Expression of estrogen receptors alpha and beta in the corpus luteum and uterus from non-pregnant and pregnant llamas

Because estrogen may be involved in maternal recognition of pregnancy and embryonic migration in llamas, expression of estrogen receptor subtypes alpha (ERalpha) and beta (ERbeta) was evaluated in corpus luteum (CL), endometrium, and uterus using relative RT-PCR. Tissues were recovered from sterile-mated (SM) and pregnant (PG) females during Days 7-11 and 7-13 (Day 0 = day of mating), respectively, and follicular phase and juvenile females. Luteal expression of ERalpha and beta was similar (P > 0.10) in SM and PG females and within Days 7-11, however, expression of ERalpha in ovarian tissue from follicular phase females was greater (P < 0.05) than Days 7 and 9 CL. Uterus expressed less ERalpha and beta compared to endometrium (P = 0.07 and P < 0.01, respectively). Expression of ERalpha was greater (P < 0.05) in Day 7 and follicular phase uteri than Days 9 and 11, Day 13 PG and juvenile uteri. Uterine ERbeta expression was greater (P = 0.09) in PG versus SM females and in mated compared to follicular phase females (P < 0.05). Endometrial expression of ERalpha and beta did not differ (P > 0.10) between SM and PG females or by day. The presence of luteal ER during this period may mean a role for estradiol in maternal recognition of pregnancy. Observed increases in uterine ER expression with no changes in endometrium suggest expression increased in myometrium and/or perimetrium. Upregulation of myometrial ERbeta in PG females may be involved in supporting uterine migration of the embryo.     

Messenger RNA levels of estrogen receptors alpha and beta and progesterone receptors in the cyclic and inseminated/early pregnant sow uterus

The aim of the present study was to investigate differences in the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus at different stages of the estrous cycle as well as in inseminated sows at estrus and during early pregnancy by use of solution hybridization and in relation to plasma levels of estradiol and progesterone. Uterine samples were collected at different stages of the estrous cycle and after insemination/early pregnancy. In the endometrium, the expression of ERalpha mRNA and PR mRNA was similar for cyclic and early pregnant groups. Both were highest at early diestrus/70 h after ovulation and ERalpha mRNA was lowest at late diestrus/d 19 while PR mRNA was lowest at diestrus and late diestrus/d 11 and d 19. The expression of endometrial ERbeta was constantly low during the estrous cycle but higher expression was found in inseminated/early pregnant sows at estrus and 70 h after ovulation. In the myometrium, high expression of ERalpha mRNA and PR mRNA was observed at proestrus and estrus in cyclic sows and at estrus in newly inseminated sows. Higher expression of myometrial ERbeta mRNA was found in inseminated/early pregnant sows compared with cyclic sows, although significant only at estrus. In conclusion, the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus differed between endometrium and myometrium as well as with stages of the estrous cycle and early pregnancy. In addition to plasma steroid levels, the differences between cyclic and inseminated/early pregnant sows suggest that other factors, e.g. insemination and/or the presence of embryos, influence the expression of these steroid receptor mRNAs in the sow uterus.     

Expression of estrogen receptors alpha and beta in the baboon fetal ovary

In adult mammals, estrogen regulates ovarian function, and estrogen receptor (ER) is expressed in granulosa cells of antral follicles of the adult baboon ovary. Because the foundation of adult ovarian function is established in utero, the present study determined whether ERalpha and/or ERbeta were expressed in fetal ovaries obtained on Days 100 (n = 3) and 165-181 (n = 5) of baboon gestation (term = Day 184). On Day 100, ERalpha protein was detected by immunocytochemistry in surface epithelium and mesenchymal-epithelial cells but not oocytes in germ cell cords. ERbeta protein was also detected by immunocytochemistry on Day 100 of gestation and was abundantly expressed in mesenchymal-epithelial cells in germ cell cords, lightly expressed in the germ cells, but was not detected in the surface epithelium. On Days 165-180 of gestation, ERalpha expression was still intense in the surface epithelium, in mesenchymal-epithelial cells throughout the cortex, and in nests of cells between follicles. ERalpha expression was lighter in granulosa cells and was not observed in all granulosa cells, particularly in follicles close to the cortex. In contrast, ERbeta expression was most intense in granulosa cells, especially in flattened granulosa cells, was weaker in mesenchymal-epithelial cells and nests of cells between follicles, and was absent in the surface epithelium. Using an antibody to the carboxy terminal of human ERbeta, ERbeta protein was also detected by Western immunoblot with molecular sizes of 55 and 63 kDa on Day 100 and primarily 55 kDa on Day 180. The mRNAs for ERalpha and ERbeta were also detected by Northern blot analysis in the baboon fetal ovary. These results are the first to establish that the ERalpha and ERbeta mRNAs and proteins are expressed and exhibit changes in localization in the primate fetal ovary between mid and late gestation. Because placental estrogen production and secretion into the baboon fetus increases markedly during advancing pregnancy, we propose that estrogen plays an integral role in programming fetal ovarian development in the primate.     

