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Antagonists of Myosin Light Chain Kinase and of Myosin II Inhibit Specific Events of Egg Activation in Fertilized Mouse Eggs¹

Department of Obstetrics and Gynecology,⁴ Tufts-New England Medical Center, Boston, Massachusetts 02111 Sackler School of Biomedical Sciences,⁵ Program in Cell, Molecular, and Developmental Biology, Tufts University School of Medicine, Boston, Massachusetts 02111

ABSTRACT

Although recent studies have demonstrated the importance of calcium/calmodulin (Ca²⁺/CAM) signaling in mammalian fertilization, many targets of Ca²⁺/CAM have not been investigated and represent potentially important regulatory pathways to transduce the Ca²⁺ signal that is responsible for most events of egg activation. A well-established Ca²⁺/CAM-dependent enzyme is myosin light chain kinase (MYLK2), the downstream target of which is myosin II, an isoform of myosin known to be important in cytokinesis. In fertilized mouse eggs, established inhibitors of MYLK2 and myosin II were investigated for their effects on events of egg activation. The MYLK2 antagonist, ML-7, did not decrease the activity of Ca²⁺/CAM protein kinase II or the elevation of intracellular Ca²⁺, and it did not delay the onset of Ca²⁺ oscillations. In contrast, ML-7 inhibited second polar body (PB) formation in a dose-dependent manner and reduced cortical granule (CG) exocytosis by a mean of approximately 50%. The myosin II isoform-specific inhibitor, blebbistatin, had similar inhibitory effects. Although both antagonists had no effect on anaphase onset, they inhibited second PB formation by preventing spindle rotation before telophase II and normal contractile ring constriction. To our knowledge, this is the first report that MYLK2 and myosin II are involved in regulating the position of the meiotic spindle, formation of the second PB, and CG exocytosis. The present results suggest that MYLK2 is one of a family of CAM-dependent proteins that act as multifunctional regulators and transduce the Ca²⁺ signal at fertilization.

calcium, gamete biology, in vitro fertilization, meiosis, signal transduction

¹ Supported by NICHD grants HD-24191 and HD-43363.

² Correspondence: Tom Ducibella, Department of Obstetrics and Gynecology, Box 36, Ziskind Building, Room 405, Tufts-New England Medical Center, 750 Washington Street, Boston, Massachusetts 02111. FAX: 617 636 5087; tducibella{at}tufts-nemc.org

³ These authors contributed equally to this work.