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

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.     

Localization of fodrin during fertilization and early development of sea urchins and mice

Fodrin, a spectrin-like protein, is localized in gametes, zygotes, and embryos from sea urchins and mice. Mammalian fodrin comprises two polypeptides with molecular weights of approximately 240 kDa (alpha) and 235 kDa (beta). An antibody specific for mammalian alpha-fodrin cross-reacted with a 240-kDa polypeptide from sea urchin egg extracts. This indicates that sea urchins contain a protein of similar electrophoretic mobility and immunological properties to mammalian alpha-fodrin. When this antibody was used to stain the sea urchin gametes with indirect immunofluorescence, fodrin-specific fluorescence was localized to the acrosome of the sperm and was distributed over the entire egg near the surface in a punctate pattern similar to the distribution of polymeric actin. During sperm incorporation, the fodrin-specific fluorescence is found at the site of sperm incorporation, in the fertilization cone. After fertilization, the intensity of fodrin fluorescence increases. During mitosis and cytokinesis in sea urchins, the entire surface of the egg remains stained; the cleavage furrow also was stained but no more intensely than was the rest of the egg surface. Antibody labeling with colloidal gold followed by electron microscopy showed that fodrin was loated in the cytoplasm immediately beneath the plasma membrane. In unfertilized mouse oocytes, both actin and fodrin were stained most intensely beneath the membrane adjacent to the meiotic spindle. After insemination, the cell surfaces of the pronucleate egg and the second polar body were stained; however, the actin matrix surrounding the apposed pronuclei did not bind the fodrin antibody. During cytokinesis in the mouse, the cleavage furrow stained more intensely than did the rest of the egg cortex, and in embryos the cell borders were delineated. These results indicate that organisms as unrelated to mammals as sea urchins have fodrin-like proteins; the rearrangements of such proteins suggest that they participate in the actin-mediated events at the cell surface during fertilization and early development in both mice and sea urchins.     

Fertilization cone formation in starfish oocytes: The role of the egg cortex actin microfilaments in sperm incorporation

The process of sperm incorporation into starfish (Asterias amurensis) oocytes was examined by electron and fluorescence microscopy. The fertilization cone began to form at the place where the acrosomal process fused with the egg surface and developed into an inverted conical mass containing a small amount of electron-dense cytoplasm. Microfilaments, which stained with NBD-phallacidin, were detected in the fertilization cone. Microvillar protrusions from the fully grown fertilization cone engulfed the sperm head outside the fertilization membrane. The sperm organelles were incorporated into the egg cortex with the absorption of the protrusions. Cytochalasin B inhibited sperm incorporation, fertilization cone formation, and actin filament organization. It is suggested that the development and reduction of the fertilization cone, which depend on the functioning of microfilaments, are necessary for sperm incorporation in starfish.     

Microfilament stabilization by jasplakinolide arrests oocyte maturation, cortical granule exocytosis, sperm incorporation cone resorption, and cell-cycle progression, but not DNA replication, during fertilization in mice

Jasplakinolide (JAS), which induces microfilament polymerization and stabilization, inhibits microfilament-mediated events in murine oocyte maturation and fertilization in a fashion unlike the effects of cytochalasin B (CCB) and latranculin A (LAT A). JAS prevents egg polar body emission at a much lower concentration than either CCB or LAT A. Microfilament bundles were detected on the entire egg cortex after JAS exposure. Conversely, microfilament patterns did not change after exposure to CCB, and few microfilaments were observed after exposure to LAT A. Eggs that were allowed to recover from JAS were unable to recover normal microfilament organization. During oocyte maturation, JAS prevented both spindle migration to the oocyte cortex and first polar body emission. During in vitro fertilization, sperm head entered the eggs and formed pronuclei, but sperm tail entry, pronuclear centration, and second polar body emission were not detected. DNA synthesis occurs in these JAS-treated zygotes. JAS inhibited not only the formation, but also the disassembly, of incorporation cones. JAS was also found to prevent cortical granule exocytosis following artificial activation, and cortical granules were still beneath the plasma membrane even after activation. Finally, incorporation of microinjected nonmuscle actin into the microfilament network of mice eggs was delayed by JAS. We conclude that JAS acts as a microfilament inhibitor during maturation and fertilization and is more powerful than other inhibitors. Its mechanism differs in that it promotes assembly and stabilization of microfilaments. JAS is a novel cell permeable tool for the investigation of microfilament-dependent events in early mammalian development.     

