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.     

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.     

Characterization of the increased estrogen synthesis in skin fibroblasts from the Sebright bantam

We have utilized the Sebright bantam chicken as a model system to explore the regulation of estrogen formation in peripheral tissues. As the result of a gene mutation, Sebright males develop a female feathering pattern associated with an increase in estrogen synthesis in skin and in fibroblasts cultured from skin. To provide insight into the mechanisms by which estrogen synthesis is increased we examined several parameters of the aromatase reaction in homogenates from control ovaries, Sebright ovaries and fibroblasts grown from Sebright skin: the pH optima; the apparent Km's for testosterone, 19-nortestosterone, androstenedione, 16-hydroxyandrostenedione, and NADPH; the apparent Ki's for 4-hydroxyandrostenedione, aminoglutethimide, dehydroepiandrosterone, and 19-hydroxytestosterone; and the inactivation of the reaction by heating. No qualitative differences were identified in the enzyme from Sebright and control birds.     

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.     

Proteomic analysis of hypothalamic proteins of high and low egg production strains of chickens

Two slow-growth local chicken strains, derived from a common base population, were bi-directionally selected over twenty generations for carcass traits (B strain) and egg production (L2 strain). The objective of the present study was to identify hypothalamic proteins associated with high egg production (by taking advantage of the similar genetic background of these two strains). Prior to and during egg laying, hypothalamic proteins of B and L2 hens were analyzed with two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Approximately 430 well-resolved spots, ranging from 10 to 40 kDa, pH 5-9, were quantified by image processing. Eight protein spots differed in quantity between B and L2 strains at either stage. Using LC-MS/MS, we identified six of eight protein spots, including proteins known for regulating gene expression, signal transduction and lipid metabolism. The mRNA expression levels of these six proteins were then evaluated by quantitative RT-PCR in five strains of hens, including B, L2 and another three commercial strains; heterogeneous nuclear ribonucleoprotein H3 (HNRPH3) was higher in L2 than in the B strain (consistent with the findings in 2-DE). Increased levels of HNRPH3 mRNA were also present in the hypothalamus of high-egg-yield White Leghorn layers, but were absent in other domestic commercial strains with low egg production rates. In conclusion, the expression level of HNRPH3 may be a new molecular marker to screen for high egg production in slow-growth local chickens.     

Molecular characterization and expression of the cholesteryl ester transfer protein gene in chickens

The cDNA for cholesteryl ester transfer protein (CETP), a protein that catalyzes cholesteryl ester transfer between very low density and high density lipoproteins in plasma, was isolated from chicken liver. When the recombinant protein was overexpressed in HEK293 cells, cholesteryl ester transfer activity was observed in media and cell lysates. By Northern blot analysis, chicken CETP mRNA expression was detected in liver, brain, heart, and spleen. Changes in chicken CETP mRNA expression and plasma CETP activity with nutritional state were examined and found to increase following dietary supplementation with cholesterol in a similar way as in humans. Both the hepatic CETP mRNA levels and plasma CETP activity were significantly lower in mature (i.e egg-laying) hens than in immature female chickens, but were unaffected by age in male animals. Similar changes to those observed in female chickens were observed upon estradiol administration of males. The present study is the first to report the molecular characterization of an avian CETP, and the impairments of CETP gene and activity, which might be regulated by estrogen, play an important role in egg production in laying hens, demonstrating species-specific differences in the lipid metabolism of avian and mammalian species.     

LUTEAR CELLS AND HEN-FEATHERING

The experimental evidence had made clear that some substance is produced in the testis of the male Sebright that suppresses in him the development of the secondary sexual plumage of the cock of his species. The detection in his testis of lutear cells like those in hens makes the conclusion highly probable that it is these cells that cause the suppression of cock-feathering in both the Sebright male and in hens of all fowls. Genetic work by Morgan had shown that one or two Mendelian factor-differences are responsible for hen-feathering in the Sebright. These factor-differences produce their effects through the testes. The presence of these genetic factors, we now see, causes the testes of the Sebright to produce a kind of secretory cell that is ordinarily only produced in the female, or possibly to a slight extent in young males (Boring), or in numbers insufficient to suppress the male plumage in the testes of some ordinary cock birds (Reeves).     

Avian SERPINB12 Expression in the Avian Oviduct Is Regulated by Estrogen and Up-Regulated in Epithelial Cell-Derived Ovarian Carcinomas of Laying Hens

Serine protease inhibitors (SERPINs) are involved in a variety of biological processes such as blood clotting, angiogenesis, immune system, and embryogenesis. Although, of these, SERPINB12 is identified as the latest member of clade B in humans, little is known of it in chickens. Thus, in this study, we investigated SERPINB12 expression profiles in various tissues of chickens and focused on effects of steroid hormone regulation of its expression. In the chicken oviduct, SERPINB12 mRNA and protein are abundant in the luminal (LE) and glandular (GE) epithelia of the magnum in response to endogenous or exogenous estrogen. Furthermore, SERPINB12 mRNA and protein increase significantly in GE of cancerous ovaries of laying hens with epithelia-derived ovarian cancer. Collectively, these results indicate that SERPINB12 is a novel estrogen-stimulated gene that is up-regulated by estrogen in epithelial cells of the chicken oviduct and that it is a potential biomarker for early detection of ovarian carcinomas in laying hens and women.     

