Human Endometrial Extracellular Vesicles Functionally Prepare Human Trophectoderm Model for Implantation: Understanding Bidirectional Maternal‐Embryo Communication

Embryo implantation into maternal endometrium is critical for initiation and establishment of pregnancy, requiring developmental synchrony between endometrium and blastocyst. However, factors regulating human endometrial–embryo cross talk and facilitate implantation remain largely unknown. Extracellular vesicles (EVs) are emerging as important mediators of this process. Here, a trophectoderm spheroid‐based in vitro model mimicking the pre‐implantation human embryo is used to recapitulate important functional aspects of blastocyst implantation. Functionally, human endometrial EVs, derived from hormonally treated cells synchronous with implantation, are readily internalized by trophectoderm cells, regulating adhesive and invasive capacity of human trophectoderm spheroids. To gain molecular insights into mechanisms underpinning endometrial EV‐mediated enhancement of implantation, quantitative proteomics reveal critical alterations in trophectoderm cellular adhesion networks (cell adhesion molecule binding, cell–cell adhesion mediator activity, and cell adherens junctions) and metabolic and gene expression networks, and the soluble secretome from human trophectodermal spheroids. Importantly, transfer of endometrial EV cargo proteins to trophectoderm to mediate changes in trophectoderm function is demonstrated. This is highlighted by correlation among endometrial EVs, the trophectodermal proteome following EV uptake, and EV‐mediated trophectodermal cellular proteome, important for implantation. This work provides an understanding into molecular mechanisms of endometrial EV‐mediated regulation of human trophectoderm functions—fundamental in understanding human endometrium–embryo signaling during implantation.     

Gene replacement reveals a specific role for E-cadherin in the formation of a functional trophectoderm

During mammalian embryogenesis the trophectoderm represents the first epithelial structure formed. The cell adhesion molecule E-cadherin is ultimately necessary for the transition from compacted morula to the formation of the blastocyst to ensure correct establishment of adhesion junctions in the trophectoderm. Here, we analyzed to what extent E-cadherin confers unique adhesion and signaling properties in trophectoderm formation in vivo. Using a gene replacement approach, we introduced N-cadherin cDNA into the E-cadherin genomic locus. We show that the expression of N-cadherin driven from the E-cadherin locus reflects the expression pattern of endogenous E-cadherin. Heterozygous mice co-expressing E- and N-cadherin are vital and show normal embryonic development. Interestingly, N-cadherin homozygous mutant embryos phenocopy E-cadherin-null mutant embryos. Upon removal of the maternal E-cadherin, we demonstrate that N-cadherin is able to provide sufficient cellular adhesion to mediate morula compaction, but is insufficient for the subsequent formation of a fully polarized functional trophectoderm. When ES cells were isolated from N-cadherin homozygous mutant embryos and teratomas were produced, these ES cells differentiated into a large variety of tissue-like structures. Importantly, different epithelial-like structures expressing N-cadherin were formed, including respiratory epithelia, squamous epithelia with signs of keratinization and secretory epithelia with goblet cells. Thus, N-cadherin can maintain epithelia in differentiating ES cells, but not during the formation of the trophectoderm. Our results point to a specific and unique function for E-cadherin during mouse preimplantation development.     

囊胚形成的基因表达与调控(英文)

囊胚形成是胚胎早期发育过程中一个重要阶段 ,涉及几个重要的生理事件 ,即细胞融合 (compaction ,亦称致密化作用 )、囊胚腔出现、囊胚腔扩张及滋养层和内细胞团的分化。在细胞间连接蛋白的作用下 ,各种细胞间连接方式逐步建立起来 ,在合子型基因组表达调控下 ,促进了最终囊胚的形成。细胞间连接蛋白和细胞粘附相关蛋白参与组建各种细胞间连接 ,参与细胞融合、囊胚腔形成、滋养层分化和囊胚扩张等过程。通过顶部的紧密连接、侧部的缝隙连接和桥粒 ,建立起细胞的连接复合体。在人胚胎 8 细胞之前 ,卵裂球细胞界限明显 ,可能以中间连接方式相互作用 ;8 细胞期发生致密化作用 ,通过紧密连接将细胞分成顶部和基部 ,使得胚胎处于半封闭状态 ,促进胚胎内部积液 ,形成囊胚腔。细胞融合的同时也产生缝隙连接。桥粒最初出现在人胚胎达到 3 2 细胞阶段 ,桥粒连接参与囊胚腔形成以及在囊胚扩张时维持滋养层的稳定性。桥粒由一些跨膜粘蛋白组成 ,包括参与细胞内粘附的桥粒子和桥粒球以及一些细胞质内蛋白 (如desmoplakins,plakoglobin ,plakophilin) ,由细胞内蛋白质形成空斑结构并介导细胞角蛋白丝固定。对植入前牛胚胎的研究表明 ,只有DcII,DcIII和plako三种桥粒蛋白参与桥粒组建。在鼠囊胚中DcII的表达部位位于     

