The non-aromatizable androgen, dihydrotestosterone, induces antiestrogenic responses in the rainbow trout

In order to satisfy government mandates, numerous studies have been performed categorizing potential endocrine disrupting chemicals as (anti)estrogens or (anti)androgens. We report here that dihydrotestosterone (DHT), a potent, non-aromatizable androgen receptor agonist, induces antiestrogenic responses through direct and/or indirect modulation of vitellogenin (Vg), steroid hormone and total cytochrome P450 levels. DHT and two weak, aromatizable androgens, DHEA and androstenedione (0.05-50 mg/kg per day), were fed to juvenile trout for 2 weeks. DHEA and androstenedione significantly increased blood plasma Vg by up to 30- and 45-fold, respectively (P<0.05, t-test). 17beta-Estradiol (E2) increases were also observed with both androgens, albeit with lower sensitivity. DHT markedly decreased Vg and E2 levels, suggesting that DHEA and androstenedione increased Vg and E2 via conversion to E2 and not by estrogen receptor agonism. DHEA and androstenedione had no effect on total cytochrome P450 content, while DHT significantly decreased P450 content in a dose dependent fashion. These results indicate that alterations in metabolism mediated by androgen receptor binding may be responsible for the Vg and E2 decreases by DHT. In an attempt to decipher between receptor and non-receptor androgenic mechanisms of the observed DHT effects, DHT (0, 50 or 100 mg/kg per day) and flutamide (0-1250 mg/kg per day), an androgen receptor antagonist, were fed to juvenile rainbow trout for 2 weeks. Flutamide alone was as effective as DHT in decreasing E2 and Vg levels in males but did not significantly reverse DHT induced Vg decreases in either sex (P>0.05, F-test). DHT decreases in total P450 content were partially attenuated in males by flutamide co-treatment, but not females, suggesting a partial androgenic mechanism to the P450 decreases as well as a fundamental sex difference responding to androgen receptor binding. Moreover, flutamide alone decreased P450 content by up to 30% in males and 40% in females. These effects may be mediated through direct androgen receptor binding irrespective of whether the binding is agonistic or antagonistic. This study indicates that androgen receptor agonists/antagonists can elicit significant antiestrogenic effects that may not necessarily be mediated through classic receptor binding mechanisms and signal transduction pathways.     

Stage-Specific Effects of Androgens and Estradiol-17beta on the Development of Late Primary and Early Secondary Ovarian Follicles of Coho Salmon (Oncorhynchus kisutch) In Vitro

An in vitro system was used to analyze the effects of sex steroids on the development of primary (late perinucleolar stage) and early secondary, previtellogenic (early cortical alveolus stage) ovarian follicles of coho salmon cultured for up to 21 days. Late perinucleolar-stage follicles increased significantly in size after 7 days of treatment with low concentrations of 11-ketotestosterone (11-KT), a nonaromatizable androgen. An androgen receptor antagonist (flutamide) inhibited this growth-promoting effect, and the highest concentration resulted in atresia of follicles, implicating androgens as survival factors at this stage. Testosterone (T) was less effective than 11-KT in promoting growth, but blocking aromatization with exemestane resulted in a growth response similar to that of 11-KT. Estradiol-17beta (E2) had no effect on growth at this stage. After 21 days of culture, E2 was the most potent steroid in increasing the number of follicles containing cortical alveoli and the number of cortical alveoli within those follicles. At the early cortical alveolus stage, low doses of E2 promoted growth and strongly stimulated synthesis of cortical alveoli, actions that were inhibited by an estrogen receptor antagonist (tamoxifen). 11-KT displayed moderate growth-promoting effects, and 11-KT and T stimulated moderate to substantial increases in abundance of cortical alveoli. This study shows that the predominant role of androgens is the promotion of growth of late perinucleolar-stage follicles, while E2 stimulates both the growth and accumulation of cortical alveoli in early cortical alveolus-stage follicles.     

