Biomedical subjects

Susan J Kimber

Publications and source records attributed to Susan J Kimber.

3 recordsLinked to original sources

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↗