The Bysl gene product, bystin, is essential for survival of mouse embryos

Human bystin is a cytoplasmic protein directly binding to trophinin, a cell adhesion molecule potentially involved in human embryo implantation. The present study shows that bystin is expressed in luminal and glandular epithelia in the mouse uterus at peri-implantation stages. In fertilized embryos, bystin was not seen until blastocyst stage. Bystin expression started during hatching and increased in expanded blastocyst. However, bystin apparently disappeared from the blastocyst during implantation. After implantation bystin re-appeared in the epiblast. Targeted disruption of the mouse bystin gene, Bysl, resulted in embryonic lethality shortly after implantation, indicating that bystin is essential for survival of mouse embryos.     

Effects of leukaemia inhibitory factor on embryo implantation in the mouse

Leukaemia inhibitory factor (LIF) is a pleiotrophic cytokine. Recent reports indicate that LIF is relevant to murine embryo implantation. In this work, results of indirect immunofluorescence under a confocal microscope illustrated that LIF was mainly located in the uterine lumen and uterine epithelial cells in pregnant mice on day 4. The number of embryos implanted in pregnant mice on day 8 decreased significantly after injection of 3 microg LIF antibodies into a uterine horn (P<0.001), which demonstrated again that LIF is a critical factor for embryo implantation. In a co-culture system, LIF (0.1 ng/ml, 1 ng/ml, 10 ng/ml and 100 ng/ml) significantly enhanced the blastocyst outgrowth after 24, 48 or 72 h of co-culture, and outgrowth areas after 72 h of co-culture. Conversely, 5 microg/ml and 10 microg/ml, but not 1 microg/ml, LIF antibodies decreased the percentage of blastocysts with outgrowth; only 10 microg/ml LIF antibody inhibited blastocyst outgrowth area significantly (P<0.001). However, neither LIF nor its antibodies changed embryo attachment. Analysis of correlation showed that the effects of LIF or its antibodies on the blastocyst outgrowth were dose-dependent. In summary, different pathways may exist to regulate the blastocyst attachment and outgrowth on a monolayer of uterine epithelial cells. LIF protein from the maternal uterus exerts an essential role in embryo implantation in the mouse, which is mediated by stimulating trophoblast outgrowth, but not by promoting the attachment.     

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.     

Dynamic distribution of epidermal growth factor during mouse embryo peri-implantation

Embryo implantation depends on the synchronized development of the blastocyst and the endometrium. This process is highly controlled by the coordinated action of the steroid hormones: estrogen and progesterone. By autocrine, paracrine or juxtacrine routes, some growth factors or cytokines are involved in this steroidal regulation pathway. Here we report the effects of epidermal growth factor (EGF) on embryo implantation in the mouse, the expression and distribution patterns of EGF protein in the mouse blastocyst, ectoplacental cone (EPC) and peri-implantation uterus on days 1-8 of gestation.By RT-PCR and dot blot, we found that EGF and its receptor (EGFR) are co-expressed in the blastocyst and peri-implantational uteri of pregnant days 2-8 (D2-D8) mice. Injection of EGF antibody into a uterine horn on the third day of pregnancy (D3) significantly reduced the number of mouse embryos that implanted on D8, indicating EGF have a function in the mouse embryo implantation.Further investigation by using indirect immunofluorescence and confocal microscope was made to trace EGF and EGFR protein localization during the mouse embryo implantation. EGF and EGFR are co-localized in the blastocyst, and in the secondary trophoblastic giant cells (SGC) of the EPC. At the pre-implantation stage, the distribution of EGF protein in the mouse uterus changes from epithelium to stroma. On D1 of pregnancy, EGF is mainly distributed in uterine stroma and myometrium. On D2, it is present in the uterine epithelium. On D3, it changes again from the uterine epithelium to the stroma. By D4, EGF is predominantly in the stroma. This dynamic distribution correlates with the proliferation activity of uterine cells at each period. On D6-D8 of embryo implantation, EGF 3 protein accumulates at the uterine mesometrial pole, a region that contributes to the trophoblastic invasiveness and placentation.This temporal and spatial localization of EGF protein in the mouse uterus implicates the cytokine in the regulation of trophoblastic invasiveness and uterine receptiveness.     