Uterine histotroph and conceptus development: select nutrients and secreted phosphoprotein 1 affect mechanistic target of rapamycin cell signaling in ewes

Interferon tau (IFNT), the pregnancy recognition signal in ruminants, abrogates the uterine luteolytic mechanism to ensure maintenance of function for the corpora lutea to produce progesterone (P4). IFNT also suppresses expression of classical IFN-stimulated genes by uterine lumenal epithelium (LE) and superficial glandular (sGE) epithelium but, acting in concert with progesterone, affects expression of a multitude of genes critical to growth and development of the conceptus. The LE and sGE secrete proteins and transport nutrients into the uterine lumen necessary for conceptus development, pregnancy recognition signaling, and implantation. Secretions include arginine and secreted phosphoprotein 1 (SPP1). Arginine can be metabolized to nitric oxide and to polyamines or act directly to activate the mechanistic target of rapamycin cell signaling pathway to stimulate proliferation, migration, and mRNA translation in trophectoderm cells. SPP1 binds alphavbeta3 and alpha5beta1 integrins to induce focal adhesion assembly, adhesion, and migration of conceptus trophectoderm cells during implantation. Thus, arginine and SPP1 mediate growth, migration, cytoskeletal remodeling, and adhesion of trophectoderm essential for pregnancy recognition signaling and implantation. This minireview focuses on components of histotroph that affect conceptus development in the ewe.     

Simulating the mammalian blastocyst--molecular and mechanical interactions pattern the embryo

Mammalian embryogenesis is a dynamic process involving gene expression and mechanical forces between proliferating cells. The exact nature of these interactions, which determine the lineage patterning of the trophectoderm and endoderm tissues occurring in a highly regulated manner at precise periods during the embryonic development, is an area of debate. We have developed a computational modeling framework for studying this process, by which the combined effects of mechanical and genetic interactions are analyzed within the context of proliferating cells. At a purely mechanical level, we demonstrate that the perpendicular alignment of the animal-vegetal (a-v) and embryonic-abembryonic (eb-ab) axes is a result of minimizing the total elastic conformational energy of the entire collection of cells, which are constrained by the zona pellucida. The coupling of gene expression with the mechanics of cell movement is important for formation of both the trophectoderm and the endoderm. In studying the formation of the trophectoderm, we contrast and compare quantitatively two hypotheses: (1) The position determines gene expression, and (2) the gene expression determines the position. Our model, which couples gene expression with mechanics, suggests that differential adhesion between different cell types is a critical determinant in the robust endoderm formation. In addition to differential adhesion, two different testable hypotheses emerge when considering endoderm formation: (1) A directional force acts on certain cells and moves them into forming the endoderm layer, which separates the blastocoel and the cells of the inner cell mass (ICM). In this case the blastocoel simply acts as a static boundary. (2) The blastocoel dynamically applies pressure upon the cells in contact with it, such that cell segregation in the presence of differential adhesion leads to the endoderm formation. To our knowledge, this is the first attempt to combine cell-based spatial mechanical simulations with genetic networks to explain mammalian embryogenesis. Such a framework provides the means to test hypotheses in a controlled in silico environment.     