Effects of atorvastatin on lipid metabolism in normolipidemic and hereditary hyperlipidemic, non-laying hens

As a result of a hereditable point mutation in the oocyte very low density lipoprotein (VLDL) receptor, sexually mature restricted ovulator (RO) female chickens (Gallus gallus), first described as a non-laying strain, exhibit endogenous hyperlipidemia and develop atherosclerotic lesions. In a 20-day study, RO hens and their normolipidemic (NL) siblings were fed either a control diet, or the control diet supplemented with 0.06% atorvastatin (AT), a potent 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) inhibitor. Compared to NL hens, RO birds exhibited greatly elevated baseline plasma total cholesterol (CHOL) and triglyceride (TG) concentrations (1.56 vs. 4.55 g/l and 30.7 vs. 138.4 g/l, respectively). AT attenuated plasma CHOL and TG concentrations by 60.3% and 70.1%, respectively, in NL hens and by 45.1% and 34.3%, respectively, in RO hens. Messenger RNA levels of several key genes involved in hepatic VLDL assembly were suppressed in RO vs. NL hens, but were unaffected by AT. In contrast, AT elevated liver HMGR mRNA levels in NL and RO birds, but only NL hens exhibited an AT-associated increase in hepatic HMGR immunoreactive protein levels. Down-regulation of HMGR gene expression due to higher baseline levels of circulating CHOL may explain why RO birds responded less robustly than NL hens to AT administration.     

Aromatase in endometriosis and uterine leiomyomata

Endometrial tissue from uterine disease-free women does not exhibit aromatase activity. In contrast, aromatase enzyme activity and mRNA levels are readily detectable in endometriosis. PGE₂ stimulates both aromatase expression and activity in endometriotic stromal cells via promoter II region of the aromatase gene. This results in local production of estradiol, which induces PGE₂ formation and establishes a positive feedback cycle. This mechanism seems to contribute to continuous production of estradiol and PGE₂. Aromatase mRNA levels and enzyme activity are also present in uterine leiomyomata that are estrogen-dependent benign tumors of the myometrium. Successful treatment of endometriosis and uterine leiomyomata using aromatase inhibitors by recent pilot trials underscores the clinical significance of these molecular studies.     

Insulin alters the effects of follicle stimulating hormone on aromatase in bovine granulosa cells in vitro

It is known that follicle-stimulating hormone (FSH) and insulin stimulate estradiol secretion from cultured non-luteinizing granulosa cells. The interaction between these hormones is less well understood. Granulosa cells from small (2-4 mm) bovine follicles were cultured in serum-free medium to determine if cytochrome P450 aromatase activity is regulated by FSH in the presence of different concentrations of insulin. Insulin significantly stimulated aromatase activity in the absence of FSH. There was a significant interaction between insulin and FSH on aromatase activity, such that FSH stimulated activity at low (0.5, 1 and 10 ng/ml) doses of insulin, whereas at higher (100 ng/ml) doses of insulin FSH failed to stimulate aromatase activity. To determine if the lack of a response to FSH with higher doses of insulin is related to gene expression, the effect of FSH on P450 aromatase mRNA levels was measured. An 'uncoupling' of mRNA and enzyme activity was observed for cells cultured with 100 ng/ml insulin, as FSH significantly increased P450 aromatase mRNA abundance without affecting estradiol secretion or aromatase activity. We conclude that in the presence of high doses of insulin, FSH decreases aromatase activity, and an uncoupling of P450 aromatase mRNA and aromatase activity occurs. This may have implications for infertility treatments when there is a risk of hyperinsulinemia.     

