Expression of calbindin-D28k and its regulation by estrogen in the human endometrium during the menstrual cycle

Human endometrium resists embryo implantation except during the 'window of receptivity'. A change in endometrial gene expression is required for the development of receptivity. Uterine calbindin-D28k (CaBP-28k) is involved in the regulation of endometrial receptivity by intracellular Ca2+. Currently, this protein is known to be mainly expressed in brain, kidneys, and pancreas, but potential role(s) of CaBP-28k in the human uterus during the menstrual cycle remain to be clarified. Thus, in this study we demonstrated the expression of CaBP-28k in the human endometrium in distinct menstrual phases. During the human menstrual cycle, uterine expression levels of CaBP-28k mRNA and protein increased in the proliferative phase and fluctuated in these tissues, compared with that observed in other phases. We assessed the effects of two sex-steroid hormones, 17beta-estradiol (E2) and progesterone (P4), on the expression of CaBP-28k in Ishikawa cells. A significant increase in the expression of CaBP-28k mRNA was observed at the concentrations of E2 (10(-9 to -7) M). In addition, spatial expression of CaBP-28k protein was detected by immunohistochemistry. CaBP-28k was abundantly localized in the cytoplasm of the luminal and glandular epithelial cells during the proliferative phases (early-, mid-, late-) and early-secretory phase of menstrual cycle. Taken together, these results indicate that CaBP-28k, a uterine calcium binding protein, is abundantly expressed in the human endometrium, suggesting that uterine expression of CaBP-28k may be involved in reproductive function during the human menstrual cycle.     

Coexpression and estrogen-mediated regulation of TRPV6 and PMCA1 in the human endometrium during the menstrual cycle

Maintenance of calcium balance in the uterus is essential for many of its functions, including embryo implantation. The plasma membrane Ca²⁺‐pumping ATPase proteins are encoded by four genes designated PMCA1‐4, and PMCA1 is expressed in the uterus of rats during the estrous cycle. Although transient receptor potential cation channel subfamily V, member 6 (TRPV6), has been detected in the human placenta, pancreas and the prostate gland, expression patterns of uterine TRPV6 and PMCA1 and their potential roles in the human endometrium remain to be elucidated. In the present study, the expression patterns of TRPV6 and PMCA1 were examined to predict their potential roles in the human endometrium during the menstrual cycle. Human classified endometrial tissues (total n = 40) were separated into three groups according to menstrual cycle phase: menstrual, proliferative (early‐, mid‐, late), and secretory phase (early‐, mid‐, late). The expression of TRPV6 and PMCA1 mRNA and protein in the uterine endometrium during the menstrual cycle increased by 1.5‐ to 1.8‐fold at the proliferative phase (early‐, mid‐, and late‐) in comparison to the other phases. Estrogen treatment caused a significant increase in TRPV6 and PMCA1 mRNA expression. Immunohistochemical analysis of the distribution of TRPV6 and PMCA1 in the uterus revealed that both proteins are abundantly expressed in the cytoplasm of endometrial and glandular epithelial cells during menstrual phases. Taken together, these results suggest that uterine expression of TRPV6 and PMCA1 may be involved in human reproductive function. Mol. Reprod. Dev. 78:274–282, 2011. © 2011 Wiley‐Liss, Inc.     

Serum estradiol-17 beta, progesterone and respective uterine cytosol receptor concentrations in bitches with spontaneous pyometra