Effects of androgens on serum concentrations of gonadotropins and ovarian steroids in gilts

To examine how androgens affect endocrine events associated with increased ovulation rate, gilts were injected with androgen receptor agonists, an antagonist, or a combination of both. Blood samples were collected hourly from Day 13 to estrus (Day 0 = onset of estrus) coincident with gilts (n = 6 per treatment) receiving daily treatments of vehicle (corn oil), 10 mg of testosterone, 10 mg of 5 alpha-dihydrotestosterone (dihydrotestosterone), 1.5 g of flutamide (an androgen receptor antagonist), testosterone plus flutamide, or dihydrotestosterone plus flutamide. Treatment of gilts with testosterone or dihydrotestosterone alone increased (P < 0.05) concentrations of FSH in serum, and these effects were blocked by cotreatment with flutamide. Estradiol-17beta and androstenedione concentrations in serum were increased (P < 0.05) at 2 h after injection of testosterone or testosterone plus flutamide but not after dihydrotestosterone treatment, probably because of the role of testosterone as a substrate for estradiol-17beta and androstenedione synthesis. There were no effects of the six treatments on serum concentrations of progesterone during luteolysis, but treating gilts with testosterone shortened (P < 0.05) the proestrous period. Total embryonic loss by Day 11 in gilts treated with dihydrotestosterone was reversed when gilts were cotreated with dihydrotestosterone plus flutamide. Results of this experiment indicated that androgen actions both increased FSH secretion and reduced embryonic survival by a mechanism(s) dependent on the androgen receptor.     

Regulation of sexual odor preference by sex steroids in the posterodorsal medial amygdala in female rats

Our previous study in male rats demonstrated that bilateral administration of flutamide, an androgen receptor (AR) antagonist, into the posterodorsal medial amygdala (MePD) increased the time sniffing male odors to as high as that sniffing estrous odors, eliminating the preference for estrous odors over male odors. This made us speculate that under blockade of AR in the MePD, testosterone-derived estrogen acting on the same brain region arouses interest in male odors which is otherwise suppressed by concomitant action of androgen. In cyclic female rats, endogenous androgen has been thought to be involved in inhibitory regulation of estrogen-activated sexual behavior. Thus, in the present study, we investigated the possibility that in female rats the arousal of interest in male odors is also normally regulated by both estrogen and androgen acting on the MePD, as predicted by our previous study in male rats. Implantation of either the estrogen receptor blocker tamoxifen (TX) or a non-aromatizable androgen 5α-dihydrotestosterone (DHT) into the MePD of ovariectomized, estrogen-primed female rats eliminated preference for male odors over estrous odors by significantly decreasing the time sniffing male odors to as low as that sniffing estrous odors. The subsequent odor discrimination tests confirmed that the DHT and TX administration did not impair the ability to discriminate between male and estrous odors. These results suggest that in estrous female rats estrogen action in the MePD plays critical roles in the expression of the preference for male odors while androgen action in the same brain region interferes with the estrogen action.     

Estrogen and progesterone modulation of eosinophilic infiltration of the rat uterine cervix

Ripening of the rat cervix involves widespread collagenolysis that follows an eosinophilic leukocyte infiltration. The hormonal control of these events is not well understood. The aims of this study were to investigate the mechanism through which progesterone (P) and 17beta-estradiol (E(2)) modulate eosinophilic invasion and to determine if this event is protein synthesis mediated. Cervical eosinophilic invasion was measured in intact rats during the second half of pregnancy and compared with values from ovariectomized (O) pseudopregnant (PSP) rats treated with P and E(2) in doses that mimicked the levels of pregnancy. Other O-PSP rats were treated with an E(2) antagonist (tamoxifen) and the antiprogestin RU-486. To study the role of protein synthesis in eosinophilic invasion of the cervix, rats were treated with actinomycin-D (an inhibitor of mRNA synthesis), and animals were sacrificed on D21 or D22 to evaluate eosinophilic invasion. Rats treated with E(2) showed high levels of infiltration and tamoxifen blocked this E(2) effect. On the other hand, P antagonized the stimulatory effects of E(2) on eosinophilic invasion, however when the P and E(2) treated rats were injected with RU-486 the inhibitory effect of P was reversed. In intact pregnant rats a sharp rise in eosinophilic infiltration was detected on D23, 20 h after the fall of serum P. Finally, E(2) treated rats injected with actinomycin-D had no invasion of eosinophils. In conclusion, the estrogen-triggered eosinophil invasion is affected by the classic estrogen receptor antagonist tamoxifen and by the mRNA synthesis blocker actinomycin-D suggesting a genomic action of E(2). Furthermore, the estrogen effect is blocked by P and this inhibition is reversed by RU-486.     