Leukaemia inhibitory factor and the regulation of pre-implantation development of the mammalian embryo

This paper reviews the evidence that certain growth factors, particularily leukaemia inhibitory factor (LIF), play a crucial role in regulating the development of the pre-implantation mammalian embryo. LIF was originally implicated in regulating the early development of the mouse embryo because it inhibited the differentiation of embryonic stem (ES) cells, pluripotential cells derived from the inner cell mass of the blastocyst. Subsequent studies on its role in vivo revealed, surprisingly, that it is essential for the growth rather than the differentiation of the blastocyst. In vivo, overtly normal blastocysts can be produced in a LIF-deficient environment that are capable of forming viable fertile adults. However, in the absence of LIF, they fail to implant and enter into a state resembling that exhibited by blastocysts undergoing delayed implantation, which is characterized by a cessation of cell proliferation. This failure to implant occurs because the principle sites of LIF production are the endometrial glands of the uterus. These synthesize and secrete LIF at implantation, with LIF synthesis essential for implantation. Preliminary evidence indicates that LIF synthesis is required both by the uterus for it to undergo decidualization and by the blastocyst for implantation. These data indicate that the maternal environment plays a crucial role in the development and growth of the pre-implantation embryo, by supplying factors that regulate these processes in the embryo. © 1994 Wiley-Liss, Inc.     

Steroids modulate the expression of alpha4 integrin in mouse blastocysts and uterus during implantation

Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and the maternal uterine milieu, which are controlled at the embryo-maternal interface by the coordinated interplay of a variety of growth factors, cytokines, hormones, and cell adhesion molecules expressed by both the decidualized endometrium and the trophoblast cells. Proper implantation of the embryo is solely dependent on the initial endometrial receptivity and the preparation of the blastocyst to glue itself to the uterine wall. Both these events are considered to be mediated by cell adhesion molecules and integrins expressed by the blastocyst as well by as the maternal endometrium. Integrin expression by the blastocyst and the uterus is a dynamic process. However, reports on the expression and the hormonal modulation of integrins and their role in blastocyst activation and uterine receptivity during implantation are meager. The present study investigates the expression and hormonal regulation of alpha4beta1 integrin by steroid hormones in the blastocyst and the receptive uterus using an in vivo, delayed-implantation mouse model system. The dormant and activated blastocysts as well as the uteri were recovered from ovariectomized mice after progesterone-alone and progesterone-plus-estrogen therapy, respectively. Immunolocalization of protein expression of alpha4 and beta1 integrin subunits indicate that steroids modulate the expression of alpha4beta1 integrin receptor in the mouse blastocyst as well as the uterus and that a differential expression is observed with exposure to progesterone and estrogen. Intrauterine blocking of alpha4 integrin by specific antibody resulted in implantation failure in normal as well as in delayed-implantation mice. Based on our data, we propose here, to our knowledge for the first time, that alpha4beta1 integrin, which is responsible for binding to fibronectin and vascular cell adhesion molecule-1, is induced by estradiol and is down-regulated by progesterone in mice during implantation. Furthermore, the results also indicate the direct role of alpha4 integrin in the process of implantation.     

Uterine RGS2 expression is regulated by exogenous estrogen and progesterone in ovariectomized mice,and downregulation of RGS2 expression in artificial decidualized ESCs inhibits trophoblast spreading in vitro

Embryo implantation is a complicated event that relies on two critical factors: the competent blastocyst and the receptive uterus. Successful implantation results from tight coordination of these two factors. The maternal hormone environment of the uterus and molecular cross‐talk between the embryo and uterine tissue play pivotal roles in implantation. Here we showed that regulator of G‐protein signaling 2 (RGS2), a member of ubiquitous family of proteins that regulate G‐protein activation, plays an important role in embryo implantation by interfering in the cross‐talk between the embryo and uterine tissue. RGS2 expression increased during the implantation process, and was higher in the implant site than at the nonimplantation site. Meanwhile, ovariectomized (OVX) mice exhibited higher expression of RGS2 in the uterus. Exogenous 17β‐estradiol and progesterone in OVX mice downregulated the expression of RGS2. Treatment with exogenous 17β‐estradiol alone caused uterine RGS2 messenger RNA levels of OVX mice to return to those of normal female mice; when these mice were treated with progesterone or 17β‐estradiol plus progesterone, RGS2 levels rose. Downregulation of Rgs2 by small interfering RNA in an in vitro coculture system of decidualized endometrial stromal cells and blastocysts inhibited blastocyst outgrowth by restricting trophoblast spreading, suggesting a mechanism by which RGS2 regulates embryo implantation.     