The role of serum estradiol-17 beta (E(2)) and progesterone (P(4)) in relation to uterine estrogen (ER) and progesterone receptors (PR) was investigated in canine cystic endometrial hyperplasia-pyometra (CEH-P). Blood and uterine samples were collected pre- and post-ovariohysterectomy, respectively, from 54 bitches presenting spontaneous CEH-P and 25 healthy control bitches. Competitive enzyme immunoassays (EIA) and enzyme ligand immunoassays (ELIA) were applied to estimate serum hormones and uterine cytosol active receptors, respectively. Animals were classified in the stages of first half of diestrus, second half of diestrus and early anestrus on the basis of reproductive history, clinical signs, uterine and ovarian macro- and microscopic inspection and serum P(4) concentration. Bitches with CEH-P, compared to their respective stage controls, exhibited (a) similar P(4) fluctuations, (b) higher E(2) concentrations, (c) lower PR concentrations during diestrus first and second half and (d) lower ER concentrations during diestrus first half and early anestrus. Negative correlation was detected between P(4) and ER within both CEH-P and control groups. It was concluded that P(4) was the main uterine receptor regulator for both PR and ER during diestrus and early anestrus in healthy and affected uteri. However, in CEH-P bitches, high P(4) levels in diestrus appeared to over-activate uterine PRs, leading to stronger PR self-down regulation and ER suppression. These findings indicate an increased sensitivity of CEH-P uterus to P(4) action. During early anestrus, a complementary role of endogenous E(2) was considered, since reduction of P(4) action appeared to permit uterine ER replenishment and activation by relatively high E(2) levels.     

Gastrin-releasing peptide (GRP) in the ovine uterus: regulation by interferon tau and progesterone

Gastrin-releasing peptide (GRP) is abundantly expressed by endometrial glands of the ovine uterus and processed into different bioactive peptides, including GRP1-27, GRP18-27, and a C-terminus, that affect cell proliferation and migration. However, little information is available concerning the hormonal regulation of endometrial GRP and expression of GRP receptors in the ovine endometrium and conceptus. These studies determined the effects of pregnancy, progesterone (P4), interferon tau (IFNT), placental lactogen (CSH1), and growth hormone (GH) on expression of GRP in the endometrium and GRP receptors (GRPR, NMBR, BRS3) in the endometrium, conceptus, and placenta. In pregnant ewes, GRP mRNA and protein were first detected predominantly in endometrial glands after Day 10 and were abundant from Days 18 through 120 of gestation. Treatment with IFNT and progesterone but not CSH1 or GH stimulated GRP expression in the endometrial glands. Western blot analyses identified proGRP in uterine luminal fluid and allantoic fluid from Day 80 unilateral pregnant ewes but not in uterine luminal fluid of either cyclic or early pregnant ewes. GRPR mRNA was very low in the Day 18 conceptus and undetectable in the endometrium and placenta; NMBR and BRS3 mRNAs were undetectable in ovine uteroplacental tissues. Collectively, the present studies validate GRP as a novel IFNT-stimulated gene in the glands of the ovine uterus, revealed that IFNT induction of GRP is dependent on P4, and found that exposure of the ovine uterus to P4 for 20 days induces GRP expression in endometrial glands.     

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

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

Synthesis of uterine endometrial proteins during early diestrus in the cyclic and pregnant dog, and after estrogen and progesterone treatment.

The objectives of this study were to identify and characterize dog uterine endometrial proteins synthesized de novo in explant culture during early luteal phase, to examine distribution of these proteins prior to the embryo's entering the uterus and during its free-floating period prior to implantation, and to examine regulation of endometrial proteins by estrogen and progesterone (P4) treatments. Uterine endometrium was collected from cyclic and pregnant bitches on diestrus Days 3, 7, and 10 as determined by loss of cornification of vaginal epithelium, and from ovariectomized dogs after treatment with corn oil, estrogen, P4, or estrogen followed by 1 or 2 wk of P4. Tissue was incubated in an explant culture system in the presence of [3H]leucine or [35S]methionine. The rate of incorporation of [3H]leucine into nondialyzable macromolecules indicated no significant change in rates of incorporation by status (pregnant vs. nonpregnant), day, or steroid treatment. Uterine endometrial-conditioned culture medium, analyzed by two-dimensional SDS-PAGE and fluorography, revealed a complex array of at least ten proteins or protein complexes in cyclic and pregnant bitches. No difference in protein pattern was detected by status; however, differences in distribution were apparent by day of cycle or early pregnancy. Two major proteins, cP5 (M(r) 54,686) and cP6 (M(r) 23,010) appeared to be differentially expressed. Expression of cP5, maximal on diestrus Day 3, decreased as the cycle or pregnancy progressed to diestrus Day 10. In contrast, expression of cP6, a minor protein on diestrus Day 3, appeared to be up-regulated for each status to Day 10, with increased intensity and multiple isoelectric and molecular-weight variants. In ovariectomized steroid-treated dogs, two-dimensional SDS-PAGE showed that pattern and distribution of specific proteins were affected by treatment. Acidic protein cP1 (M(r) 87,600), synthesized after corn oil and P4 treatment, was suppressed with estradiol (E2). Proteins cP2 (M(r) 40,000 and M(r) 42,000), present with all treatments, were intensified with P4. A high-M(r) basic protein complex (cP3) and acidic protein cP4 were expressed with E2 and maintained with P4 treatment. Proteins cP5 and cP6, while not induced by E2 or P4 alone, required E2 priming for P4 induction. Protein cP5 was down-regulated while cP6 was up-regulated with P4 for 2 wk. Proteins induced by estrogen followed by 1 or 2 wk of P4 treatments were similar to those released by endometrial explants collected from pregnant and cyclic bitches on Days 3, 7, and 10 of spontaneous diestrus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of galectin-1 mRNA in the mouse uterus is under the control of ovarian steroids during blastocyst implantation