Testosterone inhibits estrogen-induced mammary epithelial proliferation and suppresses estrogen receptor expression.

This study investigated the effect of sex steroids and tamoxifen on primate mammary epithelial proliferation and steroid receptor gene expression. Ovariectomized rhesus monkeys were treated with placebo, 17beta estradiol (E2) alone or in combination with progesterone (E2/P) or testosterone (E2/T), or tamoxifen for 3 days. E2 alone increased mammary epithelial proliferation by approximately sixfold (P:<0.0001) and increased mammary epithelial estrogen receptor (ERalpha) mRNA expression by approximately 50% (P:<0.0001; ERbeta mRNA was not detected in the primate mammary gland). Progesterone did not alter E2's proliferative effects, but testosterone reduced E2-induced proliferation by approximately 40% (P:<0.002) and entirely abolished E2-induced augmentation of ERalpha expression. Tamoxifen had a significant agonist effect in the ovariectomized monkey, producing a approximately threefold increase in mammary epithelial proliferation (P:<0.01), but tamoxifen also reduced ERalpha expression below placebo level. Androgen receptor (AR) mRNA was detected in mammary epithelium by in situ hybridization. AR mRNA levels were not altered by E2 alone but were significantly reduced by E2/T and tamoxifen treatment. Because combined E2/T and tamoxifen had similar effects on mammary epithelium, we investigated the regulation of known sex steroid-responsive mRNAs in the primate mammary epithelium. E2 alone had no effect on apolipoprotein D (ApoD) or IGF binding protein 5 (IGFBP5) expression, but E2/T and tamoxifen treatment groups both demonstrated identical alterations in these mRNAs (ApoD was decreased and IGFBP5 was increased). These observations showing androgen-induced down-regulation of mammary epithelial proliferation and ER expression suggest that combined estrogen/androgen hormone replacement therapy might reduce the risk of breast cancer associated with estrogen replacement. In addition, these novel findings on tamoxifen's androgen-like effects on primate mammary epithelial sex steroid receptor expression suggest that tamoxifen's protective action on mammary gland may involve androgenic effects.     

Effects of testosterone and 17-beta-estradiol on TNF-alpha-induced E-selectin and VCAM-1 expression in endothelial cells. Analysis of the underlying receptor pathways

This study investigated the effects of testosterone and 17-beta-estradiol on tumor necrosis factor-alpha (TNF-alpha)-induced endothelial expression of E-selectin and vascular cell adhesion molecule-1 (VCAM-1) and the potential roles of hormone receptors involved in these actions. Human umbilical vein endothelial cells (HUVEC) were stimulated with TNF-alpha in the presence or absence of testosterone or 17-beta-estradiol, and the expression of E-selectin and VCAM-1 was investigated. As shown by Western blot analysis, co-administration with testosterone or 17-beta-estradiol increased the expression of E-selectin and VCAM-1 induced by TNF-alpha at 6 h and 3 h, respectively. Similarly, RT-PCR analysis revealed a significant increase in the amount of mRNA for E-selectin and VCAM-1 after co-administration with testosterone or 17-beta-estradiol in TNF-alpha-stimulated HUVEC. The presence of mRNA and proteins for androgen receptor and estrogen receptor alpha in HUVEC was verified by RT-PCR and Western blot. Flow cytometric analysis showed that preincubation with androgen receptor antagonist cyproterone and estrogen receptor antagonist tamoxifen completely abrogated the upregulating effects of testosterone and 17-beta-estradiol on TNF-alpha-induced E-selectin and VCAM-1 expression, respectively. Expression of TNF receptors in TNF-alpha-stimulated HUVEC was not influenced by testosterone and 17-beta-estradiol. The data indicate that both testosterone and 17-beta-estradiol increase TNF-alpha-induced E-selectin and VCAM-1 expression in endothelial cells via a receptor-mediated system, and expression of TNF receptors are not changed in these actions. The implications of these results for the facilitory effects of both sex hormones on immune reactions are discussed.     

