Growth-promoting effects of different fractions of extra-embryonic coelomic fluid on embryonic development

In the early stages of embryonic development, many growth-promoting molecul es must be provided by the maternal system. These factors may be supplied l ocally to the embryo, by the decidua, the placenta, or the yolk sac. In thi s study the growth-promoting potential of extra-embryonic coelomic fluid (E ECF) and its fractions was investigated. The embryonic requirement of growt h-promoting molecules may be studied by reducing the growth-supporting capa city of serum. Thus, ultrafiltration of rat serum was carried out for 8h us ing Millipore filters with a molecular weight exclusion of 30 kDa. Rat embr yos at 9.5 days of age were cultured for 8 days for anembryonic yolk sacs, and then EECF was collected and divided into three different molecular weig ht fractions by ultrafiltration. Rat embryos were cultured for 48 h in whol e rat serum and the serum retenate (which has low growth-supporting capacit y) in the presence and absence of EECF, its fractions, or in EECF only. Emb ryos grown in retenate showed severe growth retardation, and the addition o f EECF significantly improved embryonic growth. The fraction which containe d the molecules with molecular weight between 10 and 30 kDa had significant ly more effect on embryonic development than the other fractions. This frac tion of EECF was analysed by gel electrophoresis. Three of the four protein bands observed in this fraction were identified by amino-terminal sequenci ng as alpha-fetoprotein precursor (22 kDa), apolipoprotein Al precursor (24 kDa) and fetal haemoglobin Y2 chain /14kDa), none of which are likely to be responsible for the growth-promoting activity. To further investigate grow th-promoting proteins, EECF was Western-blotted to nitrocellulose membranes and probed with antisera against rat prolactin, epidermal growth factor, i nsulin-like growth factors I and II and human placental lactogen. No immuno reactive bands were detected in the EECF, suggesting that either these prot eins are not present or are present at levels too low to be detected. Altho ugh the growth-promoting effect of the EECF was demonstrated in this study, the molecules responsible remain uncharacterized.