Mutual and intercompartmental regulation of estrogen receptor and progesterone receptor expression in the mouse uterus

The epithelial and stromal compartments of the uterus undergo significant estrogen- and progesterone (P4)-induced changes during the estrous cycle. While in the adult mouse, epithelial proliferation and stromal inflammation are induced by estrogen, P4 is antiproliferative in the epithelium and both proliferative and anti-inflammatory in the stroma. In light of these compartmentally varying roles, we have immunohistochemically examined estrogen and P4 regulation of the expression of their receptors (ER and PR) and their epithelial target gene lactoferrin (LF) in wild-type and PR null mutant mice. We demonstrate that estrogen exerts compartment-specific effects on the expression of ER, resulting in decreased levels of stromal and glandular epithelial (GE) ER and increased luminal epithelial (LE) and myometrial ER. Estrogen also has dual effects on PR expression, decreasing levels in the LE while at the same time increasing levels in the stroma and myometrium. Estrogen and P4 together mediate their effects in part through the ability of P4 to selectively inhibit myometrial ER expression while preserving GE expression. We also demonstrate a general negative feedback by P4 on PR expression that is most prominent in the GE. Finally, we demonstrate using the estrogen- and P4-responsive epithelial target gene LF that the differential regulation of PR in the glandular and luminal epithelium results in different functional responses of these compartments to P4. Together, our data indicate that the pleiotropic effects of estrogen and P4 in the adult mouse uterus are mediated by complex hormonal interregulation of ER and PR in specific uterine compartments.     

Androgen regulation of the insulin-like growth factor-I and the estrogen receptor in rat uterus and liver

For the first time testosterone is shown to be an important regulator of the insulin-like growth factor-I (IGF-I) in the rat uterus under in vivo conditions. In this study the regulation of IGF-I and the estrogen receptor (ER) by gonadal steroids in the uterus and liver of female rats was monitored. The ER level was assayed by hormone binding after treatment with testosterone, 5 alpha-dihydrotestosterone or estradiol and specific mRNA species were analyzed by a solution hybridization/RNase protection assay using 35S-labeled RNA probes. Ovariectomized rats restored uterine weight after treatment with testosterone. Uterine IGF-I mRNA was more than 20-fold higher in testosterone treated rats compared to untreated ovariectomized controls after 48 h treatment. The effects of testosterone on ovariectomized animals was followed in a timecourse study. Testosterone administration increased uterine IGF-I mRNA expression during the first 48 h and the maximally induced level was maintained throughout the duration of the experiment (168 h). Since induction of IGF-I mRNA by estrogen is transient, these data indicate that androgen and estrogen increase IGF-I mRNA by different mechanisms. Regulation of IGF-I mRNA by gonadal steroids was also studied in hypophysectomized animals. The rats were given either testosterone, 5 alpha-dihydrotestosterone or estradiol, and uterine IGF-I mRNA was measured after 1 week of treatment. At this timepoint estrogen treated rats showed levels of IGF-I mRNA not significantly different from those of hypophysectomized controls. In contrast testosterone and 5 alpha-dihydrotestosterone increased the IGF-I mRNA level 30 and 40 times, respectively, relative to hypophysectomized control animals. Since 5 alpha-dihydrotestosterone is not convertable to estrogen, the induction by testosterone was considered to be a true androgenic phenomenon.     

Immunolocalization of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in rat uterus: variation across the estrous cycle and regulation by estrogen and progesterone

Glucocorticoid hormone action in several target tissues is dependent not only on the expression of the glucocorticoid receptor, but also on that of the 11beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes, 11betaHSD-1 and -2. In the uterus, glucocorticoids can exert inhibitory effects on a range of important functions, particularly in relation to the effects of estrogen. Therefore, the present study examined immunolocalization of the two 11betaHSD enzymes in the rat uterus at each stage of the estrous cycle and after ovariectomy with or without estrogen/progesterone replacement. In cycling rats 11betaHSD-1 was localized to luminal and glandular epithelial cells and to eosinophils in both the endometrial stroma and myometrium. In contrast, 11betaHSD-2 immunostaining was localized to endometrial stromal cells and myometrial cells, with no staining evident in epithelial cells or eosinophils. Immunostaining for both enzymes was cycle dependent, being maximal at proestrus and minimal at diestrus. Western blot analysis of whole uterus at proestrus showed the presence of 34- and 40-kDa immunoreactive species for 11betaHSD-1 and -2, respectively. These immunoreactive signals were almost abolished by ovariectomy, but this effect was reversed for both enzymes by estrogen replacement with or without progesterone. These effects of ovariectomy and steroid replacement were confirmed by immunocytochemical analysis, with the exception that progesterone appeared to enhance the stimulatory effects of estrogen on 11betaHSD-2 specifically within the endometrial stroma. In conclusion, these results establish the presence of both 11betaHSD-1 and -2 in the nonpregnant rat uterus and show distinct distributions for the two enzymes and cyclic variation related to positive regulation by ovarian steroids. The physiological implications of these patterns of 11betaHSD expression will ultimately depend on the reaction direction for each enzyme, but 11betaHSD-2 is likely to limit disruptive effects of glucocorticoids on the endometrial stroma, and 11betaHSD-1 may then serve to selectively reactivate glucocorticoids in epithelial cells.     