Sperm Chromatin-Induced Ectopic Polar Body Extrusion in Mouse Eggs after ICSI and Delayed Egg Activation

Meiotic chromosomes in an oocyte are not only a maternal genome carrier but also provide a positional signal to induce cortical polarization and define asymmetric meiotic division of the oocyte, resulting in polar body extrusion and haploidization of the maternal genome. The meiotic chromosomes play dual function in determination of meiosis: 1) organizing a bipolar spindle formation and 2) inducing cortical polarization and assembly of a distinct cortical cytoskeleton structure in the overlying cortex for polar body extrusion. At fertilization, a sperm brings exogenous paternal chromatin into the egg, which induces ectopic cortical polarization at the sperm entry site and leads to a cone formation, known as fertilization cone. Here we show that the sperm chromatin-induced fertilization cone formation is an abortive polar body extrusion due to lack of spindle induction by the sperm chromatin during fertilization. If experimentally manipulating the fertilization process to allow sperm chromatin to induce both cortical polarization and spindle formation, the fertilization cone can be converted into polar body extrusion. This suggests that sperm chromatin is also able to induce polar body extrusion, like its maternal counterpart. The usually observed cone formation instead of ectopic polar body extrusion induced by sperm chromatin during fertilization is due to special sperm chromatin compaction which restrains it from rapid spindle induction and therefore provides a protective mechanism to prevent a possible paternal genome loss during ectopic polar body extrusion.     

Latrunculin inhibits the microfilament-mediated processes during fertilization, cleavage and early development in sea urchins and mice

Latrunculin A, a marine toxin from a Red Sea sponge, is a potent inhibitor of the microfilament-mediated processes of fertilization and early development in sea urchins and in mice. Sperm from sea urchins, but not those from Limulus or mice, were affected by latrunculin, and fertilization in both sea urchins and in mice was arrested but at different stages. Sea urchin sperm treated with 2.6 microM latrunculin are unable to assemble acrosomal processes and their ability to fertilize eggs is impaired. The unwinding of the Limulus sperm acrosomal process occurs in the presence of latrunculin. Treated mouse sperm are able to fertilize mouse oocytes in vitro, suggesting that microfilaments may not be required in this mammalian sperm. In sea urchin eggs, sperm incorporation, microvillar elongation and cytokinesis are inhibited. Microtubule-mediated motility occurs normally. 20 nM latrunculin prevents the morphogenetic movements during gastrulation. It reduces the viscosity of actin gels from sea urchin egg homogenates. In unfertilized mouse oocytes, it prevents the colcemid-induced dispersion of the meiotic chromosomes; accumulations of cortical actin are noted adjacent to the scattered chromosomes. Sperm incorporation during mouse fertilization in vitro is unaffected suggesting that sperm entry may occur independent of microfilament activity in mammals. However, the apposition of the pronuclei at the center of the egg cytoplasm does not occur, providing evidence that cytoplasmic microfilaments may be required for the motions leading to pronuclear union during mouse fertilization. It inhibits the second polar body formation and cytokinesis. These results indicate that latrunculin is a potent inhibitor of microfilament-mediated processes in sperm, eggs and embryos, and that it may prove to be a powerful new drug for exploring the cellular behavior of microfilaments in the maintenance of cell shape and during motility.     

Cytoskeletal organization in the oocyte,zygote, and early cleaving embryo of the stripe-faced dunnart (Sminthopsis macroura)

Ovulation occurs in Sminthopsis macroura approximately 160 hr after administration of 1.3 IU PMSG, and yields significantly more oocytes than does spontaneous ovulation (P = 0.001). Germinal vesicle (GV)-stage oocytes have a thin cortical rim of microfilaments, which is disrupted by exposure to cytochalasin D. After GV breakdown, the first meiotic spindle forms subcortically and parallel to the oolemma. It rotates during anaphase and telophase to extrude the first polar body. This rotation is associated with a local cortical concentration of microfilaments, which is extruded in the first polar body. The second meiotic spindle is orthogonal to the surface, and extrusion of the second polar body is not associated with obvious local changes in cortical actin, resulting in a polar body containing little polymerized actin. The sites of second polar body emission and sperm entry are always in the half of the oocyte opposite the concentrating yolk mass, and are within 60° of each other in most oocytes. During the concentration and eccentric movement of the yolk, microfilaments condense around it. During yolk expulsion, these microfilaments become continuous with those located subcortically. During early cleavage, the cytocortex of the zygote, but not of the extruded yolk mass, stains heavily for polymerised actin. Multiple sites of pericentriolar material are detectable in the cytoplasm of some secondary unfertilized oocytes which, in the presence of taxol, generate large cytasters and pseudospindle structures. After fertilization, a large aster is formed in association with the sperm entry point and serves as the center of an extensive cytoplasmic network of microtubules which surrounds but does not enter the yolk mass. Taxol treatment generates small cytasters within this meshwork and promotes selective stabilization of some periyolk microtubules opposite to the sperm aster. © 1995 Wiley-Liss, Inc.     

