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Development, Vol 121, Issue 2 299-307, Copyright © 1995 by Company of Biologists
JOURNAL ARTICLES |
CM Chia, RM Winston and AH Handyside
Institute of Obstetrics and Gynaecology, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK.
Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) through their common receptor, epidermal growth factor receptor (EGFR) are known to enhance mitogenesis, development and implantation in several species. In the mouse, co-culture of grouped embryos in microdrops increases the cell number and proportion developing to the blastocyst stage. A similar effect is observed with culture of single embryos in medium supplemented with EGF or TGF-alpha highlighting their embryotrophic effects. To study the role of EGF, TGF-alpha and EGFR in early human development, two methods applicable for analysis of expression at the single embryo level have been employed. In the first method, reverse transcription-polymerase chain reaction has been used to examine the presence of transcripts. Following reverse transcription, strategically designed nested primers, optimised for specificity, were used for amplification from the cDNA equivalent of a single embryo. The products were then verified by restriction enzyme digestion and sequence analysis. In the second method, immunocytochemistry has been used to colocalise the expressed proteins. Individual embryos were paraffin embedded and serial sectioned, allowing adjacent sections to be examined with different antibodies and controls. Monoclonal TGF-alpha and polyclonal EGF and EGFR primary antibodies were used. Staining was performed by peroxidase-conjugated avidin-biotin immunocytochemistry with the appropriate controls. The combination of these two methods can potentially be used for simultaneous analysis of several growth factors and/or their receptors in the same human embryos. Transcripts for EGF, TGF-alpha and EGFR were detected in unfertilized oocytes and embryos between 8-cell and blastocyst stages on day 3 to 6 post-insemination.(ABSTRACT TRUNCATED AT 250 WORDS)
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