Hormonal regulation of apoptosis in the endometrium of common marmosets (Callithrix jacchus)

Phase-dependent apoptotic changes in the human endometrium during an ovarian cycle imply a potential role of steroids in the regulation of apoptosis. The present study was undertaken to determine the direct role of hormones in endometrial apoptosis in marmosets (Callithrix jacchus), a primate species which shows similarity to humans in terms of the cycle length and pattern. Endometrial apoptosis was detected by 3'-end labeling (TUNEL) in various phases of ovarian cycle in naturally cycling healthy marmosets (n=14) and also in ovariectomized marmosets (n=13) treated with either estradiol alone (E) or progesterone alone (P) or estradiol followed by progesterone (E+P). Expressions of apoptosis associated genes such as Bcl-2 family members (Bax and Bcl-2), proliferating cell nuclear antigen (PCNA)--a proliferation marker and steroid receptors, ERalpha and PR A were analysed by immunohistochemical methods. Apoptosis was intense in the glandular epithelial cells of endometrium during the mid-luteal phase as compared to other phases in naturally cycling animals; in the E+P group as compared to other groups of ovariectomized animals (P<0.05). Pronounced apoptosis in the mid-luteal phase was accompanied by the increased expression of Bax in glandular epithelial cells; while Bcl-2 immunoreactivity remained unchanged. PCNA expression was higher in the naturally cycling animals in the follicular phase and in the E group of the ovariectomized animals as compared those in the other groups. Immunoreactive ERalpha and PR A in glandular epithelial cells were most abundant during early follicular phase in naturally cycling animals and in both E and E+P groups among the ovariectomized animals. The present study highlights the importance of apoptosis in endometrial remodeling during the ovarian cycle and secondly, the role of both estradiol and progesterone in the regulation of apoptosis.     

Immunocytochemical analysis of estrogen and progestin receptors in uteri of steroid-treated and pregnant cats.

The purpose of this study was to determine the distribution of estrogen receptors (ER) and progestin receptors (PR) in specific uterine cell populations during various steroid hormone treatment regimens, and to determine if ER and PR distribution in the uterus is altered during implantation and the establishment of pregnancy in the cat. The tissues were processed for indirect immunocytochemical localization of receptors using specific monoclonal antibodies against ER and PR. ER were present in the nuclei of all epithelial cells and stromal fibroblasts in endometrium obtained from ovariectomized animals, whereas PR were only detectable in the nuclei of stromal fibroblasts. There was an apparent increase in the staining intensity and number of nuclei that stained positively for both ER and PR in all cell populations after 14 days of estradiol treatment. The administration of progesterone for 14 and 21 days, in the presence or absence of continuous estradiol, reduced the apparent intensity of staining and the number of nuclei staining positively for both ER and PR. ER were undetectable in the luminal epithelium, but remained in the glandular epithelial cells and stromal fibroblasts, whereas PR were only detectable in stromal fibroblasts. ER and PR localization in the endometrium obtained from estrus animals was similar to that observed in the estradiol-treated animals. A general decrease in intensity of staining for both ER and PR was evident by Day 5 postcoitus in pregnant animals. This decrease in intensity of staining continued until Day 12 postcoitus, when the distributions of ER and PR were similar to those observed in the ovariectomized estradiol-primed, progesterone-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Paracrine regulation of epithelial progesterone receptor and lactoferrin by progesterone in the mouse uterus

The objective of this study was to determine whether uterine stromal and/or epithelial progesterone receptor (PR) is required for the antagonism by progesterone (P(4)) of estradiol-17beta (E(2)) action on expression of PR and lactoferrin in uterine epithelium. Uterine tissue recombinants were prepared with epithelium (E) and stroma (S) from wild-type (wt) and PR knockout (PRKO) mice: wt-S+wt-E and PRKO-S+wt-E. P(4) action on epithelial PR expression was studied in wt-S+wt-E and PRKO-S+wt-E tissue recombinants. E(2) down-regulated epithelial PR in both types of tissue recombinants, but P(4) blocked E(2)-induced down-regulation of epithelial PR only in wt-S+wt-E tissue recombinants. Thus, P(4) requires stromal PR to inhibit E(2)-induced down-regulation of epithelial PR. Epithelial PR is not sufficient in itself. The inhibitory effect of P(4) on lactoferrin expression was studied in 4 types of tissue recombinants (wt-S+wt-E, PRKO-S+wt-E, wt-S+PRKO-E, and PRKO-S+PRKO-E). E(2) induced lactoferrin in all 4 types of tissue recombinants. P(4) blocked E(2)-induced lactoferrin expression only in wt-S+wt-E tissue recombinants. In wt-S+PRKO-E tissue recombinants, P(4) inhibited lactoferrin expression only partially. P(4) failed to block E(2)-induced lactoferrin expression in PRKO-S+wt-E and PRKO-S+PRKO-E tissue recombinants. Thus, both epithelial and stromal PR are essential for full P(4) inhibition of E(2)-induced lactoferrin expression.     

