Distribution and Regulation of ENaC Subunit and CFTR mRNA Expression in Murine Female Reproductive Tract

The present study investigated the regional distribution and cyclic changes in the mRNA expression of epithelial Na+ channel (ENaC) subunit and cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel, in adult female mouse reproductive tract. In situ hybridization revealed that in contrast to the abundant expression of CFTR, ENaC (alpha, beta, gamma) mRNA signal was not detected throughout the estrus cycle in the ovary and oviduct. Messenger RNA for all ENaC subunits was abundantly detected in the cervical and vaginal epithelia throughout the estrus cycle but for CFTR, mRNA was found only at proestrus. In the uterine epithelium, alphaENaC mRNA was detected at diestrus but not found at any other stage, while CFTR mRNA was only detected at early estrus but not other stages. Semi-quantitative RT-PCR detected mRNA for all ENaC subunits in the uterus throughout the cycle with maximal expression at diestrus and CFTR mRNA was only found in the early stages of the cycle. The involvement of ENaC and CFTR in Na+ absorption and Cl- secretion was demonstrated in cultured endometrial epithelia using the short-circuit current technique and found to be influenced by ovarian hormones. Taken together, these data indicate a main secretory role of the ovary and oviduct and a predominantly absorptive role of the cervix and vagina. The present results also suggest an ability of the uterus to secrete and absorb at different stages of the estrus cycle. Variations in the fluid profiles may be dictated by the regional and cyclic variations in expression of ENaC and CFTR and are likely to contribute to various reproductive events in different regions of the female reproductive tract.     

Beta-oestradiol rescues DeltaF508CFTR functional expression in human cystic fibrosis airway CFBE41o- cells through the up-regulation of NHERF1.

BACKGROUND INFORMATION: CF (cystic fibrosis) is a disease caused by mutations within the CFTR (CF transmembrane conductance regulator) gene. The most common mutation, DeltaF508 (deletion of Phe-508), results in a protein that is defective in folding and trafficking to the cell surface but is functional if properly localized in the plasma membrane. We have recently demonstrated that overexpression of the PDZ protein NHERF1 (Na(+)/H(+)-exchanger regulatory factor 1) in CF airway cells induced both a redistribution of DeltaF508CFTR from the cytoplasm to the apical membrane and the PKA (protein kinase A)-dependent activation of DeltaF508CFTR-dependent chloride secretion. In view of the potential importance of the targeted up-regulation of NHERF1 in a therapeutic context, and since it has been demonstrated that oestrogen treatment increases endogenous NHERF1 expression, we tested the hypothesis that oestrogen treatment can increase NHERF1 expression in a human bronchiolar epithelial CF cell line, CFBE41o(-), with subsequent rescue of apical DeltaF508CFTR chloride transport activity. RESULTS: We found that CFBE41o(-) cells do express ERs (oestrogen receptors) in the nuclear fraction and that beta-oestradiol treatment was able to significantly rescue DeltaF508CFTR-dependent chloride secretion in CFBE41o(-) cell monolayers with a peak between 6 and 12 h of treatment, demonstrating that the DeltaF508CFTR translocated to the apical membrane can function as a cAMP-responsive channel, with a significant increase in chloride secretion noted at 1 nM beta-oestradiol and a maximal effect observed at 10 nM. Importantly, knock-down of NHERF1 expression by transfection with siRNA (small interfering RNA) for NHERF1 inhibited the beta-oestradiol-dependent increase in DeltaF508CFTR protein expression levels and completely prevented the beta-oestradiol-dependent rescue of DeltaF508CFTR transport activity. CONCLUSIONS: These results demonstrate that beta-oestradiol-dependent up-regulation of NHERF1 significantly increases DeltaF508CFTR functional expression in CFBE41o(-) cells.     

