Sexually dimorphic androgen and estrogen receptor mRNA expression in the brain of túngara frogs

Sex steroid hormones are potent regulators of behavior and they exert their effects through influences on sensory, motor, and motivational systems. To elucidate where androgens and estrogens can act to regulate sex-typical behaviors in the túngara frog (Physalaemus pustulosus), we quantified expression of the androgen receptor (AR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ) genes in the brains of male and females. To do so, we cloned túngara-specific sequences for AR, ERα, and ERβ, determined their distribution in the brain, and then quantified their expression in areas that are important in sexual communication. We found that AR, ERα, and ERβ were expressed in the pallium, limbic forebrain (preoptic area, hypothalamus, nucleus accumbens, amygdala, septum, striatum), parts of the thalamus, and the auditory midbrain (torus semicircularis). Males and females had a similar distribution of AR and ER expression, but expression levels differed in some brain regions. In the auditory midbrain, females had higher ERα and ERβ expression than males, whereas males had higher AR expression than females. In the forebrain, females had higher AR expression than males in the ventral hypothalamus and medial pallium (homolog to hippocampus), whereas males had higher ERα expression in the medial pallium. In the preoptic area, striatum, and septum, males and females had similar levels of AR and ER expression. Our results suggest that sex steroid hormones have sexually dimorphic effects on auditory processing, sexual motivation, and possibly memory and, therefore, have important implications for sexual communication in this system.     

Expression of estrogen receptors,androgen receptor and steroid receptor coactivator-3 is negatively correlated to the differentiation of astrocytic tumors

Astrocytic tumors are the most common primary brain tumors. It has been reported that androgen receptor (AR), estrogen receptors alpha (ERα) and beta (ERβ) and their coactivator SRC-1 and SRC-3 are involved in the regulation of the growth and development of many tumors, but their expression profiles and significances in the astrocytic tumors remain largely unknown. In this study, the expression of AR, ERs, and SRCs, and the possible roles of them in astrocytic neoplasm were evaluated and compared to normal brain tissues by nickel-intensified immunohistochemistry with tissue microarrays. The results showed that there were no age- or gender-differences regarding to the levels of these receptors or coactivators in astrocytic or normal brain tissues. In the high-grade astrocytic tissue, the levels of AR, ERs and SRC-3 were significantly decreased when compared to the low-grade astrocytic tissues, but the levels of SRC-1 remain unchanged. Correlation analysis revealed that the levels of AR, ERs and SRC-3 were negatively correlated to tumor differentiation, and the levels of SRC-3 were positively correlated to that of ERα. Furthermore, the decreased levels of SRC-3 were associated with an increase of ERβ in astrocytic tumors when compared to that of normal brain tissues. These above results indicate a combination of decreased expression of ERs, AR and SRC-3 but not SRC-1 may be involved in the tumorigenesis of gliomas, ERα/SRC-3 axis may play central role in the regulation these tumors.     

‘Specific receptor binding’ of radioactively labeled products of radiolysis of the estrogen receptor ligand R 2858 (17α-ethylnyl-11β-methoxy-estradiol-17β)

Radioactively labeled steroids undergo decomposition processes, which are dependent on time, storage conditions (temperature, solvents, etc.), degree of labeling etc. This communication shows that several decomposition products of 17α-ethynyl-11β-methoxy-estradiol-17β (R 2858 0) bind to rat uterine cytosol in a way that would be interpreted as ‘specific receptor binding’ if some of these compounds were present in the ligand solution used for estrogen receptor determination. Thys, the binding was charcoal-resistant and displaceable with an excess of unlabeled R 2858, and the percentage of binding was of significant magnitude to seriously interfere with receptor measurements.     

Myometrial effects of selective estrogen receptor modulators on estradiol-responsive gene expression are gene and cell-specific

We examined in vivo effects of selective estrogen receptor modulators (SERMs) 4-OH-tamoxifen (Tam), GW 5638 (GW) and EM-800 (EM) on myometrial gene expression. The uteri of ovariectomized ewes were infused with 10⁻⁷ M of one SERM via indwelling catheters for 24 h preceding hysterectomy. Half of the ewes in each SERM group received an intramuscular injection of 50 μg 17β-estradiol (E2) 18 h prior to hysterectomy. Northern blot analysis and in situ hybridization demonstrated that E2 increased estrogen receptor (ER), progesterone receptor (PR) and cyclophilin (CYC) gene expression in the cells of both inner layer of myometrium (IM) and outer layer of myometrium (OM) as well as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression in OM. Tam also increased ER mRNA levels in OM. EM appeared to increase ER gene expression, but antagonized E2’s up-regulation of PR and CYC gene expression in both IM and OM. Tam and GW also antagonized E2 up-regulation of PR gene expression in OM but not IM. No SERM affected GAPDH gene expression with or without E2. Immunohistochemistry indicated that E2 increased nuclear ER and PR protein levels in both IM and OM. EM was unique in up-regulating ER protein levels, opposite to its effects in endometrial cells. All SERMs tested antagonized this increase in PR immunostaining preferentially in OM compared to the IM layer. These results illustrate gene and cell layer-specific effects of SERMs in sheep myometrium.     

