Chorionic gonadotropin and uterine dialogue in the primate

Implantation is a complex spatio-temporal interaction between the growing embryo and the mother, where both players need to be highly synchronized to be able to establish an effective communication to ensure a successful pregnancy. Using our in vivo baboon model we have shown that Chorionic Gonadotropin (CG), as the major trophoblast derived signal, not only rescues the corpus luteum but also modulates the uterine environment in preparation for implantation. This response is characterized by an alteration in both the morphological and biochemical activity in the three major cell types: luminal and glandular epithelium and stromal fibroblasts. Furthermore, CG and factors from the ovary have a synergistic effect on the receptive endometrium. Novel local effects of CG which influence the immune system to permit the survival of the fetal allograft and prevent endometrial cell death are also discussed in this review. An alternate extracellular signal-regulated kinase (ERK) activation pathway observed in epithelial endometrial cells and the possibility of differential expression of the CG/LH-R isoforms during gestation, open many questions regarding the mechanism of action of CG and its signal transduction pathway within the primate endometrium.     

Light and electron microscopic radioautography of DNA synthesis in the endometria of pregnant-ovariectomized mice during activation of implantation window.

Pregnant mice were ovariectomized at pre-implantation stage and exogenous nidatory estradiol was administered to evaluate the DNA synthesis of the endometrial cells during activation of uterine receptivity for blastocyst implantation. After 0, 3, 6, 12 and 18 hrs. of estradiol treatment, the animals received 3H-thymidine injection, sacrificed 1 hr. later, and the uteri were prepared for light and electron microscopic radioautography. At time 0, no labelled stromal or epithelial cells was found in the endometrium. According to the time-lapse after estradiol induction, a gradual increase of labelled stromal and endothelial cells was seen in the endometrium. The highest labeling index was observed at the antimesometrial side of the implantation sites and the lowest value was found at the interimplantation site. The cells found at mesometrial side of the implantation site showed an intermediate labeling index. Eighteen hrs. after estradiol treatment, the labelled stromal cells found near the implantation chamber resembled the morphology of decidual cells while those labelled cells localized at the interimplantation sites were similar to the fibroblast. The uterine luminal epithelial cells showed low DNA synthesis after estradiol treatment resulting in only a few labelled cells at the interimplantation sites and no labelled cells at the implantation sites. A similar labeling pattern was seen in the glandular epithelium. The distribution of labelled cells seen among the regions of pregnant endometrium under estradiol effect suggest that DNA synthesis related to uterine activation for blastocyst implantation is a focal reaction, where the luminal epithelium does nt proliferate while the stromal and endothelial cells around the conceptus increase the DNA synthesis to prepare the endometrial decidualization.     

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.     

Endometriosis is associated with progesterone resistance in the baboon (Papio anubis) oviduct: evidence based on the localization of oviductal glycoprotein 1 (OVGP1)

Endometriosis has been associated with a reduced response to progesterone in both the eutopic and ectopic endometrium. In this study we evaluated OVGP1 and steroid receptor expression in oviducts of baboons with endometriosis during the midsecretory phase and determined whether progesterone resistance associated with endometriosis also occurs in the oviduct. Oviducts obtained during the window of uterine receptivity (Day 10 postovulation [PO]) from animals with induced and spontaneous disease were compared to control animals during the proliferative stage and in the implantation window as well as animals treated with the progesterone receptor (PGR) antagonist ZK 137.299 (ZK). OVGP1 was significantly higher in animals with endometriosis compared with Day 10 PO controls and was similar to that seen in the late proliferative phase and in ZK-treated animals. Baboons with spontaneous endometriosis also showed a similar persistence of OVGP1, which was correlated with the maintenance of estrogen receptor 1 (ESR1) in the epithelial cells of animals with endometriosis. However, epithelial cell height and the percentage of ciliation were not affected by endometriosis. These data imply that the normal antagonism of progesterone on ESR and OVGP1, which results in their downregulation during the window of implantation, is absent in animals with endometriosis. This was confirmed further when the action of PGR was antagonized in animals without disease, which also resulted in the persistence of ESR1 and OVGP1. These studies suggest that an aberrant oviductal environment may be an additive factor that contributes to endometriosis-associated infertility.     

