Differential Expression and Functions of Cortical Myosin IIA and IIB Isotypes during Meiotic Maturation, Fertilization, and Mitosis in Mouse Oocytes and Embryos

Abstract

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome-cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development. INTRODUCTIONThe cortex of mature mouse oocytes is polarized: the area adjacent to the eccentrically positioned second meiotic spindle is devoid of cortical granules and surface microvilli, diminished in concanavalin A lectin binding and demonstrates an increase in cortical actin filaments (reviewed by Longo, 1989). Similar events are observed in the cortex and plasma membrane in the vicinity of the incorporating sperm head (Nicosia et al., 1977(Nicosia et al., , 1978Shalgi et al., 1978). The induction of these cortical and cell surface modifications are ‡ Corresponding author: Oregon Regional Primate Research Center and Oregon Health Science University, 505 N.W. 185th Avenue, Beaverton, OR 97006-3499. E-mail: schatten@ohsu.edu.© 1998 by The American Society for Cell Biology 2509 strongly correlated to the presence of meiotic chromosomes or demembranated sperm DNA (Longo and Chen, 1985;Maro et al.,...

Proline-rich tyrosine kinase2 is involved in F-actin organization during in vitro maturation of rat oocyte

“…In telophase, bundles of F-actin form a contractile ring and create a cleavage furrow to partition the oocyte into two daughter cells. Myosin II proteins also concentrate in the second polar body cleavage furrows of mouse egg (Simerly et al 1998). Distinct Pyk2 staining is observed in meiosis I cleavage furrows in our study.…”

Polar body emission

“…Schuh et al [Schuh and Ellenberg, 2008], on the other hand, propose that the leading spindle pole-based myosin II travels toward the cortex, pulling the spindle along. The pulling model is consistent with an earlier study reporting that antibodies against myosin II inhibits metaphase I spindle migration to the cortex [Simerly et al, 1998]. How myosin II is anchored at the spindle pole remains unknown.…”

Polar body cytokinesis

“…The size of the polarized region is proportional to the amount of DNA injected and is inversely correlated to the distance between the chromatin and the closest cortex [Deng et al, 2007]. Interestingly, sperm chromatin also induces formation of a similar cortical structure during fertilization [Maro et al, 1984;Simerly et al, 1998;Deng et al, 2003Deng et al, , 2005. Thus, chromatin, and not spindle microtubules, is responsible for division site determination in oocytes.…”

“…Dispersal of the chromatin using nocodazole induces the formation of a cortical differentiated zone above each chromosome clump. Theurkauf et al, 1992Theurkauf et al, , 1993Liu et al, 1999;Lei and Warrior, 2000;Swan et al, 1999 Nuclear anchoring F-actin, Anc related protein Msp-300, bZIP transcription factor Yu et al, 2006;Guichet et al, 2001 Meiotic Alexandre et al, 1989;Brunet et Maro, 2007 Meiotic maturation Metaphase I Spindle migration F-actin, Formin-2, Myosin II, Mos/MAP kinase pathway,-Golgi traficking, ARF1, Spire, ARP2/3, WAVE 2, JMY Longo and Chen, 1985;Verlhac et al, 2000;Terada et al, 2000;Li et al, 2008;Leader et al, 2002;Dumont et al, 2007a;Simerly et al, 1998;Schuh and Ellenberg, 2008;Azoury et al, 2008;Wang et al, 2008, 2009, Sun et al, 2011aPfender et al, 2011 (Continued) microtubule dynamics and the motor protein kinesin-1 are required for spindle mig...…”