Hormonal regulation of the estrogen receptor in primary cultures of hepatocytes from female rats

Estrogen treatment affects the hepatic synthesis and/or secretion of several proteins involved in clinically important pathological processes such as atherosclerosis, hypertension, and thrombosis. The endocrine regulation of the estrogen receptor (ER) concentration in primary cultures of rat hepatocytes was studied. Human growth hormone (hGH) and dexamethasone (DEX) in combination increased ER concentration 6-fold and ER mRNA levels 2.5-fold. These effects were not significantly different from those observed after treatment with the purely somatogenic bovine growth hormone (GH) in combination with DEX. Treatment with the lactogen ovine prolactin in the presence or absence of DEX did not significantly affect ER or ER mRNA concentrations. Triiodothyronine treatment at the most effective concentration (50 nM) increased ER and ER mRNA levels twofold. Medium supplementation with estradiol (0.1 nM) throughout the experiment did not affect the response to treatment with hGH and DEX. Treatment with high concentrations of ethinylestradiol in combination with hGH and DEX, however, increased the ER level twice as much as hGH and DEX without addition of estradiol or ethinylestradiol, whereas the ER mRNA concentration was the same in both the GH+DEX group and GH+ DEX+ (estradiol or ethinylestradiol) groups. These data indicate the importance of GH in combination with glucocorticoids for the maintenance of ER concentrations in the rat liver. Thyroid hormones may be of some, although minor importance, whereas the data suggest that prolactin is not directly involved in hepatic ER regulation.     

Cloning and expression of the Moraxella catarrhalis lactoferrin receptor genes

The lactoferrin receptor genes from two strains of Moraxella catarrhalis have been cloned and sequenced. The lfr genes are arranged as lbpB followed by lbpA, a gene arrangement found in lactoferrin and transferrin receptor operons from several bacterial species. In addition, a third open reading frame, orf3, is located one nucleotide downstream of lbpA. The deduced lactoferrin binding protein A (LbpA) sequences from the two strains were found to be 99% identical, the LbpB sequences were 92% identical, and the ORF3 proteins were 98% identical. The lbpB gene was PCR amplified and sequenced from a third strain of M. catarrhalis, and the encoded protein was found to be 77% identical and 84% similar to the other LbpB proteins. Recombinant LbpA and LbpB proteins were expressed from Escherichia coli, and antisera raised to the purified proteins were used to assess antigenic conservation in a panel of M. catarrhalis strains. The recombinant proteins were tested for the ability to bind human lactoferrin following gel electrophoresis and electroblotting, and rLbpB, but not rLbpA, was found to bind lactoferrin. Bactericidal antibody activity was measured, and while the anti-rLbpA antiserum was not bactericidal, the anti-rLbpB antisera were found to be weakly bactericidal. Thus, LbpB may have potential as a vaccine candidate.     

Interactions of 16alpha,17alpha-cyclohexane derivatives of progesterone with the progesterone receptor from rat uterus

Pregna-D'-pentaranes, 16alpha,17alpha-cyclohexanoprogesterone and 6alpha-methyl-16alpha,17alpha-cyclohexanoprogestero ne, were found to specifically interact with the progesterone receptor of soluble fraction from rat uterus. The formation of complexes between 3H-labeled derivatives of these steroids and the protein was complete within 1 to 3 h at 0-4 degreesC. The dissociation of these complexes was a two-phase process, the contribution of the fast dissociating complexes decreasing with increasing preincubation time. The dissociation constant (k-1) values for progesterone, 16alpha, 17alpha-cyclohexanoprogesterone, and 6alpha-methyl-16alpha, 17alpha-cyclohexanoprogesterone complexes with the protein after 1 h preincubation were 6.5 +/- 0.8, 8.8 +/- 5.5, and (16.6 +/- 5.6).10(-4) sec(-1) for the fast phase and 5.1 +/- 0.5, 3.5 +/- 0.8, and (2.8 +/- 0.6).10(-5) sec(-1) for slow phase, respectively. The equilibrium Kd values were 11.7 +/- 2.1, 19.0 +/- 2.0, and 66.1 +/- 14.6 nM for progesterone, 16alpha,17alpha-cyclohexanoprogesterone, and 6alpha-methyl-16alpha,17alpha-cyclohexanoprogestero ne, respectively. The steroids mutually inhibited the binding of their 3H-labeled derivatives to the protein, the inhibition being of competitive type. In the case of [3H]6alpha-methyl-16alpha, 17alpha-cyclohexanoprogesterone, the inhibitory efficacy of progesterone declined with an increase of its concentration; this points to possible heterogeneity of binding sites for the 3H-labeled ligand. The comparison of the results with those obtained by us earlier (Biochemistry (Moscow), 1996, 61, 1034-1041) suggests the existence of significant species differences in progesterone receptor structure within or near the region that interacts with the D-ring of a hormone molecule.     

