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.     

Estradiol and a selective estrogen receptor modulator affect steroid hormone receptor messenger RNA levels and turnover in explant cultures of sheep endometrium

Estrogens upregulate estrogen receptor (ER) and progesterone receptor (PR) gene expression in endometrium immediately before ovulation to prepare it for nurturing embryos. Most in vitro model systems have lost the ability to upregulate expression of the ER gene in response to estradiol (E2) or the ability to express the ER gene at all. Here, we used explant cultures from control and E2-treated ewes and assessed expression of four genes (ER, PR, glyceraldehyde 3-phosphate dehydrogenase [GAPDH], and cyclophilin [CYC] genes) that are upregulated by E2 in vivo on Northern blots. In cultures from control and E2-treated ewes, ER and PR messenger ribonucleic acid (mRNA) levels dropped significantly during 24 h of culture in the absence of E2. Glyceraldehyde 3-phosphate dehydrogenase mRNA levels increased 300% in explants from control ewes to match the higher levels in the endometrium of the E2-treated ewe (in vivo and in explant culture). The only effect of E2 in the explant cultures was to prevent the decrease in PR mRNA. The new selective ER modulator, EM-800 (EM), decreased ER and PR mRNA levels in explants from control ewes but upregulated GAPDH and CYC mRNA levels. The EM treatment in vitro mimicked that of E2 by increasing the half-life of ER mRNA in endometrial explants. These data illustrate distinct, gene-specific effects of the explant culture process, E2, and EM on the expression of endometrial genes.     

Cell-specific expression of estrogen-responsive genes in the uteri of cyclic, early pregnant and ovariectomized ewes

A single physiological dose of estradiol up-regulates estrogen receptor-alpha(ER), progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), c-fos, cyclophilin, and actin mRNAs in the endometrium of ovariectomized ewes. Therefore, we hypothesized that these genes would be up-regulated by the preovulatory surge of estrogen which occurs on the evening of Day 15 in the estrous cycle of sheep. ER and PR mRNA concentrations increased between Day 15 and Day 1 in cyclic ewes in most endometrial epithelial cells, while GAPDH mRNA increased in epithelial and stromal cells in the deep endometrium. Day 15 pregnant ewes had lower expression of ER, PR, GAPDH, cyclophilin and actin genes. For ER and GAPDH mRNAs, the greatest reduction occurred in the superficial endometrium. Ovariectomized ewes demonstrated concentrations of ER, PR, and GAPDH mRNAs that were similar to those in the cyclic ewes. While concentrations of c-fos mRNA did not differ between groups, those of cyclophilin and actin mRNAs were lower in the pregnant and ovariectomized ewes. In conclusion, ER, PR and GAPDH gene expression rose during estrus in endometrial cells with the highest ER gene expression and were repressed in pregnant ewes in superficial endometrial cells with the greatest PR gene expression.     

The effects of estradiol and selective estrogen receptor modulators on gene expression and messenger RNA stability in immortalized sheep endometrial stromal cells and human endometrial adenocarcinoma cells

The purpose of this study was to identify an endometrial cell line that maintained the E2 up-regulation of estrogen receptor (ER) mRNA by enhanced message stability and to assess its dependence on ER protein. Estradiol (E2) effects on gene expression were measured in three cell lines: one immortalized from sheep endometrial stroma (ST) and two from human endometrial adenocarcinomas (Ishikawa and ECC-1). E2 up-regulated ER mRNA levels in ST and Ishikawa cells, but down-regulated ER mRNA levels in ECC-1 cells. E2 up-regulated progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and transforming growth factor-alpha (TGF-alpha) in both Ishikawa and ECC-1 cells. The selective estrogen receptor modulator ICI 182,780 antagonized the E2-induced up-regulation of ER and/or PR mRNA levels in all three cells, while another, GW 5638, antagonized the up-regulation of PR mRNA in Ishikawa and ECC-1 cells. In mechanistic studies, E2 had no effect on ER mRNA stability in ST cells and it destabilized ER mRNA in ECC-1 cells. Thus, Ishikawa cells appear to be the most physiologically relevant cell line in which to study the up-regulation of ER mRNA levels by enhanced mRNA stability. Its antagonism by ICI 182,780 reveals that ER protein is involved in this E2 response.     

