Early pregnancy factor: large scale isolation of rosette inhibition test-active polypeptides from ovine placental...

Protocols are described for the isolation of substantial (mg) amounts of a rosette inhibition test (RIT)-active polypeptide fraction from ovine placental extracts. The main component of the preparation is a 12K polypeptide which contains a highly reactive thiol group. Oxidation may occur during isolation with the result that the final preparation is a mixture of the 12K polypeptide and a 25K disulphide linked dimer. The highly reactive thiol group was found to be directly involved in activity expression since gentle reduction followed by iodoacetylation resulted in a complete loss of activity. Antisera were prepared and the antibodies removed all the RIT activity from fresh ovine placental extracts, indicating that molecules related to those in the isolated preparation were responsible for all the activity in crude extracts. The antibodies also removed all the RIT activity from ovine and murine pregnancy sera, obtained both before and after implantation. Since EPF is defined as an RIT activity detected in pregnancy serum, these results establish that EPF activity is due to molecules similar to those isolated from the placental extracts. The availability of the preparative protocol and antibodies should hasten the biochemical definition of the EPF phenomenon.