Androgen receptor-mediated inhibition of estrogen-induced uterine peroxidase

Flutamide, an anti-androgen known to act through the androgen receptor, abolished the inhibitory action of testosterone on the induction of peroxidase in immature rat uteri without affecting inhibition produced by progesterone. The time course of the androgen effect on estrogen-induced uterine peroxidase, uterine weight and glucose 6-phosphate dehydrogenase activity was also determined together with the effect of flutamide on these steroid hormone-sensitive parameters. The possible mechanism of action of these compounds is discussed, particularly in the light of estrogen-induced eosinophilia. It is proposed that the observed interaction between testosterone and estradiol is mediated through their own specific receptors and not by illicit occupation of the estrogen receptor by the androgen. 5-Androstene-3 beta, 17 beta-diol (Adiol), an androgen known to exert estrogenic effects through the estrogen receptor, induced uterine peroxidase and was without significant effect on the action of estradiol, in contrast to testosterone.     

Tamoxifen elicits its anti-estrogen effects in growth plate chondrocytes by inhibiting protein kinase C

17 beta-Estradiol (E(2)) regulates growth plate cartilage cells via classical nuclear receptor mechanisms, as well as by direct effects on the chondrocyte membrane. These direct effects are stereospecific, causing a rapid increase in protein kinase C (PKC) specific activity, are only found in cells from female rats and are mimicked by E(2)-bovine serum albumin (BSA), which cannot penetrate the cell membrane. E(2) and E(2)-BSA stimulate alkaline phosphatase specific activity and proteoglycan sulfation in female rat costochondral cartilage cell cultures, but traditional nuclear receptors do not appear to be involved. This study examined the effect of the anti-estrogen tamoxifen on these markers of chondrocyte differentiation; the gender-specificity of tamoxifen's effect on PKC, if tamoxifen has an effect on vitamin D metabolite-stimulated PKC, which is mediated via specific membrane receptors (1,25-mVDR; 24,25-mVDR) and whether the effect of tamoxifen is mediated by nuclear estrogen receptors. Tamoxifen dose-dependently inhibited the effect of E(2)-BSA on PKC, alkaline phosphatase and proteoglycan sulfation in confluent cultures of female resting zone (RC) cells and growth zone (GC) (prehypertrophic/upper hypertrophic zones) cells, suggesting that its action is at the membrane and not cell maturation-dependent. Neither the estrogen receptor (ER) antagonist ICI 182780 nor the ER agonist diethylstilbesterol affected E(2) or E(2)-BSA-stimulated PKC in female chondrocytes. Tamoxifen also inhibited the increase in PKC activity due to 1 alpha,25-(OH)(2)D(3) or 24R,25-(OH)(2)D(3) in growth plate cells derived from either female or male rats. Inhibition of PKC by tamoxifen may be a general property of membrane receptors involved in rapid responses to hormones.     