Differential expression of ezrin/radixin/moesin (ERM) and ERM-associated adhesion molecules in the blastocyst and uterus suggests their functions during implantation

Development of the blastocyst to implantation competency, differentiation of the uterus to the receptive state, and a cross talk between the implantation-competent blastocyst and the uterine luminal epithelium are all essential to the process of implantation. In the present investigation, we examined the possibility for a potential cross talk between the blastocyst and uterus involving the ezrin/radixin/moesin (ERM) proteins and ERM-associated cytoskeletal cross-linker proteins CD43, CD44, ICAM-1, and ICAM-2. In normal Day 4 blastocysts and after rendering dormant blastocysts to implantation-competent by estrogen in vivo (activated), the outer surface of mural trophectoderm cells showed much higher levels of radixin as compared to those in the polar trophectoderm cells, inner cell mass (ICM), and primitive endoderm. In contrast, ezrin was present on both the mural and the polar trophectoderm cell surfaces of normal Day 4 and activated blastocysts at higher intensity than dormant blastocysts. A distinct localization was noted in the primitive endoderm of dormant blastocysts that was not apparent in activated or normal Day 4 blastocysts. The expression of moesin was modestly higher at the mural trophectoderm of implantation-competent blastocysts, while the localization appeared to be present primarily on the polar trophectoderm cell surface of Day 4 blastocysts. The localization of ERM-associated adhesion molecules CD43, CD44, and ICAM-2 was more intense in the implantation-competent blastocysts compared with the dormant blastocysts. However, while CD44 was present both in the trophectoderm and in ICM, CD43 and ICAM-2 were localized primarily to the trophectoderm. The signal for ICAM-1 was very intense in the ICM but was modest in the trophectoderm. No significant changes in fluorescence intensity were noted between activated and dormant blastocysts. In the receptive uterus on Day 4 of pregnancy, ERM proteins were localized to the uterine epithelium, while on Day 5 the localization, especially of radixin and moesin, extended to the stroma surrounding the implantation chamber. With respect to ERM-associated adhesion molecules, while CD44 and ICAM-1 were exclusively localized in the stroma on Day 4, CD43 and ICAM-2 were localized to the epithelium. On Day 5, the localization of CD44 and ICAM-1 became highly concentrated in the antimesometrial stroma of the implantation chamber. The localization of CD43 and ICAM-2 remained mostly epithelial, although some stromal localization of CD43 was noted on Day 5. These results suggest that differential expression and distribution of ERM proteins and ERM-associated adhesion molecules are involved in the construction of the cellular architecture necessary for blastocyst activation and uterine receptivity leading to successful implantation.     

Development of tight junctions de novo in the mouse early embryo: control of assembly of the tight junction-specific protein, ZO-1

Tight junction development during trophectoderm biogenesis in the mouse preimplantation embryo has been examined using monoclonal antibodies recognizing the tight junction-specific peripheral membrane protein, ZO-1. In immunoblots, mouse embryo ZO-1 had a molecular mass (225 kD) equivalent to that in mouse liver, was barely detectable in four-cell embryos although later stages exhibited increasing levels. ZO-1 was first detected immunocytochemically at the compacting eight-cell stage, coincident with or just after the expression of basolateral cell adhesion and apical microvillous polarity. Initially, ZO-1 was present as a series of spots along the boundary between free and apposed cell surfaces in intact embryos or cell couplets, but subsequently staining became more linear with blastocyst trophectoderm cells being bordered by a continuous ZO-1 belt. Inhibition of cell adhesion at the 8-cell stage delayed ZO-1 appearance and randomized its surface distribution in a reversible manner. Microfilament disruption, but not microtubule depolymerization, produced major disturbances in ZO-1 distribution. ZO-1 assembly de novo appeared to be independent of proximate DNA and RNA synthesis but was inhibited substantially in the absence of protein synthesis during the eight-cell stage, a treatment that did not prevent intercellular adhesion and polarization. ZO-1 surface assembly, but not adhesion and polarization, was also perturbed when single eight-cells were combined with single four-cells. The results suggest that tight junction development in mouse embryos is a secondary event in epithelial biogenesis, being dependent upon cell adhesion and cytoskeletal activity for normal expression, and can be disrupted without disturbing the generation of a stably polarized phenotype.     

