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Copyright of Forum: Qualitative Social Research / Qualitative Sozialforschung is the property of Forum Qualitative Social Research and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.09.037 Byline: Judith J. Eckert (a)(b), Amanda McCallum (a), Andrew Mears (a), Martin G. Rumsby (c), Iain T. Cameron (b), Tom P. Fleming (a) Keywords: Protein kinase C; Mouse embryo; Trophectoderm; Tight junction; ZO-1; Cell contact pattern; Inner cell mass; ZO-2 Abstract: In mouse early development, cell contact patterns regulate the spatial organization and segregation of inner cell mass (ICM) and trophectoderm epithelium (TE) during blastocyst morphogenesis. Progressive membrane assembly of tight junctional (TJ) proteins in the differentiating TE during cleavage is upregulated by cell contact asymmetry (outside position) and suppressed within the ICM by cell contact symmetry (inside position). This is reversible, and immunosurgical isolation of the ICM induces upregulation of TJ assembly in a sequence that broadly mimics that occurring during blastocyst formation. The mechanism relating cell contact pattern and TJ assembly was investigated in the ICM model with respect to PKC-mediated signaling and gap junctional communication. Our results indicate that complete cell contact asymmetry is required for TJ biogenesis and acts upstream of PKC-mediated signaling. Specific inhibition of two PKC isoforms, PKC[delta] and I[paragraph], revealed that both PKC activities are required for membrane assembly of ZO-2 TJ protein, while only PKCI[paragraph] activity is involved in regulating ZO-1[alpha]+ membrane assembly, suggesting different mechanisms for individual TJ proteins. Gap junctional communication had no apparent influence on either TJ formation or PKC signaling but was itself affected by changes of cell contact patterns. Our data suggest that the dynamics of cell contact patterns coordinate the spatial organization of TJ formation via specific PKC signaling pathways during blastocyst biogenesis. Author Affiliation: (a) University of Southampton, School of Biological Sciences, Bassett Crescent East, Southampton, SO16 7PX, UK (b) University of Southampton, Developmental Origins of Health and Disease Division, School of Medicine, Coxford Road, Southampton, SO16 5YA, UK (c) University of York, Department of Biology, York, YO1 5DD, UK Article History: Received 19 May 2005; Revised 1 September 2005; Accepted 22 September 2005

During early mammalian development, blastocyst morphogenesis is achieved by epithelial differentiation of trophectoderm (TE) and its segregation from the inner cell mass (ICM). Two major interrelated features of TE differentiation required for blastocoel formation include intercellular junction biogenesis and a directed ion transport system, mediated by [Na.sup.+]/[K.sup.+] ATPase. We have examined the relative contribution of intercellular signalling mediated by protein kinase C (PKC) and gap junctional communication in TE differentiation and blastocyst cavitation. The distribution pattern of four ([delta], [theta], [iota]/[lambda], [zeta]) PKC isoforms and PKC[micro]/PKD1 showed partial colocalisation with the tight junction marker ZO-1[alpha]+ in TE and all four PKCs ([delta], [theta], [iota]/[lambda], [zeta]) showed distinct TE/ICM staining patterns (predominantly at the cell membrane within the TE and cytoplasmic within the ICM), indicating their potential contribution to TE differentiation and blastocyst morphogenesis. Specific inhibition of PKC[delta] and [zeta] activity significantly delayed blastocyst formation. Although modulation of these PKC isoforms failed to influence the already established programme of epithelial junctional differentiation within the TE, [Na.sup.+]/[K.sup.+] ATPase [alpha]1 submit was internalised from membrane to cytoplasm. Inhibition of gap junctional communication, in contrast, had no influence on any of these processes. Our results demonstrate for the first time that distinct PKC isotypes contribute to the regulation of cavitation in preimplantation embryos via target proteins including [Na.sup.+]/[K.sup.+] ATPase. Keywords: Protein kinase C; Mouse embryo; Blastocyst; Trophectoderm; Inner cell mass; Tight junction; ZO-1; [Na.sup.+]/[K.sup.+] ATPase; Cavitation

Adverse programming of adult non-communicable disease can be induced by poor maternal nutrition during pregnancy and the periconception period has been identified as a vulnerable period. In the current study, we used a mouse maternal low-protein diet fed either for the duration of pregnancy (LPD) or exclusively during the preimplantation period (Emb-LPD) with control nutrition provided thereafter and postnatally to investigate effects on fetal bone development and quality. This model has been shown previously to induce cardiometabolic and neurological disease phenotypes in offspring. Micro 3D computed tomography examination at fetal stages Embryonic day E14.5 and E17.4, reflecting early and late stages of bone formation, demonstrated LPD treatment caused increased bone formation of relative high mineral density quality in males, but not females, at E14.5, disproportionate to fetal growth, with bone quality maintained at E17.5. In contrast, Emb-LPD caused a late increase in male fetal bone growth, proportionate to fetal growth, at E17.5, affecting central and peripheral skeleton and of reduced mineral density quality relative to controls. These altered dynamics in bone growth coincide with increased placental efficiency indicating compensatory responses to dietary treatments. Overall, our data show fetal bone formation and mineral quality is dependent upon maternal nutritional protein content and is sex-specific. In particular, we find the duration and timing of poor maternal diet to be critical in the outcomes with periconceptional protein restriction leading to male offspring with increased bone growth but of poor mineral density, thereby susceptible to later disease risk. [ABSTRACT FROM AUTHOR]

Copyright of Forum: Qualitative Social Research / Qualitative Sozialforschung is the property of Forum Qualitative Social Research and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Background The formation of two distinctive cell lineages in preimplantation mouse embryos is characterized by differential gene expression. The cells of the inner cell mass are pluripotent and express high levels of Oct4 mRNA, which is down-regulated in the surrounding trophectoderm. In contrast, the trophectoderm of female embryos contains Xist mRNA, which is absent from cells of the inner mass. Prior to blastocyst formation, all blastomeres of female embryos still express both of these RNAs. We, thus, postulated that simultaneous quantification of Oct4 and Xist transcripts in individual blastomeres at the 8-cell stage could be informative as to their subsequent fate. Testing this hypothesis, however, presented numerous technical challenges. We overcame these difficulties by combining PurAmp, a single-tube method for RNA preparation and quantification, with LATE-PCR, an advanced form of asymmetric PCR. Results We constructed a duplex RT-LATE-PCR assay for real-time measurement of Oct4 and Xist templates and confirmed its specificity and quantitative accuracy with different methods. We then undertook analysis of sets of blastomeres isolated from embryos at the 8-cell stage. At this stage, all cells in the embryo are still pluripotent and morphologically equivalent. Our results demonstrate, however, that both Oct4 and Xist RNA levels vary in individual blastomeres comprising the same embryo, with some cells having particularly elevated levels of either transcript. Analysis of multiple embryos also shows that Xist and Oct4 expression levels are not correlated at the 8-cell stage, although transcription of both genes is up-regulated at this time in development. In addition, comparison of data from males and females allowed us to determine that the efficiency of the Oct4/Xist assay is unaffected by sex-related differences in gene expression. Conclusion This paper describes the first example of multiplex RT-LATE-PCR and its utility, when combined with PurAmp sample preparation, for quantitative analysis of transcript levels in single cells. With this technique, copy numbers of different RNAs can be accurately measured independently from their relative abundance in a cell, a goal that cannot be achieved using symmetric PCR. The technique illustrated in this work is relevant to a wide array of applications, such as stem cell and cancer cell analysis and preimplantation genetic diagnostics.