (Bu)2 cAMP. At infantile stages, aromatase activity was stimulated 1.3-3.5-fold, which was close to the 3.9-fold stimulation obtained with (Bu)2cAMP. LH was ineffective at both foetal and infantile stages.     

New data on ovulatory action of estrogens during 5 day cycles in rats

A peculiar laboratory strain W1 of Wistar rats has 5 day cycles and can be made precociously receptive to a male by an injection of estradiol 10 mcg sc at 1500 of Cycle Day 2 (Diestrus III). In the course of repeating previous work the authors noted that receptivity increased from 25 to 63%, and they investigated the mechanism by checking ovaries of estrogen treated females histologically for ovulation, in comparison with ovaries of females exposed to males during the night of Diestrus III. In this experiment 56% of estrogen treated rats ovulated. 41 out of 60 (68%) of the paired rats accepted a male (verified by sperm in vaginal smear), and 93% of these ovulated. Of the 19 who were unreceptive, 1 (5.3%) ovulated (p.001). Thus estrogen treatment is much more effective than previously observed; 5 day cycling rats have a related precocity of sexual receptivity and capacity to ovulate under this stimulus.     

In vitro and in vivo studies with aromatase inhibitor 4-hydroxy-androstenedione

Studies with 4-hydroxyandrostenedione (4-OHA) are described which demonstrate inhibition of aromatase in human placentra and rat ovaries. In animal experiments, the compound was compared with aminoglutethimide (AG) for antitumor activity and effects on plasma hormone levels. 4-OHA was more effective than AG in causing regression of DMBA-induced hormone dependent tumors in the rat. Although estradiol concentrations in ovarian vein blood were reduced initially by both compounds, there is a reflex rise in LH and estradiol levels during long-term treatment with AG, whereas hormone levels in 4-OHA treated animals remained suppressed. Further studies in ovariectomized rats indicated that during long-term treatment, 4-OHA acts as a weak androgen (the compound has less than 1% the activity of testosterone) to directly inhibit the post-castrational rise in gonadotropin levels. This antigonadotropin action of the steroidal aromatase inhibitor may help maintain reduced ovarian estrogen secretion and thus contribute to the antitumor activity of 4-OHA.     

Formation and metabolism of 5(10)-estrene-3 beta,17 beta-diol, a novel 19-norandrogen produced by porcine granulosa cells from C19 aromatizable androgens

The biosynthesis of non-aromatic 19-norsteroids has been studied using primary cultures of porcine granulosa cells. Formation of 5(10)-estrene-3 beta,17 beta-diol, a novel 19-norsteroid, from androstenedione and 19-hydroxyandrostenedione by porcine granulosa cells is reported for the first time. The structure was deduced from (i) comparison of its elution times on C18 reverse phase HPLC with authentic 5(10)-estrene-3 beta,17 beta-diol (ii) identification with 5(10)-estrene-3 beta,17 beta-diol-diacetate after acetylation (iii) oxidation/acid catalysed isomerization to 19-norandrostenedione. Serum or serum plus FSH significantly stimulated (seven fold increase) formation of 5(10)-estrene-3 beta,17 beta-diol from androstenedione and 19-hydroxyandrostenedione. Formation of 5(10)-estrene-3 beta,17 beta-diol from both substrates was significantly (p less than 0.01) reduced by the aromatase inhibitors 4-hydroxyandrostenedione (15 microM) and aminoglutethimide phosphate (10(-4)M). These results suggest that 5(10)-estrene-3 beta,17 beta-diol (and 19-norandrostenedione) may be formed by enzymes similar to the aromatase complex required for estradiol-17 beta biosynthesis. 5(10)-Estrene-3 beta,17 beta-diol is converted by granulosa cells to four metabolites. 19-Norandrostenedione was identified by crystallization to constant specific activity; 19-nortestosterone is a minor product. Production of 19-norandrostenedione and 19-nortestosterone indicates that granulosa cells possess the enzymes necessary for the transformation of 5(10)-estrene-3 beta,17 beta-diol and other 3-hydroxy-5(10)-estrenes to 19-nor-4-ene-3-ketosteroids. The formation of 5(10)-estrene-3 beta,17 beta-diol and 19-norandrostenedione as substantial metabolites of androstenedione suggest a physiological role for these 19-norsteroids in ovarian follicular development.     

Up-regulation of alpha-inhibin expression in the fetal ovary of estrogen-suppressed baboons is associated with impaired fetal ovarian folliculogenesis

We recently demonstrated that the number of primordial follicles was significantly reduced in the ovaries of near-term baboon fetuses deprived of estrogen in utero and restored to normal in animals administered estradiol. Although the baboon fetal ovary expressed estrogen receptors alpha and beta, the mechanism(s) of estrogen action remains to be determined. It is well established that inhibin and activins function as autocrine/paracrine factors that impact adult ovarian function. However, our understanding of the expression of these factors in the primate fetal ovary is incomplete. Therefore, we determined the expression of alpha-inhibin, activin beta(A), activin beta(B), and activin receptors in fetal ovaries obtained at mid and late gestation from untreated baboons and at late gestation from animals in which fetal estrogen levels were reduced by >95% by maternal administration of the aromatase inhibitor CGS 20267 or restored to 30% of normal by treatment with CGS 20267 and estradiol benzoate. Immunocytochemical expression of alpha-inhibin was minimal to nondetectable in fetal ovaries from untreated baboons. In contrast, in baboons depleted of estrogen, alpha-inhibin was abundantly expressed in pregranulosa cells of interfollicular nests and granulosa cells of primordial follicles. Thus, the number (mean +/- SEM) per 0.08 mm2 of fetal ovarian cells expressing alpha-inhibin, determined by image analysis, was similar at mid and late gestation and increased approximately 8-fold (P < 0.01) near term in baboons treated with CGS 20267 and was restored (P < 0.01) to normal in baboons treated with CGS 20267 plus estradiol. Activin beta(A) was detected in oocytes and pregranulosa cells at midgestation and in oocytes and granulosa cells of primordial follicles at late gestation. Activin beta(B) was also expressed in pregranulosa cells and granulosa cells at mid and late gestation, respectively, but was not detected in oocytes. Neither the pattern nor the apparent level of expression of activin beta(A) or beta(B) were altered in fetal ovaries of baboons treated with CGS 20267 or CGS 20267 and estrogen. Activin receptors IA, IB, IIA, and IIB were detected by Western blot analysis in fetal ovaries at mid and late gestation, and expression was not altered by treatment with CGS 20267 or CGS 20267 and estrogen. Activin receptors IB and IIA were localized to oocytes and pregranulosa cells at midgestation and to granulosa cells and oocytes of primordial follicles at late gestation. Thus, the decrease in the number of follicles in the primate fetal ovary of baboons deprived of estrogen in utero was associated with increased expression of alpha-inhibin. Therefore, we propose that estrogen regulates fetal ovarian follicular development by controlling alpha-inhibin expression and, thus, the intraovarian inhibin:activin ratio.