Expression of Betacellulin and Epiregulin Genes in the Mouse Uterus Temporally by the Blastocyst Solely at the Site of Its Apposition Is Coincident with the “Window” of Implantation

In the mouse, the process of implantation is initiated by the attachment reaction between the blastocyst trophectoderm and uterine luminal epithelium that occurs at 2200–2300 h on day 4 (day 1 = vaginal plug) of pregnancy. Several members of the EGF family are considered important in embryo–uterine interactions during implantation. This investigation demonstrates that the expression of two additions to the family, betacellulin and epiregulin, are exquisitely restricted to the mouse uterine luminal epithelium and underlying stroma adjacent to the implanting blastocyst. These genes are not expressed during progesterone-maintained delayed implantation, but are rapidly switched on in the uterus surrounding the implanting blastocyst following termination of the delay by estrogen. These results provide evidence that expression of betacellulin and epiregulin in the uterus requires the presence of an active blastocyst and suggest an involvement of these growth factors in the process of implantation.     

Peroxisome proliferator-activated receptor delta expression and regulation in mouse uterus during embryo implantation and decidualization

The aim of this study was to examine the expression and regulation of peroxisome proliferator-activated receptor (PPAR) PPARdelta gene in mouse uterus during early pregnancy by in situ hybridization and immunohistochemistry. PPARdelta expression under pseudopregnancy, delayed implantation, hormonal treatment, and artificial decidualization was also investigated. There was a very low level of PPARdelta expression on days 1-4 of pregnancy. On day 5 when embryo implanted, PPARdelta expression was exclusively observed in the subluminal stroma surrounding the implanting blastocyst. No corresponding signals were seen in the uterus on day 5 of pregnancy. There was no detectable PPARdelta signal under delayed implantation. Once delayed implantation was terminated by estrogen treatment and embryo implanted, a strong level of PPARdelta expression was induced in the subluminal stroma surrounding the implanting blastocyst. Estrogen treatment induced a moderate level of PPARdelta expression in the glandular epithelium, while progesterone treatment had no effects in the ovariectomized mice. A strong level of PPARdelta expression was seen in the decidua on days 6-8 of pregnancy. PPARdelta expression was also induced under artificial decidualization. These data suggest that PPARdelta expression at implantation sites require the presence of an active blastocyst and may play an essential role for blastocyst implantation.     

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.     

Epidermal growth factor-like ligands and erbB genes in the peri-implantation rabbit uterus and blastocyst

Molecular cloning of the partial cDNA coding sequences of the four erbB receptors and the epidermal growth factor (EGF)-like ligands EGF, transforming growth factor alpha (TGF), and heparin-binding EGF (HB-EGF) has provided the basis for a comprehensive analysis of the spatiotemporal expression pattern of the EGF receptor/ligand system during the peri-implantation period in the rabbit. Employing nonradioactive in situ hybridization and immunolocalization, we observed differential expression of erbB1-erbB3 within the trophectoderm of the blastocyst. ErbB1 was strongly expressed in the cytotrophoblast but was downregulated upon syncytium formation. ErbB3 was a product of both the cyto- and syncytiotrophoblast. Despite the expression of erbB2 mRNA, the trophectoderm was devoid of immunoreactive ErbB2. ErbB4 gene activity was exclusively detected in the trophoblast at midpregnancy. The luminal and glandular epithelium and stroma of the nonpregnant, pseudopregnant, and pregnant rabbit uterus at Day 6 of gestation also expressed ErbB1-ErbB3. In the peri-implantation period, gene activities of erbB1-erbB3 were upregulated upon decidualization. At the site of implantation, uterine luminal epithelial cells apposing the preimplantation blastocyst displayed a distinct membrane immunolocalization of ErbB2, identifying the uterine epithelium as target for EGF, TGFalpha, and HB-EGF derived from both the embryonic trophectoderm and the uterine epithelium. In the luminal epithelium at the antimesometrial uterine site, HB-EGF gene activity was upregulated at the time of blastocyst attachment, but this upregulation was not reflected in an increase in immunoreactive HB-EGF. The detection of tyrosine phosphorylated ErbB2 in the rabbit placenta indicated the presence of a functional ErbB/EGF-like system in the pregnant rabbit uterus. This study provides strong evidence for a role of the ErbB/EGF-like system in embryo/maternal interactions during the peri-implantation period in the rabbit.     

