The effect of dihydrotestosterone on the estrogen-induced cytodifferentiation of the chick oviduct

The androgenic effects on the estrogen-induced cytodifferentiation of the chick oviduct epithelium were investigated. Dihydrotestosterone was shown to have an effect on the organization of stromal cells. Since these cells contained androgen receptor (AR), it is reasonable to assume an involvement of androgens in the differention and functioning of these cells through a direct action. Immunohistochemical analysis revealed a wide distribution of AR. AR was shown to be expressed in both the endothelial and smooth musle cells of blood vessels. In the immature oviduct AR was located in the epithelial, mesenchymal and mesothelial cells. In the differentiating oviduct, whether induced by exogenous estrogen or normally by endogenous hormones, AR was also expressed by the tubular gland cells. Dihydrotestosterone alone had no effect on the morphology of the immature oviduct, suggesting the involvement of the determinants of differentiation in the action of androgen together with estrogen.     

Postnatal mammary ductal growth: three-dimensional imaging of cell proliferation,effects of estrogen treatment,and expression of steroid receptors in prepubertal calves

Cows may provide insights into mammary development that are not easily obtained using mouse models. Mammary growth in control and estrogen-treated calves was investigated to evaluate general patterns of proliferation and relationship to estrogen receptor (ER) expression. After in vivo labeling with bromodeoxyuridine (BrdU), serial histological sections of mammary tissue were used to generate three-dimensional reconstructions. BrdU-labeled cells were present throughout the highly branched terminal ducts. ER and progesterone receptors (PR) were colocalized in nuclei of ductal epithelial cells. However, basal cells and epithelial cells that were located in the central region of epithelial cords and those that lined the lumen of patent ducts were ER- and PR-negative, as were stromal cells. Cells along the basal portion of the epithelium were not myoepithelial. ER in mammary epithelial cells but not stromal cells is analogous to patterns in human breast but contrasts with localization in murine mammary gland. After estrogen stimulation, 99% of BrdU-labeled (and Ki67-labeled) epithelial cells were ER-negative. Data suggest that proliferation in response to estrogen treatment was initiated within ER-positive epithelial cells of the developing mammary gland and the signal was propagated in paracrine fashion to stromal elements and ER-negative epithelial cells.     

Progesterone receptor is constitutively expressed in chicken intestinal mesothelium and smooth muscle

We have previously shown that progesterone receptor (PR) is expressed in the mesothelium of the chick oviduct and ovary and in the smooth muscle cells of the oviduct and the bursa of Fabricius. Here, we investigated the presence of PR in different parts of the peritoneum and abdominal organs using an immunohistochemical staining based on monoclonal antibodies against chicken PR. In 4-week-old sexually immature chicks, PR expression was located in the mesothelial cells of different parts of the peritoneum, in a thin layer of muscle cells of the ileum and throughout the muscle tissue of the colon and cloaca. In chicks of the same age treated with estrogen, PR was demonstrated similarly in the peritoneum and in the smooth muscle cells of the ileum, colon and cloaca. Using 25-week-old mature chickens, PR was also detected in identical tissues. Immunoblotting of the cloacal cytosol revealed the B form, but no A form of PR, both of which were found in the oviduct samples. Muscle cells of the duodenum and jejunum were not found to contain PR. Estrogen treatment was not needed to stimulate the production of PR in any of the tissues examined. We therefore conclude that the B form of PR is constitutively expressed in the mesothelial cells in different parts of the peritoneum and also in the smooth muscle cells of the ileum, colon and cloaca.     

