Sex steroid receptor expression in the oviduct and uterus of sheep with estrus synchronized with progestagen or prostaglandin analogues

The objective of this study was to investigate differences in the expression of estrogen receptor-alpha (ERalpha), progesterone receptor (PR) and the proliferative indexes (Ki-67), in the uterus and oviduct of sheep with estrus synchronized either by prostaglandin analogues (Group PA, n=27) or by treatment with progestagens (Group P, n=29) on days 4 and 7 (day 0=estrus), when the embryos were collected. Immunohistochemical methods were used to quantify ERalpha, PR and Ki-67 in six superficial and deep compartments in the uterus and oviduct. The expression of ERalpha was significantly (P<0.01) lower in progestagen treated ewes than in prostaglandin analogues treated group in the luminal epithelium, superficial glands and superficial stroma in the uterus on day 4. The expression of PR was significantly lower in progesterone treated ewes than in the PA Group in the superficial gland (P<0.05) in both days studied. The lowest expression of PR was observed in the luminal caruncular epithelium and superficial glands in both treatments, obtaining the lowest levels on day 4 (P<0.05). There were significant differences between days 4 and 7 in the Ki-67 immunostaining in the luminal epithelium (P<0.01) and superficial glands (P<0.05). A higher cell proliferation was observed in the uterine epithelium (P<0.05) on day 4 in the animals treated with progestagens. Results indicate that sheep with synchronization of estrus with progestagens showed a reduction of ERalpha and PR protein expression in most of oviductal and uterine cells.     

Paracrine regulation of epithelial progesterone receptor by estradiol in the mouse female reproductive tract

Regulation of progesterone receptor (PR) by estradiol-17beta (E(2)) in mouse uterine and vaginal epithelia was studied. In ovariectomized mice, PR expression was low in both vaginal stroma and epithelium, but high in uterine epithelium. E(2) induced PR in vaginal epithelium and stroma, but down-regulated PR in uterine epithelium. Analysis of estrogen receptor alpha (ERalpha) knockout (ERKO) mice showed that ERalpha is essential for E(2)-induced PR expression in both vaginal epithelium and stroma, and for E(2)-induced down-regulation, but not constitutive expression of PR in uterine epithelium. Regulation of PR by E(2) was studied in vaginal and uterine tissue recombinants made with epithelium and stroma from wild-type and ERKO mice. In the vaginal tissue recombinants, PR was induced by E(2) only in wild-type epithelium and/or stroma. Hence, in vagina, E(2) induces PR directly via ERalpha within the tissue. Conversely, E(2) down-regulated epithelial PR only in uterine tissue recombinants constructed with wild-type stroma. Therefore, down-regulation of uterine epithelial PR by E(2) requires stromal, but not epithelial, ERalpha. In vitro, isolated uterine epithelial cells retained a high PR level with or without E(2), which is consistent with an indirect regulation of uterine epithelial PR in vivo. Thus, E(2) down-regulates PR in uterine epithelium through paracrine mechanisms mediated by stromal ERalpha.     

The activation function-1 domain of estrogen receptor alpha in uterine stromal cells is required for mouse but not human uterine epithelial response to estrogen

The activation function-1 (AF-1) domain of the estrogen receptor alpha (ERalpha) in stromal cells has been shown to be required for epithelial responses to estrogen in the mouse uterus. To investigate the role of the stroma in estrogenic responses of human uterine epithelium (hUtE), human/mouse chimeric uteri composed of human epithelium and mouse stroma were prepared as tissue recombinants (TR) that were grown in vivo under the renal capsule of female nude mouse hosts. In association with mouse uterine stroma (mUtS), hUtE formed normal glands surrounded by mouse endometrial stroma and the human epithelium influenced the differentiation of stroma into myometrium, such that a histologically normal appearing uterine tissue was formed. The hUtE showed a similar proliferative response and increase in progesterone receptors (PR) in response to 17beta-estradiol (E2) in association with either human or mUtS, as TRs. However, under identical endocrine and micro-environmental conditions, hUtE required 5-7 days exposure to E2 rather than 1 day, as shown for mouse uterine epithelium, to obtain a maximal proliferative response. Moreover, this extended length of E2 exposure inhibited mouse epithelial proliferation in the presence of mouse stroma. In addition, unlike the mouse epithelium, which does not proliferate or show regulation of PR expression in response to E2 in association with uterine stroma derived from mice that are null for the AF-1 domain of ERalpha, hUtE proliferates and PR are up-regulated in response to E2 in association genetically identical ERalpha knock-out mouse stromal cells. These results clearly demonstrate fundamental differences between mouse and human uterine epithelia with respect to the mechanisms that regulate estrogen-induced proliferation and expression of PR. Moreover, we show that genetically engineered mouse models could potentially aid in dissecting molecular pathways of stromal epithelial interactions in the human uterus.     