Galectin-1 is a member of β-galactoside-binding lectins expressed in a variety of mammalian tissues. We report here that galectin-1 mRNA is abundantly expressed in the mouse reproductive organs such as the uterus and ovary. Uterine expression of galectin-1 mRNA is specifically regulated in the embryonic implantation process. Its expression increased at a high level on the fifth day post coitum (dpc 5) when embryos hatched into the endometrial epithelial cells. In the absence of embryos, however, galectin-1 expression in the mouse uterus decreased on dpc 5. In the delayed implantation mice, galectin-1 mRNA level was augmented by the termination of the delay of implantation. Ovarian steroids progesterone and estrogen differentially regulated galectin-1 mRNA level in uterine tissues. Treatment with RU486, a progesterone receptor antagonist, blocked progesterone-induced galectin-1 mRNA level in uterine tissues of ovariectomized mouse. ICI182780, a pure estrogen receptor antagonist, clearly blocked the estrogen effect. Taken together, galectin-1 gene expression in the uterine tissues was regulated by ovarian steroids and this regulation correlated with the implantation process. Mol. Reprod. Dev. 48:261–266, 1997. © 1997 Wiley-Liss, Inc.     

基质金属蛋白酶-2、-9及其组织抑制因子(TIMPs)在动情周期大鼠子宫中的表达(英文)

基质金属蛋白酶（ＭＭＰｓ）家族的作用是降解所有细胞外基质,其活性受其特异性组织抑制因子（ＴＩＭＰｓ）的抑制。细胞外基质成分的降解与重组在动物生殖生长过程中起重要作用,其变化可以通过ＭＭＰｓ和ＴＩＭＰｓ两者表达水平的变化进行监测。大鼠虽然没有月经形成,但是在其子宫内膜也出现类似灵长类的生殖生物学变化。本文从ＭＭＰｓ和ＴＩＭＰｓ两者的表达水平,对大鼠子宫内膜的这些变化进行了研究。于大鼠动情周期的不同时期,将其处死、取子宫制备酶粗提液和组织切片,采用酶谱法（ｚｙｍｏｙｒａｎｈｎ）和原位杂交方法研究动情周期大鼠子宫中ＭＭＰ－２和－９的活性变化以及ＭＭＰ－２、－９和ＴＩＭＰ－１、－２、－３ｍＲＮＡ的表达。并通过光密度扫描方法对酶谱结果进行半定量分析。所用杂交探针见Ｔａｂｌｅ１。酶谱结果显示：在动情周期大鼠子宫中只检测到６７ｋＤａ的ＭＭＰ－２活性,而没有检测到ＭＭＰ－９的活性（Ｆｉｇ．１）。ＭＭＰ－２的活性在动情前期最高,动情期和动情后期次之,间情期最低（Ｆｉｇ．２）。原位杂交结果显示：ＭＭＰ－２、－９、ＴＩＭＰ－１、－２、－３ｍＲＮＡ主要在子宫内膜基底部的基质细胞中表达。ＭＭＰ－２和－９ｍＲＮＡ在动情前期、动情期和动     

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.     