Target specificity of selective estrogen receptor modulators within human endometrial cancer cells

Selective estrogen receptor modulators (SERMs) are estrogen receptor (ER) ligands that function as antagonists in some tissues, but have either partial or full agonist activity in others. SERMs often display variable partial agonist activity in uterine tissues and this activity can be displayed in uterine cell lines such as the human Ishikawa endometrial adenocarcinoma cell line. In this study, we compared the effects of several ER ligands including some SERMs on alkaline phosphatase (AP) activity and the expression of an ER target gene, the progesterone receptor (PR), in Ishikawa cells. As expected, estradiol (E2) was a potent and efficacious activator of both AP activity and PR mRNA expression. 4-Hydroxytamoxifen (4OHT) stimulated AP activity to a level 47% of that of E2 (100nM), while CP 336156 (lasofoxifene) increased AP activity 18%. A benzothiophene, such as LY 117018, a raloxifene analog, stimulated AP even less with values approximately 11% of E2-stimulated levels. A pure antiestrogen, ICI 182,780 did not stimulate AP activity. Interestingly, when we examined the ability of these compounds to increase the expression of the ER target gene, PR, a different rank order of efficacy was detected. After E2, CP 336156 was the most efficacious in increasing PR mRNA with a maximal stimulation of 20% of E2 levels, while 4OHT stimulated only 17%. LY 117018 increased PR mRNA expression 8% while ICI 182,780 did not increase PR mRNA expression at all. These data illustrate the target specificity that a SERM is able to display within a single cell type independent of "tissue specificity" and differential levels of expression of various cofactors. While 4OHT is 160% more active than CP 336156 in terms of inducing AP activity in the Ishikawa cells, CP 336156 has equivalent activity as 4OHT when one examines the ability of these SERMs to induce PR mRNA expression. Since the stimulation of Ishikawa cells by ER ligands is often used to assess the potential in vivo uterotrophic activity, these data indicate that examination of several endpoints in these cells may be necessary in order to fully characterize the activity of SERMs.     

Oestradiol Up-regulates Oestrogen Receptor, Cyclophilin, and Glyceraldehyde Phosphate Dehydrogenase mRNA Concentrations in Endometrium, but Down-regulates Them in Liver

Oestradiol regulates reproductive physiology and cardiovascular health in women. In the endometrium of ovariectomized ewes, previous work demonstrated that a single dose of oestradiol (50 μg) up-regulates oestrogen receptor- (ER) and progesterone receptor (PR) gene expression within 24 h. Here we compared responses to different doses of oestradiol and different dosing regimens in two diverse tissues: endometrium and liver. ER, c-fos, cyclophilin and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA concentrations were analyzed on replicate RNA slot blots in both tissues, while PR and apolipoprotein AI (apo AI) mRNA concentrations were only analyzed in endometrium or liver, respectively. Along with ER mRNA, oestradiol strongly up-regulated GAPDH and cyclophilin mRNA concentrations in endometrium. In liver, however, oestradiol down-regulated them, along with apo AI mRNA. Responses to different doses and dose regimens, including repeated 50 μg doses, were similar to those evoked by a single 50 μg dose of oestradiol. Thus, oestradiol appears to have all-or-none effects which include up-regulation of ER, cyclophilin and GAPDH gene expression in endometrium and down-regulation of ER, apo AI, cyclophilin and GAPDH gene expression in liver. These results illustrate the sharp contrast between two mammalian tissues in their responses to physiological levels of oestradiol.     

ICI 182,780 acts as a partial agonist and antagonist of estradiol effects in specific cells of the sheep uterus

We assessed the ability of ICI 182,780 (ICI) to block the estradiol (E2) responses of genes within the sheep uterus. Ovariectomized ewes in the ‘ICI+E2’ treatment group received a uterine infusion with 10⁻⁷ M ICI for 14 h, an injection of 50 μg E2 6 h after the infusion started, and were hysterectomized 18 h postinjection. Other groups received only ICI or E2, or neither treatment (‘Con’). Both E2 and ICI increased the wet weight of dissected endometrium: averaging 10.0±1.2 g for ICI+E2, ICI, and E2 groups compared to 6.8±0.6 g for Con. Slot blot analyses of endometrial RNA showed that estrogen receptor- (ER), progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), cyclophilin, actin and c-fos mRNAs responded to E2 treatment: the first five increased an average of 60% while the last decreased 38%. In situ hybridization identified more subtle ICI effects: agonistic up-regulation of GAPDH mRNA in superficial endometrial cells, and antagonistic down-regulation of ER and PR mRNAs in the inner layer of the myometrium. Thus, we conclude that the agonist versus antagonist effects of ICI relative to those of E2 are a function of the gene examined as well as the specific cell within the uterus.     

