Characterisation of Osteopontin in an In Vitro Model of Embryo Implantation

At the onset of pregnancy, embryo implantation is initiated by interactions between the endometrial epithelium and the outer trophectoderm cells of the blastocyst. Osteopontin (OPN) is expressed in the endometrium and is implicated in attachment and signalling roles at the embryo–epithelium interface. We have characterised OPN in the human endometrial epithelial Ishikawa cell line using three different monoclonal antibodies, revealing at least nine distinct molecular weight forms and a novel secretory pathway localisation in the apical domain induced by cell organisation into a confluent epithelial layer. Mouse blastocysts co-cultured with Ishikawa cell layers served to model embryo apposition, attachment and initial invasion at implantation. Exogenous OPN attenuated initial, weak embryo attachment to Ishikawa cells but did not affect the attainment of stable attachment. Notably, exogenous OPN inhibited embryonic invasion of the underlying cell layer, and this corresponded with altered expression of transcription factors associated with differentiation from trophectoderm (Gata2) to invasive trophoblast giant cells (Hand1). These data demonstrate the complexity of endometrial OPN forms and suggest that OPN regulates embryonic invasion at implantation by signalling to the trophectoder

Osmotic stress induces JNK-dependent embryo invasion in a model of implantation

In vitro culture during assisted reproduction technologies (ARTs) exposes pre-implantation embryos to environmental stressors, such as non-physiological nutritional, oxidative and osmotic conditions. The effects on subsequent implantation are not well understood but could contribute to poor ART efficiency and outcomes. We have used exposure to hyperosmolarity to investigate the effects of stress on the ability of embryos to interact with endometrial cells in an in vitro model. Culturing mouse blastocysts for 2 h in medium with osmolarity raised by 400 mosmol induced blastocoel collapse and re-expansion, but did not affect subsequent attachment to, or invasion of, the endometrial epithelial Ishikawa cell line. Inhibition of stress-responsive c-Jun N-terminal kinase (JNK) activity with SP600125 did not affect the intercellular interactions between these embryos and the epithelial cells. Four successive cycles of hyperosmotic stress at E5.5 had no effect on attachment, but promoted embryonic breaching of the epithelial cell layer by trophoblast giant cells in a JNK-dependent manner. These findings suggest that acute stress at the blastocyst stage may promote trophoblast breaching of the endometrial epithelium at implantation and implicates stress signalling through JNK in the process of trophectoderm differentiation into the invasive trophoblast necessary for the establishment of pregnancy. The data may lead to increased understanding of factors governing ART success rates and safety

High pressure phase diagrams of CeRhIn5_5 and CeCoIn5_5 studied by ac calorimetry

The pressure-temperature phase diagrams of the heavy fermion antiferromagnet CeRhIn$_5$ and the heavy fermion superconductor CeCoIn$_5$ have been studied under hydrostatic pressure by ac calorimetry and ac susceptibility measurements using diamond anvil cells with argon as pressure medium. In CeRhIn$_5$, the use of a highly hydrostatic pressure transmitting medium allows for a clean simultaneous determination by a bulk probe of the antiferromagnetic and superconducting transitions. We compare our new phase diagram with the previous ones, discuss the nature (first or second order) of the various lines, and the coexistence of antiferromagnetic order and superconductivity. The link between the collaps of the superconducting heat anomaly and the broadening of the antiferromagnetic transition points to an inhomogeneous appearence of superconductivity below $P_c \approx 1.95$ GPa. Homogeneous bulk superconductivity is only observed above this critical pressure. We present a detailed analysis of the influence of pressure inomogeneities on the specific heat anomalies which emphasizes that the observed broadening of the transitions near $P_c$ is connected with the first order transition. For CeCoIn$_5$ we show that the large specific heat anomaly observed at $T_c$ at ambient pressure is suppressed linearly at least up to 3 GPa

Apposition to endometrial epithelial cells activates mouse blastocysts for implantation.