Stimulation and selective inhibition of protein synthesis by 17 beta-estradiol, in the uterus of immature rats

The in vitro translation of RNAs extracted from immature female rat uteri 24 hrs. after one or four daily injections of 17 beta-oestradiol was carried out. The proteins labelled with (35S) methionine were separated by polyacrylamide gel electrophoresis and the resulting autoradiograms were scanned. The densitometric profile of uterine proteins obtained after a single injection of oestrogen showed a considerable increase in the majority of them, compared with controls. Following four injections of 17 beta-oestradiol the synthesis of all but one of the uterine proteins was observed. The Mr of the protein induced by repeated administration of oestrogens was about 105 kdaltons. This protein could possibly be involved in the inhibition of protein synthesis observed in the rat uterus after reiterated injections of 17 beta-oestradiol to female rats.     

Inhibition of non-genomic responses to oestrogen in the rat uterus by testosterone propionate

Testosterone propionate (50 mg/kg), administered together with oestradiol, inhibited the oestrogen-induced uterine eosinophilia, deep endometrial oedema and the increase in uterine wet weight, 6 h after treatment. The same dose of the androgen decreased the number of eosinophils in the blood and increased their degranulation, explaining the effect of testosterone in the uterus. The high doses of the androgen used were in the range of the doses reported by others to block selectively the oestrogen-induced increase in uterine peroxidase content but not other responses to oestrogen or the cytosolic oestrogen receptor translocation to the nucleus. The dissociation by high doses of testosterone of the oestrogen-induced uterine eosinophilia, wet weight increase and oedema from other responses to oestrogen in the absence of any measurable effect of testosterone upon cytosolic-nuclear oestrogen receptors supports the idea that uterine eosinophilia and oedema are oestrogenic responses regulated by mechanisms different from those of the genomic responses, and is in agreement with the hypothesis of the mediation of uterine oedema by eosinophils.     

Effect of dexamethasone on uterine cell death

Oestrogen, progesterone and androgen inhibit uterine cell death after the depletion of oestrogen. In the present study, we investigated effects of glucocorticoid on death of mouse uterine cells. Castrated female mice were given a daily injection of 17 beta-oestradiol (0.2 microgram/mouse/day) for 3 days, and then an injection of 5'-[125I]iodo-2'-deoxyuridine ([125I]IdUrd) to label DNAs of uterine cells with 125I. Mice were killed at intervals during subsequent treatments, and the retention of [125I]IdUrd incorporated into the whole uterus was determined. On subsequent injection of vehicle only, the 125I-radioactivity retained in the whole uterus rapidly decreased. Injections of dexamethasone (50 micrograms/mouse/day) reduced the loss of 125I-radioactivity slightly but significantly. Dexamethasone also showed synergistic effects on the retention of 125I-radioactivity when it was daily injected together with 17 beta-oestradiol, progesterone or 5 alpha-dihydrotestosterone. The present results suggest that glucocorticoid may affect the processes involved in the uterine cell death, in a manner such as inhibiting the uterine cell death or delaying the removal of DNAs of dead cells from the uterus.     

New Concepts on the Action of Oestrogens in the Uterus and the Role of the Eosinophil Receptor System

Two receptor systems for oestrogens have been demonstrated in the uterus: the cytosol-nuclear receptor system and the eosinophil receptor system. It has been proposed that the cytosol-nuclear receptor system mediates the genomic response to oestrogens in the uterus, while the eosinophil receptor system is thought to mediate the uterine edema and other early oestrogenic responses in the uterus. Cortisol is known to decrease drastically the number of eosinophils in the blood and therefore to limit their availability for migration to the uterus. The present results show that cortisol also drastically reduces both the oestrogen-induced uterine eosinophilia and the uterine wet weight responses, but does not interfere with the oestrogen-induced uterine RNA and protein increases. Oestradiol-17 beta has a higher affinity than oestriol for the cytosol-nuclear receptors and is now found to be the more potent oestrogen in inducing the genomic activation in the uterus. Estriol has a higher affinity than oestradiol-17 beta for the eosinophil receptors, and therefore, oestriol is the stronger oestrogen in inducing those oestrogenic effects which are mediated by the eosinophil receptor system. We conclude that the eosinophil receptor system for oestrogens is a new system, independent of Jensen's cytosol-nuclear receptor system, and this eosinophil receptor system is involved in the mechanism of oestrogen action in the uterus.     

