Expression of nuclear progesterone receptor and progesterone receptor membrane components 1 and 2 in the oviduct of cyclic and pregnant cows during the post-ovulation period

ABSTRACT: BACKGROUND: Progesterone (P4) may modulate oviductal functions to promote early embryo development in cattle. In addition to its nuclear receptor (PR), P4 may mediate its actions through P4 receptor membrane component 1 (PGRMC1) and its relative, PGRMC2. Two successive experiments were undertaken to characterise the expression of PR, PGRMC1 and PGRMC2 in the bovine oviduct during the post-ovulation period, and to relate their expression to the presence of an embryo, the proximity of the CL and to the region of the oviduct. METHODS: In the first experiment (Exp. I), whole oviduct sections were collected from Holstein cows at Day 1.5, Day 4 and Day 5 post-ovulation (n = 2 cows per stage). The expression of PR, PGRMC1 and PGRMC2 was studied in the ampulla and isthmus by RT-PCR, western-blot and immunohistochemistry. In Exp. II, oviduct epithelial cells were collected from cyclic and pregnant Charolais cows (n = 4 cows per status) at Day 3.5 post-ovulation and mRNA expression of PR, PGRMC1 and PGRMC2 was examined in the ampulla and isthmus by real-time quantitative PCR. RESULTS: In Exp. I, PR, PGRMC1 and PGRMC2 were expressed in all oviduct samples. PGRMC1 was mainly localised in the luminal epithelium whereas PR and PGRMC2 were localised in the epithelium as well as in the muscle and stroma layers of the oviduct. The expression was primarily nuclear for PR, primarily cytoplasmic for PGRMC1 and both nuclear and cytoplasmic for PGRMC2. In Exp. II, mRNA levels for PR, PGRMC1 and PGRMC2 were not affected by either the pregnancy status or the side relative to the CL. However, the expression of PR and PGRMC2 varied significantly with the region of the oviduct: PR was more highly expressed in the isthmus whereas PGRMC2 was more highly expressed in the ampulla. CONCLUSIONS: This is the first evidence of PGRMC2 expression in the bovine oviduct. Our findings suggest that P4 regulates the functions of the bovine oviduct in a region-specific manner and through both classical and non classical pathways during the post-ovulation period.     

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.     

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.     

Localization of estrogen and progesterone receptors in the endometrium of common marmosets Callithrix jacchus

In the present study, changes in the immunohistochemical localization of endometrial estrogen receptor (ER) and progesterone receptor (PR) during various stages of the ovarian cyclicity in common marmoset, have been reported. Ovarian cyclicity was monitored by estimating plasma estradiol and progesterone. During the early follicular phase, weak ER immunolocalization was observed in the endometrial stroma. During the late follicular phase under the influence of rising estradiol levels, stromal ER localization was intense. During the luteal phase, ER localization was absent in the stroma indicating that high concentrations of progesterone suppressed ER. PR localization was not observed in the stroma during the early follicular phase, while weak staining was seen in the stroma during the late follicular phase. PR localization was maximum during the mid luteal phase. However in marmoset, endometrial ER and PR localization was restricted only to the stroma. This unique feature may be due to the characteristic reproductive profile of this nonmenstruating species and needs to be studied further. Thus it can be hypothesized that in the marmoset endometrium, steroid hormone mediated effects possibly occur directly in the stroma and are then transmitted to the epithelium by autocrine/paracrine action of growth factors and cytokines.     

Cellular turnover in the rat uterine cervix and its relationship to estrogen and progesterone receptor dynamics

Histoarchitectural changes of the uterine cervix allow its successful adaptation to different physiological conditions. In this study, we evaluated cell turnover in each cellular compartment of the uterine cervix in association with steroid hormone receptor expression in order to establish the range of physiological changes. Proliferation, apoptosis, and progesterone receptor (PR) and estrogen receptor alpha (ERalpha) expression were evaluated in cycling, pregnant, and postpartum rats. In estrus and diestrus II, ERalpha and PR expression exhibited variations according to the region evaluated. Proliferation and apoptosis showed a reciprocal pattern, the epithelium being the region with higher cell turnover. High apoptotic index (AI) in estrus was associated with the lowest ERalpha and the highest PR scores. During pregnancy, proliferation of the epithelium was the predominant event and AI was low. On Postpartum Day 1 (PPD1), proliferation decreased while apoptosis increased. As described for the estrous cycle, during pregnancy and PPD1, AI and ERalpha were negatively correlated. In the fibroblastic stroma, low proliferation was observed throughout pregnancy; however, there was a net increase in cell number because very few cells underwent apoptosis. No difference in ERalpha was observed in fibroblastic cells during pregnancy and postpartum; however, a great decrease of this receptor in the epithelial compartment was observed after delivery. Unlike cervical epithelium, PR was highly expressed in stromal cells. At term, a dramatic increase in epithelial PR was observed. While epithelial PR remained high on PPD1, a decrease was observed in muscle stroma. These results show that, in all stages studied, 1) ERalpha and PR have different patterns of expression with differential responses to signals that modulate proliferation and/or apoptosis depending on the cellular compartment, and 2) even though the epithelium is the region with the highest cell turnover, the fibroblastic and muscle stroma are active regions that have their own patterns of behavior.     

