Molecular Human Reproduction, Vol. 9, No. 5, 245-252,
May 2003
© 2003 European Society of Human Reproduction and Embryology
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Gene expression regulating epithelial intercellular junction biogenesis during human blastocyst development in vitro
Submitted on July 17, 2002; accepted on January 24, 2003
1 Division of Cell Sciences, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, 2 Department of Biology, University of York, P.O.Box No. 373, York YO10 5YW, 3 Academic Unit of Paediatrics, Obstetrics and Gynaecology, University of Leeds, D Floor, Clarendon Wing, Leeds General Infirmary, Belmont Grove, Leeds LS2 9NS, UK 4 Current address: Division of Infection, Inflammation and Repair, School of Medicine, University of Southampton, Southampton General Hospital, Southampton SO16 6YD 5 Current address: Fetal Origins of Adult Disease Division, School of Medicine, University of Southampton, Princess Anne Hospital, Southampton S016 5YA, UK
6 To whom correspondence should be addressed. e-mail: tpf{at}soton.ac.uk
We investigated gene expression associated with trophectoderm epithelial intercellular junction formation in single human embryos at different stages of cleavage using RT–PCR methods based upon magnetic bead separation of polyA+ RNA. Trophectoderm tight junction (TJ) and desmosome biogenesis contribute to intercellular sealing and tissue integrity, critical for vectorial transport and blastocoel cavity formation. Expression of the various genes throughout human preimplantation development showed differing levels of sensitivity of detection; these genes included claudin-1, occludin (TM4+ and TM4 isoforms), ZO-1 (ZO-1
+ and ZO-1– isoforms), ZO-2 and JAM (junction adhesion molecule), and the desmosome junction gene, DSC2 (desmocollin 2). Some transcripts appeared to be expressed throughout preimplantation development (claudin-1, JAM, occludin TM4+ and TM4, ZO-1- isoform) while others tended to be expressed preferentially in later cleavage and associated with blastocyst formation (ZO-2, ZO-1+ isoform, DSC-2), illustrating an expression pattern broadly similar to mouse cleavage stages. Human embryo transcript detection was significantly decreased when reverse transcription was performed in solid phase to generate a bead/cDNA transient library rather than after mRNA elution from beads. Transcript detection tended to be positively correlated with embryo morphological grade using the solid phase method. In blastocysts, occludin TM4–, ZO-1+ and DSC2 transcripts were the most susceptible to failure of detection, indicative of low levels of expression which may impact on trophectoderm differentiation competence. Immunoconfocal microscopy analysis of selected adhesion and TJ proteins in human embryos indicated poor membrane assembly compared with mouse blastocysts, which may further affect embryo viability.
Key words: blastocyst/epithelium/human embryo/tight junction/trophectoderm
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