title: Chromosome segregation regulation in human zygotes: altered mitotic histone phosphorylation dynamics underlying centromeric targeting of the chromosomal passenger complex.
abstract: OBJECTIVE
  Are the kinase feedback loops that regulate activation and centromeric targeting of the chromosomal passenger complex (CPC), functional during mitosis in human embryos?
  
  
  CONCLUSIONS
  Investigation of the regulatory kinase pathways involved in centromeric CPC targeting revealed normal phosphorylation dynamics of histone H2A at T120 (H2ApT120) by Bub1 kinase and subsequent recruitment of Shugoshin, but phosphorylation of histone H3 at threonine 3 (H3pT3) by Haspin failed to show the expected centromeric enrichment on metaphase chromosomes in the zygote.
  
  
  BACKGROUND
  Human cleavage stage embryos show high levels of chromosomal instability. What causes this high error rate is unknown, as mechanisms used to ensure proper chromosome segregation in mammalian embryos are poorly described.
  
  
  METHODS
  In this study, we investigated the pathways regulating CPC targeting to the inner centromere in human embryos. We characterized the distribution of the CPC in relation to activity of its two main centromeric targeting pathways: the Bub1-H2ApT120-Sgo-CPC and Haspin-H3pT3-CPC pathways.
  
  
  METHODS
  The study was conducted between May 2012 and March 2014 on human surplus embryos resulting from in vitro fertilization treatment and donated for research. In zygotes, nuclear envelope breakdown was monitored by time-lapse imaging to allow timed incubations with specific inhibitors to arrest at prometaphase and metaphase, and to interfere with Haspin and Aurora B/C kinase activity. Functionality of the targeting pathways was assessed through characterization of histone phosphorylation dynamics by immunofluorescent analysis, combined with gene expression by RT-qPCR and immunofluorescent localization of key pathway proteins.
  
  
  RESULTS
  Immunofluorescent analysis of the CPC subunit Inner Centromere Protein revealed the pool of stably bound CPC proteins was not strictly confined to the inner centromere of prometaphase chromosomes in human zygotes, as observed in later stages of preimplantation development and somatic cells. Investigation of the regulatory kinase pathways involved in centromeric CPC targeting revealed normal phosphorylation dynamics of histone H2A at T120 (H2ApT120) by Bub1 kinase and subsequent recruitment of Shugoshin. However, phosphorylation of histone H3 at threonine 3 (H3pT3) by Haspin kinase failed to show the expected centromeric enrichment on metaphase chromosomes in the zygote, but not at later stages. Inhibition of Haspin revealed this activity to be essential for proper mitotic checkpoint complex activation in human zygotes, thus demonstrating an active mitotic checkpoint under normal conditions. Abolishment of H3pT3 during zygotic prometaphase further shows that centromeric H2ApT120 alone is not sufficient for proper shugoshin and CPC localization. As the removal of H3pT3 from the chromosome arms during prometaphase normally contributes to further centromeric enrichment of the CPC in somatic cells, CPC targeting may be less accurate in human zygotes.
  
  
  CONCLUSIONS
  Owing to ethical limitations, tripronuclear zygotes were used in functional experiments. Although these represent the best available models, it is unknown if they are completely representative for dipronuclear zygotes. In addition, further research is needed to determine to what extent the differences we observed in H3T3 phosphorylation dynamics and CPC localization affect chromosome attachment.
  
  
  CONCLUSIONS
  In the zygote, paternal and maternal chromosomes coming from two separate pronuclei, and with contrasting epigenetic signatures, need to be aligned on a single metaphase plate. Our results suggest that adaptations in mechanisms regulating CPC targeting exist in the human zygote, to ensure symmetric recruitment despite the epigenetic asymmetry between maternal and paternal chromosomes. This adaptation may come at a price regarding chromosome segregation fidelity.
  
  
  BACKGROUND
  This study was funded by the Portuguese Fundação para a Ciência e Tecnologia and the Netherlands Organization for Scientific Research. The authors have no conflicts of interest to declare.
authors.affiliation: Division of Reproductive Medicine, Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Center, 3000 CA Rotterdam, The Netherlands.
authors.forename: C
authors.initials: C
authors.lastname: van de Werken
authors.affiliation: Division of Reproductive Medicine, Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Center, 3000 CA Rotterdam, The Netherlands Department of Reproductive Medicine and Gynaecology, University Medical Center, 3584 CX Utrecht, The Netherlands Present address: IVF Department, Reinier de Graaf Groep, locatie Diaconessenhuis, 2275 CX Voorburg, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Avo Santos
authors.affiliation: Division of Reproductive Medicine, Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Center, 3000 CA Rotterdam, The Netherlands.
authors.forename: J S E
authors.initials: JS
authors.lastname: Laven
authors.affiliation: Division of Reproductive Medicine, Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Center, 3000 CA Rotterdam, The Netherlands.
authors.forename: C
authors.initials: C
authors.lastname: Eleveld
authors.affiliation: Department of Reproductive Medicine and Gynaecology, University Medical Center, 3584 CX Utrecht, The Netherlands.
authors.forename: B C J M
authors.initials: BC
authors.lastname: Fauser
authors.affiliation: Department of Molecular Cancer Research, University Medical Center, 3584 CX Utrecht, The Netherlands.
authors.forename: S M A
authors.initials: SM
authors.lastname: Lens
authors.affiliation: Division of Reproductive Medicine, Department of Obstetrics and Gynaecology, Erasmus MC, University Medical Center, 3000 CA Rotterdam, The Netherlands e.baart@erasmusmc.nl.
authors.forename: E B
authors.initials: EB
authors.lastname: Baart
mesh_terms: D064107:Aurora Kinase B / Q000378:metabolism; D064108:Aurora Kinase C / Q000378:metabolism; D001755:Blastocyst / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002503:Centromere / Q000648:ultrastructure*; D020090:Chromosome Segregation; D002875:Chromosomes / Q000378:metabolism / Q000648:ultrastructure; D018507:Gene Expression Regulation, Developmental; D006657:Histones / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008938:Mitosis; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D059008:Time-Lapse Imaging; D015053:Zygote / Q000378:metabolism* / Q000502:physiology
chemical_list: D018797:Cell Cycle Proteins; D006657:Histones; D047908:Intracellular Signaling Peptides and Proteins; C496644:SGO1 protein, human; D064107:Aurora Kinase B; D064108:Aurora Kinase C; C584277:BUB1 protein, human; C530656:HASPIN protein, human; D017346:Protein Serine-Threonine Kinases
keywords: Aurora kinase; Bub1 kinase; Haspin kinase; aneuploidy; chromosome segregation; histone phosphorylation; human embryo research; zygote