Embryogenesis-promoting factors in rat serum.
Regarding whole rat embryo cultures in vitro, rat serum as a culture medium is known to support the normal growth of rat embryos in the organogenesis phase. The purpose of the present study was to isolate the embryogenesis-promoting factors from rat serum as a first step in the development of a defined serum-free medium for a whole embryo culture system. Pooled rat serum after heat inactivation was fractionated into three major peaks (frA, containing a region of void volume, frB, and frC) by gel filtration. The 9.5-day rat embryos that were cultivated for 48 hr in essential salt medium containing frB (with a molecular size range of 100-500 kDa) revealed normal growth. Three proteins (27 kDa, 76 kDa, and 190 kDa) that had the embryogenesis-promoting effects were isolated from 3-hr delayed centrifuged rat serum by the ion exchange chromatography. The 76-kDa protein was found to be rat transferrin by immunoblotting. The 27-kDa protein was identified as apo-AI (the major apoprotein of high-density lipoprotein) by immunoblotting. High-density lipoprotein obtained from pooled rat serum by a NaBr density gradient ultracentrifugation was found to have a positive effect on embryogenesis. The 10-kDa protein was also identified as alpha 1-inhibitor 3 by immunoblotting. In addition, the embryogenesis-promoting effect of the fraction containing 27-kDa and 190-kDa proteins declined within a short period of storage at -20 degrees C. This decrease was countered by supplementing its fraction (D-2) with albumin isolated from rat serum. These results in the present study suggest that transferrin, high-density lipoprotein, and alpha 1-inhibitor 3 in rat serum may be embryogenesis-promoting factors, and that albumin appeared to play a role in the embryogenesis of rat embryos in whole embryo cultures.
Katoh
M
M
Department of Embryology, Aichi Human Service Center, Japan. miyuki@nucc.cc.nagoya-u.ac.jp
Kimura
R
R
Shoji
R
R
D000818:Animals; D001798:Blood Proteins / Q000302:isolation & purification* / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D004622:Embryo, Mammalian / Q000187:drug effects; D005314:Embryonic and Fetal Development / Q000502:physiology*; D066298:In Vitro Techniques; D008075:Lipoproteins, HDL / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012709:Serum Albumin / Q000378:metabolism*; D014168:Transferrin / Q000302:isolation & purification / Q000378:metabolism
D001798:Blood Proteins; D003470:Culture Media; D008075:Lipoproteins, HDL; D012709:Serum Albumin; D014168:Transferrin