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PubMed2003

The combination of a novel selective estrogen receptor modulator with an estrogen protects the mammary gland and uterus in a rodent model: the future of postmenopausal women's health?

The Women's Health Initiative Study and other reports have created major uncertainty among postmenopausal women and physicians concerning hormone replacement therapy. We have thus investigated the possibility of replacing the progestin in hormone replacement therapy by a novel selective estrogen receptor (ER) modulator having potent and pure antiestrogenic activity in the mammary gland and uterus. As measured by changes in histology and Cdc47 labeling in the rat model, the present study shows that the stimulatory effect of estradiol in the mammary gland and uterus is efficiently blocked by simultaneous administration of the novel selective ER modulator EM-652, but bone mineral density is preserved and serum cholesterol is decreased. After the administration of 14C-labeled EM-652, we observed that there is no detectable radioactivity in the brain. Moreover, ER alpha immunoreactivity remained constant in the hypothalamus after EM-652 treatment, whereas ER alpha became almost undetectable in the mammary gland and uterus. The present data show the poor or absent access of EM-652 to the brain, whereas the effects of estrogens are efficiently neutralized in the mammary gland and uterus. Such data support the exciting possibility of a novel approach that could meet most of the needs of women's health at menopause, namely control of hot flushes and prevention of breast, uterine, and ovarian cancer as well as osteoporosis and potentially helping brain function and preventing Alzheimer's disease with no identifiable risk or negative effect.

Animals↗
PubMed2003

Estrogen induces CCN5 expression in the rat uterus in vivo.

Estrogen plays an important role in the normal physiology as well as various pathologies of the uterus. Given the nature of uterine remodeling during the reproductive cycle and pregnancy, we sought to determine whether CCN5, a gene that we have shown to be important in smooth muscle cell proliferation and migration, is an estrogen-induced gene in the uterus. In the present study, we demonstrate that levels of CCN5 mRNA and protein expression were 5-fold higher in uteri from proestrous females relative to metestrous females, a finding consistent with estrogen induction of the CCN5 gene. Ovariectomized rats treated with exogenous estrogen or estrogen and progesterone exhibited 4- to 8-fold higher levels of CCN5 mRNA and protein than animals treated with either progesterone or vehicle alone. Analysis of rat uterine sections using immunohistochemistry demonstrates CCN5 localization throughout the uterus, including the endometrium and endometrial glands as well as the myometrium. Thus, our data indicate that CCN5 is positively regulated by estrogen in the rat uterus and suggests that this gene may play an important role in maintaining normal uterine physiology.

Animals↗
PubMed2004

Estradiol regulates the thioredoxin antioxidant system in the mouse uterus.

The rodent uterus responds to acute estradiol (E2) treatment with a series of well characterized physiological responses. In a recent screen for genes involved in this response, we found that several genes in the thioredoxin (Txn) pathway were rapidly modified after E2 treatment in the mouse uterus. Txn is a 12-kDa protein with multiple roles in the cell, including protection against oxidative stress and apoptosis, regulation of transcription factor activity, and regulation of cellular proliferation. Txn in combination with Txn reductase (Txnrd) and Txn-interacting protein (Txnip) constitute the mammalian Txn pathway. This pathway exists in multiple locations in the cell, including the cytosol and mitochondria. To analyze the levels of Txn, Txnrd, and Txnip in the uterus, we treated ovariectomized adult mice with a time course of E2 and analyzed mRNA levels by real-time PCR. E2 rapidly decreased the expression of Txnip, but increased the levels of cytosolic Txn1 and Txnrd1 as well as mitochondrial Txn2. Using the ER antagonist, ICI 182,780, and mice lacking functional estrogen receptor alpha (ERalpha), we demonstrate that these E2-mediated changes require ERalpha, but not ERbeta. The repression of Txnip by E2 was also demonstrated in vitro in MCF-7 human breast cancer cells. This repression was blocked by treatment with the histone deacetylase inhibitor, trichostatin A, suggesting that repression by E2 may involve regulation of histone acetylation. We conclude that the rapid E2-mediated activation of the Txn pathway is an important step in the response of the mammalian uterus to estrogen.