Endometrial effects of selective estrogen receptor modulators (SERMs) on estradiol-responsive gene expression are gene and cell-specific

Three selective estrogen receptor modulator (SERM) drugs which included 4-OH-tamoxifen (Tam), EM-800 (EM) and GW 5638 (GW) were investigated to determine their ability to inhibit estradiol-responsive gene expression in sheep endometrium. The uteri of ovariectomized ewes (10 ewes per SERM group) were infused with 10⁻⁷ M SERMs for 24 h prior to hysterectomy. Five ewes from each group received 50 μg 17β-estradiol (E2) and the remaining five ewes received vehicle 18 h prior to hysterectomy. Northern blot analyses and in situ hybridization demonstrated that E2 treatment increased estrogen receptor (ER), progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and cyclophilin (CYC) mRNA levels in most endometrial cells examined. Tam and GW exhibited characteristics similar to E2 by increasing ER gene expression, but they antagonized the E2-induced increases in PR and CYC mRNA levels. EM acted as an E2-agonist of GAPDH gene expression, but antagonized the E2 up-regulation of ER, PR and CYC gene expression in most endometrial cells. Immunohistochemistry determined that EM decreased ER protein levels in the glandular epithelium, and the SERMs investigated antagonized increases in PR protein levels in endometrium. In conclusion, GW and EM exhibit fewer agonist effects than Tam on endometrial gene expression. EM demonstrated the greatest antagonism of E2-enhanced levels of ER, PR and CYC, likely due to the inhibition of ER gene expression at both mRNA and protein levels.     

Heterogeneity of progesterone receptor expression in epithelial cells of immature and differentiating quail oviduct

The localization of progesterone receptor (PR) in the quail oviduct was investigated before and after the onset of sexual maturation using an immunohistochemical technique. PR was revealed exclusively in nuclei of target cells whatever the hormonal state of the tissue (immature or not, pretreated or not with progesterone). In the immature or ovariectomized quail oviduct, PR was principally localized in the undifferentiated epithelial cells; some mesothelial cells and a very few stromal cells expressed the PR. Only 40-45% of the epithelial cells were immunoreactive. These positive cells were mainly localized in the furrows of the villi where further evagination of the epithelium will occur to form the tubular glands. The onset of sexual maturation was accompanied by an increase of the proportion of positive epithelial cells and stromal cells. In estradiol-treated animals, more than 90% of the tubular gland cells were strongly stained while only 40% of the luminal epithelial cells were immunoreactive. Our results show that there are two subpopulations of epithelial cells: those expressing the PR before the onset of sexual maturation even in ovariectomized quails (constitutive expression) and those expressing the PR during sexual maturation or after estrogen injection (inductive expression). These results, associated with previously published studies dealing with the cytodifferentiation of epithelial cells during natural development or after various hormonal treatments in ovariectomized animals, suggest that the first are the progenitors of tubular gland cells, and the second the progenitors of ciliated and goblet cells. In stromal cells, PR expression is also inducuible.     

Progesterone receptor repression by estrogens in rat uterine epithelial cells

Measurements performed using cell lines or animal tissues have shown that the progesterone receptor (PR) can be induced by estrogens. By use of immunohistochemistry we studied the effects of estrogens on the PR levels in the individual cell types of the target organs uterus and breast. In the uteri of rats, ovariectomy induced a decrease in PR immunoreactivity within the myometrium and outer stromal cell layers. In contrast, in the uterine luminal and glandular epithelium and surrounding stromal cell layers the PR immunoreactivity was significantly enhanced. The same picture emerged when intact rats were treated with the pure estrogen receptor antagonist, ZM 182780 (10 mg/kg/d). Treatment of ovariectomized rats with estradiol resulted in high PR levels in the myometrium and stroma cells but low PR immunoreactivity in the epithelial cells. The ER-mediated repression of the PR immunoreactivity was evidently restricted to the uterine epithelium, as we found that in the epithelial cells of the mammary gland and in cells of N-nitrosomethylurea-induced mammary carcinomas the PR expression was induced by estrogens and was blocked by the pure antiestrogen ZM 182780. These results clearly show that in the rat the activated ER induces diverging effects on PR expression in different cell types even within the same organ.     

