Biomedical subjects

Susan J Kimber

Publications and source records attributed to Susan J Kimber.

11 recordsLinked to original sources

PubMed2006

The dynamic nature of mollusc egg surface architecture and its relation to the microtubule network.

Dynamic changes in the surface architecture pattern of embryos of the slipper limpet (Crepidula fornicata, Mollusca) were found in this study to correlate with the dynamic activity and pattern of the underlying mitotic spindle microtubule network, revealed by fluorescent labelling and confocal imaging techniques. Examination of a series of optical sections indicate that this network appears to be spatially co-ordinated together as a whole throughout the embryo. The microtubule pattern also associated with abnormal multipolar spindles resulting from an applied static magnetic field, indicating that the pattern may be generated by a natural endogenous field source. The patterning characteristics of the surface and microtubule network together provide further morphological evidence for a primary morphogenetic or developmental field system which organises the primary body axis and co-ordinates the pattern of cleavage.

Animals↗

PubMed2005

PTHrP induces changes in cell cytoskeleton and E-cadherin and regulates Eph/Ephrin kinases and RhoGTPases in murine secondary trophoblast cells.

The differentiation of murine trophoblast giant cells (TGCs) is well characterised at the molecular level and, to some extent, the cellular level. Currently, there is a rudimentary understanding about factors regulating the cellular differentiation of secondary TGCs. Using day 8.5 p.c.-ectoplacental cone (EPC) explant in serum-free culture, we have found parathyroid hormone-related protein (PTHrP) to regulate cellular changes during TGC differentiation. PTHrP greatly stimulated the formation and organisation of actin stress fibres and actin expression in trophoblast outgrowth. This coincided with changing cell shape into a flattened/fibroblastic morphology, suppression of E-cadherin expression, and increased cell spreading in culture. PTHrP also increased the nuclear staining of beta-catenin and, similar to activator protein-2gamma (AP-2gamma), showed microtubule-dependent nuclear localisation in vitro. These cellular and behavioural changes correlated with changes in the expression of RhoGTPases and in both expression and phosphorylation of Eph/Ephrin kinases. The effects of PTHrP on trophoblast cellular differentiation were abolished after blocking its action. In conclusion, PTHrP provides an excellent example of the extrinsic factors that, through their network of activities, plays an important role in cellular differentiation of secondary TGCs.

Actins↗

PubMed2005

PTHrP promotes murine secondary trophoblast giant cell differentiation through induction of endocycle, upregulation of giant-cell-promoting transcription factors and suppression of other trophoblast cell types.

The murine trophoblast cell lineage represents an intriguing experimental cell model as it is composed of four trophoblast stem (TS)-derived cell types: trophoblast giant cells (TGCs), spongiotrophoblast, syncytotrophoblast, and glycogen trophoblast cells. To investigate the role of parathyroid hormone-related protein (PTHrP) in TGC differentiation, we analyzed the effect of exogenous PTHrP on secondary TGCs of day 8.5 p.c. ectoplacental cone explant culture. Secondary TGCs expressed PTHrP and PTHR1 receptor in vivo and in vitro. TGCs treated with PTHrP had reduced proliferation and decreased apoptosis starting from day 2 in culture, and enhanced properties of giant cell differentiation: increased DNA synthesis, number of cells with giant nuclei and expression of placental lactogen-II (PL-II). The induction of TGC formation by PTHrP correlated with downregulation of cyclin B1 and mSNA expression, but upregulation of cyclin D1, thus allowing mitotic-endocycle transition. Moreover, PTHrP treatment influenced TGC differentiation by inducing the expression of transcription factors known to stimulate giant cell formation: Stra13 and AP-2gamma, and inhibiting the formation of other trophoblast cell types by suppressing trophoblast progenitors and spongiotrophoblast-promoting factors, Eomes, Mash-2, and mSNA. Taken together with the spatial and temporal patterns of TGC formation and PTHrP synthesis in vivo, these findings indicate an important role for PTHrP in the differentiation of secondary TGCs during placentation.

Animals↗

PubMed2005

Leukaemia inhibitory factor in implantation and uterine biology.

Leukaemia inhibitory factor (LIF) is one of the most important cytokines in the reproductive tract. Without expression of LIF in the uterus, implantation of a blastocyst cannot begin. Yet, 13 years after publication of the phenotype of the LIF knockout mouse we are only just beginning to understand how LIF functions in the uterus. This review addresses our knowledge of the role of LIF in regulating implantation through its influence on the luminal epithelium and stromal decidualization, but also its influence on reproductive tract cells such as leukocytes and glandular epithelium, during the pre-implantation phase of pregnancy.

Animals↗

PubMed2004

Leukocyte subpopulations in the uteri of leukemia inhibitory factor knockout mice during early pregnancy.