Animals↗
PubMed2006

Overlapping and distinct expression of progesterone receptors A and B in mouse uterus and mammary gland during the estrous cycle.

In rodents, progesterone receptors (PRs) A and B have different and often nonoverlapping roles, and this study asked whether different activities of the PR proteins in mouse are related to differences in their expression in reproductive tissues. The individual expression of PRA and PRB was determined immunohistochemically in mammary gland and uterus during the estrous cycle or in response to endocrine manipulation. In the mammary gland, PRA and PRB were colocated in PR+ epithelial cells, with little change during the estrous cycle. In the uterus, PRA was not detected in luminal epithelium at any stage of the cycle, and PR+ luminal cells expressed only PRB. In the stroma and myometrium, PRA and PRB levels fluctuated with cyclical systemic hormone exposure. Observation of functional end points suggested that augmented stromal and/or myometrial PRA in proestrus inhibited estrogen receptor expression and epithelial proliferation. Colocation of PRA and PRB was hormonally regulated, and ovariectomy did not reproduce the expression of PRA and PRB in the uterus during the estrous cycle. Whereas PRB was the only PR in the luminal epithelium in cycling mice, ovariectomy restored PRA expression, resulting in PRA-PRB colocation. In stroma and myometrium, PRA and PRB colocated in PR+ cells, but ovariectomy reduced PRA levels more than PRB, resulting in PRB-only-expressing cells. This study has shown that nonoverlapping PRA and PRB expression in the uterus, in particular the lack of PRA, and expression of PRB only in the luminal epithelium throughout the estrous cycle, is likely to contribute to the distinct roles of PRA and PRB in the adult mouse.

Animals↗
PubMed1980

In vivo uptake of estrone sulfate by rabbit uterus.

The capability of rabbit uterus and liver to hydrolyze estrone sulfate (E1S) to estrone (E1) was demonstrated in vitro by superfusion of tissue slices with mixtures of [3H]-E1S and [14C]E1. However, capillary barriers or binding factors may prevent the utilization of the blood-borne estrogen sulfate. To evaluate the extent of uptake of circulating E1S by uterine cells, a mixture of [3H]E1S and [14C]E1 was injected into the abdominal aorta of anesthetized rabbits. Shortly after the injection, samples of blood, uterine tissue, and liver were simultaneously taken. Isotope ratios in E1 isolated from these samples were measured and compared. The 3H to 14C ratios in E1 isolated from the uterus and from plasma were similar. It was concluded from these results that blood-borne E1S was not taken up directly by the uterus, since, otherwise, uterine E1 would be expected to have a higher 3H to 14C ratio than plasma E1. The isotope ratios in hepatic E1 were higher than those in E1 isolated from plasma or uterus, probably as a result of direct uptake of [3H]E1S by the liver.

Animals↗
PubMed1980

The effect of 17 beta-estradiol on collagen and noncollagenous protein synthesis in the uterus and some periodontal tissues.

The effect of a single dose of 17 beta-estradiol on collagen metabolism in the uterus and various oral tissues was determined in ovariectomized nulliparous rats. Eight days after ovariectomy, 200-g rats were given 100 micrograms estradiol ip. Forty hours later, 2 mCi [3H]proline were administered by ip injection, and the animals were killed 2 h later. The specific radioactivities of hydroxyproline and proline in both salt-soluble and salt-insoluble proteins were used as a measure of collagen and noncollagenous protein metabolism. Estradiol was found to induce a 2-fold increase in the specific radioactivity of newly synthesized collagen and an 8-fold increase in the specific radioactivity of the insoluble collagen in the uterus. The discrepancy between these results could be largely accounted for by a 2- to 3-fold increase in the size of the newly synthesized collagen pool. Stimulation of noncollagenous protein synthesis was also observed. The total collagen content of the uterus was not significantly altered in estradiol-treated animals, suggesting that estradiol stimulates both the synthesis and degradation of collagen. Using [14C]glycine as a precursor, the nature of the collagens synthesized in the uterus was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Estradiol was found to stimulate the synthesis of both type I and type III collagens, but no change in the pattern of procollagen conversion could be discerned. In contrast to the uterine tissues, the only significant effect of estradiol on protein metabolism in the oral tissues was a decrease in the newly synthesized collagen specific radioactivity in the molar periodontal ligament.