Estradiol stabilizes estrogen receptor messenger ribonucleic acid in sheep endometrium via discrete sequence elements in its 3'-untranslated region

The preovulatory surge of estrogen up-regulates estrogen receptor-alpha (ER) gene expression in the uterus during the estrous/menstrual cycles of female mammals. Previously, we demonstrated that the 5-fold increase in ER mRNA levels in endometrium of ovariectomized ewes treated with a physiological dose of estradiol (E2) is entirely due to an increase in ER mRNA stability. Our current work confirms that the E2 effect is specific to ER mRNA. The sequence of ER mRNA, cloned from sheep endometrium, shows a high degree of conservation with those of other species, even in the 5'- and the very long 3'-untranslated regions. In a cell-free assay, ER mRNA demonstrates greater stability with endometrial extracts from E2-treated ewes compared with those from untreated ovariectomized ewes. The E2-enhanced stability of ER mRNA was ablated by prior treatment of the extracts with proteinase K, 70 C heat, and oxidizing and alkylating reagents, indicating that a protein is responsible for stabilization of the message. The 3'-untranslated region of ER mRNA contains discrete sequences required for E2-enhanced stability, four of which were identified by extensive deletion mutant analyses. Transfer of two of the four minimal E2-modulated stability sequences conferred E2-enhanced stability to a heterologous RNA. These minimal E2-modulated stability sequences contain a common 10-base, uridine-rich sequence that is predicted to reside in a loop structure. Throughout our studies, estrogen stabilization of ER mRNA in sheep endometrium resembled that of vitellogenin mRNA in frog liver, indicating conservation of this ancient mechanism for enhancing gene expression in response to estrogen.     

Changes in equine endometrial oestrogen receptor alpha and progesterone receptor mRNAs during the oestrous cycle, early pregnancy and after treatment with exogenous steroids

Two experiments were performed to determine changes in the abundance of oestrogen and progesterone receptor (ER alpha and PR) mRNAs in equine endometrium during the oestrous cycle and early pregnancy, and under the influence of exogenous steroids. In Expt 1, endometrial biopsies were obtained from non-mated mares during oestrus and at days 5, 10 and 15 after ovulation, and from pregnant mares at days 10, 15 and 20 after ovulation. There were overall effects of day on the abundance of ER alpha (P = 0.0001) and PR (P = 0.0014) mRNAs. The amount of ER alpha mRNA decreased at day 10 of pregnancy, and PR mRNA was reduced at day 5 in non-mated mares and at day 15 of pregnancy, compared with oestrous values. Experiment 2 was conducted to determine the effects of exogenous steroids on endometrial ER alpha and PR mRNAs. Endometrial biopsies were obtained from 19 anoestrous mares that had been treated with vehicle, oestradiol, progesterone, or oestradiol followed by progesterone for either a short or a long duration. The steroid treatment affected the abundance of ER alpha mRNA (P = 0.0420), which was higher (P < 0.05) in the oestradiol group than in the group treated with oestradiol followed by long duration progesterone. The steroid treatment did not affect the abundance of PR mRNA. These results demonstrate that the amount of steroid receptor mRNA changes with the fluctuating steroid environment in the uterine endometrium of cyclic and early pregnant mares, and that the duration of progesterone dominance may affect ER alpha gene expression. In addition, factors other than steroids may regulate ER alpha and PR gene expression in equine uterine endometrium.     

Progesterone modulation of osteopontin gene expression in the ovine uterus

Osteopontin (OPN) is an acidic phosphorylated glycoprotein component of the extracellular matrix that binds to integrins at the cell surface to promote cell-cell attachment and cell spreading. This matrix constituent is a ligand that could potentially bind integrins on trophectoderm and endometrium to facilitate superficial implantation and placentation. OPN mRNA increases in the endometrial glandular epithelium (GE) of early-pregnant ewes, and OPN protein is secreted into the uterine lumen. Therefore, progesterone and/or interferon-tau (IFNtau) may regulate OPN expression in the uterine GE. Cyclic ewes were ovariectomized and fitted with intrauterine (i. u.) catheters on Day 5 and treated daily with steroids (i.m.) and protein (i.u.) as follows: 1) progesterone (P, Days 5-24) and control serum proteins (CX, Days 11-24); 2) P and ZK 136.317 (ZK; progesterone receptor [PR] antagonist, Days 11-24) and CX proteins; 3) P and recombinant ovine IFNtau (roIFNtau, Days 11-24); or 4) P and ZK and roIFNtau. All ewes were hysterectomized on Day 25. Progesterone induced the expression of endometrial OPN mRNA in the GE and increased secretion of a 45-kDa OPN protein from endometrial explants maintained in culture for 24 h. Administration of ZK ablated progesterone effects. Intrauterine infusion of roIFNtau did not affect OPN gene expression or secretion in any of the steroid treatments. Interestingly, OPN mRNA-positive GE cells lacked detectable PR expression, although PR were detected in the stroma. Results indicate that progesterone regulates OPN expression in GE through a complex mechanism that includes PR down-regulation, and we suggest the possible involvement of a progesterone-induced stromal cell-derived growth factor(s) that acts as a progestamedin.     

