High molecular weight glycoproteins released by expanding, pre-attachment sheep, pig and cow blastocysts in culture

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Abstract
Blastocysts isolated from sheep (day 14-16), pigs (day 16) and cows (day 19) during the pre-attachment elongation phase were cultured for up to 30 h in a modified MEM [mimimal essential medium] in the presence of radioactive amino acids (L-[14C]leucine or L-[35S]methionine) to label protein and D-[3H]glucosamine to label complex saccharides. All the blastocysts released considerable quantities of non-dialyzable radioactive material into the medium at an approximately linear rate over the course of the incubation. Ion-exchange chromatography on DEAE cellulose at pH 8.2 revealed that the major glucosamine-labeled product in the medium was a non-sulfated glycoprotein which eluted early in the salt gradient. None of the blastocysts produced any detectable glycosaminoglycan-like materials such as hyaluronic acid. The glycoprotein was purified by ion-exchange and gel filtration chromatography and had a MW > 660,000. Up to 100 .mu.g of this material could be isolated from incubations of 2 sheep conceptuses. Up to 100 .mu.g of this material could be isolated from incubations of 2 sheep conceptuses. It was relatively resistant to protease hydrolysis and consisted of .apprx. 50% carbohydrate and 50% protein. The main monosaccharide constituents, as revealed by gas-liquid chromatography, were galactose and N-acetylglucosamine plus some mannose and fucose. No sialic acid was present. The linkages between the carbohydrate chains and the peptide appeared to be resistant to alkaline borohydride cleavage and were probably, therefore, N-glycosidic.