Animals↗
PubMed1980

Temporal aspects of the involvement of the uterus and prolactin in the establishment of luteinizing hormones-dependent progesterone secretion in the rat.

This paper deals with some of the temporal aspects of the inhibition by PRL and the promotion by the uterus of the development of a crucial need for LH (LH dependency) in the maintenance of progesterone secretion by the rat corpus luteum. A severe and permanent fall in the concentration of progesterone in the serum and a shortening of the duration of the vaginal diestrus in response to 0.5 ml of a specific antiserum to LH, injected sc on the morning of day 9 of pseudopregnancy (day 1 = ovulation), confirmed the presence of LH dependency. In rats hysterectomized before the induction of pseudopregnancy or on days 2, 3, or 4 of pseudopregnancy, LH antiserum did not induce luteolysis, but in rats hysterectomized on day 5, it did. In rats subjected to chronic PRL treatment (through the homotransplantation of a rat pituitary beneath the kidney capsule) from day 2 onward, LH antiserum did not induce luteolysis if the uterus was removed on day 5, 6, or 7; if the uterus remained in place until at least day 8, however, LH antiserum induced luteolysis. In rats hysterectomized on day 5, the presence of a pituitary homotransplant only between 2 and 5 or only from day 5 onward prevented the luteolytic response to LH antiserum; if the pituitary was not transplanted until day 8, however, LH antiserum induced luteolysis. These results imply that there probably is a quantitative relationship between the duration (as well as time of onset) of the mutually opposing effects of PRL and the uterus on the appearance of LH dependency.

Animals↗
PubMed1980

Immunocytochemical localization of catechol-O-methyltransferase in the pregnant rat uterus.

Light microscopic immunocytochemical observations of catechol-O-methyl-transferase (COMT) localization in the pregnant rat uterus were made with a specific antibody to soluble rat liver COMT and the peroxidase-antiperoxidase technique. The changes in cellular localization of COMT were followed from the onset of pregnancy to 12 h after delivery of the last fetus. COMT first appeared on day 5 of pregnancy in endometrial epithelial cells of the uterus. On day 7, COMT appeared in the decidual cells on the antimesometrial side of the uterus close to the implanting blastocyst. Two morphologically distinct, COMT-positive, decidual cells were observed: polygonal cells and stellate cells. After parturition, COMT-positive decidual cells remained in the decidua basalis. The possible physiological roles of COMT in the pregnant uterus are discussed.

Animals↗
PubMed1981

Progesterone metabolism by the hypothalamus, pituitary, and uterus of the rat during pregnancy.

