EGF, TGF-ALPHA AND EGFR EXPRESSION IN HUMAN PREIMPLANTATION EMBRYOS

Epidermal growth factor (EGF) and transforming growth factor alpha (TG F-alpha) through their common receptor, epidermal growth factor recept or (EGFR) are known to enhance mitogenesis, development and implantati on 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 thei r embryotrophic effects. To study the role of EGF, TGF-alpha and EGFR in early human development, two methods applicable for analysis of exp ression at the single embryo level have been employed. In the first me thod, reverse transcription-polymerase chain reaction has been used to examine the presence of transcripts. Following reverse transcription, strategically designed nested primers, optimised for specificity, wer e used for amplification from the cDNA equivalent of a single embryo. The products were then verified by restriction enzyme digestion and se quence analysis. In the second method, immunocytochemistry has been us ed to co-localise the expressed proteins. Individual embryos were para ffin embedded and serial sectioned, allowing adjacent sections to be e xamined with different antibodies and controls. Monoclonal TGF-alpha a nd polyclonal EGF and EGFR primary antibodies were used. Staining was performed by peroxidase-conjugated avidin-biotin immunocytochemistry w ith the appropriate controls. The combination of these two methods can potentially be used for simultaneous analysis of several growth facto rs and/or their receptors in the same human embryos. Transcripts for E GF, TGF-alpha and EGFR were detected in unfertilized oocytes and embry os between 8-cell and blastocyst stages on day 3 to 6 post-inseminatio n. Similarly, at the protein level, all three were detected in unferti lized oocytes and throughout preimplantation development to day 8. At the blastocyst stage, expression was observed in both the trophectoder m and inner cell mass but decreased in more advanced blastocysts excep t in the polar trophectoderm and inner cell mass. This pattern of expr ession contrasts with the murine and bovine species in which TGF-alpha and EGFR but not EGF are expressed at preimplantation stages. The con current co-expression of these growth factors and their receptor sugge sts a role for autocrine stimulation in preimplantation development. I n the human, reduced protein levels of EGF, TGF-alpha and EGFR in adva nced blastocysts may reflect a switch to dependence on paracrine stimu lation. This may explain the inadequacy of simple culture media to mai ntain the development of human embryos at later stages and the benefit s of co-culture with somatic cells. The continuous expression of EGF, TGF-alpha and EGFR in the polar trophectoderm may be significant for i mplantation.