Estrogen induces insulin-like growth factor-I expression in the rat uterus

The inability to convincingly demonstrate a mitogenic effect of estrogen on isolated uterine cells in culture suggests that autocrine or paracrine growth factors may be important in the estrogen-induced uterine proliferative response. Here we report that uterine expression of insulin-like growth factor-I (IGF-I), an important mediator of GH action, is increased after 17 beta-estradiol (5 micrograms/100 g bw, ip) administration to ovariectomized prepubertal rats. An increase in uterine IGF-I mRNA abundance, approximately 14-fold above untreated controls, was apparent 6 h after estrogen administration and the level achieved exceeded that seen in the uterus from intact mature rats during diestrus. In contrast to the increase in IGF-I expression in the uterus, no significant change in serum IGF-I concentration or hepatic or renal IGF-I mRNA abundance was demonstrable after 17 beta-estradiol injection of ovariectomized prepubertal rats. The increase in uterine IGF-I expression, was similar in both pituitary-intact and hypophysectomized, ovariectomized rats. We believe this is the first report of induction of IGF-I expression by estrogen in vivo. As such, the finding expands the role and significance of IGF-I as a mediator of growth beyond that related to GH.     

Regulation of expression of the retinoic acid metabolizing enzyme CYP26A1 in uteri of ovariectomized mice after treatment with ovarian steroid hormones

The retinoic acid (RA) synthesizing enzymes, retinaldehyde dehydrogenases (RALDH), are expressed in specific spatial and temporal patterns in uterine tissues during estrous cycle and early pregnancy in mice. Expression of RALDH1 and 2 has been shown to be induced by estrogen treatment within the uterus. In this study, we determined the influence of progesterone and 17-ss-estradiol on the uterine expression of the RA-metabolizing enzyme CYP26A1 after specific time intervals (1, 4, 24, and 48 hr after treatment of ovariectomized mice). In a following experiment, we investigated the influence of gestagen (promegestone 0.3 mg/kg body weight), estrogen (estradiol 3 microg/kg), their combination, as well as the antagonizing anti-progesterone hormone (RU 486 10 mg/kg) on the uterine expression of CYP26A1. Expression of CYP26A1 was localized using in situ hybridization and quantified using RT-PCR. CYP26A1 mRNA expression was strongly--although transiently--induced in uterine endometrial epithelial and glandular cells after administration of gestagen or the combination of gestagen + estrogen, but not by estrogen alone. These observations were confirmed by semi-quantitative RT-PCR experiments on whole uteri. Thus, we show that the expression of CYP26A1 in endometrial epithelial cells is regulated by progesterone and not significantly influenced by co-administration of estrogen. These data indicate an additional level of hormonal control of endogenous RA levels in the mouse uterus, where its synthesis would rely on estrogen-dependent expression of RALDH enzymes, whereas its active metabolism would be triggered by progesterone-induced CYP26A1 expression.     

Selective oestrogen receptor modulators

Selective oestrogen receptor modulators (SERMs) are compounds which act like oestrogens in some target tissues but which antagonise their effects in others. The first example of a SERM (referred to as a first-generation compound) was tamoxifen, for which oestrogen-like agonist activity on bone was seen to occur simultaneously with oestrogen antagonist activity on the breast. An unwanted effect of tamoxifen was its oestrogen-like action on the endometrium. Second-generation compounds have since been developed, most notably raloxifene, which has oestrogen-like actions on bone, lipids and the coagulation system, and oestrogen antagonist effects on the breast and uterus. Raloxifene has undergone very extensive, prospective, placebo-controlled, randomised trial evaluation, in which anti-fracture efficacy (to date only for vertebral fracture) has been accompanied by a major reduction in the incidence of new breast cancer. The compound is similar to placebo in its uterine effects, and similar to oestrogen in causing a two- to threefold increase in the risk of venous thromboembolism. Its lipid effects are similar to those of oestrogen, except for a relatively small effect on high-density lipoprotein cholesterol, and no significant effect on triglycerides. Data on cardiovascular event rates are not yet available; data on cognitive function are preliminary and, to date, reassuring. The mechanisms by which the same compound can exert oestrogen agonist effects on one target and antagonist effects on another are still being clarified. Important aspects include the fact that the oestrogen receptor undergoes different conformational changes according to the ligand. Thus the crystal structure of oestradiol bound to the oestrogen receptor differs from that of raloxifene bound to the same receptor. The existence of two oestrogen receptor subtypes may also be relevant. Mechanisms include differing interactions with various domains of the oestrogen receptor, and tissue-specific recruitment of steroid receptor co-activators and co-repressors may underlie some of the tissue-specific effects. The SERMs may be the prototype for other selective steroid receptor modulators, for example the androgen and progesterone receptors. The development of tissue target-specific agents is an exciting advance in endocrine pharmacology and can be extended to agents, such as tibolone, which exert some of their tissue specificity through their metabolites.     