Metabolites of [3H]progesterone were quantitated from incubations of hypothalamus, pituitary, and uterus of rats during different stages of pregnancy. The hypothalamus, anterior pituitary, and a section of uterus from five rats on Days 1, 8, 15, and 21 of pregnancy were incubated individually with [3H]progesterone and analyzed for metabolite formation by reverse isotopic dilution analysis. The radioactive metabolites present were 5 alpha-pregnane-3,20-dione (5 alpha-DHP), 3 alpha-hydroxy-5 alpha-pregnan-20-one, 20 alpha-hydroxy-4-pregnen-3-one, 20 alpha-hydroxy-5 alpha-pregnan-3-one, and 5 alpha-pregnane-3 alpha, 20 alpha-diol. The major metabolite formed by the hypothalamus and pituitary was 5 alpha-DHP. In the pituitary samples, formation of 5 alpha-DHP was decreased on Days 15 and 21 of pregnancy compared to Day 1, and formation of 20 alpha-hydroxy-5 alpha-pregnan-3-one was decreased on Day 21 compared to Day 1. In the uterine samples, 3 alpha-hydroxy-5 alpha-pregnan-20-one was the major metabolite formed at all stages of pregnancy. The formation of all metabolic products of progesterone by the uterus was increased on Day 21 compared to Days 1, 8, and 15 of pregnancy. No changes in the formation of progesterone metabolites were observed in the hypothalamic samples during pregnancy. It is concluded that there are different profiles in the in vitro metabolism of [3H]progesterone by the hypothalamus, pituitary, and uterus of the rat during the course of pregnancy.

Animals↗
PubMed1981

Effects of nafoxidine on the luteinizing hormone surge: temporal distribution of estrogen receptors and induction of cytoplasmic progestin receptors in the hypothalamus-preoptic area, pituitary, and uterus of the immature rat.

The antiestrogen nafoxidine 1-(2-[P-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]pyrrolidine hydrochloride; 2 mg/rat), blocked LH surges induced by Silastic implants containing estradiol (E2) in oil (150 micrograms/ml) in the immature female rat and had no stimulatory effect on gonadotropin secretion by itself. Furthermore, the administration of progesterone (P) to rats primed for 24 h with nafoxidine alone or nafoxidine plus E2 did not lead to premature and enhanced LH surges (facilitation) as it does in E2-primed animals. Pituitary LH content was depleted by 30-50% after E2-induced or P-facilitated LH surges, but was unchanged after the administration of nafoxidine or nafoxidine plus E2. To investigate the cellular mechanisms involved, levels of total cytoplasmic and nuclear estrogen receptors and cytoplasmic progestin receptors in the hypothalamus-preoptic area (HPOA), pituitary, and uterus were measured by [3H]E2 or [3H]R5020 (3H-labeled 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) exchange assays at various times after the administration of E2 and /or nafoxidine. The ability of nafoxidine to bind in vitro to cytoplasmic or nuclear estrogen receptors was, respectively, 4% and 2% (HPOA), 5% and 6% (pituitary), and 1% and 2% (uterus) relative to E2 (100%). After the injection of nafoxidine or the insertion of E2 implants, cytoplasmic estrogen receptors were depleted with a similar time course and remained depressed for at least 48 h in both the HPOA and pituitary. In the uterus, the antiestrogen prevented the replenishment of cytoplasmic estrogen receptors observed in response to E2. Nuclear estrogen receptor levels in the HPOA and pituitary peaked 1 h after E2 and subsequently declined to a plateau from 24-48 h (at 2-4 times the levels found in untreated rats). After nafoxidine, accumulation of these receptors in the nucleus was more gradual and prolonged. Absolute levels of estrogen receptors translocated to the nucleus by E2 or nafoxidine were comparable. E2 treatment led to a substantial induction of cytoplasmic progestin receptors (approximately 2-fold in the HPOA, approximately 4-fold in the pituitary, and approximately 7-fold in the uterus after 48 h), and this process was considerably inhibited by nafoxidine. These results support the notions that antagonism by nafoxidine of estrogen action may be due to a defective association of the antiestrogen-receptor complex with nuclear sites, ad progestin receptor induction may be a prerequisite for the facilitation of gonadotropin surges by P.

Animals↗
PubMed1983

Analysis of binding of [3H]Estradiol to the cytosol fraction of rat pancreas: comparison with sites in the cytosol of uterus.