Implantation-induced changes in uterine arylamidase localization in the rabbit, rat, hamster and guinea-pig

Localization of uterine arylamidase activity varied between species: arylamidase was found primarily in the apical aspect of uterine epithelial cells in the rabbit, hamster and non-pregnant rat; only moderate staining was observed in these animals in the endometrial stroma. By contrast, arylamidase localization was primarily stromal in the guinea-pig at all stages studied while the luminal epithelium was devoid of reactivity. In all species, uterine enzyme activity increased before implantation but decreased in the vicinity of the blastocyst once implantation had begun. A generalized increase over the entire length of the uterus was seen during the preimplantation phase in the uterine epithelium of the rabbit and in the endometrial stroma of the guinea-pig. Increase in stromal activity appeared to indicate predecidual transformations which were embryo-dependent (i.e. localized to the implantation site) in the rat, or embryo-independent (i.e. occurring throughout the uterus) in the guinea-pig. A subsequent decrease in enzyme activity occurred in the vicinity of the implanting embryo irrespective of the cell type involved (epithelium in the rabbit, stroma/decidua in the rat and guinea-pig). Since arylamidases of the type studied here are integrated membrane proteins, the uniformity of changes observed in different species may reflect profound changes in membrane properties of endometrial cells as an element of the implantation reaction.     

Osteopontin Is Expressed in the Mouse Uterus during Early Pregnancy and Promotes Mouse Blastocyst Attachment and Invasion In Vitro

Embryo implantation into the maternal uterus is a decisive step for successful mammalian pregnancy. Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein family and participates in cell adhesion and invasion. In this study, we showed that Opn mRNA levels are up-regulated in the mouse uterus on day 4 and at the implantation sites on days 5 and 8 of pregnancy. Immunohistochemistry localized the OPN protein to the glandular epithelium on day 4 and to the decidual zone on day 8 of pregnancy. OPN mRNA and proteins are induced by in vivo and in vitro decidualization. OPN expression in the endometrial stromal cells is regulated by progesterone, a key regulator during decidualization. As a secreted protein, the protein level of OPN in the uterine cavity is enriched on day 4, and in vitro embryo culturing has indicated that OPN can facilitate blastocyst hatching and adhesion. Knockdown of OPN attenuates the adhesion and invasion of blastocysts in mouse endometrial stromal cells by suppressing the expression and enzymatic activity of matrix metalloproteinase-9 in the trophoblast. Our data indicated that OPN expression in the mouse uterus during early pregnancy is essential for blastocyst hatching and adhesion and that the knockdown of OPN in mouse endometrial stroma cells could lead to a restrained in vitro trophoblast invasion.     

Non-identical Distribution Pattern of Epidermal Growth Factor and Platelet-derived Growth Factor in the Mouse Uterus During the Oestrous Cycle and Early Pregnancy

In the present study, we examined by immunohistochemistry the cell-specific distribution of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) in the mouse uterus during the oestrous cycle and throughout the first 7 days of pregnancy. Paraffin-embedded tissue samples were immunostained using the avidin–biotin peroxidase technique and then examined by light microscopy. Our results showed that immunostaining for EGF was detected in the stroma but not in the luminal or glandular epithelium. A high concentration of EGF was detected in the stroma around the time of embryo implantation at days 3, 4 and 5 of pregnancy. The implanted embryo at day 7 of gestation showed immunostaining for EGF between the ectoderm and endoderm layers. The cell distribution pattern for PDGF was found to be different from that observed with EGF. Luminal and glandular epithelia displayed PDGF immunostaining throughout the first 7 days of pregnancy, with the highest intensity at days 4 and 5 of gestation. In contrast, no immunostaining was observed in the luminal and glandular epithelia at post-oestrus, dioestrus and pro-oestrus stages. However, a weak reaction started to appear at oestrus. The embryo at the blastocyst stage displayed a strong immunoreaction for antibody against PDGF. In addition, the decidual boundary zone surrounding the implanted embryo at days 5, 6 and 7 of gestation also showed an immunostaining for PDGF. The present observations demonstrate clearly the presence of EGF and PDGF in the mouse uterus in high concentrations at the peri- implantation period. Thus, our results, together with what is known about the effect of EGF and PDGF in controlling the growth, differentiation and activation of a variety of cell types, suggest a possible role for these growth factors during the preparation of the endometrium for implantation in controlling the proliferation activity of stromal and/or epithelial cells.     

Bmp2 is critical for the murine uterine decidual response

The process of implantation, necessary for all viviparous birth, consists of tightly regulated events, including apposition of the blastocyst, attachment to the uterine lumen, and differentiation of the uterine stroma. In rodents and primates the uterine stroma undergoes a process called decidualization. Decidualization, the process by which the uterine endometrial stroma proliferates and differentiates into large epithelioid decidual cells, is critical to the establishment of fetal-maternal communication and the progression of implantation. The role of bone morphogenetic protein 2 (Bmp2) in regulating the transformation of the uterine stroma during embryo implantation in the mouse was investigated by the conditional ablation of Bmp2 in the uterus using the (PR-cre) mouse. Bmp2 gene ablation was confirmed by real-time PCR analysis in the PR-cre; Bmp2fl/fl (termed Bmp2d/d) uterus. While littermate controls average 0.9 litter of 6.2+/-0.7 pups per month, Bmp2d/d females are completely infertile. Analysis of the infertility indicates that whereas embryo attachment is normal in the Bmp2d/d as in control mice, the uterine stroma is incapable of undergoing the decidual reaction to support further embryonic development. Recombinant human BMP2 can partially rescue the decidual response, suggesting that the observed phenotypes are not due to a developmental consequence of Bmp2 ablation. Microarray analysis demonstrates that ablation of Bmp2 leads to specific gene changes, including disruption of the Wnt signaling pathway, Progesterone receptor (PR) signaling, and the induction of prostaglandin synthase 2 (Ptgs2). Taken together, these data demonstrate that Bmp2 is a critical regulator of gene expression and function in the murine uterus.     