The sperm entry site during fertilization of the zebrafish egg: localization of actin.

The sperm entry site (SES) of zebrafish (Brachydanio rerio) eggs was studied before and during fertilization by fluorescence, scanning, and transmission electron microscopy. Rhodamine phalloidin (RhPh), used to detect polymerized filamentous actin, was localized to microvilli of the SES and to cytoplasm subjacent to the plasma membrane in the unfertilized egg. The distribution of RhPh staining at the SES correlated with the ultrastructural localization of a submembranous electrondense layer of cortical cytoplasm approximately 500 nm thick and containing 5- to 6-nm filaments. Actin, therefore, was organized at the SES as a tightly knit meshwork of filaments prior to fertilization. Contact between the fertilizing sperm and the filamentous actin network was observed by 15-20 sec postinsemination or just before the onset of fertilization cone formation. Growing fertilization cones of either artificially activated or inseminated eggs exhibited intense RhPh staining and substantial increase in thickness of the actin meshwork. Collectively, TEM and RhPh fluorescence images of inseminated eggs demonstrated that the submembranous actin became rearranged in fertilization cones to form a thickened meshwork around the sperm nucleus during incorporation. The results reported here suggest that activation of the egg triggers a dramatic polymerization of actin beneath the plasma membrane of the fertilization cone. Furthermore, the actin involved in sperm incorporation is sensitive to the action of cytochalasins.     

Immunocytochemical localization of actin in the sperm head of Talpa europaea (Insectivora)

Actin in the sperm head of Talpa europaea was observed by immunofluorescence and immunoelectron microscopy. The indirect immunofluorescence technique, using both anti-actin and DNase anti-DNase methods, showed a shining fluorescent band around the sperm head in some spermatozoa, whereas in others the fluorescence was found in the postacrosomal region. Since no labeling was detected in sperms treated with NBD-phallacidin, it is likely that mature mole sperms contain G-actin but not F-actin. The results of electron microscopy indicated the deposition of the anti-actin antibodies in two places in mole spermatozoa: the postacrosomal region and the nuclear segment of the acrosome. In the first case, the actin was localized in the space between the outer surface of the postacrosomal sheath and the plasma membrane; in the second one, the actin was localized in the space between the outer acrosomal membrane and the plasma membrane. The significance of the presence of actin and its role(s) during fertilization are discussed.     

Evidence for a role of capillary pericytes in vascular growth of the developing ovine corpus luteum

The role of microfilaments, microtubules, and mitogen-activated protein (MAP) kinase in regulation of several important dynamic events of porcine oocyte maturation and fertilization is described. Fluorescently labeled microfilaments, microtubules, and cortical granules were visualized using either epifluorescence microscopy or laser scanning confocal microscopy. Mitogen-activated protein kinase phosphorylation was revealed by Western immunoblotting. We showed that 1) microfilament disruption did not affect meiosis resumption and metaphase I meiotic apparatus formation but inhibited further cell cycle progression (chromosome separation) even though MAP kinase was phosphorylated; 2) cortical granule (CG) migration was driven by microfilaments (but not microtubules), and once the chromosomes and CGs were localized beneath the oolemma their anchorage to the cortex was independent of either microfilaments or microtubules; 3) neither microfilaments nor microtubules were involved in CG exocytosis during oocyte activation; 4) sperm incorporation was mediated by microfilaments, while pronuclear (PN) syngamy was controlled by microtubules rather than microfilaments; 5) spindle microtubule organization was temporally correlated with MAP kinase phosphorylation, while the extensive microtubule organization in the sperm aster that is required for PN apposition and syngamy occurred in the absence of MAP kinase activation; and 6) MAP kinase phosphorylation did not change either when microtubules were disrupted by nocodazole or when cytoplasmic microtubule asters were induced by taxol. The present study suggests that the role of the cytoskeleton during porcine oocyte maturation is similar to that of rodents, while the mechanisms of fertilization in pig resemble those of lower vertebrates.     