Steroid receptors and proliferation in the human breast

Despite recent gains in our knowledge of the hormonal control of proliferation and differentiation in the rodent mammary gland, the factors regulating these processes in the human are poorly understood. We have developed a model in which intact normal human breast tissue is grafted subcutaneously into adult female athymic nude mice and treated with oestrogen (E) and/or progesterone (P) at human physiological serum levels. We have shown that (i) E and not P is the major epithelial cell mitogen in the adult non-pregnant, non-lactating breast, (ii) E induces progesterone receptor (PR) expression and (iii) PR expression is maximally induced at low E concentrations while a higher amount of E is required to stimulate proliferation. These data raised the question of whether one cell type demonstrated two different responses to the two different E concentrations or whether PR expression and proliferation occurred in separate cell populations. Using dual label immunofluorescence, we showed that steroid receptor expression and proliferation (Ki67 antigen) are detected in separate cell populations in normal human breast epithelium, and that cells expressing the oestrogen receptor-alpha (ERalpha) invariably contained the PR. We also reported that this separation between steroid receptor expression and proliferation observed in the normal human epithelium is disrupted at an early stage in breast tumourigenesis. One interpretation supported by our recent findings is that some ERalpha/PR-positive epithelial cells are quiescent breast stem cells that act as "steroid hormone sensors". Such hormone sensor cells might secrete positive or negative paracrine/juxtacrine factors dependent on the prevailing E or P concentration to influence the proliferative activity of adjacent ERalpha/PR-negative epithelial cells.     

Messenger RNA levels of estrogen receptors alpha and beta and progesterone receptors in the cyclic and inseminated/early pregnant sow uterus

The aim of the present study was to investigate differences in the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus at different stages of the estrous cycle as well as in inseminated sows at estrus and during early pregnancy by use of solution hybridization and in relation to plasma levels of estradiol and progesterone. Uterine samples were collected at different stages of the estrous cycle and after insemination/early pregnancy. In the endometrium, the expression of ERalpha mRNA and PR mRNA was similar for cyclic and early pregnant groups. Both were highest at early diestrus/70 h after ovulation and ERalpha mRNA was lowest at late diestrus/d 19 while PR mRNA was lowest at diestrus and late diestrus/d 11 and d 19. The expression of endometrial ERbeta was constantly low during the estrous cycle but higher expression was found in inseminated/early pregnant sows at estrus and 70 h after ovulation. In the myometrium, high expression of ERalpha mRNA and PR mRNA was observed at proestrus and estrus in cyclic sows and at estrus in newly inseminated sows. Higher expression of myometrial ERbeta mRNA was found in inseminated/early pregnant sows compared with cyclic sows, although significant only at estrus. In conclusion, the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus differed between endometrium and myometrium as well as with stages of the estrous cycle and early pregnancy. In addition to plasma steroid levels, the differences between cyclic and inseminated/early pregnant sows suggest that other factors, e.g. insemination and/or the presence of embryos, influence the expression of these steroid receptor mRNAs in the sow uterus.     

Paracrine regulation of apoptosis by steroid hormones in the male and female reproductive system

In males, androgens are essential in maintaining the integrity of the prostate. Androgen-ablation induces apoptosis of the prostatic epithelium. In females, ovariectomy induces apoptosis in uterine epithelium while progesterone inhibits this process. The objective of this study was to determine whether androgen and progesterone inhibit apoptosis, respectively, in mouse prostatic and uterine epithelia via steroid receptors in the epithelium or in the stroma. To address this question, prostatic tissue recombinants were prepared with rat urogenital sinus mesenchyme plus bladder epithelium from wild-type or testicular feminization mutant (Tfm) mice. Thus, prostatic tissue was generated having androgen receptor (AR) in both epithelium and stroma or in the stroma only. Castration of hosts induced apoptosis in the AR-negative Tfm prostatic epithelium with an epithelial apoptotic index virtually identical to prostatic tissue recombinants containing wild-type epithelium. Moreover, this castration-induced prostatic epithelial apoptosis was blocked by testosterone and dihydrotestosterone in both wild-type and Tfm prostatic tissue recombinants. Likewise, uterine tissue recombinants were prepared in which epithelium and/or stroma was devoid of progesterone receptor (PR) by using uterine epithelium and stroma of wild-type and PR knockout mice. Progesterone inhibited uterine epithelial apoptosis only in tissue recombinants prepared with PR-positive stroma. The PR status of the epithelium did not affect epithelial apoptotic index. Therefore, the apoptosis in prostatic and uterine epithelia is regulated by androgen and progesterone via stromal AR and PR, respectively. In both cases, epithelial AR or PR is not required for hormonal regulation of epithelial apoptosis in prostatic and uterine epithelium.     