Leukemia inhibitory factor (LIF) is transiently expressed on Day (D) 1 of pregnancy by the uterine epithelium and on D4 specifically by the glandular epithelium. The Lif knockout female mice are infertile because of uterine defects that affect embryo implantation, but pregnancy can be rescued in these mice by injections of LIF on D4 of pregnancy. Many of the specific actions of LIF in the uterus are unknown, especially with regard to uterine cell biology. Leukocytes, such as macrophages, natural killer (NK) cells, and eosinophils, are present in the pregnant uterus and are thought to be beneficial, because alterations in their proportions can adversely affect pregnancy. Immunocytochemistry and cell counting were used to compare the distributions and dynamics of leukocyte subpopulations in wild-type and Lif knockout mice. The percentage of macrophages was reduced by more than half in the Lif knockout mice on D3 of pregnancy, and their distribution was disrupted, suggesting that LIF is a chemokine for these cells. The NK cells were detected as early as D3 of pregnancy, but the Lif knockout mice had double the percentage of NK cells compared to wild-type mice at this time, indicating that LIF restricts the migration of NK cells to the uterus. The Lif knockout mice also had significantly higher percentages of eosinophils in the outer stroma on D3, and in the midstroma on D4, of pregnancy, suggesting that LIF also may restrict eosinophil migration to the uterus. These alterations of the uterine leukocyte subpopulations in Lif knockout mice may disrupt pregnancy and contribute to failure of implantation.

Animals↗

PubMed2004

Trophoblast-uterine interactions at implantation.

Implantation of the embryo in the uterus is a critical and complex event and its failure is widely considered an impediment to improved success in assisted reproduction. Depending on whether placentation is invasive or superficial (epitheliochorial), the embryo may interact transiently or undergo a prolonged adhesive interaction with the uterine epithelium. Numerous candidate interactions have been identified, and there is good progress on identifying gene networks required for early placentation. However no molecular mechanisms for the epithelial phase are yet firmly established in any species. It is noteworthy that gene ablation in mice has so far failed to identify obligatory initial molecular events.

Animals↗

PubMed2004

Expression of connexins in human preimplantation embryos in vitro.

Intercellular communication via gap junctions is required to coordinate developmental processes in the mammalian embryo. We have investigated if the connexin (Cx) isoforms known to form gap junctions in rodent preimplantation embryos are also expressed in human embryos, with the aim of identifying species differences in communication patterns in early development. Using a combination of polyA PCR and immunocytochemistry we have assessed the expression of Cx26, Cx31, Cx32, Cx40, Cx43 and Cx45 which are thought to be important in early rodent embryos. The results demonstrate that Cx31 and Cx43 are the main connexin isoforms expressed in human preimplantation embryos and that these isoforms are co-expressed in the blastocyst. Cx45 protein is expressed in the blastocyst but the protein may be translated from a generally low level of transcripts: which could only be detected in the PN to 4-cell embryos. Interestingly, Cx40, which is expressed by the extravillous trophoblast in the early human placenta, was not found to be expressed in the blastocyst trophectoderm from which this tissue develops. All of the connexin isoforms in human preimplantation embryos are also found in rodents pointing to a common regulation of these connexins in development of rodent and human early embryos and perhaps other species.

Blastocyst↗

PubMed2004

Expression of 11 members of the BCL-2 family of apoptosis regulatory molecules during human preimplantation embryo development and fragmentation.

Apoptosis during preimplantation development has received much interest because of its potential role in eliminating defective cells. Although development in humans is characterised by a high degree of genetic abnormality, little is known of the regulation of apoptosis in embryos. By PolyA PCR we analysed expression of 11 BCL-2 genes in individual human embryos representative of normal development and in severely fragmented embryos. We demonstrate constitutive expression of BAX in virtually all embryos at all stages of development, and variable expression of BCL2, BCL-XL, BCL-W, MCL-1 BAK, BAD, BOKL, BID, BIK, and BCL-XS. The frequency of expression of pro- and anti-apoptotic BCL-2 members was similar throughout development, except at the two-cell stage where pro-apoptotic genes predominated. Protein expression was confirmed for BCL-2, MCL-1, BCL-X, BAX, BAD, and activated caspase 3. BCL-2 protein was associated with mitochondria but expressed inconsistently in the blastocyst inner cell mass. Consistent differences between morphologically intact and fragmented embryos included the expression of BAK in fragmented but not intact four-cell embryos. Our study addresses the importance of examining single human embryos representative of the viable population for a large number of genes, in order to establish meaningful expression profiles and provide information on overlapping function in a large gene family.

Apoptosis↗

PubMed2003

Amplification of representative cDNA pools from single human oocytes and pronucleate embryos.

