Diverse functions of HBEGF during pregnancy

The establishment of pregnancy requires an intimate physical interaction and a molecular dialogue between the conceptus and the maternal reproductive tract that commences at implantation and continues until the placenta is formed and fully functional. Failure of the regulatory processes that ensure the fidelity of this relationship can precipitate a catastrophic pregnancy loss. One of the earliest identified molecular mediators of blastocyst implantation is heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF), which signals between the endometrium and implanting trophoblast cells to synchronize their corresponding developmental programs. HBEGF expression by trophoblast cells of the developing placenta appears to regulate extravillous differentiation and provide cytoprotection in a sometimes-hostile environment. This versatile member of the EGF signaling system will be examined in light of its associations with key events during early pregnancy. Mol. Reprod. Dev. 76: 1116–1127, 2009. © 2009 Wiley-Liss, Inc.     

Morphological changes in the blastocyst of the western spotted skunk during activation from delayed implantation

Blastocysts collected from the spotted skunk during delay of implantation, early activation and late activation demonstrate three-tiered growth and developmental changes. The slow-growing blastocyst from the several months of delay is small (less than 1.1 mm) with a rounded inner cell mass consisting of clusters of rounded, lipid-filled cells. During the several days of early activation, the lipid in both inner cell mass and trophoblast diminishes, polyribosomes increase in number, and the endodermal layer differentiates as the blastocyst grows (1.2-1.6 mm). At activation the inner cell mass flattens, becomes uncovered by polar trophoblast, and forms a disc of columnar epiblast cells. The blastocyst expands rapidly during the last 24-48 h prior to implantation to 1.7-2.0 mm, and the trophoblast becomes cuboidal with a marked endocytotic apparatus. The morphological evidence, together with previous studies of protein and RNA synthesis, suggests a tooling-up period during early activation with progressive increases in rates of growth and differentiation in the last hours as implantation approaches.     

Trophoblast-specific expression and function of the integrin alpha 7 subunit in the peri-implantation mouse embryo.

For implantation and placentation to occur, mouse embryo trophoblast cells must penetrate the uterine stroma to make contact with maternal blood vessels. A major component of the uterine epithelial basement membrane and underlying stromal matrix with which they interact is the extracellular matrix protein laminin. We have identified integrin alpha 7 beta 1 as a major receptor for trophoblast-laminin interactions during implantation and yolk sac placenta formation. It is first expressed by trophectoderm cells of the late blastocyst and by all trophectoderm descendants in the early postimplantation embryo through E8.5, then disappears except in cells at the interface between the allantois and the ectoplacental plate. Integrin alpha 7 expression is a general characteristic of the early differentiation stages of rodent trophoblast, given that two different cultured trophoblast cell lines also express this integrin. Trophoblast cells interact with at least three different laminin isoforms (laminins 1, 2/4, and 10/11) in the blastocyst and in the uterus at the time of implantation. Outgrowth assays using function-blocking antibodies show that alpha 7 beta 1 is the major trophoblast receptor for laminin 1 and a functional receptor for laminins 2/4 and 10/11. When trophoblast cells are cultured on substrates of these three laminins, they attach and spread on all three, but show decreased proliferation on laminin 1. These results show that the alpha 7 beta 1 integrin is expressed by trophoblast cells and acts as receptor for several isoforms of laminin during implantation. These interactions are not only important for trophoblast adhesion and spreading but may also play a role in regulating trophectoderm proliferation and differentiation.     

Exogenous amino acids regulate trophectoderm differentiation in the mouse blastocyst through an mTOR-dependent pathway.

At the late blastocyst stage, the epithelial trophectoderm cells of the mammalian embryo undergo a phenotypic change that allows them to invade into the uterine stroma and make contact with the maternal circulation. This step can be regulated in vitro by the availability of amino acids. Embryos cultured in defined medium lacking amino acids cannot form trophoblast cell outgrowths on fibronectin, an in vitro model of implantation, but remain viable for up to 3 days in culture and will form outgrowths when transferred into complete medium. The amino acid requirement is a developmentally regulated permissive event that occurs during a 4- to 8-h period at the early blastocyst stage. Amino acids affect spreading competence specifically by regulating the onset of protrusive activity and not the onset of integrin activation. Rapamycin, a specific inhibitor of the kinase mTOR/FRAP/RAFT1, blocks amino acid stimulation of embryo outgrowth, demonstrating that mTOR is required for the initiation of trophectoderm protrusive activity. Inhibition of global protein translation with cycloheximide also inhibits amino acid-dependent signals, suggesting that mTOR regulates the translation of proteins required for trophoblast differentiation. Our data suggest that mTOR activity has a developmental regulatory function in trophectoderm differentiation that may serve to coordinate embryo and uterus at the time of implantation.     

