Nuclear and cytosol steroid hormone receptor levels in the myometrium during pregnancy in the sheep

Methods for the measurement of nuclear receptors for oestradiol and progesterone in sheep myometrium have been established. Scatchard analysis of nuclear receptors gave dissociation constants (nM) on days 0 and 112 of pregnancy of 1.95 and 1.76 for oestradiol and 4.20 and 4.12 for progesterone, respectively. The concentration of nuclear and cytosol high-affinity receptors for oestradiol and progesterone has been determined during the first 112 days of gestation; and possible roles of oestradiol and progesterone in the regulation of myometrial hypertrophy and function are discussed.The rate of hypertrophy, as measured by changes in protein : DNA and RNA : DNA ratios, was maximal during days 56–84 and declined thereafter. The level of cytosol oestradiol receptor decreased rapidly between day 0 (oestrus) and day 28, and then more slowly between days 28 and 112, when expressed per unit of cytosol protein. However, when expressed per unit of DNA the level increased after day 28 to a peak at day 84, then decreased markedly to day 112. The level of nuclear oestradiol receptor declined from a peak at oestrus to very low levels on days 56–84, then increased markedly to day 112. The concentration of cytosol progesterone receptor declined from a peak at oestrus to low levels on days 28–112. The changes in the level of nuclear progesterone receptor were more complex; the level increased between oestrus and day 28, declined markedly to day 56, then increased again to high levels on days 84–112.The data suggest that oestradiol does not have any important role in stimulating myometrial growth, since the level of nuclear receptor for oestradiol was low when the rate of hypertrophy was maximal. The changes in nuclear progesterone receptor level were less clearly separated, temporally, from changes in rate of hypertrophy, and the possible influence of progesterone on myometrial growth remains unclear.     

Uterine oxytocin receptors in cyclic and pregnant cows.

Binding of [3H]oxytocin to uterine subcellular preparations ('oxytocin receptor concentrations') was measured in uterine tissue of heifers and multiparous dairy cows at various stages of the oestrous cycle and during early pregnancy. A method for the assay of ovine uterine oxytocin receptors was optimized for use on bovine tissue. Oxytocin receptor concentrations were increased in cyclic animals around the period of luteolysis and oestrus, rising on Day 15 in endometrium and on Day 17 in myometrium while pregnant animals showed no comparable rise. Receptor concentrations then declined on Day 3 after oestrus in myometrium and on Day 5 in endometrium. Some cyclic animals did not show the expected rise in receptors in the late luteal phase; these animals had abnormally high progesterone concentrations for this stage of the cycle. In animals slaughtered on Day 18 after oestrus and/or insemination which had low oxytocin receptor levels, plasma progesterone concentrations were consistently high; while all animals showing the late luteal phase elevation in receptor values had low progesterone concentrations. Oxytocin receptor and progesterone concentrations were negatively correlated (P less than 0.05). These data support the hypothesis that oxytocin receptor level is a key factor in the process of luteolysis in cattle and that in pregnancy there is suppression of uterine oxytocin receptor at the expected time of luteolysis. We suggest that uterine oxytocin receptor levels are partly controlled by circulating steroid hormones and are suppressed during early pregnancy.     

Endometrial protein secretion during early pregnancy in entire and ovariectomized ewes

The secretion and synthesis of protein in vitro by explants of endometrium were examined in entire ewes during the first 10 days of the oestrous cycle and during an equivalent interval in ovariectomized ewes which received injections of oestradiol and progesterone. The schedule of steroid injections given was designed to simulate endogenous ovarian secretion of progesterone during the luteal phase before oestrus, of oestradiol around oestrus and of progesterone during the luteal phase after oestrus. The rate of protein synthesis and tissue RNA:DNA and protein:DNA ratios in intercaruncular and caruncular endometrium were generally higher in entire than in ovariectomized ewes. In ovariectomized ewes oestradiol increased these activities at 2-4 days after oestrus, whereas progesterone preceding oestradiol caused increases at oestrus, but not thereafter. In entire ewes and in ovariectomized ewes receiving the full steroid treatment regimen, protein secretion was high at oestrus and declined markedly during the next 4-6 days. In ovariectomized ewes not receiving progesterone before oestradiol, secretion increased between 4 and 6 days after oestrus, or during the equivalent stage of treatment in ewes which did not show oestrus. The omission of this progesterone did not modify secretion by caruncular endometrium. Oestradiol increased protein secretion by both tissues. The data suggest that progesterone given before oestradiol (or its equivalent in entire ewes) inhibits the secretion, at about 4-7 days after oestrus, of uterine proteins which may impair embryo development in ovariectomized ewes which do not receive this progesterone.     

