Expression of PIK3IP1 in the murine uterus during early pregnancy

The ovarian steroid hormones, estrogen (E2) and progesterone (P4), are essential regulators of uterine functions necessary for development, embryo implantation, and normal pregnancy. ARID1A plays an important role in steroid hormone signaling in endometrial function and pregnancy. In previous studies, using high density DNA microarray analysis, we identified phosphatidylinositol-3-kinase interacting protein 1 (Pik3ip1) as one of the genes up-regulated by ARID1A. In the present study, we performed real-time qPCR and immunohistochemistry analysis to investigate the regulation of PIK3IP1 by ARID1A and determine expression patterns of PIK3IP1 in the uterus during early pregnancy. The expression of PIK3IP1 was strong at the uterine epithelial and stromal cells of the control mice. However, expression of PIK3IP1 was remarkably reduced in the Pgr^cre/+Arid1a^f/f mice and progesterone receptor knock-out (PRKO) mice. During early pregnancy, PIK3IP1 expression was strong at day 2.5 of gestation (GD 2.5) and then slightly decreased at GD 3.5?at the epithelium and stroma. After implantation, PIK3IP1 expression was detected at the secondary decidualization zone. To determine the ovarian steroid hormone regulation of PIK3IP1, we examined the expression of PIK3IP1 in ovariectomized control, Pgr^cre/+Arid1a^f/f, and PRKO mice treated with P4 or E2. P4 treatment increased the PIK3IP1 expression at the luminal and glandular epithelium of control mice. However, the PIK3IP1 induction was decreased in both the Pgr^cre/+Arid1a^f/f and PRKO mice, compared to controls. Our results identified PIK3IP1 as a novel target of ARID1A and PGR in the murine uterus.     

雌激素和孕激素对不同发情方式小鼠子宫内膜中孕激素受体分布的影响

目的探讨雌（Estrogen,E2）、孕激素（Progesterone,P4）对同期发情与自然发情小鼠子宫内膜中孕激素受体（Progesterone receptor,PR）分布的影响。方法45只同日龄雌鼠,根据处理方式的不同随机分为5组：自然发情组（对照组）、同期发情组、卵巢摘除组、P4处理组和E2处理组,5组小鼠在见栓后第4、6、8天分别取样后,采用免疫组织化学法观察小鼠子宫内膜中PR的分布变化情况。结果免疫组织化学染色结果显示,5个处理组小鼠子宫内膜的三种细胞中都有PR存在;同期发情组小鼠子宫内膜中三种类型细胞PR的表达与自然发情组差异有显著性（P〈0.05）;P4处理组小鼠子宫内膜中三种类型细胞PR的表达在见栓第4、6天显著低于卵巢摘除组（P〈0.05）;E2处理组小鼠子宫内膜腺上皮和间质中PR在第4、6、8天时都显著高于卵巢摘除组（P〈0.05）,而在腔上皮中则显著低于卵巢摘除组（P〈0.05）。结论同期发情处理与自然发情小鼠的子宫内膜上PR的分布,都受E2和P4的特异诱导而变化。     

同期发情处理小鼠与自然发情小鼠子宫内膜中孕激素受体分布的比较

目的从孕激素受体（PR）的角度探讨同期发情处理与自然发情小鼠的子宫内膜上,孕激素受体分布是否受内源孕激素的特异诱导而变化,两者之间是否存在差异。方法27只同日龄母鼠,根据处理方式的不同随机分为三个组：自然发情假孕组（对照组）、同期发情处理假孕组和自然发情假孕第l天摘除卵巢组,3个组的小鼠在见栓后第4、6、8天分别取样后,采用免疫组织化学法观察小鼠子宫内膜中孕激素受体的分布情况。结果免疫组化结果显示,三个处理组小鼠子宫内膜的三种细胞中都有PR存在;见栓第4天时,同期发情处理组小鼠子宫腺上皮细胞和基质细胞的PR胞核阳性率显著高于自然发情组（P〈0．05）;见栓第6天时,同期发情处理组小鼠子宫内膜三种细胞中的PR胞核阳性率显著高于自然发情组（P〈0．05）;同时自然发情假孕第1天摘除卵巢组在见栓第6和8天时的阳性率均显著低于其它两组（P〈0．05）。结论同期发情处理的小鼠子宫内膜中孕激素受体分布显著高于自然发情小鼠,且两者都受其内源性孕激素的特异诱导而变化。     

