Mitotic spindle disruption in human preimplantation embryos activates the spindle assembly checkpoint but not apoptosis until Day 5 of development

作者：Jacobs K; Van de Velde H; De Paepe C; Sermon K^*; Spits C^*

来源：Molecular Human Reproduction, 2017, 23(5): 321-329.

DOI：10.1093/molehr/gax007

• spindle assembly checkpoint
• human preimplantation embryo
• aneuploidy
• apoptosis
• metaphase arrest
• checkpoint uncoupling

STUDY QUESTION: Is the spindle assembly checkpoint (SAC) active during human preimplantation development?
SUMMARY ANSWER: Mitotic spindle disruption during mitosis activates the SAC from at least Day 3 of human preimplantation development, but this does not lead to apoptosis until Day 5.
WHAT IS KNOWN ALREADY: Human preimplantation embryos frequently acquire chromosomal abnormalities, but the mechanisms behind this are poorly understood. It has been speculated that a dysfunctional SAC could be responsible. Although research has shown that the SAC components are present during early human development, functional studies are lacking.
STUDY DESIGN, SIZE, DURATION: In vitro study using human preimplantation embryos in a university research laboratory. We studied a total of 38 Day-3, 38 Day-4, 29 Day-5 and 21 Day-6 human preimplantation embryos, donated for research, during 16 h of incubation.
PARTICIPANT/MATERIALS, SETTING, METHODS: We cultured human preimplantation embryos overnight in a time-lapse imaging system, in control or in a nocodazole-containing medium that prevents the formation of a proper mitotic spindle. The embryos were subsequently fixed and analysed by immunocytochemistry for tubulin or mitotic and apoptotic markers, or by FISH.
MAIN RESULTS AND THE ROLE OF CHANCE: All embryos showed an increase in M-phase cells from 4.1-8.8% to 21.4-53.5% when exposed to nocodazole (P < 0.05; two-way ANOVA for all groups except Day-4 embryos, P = 0.128) suggesting SAC functionality. Apoptosis, which was rarely detected between Day 3 and Day 6 in good-quality control embryos, increased from Day 5 onwards in nocodazole-treated embryos and became statistically different from Day 6 (P < 0.01; two-way ANOVA). The FISH data suggest that in compacted Day-4 embryos, approximately one in six cells started a polyploid new cell cycle rather than to go in apoptosis after the failure to maintain the SAC-mediated M-phase arrest. These results suggest that during early embryo development, blastomeres with unresolved chromosome misalignments during M-phase can escape SAC-mediated apoptosis, continue cell division which can then result in aneuploid daughter cells.
LARGE SCALE DATA: Not applicable.
LIMITATIONS, REASONS FOR CAUTION: This study used nocodazole to inhibit microtubule polymerization, a drug that is regularly used to induce metaphase arrest and SAC activation. Results should be extrapolated to naturally occurring chromosome misalignments with care.
WIDER IMPLICATIONS OF THE FINDINGS: Our results provide functional data that can help explain the high aneuploidy rates seen in human cleavage-stage embryos and suggest that this is due to their unusual cell cycle control.

• 出版日期2017-5