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.     

Adaptations to reduction of endometrial surface available for placental development in sheep

On Day 5 of pregnancy, before the blastocyst migrates to the uterus, one uterine horn was ligated to restrict the trophoblast to the lumen ipsilateral to the corpus luteum. The numbers of placentomes (caruncles and cotyledons) were reduced by half, but neither at 120 nor at 140 days of pregnancy (term 147 days) did the weights of placentae and fetuses of treated ewes differ significantly from those of control ewes. Amongst uterus-ligated animals prepared for chronic study, the rate of uterine blood flow (electromagnetic flow transducer, ml/min) to the pregnant horn was higher than in control ewes, as was the concentration of progestagens in maternal peripheral blood. There may be a compensatory response that causes hypertrophy of placentomes and that increases blood flow to the uterine horn containing placental tissue.     

Progesterone and oestrogen receptors in the female genital tract throughout pregnancy in tammar wallabies

The tammar, Macropus eugenii, is a monovular macropodid marsupial which has a post-partum oestrus and an 11 month embryonic diapause. Progesterone and oestradiol cytosol receptors were measured by Scatchard analyses and single point analysis in the lateral vagina, endometrium and myometrium of the gravid and contralateral non-gravid uterus throughout pregnancy, immediately after parturition and during seasonal reproductive quiescence. In endometrial tissues, both progesterone and oestradiol receptors doubled in concentration in both gravid and non-gravid uteri between day 0 and day 5 of pregnancy, coinciding with previously described peak values in peripheral plasma progesterone and oestrogen. Receptor concentrations in endometrial tissue during seasonal quiescence were not significantly different from those immediately after reactivation. After day 12 of pregnancy, downregulation of both progesterone and oestradiol cytosolic receptors occurred concomitant with the increase in progesterone in the peripheral plasma. However, there was a unilateral increase in oestradiol receptor concentrations in endometrium obtained from the non-gravid uterus between day 25 of the 26.5 day gestation and immediately after parturition. Myometrial receptor concentrations mirrored those of the endometrium but were lower. Concentrations of progesterone receptor in the lateral vaginae were at the lower limit of detection, while the oestradiol cytosol receptor concentrations were even lower in this tissue. Thus, the steroid receptor concentrations provide another example of local unilateral endocrine responses in the reproductive tract of the tammar. These results also indicate that the downregulation of progesterone and oestradiol receptors that occurs in both uteri in mid- and late-pregnancy is selectively and locally reversed before parturition in the non-gravid endometrium in response to the local effects of follicular oestradiol from the ipsilateral ovary.     

Expression and localization of nodal in bovine oviduct and uterus during different functional stages of oestrus cycle and pregnancy

Members of TGF-β superfamily play a major role in the endometrial changes involved in the establishment and maintenance of pregnancy. Their deregulated expression and action could lead to absolute or partial failure of embryo implantation. Nonetheless, the precise function and mechanism of many of these cytokines remain unclear. Nodal, a transforming growth factor beta (TGF-β) superfamily member, was characterized in the human and rodent uterus and implicated in the tissue remodeling events during menstruation and embryo implantation. In order to study its possible role in the cattle reproductive process, we have analyzed Nodal expression pattern and localization in the oviduct and uterine horn during the oestrus cycle and early pregnancy (day 20). Nodal was detected both in oviduct and uterus during either the oestrus cycle or pregnancy; however, it shows a differential expression profile in the uterine horn at dioestrus and pregnancy, decreasing 1.5 and 1.4 folds in comparison with oestrus. Nodal immunostaining intensity was observed in stromal and in epithelial cells of the surface and the glandular epithelium. The staining pattern correlates with the RT-qPCR expression profile. This work is the first to evidence the presence of Nodal in the bovine reproductive tract; our data suggest that Nodal is a novel cytokine that would be involved in the remodelling occurring in the endometrium of cattle during the oestrus cycle and in the embryo implantation. The identification of new molecules that participate in endometrium cycling and/or pregnancy may be useful for predicting the ability of the uterine tissue to establish and maintain pregnancy or for detecting the infertility processes. These results highlight Nodal as a possible novel marker of the fertility process, nevertheless further studies should be done to determine its role in the reproductive system.     

