Uterine Rbpj is required for embryonic-uterine orientation and decidual remodeling via Notch pathway-independent and -dependent mechanisms

Coordinated uterine-embryonic axis formation and decidual remodeling are hallmarks of mammalian post-implantation embryo development. Embryonic-uterine orientation is determined at initial implantation and synchronized with decidual development. However, the molecular mechanisms controlling these events remain elusive despite its discovery a long time ago. In the present study, we found that uterine-specific deletion of Rbpj, the nuclear transducer of Notch signaling, resulted in abnormal embryonic-uterine orientation and decidual patterning at post-implantation stages, leading to substantial embryo loss. We further revealed that prior to embryo attachment, Rbpj confers on-time uterine lumen shape transformation via physically interacting with uterine estrogen receptor (ERα) in a Notch pathway-independent manner, which is essential for the initial establishment of embryo orientation in alignment with uterine axis. While at post-implantation stages, Rbpj directly regulates the expression of uterine matrix metalloproteinase in a Notch pathway-dependent manner, which is required for normal post-implantation decidual remodeling. These results demonstrate that uterine Rbpj is essential for normal embryo development via instructing the initial embryonic-uterine orientation and ensuring normal decidual patterning in a stage-specific manner. Our data also substantiate the concept that normal mammalian embryonic-uterine orientation requires proper guidance from developmentally controlled uterine signaling.     

The chick embryo chorioallantoic membrane as a model for tumor biology

Among the in vivo models, the chick embryo chorioallantoic membrane (CAM) has been used to implant several tumor types as well as malignant cell lines to study their growth rate, angiogenic potential and metastatic capability. This review article is focused on the major compelling literature data on the use of the CAM to investigate tumor growth and the metastatic process.     

Cripto-independent Nodal signaling promotes positioning of the A-P axis in the early mouse embryo

During early mouse development, the TGFβ-related protein Nodal specifies the organizing centers that control the formation of the anterior-posterior (A-P) axis. EGF-CFC proteins are important components of the Nodal signaling pathway, most likely by acting as Nodal coreceptors. However, the extent to which Nodal activity depends on EGF-CFC proteins is still debated. Cripto is the earliest EGF-CFC gene expressed during mouse embryogenesis and is involved in both A-P axis orientation and mesoderm formation. To investigate the relation between Cripto and Nodal in the early mouse embryo, we removed the Nodal antagonist Cerberus 1 (Cer1) and simultaneously Cripto, by generating Cer1;Cripto double mouse mutants. We observed that two thirds of the Cer1;Cripto double mutants are rescued in processes that are severely compromised in Cripto^−/− embryos, namely A-P axis orientation, anterior mesendoderm and posterior neuroectoderm formation. The observed rescue is strongly reduced in Cer1;Cripto;Nodal triple mutants, suggesting that Nodal can signal extensively in the absence of Cripto, if Cer1 is also inhibited. This signaling activity drives A-P axis positioning. Our results provide evidence for the existence of Cripto-independent signaling mechanisms, by which Nodal controls axis specification in the early mouse embryo.     

The anterior-posterior axis emerges respecting the morphology of the mouse embryo that changes and aligns with the uterus before gastrulation

BACKGROUND: When the anterior-posterior axis of the mouse embryo becomes explicit at gastrulation, it is almost perpendicular to the long uterine axis. This led to the belief that the uterus could play a key role in positioning this future body axis. RESULTS: Here, we demonstrate that when the anterior-posterior axis first emerges it does not respect the axes of the uterus but, rather, the morphology of the embryo. Unexpectedly, the emerging anterior-posterior axis is initially aligned not with the long, but the short axis of the embryo. Then whether the embryo develops in vitro or in utero, the anterior-posterior axis becomes aligned with the long axis of embryo just prior to gastrulation. Of three mechanisms that could account for this apparent shift in anterior-posterior axis orientation-cell migration, spatial change of gene expression, or change in embryo shape-lineage tracing studies favor a shape change accompanied by restriction of the expression domain of anterior markers. This property of the embryo must be modulated by interactions with the uterus as ultimately the anterior-posterior and long axes of the embryo align with the left-right uterine axis. CONCLUSIONS: The emerging anterior-posterior axis relates to embryo morphology rather than that of the uterus. The apparent shift in its orientation to align with the long embryonic axis and with the uterus is associated with a change in embryo shape and a refinement of anterior gene expression pattern. This suggests an interdependence between anterior-posterior gene expression, the shape of the embryo, and the uterus.     

