Partial purification and characterization of serum embryotrophic factor required for early postimplantation growth of rat embryos in culture.
Serum embryotrophic factor (SEF) required for the growth of cultured postimplantation rat embryos was partially purified from rat serum. Rabbit serum was used as a basal medium for the embryo culture, and embryotrophic activity was measured as embryonic protein increase. For partial purification of SEF, the rat serum globulin fraction obtained by ultracentrifugation and (NH4)2SO4 precipitation was fractionated by gel filtration, diethylaminoethyl ion-exchange chromatography, and hydroxyapatite chromatography. Partially purified SEF was characterized by stability tests and affinity chromatography. SEF was inactivated by heat, acid, dithiothreitol reduction, or trypsin digestion. SEF bound to concanavalin A but not to heparin. These results indicated that SEF was an acid-labile acidic glycoprotein with disulphide bonds and no affinity for heparin. The M(r) of SEF was estimated to be about 180 x 10(3) by gel filtration. The specific activity (U/g protein) was increased about 25-fold with 9.4% recovery by the partial purification, when 1 U of SEF was defined as the amount giving 50% embryonic protein increase. By polyacrylamide gel electrophoresis, a protein most likely to be SEF was identified as a heterodimer composed of subunits of M(r) 116 x 10(3) and 62 x 10(3) linked by disulphide bonds, and was shown to be contained in the medium at micromolar concentrations. SEF appeared to be distinct from known protein embryotrophic factors, growth factors, or cytokines.
Usami
M
M
Division of Pharmacology, National Institute of Health Sciences, Tokyo, Japan.
Ohno
Y
Y
D000818:Animals; D002845:Chromatography; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D003470:Culture Media; D017886:Durapatite; D004622:Embryo, Mammalian / Q000187:drug effects / Q000502:physiology*; D005314:Embryonic and Fetal Development; D006133:Growth Substances / Q000737:chemistry* / Q000494:pharmacology*; D009924:Organ Culture Techniques; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D012712:Serum Globulins / Q000302:isolation & purification
D003470:Culture Media; D006133:Growth Substances; D012712:Serum Globulins; D017886:Durapatite