Characterization of uterine sex steroid receptors in the pig and their variation during the oestrous cycle

The present study establishes and validates an in vitro binding and exchange assay for tissue receptors for oestradiol (E) and progesterone (P) in pig uterus. Both hormones bound to specific cytoplasmic (Rc) and nuclear (Rn) receptor proteins with high affinity. The relative concentrations of the receptors were measured in dissected samples from endometrium and myometrium obtained at late prooestrus, oestrus, and luteal phases of the oestrous cycle. The Scatchard analysis of the oestradiol and R 5020-receptor complex displayed linearity and indicated a single class of high affinity, low capacity binding sites. Significant variations were seen in the binding of E and P to their cytosolic and nuclear receptors, following the changes in the circulating levels of the hormones in blood plasma during the oestrous cycle. Both tissue components, i.e. endometrium and myometrium followed a similar pattern when related to the stage of the oestrous cycle considered. The ERc increased from prooestrus, reaching a maximum at standing oestrus, thereafter decreasing. The concentration of ERn increased from prooestrus towards the early luteal phase, with a significant reduction by day 8 of the cycle. The amounts of PRc were maximal at standing oestrus, remaining high during the early luteal phase, while the PRn showed a linear increase from oestrus onwards throughout the luteal phase.     

Placental morphology in African antelopes and giraffes

Delivered placentas from nineteen antelopes and five giraffes and placentas from five antelopes dead during parturition and eight antelopes dead during advanced pregnancy were morphologically examined. Placental length and weight, arrangement, number and size of cotyledons, and other features were recorded wherever the conditions of placentas permitted.

Cotyledon rows corresponded to the caruncle rows on uterine walls. The number of rows was usually four, although in Hippotraginae it was six to eight and in Reduncinae it was two. Numbers of cotyledons matched or were lower than total numbers of caruncles in the uteri of most species. In antelopes with uterus duplex [Hippotraginae and wildebeests (Alcelaphinae)] and in other members of the Alcelaphinae subfamily, the placenta occupied only one uterine horn and the numbers of cotyledons corresponded to the numbers of caruncles in one uterine horn. Amniotic “manes” and “plaques” were found in some placentas.     

Dominance of type 1 angiotensin II receptor in the nonpregnant and pregnant bovine uterus

The present study was undertaken to characterize, determine and localize angiotensin II receptors in the nonpregnant and pregnant bovine uterus. In addition, the concentration of active renin, which is responsible for the generation of angiotensin, was determined. Autoradiography and angiotensin II receptor binding studies showed that all compartments of the bovine uterus contained high concentrations of angiotensin II receptors. In general, the type 1 angiotensin II receptor (AT1) predominated over the AT2 receptor. In the endometrium, the highest density was found in the caruncles and the AT1 receptor was always predominant. The density of angiotensin II receptors in the endometrium increased at the beginning of pregnancy, but decreased and reached values similar to those in nonpregnant animals near term. In the myometrium, the density of angiotensin II receptors was highest at or near the endometrial-myometrial junction. In this area, the predominant type of angiotensin II receptor in the uterus of cyclic cows varied, whereas the AT1 receptor always predominated during pregnancy. Non-AT1 and non-AT2 binding sites were found in the same locations as the angiotensin II receptors, but at lower densities. With the exception of the pregnant endometrium, all compartments contained higher active renin concentrations than found in plasma, indicating local synthesis of renin. This study demonstrates a difference in the expression of types of angiotensin II receptor in the bovine uterus compared with other species. The high densities of angiotensin II receptors localized in several important areas imply that the renin-angiotensin system participates in regulation of growth and tissue function in the bovine uterus.     

Expression of the orexin system in the porcine uterus,conceptus and trophoblast during early pregnancy

Orexin A and B are hypothalamic peptides derived from the prepro-orexin (PPO) precursor. Orexins stimulate food intake and arousal. Those peptides bind and activate two G protein-coupled receptors: orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). Numerous authors have suggested that orexins play an important role in the regulation of the reproductive functions. The objective of the present study was to analyse the presence of and changes in the gene and protein expression pattern of the orexin system in the porcine uterus, conceptus and trophoblast (chorioallantois) during early pregnancy. In the endometrium, the highest PPO and OX1R gene expression was detected on days 15 to 16 of gestation. The OX2R mRNA content in the endometrium was higher on days 10 to 11 and 15 to 16 than on days 12 to 13 and 27 to 28. In the trophoblasts, PPO gene expression was higher on days 30 to 32 than on days 27 to 28. The highest PPO protein content in the endometrium was noted on days 12 to 13. The highest OX1R protein content in the endometrium was detected on days 10 to 11, whereas OX2R protein on days 15 to 16. In the trophoblasts, PPO and OX1R protein levels were more pronounced on days 27 to 28 than on days 30 to 32, but OX2R expression was higher on days 30 to 32. The expression of PPO, OX1R and OX2R was different in the conceptuses and trophoblasts during early pregnancy. Local orexin production and the presence of the specific orexin receptors suggest that the orexin system may participate in the control of porcine reproductive functions by exerting endocrine and auto/paracrine effects on the uterus, conceptuses and trophoblasts during early pregnancy. This study provides the first evidence for the presence of orexins and their receptors in the uteri, conceptuses and trophoblasts in pigs during early pregnancy. The local orexin system is dependent on the stage of pregnancy.     