The cytosol fraction of rat pancreas [100,000 X g (for 1 h) supernatant] demonstrated specific but nonsaturable binding of [3H]estradiol in the concentration range of 2-50 X 10(-9) M. Scatchard analysis of specifically bound [3H]estradiol, determined by the isotope dilution technique (competition with excess unlabeled estradiol), indicated a single class of binding sites (approximately 4.4 pmol/mg protein) with an apparent Kd of 5 X 10(-8) M. Such cytosol fractions, prepared from homogenates that contained protease inhibitors, when prelabeled with [3H]estradiol, demonstrated a single sharp eluate peak of radioactivity after Sephadex G-200 chromatography that corresponded to a molecular weight of 120,000. When protease inhibitors were omitted, [3H]estradiol was associated with material of considerably lower molecular weight. Routinely, the protease inhibitors leupeptin (1 mM), phenylsulfonyl fluoride (0.5 mM), and tosylphenylalanylchloromethyl ketone (0.05 mM) were included in the buffer used for homogenizing both the pancreas and uterus. The protein that binds [3H]estradiol in uterus differed from that in pancreas in a number of ways: 1) in the range of 10-20 X 10(-9) M [3H]estradiol, specific binding of the hormone to uterine sites was saturable, and Scatchard analysis indicated a single class of binding sites, (approximately 0.5 pmol/mg protein) having an apparent Kd of 3 X 10(-10) M; 2) the rate constants of dissociation of the [3H] estradiol-bound complexes in pancreas and uterus were 3.1 X 10(-4) sec-1 (t1/2, 37 min) and 8.7 X 10(-5) sec-1 (t1/2, 134 min), respectively; 3) the molecular weight of the estrogen-binding protein in freshly prepared uterine supernatant fractions appeared to be at least 240,000; this was unaltered regardless of whether protease inhibitors were present during initial homogenization of the tissue; and 4) when uterine supernatants prepared in the absence of protease inhibitors were kept at 8 C for 24 h and then analyzed by Sephadex G-200 chromatography, a second peak of [3H]estradiol-binding activity appeared at the same eluate volume as the low molecular weight binding fractions of pancreas. These data suggest that although the binding proteins in pancreas and uterus are different, there may be some common features at the hormone-binding locus.

Animals↗
PubMed1988

Estrous cycle- and pregnancy-related differences in expression of the proenkephalin and proopiomelanocortin genes in the ovary and uterus.

Expression of the genes for two opioid peptide precursors, proenkephalin and POMC, was examined within the female reproductive system of rodents as a function of the estrous cycle and during pregnancy. Proenkephalin RNA was found to change markedly during the estrous cycle in both the ovary and uterus (approximately 6- and 3-fold, respectively). The highest concentrations occurred at estrus in the rat ovary and at metestrus and diestrus in the rat uterus. In sharp contrast to proenkephalin RNA, the abundance of POMC RNA remained relatively constant throughout the estrous cycle in both tissues. Similar results were obtained in the cycling hamster ovary. During pregnancy, the concentrations of proenkephalin RNA in the rat ovary showed little variation, while in the uterus a 4-fold increase in this transcript was observed. The effects of pregnancy on POMC RNA were the reverse of this pattern; its abundance increased 2-fold in the ovary and did not vary substantially in the uterus. These differences in the expression of proenkephalin and POMC genes during the estrous cycle and pregnancy suggest that these two opioid peptide precursors are associated with distinct functional roles within the female reproductive system.

Animals↗
PubMed1989

Lactotransferrin gene expression in the mouse uterus and mammary gland.