Involvement of androgen receptor in 17beta-estradiol-induced cell proliferation in rat uterus

Although it is known that, in the uterus, estrogen receptor alpha (ERalpha) is involved in proliferation and progesterone receptor in differentiation, the role of the two other gonadal-hormone receptors expressed in the uterus, androgen receptor (AR) and estrogen receptor beta (ERbeta), remains undefined. In this study, the involvement of AR in 17beta-estradiol (E(2))-induced cellular proliferation in the immature rat uterus was investigated. AR levels were low in the untreated immature uterus, but 24 h after treatment of rats with E(2), there was an increase in the levels of AR and of two androgen-regulated genes, IGF-I and Crisp (cysteine-rich secretory protein). As expected, E(2) induced proliferation of luminal epithelial cells. These actions of E(2) were all blocked by both the antiestrogen tamoxifen and the antiandrogen flutamide. The E(2)-induced AR was found by immunohistochemistry to be localized exclusively in the stroma, mainly in the myometrium, where it colocalized with ERalpha but not with ERbeta. ERbeta, detected with two different ERbeta-specific antibodies, was expressed in both stromal and epithelial cells either alone or together with ERalpha. Treatment with E(2) caused down-regulation of ERalpha and ERbeta in the epithelium. The data suggest that, in E(2)-induced epithelial cell proliferation, ERalpha induces stromal AR and AR amplifies the ERalpha signal by induction of IGF-I. Because AR is never expressed in cells with ERbeta, it is unlikely that ERbeta signaling is involved in this pathway. These results indicate an important role for AR in proliferation of the uterus, where estrogen and androgen do not represent separate pathways but are sequential steps in one pathway.     

Estrogen-like effects of 7,12-dimethylbenz(a)anthracene on the female rat hypothalamo-pituitary axis

We have recently demonstrated that 7,12-dimethylbenz(a)anthracene (DMBA), a potent inducer of mammary tumors in rodents, can in vitro decrease the number of membrane dopamine D2 receptors and stimulate prolactin (PRL) release, by direct estrogen-like actions on anterior pituitary. In the present study, we tested the ability of DMBA to mimic the in vivo estradiol (17 beta E2) effects on pituitary D2 receptors and on PRL as well as LH release. We have found that DMBA, like 17 beta E2, when injected to ovariectomized rats, induced a decrease in the number of anterior pituitary D2 receptors, a release of PRL and exerted a biphasic (acute negative and longer term positive) action on LH secretion. We thus examined the ability of DMBA to interact with 17 beta E2 receptors in the hypothalamo-pituitary axis: DMBA binds to the pituitary cytosolic estrogen receptors with an affinity 0.001% that of 17 beta E2. Finally [3H]DMBA binds to hypothalamus-containing brain sections. This binding was displaced partially by RU 2858 a pure estrogen agonist and totally by tamoxifen, a purported estrogen antagonist. No competition for [3H]DMBA binding was observed with an androgen (RU 1881) or a glucocorticoid (RU 26988) agonist. From these data, it may be concluded that DMBA can act as a partial estrogen in pituitary and hypothalamic tissues.     

Effects of 17beta-estradiol and flutamide on splenic macrophages and splenocytes after trauma-hemorrhage

Since splenic immune functions are depressed in metestrus females following trauma-hemorrhage, we hypothesized that administration of the androgen receptor antagonist flutamide at the onset of resuscitation will maintain the immune function of the spleen following trauma-hemorrhage. Female C57BL6/J mice (metestrus state, 8-12 weeks old), underwent laparotomy and hemorrhagic shock (35.0+/-5.0 mm Hg for 90 min) and received 17beta-estradiol (50 microg/25 g), flutamide (625 microg/25 g) or 17beta-estradiol+flutamide. Four hours after resuscitation, the in vitro productive capacity of different cytokines (TNF-alpha, IL-6, IL-10, and IFN-gamma) by splenic MPhi and splenocytes were determined by flow cytometry. A significantly decreased cytokine production by both splenocytes and splenic MPhi was observed following trauma-hemorrhage compared to shams. Administration of 17beta-estradiol, flutamide and 17beta-estradiol+flutamide following trauma-hemorrhage resulted in a significant increase in the in vitro IL-6 release by splenic MPhi. The TNF-alpha productive capacity, however, was only restored by 17beta-estradiol and 17beta-estradiol+flutamide administration following trauma-hemorrhage. No significant effect of either treatment was observed with regard to the suppressed splenic MPhi IL-10 release. Anti-CD3 stimulation, administration of 17beta-estradiol and 17beta-estradiol+flutamide, but not the administration of flutamide alone resulted in a significant increased release of TNF-alpha, IL-6 and IFN-gamma compared to vehicle-treated animals. No significant effect of either treatment was found on IL-10 productive capacity. These results collectively suggest that flutamide administration following trauma-hemorrhage in females has beneficial effects on splenic immune function. However, flutamide administration in combination with estrogen does not provide any significant, additional effects over 17beta-estradiol administration alone.     