Endometrial effects of selective estrogen receptor modulators (SERMs) on estradiol-responsive gene expression are gene and cell-specific

Three selective estrogen receptor modulator (SERM) drugs which included 4-OH-tamoxifen (Tam), EM-800 (EM) and GW 5638 (GW) were investigated to determine their ability to inhibit estradiol-responsive gene expression in sheep endometrium. The uteri of ovariectomized ewes (10 ewes per SERM group) were infused with 10⁻⁷ M SERMs for 24 h prior to hysterectomy. Five ewes from each group received 50 μg 17β-estradiol (E2) and the remaining five ewes received vehicle 18 h prior to hysterectomy. Northern blot analyses and in situ hybridization demonstrated that E2 treatment increased estrogen receptor (ER), progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and cyclophilin (CYC) mRNA levels in most endometrial cells examined. Tam and GW exhibited characteristics similar to E2 by increasing ER gene expression, but they antagonized the E2-induced increases in PR and CYC mRNA levels. EM acted as an E2-agonist of GAPDH gene expression, but antagonized the E2 up-regulation of ER, PR and CYC gene expression in most endometrial cells. Immunohistochemistry determined that EM decreased ER protein levels in the glandular epithelium, and the SERMs investigated antagonized increases in PR protein levels in endometrium. In conclusion, GW and EM exhibit fewer agonist effects than Tam on endometrial gene expression. EM demonstrated the greatest antagonism of E2-enhanced levels of ER, PR and CYC, likely due to the inhibition of ER gene expression at both mRNA and protein levels.     

Social status and sex independently influence androgen receptor expression in the eusocial naked mole-rat brain

Naked mole-rats (Heterocephalus glaber) are eusocial rodents that live in large subterranean colonies including a single breeding female and 1-3 breeding males; all other members of the colony, known as subordinates, are reproductively suppressed. We recently found that naked mole-rats lack many of the sex differences in the brain and spinal cord commonly found in other rodents. Instead, neural morphology is influenced by breeding status, such that breeders, regardless of sex, have more neurons than subordinates in the ventromedial nucleus of the hypothalamus (VMH), and larger overall volumes of the bed nucleus of the stria terminalis (BST), paraventricular nucleus (PVN) and medial amygdala (MeA). To begin to understand how breeding status influences brain morphology, we examined the distribution of androgen receptor (AR) immunoreactivity in gonadally intact breeders and subordinates of both sexes. All animals had AR+ nuclei in many of the same regions positive for AR in other mammals, including the VMH, BST, PVN, MeA, and the ventral portion of the premammillary nucleus (PMv). We also observed diffuse labeling throughout the preoptic area, demonstrating that distribution of the AR protein in presumptive reproductive brain nuclei is well-conserved, even in a species that exhibits remarkably little sexual dimorphism. In contrast to other rodents, however, naked mole-rats lacked AR+ nuclei in the suprachiasmatic nucleus and hippocampus. Males had more AR+ nuclei in the MeA, VMH, and PMv than did females. Surprisingly, breeders had significantly fewer AR+ nuclei than subordinates in all brain regions examined (VMH, BST, PVN, MeA, and PMv). Thus, social status is strongly correlated with AR immunoreactivity in this eusocial species.     

The human brain has distinct regional expression patterns of estrogen receptor alpha mRNA isoforms derived from alternative promoters

The human estrogen receptor (ER) alpha gene is transcribed from multiple promoters, generating mRNA isoforms with unique 5' ends in the untranslated region. In the present study, alternative promoters were shown to regulate the ERalpha gene expression in different neuronal populations of the human brain. By using in situ hybridization histochemistry, the A and B promoters, but not the C promoter, in the ERalpha gene were found to be active in the human forebrain. The mRNA isoform transcribed from the A promoter was expressed in low levels in most of the brain areas where ERalpha mRNA was present. In contrast, the B promoter mRNA isoform was more restricted, localized predominantly in high-expressing ERalpha mRNA regions. The gross anatomical distribution of the different mRNA isoforms analyzed with RT-PCR generally supported the results obtained by the in situ hybridization. Estrogen is known to modulate many different brain functions, such as neuroendocrine events associated with reproduction, mood, and cognition, likely to be mediated by different neuronal populations. Thus, the current findings of alternative ERalpha promoter expression in distinct neuronal populations suggest that multiple promoter usage is a possible mechanism to achieve differentiated regulation of the ERalpha expression, dependent on the cell phenotype and consequently the functions mediated by the specific neuron.     