The mouse mammary gland and uterus expressed the gene for the secretory protein lactotransferrin under various physiological conditions. Lactotransferrin, however, was induced by estrogen in a time- and dose-dependent fashion in the uterus of the immature mouse, but was not affected by estrogen in the mammary gland. Differences were also found in the expression of lactotransferrin in mammary glands and uteri of adult females during lactation. A high level of the protein was detected by immunocytochemistry in uterine epithelial cells 1 day after parturition, but immunoreactivity disappeared quickly thereafter. Lactotransferrin message was, however, relatively abundant in the mammary gland at the end of the lactation period. The presence of lactotransferrin in various tissues also was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Two forms of immunoreactive material was detected by this method; a 70K band was found in uterine luminal fluid from the estrogen-stimulated immature mouse and in homogenates of lung, vagina, mammary gland, oviduct, spleen, lymph node, and uterus of the adult female mouse, and a 65K band was detected in submaxillary gland, kidney, ovary, and all of the above tissues. Brain and duodenum had no detectable immunoreactive material. A transient appearance of lactotransferrin was observed in the uterine luminal fluid of pseudopregnant mice. These changes in the level of lactotransferrin in the uterus and mammary gland under various physiological conditions suggest that the regulation of this protein's expression is tissue specific.

Animals↗
PubMed1989

Immunofluorescent analysis of estrogen induction of progesterone receptor in the rhesus uterus.

Estrogen (E) has been shown to induce an increase in progesterone (P) receptor (PR) concentration in uterine tissue of both rodents and primates. Because of the presence of different cell types within the uterus, we were interested in determining whether estrogen-induced PR were cell type specific in the nonhuman primate uterus (rhesus monkey). Immunofluorescent analyses of E receptor (ER) and PR were performed on fresh frozen cryostat sections (6 microns) of uterine tissue from ovariectomized (3 months) and estradiol (E2)-treated (peak level of E2 during an artificial menstrual cycle) rhesus monkeys. Antibodies to ER and PR were obtained from Abbott Laboratories (H222) and Transbio (MPR1). The avidin-biotin complex technique was used with streptavidin-conjugated Texas red for fluorescent detection. Ovariectomized monkeys showed positive fluorescence for ER in luminal and glandular epithelia, stromal cells, and smooth muscle cells of the myometrium. In contrast, positive fluorescence for PR was observed primarily in glandular epithelia, with little or no fluorescent detection in luminal epithelium, stromal cells, or myometrial smooth muscle cells. After E2 treatment strong positive fluorescence for PR was observed in luminal and glandular epithelia, stromal cells, and myometrial smooth muscle cells. Strong positive fluorescence for ER was also observed in the same cell types. Fluorescent detection of ER and PR was restricted to the nuclei of these cell types. These studies show that ER are present constitutively in all cell types of the E-withdrawn (ovariectomized) nonhuman primate uterus, whereas PR are primarily restricted to glandular epithelia. E2 treatment, which simulated the follicular phase and E2 surge, resulted in the appearance of immunofluorescent PR in luminal epithelia, stromal cells, and myometrial smooth muscle cells. These studies serve to define the cellular pattern of E2-induced PR in the primate uterus.

Animals↗
PubMed1993

Estrogen-induced calbindin-D 9k gene expression in the rat uterus during the estrous cycle: late antagonistic effect of progesterone.

Progesterone modulates estrogen-stimulated responses in the uterus. Calbindin-D 9k (CaBP9k), a 17 beta-estradiol-responsive gene expressed in the uterus, was used as a marker to examine the interactions between endogenous progesterone and estradiol in the rat. The variations in uterine CaBP9k messenger RNAs (mRNAs) during the rat estrous cycle indicated that CaBP9k gene expression was greatest during the estrogen-dominated phases (proestrus and estrus) and became totally repressed during diestrus, when progesterone predominates. Estradiol was found to be the major controlling factor of CaBP9k gene expression in vivo, progesterone antagonizing estrogen-induced CaBP9k gene expression. The inhibitory role of progesterone was further examined in two experiments. Mature cyclic rats were injected with the progesterone antagonist RU486 before the progesterone surge of proestrus, and the estrous cycle was mimicked in ovariectomized rats by sequential injections of estrogen and progestin. Progesterone did not appear to be involved in the rapid decrease in CaBP9k mRNA during estrus but was implicated in the down-regulation of the estrogen-stimulated CaBP9k gene expression at the end of estrus and during diestrus. This delayed effect of progesterone was confirmed in the ovariectomized rat model. CaBP9k mRNA accumulation in estrogen-primed ovariectomized rats was suppressed by estrogen followed 1 h later by the progesterone agonist R5020. This effect occurred more than 24 h after progestin treatment. The inhibition of the estrogen-induced CaBP9k gene expression in the rat uterus by progesterone is certainly mediated by the progesterone receptor, because progesterone had no effect without estrogen priming or when the antagonist RU486 was used. The delayed progesterone effect probably does not involve depletion of nuclear estrogen receptors, the major rapid mechanism proposed for estrogen inhibition by progesterone in the rodent uterus, or control of estrogen receptor synthesis, as shown by Northern blot analysis of estrogen receptor mRNA.