Effect of dihydrotestosterone on hydrogen peroxide-induced apoptosis of mouse embryonic stem cells

Steroid hormones have been reported to activate various signal transducers that trigger a variety of cellular responses. Among these hormones, testosterone has been identified as an antioxidant that protects against cellular damage. Therefore, using mouse embryonic stem (ES) cells as a model system, this study evaluated the effects of dihydrotestosterone (DHT), a biologically active testosterone metabolite, on H2O2-induced apoptosis. H2O2 increased the release of lactate dehydrogenase (LDH) and DNA fragmentation but reduced the cell viability in a time-dependent manner (> or =8 h). Moreover, H2O2 decreased the level of DNA synthesis and the levels of the cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4]. These effects of H2O2 were inhibited by a pretreatment with DHT. However, a treatment with flutamide (androgen receptor inhibitor, 10(-3) M) abolished the protective effects of DHT. This result was supported by the presence of the androgen receptor in mouse ES cells. The activity of the antioxidant enzyme, catalase, was increased by the DHT treatment but not by a co-treatment with DHT and flutamide. Using CM-H(2)DCFDA (DCF-DA) for the detection of intracellular H2O2, DHT decreased the intracellular H2O2 levels but flutamide blocked this effect. H2O2 also increased the level of p38 MAPK, JNK/SAPK, and NF-kappaB phosphorylation, which were inhibited by the DHT pretreatment. Catalase inhibited the effect of H2O2 on MAPKs and NF-kappaB. However, the flutamide treatment abolished the inhibitory effects of DHT on the H2O2-induced increase in the levels of p38 MAPK, JNK/SAPK, and NF-kappaB phosphorylation. DHT inhibited the H2O2-induced increase in caspase-3 expression and decreased the level of Bcl-2 and the cellular inhibitor of apoptosis protein (cIAP)-2. These effects were abolished by the flutamide treatment. In conclusion, DHT prevents the H2O2-induced apoptotic cell death of mouse ES cells through the activation of catalase and the downregulation of p38 MAPK, JNK/SAPK, and NF-kappaB via the androgen receptor.     

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.     

Sex steroids do not alter sex differences in tyrosine hydroxylase activity of dopaminergic neurons in vitro

Summary In order to distinguish the effects of genetic sex from those of sex hormones on the sexual differentiation of dopaminergic neurons, catecholamine synthesis was studied in gender-specific cultures of embryonic day-14 rat diencephalon. In addition to embryos from normal dams, embryos were used whose mothers had been treated with the estrogen antagonist tamoxifen or the testosterone antagonist cyproterone acetate on days 12 and 13 of gestation. Cultures from embryos of untreated dams were fed daily with a medium containing 17-estradiol or testosterone. After 10 days in vitro, cultures were immunostained for tyrosine hydroxylase and the accumulation of dihydroxyphenylalanine (DOPA) was measured in the presence of the DOPA decarboxylase inhibitor NSD 1015. Rates of DOPA synthesis, unlike the numbers of tyrosine hydroxylase-immunoreactive neurons, were markedly higher in female cultures under all experimental conditions. Treatment of dams with antisteroids prior to removal of the embryos had no influence on these results. Treatment of cultures with both steroids decreased DOPA formation in a dose-dependent manner without altering the sex difference. These results suggest that cultured diencephalic dopaminergic neurons develop sex differences in the activity of tyrosine hydroxylase. This sexual dimorphism is initiated independently of the action of gonadal steroid hormones. Sex hormones exert an additional modulatory influence on the activity of the enzyme but do not abolish or reverse sex differences. Therefore, the concept of a purely epigenetic mode of sexual differentiation of the mammalian brain needs to be broadened to incorporate other mechanisms, such as the cell-autonomous fulfillment of a sex-specific genetic program.     