Regulation of calcitonin gene-related peptide receptors in the rat uterus during pregnancy and labor and by progesterone.

Calcitonin gene-related peptide (CGRP) is a potent smooth muscle relaxant in a variety of tissues. We recently demonstrated that CGRP relaxes uterine tissue during pregnancy but not during labor. In the present study we examined whether uterine (125)I-CGRP binding and immunoreactive CGRP receptors are regulated by pregnancy and labor and by sex steroid hormones. We found that (125)I-CGRP binding to membrane preparations from uteri was elevated during pregnancy and decreased during labor and postpartum. Changes in immunoreactive CGRP receptors were similar to the changes in (125)I-CGRP binding in these tissues, suggesting pregnancy-dependent regulation of CGRP receptor protein. CGRP receptors were elevated by Day 7 of gestation, and a precipitous decrease in these receptors occurred on Day 22 of gestation prior to the onset of labor. Both (125)I-CGRP-binding and immunofluorescence studies indicated that CGRP receptors were localized to myometrial cells. Hormonal control of uterine CGRP receptors was assessed by the use of antiprogesterone RU-486, progesterone, and estradiol-17beta. RU-486 induced a decrease in uterine CGRP receptors during pregnancy (Day 19). On the other hand, progesterone prevented the fall in uterine CGRP receptors at term (Day 22). In addition, progesterone also increased uterine CGRP receptors in nonpregnant, ovariectomized rats, while estradiol had no effects. These hormone-induced changes in uterine CGRP receptors were demonstrated by (125)I-CGRP-binding, Western immunoblotting, and immunolocalization methods. These results indicate that CGRP receptors and CGRP binding in the rat uterus are increased with pregnancy and decreased at term. These receptors are localized to the myometrial cells, and progesterone is required for maintaining CGRP receptors in the rat uterus. Thus, the inhibitory effects of CGRP on uterine contractility are mediated through the changes in CGRP receptors and may play a role in uterine quiescence during pregnancy.     

Secretory proteins and growth factors of the baboon (Papio anubis) uterus: potential roles in pregnancy.

The primate endometrium undergoes distinct morphological changes during the menstrual cycle. These alterations are regulated by the steroid hormones, estrogen and progesterone. Several lines of evidence suggest that some of these hormonally induced changes may be modulated by growth factors. Our studies have focused on characterizing the secretory activity of the uterine endometrium associated with these hormonally regulated morphological changes during the menstrual cycle and pregnancy in the baboon. Additionally, we have also attempted to study the regulation of specific growth factors and their receptors. In this review we present evidence to indicate that growth factor receptors for insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF), and secretory proteins, insulin-like growth factor binding protein-1 (IGFBP-1) and retinol binding protein (RBP), which are present in the glandular epithelium during the menstrual cycle, undergo cell-specific changes in gene expression at the implantation site during pregnancy. We postulate that these alterations in growth factor receptor and secretory protein expression are conceptus modulated and may play important regulatory roles during trophoblast invasion and decidualization.     

Spatial and molecular aspects of estrogen and progesterone receptor expression in human uteri and uterine carcinomas

The expression of steroid hormone receptors as molecules reflecting processes of development and differentiation in the human uterine tissue was analysed in a spatial distinct fashion: tissue samples were excised at the fundus and at different, spatially distinct positions of the uterus. They were analysed for concentrations of cytosolic estrogen and progesterone receptors in supernatants from frozen sections using an isoelectric focusing technique. The spatial and molecular distinct, qualitative and quantitative pattern of their expression in the human uterus and uterine adenocarcinomas were studied by sectioning tissue sample from the functionalis through the basalis of the endometrium until reaching deep myometrial parts of the tissue: (1) Specific spatial patterns of estrogen and progesterone receptor levels were detectable throughout the menstrual cycle. (2) For proliferative endometrium from the functionalis to the basalis of the endometrium, the content of both cytosolic receptor species increased up to 6-fold. (3) Differences detectable were less pronounced in the myometrial part of the tissue. (4) Differences of steroid receptor concentrations measured in the endometrium at different uterine positions were highest between fundus and corpus of the endometrium. (5) Maximal differences were detectable around ovulation. (6) After secretory transformation of the organ, specific patterns were still detectable, however quantitative differences were less pronounced. (7) Additionally, quantitative differences measurable were accompanied by variations of molecular properties of the progesterone receptor as demonstrated in an isoelectric focusing gel. (8) In endometrial adenocarcinomas, not only significant quantitative alterations in steroid receptor content were measured, but also a significantly changed spatial pattern of receptor concentrations, also a change of the molecular properties of the progesterone receptor was resolved if these tumor parameters were compared to those detected in the normal tissue of the same organ surrounding the tumor.     