Fibromodulin gene transcription is induced by ultraviolet irradiation, and its regulation is impaired in senescent human fibroblasts

Cells undergoing replicative senescence display an altered pattern of gene expression. Senescent fibroblasts show significant changes in the expression of mRNAs encoding extracellular matrix-remodeling proteins; among these mRNAs, the mRNA encoding fibromodulin is highly decreased in these cells. To understand the molecular basis of this phenomenon, we explored the regulatory mechanisms of the human fibromodulin gene. We found that fibromodulin gene promoter contains a cis-element, crucial for its basal expression, that forms a DNA-protein complex when exposed to nuclear extracts from exponentially growing human fibroblasts and not to extracts from cells undergoing senescence by repeated in vitro passages or by mild oxidative stress. The purification of this complex showed that it contains the damage-specific DNA-binding protein DDB-1. The latter is known to be induced by UV irradiation; therefore we checked whether fibromodulin gene promoter is regulated upon the exposure of the cells to UV rays. The results showed that, in exponentially growing fibroblasts, the promoter efficiency is increased by UV irradiation and the DDB-1-containing complex is robustly enriched in cells exposed to UV light. Accordingly, in these experimental conditions the endogenous fibromodulin mRNA accumulates to very high levels. On the contrary, senescent cells did not show any activation of the fibromodulin gene promoter, any induction of the DDB-1-containing complex, or any accumulation of fibromodulin mRNA. These phenomena are accompanied in senescent cells by a decrease of the UV-damaged DNA binding activity.     

A novel role of sodium butyrate in the regulation of cancer-associated aromatase promoters I.3 and II by disrupting a transcriptional complex in breast adipose fibroblasts

The aromatase gene encodes the key enzyme for estrogen formation. Aromatase enzyme inhibitors eliminate total body estrogen production and are highly effective therapeutics for postmenopausal breast cancer. A distal promoter (I.4) regulates low levels of aromatase expression in tumor-free breast adipose tissue. Two proximal promoters (I.3/II) strikingly induce in vivo aromatase expression in breast fibroblasts surrounding malignant cells. Treatment of breast fibroblasts with medium conditioned with malignant breast epithelial cells (MCM) or a surrogate hormonal mixture (dibutyryl (Bt2)cAMP plus phorbol diacetate (PDA)) induces promoters I.3/II. The mechanism of promoter-selective expression, however, is not clear. Here we reported that sodium butyrate profoundly decreased MCM- or Bt2cAMP + PDA-induced promoter I.3/II-specific aromatase mRNA. MCM, Bt2cAMP + PDA, or sodium butyrate regulated aromatase mRNA or activity only via promoters I.3/II but not promoters I.1 or I.4 in breast, ovarian, placental, and hepatic cells. Mechanistically, recruitment of phosphorylated ATF-2 by a CRE (-211/-199, promoter I.3/II) conferred inductions by MCM or Bt2cAMP + PDA. Chromatin immunoprecipitation-PCR and immunoprecipitation-immunoblotting assays indicated that MCM or Bt2cAMP + PDA stabilized a complex composed of phosphorylated ATF-2, C/EBPbeta, and cAMP-response element-binding protein (CREB)-binding protein in the common regulatory region of promoters I.3/II. Overall, histone acetylation patterns of promoters I.3/II did not correlate with sodium butyrate-dependent silencing of promoters I.3/II. Sodium butyrate, however, consistently disrupted the activating complex composed of phosphorylated ATF-2, C/EBPbeta, and CREB-binding protein. This was mediated, in part, by decreased ATF-2 phosphorylation. Together, these findings represent a novel mechanism of sodium butyrate action and provide evidence that aromatase activity can be ablated in a signaling pathway- and cell-specific fashion.     

Sex differences and androgen-dependent regulation of aromatase (CYP19) mRNA expression in the developing and adult rat brain

Sex differences, androgen dependence and asymmetries of aromatase activity have been reported during ontogeny of the rat. It remains to be elucidated, however, whether the changes in aromatase activity are reflected by similar changes in specific mRNA levels. In addition, very little is known regarding mechanism(s) underlying such differential regulation of aromatase expression. To address these questions, we have employed the in situ hybridization (ISH) technique to examine specific mRNA levels in the brain of both male and female rats at selected stages of development. In prenatal stages of development, at gestational day (GD) 18 and 20, aromatase mRNA was detected in several hypothalamic and limbic brain regions. Semiquantitative analysis of aromatase mRNA did not reveal statistically significant sex differences in any of these regions (except in one experiment at GD20, when a sex difference was found in the medial preoptic nucleus). In contrast, clear sex differences were determined at postnatal day (PN) 2; male animals contained significantly more aromatase mRNA in the bed nucleus of the stria terminalis (BST) and thesexually dimorphic nucleus of the preoptic area (SDN) compared to female rats. Four days later in development, at PN6, sex differences of aromatase mRNA signals were observed in the BST, but were no longer detectable in the SDN. At PN15 and in adult animals, no sex differences could be determined. The effect of flutamide treatment (50 mg/kg/day) was investigated in GD20 fetuses as well as in adult rats. No statistically significant changes in aromatase mRNA expression were found in either case. In summary, our results suggest that differential regulation of aromatase mRNA expression during the critical period of sexual differentiation might, in part, account for the establishment of some of the many sexually dimorphic parameters of the rat brain. The role of androgens in the regulation of the sex-specific and developmental expression of aromatase mRNA in the rat brain remains to be clarified.