Stage dependent and androgen inductive expression of orphan receptor TR4 in rat testis

In this study, we investigated the expression of TR4 in different stages of seminiferous tubules and the relationship between TR4 and androgen in rat testis. We found that TR4 was stage-dependently expressed in rat seminiferous tubules, T withdrawal induced by high doses of testosterone undecanoate and ethane dimethane sulfonate inhibit TR4 expression in rat testis, and testosterone induced TR4 expression in co-cultured primary germ/Sertoli cells. Furthermore, we demonstrated that androgen receptor could enhance TR4-mediated transactivation activity in testis cells in the presence of testosterone. Together, these data indicate that the expression of TR4 in rat testis is stage dependent and androgen inductive, and suggest the important role of orphan receptor TR4 in spermatogenesis.     

Effect of testosterone and melatonin on social dominance and agonistic behavior in male Tscheskia triton

Social dominance and agonistic behavior play important roles in animal societies. Melatonin and testosterone are closely related to social dominance and agonistic behavior in rodents, but interactions between both of them remain unknown. In this study we investigated the effects of testosterone and melatonin by manipulating photoperiod and castration on social dominance and agonistic behavior in male Tscheskia triton. Castration significantly decreases social dominance of both short- and long-day males, suggesting that testosterone benefits social dominance of males in both breeding and non-breeding seasons. In intact conditions, long-day males tended to dominate short-day males, suggesting that the effect of testosterone on social dominance was a little stronger than melatonin. However, castrated short-day males became dominant over their castrated long-day opponents meaning that high melatonin levels obviously benefit social dominance in males. Hormone implantation indicated that testosterone had no effect on non-breeding condition, but that melatonin was important during the breeding season. Our results indicate that both testosterone and melatonin are important in determining social dominance in male hamsters, and the effect of testosterone appears to be stronger than melatonin. Testosterone is responsible for aggression and social dominance in male hamsters during the breeding season, while melatonin regulates behavior during non-breeding, probably due to the different seasonal secretory patterns of the hormones.     

Expression of estrogen receptor alpha and beta in myometrium of premenopausal and postmenopausal women

Although a clear role for estrogen receptor (ER) alpha has been established, the contribution of ERbeta in estrogen-dependent development, growth and functions of the myometrium is not understood. As a first step towards understanding the role of ERbeta, we have examined the expression of ERalpha and ERbeta in the human myometrium. With competitive RT-PCR assays, the level of ERbeta mRNA was 10-200 times lower than that of ERalpha mRNA in both premenopausal and postmenopausal myometrium. In premenopausal myometrium, the expression pattern of ERbeta mRNA during the menstrual cycle was similar to that of ERalpha mRNA, with highest levels in peri-ovulatory phase. In postmenopausal myometrium, ERbeta mRNA was significantly higher than it was in premenopausal myometrium, while the level of ERalpha mRNA was lower. The net result was a change in the ratio of ERbeta to ERalpha mRNA expression. The ratio changed from 0.6-1.5 in premenopausal to 2.5-7.6 in postmenopausal myometrium. In premenopausal women, the gonadotropin releasing hormone analogue, leuprorelin acetate, elicited a decrease in ERalpha and an increase in ERbeta mRNA expression to cause a postmenopausal receptor phenotype. Estradiol, on the other hand, reversed ERalpha and ERbeta mRNA expression and their ratio in postmenopausal myometrium to those of premenopausal myometrium. Immunohistochemical staining and Western blot analysis of ERalpha and ERbeta with semiquantitative analysis showed good agreement between mRNA and protein levels. The data indicate that coordinated expression of ERalpha and ERbeta might be necessary for normal estrogen action in myometrium. Furthermore, estrogen appears a dominant regulator of both receptors in the myometrium.     

Estradiol restores diabetes-induced reductions in sex steroid receptor expression and distribution in the vagina of db/db mouse model

Sex steroid hormones and receptors play an important role in maintaining vaginal physiology. Disruptions in steroid receptor signaling adversely impact vaginal function. Limited studies are available investigating the effects of diabetic complications on steroid receptor expression and distribution in the vagina. The goals of this study were to investigate type 2 diabetes-induced changes in expression, localization and distribution of estrogen (ER), progesterone (PR) and androgen receptors (AR) in the vagina and to determine if estradiol treatment ameliorates these changes. Eight-week-old female diabetic (db/db) mice (strain BKS.Cg-m+/+ Lepr^db/J) were divided into two subgroups: untreated diabetic and diabetic animals treated with pellets containing estradiol. Control normoglycemic littermates were subcutaneously implanted with pellets devoid of estradiol. At 16 weeks of age, animals were sacrificed, vaginal tissues excised and analyzed by Western blot and immunohistochemical methods. Diabetes produced marked reductions in protein expression of ER, PR, and AR. Diabetes also resulted in marked differences in the distribution, staining intensity and proportion of immunoreactive cells containing these steroid receptors in the epithelium, lamina propria and muscularis. Treatment of diabetic animals with estradiol restored receptor protein expression and distribution similar to those levels observed in control animals. This study demonstrates that type 2 diabetes markedly reduces steroid receptor protein expression and distribution in the vagina. Estradiol treatment of diabetic animals ameliorates these diabetes-induced changes.     

