Latent effects on in vitro development following cytochalasin B treatment of 8-cell mouse embryos

Eight-cell mouse embryos when treated with 4.0 microgram/ml cytochalasin B (CB) in vitro undergo a reversible developmental arrest. Upon rinsing of embryos and subsequent culture in control medium, normal morphogenetic processes such as compaction of 8-cell embryos, cavitation, and post-blastocyst attachment and outgrowth are restored. However, the effects of CB on mouse embryos are not completely reversible; latent post-blastocyst defects become increasingly more prevalent as CB treatment duration increases. The present study was conducted to quantitatively determine latent effects of CB on post-blastocyst embryos by comparing their ability to attach and to sustain the growth and differentiation of ICM and trophoblast tissues. Groups of 8-cell embryos were cultured in Brinster's BMOC-3 medium containing 4.0 microgram/ml cytochalasin B for 6, 12, 18, and 24 h. Following treatment, embryos were rinsed and cultured until 190 h post coitum (h.p.c.) in Eagle's MEM/10% fetal calf serum modified to contain optimal levels of essential amino acids. Blastocysts generally attached to the surface of the plastic substratum by 120 h.p.c. At selected time periods after attachment (130, 160, and 190 h.p.c.), embryos were scored for outgrowth size, ICM size, extent of peripheral hyaloplasmic fan, and number of trophoblast nuclei per outgrowth. Analyses of variance (ANOVAs) were conducted for each of the four parameters listed above. Rates of attachment were analyzed by chi2 test. Results show that the treatments affect (P less than 0.01) embryo attachment, number of trophoblast nuclei per outgrowth, hyaloplasmic fan production, and ICM growth in a duration-dependent manner. Interestingly, since treatment effects on outgrowth areas are nonsignificant apparently CB does not significantly change total outgrowth area. But CB treatment does cause abnormal fan production and decreased trophoblast nuclei numbers. However, trophoblast cells are apparently more resistant than ICM to CB as is evident by the high incidence of trophoblast outgrowths devoid of ICM. CB (4.0 microgram/ml) treatments at 8-cell stages for relatively short durations (6 and 12 h) induce latent effects on post-blastocyst embryos. Finally, there exists a definite 4.0 microgram/ml CB duration response over the 68-190 h.p.c. observation interval.