Role of early and late oestrogenic effects on implantation in the mouse

Oestrogen action in the uterus is expressed in an early phase (Phase I) and a late phase (Phase II). The role of this biphasic oestrogen action in implantation is not clear. To determine the relative importance of Phase I and II responses, triphenylethylene compounds (CI-628, LY-117018, nafoxidine, clomiphene citrate and tamoxifen) and oestrogens (oestriol and oestradiol-17 beta) were used in a physiologically relevant experimental system for studying implantation. All compounds elicited uterine water imbibition to various degrees in ovariectomized-progesterone-treated mice at 6 h (Phase I response) and their effectiveness in inducing implantation in delayed implanting mice correlated with their respective potency to increase uterine wet weight. This suggests that Phase I might be an essential component of oestrogen action in implantation and that the efficiency of a compound to elicit a Phase I response might serve as a predictive indicator of its potential action on implantation.     

Intracellular relationships of the oestrogen receptor in the rat uterus and hypothalamus during the oestrous cycle.

Simultaneous measurements were made of the specific oestrogen receptor in the nuclear and cytosol fractions prepared from the uterus and hypothalamus of 50--81-day-old female rats undergoing a 4-day oestrous cycle. In the uterus, the content of nuclear receptor fluctuated in concert with known cyclic changes in the secretion of oestrogen, being maximal at pro-oestrus. Over the period of 50--81 days, the nuclear content at all phases increased with age, again corresponding to known age-related increases in ovarian secretion of oestrogen. This age-related increase in nuclear content, averaged from the values of the different phases in each age group, was related to equivalent increases in uterine wet weight, an increase of 1 pmol of receptor being accompanied by an increase of 80--90 mg. The concentration of cytosol receptor was maintained constant, with respect to wet weight, throughout the cycle and with age, irrespective of changes in nuclear content. In the uterus of normal mature females, translocation of receptor into the nucleus did not lead to depletion of cytosol receptor, suggesting a process of continuous replenishment/synthesis. In the hypothalamus, the nuclear content of oestrogen receptor was also maximal at pro-oestrus. In contrast with the uterus, the content of hypothalamic cytosol receptor was minimal at this phase and reflects depletion of the cytosol receptor, possibly as a result of translocation. The extent of translocation was low compared with that in the uterus and did not alter with age during the age-period studied. This low nuclear binding of the receptor in vivo is discussed in relation to the presence of a cytosol factor, present in limiting amounts, which in vitro mediates the binding of cytosol receptor to oligo(dT)-cellulose. The difference in the physiological response of the uterus and of the hypothalamus to oestrogens may be related to the extent of nuclear binding of receptor.     

Antiestrogenic and antifertility actions of anordrin (2α,17ga-diethynyl-A-nor-5α-androstane-2β,17β-diol 2,17-dipropionate)

Anordrin, administered in a single s.c. dose of 62.5 μg in sesame oil, stimulated sustained uterine growth (wet weight) when measured at 24 and 72 hr, but total soluble protein and total DNA per uterus was not increased. By comparison, 3 μg of estradiol-17β under the same conditions significantly increased all three parameters of uterine growth. Both of the above steroid treatments significantly increased nuclear estrogen receptor content of the uterus, but only the estradi-ol-17β treatment resulted in significantly elevated cytosol receptor content per uterus. Anordrin binds to the 8S estrogen receptor with an affinity of about 2 × 10⁵ M^-1 as determined by competition with [³H]estradiol-17β. The abortifacient activity of Anordrin when given orally (8 mg/kg b.w.) to mice on the 7th day of pregnancy was almost completely blocked by simultaneous oral administration of estradiol-17β (0.8 mg/kg b.w.). It is concluded that the actions of Anordrin on the uterus can be attributed to its antiestrogenic activities.     

