Rat hepatoma reuber H-35 cells produce a 2-cell stage-specific inhibitor of the cleavage of mouse embryos.
We purified an embryonic stage-specific inhibitor produced by rat hepatoma Reuber H-35 cells against cleaving mouse 2-cell embryos and defined its biological properties. Zygotes obtained from CD-1 mice (a strain that shows a 2-cell block in vitro) or C57BL/6 and B6C3F1 mice (strains that do not) were cultured in media with and without 50 microM EDTA, respectively. The development of the zygotes from all strains was arrested at the 2-cell stage when zygotes were cocultured with Reuber H-35 cells. However, the embryos from C57BL/6 and B6C3F1 were less sensitive than those from CD-1 against the inhibitory effects of development. This inhibitory effect was also evident in medium conditioned with the Reuber H-35 cells. The factor from the conditioned medium was separated into its < 10 000 M(r) fraction by ultrafiltration and was further purified in fraction B-25 as a single peak by reverse-phase column chromatography. An incubation as short as 3-h during the late 2-cell stage (G2 phase) with fraction B-25 suppressed cleavage in 61.5% of the CD-1 embryos (30.3% in control culture). Although the inhibitory effect was reversible, embryos that cleaved again either degenerated or were retarded at various stages in their subsequent development. Additionally, a long-term incubation of developing zygotes with the inhibitory factor caused a significant reduction in [3H]thymidine (TdR) incorporation into the DNA of CD-1 2-cell embryos as well as developmental arrest at the interphase of the 2-cell stage. These results indicated that this factor will serve as a valuable tool with which to clarify the proliferating mechanism of the preimplantation embryo.
Kobayashi
M
M
National Institute of Animal Industry, Ibaraki, Japan.
Hirako
M
M
Minato
Y
Y
Sasaki
K
K
Horiuchi
R
R
Domeki
I
I
D000818:Animals; D002478:Cells, Cultured; D002970:Cleavage Stage, Ovum / Q000187:drug effects*; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D004247:DNA / Q000096:biosynthesis; D004492:Edetic Acid / Q000494:pharmacology; D005260:Female; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D051381:Rats; D014407:Tumor Cells, Cultured; D015053:Zygote / Q000502:physiology
D017077:Culture Media, Conditioned; D004247:DNA; D004492:Edetic Acid