Methylation of estrogen and progesterone receptor gene 5' CpG islands correlates with lack of estrogen and progesterone receptor gene expression in breast tumors

Hormonal factors have a profound influence on the development, treatment, and outcome of breast cancer. The absence of steroid hormone receptors is highly correlated with resistance to antihormonal treatments. Work in cultured human breast cancer cell lines has shown that the absence of estrogen receptor (ER) gene expression in ER- cells is associated with extensive methylation of the ER gene 5' CpG island, and treatment with agents that demethylate the ER gene CpG island results in the production of functional ER protein. The current study shows that CpG islands in the 5' region of the ER and progesterone receptor (PR) genes are methylated in a significant fraction of primary human breast cancer tissues. The ER CpG island is methylated at the methylation-sensitive NotI restriction site in 9 of 39 (25%) of primary ER- breast cancers but remains unmethylated in 53 ER+ breast cancers and 9 normal breast specimens. Three methylation-sensitive restriction sites in the PR gene CpG island are not methylated in normal breast specimens and PR+ human breast cancers but are hypermethylated in 40% of PR- human breast tumors. These data demonstrate that methylation of the ER and PR gene CpG islands is associated with the lack of ER and PR gene expression in a significant fraction of human breast cancers.     

Differential susceptibility of mouse Lyt-2 and Lyt-3 genes to negative regulation

The differences in desmosine, isodesmosine (DID), hydroxyproline and cholesterol in the human thoracic aorta from diabetic (n = 16) and non-diabetic (n = 17) autopsy subjects were investigated. DID was analyzed by the use of high performance liquid chromatography. The amount of DID, and total DID (DID+reduced DID) tended to be lower in the diabetic than in non-diabetic subjects. The ratio of DID or total DID to hydroxyproline was significantly decreased in diabetic compared to non-diabetic subjects. Amount of DID, reduced DID and total DID were significantly lower in aorta with plaque formation than that without plaque or ulcer. Multiple regression analysis showed that amount of cholesterol, DID, reduced DID and age were significantly associated with dry weight per area of the aorta. A similar association was not observed in non-diabetic subjects. Compositional changes of aortic cholesterol and elastin have a closer relationship with atherosclerosis in diabetic than in non-diabetic subjects.     

Differential regulation of progesterone and estradiol production by mouse cumulus and mural granulosa cells by A factor(s) secreted by the oocyte

Mouse oocytes secrete a factor(s) that inhibits progesterone and enhances estradiol production by cumulus granulosa cells. The purpose of this study was to investigate the mechanisms by which the production of these steroids is modulated. Mouse oocyte-cumulus cell complexes (intact) and complexes from which the oocytes were removed microsurgically (oocytectomized; OOX) were cultured for up to 48 h in the presence of FSH (150 ng/ml) and testosterone (5 x 10(-7) M). For these experiments, all cells were obtained from antral follicles of 24- to 26-day-old mice primed with eCG. Intact complexes produced primarily estradiol, with significant accumulation occurring between 24 and 48 h. In contrast, OOX complexes produced little estradiol but, starting at 18 h of culture, released significantly more progesterone than did intact complexes. Progesterone accumulation in cocultures of denuded oocytes with either OOX complexes or monolayers of mural granulosa cells was significantly reduced compared to that with OOX complexes or mural granulosa cells cultured alone. If dibutyryl cAMP replaced FSH in the cocultures, similar results were obtained, suggesting that the oocyte-secreted steroid-regulating factor acts downstream of the generation of cAMP to inhibit progesterone production. Since estradiol can inhibit progesterone production by granulosa cells, we investigated the possibility that the increased progesterone released by OOX complexes was secondary to the lower estradiol production. Intact complexes cultured in the presence of the nonaromatizable androgen, 5 alpha-dihydrotestosterone, or steroidal (4-hydroxyandrostenedione) or non-steroidal (CGS 16949A) aromatase inhibitors produced little estradiol; however, progesterone production by these complexes was no different from that of estradiol-producing intact complexes. These results suggest that the steroid-regulating factor(s) secreted by occytes acts to regulate granulosa cell production of estradiol and progesterone by independent mechanisms.     