Sex-steroid-sensitive stromal cells and oviduct differentiation

The chick oviduct differentiates during sexual maturation before the age of 20 weeks. In the present work we used immunohistochemistry to study sexual maturation associated progesterone receptor (PR) expression in the chick oviduct as an indication of progesterone sensitivity. Since the PR is estrogen inducible protein, its expression also reflects the effects of endogenous estrogens. Thus PR expression can be used as a marker for action and sensitivity of cells to these sex steroids. In the luminal epithelium and mesothelium (peritoneal epithelium) the PR was expressed in high concentrations from the time before hatching (the constitutive PR). The PR was not detectable in stromal cells of immature chicks. At the age of 7-10 weeks the PR was detected in submucosal but not in mucosal stromal cells (the inductive PR). The appearance of these PR-expressing cells was associated with an increase in luminal epithelial cell proliferation. At the age of 14-16 weeks the mucosal plicae increased in height and the PR-expressing stromal cells were seen in the center of these mucosal plicae. There were also areas in the mucosal plicae where a large number of stromal cells expressing the PR were seen in the mucosal layer. Thereafter the size of the oviduct increased rapidly and the gland formation commenced. In the fully matured oviduct (over 18 weeks of age) virtually all stromal cells both in mucosa and submucosa expressed the PR. It is concluded that the PR expression in the luminal epithelium and mesothelium was constitutive (independent of sexual maturation). In stromal cells this was expressed during sexual maturation (probably induced by endogenous estrogen) and was associated with histological changes in the oviduct. We propose that direct effects of estrogen and progesterone in the oviduct growth and glandular formation are mediated through these stromal cells.     

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.     

Immunoelectron microscopic localization of progesterone receptor in the chick oviduct

A peroxidase-anti-peroxidase (PAP) method using polyclonal anti-PR antibodies was used to localize progesterone receptor (PR) electron microscopically in the chick oviduct. The immunoreaction precipitate indicating PR was localized inside the nuclei of epithelial, glandular and stromal cells. In the estrogen withdrawn oviduct cytoplasmic immunoreaction precipitate was not seen. Inside the nucleus unoccupied PR was localized mainly like the heterochromatin. As visualized by the PAP technique, the localization of PR was not systematically changed after progesterone administration. In conclusion, we suggest that progesterone receptor in the chick oviduct is an intranuclear protein.     

Localization and regulation of estrogen, progestin and androgen receptors in the seminal vesicle of the rhesus monkey

We have used monoclonal antibodies against the estrogen (E), progestin (P) and androgen (A) receptors (R) to study receptor localization and regulation in the seminal vesicles of rhesus monkeys under different hormonal conditions. The antibodies caused substantial shifts of the appropriately labeled receptors on sucrose gradients. ER levels were lower in intact males than in immature, castrate, and estrogen-treated castrates. With immunocytochemistry, ER were detectable only in stromal and smooth muscle cells, not the epithelium. The number of ER-positive stromal cells was significantly lower in intact males than in immature, castrate, and estrogen-treated castrates, and low in a DHT-treated castrate animal. Androgen receptors were localized in epithelial as well as stromal and smooth muscle cells, and the number of AR-positive stromal cells was highest in intact adults and lowest in castrated and immature animals. Estrogen treatment at the time of castration induced PR in the ER-positive stromal cells, prevented a decline in the number of AR-positive stromal cells, and caused stromal hypertrophy. In summary, in the seminal vesicle, as in the prostate, ER is restricted to the fibromuscular stroma, is suppressed by androgens, and can mediate induction of PR on estrogen treatment. Androgen receptors are present in epithelial as well as stromal and smooth muscle cells, but variations in hormonal state appear to affect regulation of AR more in the stroma than the epithelium.     

Hormone-dependent changes in A and B forms of progesterone receptor

The influence of different estrogen and/or progesterone treatments on concentrations of A and B forms of progesterone receptor (PR-A and PR-B) in the different cell types of chick oviduct was studied. A semiquantitative immunohistochemical assay for cellular PR concentrations was developed using a computer-assisted image analysis system. The staining intensity of nuclear PR in the basal layer of epithelial cells, glandular, smooth muscle and mesothelial cells was analysed separately using two monoclonal antibodies, PR6 and PR22. The measured concentrations of PR varied between different cell types and from cell to cell. A significant decrease in PR concentration, as noted by a decrease in staining intensity, was observed in all cell types studied 2 or 6 h after a single injection of progesterone with or without simultaneous estrogen administration. The decrease was also verified with immunoblotting and an immunoenzymometric assay (IEMA) for chicken PR. After down-regulation the concentration of PR recovered to the control level within 48 h after progesterone or estrogen administration. Estrogen administration alone was observed to cause changes in the concentration of PR-A only, having little or no effect on PR-B concentration depending on the cell type studied.