Animals↗
PubMed1993

Regulation of progesterone receptor gene expression and growth in the rat uterus: modulation of estrogen actions by progesterone and sex steroid hormone antagonists.

Although the rat uterus has often been used as a model to study estrogen action, relatively little is known of the mechanism(s) by which estrogen regulates uterine progesterone receptor (PR) levels in this species. In the present study, we used immature ovariectomized rats to examine the regulation of PR gene expression and growth in the uterus by estradiol (E2) as well as hormonal modulators of E2 action, namely progesterone (P), the antiestrogen LY117018 (LY), and the antiprogestin RU486. Northern blot analyses revealed eight PR mRNA species ranging in size from 3.3-14 kilobases, with the most abundant being 7.1 kilobases. E2 treatment caused rapid time- and dose-dependent increases in the steady state levels of PR mRNA, which peaked at 24 h (6-fold increase) and declined thereafter. All eight PR mRNA transcripts increased proportionally in response to E2. Immunoblot analyses indicated that these changes were accompanied by increases in PR protein (6-fold increase by 48 h), which continued to accumulate over time, unlike PR mRNA, which decreased despite continued E2 exposure. In contrast to the stimulatory effect of E2 on PR, the levels of immunoreactive estrogen receptor were reduced to about 15% of the control value by E2 within 48 h and remained low throughout the remaining treatment period. Treatment with P blocked the stimulatory effects of E2 on both PR mRNA and protein. These antagonistic actions of P were prevented by simultaneous administration of RU486. LY, which caused a slight (approximately 2.5-fold) increase in PR mRNA when administered alone, was an effective antagonist of E2-stimulated increases in PR mRNA. However, LY was incapable of completely antagonizing E2-stimulated increases in PR protein. The differences between the profiles of the time-dependent increases in PR mRNA and protein in response to E2, as well as the different sensitivities of these two end points to the antagonistic actions of LY, highlight the lack of direct correspondence between these two end points and suggest that E2 may be acting through distinct mechanisms (transcriptional and posttranscriptional, for example) to increase the levels of PR in the rat uterus. Our results indicate that E2 rapidly increases uterine PR expression and growth, and that P as well as sex steroid hormone antagonists are important modulators of these E2 actions in the rat uterus.

Animals↗
PubMed1994

Differential expression of epidermal growth factor receptor (EGF-R) gene and regulation of EGF-R bioactivity by progesterone and estrogen in the adult mouse uterus.