In the human embryo, gene expression studies have been hindered by the scarcity of material and the fact that in vitro fertilisation (IVF) embryos available for research are usually of poor quality and are, therefore, not representative of normal development. This has led most authors to study individual human embryos, using conventional RT-PCR strategies, which permit analysis of only a few genes. Variability in the expression of genes between individual embryos is characteristic of these studies. In this study, a global RT-PCR strategy has been used, allowing the analysis of an almost infinite number of genes from a single embryo. We have used oocytes, which failed to fertilise and representative pronucleate embryos donated from cycles in which the patient conceived, to investigate possible variability in transcript abundance between individual embryos. We have screened oocytes and embryos for a panel of genes including beta-actin (expressed in 24/28 oocytes, 6/6 pronuclear embryos), the integrins beta1 (17/28 oocytes, 6/6 pronuclear embryos) and beta5 (8/28 oocytes, 5/6 pronuclear embryos), and the apoptotic regulators BCL-2 (20/28 oocytes, 2/6 pronuclear embryos) and BAX (21/28 oocytes, 5/6 pronuclear embryos). The expression of the pro-apoptotic regulator BAX increased in human oocytes following prolonged periods of culture. Overall, patterns of gene transcript presence showed variation between embryos and this was independent of either zona removal or lysis conditions. Pronucleate embryos showed less variation, however, even sibling embryos from the patient did not express an identical subset of genes.

DNA, Complementary↗

PubMed2002

Human Hand1 basic helix-loop-helix (bHLH) protein: extra-embryonic expression pattern, interaction partners and identification of its transcriptional repressor domains.

The basic helix-loop-helix (bHLH) transcription factor, Hand1, plays an important role in the development of the murine extra-embryonic trophoblast cell lineage. In the present study, we have analysed the expression of Hand1 in human extra-embryonic cell types and determined its binding specificity and transcriptional activity upon interaction with different class A bHLH factors. Northern blotting and in situ hybridization showed that Hand1 mRNA is specifically expressed in amnion cells at different stages of gestation. Accordingly, we demonstrate that the protein is exclusively produced in the amniotic epithelium in vivo and in purified amnion cells in vitro using a novel polyclonal Hand1 antiserum. Reverse transcriptase-PCR and immunohistochemical staining of blastocysts revealed the production of Hand1 mRNA and polypeptide in the trophectodermal cell layer. In the presence of E12/E47, Hand1 stimulated the transcription of luciferase reporters harbouring degenerate E-boxes, suggesting that E-proteins are potential dimerization partners in trophoblastic tumour and amnion cells. In contrast, Hand1 diminished E12/E47-dependent transcription of reporters containing perfect E-boxes by inhibiting the interaction of Hand1/E-protein heterodimers with the palindromic cognate sequence. Furthermore, we show that Hand1 down-regulated GAL-E12-dependent reporter expression, indicating that the protein can also act directly as a transcriptional repressor. Mutational analyses of GAL-Hand1 suggested that two protein regions located within its N-terminal portion mainly confer the repressing activity. In conclusion, human Hand1 may play an important role in the differentiation of the amniotic membrane and the pre-implanting trophoblast. Furthermore, the data suggest that Hand1 can act as a repressor by two independent mechanisms; sequestration of class A bHLH factors from E-boxes and inhibition of their transcriptional activity.

Basic Helix-Loop-Helix Proteins↗

PubMed2002

Apoptosis in the preimplantation mouse embryo: effect of strain difference and in vitro culture.

Cell death by apoptosis occurs predominantly in the inner cell mass (ICM) of the blastocyst, the cell population which carries the germ line and gives rise to the foetus. The frequency of apoptosis in blastocysts varies widely within outbred species such as human and cow. We have addressed the basis of this variation by examining the relative influence of strain difference and in vitro culture conditions on apoptosis, using embryos from two different strains of mice (MF1 and C57BL6/CBA) in two different culture media (M16 and kSOM). In both strains and all crosses apoptosis was first detected by nuclear fragmentation or TUNEL [Terminal deoxynucleotidyl transferase mediated d-UTP nick end-labelling] labelling at the early blastocyst stage. This was true for embryos which had developed in vivo, and in vitro in both M16 and kSOM. The apoptotic index in blastocysts was found to be significantly different between both media and strain (P < 0.0001). Blastocysts from MF1 x MF1 at equivalent stages had an apoptotic index of 32.4% in M16 and 20.3% in kSOM. Blastocysts from C57BL6/CBA x C57BL6/CBA had an apoptotic index of 19.3% in M16 and 14.4% in kSOM. When embryos of similar cell number were compared, a significantly greater apoptotic index was found for cultured MF1 x MF1 embryos with a cell number between 40 and 59 compared to similar directly flushed C57BL6/CBA embryos (P = 0.001), and MF1 embryos (P < 0.0005). MF1 x MF1 embryos and C57BL6/CBA x MF1 embryos of 60-79 cells had a greater apoptotic index in M16 than kSOM (P < 0.0005) but the difference between media was not significant for C57BL6/CBA x C57BL6/CBA. When strain was compared MF1 x MF1 embryos of 60-79 cells had a significantly greater apoptotic index than C57BL6/CBA x MF1 in both media (P < 0.0005 M16; P = 0.002 kSOM) and than C57BL6/CBA x C57BL6/CBA in M16 (P = 0.019). Our data suggest that genetic make-up and the chemical composition of simple medium are equally important in determining the level of apoptosis.

Analysis of Variance↗