Expression of insulin-like growth factor binding protein-4, insulin-like growth factor-I receptor, and insulin-like growth factor-I in the mouse uterus throughout the estrous cycle

Recent evidence suggests that a regulated insulin-like growth factor (IGF) system mediates the effects of estrogen, promoting the proliferation and differentiation of specific uterine cell types throughout the estrous cycle and during gestation in the rodent. Previous studies have shown that IGFs are differentially expressed in the mouse uterus during the periimplantation period. In the current study, we examined the expression of IGF binding protein-4 (IGFBP-4), IGF-I receptor (IGF-IR), and IGF-I in the mouse uterus throughout the estrous cycle. Ligand blot analysis was conducted on uterine homogenates using [125I]IGF-I. IGFBP-4 was detected in all uterine homogenates, varying in intensity throughout the estrous cycle. In situ hybridization studies at metestrus and diestrus demonstrated an intense IGFBP-4 mRNA signal in antimesometrial stromal cells between the luminal epithelium and the myometrium, but at proestrus and estrus, no IGFBP-4 signal was detected. No IGF-I mRNA was detected at any stage of the estrous cycle by in situ hybridization. However, by RT-PCR analysis, IGF-I mRNA was detected at all stages of the estrous cycle. RT-PCR analysis also showed IGF-IR mRNA throughout the estrous cycle. Using immunohistochemistry, IGF-IR immunostaining was detected throughout the estrous cycle and on days 2-7 of gestation, but was restricted to the glandular epithelium. These results suggest that uterine IGFBP-4 expression may not be dependent on uterine IGF-I expression. They also suggest that IGFBP-4 may play a role in uterine physiology independent of the inhibition of IGF-I action, and that IGF-IR is constitutively expressed in the mouse uterus.     

Estrogen regulation of c-fos messenger ribonucleic acid

Acute administration of 17 beta-estradiol to immature female rats elicits a rapid and striking increase in the size of the uterus. This increase in size to caused by both hypertrophy and hyperplasia in the epithelial, stromal, and myometrial cells in the uterus. Previous studies have shown that induction of mRNA for the epidermal growth factor receptor, the cellular homolog of the erb-B oncogene, occurs early during estrogen-stimulated uterine growth. We report here that estradiol causes a very rapid induction of the mRNA for the cellular oncogene c-fos in immature rat uterus. Steady state levels of c-fos mRNA reach a maximum 3 h after 17 beta-estradiol administration and slowly return to low basal levels in 15 h. Dexamethasone, progesterone, and 5 alpha-dihydrotestosterone had no effect on uterine c-fos mRNA expression. The induction of c-fos mRNA by estrogen was unaffected by the protein synthesis inhibitor puromycin but completely abolished by the RNA synthesis inhibitor actinomycin D.     

GRP78 expression and regulation in the mouse uterus during embryo implantation

The aim of this study was to investigate the spatiotemporal expression and regulation of GRP78 in the mouse uterus during the peri-implantation period. The GRP78 protein was mainly detected in the luminal and glandular epithelia on days 1–4 of pregnancy. On day 5 of pregnancy, the GRP78 protein was more highly observed around the implanted embryo at the implantation site. There was no detectable GRP78 protein signal on day 5 of pseudopregnancy. GRP78 mRNA and protein levels gradually increased on days 6–8 of pregnancy, and the expression pattern was also expanded, coinciding with the development of decidua. Similarly, GRP78 expression was also strongly expressed in decidualised cells following artificial decidualisation. Compared with the results obtained with the delayed uterus, a high level of GRP78 expression was detected in the implantation-activated uterus. In the uteri of ovariectomised mice, GRP78 expression increased and reached its highest level after injection of oestrogen, and progesterone seemed to have an antagonistic effect on oestrogen up-regulation of GRP78 expression. Our data indicate that GRP78 might play an important role during the process of mouse embryo implantation, and GRP78 expression was mainly regulated by active blastocysts and maternal oestrogen.