BIOASSAY AND ISOLATION OF CONTRACTION FACTOR(S) FROM RANA AMURENSIS EMBRYO
Jiang Chu,Li Mei-fang and Ku Kuo-yen (Shanghai Institute of Cell Biology,Academia Sinica)
In a previous short communication,it has been reported that soluble factor(s) from Rana amurensis blastomeres could induce contraction of fertilized egg surface after being injected.More detailed information is given here. Embryos of late blastula stage were ho-mogenized and centrifuged at 120,000 g.for 1.5 hr.The clear supernatant was passing through Sephacryl S-300 after pH adjustment (fig.in text).The active fraction was se-parated further by DEAE Sepharose 6B (fig.2 in text).5-6 bands with molecular weight less than 70,000 were enriched as shown by SDS-polyacrylamide slab gel electrophoresis (plate I.fig.13). Contraction factor(s) could induce con-traction (Plate I.fig.3-4) which usually appeared 10-15 min after the contraction fac-tors) being injected under the fertilized egg surface and could last half an hour.The reac-tion intensity was nearly proportional to a certain amount or concentration of the active fraction injected.In appropriate condition the cleavage furrow could bend to the direction of injected site (Plate I.fig.5-9).In some cases a secondary contraction spread to the whole egg surface after the first contraction had been disappeared.If the active fraction and cytochalasin B mixture was injected,egg surface flattened out aroud the injection site by the action of cytochalasin B and contrac-ted later beginning at the margin of the affe-cted and unaffected area then the flattened area became nipple-like shaped (Plate I.fig.10-11).This indicates that the action of con-traction factor(s) is expressed throngh micro-filaments . The contraction factor(s) was present not only in the late blastula stage and in 2-8 cell stage,but also in ovulated eggs.If the contraction factor(s) was the same as the furrowing-inducing cytoplasmic component of Sawai (1972),the furrow contraction may result from the local increase of contraction factor(s).