Expression of estrogen receptors alpha and beta in term human myometrium

We used immunohistochemistry to compare the expression of estrogen receptors (ERalpha and ERbeta) in term myometria of 32 pregnant women divided in two groups. Group I comprised of 16 women in labour and group II included 16 non-laboring gravidas. We observed cytoplasmatic localization of both ER isoforms and no differences in the ER expression between the two groups of patients. The abundance and specific localization of ERs in human term myometrium seems to be independent of its contractile activity which may point to the specific role of those receptors in late pregnancy myometrium.     

Subcellular distribution of native estrogen receptor alpha and beta isoforms in rabbit uterus and ovary

The association of estrogen receptors with non-nuclear/cytoplasmic compartments in target tissues has been documented. However, limited information is available on the distribution of estrogen receptor isoforms, specially with regard to the newly described beta isotype. The subcellular localization of estrogen receptor alpha and beta isoforms was investigated in rabbit uterus and ovary. Native alpha and beta subtypes were immunodetected using specific antibodies after subjecting the tissue to fractionation by differential centrifugation. The ovary expressed alpha and beta estrogen receptors in predominant association to cytosolic components. However, in the uterus, a substantial proportion of the total estrogen binding capacity and coexpression of the two isoforms was detected in mitochondria and microsomes. The mitochondrial-enriched subfraction represented an important source of 17beta-estradiol binding, where the steroid was recognized in a stereospecific and high affinity manner. The existence of mitochondrial and membrane estrogen binding sites correlated with the presence of estrogen receptor alpha but mainly with estrogen receptor beta proteins. Using macromolecular 17beta-estradiol derivatives in Ligand Blot studies, we could confirm that both alpha and beta isoforms were expressed as the major estrogen binding proteins in the uterus, while estrogen receptor alpha was clearly the dominant isoform in the ovary. Other low molecular weight estrogen receptor alpha-like proteins were found to represent an independent subpopulation of uterine binding sites, expressed to a lesser extent. This differential cellular partitioning of estrogen receptor alpha and beta forms may contribute to the known diversity of 17beta-estradiol effects in target organs. Both estrogen receptor alpha and beta expression levels and cellular localization patterns among tissues, add complexity to the whole estrogen signaling system, in which membrane and mitochondrial events could also be implicated.     

Contribution of estrogen receptors alpha and beta to the effects of estradiol in the brain

Clinical and experimental studies show a modulatory role of estrogens in the brain and suggest their beneficial action in mental and neurodegenerative diseases. The estrogen receptors ER and ERβ are present in the brain and their targeting could bring selectivity and reduced risk of cancer. Implication of ERs in the effect of estradiol on dopamine, opiate and glutamate neurotransmission is reviewed. The ER agonist, PPT, is shown as estradiol to modulate hippocampal NMDA receptors and AMPA receptors in cortex and striatum of ovariectomized rats whereas the ERβ agonist DPN is inactive. Striatal DPN activity suggests implication of ERβ in estradiol modulation of D2 receptors and transporters in ovariectomized rats and is supported by the lack of effect of estradiol in ERβ knockout (ERKOβ) mice. Both ER and ERβ agonists modulate striatal preproenkephalin (PPE) gene expression in ovariectomized rats. In male mice PPT protects against MPTP toxicity to striatal dopamine; this implicates Akt/GSK3β signaling and the apoptotic regulators Bcl2 and Bad. This suggests a role for ER in striatal dopamine neuroprotection. ERKO mice are more susceptible to MPTP toxicity and not protected by estradiol; differences in ERKOβ mice are subtler. These results suggest therapeutic potential for the brain of ER specific agonists.     

