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 α1‐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°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 α1‐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. J. Exp. Zool. 281:188–200, 1998. © 1998 Wiley‐Liss, Inc.