Progesterone and Wnt4 control mammary stem cells via myoepithelial crosstalk

Ovarian hormones increase breast cancer risk by poorly understood mechanisms. We assess the role of progesterone on global stem cell function by serially transplanting mouse mammary epithelia. Progesterone receptor (PR) deletion severely reduces the regeneration capacity of the mammary epithelium. The PR target, receptor activator of Nf‐κB ligand (RANKL), is not required for this function, and the deletion of Wnt4 reduces the mammary regeneration capacity even more than PR ablation. A fluorescent reporter reveals so far undetected perinatal Wnt4 expression that is independent of hormone signaling. Pubertal and adult Wnt4 expression is specific to PR⁺ luminal cells and requires intact PR signaling. Conditional deletion of Wnt4 reveals that this early, previously unappreciated, Wnt4 expression is functionally important. We provide genetic evidence that canonical Wnt signaling in the myoepithelium required PR and Wnt4, whereas the canonical Wnt signaling activities observed in the embryonic mammary bud and in the stroma around terminal end buds are independent of Wnt4. Thus, progesterone and Wnt4 control stem cell function through a luminal–myoepithelial crosstalk with Wnt4 acting independent of PR perinatally.     

A comparative study of estrogen receptors alpha and beta in the rat uterus.

The uterus is an important target organ for steroid hormones. The effects of these hormones are mediated via specific receptors. The aim of this study was to compare the expression, distribution, and regulation of estrogen receptor (ER) alpha and beta in the rat uterus in order to establish possible different biological roles for the two receptor forms. Ovariectomized rats were treated with either estradiol (E(2)), progesterone (P(4)), or combinations of these for 24 or 48 h. The mRNA levels were measured by solution hybridization. Distribution of the mRNAs and receptor proteins was detected by in situ hybridization and immunohistochemistry. The results showed that ERalpha is the dominating subtype in the rat uterus. E(2) seemed to increase the ERalpha mRNA level in the glandular and luminal epithelium, but it caused a decrease of the immunostaining intensity in the glandular epithelium. P(4) reduced ERalpha expression in luminal epithelium whereas no effect was seen in the glandular epithelium. E(2) or P(4) did not alter the expression of ERbeta, on either the mRNA or protein level. In conclusion, the distribution and regulation of ERalpha and ERbeta differ in the different compartments of the rat uterus. The complex uterine responses to E(2) and P(4) are directly or indirectly mediated by differential cell-specific expression of their receptors. The low expression in the uterus and the limited regulation by gonadal steroids in this study suggest that ERbeta probably plays a minor role in the regulation of uterine physiology.     

The Wnt Co-Receptor Lrp6 Is Required for Normal Mouse Mammary Gland Development

Canonical Wnt signals are transduced through a Frizzled receptor and either the LRP5 or LRP6 co-receptor; such signals play central roles during development and in disease. We have previously shown that Lrp5 is required for ductal stem cell activity and that loss of Lrp5 delays normal mammary development and Wnt1-induced tumorigenesis. Here we show that canonical Wnt signals through the Lrp6 co-receptor are also required for normal mouse mammary gland development. Loss of Lrp6 compromises Wnt/β-catenin signaling and interferes with mammary placode, fat pad, and branching development during embryogenesis. Heterozygosity for an inactivating mutation in Lrp6 is associated with a reduced number of terminal end buds and branches during postnatal development. While Lrp6 is expressed in both the basal and luminal mammary epithelium during embryogenesis, Lrp6 expression later becomes restricted to cells residing in the basal epithelial layer. Interestingly, these cells also express mammary stem cell markers. In humans, increased Lrp6 expression is associated with basal-like breast cancer. Taken together, our results suggest both overlapping and specific functions for Lrp5 and Lrp6 in the mammary gland.     

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.     

Role of secretory protease inhibitor SPINK3 in mouse uterus during early pregnancy

Successful embryo implantation depends on intricate epithelial-stromal cross-talk. However, molecular modulators involved in this cellular communication remain poorly elucidated. Using multiple approaches, we have investigated the spatiotemporal expression and regulation of serine protease inhibitor Kazal type 3 (SPINK3) in mouse uterus during the estrous cycle and early pregnancy. In cycling mice, both SPINK3 mRNA and protein are only expressed during proestrus. In the pregnant mouse, the expression levels of both SPINK3 mRNA and protein increase on days 5-8 and then decline. Spink3 mRNA is expressed exclusively in the uterine glandular epithelium, whereas SPINK3 protein is localized on the surface of both luminal and glandular epithelium and in the decidua. Moreover, SPINK3 in the decidua has been observed in the primary decidual zone on day 6 and the secondary decidual zone on days 7-8; this is tightly associated with the progression of decidualization. SPINK3 has also been found in decidual cells of the artificially decidualized uterine horn but not control horn, whereas Spink3 mRNA localizes in the glands of both horns. The expression of endometrial Spink3 is not regulated by the blastocyst according to its expression pattern during pseudopregnancy and delayed implantation but is induced by progesterone and further augmented by a combination of progesterone and estrogen in ovariectomized mice. Thus, uterine-gland-derived SPINK3, as a new paracrine modulator, might play an important role in embryo implantation through its influence on stromal decidualization in mice.     