Immunohistochemical detection of androgen receptors in the canine uterus throughout the estrus cycle

Serum androgen levels in the bitch increase during proestrus and remain elevated until metestrus. To find out whether androgens can have a direct impact on the canine uterus, androgen receptors (AR) were identified immunohistochemically in uterine tissue. Androgen receptor distribution in the uterine horns, body and cervix was described during different cycle stages, during pregnancy and in the postpartum period. Nuclear staining for AR was observed in cells of the surface epithelium, glandular ducts, basal glands and stroma of the endometrium, and in myometrial smooth muscle cells. In addition, cytoplasmic staining was observed in epithelial cells from proestrus to early metestrus, when the cells were secretory active, and in stroma cells during pregnancy, suggesting a role for androgens in decidualization. During pregnancy and in the postpartum period nuclear staining for AR was nearly absent. During the estrus cycle stroma cells stained with higher intensities for AR than epithelial cells, supporting the idea that stroma cells mediate some effects of steroid hormones on epithelial cells in the genital tract. In contrast with earlier findings on estrogen receptor-alpha and progesterone receptors, no significant changes in androgen receptor expression were observed during the estrus cycle. Few correlations were found between the staining for AR and serum levels of the sex steroids. The present findings suggest that there is a basal expression of AR in the canine uterus throughout the estrus cycle that may not be influenced by sex steroid hormones.     

Differences in uterine immunoexpression of PR, ERα and OTR when comparing prostaglandin- to progestagen-based protocols for ovine estrus synchronization

The aim of this work was to compare PR, ERα and OTR uterine expression between days 9 and 21 of pregnancy in ewes whose estrus had been synchronized with two different protocols. Sixty-four adult Manchega ewes were synchronized with either conventional progestagens (P) or prostaglandin analogues (PG), and mated. Uterine samples were obtained from pregnant animals (group P, n=24; group PG, n=25) on days 9 post coitus (pc), 13pc, 15pc, 17pc and 21pc. Immunohistochemical detection of progesterone receptor (PR), estrogen receptor-α (ERα) and oxytocin receptor (OTR) was assessed in different uterine cell compartments including luminal and glandular epithelium, stroma and myometrium. Interaction day × treatment was obtained when assessing PR expression in the caruncular stroma (P=0.027) and myometrium (P=0.000), as well as for ERα in the superficial stroma (P=0.05). Significant "day post coitus" effect was found regarding to PR (P<0.01, with the exception of the superficial stroma, deep stroma and myometrium), ERα (P<0.01), and OTR (P<0.05, except in the deep compartments). No significant "treatment" effect was found for PR, ERα or OTR protein immunoexpression. This study supports the implication of PR, ERα and OTR within days 9-21 of the ovine pregnancy. Moreover, different expression pattern of PR and ERα proteins has been found between treatments in various compartments studied. Collectively, these results indicate that PR, ERα and OTR expression during early pregnancy is similar between ewes treated with either progestagens or prostaglandin analogues-based protocols for estrus synchronization.     

Regional differences in expression of progesterone receptor in oviduct and uterus of rabbit during early pregnancy

We characterized the expression pattern of progesterone receptor (PR) in two regions of the oviduct (ampullae and isthmus), and the uterus (epithelium and stroma) of the rabbit (Oryctolagus cuniculus) during early pregnancy (1-4 days) by RT-PCR and immunohistochemistry. We observed a significant increase in the expression of PR at mRNA level in the uterus on days 1 and 2 of pregnancy, followed by a decrease on days 3 and 4. These changes were also observed at protein level in the uterine epithelium. Interestingly, PR immunoreactivity decreased in stromal cells in all days of pregnancy as compared with non-pregnant rabbits (NG). In the isthmus PR mRNA expression significantly increased on day 2 of pregnancy and diminished on days 3 and 4, whereas no significant changes were observed in the ampullae. In epithelial and stromal cells of the isthmus, PR immunostaining was reduced through pregnancy as compared with NG group. In contrast, a reduction in PR immunostaining was observed on days 1-3 with an increase on day 4 in epithelial and stromal cells of the ampullae. The overall results suggest that PR exhibit a differential expression pattern in the oviduct and the uterus during early pregnancy of the rabbit, and that these differences are related to different functions of PR in the reproductive tract during early pregnancy.     

