Journal
MOLECULAR REPRODUCTION AND DEVELOPMENT
Volume 74, Issue 2, Pages 178-188Publisher
WILEY-LISS
DOI: 10.1002/mrd.20495
Keywords
chromatin segregation; cytokinesis; embryo; glycogen synthase kinase-3
Funding
- NICHD NIH HHS [T32-HD070048, HD35125] Funding Source: Medline
- NIGMS NIH HHS [T32-GM08322] Funding Source: Medline
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Glycogen synthase kinase-3 (GSK-3) is a highly conserved serine/threonine protein kinase implicated in diverse cellular processes. Activity of GSK-3 is essential for meiotic chromatin segregation in oocytes, yet expression and/or function of GSK-3 have not been reported in mammalian preimplantation embryos. Objectives of this study were to characterize GSK-3 protein expression/phosphorylation in mouse preimplantation embryos, to assess the effect of GSK-3 activity inhibition on early mitotic events, and to differentiate nuclear and cytoplasmic anomalies in GSK-3 inhibited embryos. Both GSK-3 isoforms were expressed during embryo development, with a differential expression of alpha versus P. Phosphorylation of GSK-3 alpha/beta at residues Y279/Y216 indicated constitutive activation throughout preimplantation development. Phosphorylation at N-terminal residues S21/S9 indicated inhibition of GSK-3 alpha/beta activity that was differentially regulated during early development; both alpha and beta isoforms were phosphorylated during early divisions, whereas at the blastocyst stage, only beta was phosphorylated. Cytoplasmic microinjection of zygotes with anti-GSK-3 alpha/beta antibody significantly compromised embryonic development past the two-cell stage compared to controls. Reversibility of developmental block was tested via pharmacological inhibitors of GSK-3, lithium chloride (LiCI) and alsterpaullone. Similar to immunoneutralization, significantly fewer zygotes cultured with either LiCl or alsterpaullone developed past the two-cell stage compared to controls and this mitotic block was not reversible. Inhibition of GSK-3 activity significantly compromised timing of pronuclear membrane breakdown and mitosis initiation, nuclear development, and cytolkinesis. Inhibition of GSK-3 also resulted in abnormal chromatin segregation, evidenced by incomplete karyokinesis and micronuclei formation. These results suggest that GSK-3 activity is critical for early preimplantation embryonic development.
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