Induction of trophinin in human endometrial surface epithelia by CGbeta and IL-1beta

During embryo implantation, trophinin mediates cell adhesion by homophilic binding at the apical surfaces of trophectoderm and endometrium. Trophinin is expressed on the human endometrial epithelia in rare occasions. We developed hCG-coated agarose beads that mimic the physical and physiological features of an implantation-stage human blastocyst. When hCG-coated beads were applied to human endometrial epithelial cells in the presence of IL-1beta, endometrial cells acquired strong trophinin expression and the ability for apical cell adhesion with trophinin-expressing human trophoblastic cells. These results provide a mechanism for trophinin-mediated adhesion of human blastocyst to endometrium by a spatially and temporally restricted paracrine effect of hCG derived from the blastocyst.     

Differentiation of an epithelium: factors affecting the polarized distribution of Na+,K(+)-ATPase in mouse trophectoderm

Na+,K(+)-ATPase is a marker of the basolateral plasma membrane domain of polarized epithelial cells, including the mural trophectoderm of the mammalian blastocyst (Watson and Kidder (1988). Dev. Biol. 126, 80-90). We have used this marker to explore the factors governing the establishment and maintenance of apical/basolateral polarity during differentiation of trophectoderm. A polyclonal antiserum (anti-GP80) against human cell-CAM 120/80, a homolog of the mouse cell-cell adhesion protein, uvomorulin, was used to prevent cell flattening (compaction) and formation of the epithelial junctional complex. The majority of treated embryos failed to develop a blastocoel; instead their blastomeres developed fluid-filled cavities that expanded while untreated control embryos were cavitating. Immunocytochemistry revealed that the catalytic subunit of Na+,K(+)-ATPase was contained within the membranes lining these cavities, as well as within numerous punctate foci in the cytoplasm. The down-regulation of expression of the enzyme that normally occurs in the ICM and polar trophectoderm did not take place, since the immunoreactivity remained equally strong in all blastomeres. The enzyme could not be detected in plasma membranes. We conclude that uvomorulin-mediated cell adhesion is involved in spatially restricting the expression of the catalytic subunit and is a prerequisite for the insertion of enzyme-laden vesicles into plasma membranes, but not for expression of the catalytic subunit gene. When fully developed blastocysts were treated with cytochalasins to disrupt the epithelial junctional complex, the catalytic subunit shifted from the basolateral to the apical plasma membrane. This finding suggests a primary role for the apical plasma membrane in the process of polarization, and implies that tight junctions are a manifestation of polarity that serve to maintain the separation between apical and basolateral markers.     

Self‐organization of human iPS cells into trophectoderm mimicking cysts induced by adhesion restriction using microstructured mesh scaffolds

Cellular dynamics leading to the formation of the trophectoderm in humans remain poorly understood owing to limited accessibility to human embryos for research into early human embryogenesis. Compared to animal models, organoids formed by self‐organization of stem cells in vitro may provide better insights into differentiation and complex morphogenetic processes occurring during early human embryogenesis. Here we demonstrate that modulating the cell culture microenvironment alone can trigger self‐organization of human induced pluripotent stem cells (hiPSCs) to yield trophectoderm‐mimicking cysts without chemical induction. To modulate the adhesion microenvironment, we used the mesh culture technique recently developed by our group, which involves culturing hiPSCs on suspended micro‐structured meshes with limited surface area for cell adhesion. We show that this adhesion‐restriction strategy can trigger a two‐stage self‐organization of hiPSCs; first into stem cell sheets, which express pluripotency signatures until around day 8–10, then into spherical cysts following differentiation and self‐organization of the sheet‐forming cells. Detailed morphological analysis using immunofluorescence microscopy with both confocal and two‐photon microscopes revealed the anatomy of the cysts as consisting of a squamous epithelial wall richly expressing E‐cadherin and CDX2. We also confirmed that the cysts exhibit a polarized morphology with basal protrusions, which show migratory behavior when anchored. Together, our results point to the formation of cysts which morphologically resemble the trophectoderm at the late‐stage blastocyst. Thus, the mesh culture microenvironment can initiate self‐organization of hiPSCs into trophectoderm‐mimicking cysts as organoids with potential application in the study of early embryogenesis and also in drug development.     

Ovine osteopontin: II. Osteopontin and alpha(v)beta(3) integrin expression in the uterus and conceptus during the periimplantation period.