Effects of recombinant ovine interferon tau, placental lactogen, and growth hormone on the ovine uterus

Studies were conducted to determine effects of intrauterine administration of recombinant ovine interferon tau (IFNtau), placental lactogen (PL), and growth hormone (GH) on endometrial function. In the first study, administration of IFNtau to cyclic ewes for one period (Days 11-15) resulted in an interestrous interval (IEI) of approximately 30 days, whereas administration for two periods (Days 11-15 and Days 21-25) extended the IEI to greater than 50 days. Administration of IFNtau from Days 11 to 15 and of PL or GH from Days 21 to 25 failed to extend the IEI more than for IFNtau alone. In the second study, effects of IFNtau, PL, and GH on endometrial differentiation and function were determined in ovariectomized ewes receiving ovarian steroid replacement therapy. Endometrial expression of mRNAs for estrogen receptor (ER), progesterone receptor (PR), and oxytocin receptor (OTR) were not affected by PL or GH treatment; however, uterine milk protein mRNA levels and stratum spongiosum gland density were increased by both PL and GH treatments. Collectively, results indicated that 1) PL and GH do not regulate endometrial PR, ER, and OTR expression or affect corpus luteum life span; 2) down-regulation of epithelial PR expression is requisite for progesterone induction of secretory gene expression in uterine glandular epithelium; 3) effects of PL and GH on endometrial function require IFNtau; and 4) PL and GH regulate endometrial gland proliferation and perhaps differentiated function.     

Endometrial expression of progesterone receptor and uteroglobin genes during early pregnancy in the rabbit

The progesterone receptor (PR) plays a pivotal role in the maturation process of the secretory endometrium, implantation and maintenance of pregnancy in rabbits. To determine the dynamics of PR gene expression and its physiological significance, the endometrial expression of PR and PR mRNA were evaluated and compared with the expression of the progesterone-regulated uteroglobin (UG) gene during 0–5 days post-coitus in rabbits. The results of immunoblot experiments indicated the presence of PR in endometrial cell extracts from days 1–4 of pregnancy with maximum PR immunostaining on day 2, followed by a marked diminution until its complete disappearance on day 5. When endometrial PR mRNA content was assessed by Northern blots, the results were similar to those of PR immunostaining, with maximal concentrations on the second day after mating. However, PR mRNA levels were still high on day 3, despite the concomitant decrease in immunostainable PR. Endometrial UG gene expression, on the other hand, exhibited a different time sequence. Thus, the UG content in uterine flushings progressively increased from day 3 after mating, reaching maximal levels on the fifth day. The endometrial UG mRNA content presented a similar profile, as its maximum concentration occurred on days 4–5. The overall results indicate that endometrial PR is down-regulated at both the mRNA and protein levels, possibly by endogenous progesterone during early pregnancy. The striking observation that maximal expression of endometrial UG gene products occurred when PR and its mRNA are no longer detectable suggests an important role for this progesterone-binding uterine protein during the preimplantation period. © 1993 Wiley-Liss, Inc.     

Regulation of gene expression and cellular localization of prostaglandin synthase by oestrogen and progesterone in the ovine uterus