How do interactions between blastocyst-stage embryos and endometrial epithelial cells regulate the early stages of implantation in an in vitro model?Mouse blastocyst apposition with human endometrial epithelial cells initiates trophectoderm differentiation to trophoblast, which goes on to breach the endometrial epithelium.In vitro models using mouse blastocysts and human endometrial cell lines have proven invaluable in the molecular characterisation of embryo attachment to endometrial epithelium at the onset of implantation. Genes involved in embryonic breaching of the endometrial epithelium have not been investigated in such in vitro models.This study used an established in vitro model of implantation to examine cellular and molecular interactions during blastocyst attachment to endometrial epithelial cells.Mouse blastocysts developed from embryonic day (E) 1.5 in vitro were hatched and co-cultured with confluent human endometrial adenocarcinoma-derived Ishikawa cells in serum-free medium. A scale of attachment stability based on blastocyst oscillation upon agitation was devised. Blastocysts were monitored for 48 h to establish the kinetics of implantation, and optical sectioning using fluorescence microscopy revealed attachment and invasion interfaces. Quantitative PCR was used to determine blastocyst gene expression. Data from a total of 680 mouse blastocysts are reported, with 3-6 experimental replicates. T-test and ANOVA analyses established statistical significance at P < 0.05, P < 0.01 and P < 0.001.Hatched E4.5 mouse blastocysts exhibited weak attachment to confluent Ishikawa cells over the first 24 h of co-culture, with intermediate and stable attachment occurring from 28 h (E5.5 + 4 h) in a hormone-independent manner. Attached embryos fixed after 48 h (E6.5) frequently exhibited outgrowths, characterised morphologically and with antibody markers as trophoblast giant cells (TGCs), which had breached the Ishikawa cell layer. Beginning co-culture at E5.5 also resulted in intermediate and stable attachment from E5.5 + 4 h; however, these embryos did not go on to breach the Ishikawa cell layer, even when co-culture was extended to E7.5 (P < 0.01). Blastocysts cultured from E4.5 in permeable transwell inserts above Ishikawa cells before transfer to direct co-culture at E5.5 went on to attach but failed to breach the Ishikawa cell layer by E6.5 (P < 0.01). Gene expression analysis at E5.5 demonstrated that direct co-culture with Ishikawa cells from E4.5 resulted in downregulation of trophectoderm transcription factors Cdx2 (P < 0.05) and Gata3 (P < 0.05) and upregulation of the TGC transcription factor Hand1 (P < 0.05). Co-culture with non-endometrial human fibroblasts did not alter the expression of these genes.None.The in vitro model used here combines human carcinoma-derived endometrial cells with mouse embryos, in which the cellular interactions observed may not fully recapitulate those in vivo. The data gleaned from such models can be regarded as hypothesis-generating, and research is now needed to develop more sophisticated models of human implantation combining multiple primary endometrial cell types with surrogate and real human embryos.This study implicates blastocyst apposition to endometrial epithelial cells as a critical step in trophoblast differentiation required for implantation. Understanding this maternal regulation of the embryonic developmental programme may lead to novel treatments for infertility.This work was supported by funds from the charities Wellbeing of Women (RG1442) and Diabetes UK (15/0005207), and studentship support for SCB from the Anatomical Society. No conflict of interest is declared

Pecan Orchard Management.

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Metabolism of the viable mammalian embryo: quietness revisited

This review examines the ‘Quiet Embryo Hypothesis’ which proposes that viable preimplantation embryos operate at metabolite or nutrient turnover rates distributed within lower ranges than those of their less viable counterparts. The ‘quieter’ metabolism consistent with this hypothesis is considered in terms of (i) ‘functional’ quietness; the contrasting levels of intrinsic metabolic activity in different cell types as a consequence of their specialized functions, (ii) inter-individual embryo/cell differences in metabolism and (iii) loss of quietness in response to environmental stress. Data are reviewed which indicate that gametes and early embryos function in vivo at a lower temperature than core body temperature, which could encourage the expression of a quiet metabolism. We call for research to determine the optimum temperature for mammalian gamete/embryo culture. The review concludes by examining the key role of reactive oxygen species, which can induce molecular damage, trigger a cellular stress response and lead to a loss of quietness

