ABSTRACT
Little is understood about the regulation of gene expression in human preimplantation embryos. We set out to examine the expression in human preimplantation embryos of a number of genes known to be critical for early development of the murine embryo. The expression profile of these genes was analysed throughout preimplantation development and in response to growth factor (GF) stimulation. Developmental expression of a number of genes was similar to that seen in murine embryos (OCT3B/4, CDX2, NANOG). However, GATA6 is expressed throughout preimplantation development in the human. Embryos were cultured in IGF-I, leukaemia inhibitory factor (LIF) or heparin-binding EGF-like growth factor (HBEGF), all of which are known to stimulate the development of human embryos. Our data show that culture in HBEGF and LIF appears to facilitate human embryo expression of a number of genes: ERBB4 (LIF) and LIFR and DSC2 (HBEGF) while in the presence of HBEGF no blastocysts expressed EOMES and when cultured with LIF only two out of nine blastocysts expressed TBN. These data improve our knowledge of the similarities between human and murine embryos and the influence of GFs on human embryo gene expression. Results from this study will improve the understanding of cell fate decisions in early human embryos, which has important implications for both IVF treatment and the derivation of human embryonic stem cells.
Subject(s)
Blastocyst/metabolism , Gene Expression Regulation, Developmental , Intercellular Signaling Peptides and Proteins/pharmacology , Cell Differentiation/genetics , Cells, Cultured , Cleavage Stage, Ovum/metabolism , Embryonic Development/drug effects , Embryonic Development/physiology , Female , Gene Expression , Gene Expression Profiling , Gene Expression Regulation, Developmental/drug effects , Heparin-binding EGF-like Growth Factor , Humans , Immunohistochemistry , Insulin-Like Growth Factor I/pharmacology , Leukemia Inhibitory Factor/pharmacology , Morula/metabolism , Polymerase Chain Reaction/methods , Pregnancy , Zygote/metabolismABSTRACT
Formation of a fully differentiated, implantation competent blastocyst requires the expression of a complex repertoire of molecules. However, the events that drive morphogenesis are poorly elucidated in the human embryo. In this work, we describe the amplification of representative cDNAs from morphologically and developmentally normal, individual human embryos at all stages from pronucleate to blastocyst. These cDNAs were probed to reveal the temporal expression pattern of cell adhesion molecules thought to play a key role in murine preimplantation embryo development. We demonstrated constitutive expression of beta actin, beta 1 and alpha 6 integrins, ZO-1 and E-cadherin, as shown previously in mouse embryos. No expression of beta 3, alpha 2, alpha 3 or alpha 7 integrins nor of L or P selectin was detected at any stage of preimplantation development. beta 5 integrin showed a regulated pattern of expression and was not expressed in blastocysts, while desmocollin-2 could only be detected at the blastocyst stage. Expression and localization of beta 1, beta 5 and alpha 6 integrins and ZO-1 and E-cadherin proteins was confirmed in blastocyst stage embryos by immunocytochemistry. We have identified differences in the expression of integrin molecules between mouse and human embryos, and propose a role for alpha v beta 5 and alpha 6 beta 1 integrin dimers in the human embryo at implantation.
Subject(s)
Blastocyst/metabolism , Cell Adhesion Molecules/genetics , Embryonic Development/physiology , Gene Expression , Cell Adhesion Molecules/metabolism , Embryonic and Fetal Development , Female , Humans , Immunohistochemistry/methods , Organ Culture Techniques , PregnancyABSTRACT
The tufA gene, which encodes the bacterial elongation factor Tu of E. coli, was isolated as a 1226 bp DNA fragment using the polymerase chain reaction(1). EcoR1 restriction enzyme digestion sites were incorporated at the 3' and 5' termini of the gene. The tufA gene was ligated into the plasmid vector pUC19 and was sequenced fully. A plasmid shown to contain the tufA gene in the correct orientation with respect to the lac promoter of pUC19 was then used to transform E.coli strain JM105 and expression of EFTu in induced cultures of the resulting clone, JM105 pJBDB01, was measured using a continuous monitoring procedure. The clone was shown to produce up to 95 mg of bacterial elongation factor Tu per litre of cell culture.