Relevance of ovarian signaling for the early behavioral transition from estrus to pregnancy in the female rabbit

During estrus, the female domestic rabbit (Oryctolagus cuniculus) displays scent marking behavior (chinning), which is immediately inhibited after mating, temporarily recovers, and then declines and remains inhibited across pregnancy. Chinning is inhibited by progesterone (P) and the activation of the progesterone receptor (PR), but it is unlikely that P participates in the “acute” (immediate) or “early” inhibition of chinning (24 to 96 h post-mating, before plasma P levels rise). Since PR is activated in a ligand-independent manner by a variety of signaling molecules, some of which (e.g., GnRH) are also associated with reflexive ovulation in this species, we hypothesized that neurochemical/neuroendocrine signals associated with mating activate PR, resulting in the inhibition of chinning. In Experiment 1, we tested whether the PR antagonist, RU486 (20 mg, injected s.c. at − 1 h, or at − 7 h and + 3 h relative to mating) prevented the post-mating inhibition of chinning in intact females. RU486 did not prevent the post-mating decline in chinning, indicating that PR activation associated with mating is not necessary for this effect. In Experiment 2, we used ovariectomized (OVX), estradiol benzoate (EB)-treated females to test the hypothesis that ovarian signaling is necessary for the post-mating inhibition of chinning. The acute inhibition of chinning occurred in OVX females, but the early inhibition was absent. We conclude that ovarian signaling is necessary for the early, but not acute, post-mating inhibition of chinning. The PR seems not to participate in either of these phases.     

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.     

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.     

Retinoic acid synthesis and metabolism are concurrent in the mouse uterus during peri-implantation

Vitamin A (retinol) and its active metabolite, retinoic acid (RA), serve dual roles in the female reproductive tract. Cytochrome P450 26A1 (Cyp26a1), an RA-metabolizing enzyme, is involved in mammalian early pregnancy. In order to investigate the role of RA synthesis and metabolism during embryo implantation, we first investigated the spatiotemporal expression of RA-signal in the mouse uterus during the peri-implantation period. RA-signal-related molecules, including binding proteins, synthesizing enzymes, catabolizing enzymes and receptors, were all expressed in the mouse uterus during embryo implantation. The locations of the RA synthetic system (Aldh1a1, Aldh1a2, CRBP1) and catabolizing enzyme (Cyp26a1) were distinctive in the mouse uterus during the peri-implantation period. Aldh1a1 was located in the gland epithelium, whereas Aldh1a2 and CRBP1 were located in the stroma and Cyp26a1 was expressed in the luminal and glandular epithelium. These results demonstrate that RA synthesis occurs in the stroma, whereas RA metabolism takes place in the endometrial epithelium. When endometrial epithelial cells were isolated on day 4.5 of pregnancy and treated with E₂ (17beta-estradiol) or a combination of E₂ and progesterone, all-trans-RA (10???M) significantly down-regulated the expression of LIF, HB-EF and CSF-1 in these cells in vitro. Taken together, these results suggest that the accumulation of RA in the stroma during mouse embryo implantation has an inhibitory effect on the expression of the three implantation-essential genes, LIF, HB-EGF and CSF-1. Therefore, the expression of Cyp26a1 in luminal and glandular epithelium might block the adverse effect of RA in order to promote successful embryo implantation.     

Effects of genistein or soy milk during late gestation and lactation on adult uterine organization in the rat

In utero and lactational exposure to estrogenic agents has been shown to influence morphological and functional development of reproductive tissues. Thus, consumption of dietary phytoestrogens, such as isoflavones, during pregnancy and lactation could influence important periods of development, when the fetus and neonate are more sensitive to estrogen exposure. In this study, reproductive outcomes after developmental exposure to isoflavones were examined in Long-Evans rats maternally exposed to isoflavones via a commercial soy beverage or as the isolated isoflavone, genistein. Most reproductive endpoints examined at birth, weaning, and 2 months of age were not significantly modified in pups of either sex after lactational exposure to soy milk (provided to the dams in place of drinking water) from birth until weaning. However, soy milk exposure induced a significant increase in progesterone receptor (PR) in the uterine glandular epithelium of the 2-month-old pups. In pregnant dams treated with genistein (GEN; 15 mg/kg body weight) by gavage, from Gestational Day 14 through weaning, PR expression in the uterine glandular epithelium from 2-month-old GEN-treated females (postexposure) was also significantly increased. Diethylstilbesterol (DES) also stimulated uterine PR expression only in the glandular but not luminal epithelial cells. However, unlike DES, in utero/lactational exposure to GEN did not increase expression of the proliferation marker, proliferating cell nuclear antigen (PCNA), in the luminal epithelial cells of the 2-month-old rat uteri. These experiments demonstrate that developmental exposure to dietary isoflavones, at levels comparable to the ranges of human exposure, modify expression of the estrogen-regulated PR in the uterus of sexually mature rats weeks after exposure ended. Since the PR is essential for regulating key female reproductive processes, such as uterine proliferation, implantation, and maintenance of pregnancy, its increased expression suggests that soy phytoestrogen exposure during reproductive development may have long-term reproductive health consequences.     