纤粘连蛋白对小鼠胚胎体外发育和体外着床的作用

应用小鼠胚泡和外胎盘锥体外培养的方法,研究了纤粘连蛋白对小鼠胚泡发育及胚泡或外胎盘锥粘附和扩展的影响。结果显示,纤粘连蛋白对小鼠胚泡发育有一定的促进作用;对胚泡及外胎盘锥的粘附和胚泡初生滋养层细胞及外胎盘锥次生滋养层细胞扩展均有显著促进作用。纤粘连蛋白分子活性位点的合成肽段精－苷－天冬－丝氨酸可有效抑制纤粘连蛋白对胚泡或外胎盘锥发育、粘附和扩展的促进作用。结果表明,纤粘连蛋白在小鼠胚胎发育和着床过     

Differential hormonal regulation of leukemia inhibitory factor (LIF) in rabbit and mouse uterus

Leukemia inhibitory factor (LIF) has been shown to be essential for the implantation of mouse blastocysts. The present study was designed to determine how LIF protein was hormonally regulated in rabbit and mouse uterus using immunohistochemistry. In unmated rabbits, LIF protein was at a low level in the uterine epithelium and glands, and up-regulated by progesterone alone or estradiol-17β and progesterone combined. Estradiol-17β alone had no apparent effect. In ovariectomized mice, the level of LIF protein was very low in the uterine epithelium and glands, and was up-regulated by estradiol-17β alone or estradiol-17β and progesterone combined. Progesterone alone had no apparent effect. These results suggest that LIF protein is differentially regulated in rabbit and mouse uterus by progesterone and estrogen, respectively. This would explain the high level of LIF protein observed in uterine epithelium and glands prior to blastocyst implantation in the two species with different hormonal requirements for implantation. © 1996 Wiley-Liss, Inc.     

Physiological and molecular determinants of embryo implantation

Embryo implantation involves the intimate interaction between an implantation-competent blastocyst and a receptive uterus, which occurs in a limited time period known as the window of implantation. Emerging evidence shows that defects originating during embryo implantation induce ripple effects with adverse consequences on later gestation events, highlighting the significance of this event for pregnancy success. Although a multitude of cellular events and molecular pathways involved in embryo–uterine crosstalk during implantation have been identified through gene expression studies and genetically engineered mouse models, a comprehensive understanding of the nature of embryo implantation is still missing. This review focuses on recent progress with particular attention to physiological and molecular determinants of blastocyst activation, uterine receptivity, blastocyst attachment and uterine decidualization. A better understanding of underlying mechanisms governing embryo implantation should generate new strategies to rectify implantation failure and improve pregnancy rates in women.     

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.     

Expression and regulation of cytosolic prostaglandin E synthase in mouse uterus during the peri-implantation period

Prostaglandin E(2) (PGE(2)) is considered important for blastocyst spacing, implantation, and decidualization in rodent uteri. PGE synthase (PGES) catalyzes the isomerization of PGH(2) to PGE(2). Two isoforms of PGES exist: microsomal PGES (mPGES) and cytosolic PGES (cPGES); however, the expression and regulation of cPGES in the mammalian uterus during early pregnancy are still unknown. The aim of this study was to investigate the differential expression of cPGES in mouse uterus during early pregnancy and its regulation under different conditions using in situ hybridization and immunohistochemistry. A strong level of cPGES mRNA signal was exhibited in the stromal cells at the implantation site on Day 5 of pregnancy, whereas cPGES immunostaining was strongly detected in the luminal epithelium. The signals for both cPGES mRNA and immunostaining were strongly detected in the decidualized cells from Days 6-8 of pregnancy. A basal level of cPGES mRNA signal and immunostaining was exhibited in the uterus in delayed implantation. After delayed implantation was terminated by estrogen treatment and embryo implantation was initiated, cPGES mRNA signal was strongly detected in the stroma underlying the luminal epithelium at the implantation site, and cPGES immunostaining was strongly observed in the luminal epithelium surrounding the implanting blastocyst. A strong cPGES mRNA signal and immunostaining were detected in decidualized cells under artificial decidualization, whereas only a basal level of cPGES mRNA signal and immunostaining were observed in the control horn. Our data suggest that cPGES may play an important role during implantation and decidualization.