Histochemical localization of estrogen and progesterone receptors: evaluation of a method

A histochemical method for the detection of estrogen (ER) and progesterone (PR) receptors in human endometrium, using estrogen and progesterone derivatives linked to fluorochrome-labeled bovine serum albumin (E2-BSA-fluorescein isothiocyanate (FITC) and progesterone-BSA-tetramethylrhodamine isothiocyanate (TMRITC], has been evaluated. The fluorochrome-labeled steroids were bound to the cytoplasm--preferably in glandular epithelial cells but to a lesser extent also to stromal cells. The steroid specificity of the observed binding was studied by preincubating the sections with a series of unlabled steroids and nonsteroidal, hormonally active compounds (estradiol-17 beta, diethylstilbestrol, tamoxifen, 5 alpha-dihydrotestosterone and R 1881 for ER and ORG 2058, R 5020, dexamethasone, cortisol and 5 alpha-dihydrotestosterone for PR). The inhibition studies indicated that E2-BSA-FITC and progesterone-BSA-TMRITC bind to ER and PR in human endometrium with a reasonable degree of specificity. The method was reproducible and various procedural steps were tested, showing satisfactory technical stability. The method is applicable to small tissue samples, and is a valuable complement to quantitative biochemical receptor assays, as it localizes the receptors in tissue slices.     

Steroid receptors in canine endometrial cells can be regulated by estrogen and progesterone under in vitro conditions

The expression of estrogen (ER) and progesterone receptors (PR) in the endometrium is regulated by steroid hormones. An increase in plasma estrogen leads to upregulation of the number of both steroid receptors, whereas a decrease in both receptors population is due to high concentration of plasma progesterone. To study the exact effect of different concentrations of beta-estradiol and progesterone on canine epithelial and stromal endometrial cells an in vitro model from dog uterus was developed and kept for 20 days. Material was obtained from healthy dogs, undergoing ovariohysterectomy. Endometrial epithelial and stromal cells were gained after collagenase treatment, followed by filtration steps. Electron microscopy and immunolabeling were used to study cell morphology and differentiation. Immunocytochemistry was used to determine proliferation rate (Ki-67), ER and PR status on Days 3, 8, 10, 13, and 20. Mitotic activity of both cells was stimulated with different concentrations of steroids and revealed high values until cells reached confluency. ER and PR expression in confluent layer from epithelial and stromal cells was upregulated with beta-estradiol. In addition progesterone significant downregulated both receptors population in stromal cells, whereas the reduction was less pronounced in epithelial cells. Results showed that our in vitro system is a useful tool to study the influence of beta-estradiol and progesterone on cell proliferation rate, ER and PR expression. The primary cell culture model helps to avoid experiments on living animals.     

Immunocytochemical analysis of oestrogen receptors and progesterone receptors in the human uterus throughout the menstrual cycle and after the menopause.

To obtain more insight into the relationship between cyclic and regional changes in steroid receptor expression and function-related changes in the various types of cell of the normal human uterus, we performed an immunocytochemical study on paraffin-embedded sections. The distribution and intensity of immunostaining for the oestrogen receptor and the progesterone receptor in the various types of cell were semiquantitatively scored. The data were statistically compared for the different phases of the menstrual cycle and after the menopause, and for the different regions of the corpus and (endo)cervix uteri. During the menstrual cycle, significant changes in oestrogen receptor score were observed in glandular and stromal cells of endometrium basalis and functionalis and in smooth muscle cells of the myometrium. In all types of cell, oestrogen receptor expression reached a maximum in the late proliferative phase. During the early secretory phase, oestrogen receptor staining declined sharply in stromal and smooth muscle cells, whereas, in glandular epithelium, oestrogen receptor expression decreased more gradually. During mid- and late-secretory phases, an increase in oestrogen receptor staining was also observed in predecidualizing stromal cells and smooth muscle cells. Progesterone receptor numbers changed significantly in glandular epithelium but not in stromal and smooth muscle cells. Glandular progesterone receptor expression reached a maximum in the early secretory phase and was then drastically reduced. During mid- and late-secretory phases stromal cells were moderately stained for progesterone receptor in contrast to epithelial gland cells which showed no or very weak staining. No regional variations in steroid receptor distribution in endometrium and myometrium were found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decidualization regulates the expression of the endometrial chorionic gonadotropin receptor in the primate

Chorionic gonadotropin (CG) plays an important role in establishing a receptive endometrium by directly modulating the function of both endometrial stromal and epithelial cells in the baboon. The focus of this study was to characterize changes in CG receptor (LHCGR, also known as CG-R) expression during the menstrual cycle and early pregnancy, particularly during decidualization. LHCGR was localized by using a peptide-specific antibody generated against the extracellular domain. Immunostaining was absent in any of the cell types during the proliferative phase of the cycle. In contrast, during the secretory phase, both luminal and glandular epithelial cells stained positively. Stromal staining was confined to the cells around spiral arteries (SAs) and in the basalis layer. This stromal staining pattern persisted at the implantation site between Days 18 and 25 of pregnancy and after CG infusion. However, as pregnancy progressed (Days 40 to 60), staining for LHCGR was dramatically decreased in the stromal cells. These data were confirmed by nonisotopic in situ hybridization. To confirm whether the loss of LHCGR was associated with a decidual response, stromal fibroblasts were decidualized in vitro, and cell lysates obtained after 3, 6, and 12 days of culture were analyzed by Western blotting. LHCGR protein decreased with the onset of decidualization in vitro, confirming the in vivo results. Addition of CG to decidualized cells resulted in the reinduction of LHCGR in the absence of dbcAMP. We propose that CG acting via its R on stromal cells modulates SA in preparation for pregnancy and trophoblast invasion. As pregnancy progresses, further modification of SA by migrating endovascular trophoblasts and subsequent decidualization results in the downregulation of LHCGR. This inhibition of LHCGR expression also coincides with the decrease of measurable CG in peripheral circulation.     