Detection of estrogen alpha and progesterone receptors and cell proliferation in the uterus during early pregnancy in the goat

The objectives of this study were to investigate in the goat uterus the expression of estrogen-alpha (ER alpha) and progesterone receptors (PR) and their relationship to proliferation indices (Ki-67) during peri-implantation on Days 22 to 30 post coitum (pc). Immunohistochemical methods were used to quantify ER alpha and PR for luminal and deep regions of the endometrium and of the myometrium. On Day 22 pc cell proliferation was only observed in the luminal epithelium. On Day 24 pc, high cell proliferation indices were seen in luminal epithelium and proliferation began in the luminal stroma and glands. There was a positive correlation between Ki-67 and total ER alpha score in the luminal epithelium (r = 0.53, P < 0.01). Levels of PR scores were highly correlated with Ki-67 indices in luminal epithelium (r = 0.74, P < 0.01) and stroma (r = 0.70, P < 0.01). No Ki-67 expression was observed in deep glands, stroma or myometrium on any of the days studied. Results indicate that patterns of ER alpha and PR expression differ markedly, and that there was a high correlation between PR expression and cell proliferation in the caprine uterus during the peri-implantation period.     

Regulation of progesterone receptors and decidualization in uterine stroma of the estrogen receptor-alpha knockout mouse

Regulation of progesterone receptor (PR) in uterine stroma (endometrial stroma plus myometrium) by estrogen was investigated in estrogen receptor-alpha (ERalpha) knockout (alphaERKO) mice. 17 beta-Estradiol (E(2)) increased PR levels in uterine stroma of ovariectomized alphaERKO mice, and ICI 182 780 (ICI) inhibited this E(2)-induced PR expression. Estrogen receptor-beta(ER beta) was detected in both uterine epithelium and stroma of wild-type and alphaERKO mice by immunohistochemistry. In organ cultures of alphaERKO uterus, both E(2) and diethylstilbestrol induced stromal PR, and ICI inhibited this induction. These findings suggest that estrogen induces stromal PR via ERbeta in alphaERKO uterus. However, this process is not mediated exclusively by ERbeta+, because in ERbeta knockout mice, which express ERalpha, PR was up-regulated by E(2) in uterine stroma. In both wild-type and alphaERKO mice, progesterone and mechanical traumatization were essential and sufficient to induce decidual cells, even though E(2) and ERalpha were also required for increase in uterine weight. Progesterone receptor was strongly expressed in decidual cells in alphaERKO mice, and ICI did not inhibit decidualization or PR expression. This study suggests that up-regulation of PR in endometrial stroma is mediated through at least three mechanisms: 1) classical estrogen signaling through ERalpha, 2) estrogen signaling through ERbeta, and 3) as a result of mechanical stimulation plus progesterone, which induces stromal cells to differentiate into decidual cells. Each of these pathways can function independently of the others.     

Rac1 deletion causes thymic atrophy

The thymic stroma supports T lymphocyte development and consists of an epithelium maintained by thymic epithelial progenitors. The molecular pathways that govern epithelial homeostasis are poorly understood. Here we demonstrate that deletion of Rac1 in Keratin 5/Keratin 14 expressing embryonic and adult thymic epithelial cells leads to loss of the thymic epithelial compartment. Rac1 deletion led to an increase in c-Myc expression and a generalized increase in apoptosis associated with a decrease in thymic epithelial proliferation. Our results suggest Rac1 maintains the epithelial population, and equilibrium between Rac1 and c-Myc may control proliferation, apoptosis and maturation of the thymic epithelial compartment. Understanding thymic epithelial maintenance is a step toward the dual goals of in vitro thymic epithelial cell culture and T cell differentiation, and the clinical repair of thymic damage from graft-versus-host-disease, chemotherapy or irradiation.     

Apoptosis in endometrium of mouse during estrous cycle

The present study was carried out to evaluate apoptosis in endometrium and to correlate these changes with the circulating levels of estradiol and progesterone in the mouse. Apoptosis was observed in various compartments of mouse uterus i.e. stroma, glandular epithelium and luminal epithelium depending on the stage of cycle. Stromal cell apoptosis was observed during various stages of cyclicity except on estrus day. Luminal epithelial cells showed apoptotic changes during all stages of cyclicity except on diestrus day. During metestrus, apoptosis was observed in glandular and luminal epithelia as well as stromal cells. Steroid antagonists such as tamoxifen and onapristone altered the apoptotic changes in the uterus. The results suggest that epithelial cell apoptosis is regulated by estrogen while stromal cell apoptosis is under the control of progesterone.     