Numerical simulation of embryo transfer: how the viscosity of transferred medium affects the transport of embryos

Background

Embryo transfer (ET) is a key step of assisted reproductive procedures, where the transferred medium containing the embryos is injected into the uterine cavity through a transcervical catheter and blended with intrauterine fluid in the uterine cavity. This procedure determines the delivery sites of embryos in the uterine cavity and has crucial impact on the implantation. Due to practical restrictions and ethical issues, it is often difficult to perform an in vivo study in humans to examine factors that affect the motions and delivery of embryos during ET. Alternatively, mathematical modeling is a powerful tool to that end.

Results

A computational model is developed to simulate the intrauterine mixing flow and track the embryo motions. Two important factors affecting the intrauterine flow are studied via this model: the viscosity of the transferred medium and the injection speed. Numerical results show that the dispersion pattern and the final delivery sites of the embryos are significantly influenced by the viscosity of the transferred medium. Specially, increasing the transferred medium viscosity close to that of the uterine fluid can enhance the probability that the embryos are delivered close to the fundus and keep them from being dragged backward to the cervix during catheter withdrawal. In addition, a slow injection speed can lower the driving force on the embryo during ET, which can prevent the embryo from being injured.

Conclusions

Based on our study, the practice of using a transferred medium with similar viscosity to that of the uterine fluid and a slow injection speed is recommended for real embryo transfer procedures in clinic.

     

Spatial and temporal alterations of phospholipids determined by mass spectrometry during mouse embryo implantation

Molecular events involved in successful embryo implantation are not well understood. In this study, we used MALDI imaging mass spectrometry (IMS) technologies to characterize the spatial and temporal distribution of phospholipid species associated with mouse embryo implantation. Molecular images showing phospholipid distribution within implantation sites changed markedly between distinct cellular areas during days 4–8 of pregnancy. For example, by day 8, linoleate- and docosahexaenoate-containing phospholipids localized to regions destined to undergo cell death, whereas oleate-containing phospholipids localized to angiogenic regions. Arachidonate-containing phospholipids showed different segregation patterns depending on the lipid class, revealing a strong correlation of phosphatidylethanolamines and phosphatidylinositols with cytosolic phospholipase A_2α and cyclooxygenase-2 during embryo implantation. LC-ESI-MS/MS was used to validate MALDI IMS phospholipid distribution patterns. Overall, molecular images revealed the dynamic complexity of lipid distributions in early pregnancy, signifying the importance of complex interplay of lipid molecules in uterine biology and implantation.     

The chick embryo: hatching a model for contemporary biomedical research

Animal models play a crucial role in fundamental and medical research. Progress in the fields of drug discovery, regenerative medicine and cancer research among others are heavily dependent on in vivo models to validate in vitro observations, and develop new therapeutic approaches. However, conventional rodent and large animal experiments face ethical, practical and technical issues that limit their usage. The chick embryo represents an accessible and economical in vivo model, which has long been used in developmental biology, gene expression analysis and loss/gain of function experiments. It is also an established model for tissue/cell transplantation, and because of its lack of immune system in early development, the chick embryo is increasingly recognised as a model of choice for mammalian biology with new applications for stem cell and cancer research. Here, we review novel applications of the chick embryo model, and discuss future developments of this in vivo model for biomedical research.     

Mucin and proteoglycan functions in embryo implantation

Embryo implantation is a complex series of events that involves changes in pattern of expression of embryonic as well as uterine cell surface components. In the case of the embryo, these changes are driven by the developmental program. In the case of the uterus, these changes are triggered by both maternal hormonal influences as well as embryo-derived factors. Aspects of the implantation process vary among species; however, interaction between the external surface of the embryonic trophectoderm and the apical surface of the lumenal uterine epithelium is a common event. Progress is being made in defining the molecular players in these cell surface interactions. Large-molecular-weight mucin glycoproteins such as MUC1 are present at the apical surface of the uterine epithelium under most conditions. Under most circumstances, these mucins appear to protect the mucosal surface from infection and the action of degradative enzymes. These mucins are antiadhesive and also appear to represent a barrier to embryo attachment. Consistent with this model, reduction of mucin expression is observed in uterine lumenal epithelia in many species. Nonetheless, mucin expression persists in the human uterus during the proposed receptive phase. It is possible that mucin loss is localized to implantation sites in humans. Alternatively, mucins may function differently within the context of human implantation than in other species. Studies primarily performed in mice indicate that heparan sulfate proteoglycans, in particular, perlecan, appears on the exterior trophectodermal surface coincident with the acquisition of attachment competence. Various in vitro studies indicate that heparan sulfate proteoglycans support embryo attachment activity that may represent an early event in embryo–uterine interaction. Uterine epithelia cells express several complementary heparan sulfate-binding proteins that may participate in these attachment processes. Use of molecular genetic approaches in mouse models, as well as careful studies of the expression and function of these molecules in the context of implantation in various species are beginning to shed light on the key molecular events of implantation. BioEssays 20 :577–583, 1998. © 1998 John Wiley & Sons Inc.     