Differential regulation by MK801 of immediate-early genes, brain-derived neurotrophic factor and trk receptor mRNA induced by a kindling after-discharge

Transient changes in immediate-early genes and neurotrophin expression produced by kindling stimulation may mediate secondary downstream events involved in kindling development. Recent experiments have demonstrated conclusively that both kindling progression and mossy fibre sprouting are significantly impaired by administration of the N-methyl-D-aspartate (NMDA) receptor antagonist MK801. To further examine the link between kindling, changes in gene expression and the NMDA receptor, we examined the effects of MK801 on neuronal induction of immediate-early genes, brain-derived neurotrophic factor (BDNF) and trk receptor mRNA expression produced by a single electrically induced hippocampal after-discharge in rats. The after-discharge produced a rapid (after 1 h) increase in Fos, Jun-B, c-Jun, Krox-24 mRNA and protein and Krox-20 protein in dentate granule neurons and a delayed, selective expression of Fos, Jun-D and Krox-24 in hilar interneurons. MK801 pretreatment produced a very strong inhibition of Fos, Jun-D and Krox-20 increases in dentate neurons but had a much smaller effect on Jun-B and c-Jun expression. MK801 did not inhibit Krox-24 expression in granule neurons or the delayed expression of Fos, Jun-D and Krox-24 in hilar interneurons. BDNF protein and trk B and trk C mRNA expression were also strongly induced in dentate granule cells 4 h following an after-discharge. MK801 abolished the increase in BDNF protein and trk B, but not trk C mRNA in granule cells at 4 h. These results demonstrate that MK801 differentially regulates the AD-increased expression of a group of genes previously identified as being likely candidates for an involvement in kindling. Because MK801 significantly retards the development of kindling and mossy fibre sprouting, it can be argued that those genes whose induction is not significantly attenuated by MK801 are unlikely to play an important role in the MK801-sensitive component of kindling and the changes in neural connectivity (mossy fibre sprouting) associated with kindling. Conversely, the role in kindling of those genes whose expression was significantly attenuated by MK801 (Fos, Jun-D, Krox-20, trkB and BDNF) requires further examination.     

Relationships between estrogen receptor and estradiol-stimulated progesterone synthesis in the rabbit corpus luteum

The effects of estradiol on luteal estrogen receptor and steroidogenesis were examined on Days 9 through 11 of pseudopregnancy. In normal pseudopregnant rabbits, unoccupied cytoplasmic and total nuclear estrogen receptor were 2.6 +/- 0.4 fmol/microgram DNA and 0.4 +/- 0.1 fmol/microgram DNA, respectively, on Day 10 of pseudopregnancy. An i.v. injection of 4 micrograms of estradiol caused the translocation of cytoplasmic receptor and a 6.6-fold increment in total nuclear receptor within 15 min, which was followed by rapid processing of the nuclear receptor. Both cytoplasmic and nuclear estrogen receptor returned to normal values within 24 h, and during this period, serum progesterone did not change significantly. Withdrawal of an estradiol implant from animals on Day 9 of pseudopregnancy initiated a marked decline in serum progesterone within 24 h. Following an injection of saline or of 4 micrograms estradiol on Day 10, unoccupied cytoplasmic estrogen receptor in saline- and estradiol-injected rabbits was 1.0 +/- 0.1 fmol/microgram DNA and 1.9 +/- 0.1 fmol/microgram DNA, respectively, on Day 11 of pseudopregnancy. Associated with the increase in cytoplasmic receptor there was an increase in serum progesterone (8.2 +/- 1.5 ng/ml), in contrast to saline-injected animals in which serum progesterone continued to decline (1.6 +/- 0.4 ng/ml). Despite the significant differences in cytoplasmic receptor and in progesterone synthesis, total nuclear estrogen receptor was not different in these animals. These data suggest that in corpora lutea already secreting progesterone at high rates during midpseudopregnancy, the rapid translocation of available estrogen receptor does not cause further stimulation of progesterone synthesis. However, if steroidogenesis is first reduced experimentally, then an injection of 4 micrograms of estradiol can readily stimulate progesterone synthesis, and this stimulation is associated with an increase in cytoplasmic receptor.     

