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 muCi L-[3H]leucine) into non-dialysable macromolecules which were released into the medium. Two-dimensional polyacrylamide gel electrophoresis revealed that at Day 13 there was only one major product (Protein X), consisting of three closely similar isoelectric species of (pI of denatured polypeptides about 5.5), each with molecular weights of 17 000. Between Days 14 and 21 additional proteins were detected. One of these was of high molecular weight (greater than 660 000) and did not appear on the two-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 micrograms per 24 h) by the trophoblast but not the yolk sac of Day-14 and Day-16 conceptuses, but was present in very low amounts in the tissues. Protein X from the incubation medium of Day-14 and Day-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.

Maintenance of the corpus luteum after uterine transfer of trophoblastic vesicles to cyclic cows and ewes

“…Because of the abundance of trophoblastic cells, as compared to the very small number of endodermic cells, and because of their very high secretory activity, the secretion of signals inhibiting luteolysis seems likely to take place in the trophectoderm, the endodermic tissue playing the essential role of sus¬ taining the trophectoderm, although endodermic cells are probably also necessary for continued growth of trophectoderm as shown in the mouse (Ilgren, 1981). Amongst these signals, trophoblastin is an antiluteolytic protein whose biochemical nature is still unknown (Martal et al, 1979) although it might correspond to the protein X of Godkin, Bazer, Moffatt, Sessions & Roberts (1982).…”

Interferon-tau, a Type 1 interferon involved in maternal recognition of pregnancy

“…Accordingly, conceptuses were cultured under serum-free conditions in presence of a radioactive amino acid for about 24 hours, and proteins released into the medium analyzed by two-dimensional PAGE. These experiments immediately revealed that the major secretory product released by the unattached sheep conceptus was a protein of molecular weight approximately 18,000, which consisted of several forms differing slightly in isoelectric point (7). The production of this protein, which was maximal at about day 15, became undetectable after the end of the third week of pregnancy.…”

Protein production by cultures established from Day-14-16 sheep and pig conceptuses

“…3). Only in the latter preparation did we detect ovine trophoblast protein-1 which we have previously shown to be the major product secreted into culture medium by whole sheep blastocysts (Godkin et al, 1982a;Godkin, Bazar & Roberts, 1984). The polypeptides (numbered 1-4) produced in major amounts by all the preparations were identical in mobility to polypeptides found in fetal sheep plasma (PI.…”

“…The proteins synthesized in vitro by sheep and pig blastocysts during the pre-, peri-and post-attachment periods have been described and some of the proteins that were released into the culture medium have been purified and characterized (Godkin, Bazer, Moffatt, Sessions & Roberts, 1982a;Godkin, Bazer, Geisert, Lewis & Roberts, 1982b;Masters et al, 1983). However, the yield of such proteins is relatively small and their preparation is time consuming and expensive.…”