Quantification of basigin mRNA in mouse oocytes and preimplantation embryos by competitive RT-PCR

Basigin is a member of the immunoglobulin superfamily and a key molecule related to mouse blastocyst implantation. Whether preimplantation mouse embryos express basigin mRNA is still unknown. The aim of this study was to use a quantitative competitive polymerase chain reaction to assess quantitatively the levels of basigin mRNA in mouse oocyte and preimplantation embryos. Basigin mRNA was detected in the oocyte and all the stages of preimplantation embryos. The levels of basigin mRNA were 0.0606 +/- 0.0282 in the oocyte, 0.0102 +/- 0.0036 in the zygote, 0.0007 +/- 0.0003 in the 2-cell embryo, 0.0031 +/- 0.0017 in the 4-cell embryo, 0.0084 +/- 0.0024 in the 8-cell embryo, 0.0537 +/- 0.0121 in the morula and 0.0392 +/- 0.0161 attomoles in the blastocyst, respectively. The levels of basigin mRNA in the oocyte, morula and blastocyst were significantly higher than those in the zygote and embryos at the 2-cell, 4-cell and 8-cell stages. The high level of basigin expression in the blastocyst may play a role during embryo implantation.     

Effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation

Mouse embryo implantation depends on the complex interaction between the embryo trophoblast cells and the uterine environment, which deposits an extracellular matrix with abundant amounts of laminin. Intrauterine injection and blastocyst or ectoplacental cone culture models were used to study the effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation in vivo and in vitro. Intrauterine injection of 32/67 kDa laminin-binding protein antibody (0.4 mg in 1 ml Ham's F-10 medium, 5 microl per mouse) into the left uterine horns of mice (n = 22) on day 3 of pregnancy inhibited embryo implantation significantly (P < 0.001) compared with the contralateral horns that had been injected with normal rabbit IgG. A continuous section study on day 5 after injection showed that the embryos in the control uteri implanted normally and developed healthily, but there were no embryos or the remaining embryos had disintegrated in the uteri injected with 32/67 kDa laminin-binding protein antibody. Blastocysts or ectoplacental cones were cultured in media containing 32/67 kDa laminin-binding protein antibody (0.2 mg ml(-1)) on laminin-coated dishes with normal rabbit IgG at the same concentration as in the controls. The 32/67 kDa laminin-binding protein had no effect on blastocyst or ectoplacental cone attachment, but prohibited the blastocyst or ectoplacental cone outgrowth and primary or secondary trophoblast giant cell migration. These results indicate that 32/67 kDa laminin-binding protein antibody blocked mouse embryo implantation by preventing embryo trophoblast cell invasion and migration through the uterine decidual basement membrane-like extracellular matrix which has a high laminin content.     

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.     

Significant differences between mouse and human trophinins are revealed by their expression patterns and targeted disruption of mouse trophinin gene.

Trophinin has been identified as a membrane protein mediating apical cell adhesion between two human cell lines: trophoblastic HT-H cells, and endometrial epithelial SNG-M cells. Expression patterns of trophinin in humans suggested its involvement in embryo implantation and early placental development. The mouse trophinin gene maps to the distal part of the X chromosome and corresponds to human chromosome Xp11.21-22, the locus where the human trophinin gene maps. Western blot analysis indicates that the molecular weight of mouse trophinin is 110 kDa, which is consistent with the calculated value of 107 kDa. Positive signals for trophinin proteins were detected in preimplantation mouse embryos at the morula and blastocyst stages. Implanting blastocysts do not show detectable levels of trophinin protein, demonstrating that trophinin is not involved in blastocyst adhesion to the uterus in the mouse. Mouse embryo strongly expressed trophinin in the epiblast 1 day after implantation. Trophinin protein was not found in the mouse uteri and placenta after 5.5 days postcoitus (dpc). Targeted disruption of the trophinin gene in the mouse showed a partial embryonic lethality in a 129/SvJ background, but the cause of this lethality remains undetermined. The present study indicates significant differences between mouse and human trophinins in their expression patterns, and it suggests that trophinin is not involved in embryo implantation and placental development in the mouse.     