Estrogen- and Antiestrogen-responsiveness of HEC1A Endometrial Adenocarcinoma Cells in Culture

HEC1A endometrial cancer cells express the wild-type form of the estrogen receptor (ER) and 17β-estradiol (E2) induces proliferation of these cells. In contrast, tamoxifen only causes a minimal increase (<20%) in cell proliferation. In HEC1A cells transiently transfected with the C3-Luc plasmid derived from the complement C3 gene, both E2 and tamoxifen exhibited ER agonist activity and tamoxifen was also a partial antagonist for this response. The relative ER agonist/antagonist activities of E2, tamoxifen and ICI 182,780 were also investigated in HEC1A1 cells transiently transfected with two E2-responsive plasmids, pCATHD-CAT and pCKB-CAT which contain 5′-promoter inserts from the cathepsin D and creatine kinase B genes, respectively. The results showed that E2 and tamoxifen induced reporter gene activity in cells transiently transfected with both constructs. ICI 182,780 exhibited partial ER agonist activity only in cells transiently transfected with pCKB-CAT and antagonized E2-induced reporter gene activity using both the CKB- and CATHD-derived constructs. These results demonstrate that HEC1A endometrial cancer cells are E2-responsive and represent a useful cell culture model for understanding hormone/antihormone-induced endometrial cell responses.     

Effects of 17 alpha-methyltestosterone on uterine morphology and heat shock protein expression are mediated through estrogen and androgen receptors

Testosterone and the synthetic androgen, 17 alpha-methyltestosterone (MT), have been shown to increase uterine weights and alter uterine morphology. However, whereas the mechanism of action of testosterone in the uterus has been studied, it is not known if the actions of MT are mediated through androgen (AR) or estrogen (ER) receptors. In the present study, we have shown that MT, at 0.5 or 10 mg/kg per day, increases uterine weight and alters uterine morphology in a dose-dependent manner. Co-administration of the anti-androgen, flutamide, or the anti-estrogen, ICI 182,780, with MT revealed that the effects of the low dose of MT are mediated through the ER, whereas those of the high dose are mediated through both the ER and AR. In addition, we have studied the effects of MT on uterine heat shock proteins (hsps), a group of estrogen-regulated proteins whose levels increase in response to growth signals and protein damage. MT increased levels of hsp90 alpha, hsp72, and grp94. All effects on uterine hsp levels were antagonized by the anti-estrogen and not the anti-androgen. Collectively, the results of the present study indicate that the effects of MT in the uterus are mediated through the AR and ER.     

Androgens on the estrogen receptor II — correlation between nuclear translocation and uterine protein synthesis

In the immature rat uterus, occupation of the androgen and estrogen receptor sites after injection of 5 α dihydrotestosterone (DHT = 17β-hydroxy-5α-androstan-3-one) were compared to the biological responses induced by the androgen on protein synthesis. Injection of 100 μg DHT induced a maximal occupation of androgen receptor sites (RA) but was totally ineffective in translocating the estrogen receptor sites and in increasing general protein synthesis. Conversely, high doses of androgen (? 3 mg) translocated the estrogen receptor (RE) and stimulated general protein synthesis. In addition, these high doses induced a specific uterine protein undistinguishable from that induced by estradiol treatment (IP). These results strongly suggest that the uterotrophic response of the uterus to androgen is correlated with the nuclear translocation of the estrogen receptor and not with that of the androgen receptor which is present in much smaller amounts.