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.     

Expression of progesterone receptor membrane component-1 in bovine reproductive system during estrous cycle

Several reports suggest the participation of progesterone receptor membrane component 1 (PGRMC1) in progesterone signaling in the reproductive system. This study aimed at investigating the presence and localization of PGRMC1 in bovine ovary, oviduct and uterus, during the follicular and luteal phases of the estrous cycle. In the ovary, PGRMC1 has been detected in surface germinal epithelium, granulosa cells, theca cells and in the germinal vesicle of the oocytes at all stages of folliculogenesis. In the corpus luteum the expression of PGRMC1 was influenced by the stage of the estrous cycle. In the oviducts and in the uterus horns, PGRMC1 was immunolocalized in the luminal epithelium, in the muscle layer cells and in the endothelial cells. In the uterus, PGRMC1 was intensely localized also in the glandular endometrium. However, in the oviducts and in the uterus horns, the localization of PGRMC1 was independent on the stage of the estrous cycle and on whether evaluating the ipsilateral or the contralateral organ. In conclusion, the present immunohistochemical study showed that PGRMC1 is located in various compartments of the bovine female reproductive organs. With the exception of the corpora lutea, PGRMC1 localization showed similar pattern during different stages of the estrous cycle.     

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.     

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.     

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.     

Distribution of estrogen receptor α and progesterone receptor B in the bovine oviduct during the follicular and luteal phases of the sexual cycle: an immunohistochemical and semi-quantitative study

Abstract

The aim of our study was to determine the distribution of estrogen receptor α (ERα) and progesterone receptor B (PR-B) in the bovine oviduct during the follicular and luteal phases. Bovine oviducts from 23 animals were obtained from a local slaughterhouse. Blood samples from these animals also were taken before death to measure estrogen and progesterone levels. The serum levels of estradiol-17β and progesterone changed during the estrous cycle. Tissue distribution of ERα and PR-B was examined using immunohistochemical techniques and the results showed that ERα and PR-B were stained in nuclei of cells and could be detected in all compartments along the entire oviduct during both the follicular and luteal phases. During the follicular phase, no significant differences were found between ERα and PR-B distribution (p < 0.05), while significant differences were found between ERα and PR-B during the luteal phase (p < 0.05). We results indicated that the frequency and intensity of ERα and PR-β immunoreactivity in the oviduct of bovines varied according to the oviductal cell types and the phases of the sexual cycle.     

Relationship between cellular DNA synthesis,PCNA expression and sex steroid hormone receptor status in the developing mouse ovary,uterus and oviduct

The proliferative activities of the different cellular compartments of the developing mouse ovary, uterus, and oviduct were studied by radioautographic assessment of DNA synthesis with [³H]-thymidine labeling and by immunohistochemical staining of proliferating cell nuclear antigen (PCNA). The distributions of estrogen and progesterone receptors (ER and PR) were studied by immunohistochemical staining. The values of the PCNA positive staining indices were a little higher than that of the radioautographic labeling indices. However, linear relations were shown for the two indices. The proliferative activities were high from postnatal day 1–7 and decreased from day 14 in the different cellular compartments of the ovary. The proliferative activities were high on days 1, 3 and decreased from day 7 in the uterus and oviduct. Staining of ER and PR was very weak in the surface epithelium, stroma and large follicles of the ovary. Positive staining for ER occurred from day 14 in the uterine epithelium and from day 7 in oviductal epithelium. Positive staining for PR was observed from day 1 in both the uterine and oviductal epithelium. However, the positivity of both ER and PR occurred from postnatal day 1 in the stromal cells of the uterus and oviduct. These results suggest that the appearance of the steroid receptors differ between the different cellular compartment of the reproductive organs. The proliferative activities have an inverse relation with the expression of the steroid hormone receptors in the female reproductive organs during developmental stages. Therefore, we propose that there is an autonomous proliferation mechanism in the development of the reproductive organs or that the proliferation is moderated by factors other than steroid hormones.     

FSH促进牦牛输卵管上皮细胞分泌输卵管特异性糖蛋白的研究

为在胚胎共培养过程中添加相关激素提高哺乳动物胚胎发育的研究提供理论依据,本研究探讨了促卵泡生成素(FSH)对牦牛输卵管上皮细胞分泌特异性糖蛋白的影响。体外分离培养牦牛输卵管上皮细胞,并在细胞中添加不同浓度的FSH,作用6 h后运用荧光定量PCR分析输卵管特异性糖蛋白mRNA的表达水平,并用细胞免疫标记对其分泌输卵管蛋白的部位进行分析。结果显示,FSH的浓度为0.5-5.0μg/m L时,输卵管蛋白的表达量随着FSH浓度的上升而增加,在5.0μg/m L的FSH作用后,输卵管蛋白的mRNA表达量最高;浓度超过10.0μg/m L时,输卵管蛋白基因的表达量降低。结果表明,FSH具有促进输卵管上皮细胞分泌输卵管特异性糖蛋白的作用,且具有剂量依赖性,其最佳作用浓度为5.0μg/m L,输卵管蛋白主要由细胞质分泌。