These findings indicate that estrogen and progesterone cause cell-specific changes not only to the total concentration of PR but also to the cellular ratio of PR-A and PR-B.     

Sexual maturation-associated and estrogen-induced progesterone receptor expression in the bursa of Fabricius

The expression of the progesterone receptor (PR) was studied in the chicken bursa of Fabricius (BF) in both sexes from the time of hatching until the bursal involution. Steroid binding studies, immunohistochemistry, and autoradiography were used to characterize and localize the receptor. Three different polyclonal antibodies (IgG-RB, IgG-G3, and IgG-RB2) directed against the chick oviduct progesterone receptor were used for the studies. With immunohistochemistry, no receptor-positive cells were detected in the bursae of young chicks. The first receptor-positive cells were occasionally seen at the age of 10 wk in the frozen sections, not in the paraffin sections. In older female chicks, the staining became more abundant. In males, the PR was expressed only after estradiol treatment. The staining was located in the nuclei of the subepithelial and the interfollicular cells, which were probably mesenchymal in origin. The bursal epithelium and the lymphocytes were not stained. By using a combined technique of autoradiography and immunohistochemistry, we were able to demonstrate that the same cells also concentrated tritiated ORG 2058 (a specific synthetic progestin) in their nuclei. In steroid binding studies with tritiated ORG 2058, the receptor concentration after the age of 10 wk was 50 to 120 fmol/mg protein. Low-level ORG 2058 binding was also detected in young chicks of both sexes before the age of 10 wk. The progestin-binding molecule resembled the progesterone receptor of the chick oviduct in molecular size (studied with HPLC) and binding properties. The PR expression in the BF was preceded by the expression of PR in the oviduct stromal cells and by an increase in oviduct epithelial proliferation, indicating the BF is affected by factors associated with sexual maturation. It is concluded that the subepithelial and the interfollicular stromal cells in the BF, but not the epithelial or follicular cells, are estradiol-sensitive in both sexes immediately after hatching. The endogenous estrogens, however, are not able to induce PR until after the onset of sexual maturation, and only in females. This implies that estrogen and progesterone may affect the structural organization of the BF through the stromal cells, but probably not before the onset of puberty.     

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.     

Sex steroid sensitivity of developing bursa of Fabricius

Sex steroid sensitivity of the bursa of Fabricius (BF) was studied from the early embryonic time until its regression. Expression of progesterone receptor (PR) served as a dual marker: first, as a marker for progesterone sensitivity and second, as a marker for estrogen action, since it is an estrogen-induced protein. The progesterone binding molecule in the bursa was characterized by different chromatography methods and by steroid binding studies. We showed that it fulfils the criteria of a progesterone receptor by binding, structural and immunological properties. With immunohistochemistry and with the combined techniques of immunohistochemistry and autoradiography we demonstrated two cell types which express the PR: smooth muscle cells surrounding the BF and stromal cells located under the bursal epithelium and between the lymphoid follicles. The epithelium and the cells inside the lymphoid follicles were negative. Using immunoelectron microscopy the PR-expressing stromal cells were shown to be fibroblasts. The cloacal mesenchyme, from which the BF develops, was shown to be sensitive to exogenous estrogen very early during the embryonic time. The mesenchyme around and inside the developing BF reached estrogen sensitivity a few days later. The estrogen-sensitive mesenchymal cells were first seen surrounding the bursal primordium and later in the center of the plicae. During a natural sexual maturation without exogenous estradiol an expression of the PR was detected much later, at the age of 10-12 weeks after hatching. This expression correlates with the onset of the bursal regression and with the increase of the sex steroid levels in the blood. In the oviduct stroma PR was undetectable before the onset of sexual maturation. In the oviduct stroma PR becomes detectable a few weeks earlier than in the bursa.     