Polymorphisms of estrogen receptors alpha and beta in idiopathic, infertile Brazilian men: a case-control study

Estrogen plays an important role in the human reproductive system and its action is mediated mainly by two specific receptors: α (ERα) and β (ERβ). There are polymorphic variants in both ER genes, and studies showed their association with reproductive outcomes. We aimed to determine the distribution of ERα and ERβ gene polymorphisms in idiopathic, infertile Brazilian patients in a case–control study comprising 187 idiopathic, infertile Brazilian men with nonobstructive azoospermia (NOA, n = 78) or severe oligozoospermia (SO, n = 109) and 216 fertile men. Detection of ERα (PvuII and XbaI) and ERβ (AluI and RsaI) gene polymorphisms were performed using TaqMan PCR. The results were analyzed statically, and a P‐value < 0.05 was considered significant. Single‐marker analysis revealed that neither PvuII nor XbaI polymorphisms of the ERα gene were associated either with NOA group (P = 0.662 and P = 0.527, respectively) or SO group (P = 0.777 and P = 1.0, respectively). Regarding ERβ polymorphisms, no statistical difference was observed between the AluI polymorphism and NOA group compared to controls (P = 1.0) or between SO group and controls (P = 0.423). We found similar results with the RsaI polymorphism. Statistical analysis did not reveal a difference between NOA (P = 0.740) and SO (P = 0.920) groups compared to controls. Combined genotypes of ERα and ERβ polymorphisms did not identify a haplotype associated with idiopathic infertility. Thus, in the Brazilian population, genetic variations in both estrogen receptors alpha (PvuII and XbaI) and beta (AluI and RsaI) were not relevant to idiopathic infertility. Mol. Reprod. Dev. 78:665–672, 2011. © 2011 Wiley‐Liss, Inc.     

Ligands differentially modulate the protein interactions of the human estrogen receptors alpha and beta

The interactions of human estrogen receptor subtypes ERalpha and ERbeta with DNA and a 210 amino acid residue fragment of the coactivator protein SRC-1 bearing three nuclear receptor interaction motifs were investigated quantitatively using fluorescence anisotropy in the presence of agonist and antagonist ligands. ERalpha and ERbeta were found to bind in a similar manner to DNA, and both salt and temperature affected the affinity and/or stoichiometry of these interactions. The agonist ligands estradiol, estrone and estriol did not modify the binding of ERalpha to the fluorescein-labeled target estrogen response element. However, in the case of ERbeta, these ligands led to the formation of some higher-order protein-DNA complexes and a small decrease in affinity. The partial agonist 4-hydroxytamoxifen had little effect on either ER subtype, whereas the pure antagonist ICI 182,780 led to the cooperative formation of protein-DNA complexes of higher order than dimer, as further demonstrated by competition experiments and gel mobility-shift assays. In addition to DNA binding, the interaction of both ER subtypes with the Alexa488-labeled SRC-1 coactivator fragment was investigated by fluorescence anisotropy. The agonist ligands estrone, estradiol, estriol, genistein and ethynyl estradiol exhibited distinct capacities for inducing the recruitment of SRC-1 that were not correlated with their affinity for the receptor. Moreover, estrone and genistein exhibited subtype specificity in that they induced SRC-1 recruitment to ERbeta with much higher efficiency than in the case of ERalpha. The differential coactivator recruitment capacities of the ER agonists and their receptor subtype coactivator recruitment specificity may be linked to the molecular structure of the agonists with respect to their interactions with a specific histidine residue located at the back of the ligand-binding pocket. Altogether, these quantitative in vitro studies of ER interactions reveal the complex energetic and stoichiometric consequences of changes in the chemical structures of these proteins and their ligands.