Glycogen synthase kinase-3 regulation of chromatin segregation and cytokinesis in mouse preimplantation embryos.
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 beta. Phosphorylation of GSK-3alpha/beta at residues Y279/Y216 indicated constitutive activation throughout preimplantation development. Phosphorylation at N-terminal residues S21/S9 indicated inhibition of GSK-3alpha/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-3alpha/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 (LiCl) 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 cytokinesis. 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.
Acevedo
Nicole
N
Department of Molecular, University of Michigan, Ann Arbor, Michigan, USA.
Wang
Xia
X
Dunn
Rodney L
RL
Smith
Gary D
GD
D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000201:enzymology; D002453:Cell Cycle; D002455:Cell Division; D002843:Chromatin / Q000502:physiology*; D047108:Embryonic Development; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics* / Q000378:metabolism*; D007150:Immunohistochemistry; D051379:Mice; D009865:Oocytes / Q000201:enzymology*; D008941:Spindle Apparatus
D002843:Chromatin; D038362:Glycogen Synthase Kinase 3