NUCLEAR-ENVELOPE BREAKDOWN IS UNDER NUCLEAR NOT CYTOPLASMIC CONTROL IN SEA-URCHIN ZYGOTES

Nuclear envelope breakdown (NEB) and entry into mitosis are thought to be driven by the activation of the p34(cdc2)-cyclin B kinase complex or mitosis promoting factor (MPF). Checkpoint control mechanisms that monitor essential preparatory events for mitosis, such as DNA replicat ion, are thought to prevent entry into mitosis by downregulating MPF a ctivation until these events are completed. Thus, we were surprised to find that when pronuclear fusion in sea urchin zygotes is blocked wit h Colcemid, the female pronucleus consistently breaks down before the male pronucleus. This is not due to regional differences in the time o f MPF activation, because pronuclei touching each other break down asy nchronously to the same extent. To test whether NEB is controlled at t he nuclear or cytoplasmic level, we activated the checkpoint for the c ompletion of DNA synthesis separately in female and male pronuclei by treating either eggs or sperm before fertilization with psoralen to co valently cross-link base-paired strands of DNA. When only the maternal DNA is cross-linked, the male pronucleus breaks down first. When the sperm DNA is cross-linked, male pronuclear breakdown is substantially delayed relative to female pronuclear breakdown and sometimes does not occur. Inactivation of the Colcemid after female NEB in such zygotes with touching pronuclei yields a functional spindle composed of matern al chromosomes and paternal centrosomes. The intact male pronucleus re mains located at one aster throughout mitosis. In other experiments, w hen psoralen-treated sperm nuclei are allowed to fuse with normal fema le pronuclei, over 90% of the zygote nuclei do not break down for at l east 2 h after the controls even though H-1 histone kinase activity gr adually rises close to, or higher than, control mitotic levels. The sa me is true for normal zygotes treated with aphidicolin to block DNA sy nthesis. From these results, we conclude that NEB in sea urchin zygote s is controlled at the nuclear, not cytoplasmic, level, and that mitot ic levels of cytoplasmic MPF activity are not sufficient to drive NEB for a nucleus that is under checkpoint control. Our results also demon strate that the checkpoint for the completion of DNA synthesis inhibit s NEB by acting primarily within the nucleus, not by downregulating th e activity of cytoplasmic MPF.