Purification and properties of a major, low molecular weight protein released by the trophoblast of sheep blastocysts at Day 13-21

Abstract

Sheep blastocysts (day 13-21) incubated in a modified Minimum Essential Medium released proteins into the medium at an approximately linear rate over a 24 h period. Single day 16 blastocysts converted 2-8% of the radioactivity supplied (100 .mu.Ci L-[3H]leucine) into non-dialyzable macromolecules which were released into the medium. Two-dimensional polyacrylamide gel electrophoresis revealed that at day 13 there was only 1 major product (Protein X), consisting of 3 closely similar isoelectric species (pI of denatured polypeptides .apprx. 5.5), each with MW of 17,000. Between days 14 and 21 additional proteins were detected. One of these was of high MW (> 660,000) and did not appear on the 2-dimensional gels, but Protein X continued to predominate until day 23 when it could not be detected. Explants of chorion from day 30 of pregnancy failed to secrete Protein X. Protein X was released in significant quantities (50-100 .mu.g/24 h) by the trophoblast but not the yolk sac of day 14 and 16 conceptuses, but was present in very low amounts in the tissues. Protein X from the incubation medium on day 14 and 16 conceptuses was purified by successive DEAE ion exchange and Sephacryl S-200 gel chromatography. Because Protein X and some of the other proteins are produced transiently between days 13 and 21, it is possible that they may play a role in maternal recognition of pregnancy in sheep.