Spatiotemporal expression of cyclooxygenase 1 and cyclooxygenase 2 during delayed implantation and the periimplantation period in the Western spotted skunk

Embryonic development in the western spotted skunk is arrested after blastocyst formation for about 200 days. This developmental arrest is believed to be due to insufficiency of uterine conditions to support continuous development. Implantation and decidualization are defective in cyclooxygenase 2 (Cox2)-, but not Cox1-, deficient mice. We therefore used Northern and in situ hybridization to investigate changes in uterine expression of Cox1 and Cox2 genes during various stages of pregnancy in the spotted skunk. Cox1 was constitutively expressed at all stages of pregnancy examined, but it did exhibit localized up-regulation in the trophoblast and necks of uterine glands at early implantation sites. Cox2 expression was highly regulated with little or no expression during delayed implantation. Cox2 expression was first detected in the uterus and trophoblast prior to blastocyst attachment and remained detectable for 5-6 days after blastocyst attachment. Cox2 expression was also localized in the luminal and glandular epithelia of uterine segments located between implantation chambers. Changes in Cox expression were not correlated with the abrupt increase in uterine weight that occurs simultaneously with renewed embryonic development but was correlated with an influx of serum proteins into the uterus observed in a previous study.     

Trophoblast cells exhibit differential responses to laminin isoforms

Extracellular matrix (ECM) has specific effects on cell behavior that influence many aspects of early development. In the early postimplantation mouse embryo the ECM component laminin promotes polarization and survival of the embryonic ectoderm and formation of Reichert's membrane. In addition, dynamic patterns of laminins 1 and 10/11 expression in the embryo and the uterus correlate with the progression of implantation. In the implanting blastocyst, laminin 1 is strongly expressed in the trophectoderm basement membrane, whereas laminin 10/11 is expressed only in the inner cell mass and polar trophectoderm. In the uterus, laminin 10/11 is strongly expressed in the decidualizing matrix of the stroma. We show here that laminins 1 and 10/11 have distinct effects on trophoblast cell behavior that influence the process of implantation. Laminin 1 promotes random migration and decreases spreading, whereas laminin 10/11 promotes both spreading and persistent migration. When presented as adjacent substrates, cells stop at the boundary and do not enter the region containing laminin 1. Laminin 1 also affects cell-cell adhesion through changes in the localization of vascular endothelial (VE) cadherin. Cultured cells and primary trophoblast explants become single cells or very small groups on laminin 1 and VE-cadherin localization at regions of cell-cell contact decreases dramatically. In contrast, trophoblast cells maintain strong cell-cell contacts on substrates of laminins 10/11, and exhibit strong staining of VE-cadherin in all regions of cell-cell contact. These effects, and the localization of laminin 1 in Reichert's membrane and laminin 10/11 in the surrounding decidual matrix, suggest that these laminin isoforms influence the direction and quality of invasion of trophoblast cells during implantation, and provide epigenetic cues that drive the morphogenesis of the yolk sac placenta.     

Amino acid transport regulates blastocyst implantation

Mouse blastocyst outgrowth in vitro and probably implantation in vivo require amino acid signaling via the target of rapamycin (TOR) pathway. This signaling does not simply support protein synthesis and trophoblast differentiation. Rather, it regulates development of trophoblast protrusive activity and may act as a developmental checkpoint for implantation. Moreover, intracellular amino acids per se are insufficient to elicit TOR signaling. Instead, de novo transport of amino acids, and particularly of leucine, stimulate mTOR activity at the blastocyst stage. The activity of the broad-scope and yet leucine-selective amino acid transport system B0,+ could produce such increases in intracellular amino acid concentrations. For example, system B0,+ uses a Na+ gradient to drive amino acid uptake, and the Na+ concentration in uterine secretions increases by nearly two-fold about 18 h before implantation. The resultant mTOR signaling could trigger polyamine, insulin-like growth factor II, and nitric oxide production in blastocysts and the increased cell motility sometimes associated with synthesis of these bioactive molecules.     

Alpha5beta1, alphaVbeta3 and the platelet-associated integrin alphaIIbbeta3 coordinately regulate adhesion and migration of differentiating mouse trophoblast cells

During blastocyst implantation, interaction between integrins on the apical surface of the trophoblast and extracellular matrix (ECM) in the endometrium anchors the embryo to the uterine wall. Strong adhesion of the blastocyst to fibronectin (FN) requires integrin signaling initiated by exogenous fibronectin. However, it is not known how integrin signaling enhances blastocyst adhesion. We present new evidence that the integrin, alphaIIbbeta3, plays a key role in trophoblast adhesion to fibronectin during mouse peri-implantation development. Trafficking of alphaIIb to the apical surface of the trophoblast increased dramatically after blastocysts were exposed to fibronectin, whereas other fibronectin-binding integrins, alpha5beta1 and alphaVbeta3, were resident at the apical surface before ligand exposure. Functional comparisons among the three integrins revealed that ligation of alpha5beta1 most efficiently strengthened blastocyst fibronectin-binding activity, while subsequent trophoblast cell migration was dependent primarily on the beta3-class integrins. In vivo, alphaIIb was highly expressed by invasive trophoblast cells in the ectoplacental cone and trophoblast giant cells of the parietal yolk sac. These data demonstrate that trafficking of alphaIIb regulates adhesion between trophoblast cells and fibronectin as invasion of the endometrium commences.