Modulation of the action of chorionic gonadotropin in the baboon (Papio anubis) uterus by a progesterone receptor antagonist (ZK 137. 316)

Signals from the developing mammalian blastocyst rescue the corpus luteum (CL) and modulate the uterine environment in preparation for implantation and early pregnancy. Our previous studies demonstrated that both short- and long-term administration of chorionic gonadotropin (CG) markedly alters the morphology and the biochemical activity of the receptive endometrium. Because the effects of CG were superimposed on a progesterone-primed endometrium, this study was undertaken to determine if the inhibition of progesterone action by progesterone receptor antagonists (PRa) in intact and ovariectomized baboons would alter the action of CG on the endometrium at the time of uterine receptivity. In the short-term hCG-treated baboons, the PRa reduced the epithelial plaque reaction, completely inhibited alpha-smooth muscle actin (alphaSMA) expression in stromal fibroblasts, and induced the reappearance of the progesterone (PR) and estrogen (ERalpha) receptors in epithelial cells. However, this treatment protocol had no effect on the expression of glycodelin in the glandular epithelium. In contrast, glycodelin expression in addition to alphaSMA was suppressed in the ovariectomized animals. In the long-term hCG-treated baboons, the PRa had a similar effect on both alphaSMA, PR, and ER. In addition, this treatment also resulted in an inhibition of glycodelin expression in the glandular epithelium. These results indicate that blocking the action of progesterone on the endometrium even for a short period of time has a profound effect on the hCG-induced response in stromal fibroblasts. In contrast, for the diminution of glandular epithelial function in the presence of an ovary requires prolonged inhibition of progesterone action, suggesting a potential paracrine effect on the endometrium from the CL in response to hCG.     

Zonula occludens-1 and E-cadherin are coordinately expressed in the mouse uterus with the initiation of implantation and decidualization

Two-way interactions between the blastocyst trophectoderm and the uterine luminal epithelium are essential for implantation. The key events of this process are cell-cell contact of trophectoderm cells with uterine luminal epithelial cells, controlled invasion of trophoblast cells through the luminal epithelium and the basement membrane, transformation of uterine stromal cells surrounding the blastocyst into decidual cells, and protection of the "semiallogenic" embryo from the mother's immunological responses. Because cell-cell contact between the trophectoderm epithelium and the luminal epithelium is essential for implantation, we investigated the expression of zonula occludens-1 (ZO-1) and E-cadherin, two molecules associated with epithelial cell junctions, in the mouse uterus during the periimplantation period. Preimplantation uterine epithelial cells express both ZO-1 and E-cadherin. With the initiation and progression of implantation, ZO-1 and E-cadherin are expressed in stromal cells of the primary decidual zone (PDZ). As trophoblast invasion progresses, these two molecules are expressed in stroma in advance of the invading trophoblast cells. These results suggest that expression of these adherence and tight junctions molecules in the PDZ serves to function as a permeability barrier to regulate access of immunologically competent maternal cells and/or molecules to the embryo and provide homotypic guidance of trophoblast cells in the endometrium.     

Expression of nidogens in rat uterus and embryo during decidualization and implantation

The purpose of this study was to demonstrate the expression of nidogen-1 and nidogen-2 and their possible role in decidualization and implantation events during early pregnancy in rats. The tissue samples were examined from pregnant animals between gestational days 1-8 using immunocytochemistry. The uterine luminal epithelium, the glandular epithelium, and the myometrial smooth muscle cells stained strongly from gestational days 1-8 with both nidogen antibodies. At day 4 the decidual reaction areas began to appear in the stromal matrix and immunostaining of both nidogens revealed that the basement membrane of the surface epithelium was discontinuous. The differentiation of stromal cells into decidual cells was seen at gestational day 5 and both nidogens were weakly expressed in the decidualizing cells. At day 6, nidogen-2 immunoreactivity was higher in the primary decidual cells close to the embryo than nidogen-1, and during development of the decidual tissue both nidogens appeared in the endometrial stromal cells. At day 7, while expression of both nidogens declined in the primary decidual cells, their expression was markedly observed in the secondary decidual cells close to the myometrium. At day 8, expression of both nidogens was also observed to increase in the primary decidual cells. While nidogen-2 expression was seen in the parietal endoderm and primary ectoderm of the rat embryos at this developmental stage, nidogen-1 expression was only detected in the parietal endoderm. These results indicate that nidogen-1 and nidogen-2 could play important roles during embryogenesis, decidualization, and implantation in the endometrium of rat uterus.     