Cloning and expression analysis of androgen receptor gene in chicken embryogenesis

We cloned a full-length androgen receptor (AR) cDNA from chicken (Gallus gallus) gonads. The cDNA sequence has an open reading frame of 2109 bp encoding 703 amino acids. The chicken AR (cAR) shares high homology with ARs from other species in its amino acid sequences, in particular DNA binding domain (DBD) and ligand binding domain (LBD). RT-PCR analysis revealed that cAR mRNA is expressed in several embryonic tissues of both sexes, and relatively higher expression was observed in left ovary compared with testis. The immunoreactive signal of AR was co-localized within the ovarian cell nucleus, while such nuclear localization was not detected in those of testis. To get insight on the possible role of androgen-AR signaling during gonadal development, non-steroidal AR antagonist, flutamide, was administrated in ovo. The treatment induced the disorganization of sex cords in ovarian cortex at day 12 of incubation. The effect was restored by testosterone co-treatment, implying the possibility that AR mediated signaling may be involved in ovarian morphogenesis. Furthermore, co-treatment of flutamide with estradiol-17beta (E2) also restored the phenotype, suggesting androgen-AR signaling might activate aromatase expression that is necessary for estrogen synthesis. These findings suggest androgen-AR signaling might contribute to chicken embryonic ovarian development.     

Nonylphenol modulates expression of androgen receptor and estrogen receptor genes differently in gender types of the hermaphroditic fish Rivulus marmoratus

To uncover the effect of estrogenic chemicals [4-nonylphenol (NP) and bisphenol A (BisA)] on the expression of androgen receptor (AR) and estrogen receptors (ERalpha and ERbeta) in the hermaphroditic fish Rivulus marmoratus, we cloned the full length of the cDNAs encoding AR, ERalpha, and ERbeta from gonadal tissue of R. marmoratus and analyzed the modulation of expression of these genes following exposure to estrogenic chemicals using real-time RT-PCR. R. marmoratus AR, ERalpha, and ERbeta genes showed a high similarity to the relevant fish species on amino acid residues, respectively. Rm-ERalpha and Rm-ERbeta cDNAs included a serine-rich region when compared to other teleost fish ER genes. Tissue-specific expression of Rm-AR and Rm-ERbeta mRNAs in adult hermaphrodite R. marmoratus was high in the gonad, while Rm-ERalpha mRNA was high in the liver based on real-time RT-PCR. In addition, Rm-AR and Rm-ERalpha mRNAs increased along with developmental stage from stage 3 (5 dpf) to hatching, while Rm-ERbeta mRNA increased from stage 2 (2 dpf). To uncover the effect of estrogenic chemicals on R. marmoratus, we exposed the fish to NP (300 microg/l) and BisA (600 microg/l) for 96 h. Significant down-regulation of Rm-AR, Rm-ERalpha, and Rm-ERbeta mRNA was observed in gonadal tissue after exposure to NP but not BisA. In the liver, there were gender differences in gene expression after EDC exposure. These results demonstrate that expression patterns of the Rm-AR, Rm-ERalpha, and Rm-ERbeta genes in the hermaphroditic fish, R. marmoratus, vary according to tissue and developmental stage as well as the specificity of environmental estrogenic chemicals. These genes can be useful as molecular biomarkers in assessing the potential impact of estrogenic compounds using this species as a model system.     

Differentiation of murine embryonic stem cells induces progesterone receptor gene expression

The role of steroid hormone receptors in very early embryonic development remains unknown. Clearly, expression during organogenesis is important for tissue-specific development. However, progesterone receptor (PR) and estrogen receptors (ERalpha, ERbeta) are expressed during early development through the blastocyst stage in mice and other species, and yet are not essential for embryonic viability. We have utilized the mouse embryonic stem (mES) cell model to investigate the regulated expression of these receptors during differentiation. Surprisingly, one of the earliest changes in gene expression in response to a differentiation signal observed is PR gene induction. It parallels the time course of expression for the patterning genes Hoxb1 and Hoxa5. Unexpectedly, PR gene expression is not regulated in an estrogen-dependent manner by endogenous ERs or by transiently overexpressed ERalpha. Our results suggest a potentially novel mechanism of PR gene regulation within mES cells compared to adult tissues and the possibility of unique targets of PR action during early mES cell differentiation.