Osteopontin (OPN) is an acidic 70-kDa glycoprotein that is cleaved by proteases to yield 45-kDa and 24-kDa fragments. The 70-kDa and 45-kDa proteins contain a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence that binds to cell surface integrins (primarily alpha(v)beta(3) heterodimer) to promote cell-cell attachment and cell spreading. A 70-kDa acidic protein was previously detected by two-dimensional (2D) PAGE in Day 17 pregnant endometrial cytosolic extracts using Stainsall and identified as immunoreactive OPN using Western blotting. Three forms of immunoreactive OPN proteins (70, 45, and 24 kDa) were detected by 1D PAGE and Western blot analysis of endometrial extracts. OPN protein in endometrial extracts did not differ between cyclic and pregnant ewes. However, the amount of 45-kDa OPN increased in uterine flushings from pregnant ewes between Days 11 and 17. Immunoreactive OPN was localized to luminal and glandular epithelia of both cyclic and pregnant ewes, and to trophectoderm of Day 19 conceptuses. The alpha(v) and beta(3) integrins were detected on Day 19 endometrium and conceptuses by immunofluorescence. It was reported that OPN mRNA increases in the uterine glands of pregnant ewes and secretion of OPN protein into the uterine lumen increases during early pregnancy. The present results demonstrate accumulation of OPN protein on endometrial LE and conceptus trophectoderm. Therefore, it is hypothesized that progesterone and/or interferon-tau induce expression, secretion and/or proteolytic cleavage of OPN by uterine epithelium. Secreted OPN is then available as ligand for alpha(v)beta(3) integrin heterodimer on trophectoderm and uterus to 1) stimulate changes in morphology of conceptus trophectoderm and 2) induce adhesion between luminal epithelium and trophectoderm essential for implantation and placentation.     

Signal Transduction in Human Embryo Implantation

Determining the molecular mechanism of human embryo implantation is an extremely challenging task due to the limitation of materials and significant differences in this process among mammalian species. Trophinin has been identified as an apical cell adhesion molecule with potential involvement in human embryo implantation. We found that trophinin-mediated cell adhesion triggers signal transduction in human trophoblastic cells for proliferation and invasion, implicating in trophectoderm cell activation for placental formation. Prior to cell adhesion trophinin arrests ErbB4 by binding through bystin, which prevents ErbB4 from activation. Trophinin-mediated cell adhesion causes dissociation of bystin from trophinin, freeing ErbB4 from arrest and enabling tyrosine phosphorylation. Therefore trophinin functions as an adhesion molecule on the cell surface and as a molecular switch for trophoblast activation in the cytoplasm.     

Trophinin-mediated cell adhesion induces apoptosis of human endometrial epithelial cells through PKC-δ

Trophinin is an intrinsic membrane protein expressed in trophectoderm cells of embryos and in uterine epithelial cells. Trophinin potentially mediates apical cell adhesion at human embryo implantation sites through trophinin-trophinin binding in these two cell types. Trophinin-mediated cell adhesion activates trophectoderm cells for invasion, whereas the effect of adhesion on maternal side is not known. We show that addition of GWRQ peptide, a previously established peptide that mimics trophinin-mediated cell adhesion, to human endometrial epithelial cells expressing trophinin induces their apoptosis. FAS involvement was excluded, as GWRQ did not bind to FAS, and FAS knockdown did not alter GWRQ-induced apoptosis. Immunoblotting analyses of protein kinases revealed an elevation of PKC-δ protein in GWRQ-bound endometrial epithelial cells. In the absence of GWRQ, PKC-δ associated with trophinin and remained cytoplasmic, but after GWRQ binding to the trophinin extracellular domain, PKC-δ became tyrosine phosphorylated, dissociated from trophinin and entered the nucleus. In PKC-δ knockdown endometrial cells, GWRQ did not induce apoptosis. These results suggest that trophinin-mediated cell adhesion functions as a molecular switch to induce apoptosis through the PKC-δ pathway in endometrial epithelial cells. Thus, trophinin-mediated induction of apoptosis of endometrial epithelial cells, which function as a barrier to embryo invasion, allows trophoblast invasion of maternal tissue and embryo implantation in humans.Key words: blastocyst, embryo implantation, apoptosis, cell adhesion, signal transduction