Magnetic Exciton Mediated Superconductivity in the Hidden-Order Phase of URu2Si2

We propose the magnetic exciton mediated superconductivity occurring in the enigmatic hidden-order phase of URu2Si2. The characteristic of the massive collective excitation observed only in the hidden-order phase is well reproduced by the antiferro hexadecapole ordering model as the trace of the dispersive crystalline-electric-field excitation. The disappearance of the superconductivity in the high-pressure antiferro magnetic phase can naturally be understood by the sudden suppression of the magnetic-exciton intensity. The analysis of the momentum dependence of the magnetic-exciton mode leads to the exotic chiral d-wave singlet pairing in the Eg symmetry. The Ising-like magnetic-field response of the mode yields the strong anisotropy observed in the upper critical field even for the rather isotropic 3-dimensional Fermi surfaces of this compound.Comment: 5 pages, 4 figure

Pedestrian Approach to the Two-Channel Kondo Model

We reformulate the two-channel Kondo model to explicitly remove the unscattered charge degrees of freedom. This procedure permits us to move the non-Fermi liquid fixed point to infinite coupling where we can apply a perturbative strong-coupling expansion. The fixed point Hamiltonian involves a three-body Majorana zero mode whose scattering effects give rise to marginal self-energies. The compactified model is the N=3 member of a family of "O(N)" Kondo models that can be solved by semiclassical methods in the large $N$ limit. For odd $N$, {\em fermionic} "Kink" fluctuations about the $N=\infty$ mean-field theory generate a fermionic $N$-body bound-state which asymptotically decouples at low energies. For N=3, our semi-classical methods fully recover the non-Fermi liquid physics of the original two channel model. Using the same methods, we find that the corresponding O(3) Kondo lattice model develops a spin-gap and a gapless band of coherently propagating three-body bound-states. Its strong-coupling limit offers a rather interesting realization of marginal Fermi liquid behavior.Comment: 17 pages, Revtex 3.0. Replaced with fully compiled postscript file

Magnetic Penetration Depth in Unconventional Superconductors

This topical review summarizes various features of magnetic penetration depth in unconventional superconductors. Precise measurements of the penetration depth as a function of temperature, magnetic field and crystal orientation can provide detailed information about the pairing state. Examples are given of unconventional pairing in hole- and electron-doped cuprates, organic and heavy fermion superconductors. The ability to apply an external magnetic field adds a new dimension to penetration depth measurements. We discuss how field dependent measurements can be used to study surface Andreev bound states, nonlinear Meissner effects, magnetic impurities, magnetic ordering, proximity effects and vortex motion. We also discuss how penetration depth measurements as a function of orientation can be used to explore superconductors with more than one gap and with anisotropic gaps. Details relevant to the analysis of penetration depth data in anisotropic samples are also discussed.Comment: topical review, 57 pages, 219 reference

Maki Parameter and Upper critical Field of the Heavy-Fermion Superconductor UBe13

We have performed low-temperature specific-heat measurements in magnetic fields for a single crystal UBe13. It has been observed that our sample exhibits a superconducting transition at an intermediate temperature between previously reported values for two variant samples called H type and L type. The specific heat C(T) of our sample shows a T^3 behavior in the temperature range below 0.7 Tc, which is similar to the behavior of the H-type sample, suggesting the existence of point nodes in the superconducting gap function. We have obtained the upper-critical-field curves Hc2 for the [001], [110], and [111] crystal axes, which show no anisotropy at least down to the lowest measured temperature of 0.5 K. We have also derived the Maki parameter kappa2, and it has been revealed that the kappa2 steeply decreases isotropically upon cooling just below Tc. Paramagnetic effects and the symmetry of Cooper pairing of UBe13 are discussed.Comment: 4 pages, 5 figure