The present study examined several aspects of epidermal growth factor receptor (EGF-R) in the mouse uterus during the peri-implantation period and after ovarian hormone treatment of adult ovariectomized mice. The cell-specific distribution, regulation of expression, and binding kinetics were assessed by immunohistochemistry, Northern blot analysis, and ligand binding assays, respectively. Affinity cross-linking studies ascertained the size of the EGF-R, and its bioactivity was examined by determining EGF-dependent subcellular protein tyrosine kinase activity and receptor autophosphorylation. In the intact uterus and separated cell types, EGF-R was detected in the stroma, deciduum, and myometrium, but not in the luminal or glandular epithelium. Uterine EGF-R mRNA transcript profiles showed some differences between pregnant and ovariectomized mice regardless of steroid hormone treatments. Two major [6.5- and 2.7-kilobase (kb)] and two less abundant (9.6- and 5.0-kb) transcripts were detected in pregnant uterine poly(A)+ RNA. Three additional transcripts (< 2.0 kb) were detected in decidual poly(A)+ RNA, and a larger transcript (8.0 kb) was detected in uterine poly(A)+ RNA isolated from ovariectomized mice. Scatchard analysis of EGF binding also revealed apparent differences in binding kinetics between pregnant and ovariectomized mice, although EGF was cross-linked to a 170-kilodalton protein under these conditions. Two classes (Kd, approximately 0.2 and approximately 2.0 nM) of binding sites were noted in pregnant mice, whereas a single class (Kd, approximately 1.0 nM) was found in ovariectomized mice. 17 beta-Estradiol (E2) caused a rapid transient upregulation of uterine EGF-R mRNA levels and increased the number of EGF-binding sites in ovariectomized mice, as did an injection of progesterone (P4). However, the bioactivity of EGF-R could not be detected in uteri of ovariectomized mice treated with oil or P4. E2 treatment was found to be essential for EGF-R bioactivity. Taken together, the results suggest that in the adult mouse uterus, EGF-R status is influenced by factors other than P4 and E2, the epithelium is not the direct target for the actions of EGF-related growth factors as thought previously, the mitogenic effects of these growth factors on epithelial cells in vivo are perhaps mediated by other uterine cell-types expressing EGF-R, and, lastly, these growth factors are not likely to be functional in the uterus in the absence of estrogen. The present observations are supportive of the concept of paracrine or juxtacrine interactions between EGF-related growth factor ligands of luminal epithelial origin and blastocyst EGF-R in the process of implantation.

Animals↗
PubMed1995

Cellular localization of estradiol-induced c-fos messenger ribonucleic acid in the rat uterus: c-fos expression and uterine cell proliferation do not correlate strictly.

Estrogens stimulate DNA synthesis and cell proliferation in the uterus. All major uterine cell types (luminal and glandular epithelium, stroma, and myometrium) respond to 17 beta-estradiol in the immature animal, whereas primarily epithelial cells of the uterine endometrium respond in the mature animal. Rapid activation of the c-fos protooncogene by estrogen precedes the uterine growth, suggesting that c-fos plays a role in amplifying the hormonal signal. The specific uterine cell types in which estrogen induces c-fos messenger RNA (mRNA) expression, however, have not been identified in either mature or immature animals. In this study, in situ hybridization was used to determine the cell type-specific location of mRNA encoding c-fos in the uterus. In both immature and mature castrated rats at 3 h after 17 beta-estradiol administration, c-fos expression was detected primarily in uterine luminal and glandular epithelia. Expression of c-fos returned to baseline levels by 24 h post 17 beta-estradiol treatment. There was no apparent difference in the uterine cell type-specific pattern of c-fos expression stimulated by estradiol in mature vs. immature animals. Nuclear run-on transcription assay in isolated luminal epithelial cell nuclei showed that c-fos gene transcription increased rapidly in the uterus after estradiol stimulation. Treatment of adult rats with a single injection of 16 alpha-estradiol, a short-acting, nonmitogenic estrogen, induced c-fos primarily in the uterine glandular epithelia. Progesterone is known to modify the action of estrogen on the uterus by redirecting the proliferative response from epithelia to stroma. To determine if progesterone modulation of estrogen action involves shifting of c-fos expression to stromal cells, rats were treated with progesterone for 48 h and then killed 0, 3, 6, or 12 h after an estradiol injection. In situ hybridization analysis revealed that c-fos mRNA remained localized in the uterine luminal and glandular epithelia, and expression was not shifted to the stroma. Although these results support the idea that c-fos plays a role in proliferation of uterine epithelial cells, they also invite reassessment of the role played by c-fos in both epithelial and nonepithelial uterine cell types.

Animals↗