Transforming growth factor beta isoforms regulation of Akt activity and XIAP levels in rat endometrium during estrous cycle, in a model of pseudopregnancy and in cultured decidual cells

Background  

During the estrous cycle, the rat uterine endometrium undergoes many changes such as cell proliferation and apoptosis. If implantation occurs, stromal cells differentiate into decidual cells and near the end of pregnancy, a second wave of apoptosis occurs. This process called decidual regression, is tightly regulated as is it crucial for successful pregnancy. We have previously shown that TGF-beta1, TGF-beta2 and TGF-beta3 are expressed in the endometrium during decidual basalis regression, but although we had demonstrated that TGF- beta1 was involved in the regulation of apoptosis in decidual cells, the ability of TGF- beta2 and TGF-beta3 isoforms to trigger apoptotic mechanisms in these cells remains unknown. Moreover, we hypothesized that the TGF-betas were also present and regulated in the non-pregnant endometrium during the estrous cycle. The aim of the present study was to determine and compare the specific effect of each TGF-β isoform in the regulation of apoptosis in sensitized endometrial stromal cells in vitro, and to investigate the regulation of TGF-beta isoforms in the endometrium during the estrous cycle in vivo.     

Identification by hybridization histochemistry of human endometrial cells expressing mRNAs encoding a uterine beta-lactoglobulin homologue and insulin-like growth factor-binding protein-1.

A beta-lactoglobulin homologue (beta LG/PP14) and insulin-like growth factor-binding protein-1 (IGFBP-1) are two major secretory proteins of the human endometrium. In the present study, we have shown that beta LG/PP14 mRNA is expressed in the endometrium in a cyclic manner, being hardly detectable in midcycle and most abundant during the late secretory phase. IGFBP-1 mRNA is also expressed in endometrium, but in amounts smaller than those encoding beta LG/PP14 and with maximal accumulation earlier in the secretory phase. The expression of these two mRNAs occurs in different cell types of the endometrium, as revealed by in situ hybridization techniques using single-stranded RNA probes. The glandular epithelial cells accumulate beta LG/PP14 mRNA during the late secretory phase of the cycle, whereas only the stromal cells of the late secretory endometrium express IGFBP-1 mRNA. In contrast to the endometrium, the two mRNAs are present at very low abundance in the fallopian tubes where they are expressed in the epithelial cells of the mucosa.     

Localization of ubiquitin and ubiquitin cross-reactive protein in human and baboon endometrium and decidua during the menstrual cycle and early pregnancy

We have examined the distribution of ubiquitin and the related ubiquitin cross-reactive protein (UCRP) in paraffin-embedded sections of human and baboon endometrium and decidua by immunoperoxidase or immunofluorescence cytochemistry with antibodies raised against ubiquitin, UCRP, CD45, and insulin-like growth factor-binding protein-1. Anti-ubiquitin immunoreactivity was present in the nonpregnant endometrium, particularly in the glandular epithelial cells, and up-regulated in endometrial stromal cells as they decidualized at the beginning of pregnancy. Anti-UCRP immunoreactivity was absent from nonpregnant tissue but accumulated to high levels in decidual cells during pregnancy. Western blotting indicated that immunoreactivity was primarily due to the presence of ubiquitin and UCRP conjugated to other proteins, and that although levels of ubiquitin-protein conjugates do not change substantially during pregnancy, decidualization is accompanied by the appearance of conjugates of UCRP. Baboon uterine tissues demonstrated a similar distribution of the two proteins, which indicates that the baboon may be a useful model for study of the role of the ubiquitin system and UCRP in the establishment of pregnancy in humans.     

