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.
Metcalfe
Anthony D
AD
School of Biological Sciences, University of Manchester, United Kingdom.
Bloor
Debra J
DJ
Lieberman
Brian A
BA
Kimber
Susan J
SJ
Brison
Daniel R
DR
D018076:DNA, Complementary / Q000378:metabolism*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D009865:Oocytes / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D015053:Zygote / Q000378:metabolism*
D018076:DNA, Complementary; D012313:RNA