Progesterone receptor concentration differences in the chick oviduct cells and apparent down-regulation by ligand. A semiquantitative immunohistochemical study

A semiquantitative immunohistochemical technique was developed for identification of chick progesterone receptor (PR). The mouse monoclonal antibody PR6 was used. The nuclear PR concentration was analyzed with Leitz Orthoplan MPV-3 light microscope. The target tissue was chick oviduct, with epithelial, glandular, mesenchymal, smooth muscle and peritoneal cells analyzed separately. PR concentration varied between different cell types and also from cell to cell within a single cell type. A significant decrease of PR concentration, as noted by decrease in staining, was also observed in all studied cell types, 6 h after a single injection of progesterone. This technique allows for histological identification of biochemical events that should help lead to the understanding of the role of PR changes in a variety of experimental situations.     

Localization of estrogen receptor α and progesterone receptor B in bovine cervix and vagina during the follicular and luteal phases of the sexual cycle

Abstract

The localization and distribution of estrogen receptors (ERα) and progesterone receptors (PR-B) in the cervix and vagina of sexually mature bovines during the follicular and luteal phases of the sexual cycle were studied using immunohistocehmistry. The estrous cycle stage of 23 Holstein bovines was assessed by gross and histological appearance of ovaries and blood steroid hormone values. Tissue samples from cervix and vagina were fixed in 10% formaldehyde for routine histological processing. Nuclear staining for ERα and PR-B was observed in the epithelial cells of the surface epithelium, stromal cells and smooth muscle cells. Generally, in the cervix, ERα immunoreactivity was more intense in the epithelial and smooth muscle cells during the follicular phase and in the epithelial cells during the luteal phase (p < 0.05). PR-B immunoreactivity was more intense in the epithelial and smooth muscle cells than in the superficial and deep stromal cells during the follicular and luteal phases (p < 0.05). In the vagina, ERα and PR-B immunoreactivities were more intense in the epithelial cells than in the connective tissue cells and smooth muscle cells during the follicular and luteal phases (p < 0.05). These results indicated that the frequency and intensity of ERα and PR-B immunoreactivity in the cervix and vagina of bovines varied according to the cervical and vaginal cell types and the phases of the sexual cycle.     

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.     

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.     

Distribution of estrogen and progesteron receptors in the uterus: an immunohistochemical study in the immature and adult pseudopregnant rabbit.

In order to clarify the distribution and content of estrogen (ER) and progesteron receptors (PR) under changing hormonal influences within the various cell populations of the uterus (glandular and luminal endometrial epithelium, stroma, myometrium), immunohistochemical determinations using specific monoclonal antibodies were made. To correlate the immunohistochemical findings with peripheral hormone levels and specific tasks of the endometrium, 17 beta-estradiol and progesterone serum levels were measured and cell proliferation determined by use of BrdU-labelling-immunohistochemistry. At the subcellular level ER and PR were located exclusively in the cell nuclei of female rabbits, which were either immature and lacking any peripheral hormone levels or were pseudopregnant (d0-d8 p.hCG). In the immature rabbits a general faint ER and PR immunostaining was found. In addition to a general increase in ER and PR in all cell populations estrous rabbits (d0 p.hCG) showed a significant rise of ER in the epithelial cells and of PR in the myometrium. Within the epithelial cells and the myometrium the ER dropped heavily within a few days of pseudopregnancy. The PR, however, increased sharply during the first two days of pseudopregnancy and decreased gradually following d4 p.hCG. A close relationship was observed between the high PR content and the proliferation rate of the epithelial cells on d2 p.hCG. In spite of the more rapid decrease of ER compared with PR, the glandular epithelium retained positive immunostaining. In the stroma the ER and especially PR content did not change significantly during the course of pseudopregnancy suggesting that some of the well-known differentiation events in the luminal epithelium may be mediated by the stroma.