Leptin-directed embryo implantation: leptin regulates adhesion and outgrowth of mouse blastocysts and receptivity of endometrial epithelial cells

Leptin is a 16-kDa multifunctional protein. Recent reports indicate that leptin is an important molecule during implantation and placentation, implicated in embryonic-maternal cross-talk and cytotrophoblast invasiveness, however, the role of leptin playing in the process of normal blastocyst implantation has not been well characterized. In the present study, the possible mechanisms of leptin playing in mouse blastocyst implantation were investigated. Leptin and receptor isoforms mRNAs were detected in whole mouse uteri during estrous cycle and peri-implantation periods. Immunofluorescent analysis further confirmed Ob-R protein was present in mouse uterus. The differential amounts of leptin and Ob-R isoforms suggested a role for leptin in such endometrial issues as blastocyst implantation. In vitro culture model for studying embryo implantation, leptin promoted mouse blastocyst adhesion and blastocyst outgrowth on fibronectin. Blastocysts treated with 300 ng/ml leptin had the greatest adhesion rate of 76.58+/-6.41% (P=0.046), and blastocysts treated with 30 ng/ml leptin had the greatest outgrowth rate of 78.64+/-8.48% (P=0.005). In isolated endometrial epithelial cells, leptin upregulated amounts of alpha v and beta 3 integrin, and promoted cell adhesion to such extracellular matrix proteins as fibronectin, laminin and type IV collagen, showing a dose- and time-dependent cell-adhesive capacity. Collectively, the information from the present study may partly account for leptin-induced mouse blatocyst implantation.     

Transforming growth factor alpha (TGFalpha) modulates the effect of genomic imprinting and prolongs the development of parthenogenetic murine embryos]

The effect of transforming growth factor alpha (TGF alpha) on the development of diploid parthenogenetic mouse embryos (CBA x C57BL/6)F1 was studied. The embryos were in vitro treated with the TGF alpha at the stage of morula. Upon reaching the blastocyst stage, each embryo was implanted into uterus of a pseudopregnant female. At a dose of 5 ng/ml, the TGF alpha was found to improve development of parthenogenetic embryos before implantation, increase significantly the number of developing blastocysts, and promote embryo implantation into uterus. After treatment with TGF alpha at a dose of 10 ng/ml, 4% of parthenogenetic embryos reached the stage of 30-45 somites and had forelimb and hindlimb buds; the embryo size from vertex to sacrum was 2.0 to 3.8 mm. A well-developed placenta was observed in 6% of TGF alpha-treated parthenogenetic embryos that reached the somite stages. In the parthenogenetic embryos with the most prominent development (42-45 somites) treated with 10 ng/ml of TGF alpha, the placental diameter was 4.0 to 4.2 mm on day 12 of gestation, which is close to the placental size of the normal (fertilized) 11-day-old mouse embryos. Our results suggest that endogenous TGF alpha can modulate the effects of genomic imprinting significantly improving formation of trophoblast derivatives and promoting longer postimplantation development of parthenogenetic embryos.     

Restraint Stress Inhibits Mouse Implantation: Temporal Window and the Involvement of HB-EGF,Estrogen and Progesterone

It is known that psychological stress affects reproduction in women, but it is unknown whether the effect is by impairing implantation. Although studies suggest that long periods of auditory or restraint stress may inhibit implantation in rats and mice, the exact stage of pregnancy at which stress impairs implantation is unclear. Furthermore, whether stress impairs implantation by decreasing the heparin-binding epidermal growth factor-like growth factor (HB-EGF), estrogen and/or progesterone and whether by acting on embryos or on the uterus need further investigations. In this study, a 24-h restraint stress was initiated at 15:30 of day 3 (regimen 1) or at 07:30 (regimen 2) or 15:30 of day 4 (regimen 3) of pregnancy (vaginal plug  =  day 1) to observe effects of restraint stress applied at different peri-implantation stages on implantation. Among the three regimens, whereas regimens 1 and 3 affected neither term pregnancy nor litter size, regimen 2 reduced both. Further observations indicated that regimen 2 of restraint stress also delayed blastocyst hatching and the attachment reaction, decreased serum concentrations of progesterone and estradiol, and down regulated the expression of HB-EGF in both the endometrium and blastocysts. Taken together, the results suggested that restraint stress inhibited mouse implantation in a temporal window-dependent manner and by impairing blastocyst activation and hatching and uterine receptivity via down-regulating HB-EGF, estrogen and progesterone. Thus, the stress applied within the implantation window impaired implantation by acting on both embryos and the uterus.