Germ cell migration in zebrafish is cyclopamine-sensitive but Smoothened-independent

Primordial germ cells (PGCs) are the progenitors of reproductive cells in metazoans and are an important model for the study of cell migration in vivo. Previous reports have suggested that Hedgehog (Hh) protein acts as a chemoattractant for PGC migration in the Drosophila embryo and that downstream signaling proteins such as Patched (Ptc) and Smoothened (Smo) are required for PGC localization to somatic gonadal precursors. Here we interrogate whether Hh signaling is required for PGC migration in vertebrates, using the zebrafish as a model system. We find that cyclopamine, an inhibitor of Hh signaling, causes strong defects in the migration of PGCs in the zebrafish embryo. However, these defects are not due to inhibition of Smoothened (Smo) by cyclopamine; rather, we find that neither maternal nor zygotic Smo is required for PGC migration in the zebrafish embryo. Cyclopamine instead acts independently of Smo to decrease the motility of zebrafish PGCs, in part by dysregulating cell adhesion and uncoupling cell polarization and translocation. These results demonstrate that Hh signaling is not required for zebrafish PGC migration, and underscore the importance of regulated cell-cell adhesion for cell migration in vivo.     

The effect on preimplantation embryo development of non-specific inflammation localized outside the reproductive tract

The aim of this study was to evaluate the possible effect of non-specific acute inflammation localized outside the reproductive tract on the quality of preimplantation embryos. In fertilized female mice two experimental models of inflammation were used—trinitrobenzene sulfonic acid colitis and carrageenan paw oedema. Inflammation was induced during the cleavage period of embryo development and embryos were collected at 92 h post hormonal synchronization. Stereomicroscopical evaluation of in vivo derived embryos showed that the presence of inflammation in the maternal body did not affect their basic developmental abilities, i.e. there were no significant differences in the proportion of early blastocysts, morulas, slowly developing embryos and degenerates between embryonic pools obtained from mothers with induced inflammation and control mothers. In the next step, non-degenerated embryos from all mothers were cultured in vitro under standard conditions for another 24 h, and the average cell number (fluorescence DNA staining) and the incidence of cell death (fluorescence viability staining combined with TUNEL assay) were evaluated. The majority of cultured embryos reached expanded blastocyst stage. There were no significant differences in the average cell numbers of blastocysts, but blastocysts derived from mothers with induced inflammation showed a significantly higher incidence of dead cells in both experiments. The majority of dead cells were of apoptotic origin. These results show that non-specific inflammation localized outside the reproductive tract has no detrimental effect on the preimplantation embryo growth; however it can affect the embryo quality.     

Omics insights into extracellular vesicles in embryo implantation and their therapeutic utility

Implantation success relies on intricate interplay between the developing embryo and the maternal endometrium. Extracellular vesicles (EVs) represent an important player of this intercellular signalling through delivery of functional cargo (proteins and RNAs) that reprogram the target cells protein and RNA landscape. Functionally, the signalling reciprocity of endometrial and embryo EVs regulates the site of implantation, preimplantation embryo development and hatching, antioxidative activity, embryo attachment, trophoblast invasion, arterial remodelling, and immune tolerance. Omics technologies including mass spectrometry have been instrumental in dissecting EV cargo that regulate these processes as well as molecular changes in embryo and endometrium to facilitate implantation. This has also led to discovery of potential cargo in EVs in human uterine fluid (UF) and embryo spent media (ESM) of diagnostic and therapeutic value in implantation success, fertility, and pregnancy outcome. This review discusses the contribution of EVs in functional hallmarks of embryo implantation, and how the integration of various omics technologies is enabling design of EV-based diagnostic and therapeutic platforms in reproductive medicine.