Global protein expression profiling underlines reciprocal regulation of caveolin 1 and endothelial nitric oxide synthase expression in ovariectomized sheep uterine artery by estrogen/progesterone replacement therapy

Ovariectomized (OVX) ewes were assigned to receive vehicle, progesterone (P4, 0.9-g controlled internal drug release vaginal implants), estradiol-17beta (E2, 5 microg/kg bolus + 6 microg kg(-1) day(-1)), or P4 + E2 for 10 days (n = 3/group). Uterine artery endothelial proteins were mechanically isolated on Day 10. The samples were used for protein expression profiling by the Ciphergen Proteinchip system and immunoblotting analysis of endothelial nitric oxide synthase (NOS3, also termed eNOS) and caveolin 1. Uterine artery rings were cut and analyzed by immunohistochemistry to localize NOS3 and caveolin 1 expression. With the use of the IMAC3 protein chip with loading as little as 2 microg protein/sample, many protein peaks could be detected. Compared to vehicle controls, a approximately 133.1-kDa protein was identified to be upregulated by 2- to 4-fold in OVX ewes receiving E2, P4, and their combination, whereas a approximately 22.6-kDa protein was downregulated by 2- to 4-fold in OVX ewes receiving E2 and E2/P4, but not P4 treatments. Western blot analysis revealed that E2, P4, and their combination all increased NOS3 protein, whereas E2 and its combination with P4, but not P4 alone, downregulated caveolin 1 expression. Immunohistochemical analysis revealed that NOS3 was mainly localized in the endothelium and upregulated by E2, whereas caveolin 1 was localized in both endothelium and smooth muscle and downregulated by E2. Thus, our data demonstrate that uterine artery endothelial NOS3 and caveolin 1 are regulated reciprocally by estrogen replacement therapy. In keeping with the facts that E2, but not P4, causes uterine vasodilatation and that E2 and P4 increase NOS3 expression, but only E2 decrease caveolin 1 expression, our current study suggests that both increased NOS3 expression and decreased caveolin 1 expression are needed to facilitate estrogen-induced uterine vasodilatation.     

Uterine and placental expression of TRPV6 gene is regulated via progesterone receptor- or estrogen receptor-mediated pathways during pregnancy in rodents

Transient receptor potential cation channel, subfamily V, member 6 (TRPV6) is an epithelial Ca²⁺ channel protein expressed in calcium absorbing organs. In the present study, we investigated the expression and regulation of uterine and placental TRPV6 during gestation in rodents. Uterine TRPV6 peaked at pregnancy day (P) 0.5, P5.5 and, P13.5 and was detected in uterine epithelium and glands of rats, while placental TRPV6 mRNA levels increased in mid-gestation. Uterine and placental TRPV6 mRNA levels in rats appear to cyclically change during pregnancy, suggesting that TRPV6 may participate in the implantation process. In addition, uterine TRPV6 mRNA is only expressed in placenta-unattached areas of the uterus, and uterine TRPV6 immunoreactivity was observed in luminal and glandular epithelial cells. In the placenta, TRPV6 was detected in the labyrinth and spongy zone. These results may indicate that TRPV6 has at least two functions: implantation of the embryo and maintenance of pregnancy. To investigate the pathway(s) mediating TRPV6 expression in rodents, anti-steroid hormone antagonists were injected prior to maximal TRPV6 expression. In rats, TRPV6 expression was reduced by RU486 (an anti-progesterone) through progesterone receptors, and ICI 182,780 (an anti-estrogen) blocked TRPV6 expression via estrogen receptors in mice. The juxtaposition of uterine and placental TRPV6 expressed in these tissues supports the notion that TRPV6 participates in transferring calcium ions between the maternal and fetal compartments. Taken together, TRPV6 gene may function as a key element in controlling calcium transport in the uterus between the embryo and the placenta during pregnancy.