Local increase of ovarian steroid hormone concentration in blood supplying the oviduct and uterus during early pregnancy of sows

Countercurrent transfer in the ovarian vascular pedicle elevates the concentration of steroid hormones in blood supplying the oviduct and periovarian part of the uterus during the estrous cycle in the pig. This study was conducted to determine whether during early pregnancy the arterial blood supply to the oviduct and uterus carries greater concentration of steroid hormone than systemic blood. The concentration of ovarian steroid hormones (progesterone, estradiol-17 beta, estrone, androstenedione and testosterone) was measured in 40 gilts on Days 12, 18, 25 or 35 of pregnancy. Silastic catheters were inserted: a) into the jugular vein, b) into the branch of uterine artery close to the ovary (proximal to the ovary) and c) into the branch of the uterine artery close to the cervix (distal to the ovary). On the day following surgery simultaneous blood samples from cannulated vessels were collected every 20 min for 3 hours. The concentration of steroid hormones was determined by radioimmunoassay. The mean concentrations of studied hormones in branches of the uterine artery proximal and distal to the ovary were significantly greater than in the jugular vein (P < 0.001) by 18 to 69% and 7 to 31%, respectively. The concentrations of hormones in proximal and distal to the ovary branch of the uterine artery were also significantly different (P < 0.001). The increase in concentrations of the measured hormones did not differ considerably between investigated days of pregnancy. It is concluded that during maternal recognition of pregnancy, formation of the corpus luteum of pregnancy, implantation of the embryo and the placenta elongation the oviduct and uterus are supplied with locally elevated concentration of steroid hormones compared to systemic blood.     

The influence of exogenous steroid hormones on steroid receptors, uterine histological structure and the bacterial flora of the normal bitch.

Oestrogen (ER) and progesterone (PR) receptors have been shown to vary in both concentration and distribution during the oestrous cycle of the bitch, influenced by the normal changes in endogenous reproductive hormones. The influence of exogenous steroid hormones on steroid receptors and the histological structure of the uterus was studied in two groups of parous Beagle bitches. Group A (n = 6) were treated with progesterone (P4) in oil i.m. (3 mg/kg) in late metoestrus on the day that peripheral plasma P4 concentrations were first identified as <10 ng/ml, and subsequently once weekly on three other occasions. Group B (n = 6) were treated with a single i.m. injection of MPA (50 mg, 4.2-5.6 mg/kg) following the same protocol. Full-thickness uterine wall biopsies were obtained from the mid part of one horn 2-7 days after the last (fourth) injection of P4 or MPA. During the subsequent oestrus, when peripheral plasma P4 concentrations were between 8 and 10 ng/ml, each bitch in both groups (n = 12) received a single injection of oestradiol benzoate (ODB) in oil i.m. (7.5 mg, 0.63-0.84 mg/kg). All bitches had an ovariohysterectomy 7 days later. Full-thickness uterine wall samples were obtained from the mid part of the intact horn and other parts of the uterus. Swabs were taken from the uterine lumen for bacteriological examination; all were sterile. Tissue samples were sectioned and examined for evidence of lesions, and stained for ER and PR receptors using an immunocytochemical method. The immunoreactivity was scored semiquantitatively, incorporating both the intensity and distribution of specific staining of the receptors using a simplified histoscore (H-score). At the time of ovariohysterectomy, fluid had accumulated in the isolated section of the uterine horn distal to the point of biopsy; the volume was greater in the MPA-treated bitches. There was also evidence in some sections of histological changes in the endometrium. Variations in the expression of both ER and PR were seen between bitches, which may have been due to some not being in mid-metoestrus at the time of treatment. In general, ER scores were low after P4 and MPA treatment, but following ODB there was a significant (P<0.05) increase in ER expression in all parts of the endometrium. PR scores were zero in the glandular epithelium of all 12 bitches after P4, MPA and ODB treatment, whereas in the other parts of the endometrium they were generally moderate to high. Following treatment with ODB, PR generally increased in the three regions of the endometrium where PR were present. The study shows that ER and PR distribution and expression in the endometrium of bitches can be modified by P4, MPA and ODB, with evidence of individual variation.