Chorionic gonadotropin and uterine dialogue in the primate

Implantation is a complex spatio-temporal interaction between the growing embryo and the mother, where both players need to be highly synchronized to be able to establish an effective communication to ensure a successful pregnancy. Using our in vivo baboon model we have shown that Chorionic Gonadotropin (CG), as the major trophoblast derived signal, not only rescues the corpus luteum but also modulates the uterine environment in preparation for implantation. This response is characterized by an alteration in both the morphological and biochemical activity in the three major cell types: luminal and glandular epithelium and stromal fibroblasts. Furthermore, CG and factors from the ovary have a synergistic effect on the receptive endometrium. Novel local effects of CG which influence the immune system to permit the survival of the fetal allograft and prevent endometrial cell death are also discussed in this review. An alternate extracellular signal-regulated kinase (ERK) activation pathway observed in epithelial endometrial cells and the possibility of differential expression of the CG/LH-R isoforms during gestation, open many questions regarding the mechanism of action of CG and its signal transduction pathway within the primate 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.     

Expression of nerve growth factor and its receptors NTRK1 and TNFRSF1B is regulated by estrogen and progesterone in the uteri of golden hamsters

Experiments were conducted using female golden hamsters to identify the presence of nerve growth factor (NGF) and its receptors NTRK1 and TNFRSF1B in the uteri of female animals and regulation on their expression by estrogen and progesterone. NGF and its receptor NTRK1 were immunolocalized to luminal epithelial cells, glandular cells, and stromal cells. TNFRSF1B was immunolocalized in luminal epithelial and glandular cells, with no staining found in stromal cells of the uterine horns of normal cyclic golden hamsters. Strong immunostaining of NGF and its receptors NTRK1 and TNFRSF1B was observed in uteri on the day of proestrus as compared to the other stages of the estrous cycle. Results of immunoblot analysis of NGF revealed that there was a positive correlation between uterine NGF expression and plasma concentrations of estradiol-17beta. To clarify the effects of estrogen and progesterone on NGF, NTRK1, and TNFRSF1B expression, adult female golden hamsters were ovariectomized and treated with estradiol-17beta and/or progesterone. Immunoblot analysis and immunohistochemistry indicated that estradiol-17beta stimulated expression of NGF and its two receptors in the uterus. Treatment with progesterone also increased NGF and NTRK1 expression in the uterus. However, no additive effect of these steroids on expression of NGF and its receptors was observed. Changes in uterine weights induced by estradiol-17beta and/or progesterone showed the same profile with that of NGF, suggesting that a proliferative act of NGF may be involved in uterine growth. These results suggest that NGF may play important roles in action of steroids on uterine function.     

Estrogen receptors, progestin receptors and DNA synthesis in the macaque endometrium during the luteal-follicular transition

We have suggested that in the nonhuman primate endometrium, stromal cells might play a role in mediating the effects of estrogen on the epithelium, especially during the luteal-follicular transition (LFT) when target cells normally escape from the inhibitory influence of progesterone (P). We now report that like estrogen receptors (ER), endometrial progestin receptors (PR) are detectable only in stromal cells until the fifth day of the LFT. With a technique that combined immunocytochemistry and autoradiography on the same sections, we characterized the cellular distribution of ER or PR coincidentally with the localization of [3H]thymidine taken up in vitro by endometria from monkeys undergoing an LFT. DNA synthesis in the glands of the upper endometrium was E2-dependent, but the distribution of [3H]thymidine was not positively correlated with the presence of ER or PR. Readministration of P to animals on days 3 or 4 of the LFT significantly reduced the [3H]thymidine labeling index of the glandular epithelium and caused stromal ER to decline, but P did not block the eventual appearance of ER in epithelial cells on day 5 of the LFT. Thus, E2 stimulated DNA synthesis in epithelial cells that lacked ER, and P suppressed DNA synthesis in these cells even though PR was only detected in the stroma when P treatment began. These data are consistent with a role for endometrial stromal cells in mediating the effects of E2 and P on the epithelium during the LFT.