Identification of estrogen regulated genes in Fe33 rat hepatoma cells by differential display polymerase chain reaction and their hormonal regulation in rat liver and uterus

We applied the differential display RT-PCR (ddRT-PCR) technology to identify estrogen-regulated hepatic genes in the estrogen receptor expressing rat hepatoma cell line Fe33. Three genes of known sequences were detected by the ddRT-PCR approach: IGF binding protein-1 (IGFBP-1), vitamin D-dependent calcium-binding protein (CaBP9k) and major acute phase protein (MAP). Effects of ethinyl estradiol on the mRNA levels of these genes were confirmed by "Northern-blot" analysis. If given in combination with dexamethasone and glucagon, ethinyl estradiol caused 40-, 15- and 11-fold increases in the mRNA steady state level of IGFBP-1, CaBP9k and MAP, respectively, in Fe33 cells 24 h after addition of hormone. Besides ethinyl estradiol, the partial estrogen agonist OH-tamoxifen caused dose dependent effects on expression of MAP and IGFBP-1. Estrogen regulation of the respective genes and the modulatory effects of progesterone (10 mg/animal/day) were studied in ovariectomized rats treated subcutaneously for 14 days with 1 microgram/animal/day estradiol. "Northern-blot" analysis of liver RNA revealed a 6-fold stimulation of IGFBP-1 mRNA levels in estradiol-treated compared to vehicle-treated rats and a weak but detectable increase of MAP mRNA steady state level (1.6-fold) upon estradiol administration. No effect of estradiol treatment could be monitored for CaBP9k in rat liver. Modulatory effects of progesterone on estradiol-stimulated expression in the liver could be monitored for IGFBP-1 only. In an extension of our investigation on the expression of the three genes in rat liver, we determined their expression and hormonal regulation in the uterus of the same animals. In the uterus, estradiol caused an increase in CaBP9k mRNA. In contrast, IGFBP-1 mRNA levels increased dramatically upon progesterone administration, whereas no effect of estradiol treatment could be detected. MAP mRNA levels increased only after coadministration of estradiol and progesterone. In conclusion, the ddRT-PCR proved to be a powerful method to identify estrogen-regulated genes. The study on the hormonal regulation of three genes stimulated by estrogen in Fe33 cells revealed similarities and differences in their regulation in vivo and in vitro.     

Ontogeny of estrogen receptor messenger ribonucleic acid expression in the postnatal rat uterus

During the first 2 wk of postnatal life, the rodent uterus undergoes a period of marked growth and differentiation. To further examine the role of the estrogen receptor (ER) in the mediation of uterine development, we analyzed the ontogeny of ER mRNA expression in the postnatal rat uterus using in situ hybridization. ER mRNA was present in the uterine stroma on the day of birth and progressively increased in abundance during the first 2 wk of postnatal life. In contrast, ER mRNA was not detectable in the luminal epithelium at birth and did not become abundant in this region until postnatal day (P) 7. ER mRNA abundance increased in the luminal epithelium and in the invaginating and fully formed glandular epithelium during the second week of life. At P21 ER mRNA was more abundant in the glandular epithelium than in any other uterine cell type. These results are consistent with, and extend the findings of, previous studies using uterine homogenate binding assays and immunohistochemistry to define ER ontogeny in rodents. Delineation of the temporal and cell-type specific pattern of ER mRNA ontogeny in the postnatal rat uterus furthers our understanding of the molecular basis of both endogenous and exogenous estrogen effects on uterine growth and development.     

Differential regulation of mouse embryo development and viability by amino acids

The amino acid requirements of the preimplantation mouse embryo in culture changes as development proceeds from the zygote to the blastocyst stage. Eagle's non-essential amino acids and glutamine significantly increased cleavage rates during the first four cell cycles, while Eagle's essential amino acids without glutamine did not confer any benefit to embryo development before the eight-cell stage. After the eight-cell stage, non-essential amino acids and glutamine no longer stimulated cleavage rates but significantly increased blastocoel development and blastocyst hatching. In contrast, after the eight-cell stage essential amino acids increased cleavage rates as well as stimulating development of the inner cell mass of the resultant blastocysts. Fetal development after transfer of blastocysts was also significantly increased by culture with essential amino acids from the eight-cell stage. Consequently highest rates of development in vitro and viability after transfer were achieved when embryos were cultured with non-essential amino acids and glutamine to the eight-cell stage followed by development to the blastocyst stage in the presence of all 20 amino acids. Analysis of the parameters measured revealed a significant relationship between number of blastocyst cells and inner cell mass development with viability after transfer. Blastocyst formation and hatching could not be used to assess subsequent developmental potential.