Effect of plane of nutrition on endometrial sex steroid receptor expression in ewes

The effect of plane of nutrition on progesterone receptor (PR) and estrogen receptor alpha (ERalpha) expression in ovine endometrium was investigated. Rasa Aragonesa ewes (n=26) were fed diets to provide either 1.5 (Group C) or 0.5 (Group L) times the daily maintenance requirement and were slaughtered at Days 5 or 10 of the estrous cycle (Day 0=estrus). PR and ERalpha immunoreactivity were analyzed in eight endometrial cell compartments, defined by cell type and location. Group L had less PR immunostaining on Day 5 (P<0.05), which is consistent with lesser endometrial content of progesterone found in such animals. Most cell types of Group C had down regulation of PR at Day 10, but in Group L, this pattern was observed only in three cell compartments. The lesser PR contents found at Day 5 in Group L ewes may explain the lack of inhibition of PR. No effect of treatment or day of the estrous cycle was observed in ERalpha. Results indicate that endometrial PR is affected in a cell type, in specific manner, by plane of nutrition.     

Uterine estrogen receptor alpha and progesterone receptor during the follicular and luteal phase in llamas

Estrogen receptor-alpha (ERalpha) and progesterone receptor (PR) were characterized in different endometrial cell types as luminal and glandular epithelium and stroma during the follicular (FP) and the luteal phase (LP) in llamas. Animals were examined daily by transrectal ultrasonography for the determination of the presence of an ovulatory follicle and ovulation was immediately induced by a GnRH injection (Day 0). Endometrial samples were obtained by transcervical biopsies from the left uterine horn on Day 0 (FP) and 9 days after the GnRH injection (Day 9, LP). Blood samples were collected on these days for estradiol 17beta and progesterone determination by RIA. An immunohistochemical technique was used to visualize ERalpha and PR immunostaining which was then analyzed by two independent observers. Total positive area and average staining for ERalpha were affected by the phase of the ovarian activity: in the three cell types there was more positive area and intense staining during the FP than during the LP. Similar findings were observed for PR, more positive stained areas were found during the FP than during the LP in the epithelia. In addition, the three cell types had more intense staining during the FP than during the LP. An effect of the cell type for ERalpha and PR was observed; epithelia (luminal and glandular) had more positive stained areas and greater intensity than stromal cells. In conclusion, the results of the present study suggest that in llamas, like in other ruminants, estradiol has a stimulatory effect while progesterone downregulates the ERalpha and PR and that the receptor is cell type specific.     

Estrogen induces a systemic growth factor through an estrogen receptor-alpha-dependent mechanism

Estrogen induces proliferation of uterine epithelium through a paracrine action of estrogen receptor (ERalpha) in the underlying stroma. In ovariectomized mice primed with progesterone, estrogen stimulates proliferation in both the epithelium and the stroma. We set out to test whether a paracrine mode of action is involved in estrogen-induced proliferation of the uterine stroma. Epithelial and mesenchymal tissues derived from uteri of neonatal ERalpha null mice (ERalphaKO) or wild-type mice were separated and recombined in all four possible configurations (ERalpha+ or ERalpha- epithelium with ERalpha+ or ERalpha- mesenchyme) and grafted into female athymic mice. After 5 wk, hosts were ovariectomized and challenged with hormone treatment, and cellular proliferation was monitored by thymidine autoradiography. Results showed that, although the full response of the epithelium was dependent on an ERalpha-positive mesenchyme, stromal cell proliferation was independent of tissue ERalpha. This latter observation suggests that the response of the stroma was due to a systemic factor induced in the ERalpha-positive hosts. To test this possibility, pieces of whole uterus from neonatal wild-type or ERalphaKO mice were grafted into syngeneic wild-type or ERalphaKO hosts. In these whole-uterus grafts, estradiol stimulated ERalphaKO uterine stroma when they were grown in wild-type hosts but not when grown in ERalphaKO hosts. The epithelium of whole-uterus ERalphaKO grafts did not respond to estrogen, regardless of the host phenotype. These observations suggest that treatment of progesterone-primed mice with estradiol stimulates production of a systemic factor that is capable of inducing uterine stromal cell proliferation and that this systemic factor is produced by an ERalpha-dependent mechanism.