Ulipristal Acetate Inhibits Progesterone Receptor Isoform A-Mediated Human Breast Cancer Proliferation and BCl2-L1 Expression

The progesterone receptor (PR) with its isoforms and ligands are involved in breast tumorigenesis and prognosis. We aimed at analyzing the respective contribution of PR isoforms, PRA and PRB, in breast cancer cell proliferation in a new estrogen-independent cell based-model, allowing independent PR isoforms analysis. We used the bi-inducible human breast cancer cell system MDA-iPRAB. We studied the effects and molecular mechanisms of action of progesterone (P4) and ulipristal acetate (UPA), a new selective progesterone receptor modulator, alone or in combination. P4 significantly stimulated MDA-iPRA expressing cells proliferation. This was associated with P4-stimulated expression of the anti-apoptotic factor BCL₂-L₁ and enhanced recruitment of PRA, SRC-1 and RNA Pol II onto the +58 kb PR binding motif of the BCL ₂ -L ₁ gene. UPA decreased cell proliferation and repressed BCL_2-L₁ expression in the presence of PRA, correlating with PRA and SRC1 but not RNA Pol II recruitment. These results bring new information on the mechanism of action of PR ligands in controlling breast cancer cell proliferation through PRA in an estrogen independent model. Evaluation of PR isoforms ratio, as well as molecular signature studies based on PRA target genes could be proposed to facilitate personalized breast cancer therapy. In this context, UPA could be of interest in endocrine therapy. Further confirmation in the clinical setting is required.     

Expression and hormonal regulation of calcyclin-binding protein (CacyBP) in the mouse uterus during early pregnancy

Calcyclin-binding protein (Siah-1-Interacting Protein, CacyBP/SIP), is a calcium signaling protein involved in the degradation of beta-catenin, however, little is known about its role in reproductive biology. The present study was to character its temporospatial expression pattern and regulation in mouse uterus and to investigate whether it plays a role in the regulation of normal endometrial events. While prominently expressed in both luminal and glandular epithelia, CacyBP underwent dynamic changes during early pregnancy. CacyBP expression was observed weakly from days 1-4. An intense accumulation in luminal and glandular epithelia as well as decidua surrounding the embryo at later stages (days 5-7) was observed. Most notably, CacyBP accumulation in trophoblast was pronounced at day 7. Using ovariectomized and pseudopregnant mice, we found that progesterone (P(4)) and 17beta-estradiol (E(2)) led to increased expression of CacyBP gene and this could be abolished by Ru486 and tamoxifen, respectively. Antisense oligonucleotides (ODNs) against CacyBP significantly inhibited cultured endometrial stromal cells' (ESCs) apoptosis induced by UV irradiation. Injection of antisense ODNs into mouse uterine horn severely impaired the number of implanted blastocysts. Taken together, our results suggested that CacyBP expression was positively regulated by P(4) and E(2). CacyBP may be involved in the regulation of endometrial cell apoptosis during early pregnancy and play an important role in mouse endometrial events such as pregrancy establishment.     

Expression of cellCAM-105 in the apical surface of rat uterine epithelium is controlled by ovarian steroid hormones

Affinity-purified antibodies to cellCAM-105, an adhesive cell surface glycoprotein, were used in immunohistochemical investigations of rat uteri at various functional stages: (i) the oestrous, pro-oestrous, metoestrous, and dioestrous stages of the oestrous cycle, (ii) Days 1-8 of normal pregnancy, (iii) delayed implantation, (iv) 18 h after oestrogen reactivation from delay of implantation, and (v) juvenile rats, and normal ovariectomized adults, respectively, before and after experimental injection of progesterone and/or oestrogen. CellCAM-105 was present in the apical zones of the luminal and glandular epithelium cells in a stage-specific and hormone-dependent manner. The results indicate that: (1) steroid hormones are essential for the expression of cellCAM-105 in the uterine epithelial cells; (2) progesterone induces cellCAM-105 expression in the glandular epithelium, and oestrogen induces cellCAM-105 expression in the luminal epithelium; (3) progesterone induces down-regulation of cellCAM-105 from the surface of the uterine luminal epithelium of juvenile rats; (4) cellCAM-105 is absent in the luminal epithelial cells but present in the glandular epithelial cells of the rat uterus at the time of blastocyst implantation.