Expression of the gene for prostaglandin synthase (PGS) was examined in whole endometrial tissue derived from ewes during the oestrous cycle (Days 4-14), on Day 15 of pregnancy and following ovariectomy and treatment with ovarian steroid hormones. Whilst no significant differences were seen in PGS mRNA concentrations analysed by Northern blot analysis in endometrial tissue during the oestrous cycle or in early pregnancy, treatment of ovariectomized (OVX) ewes with oestradiol-17 beta markedly reduced endometrial PGS mRNA concentration. There was no difference in PGS mRNA concentration in ewes treated with progesterone, either alone or in conjunction with oestrogen, from that in OVX controls. In contrast, differences in immunolocalization of PGS observed in uterine tissue from OVX-steroid-treated ewes were much more marked and reflected similar changes seen previously in the immunocytochemical distribution of endometrial PGS during the oestrous cycle. In OVX ewes and those treated with oestrogen, immunocytochemical staining for PGS was seen in stromal cells, but little immunoreactive PGS was located in the endometrial epithelial cells. However, in ewes treated with progesterone alone or with oestrogen plus progesterone, PGS was found in luminal and glandular epithelial cells and in stromal cells. Intensity of immunostaining for PGS in endothelial cells and myometrium did not differ between the treatments. Thus, whilst oestrogen lowers PGS mRNA in the endometrium, presumably in stroma, it may also increase the stability of the enzyme itself in the stromal cells. Although oestradiol-17 beta has no effect on PGS in endometrial epithelium, progesterone stimulates the production of PGS in endometrial epithelial cells without altering the overall abundance of PGS mRNA in the endometrium as a whole. Conceptus-induced changes in PGF-2 alpha release by ovine endometrium would not appear to be mediated via effects on PGS gene expression or protein synthesis.     

Comparative study of the short-term effects of a novel selective estrogen receptor modulator, ospemifene, and raloxifene and tamoxifen on rat uterus

To investigate the differential short-term effects of selective estrogen receptor (ER) modulators (SERMs) on uterus, we treated adult ovariectomized rats with a novel SERM, ospemifene (Osp), two previously established SERMs (tamoxifen and raloxifene (Ral)) and estradiol. The expression of two estrogen-regulated early response genes c-fos and vascular endothelial growth factor (VEGF), and DNA synthesis were analysed at 1-24 h after treatment of ovariectomized rats. Induction of c-fos mRNA by each of the SERMs showed a biphasic pattern with peaks at 3 and 20 h, respectively. The maximum level of VEGF mRNA was observed at 1 h after raloxifene and 6 h after tamoxifen or ospemifene treatment. Maximum levels of the c-fos and VEGF mRNA after raloxifene treatment were higher than those seen after treatments with E2 or a corresponding dose of tamoxifen or ospemifene. DNA synthesis was significantly increased by ospemifene, tamoxifen and raloxifene both in luminal and glandular epithelium. The stimulation was transient, peaking at 16 h. In comparison, the maximum level observed at 16 h after E2 treatment sustained at least until 24 h. DNA synthesis in stromal cells was increased by the SERMs but not by E2 at 24 h. When treated together with E2, the SERMs were able to antagonise E2-stimulated DNA synthesis at 16 h. Our results demonstrate that the initial response of uterus to ospemifene, raloxifene and tamoxifen includes activation of early response genes and even transient stimulation of DNA synthesis in spite of their different long-term effects. However, the early stimulatory events may be mediated by different mechanisms leading to diverging pathways in various tissue compartments and development of differential SERM-specific long-term responses of uterus.     

Steroid hormones acutely regulate expression of a Nudix protein-encoding gene in the endometrial epithelium of sheep

Steroid hormones regulate endometrial gene expression to meet the needs of developing embryos. Our hypothesis is that steroid hormones transiently induce expression of genes in the endometrial epithelium to make the uterine environment different between the earliest days of pregnancy. We identified one such gene product using differential display-polymerase chain reactions. The gene product that was strongly induced in ewes between day 3 and 6 of the estrous cycle was cloned and sequenced to identify it as encoding a member of the Nudix family of hydrolase enzymes. Northern blot analyses indicated that NUDT16 mRNA concentrations were elevated 10-fold in the endometrium of sheep from day 5 to 9 of the estrous cycle and returned to basal levels by day 11. In assays of RNA samples from 15 different tissues from an adult ewe, the concentrations of NUDT16 mRNA were greatest in endometrium. In situ hybridization localized NUDT16 mRNA exclusively to the endometrial epithelial cells of the glands and uterine lumen. In ovariectomized ewes, NUDT16 mRNA was induced by a regimen of alternating estrogen and progesterone therapy designed to mimic the hormonal experiences of a ewe at day 6 of the estrous cycle. The final estrogen treatment in the regimen was critical to the expression of NUDT16 as well as progesterone receptor and estrogen receptor-beta genes. Characterization of the NUDT16 gene identified putative steroid hormone response elements, which can now be investigated to understand its unique pattern of regulation in the earliest days of pregnancy.