Steroidogenic enzyme and key decidualization marker dysregulation in endometrial stromal cells from women with versus without endometriosis

Identification of mechanisms underlying endometriosis pathogenesis will facilitate understanding and treatment of infertility and pain associated with this disorder. Herein, we investigated the expression of steroidogenic pathway enzymes and key decidualization biomarkers in endometrial tissue and in eutopic endometrial stromal fibroblasts (hESFs) from women with vs. those without endometriosis, and subsequently treated in vitro with 8-bromo-cAMP (8-Br-cAMP) or progesterone (P4). Real-time quantitative PCR, immunohistochemistry, ELISA, and radiometric aromatase activity assay were used. The results demonstrate significantly increased (14.5-fold; P=0.037) expression of aromatase in eutopic endometrium of women with disease. In 8-Br-cAMP-treated hESF from eutopic endometrium of women with endometriosis, the balance in estradiol (E2) and P4 biosynthetic and metabolizing enzymes is disturbed (decreased HSD3B1 and HSD17B2, and increased HSD17B1 and aromatase), with the equilibrium being shifted towards an E2-enriched milieu. However, hESF from the same group of women treated with P4 did not demonstrate such responsiveness. Lower expression of IGFBP1 and prolactin mRNA and protein was observed in hESF from women with vs. those without endometriosis in response to 8-Br-cAMP, but not P4, suggesting a blunted response of these decidual biomarkers to activation of the PKA pathway in eutopic endometrium in women with disease. The dichotomy of 8-Br-cAMP regulation of select steroidogenic enzymes leading to an enriched E2 milieu within the endometrium and a blunted response of decidual biomarkers to this decidualizing agent of hESF from women with endometriosis suggests resistance to full decidualization of the stromal fibroblasts and mechanisms underlying implantation failure and the pathophysiology of this disorder.     

小鼠围着床期子宫内膜甲基化CpG结合域蛋白2的表达规律

为研究甲基化CpG结合域蛋白2（methyl-CpG binding domain protein 2,MBD2）在围植入期小鼠子宫内膜的表达规律,通过采用实时荧光定量PCR（Real-time fluorescence quantitative PCR,qPCR）、Western blot和免疫组化技术检测未孕小鼠（d0）和不同孕天小鼠子宫MBD2的表达情况。qPCR结果显示,d0至d7的小鼠子宫内膜组织均有MBD2 mRNA表达,在d5至d7高表达。MBD2蛋白在子宫内膜的表达规律与qPCR结果相符。MBD2蛋白在孕d1到d4中度表达于腔上皮、腺上皮和基质细胞,在d5至d7基质细胞表达增强,主要表达于蜕膜区。假孕小鼠子宫内膜中,MBD2在腔上皮、腺上皮和基质细胞中中度表达,d5至d7基质细胞表达明显减弱。动物模型中,宫角注射MBD2基因反义寡聚脱氧核苷酸,可抑制MBD2的表达,降低人工诱导蜕膜化反应和蜕膜化标志物PRL的表达。MBD2在早孕小鼠子宫内膜的表达模式提示其可能参与了蜕膜化过程。     

Deregulation of the serum- and glucocorticoid-inducible kinase SGK1 in the endometrium causes reproductive failure

Infertility and recurrent pregnancy loss (RPL) are prevalent but distinct causes of reproductive failure that often remain unexplained despite extensive investigations. Analysis of midsecretory endometrial samples revealed that SGK1, a kinase involved in epithelial ion transport and cell survival, is upregulated in unexplained infertility, most prominently in the luminal epithelium, but downregulated in the endometrium of women suffering from RPL. To determine the functional importance of these observations, we first expressed a constitutively active SGK1 mutant in the luminal epithelium of the mouse uterus. This prevented expression of certain endometrial receptivity genes, perturbed uterine fluid handling and abolished embryo implantation. By contrast, implantation was unhindered in Sgk1-/- mice, but pregnancy was often complicated by bleeding at the decidual-placental interface and fetal growth retardation and subsequent demise. Compared to wild-type mice, Sgk1-/- mice had gross impairment of pregnancy-dependent induction of genes involved in oxidative stress defenses. Relative SGK1 deficiency was also a hallmark of decidualizing stromal cells from human subjects with RPL and sensitized these cells to oxidative cell death. Thus, depending on the cellular compartment, deregulated SGK1 activity in cycling endometrium interferes with embryo implantation, leading to infertility, or predisposes to pregnancy complications by rendering the feto-maternal interface vulnerable to oxidative damage.