Surface activity at the egg plasma membrane during sperm incorporation and its cytochalasin B sensitivity: Scanning electron microscopy and time-lapse video microscopy during fertilization of the sea urchin Lytechinus variegatus

Sperm incorporation and the formation of the fertilization cone with its associated microvilli were investigated by scanning electron microscopy of eggs denuded of their vitelline layers with dithiothreitol or stripped of their elevating fertilization coats by physical methods. The activity of the elongating microvilli which appear to engulf the entering spermatozoon was recorded in living untreated eggs with time-lapse video microscopy. Following the acrosome reaction, the elongated acrosomal process connects the sperm head to the egg surface. About 15 microvilli adjacent to the attached sperm elongate at a rate of 2.6 μm/min and appear to engulf the sperm head, midpiece, and sperm tail. These elongate microvilli swell to form the fertilization cone (average height, 6.7 ± 2.0 μm) and are resorbed as the sperm tail enters the egg cytoplasm 10 min after insemination. Cytochalasin B, an inhibitor of microfilament motility, completely inhibits the observed egg plasma membrane surface activity in both control and denuded eggs. These results argue for a role of the microfilaments found in the egg cortex and microvilli as necessary for the engulfment of the sperm during incorporation and indicate that cytochalasin interferes with the fertilization process at this site.     

Fertilization and the cytoskeleton in the mouse

The behaviour and roles of the microtubule network and the microfilaments following fertilization in the mouse oocyte are described. The microtubule network is organized by multiple microtubule organizing centres (MTOCs) and these play a major role in establishing spindle structure and pronuclear movement following fertilization; in contrast to sea urchin and frog eggs, the sperm centriole plays little part in organization of the post-fertilization spindle. The microfilaments are required for spindle rotation, polar body formation, certain changes in the egg cortex, and also for pronuclear movement. Influences of the chromosomes on microtubule and microfilament organisation are also discussed.     

Rotation of meiotic spindle is controlled by microfilaments in mouse oocytes

The completion of meiosis requires the spatial and temporal coordination of cytokinesis and karyokinesis. During meiotic maturation, many events, such as formation, location, and rotation of the meiotic spindle as well as chromosomal movement, polar body extrusion, and pronuclear migration, are dependent on regulation of the cytoskeleton system. To study functions of microfilaments in meiosis, we induced metaphase II (MII) mouse oocytes to resume meiosis by in vitro fertilization or parthenogenetic activation, and we treated such oocytes with cytochalasin B (CB). The changes of the meiotic spindle, as visualized in preparations stained for beta-tubulin and chromatin, were observed by fluorescent confocal microscopy. The meiotic spindle of MII oocytes was observed to be parallel to the plasmalemma. After meiosis had resumed, the spindle rotated to the vertical position so that the second polar body could be extruded into the perivitelline space. When meiosis resumed and oocytes were treated with 10 micro g/ml of CB, the spindle rotation was inhibited. Consequently, the oocyte formed an extra pronucleus instead of extruding a second polar body. These results indicate that spindle rotation is essential for polar body extrusion; it is the microfilaments that play a crucial role in regulating rotation of the meiotic spindle.     

Identification of actin in boar spermatids and spermatozoa by immunoelectron microscopy

Actin was localized in testicular spermatids and in ionophore-treated ejaculated sperm of boar by use of a monoclonal anti-actin antibody labeled with colloidal gold. With the on-grid postembedding immunostaining of Lowicryl K4M sections, actin was identified in the subacrosomal region of differentiating spermatids, in the microfilaments of the surrounding Sertoli cells, and in the myoid cells of the tubular wall. Ejaculated sperm, labeled with the preembedding method, showed actin between the plasma membrane and the outer acrosomal membrane of the equatorial segment. Indirect immunofluorescence was positive in the equatorial segment and in the acrosomal cap of intact sperm, whereas reacted sperm at the anterior head region retained fluorescence only in the inner acrosomal membrane. Rhodamine-phalloidin failed to stain intact and reacted sperm. The distribution of actin in sperm head membranes (inner acrosomal membrane, membranes of the equatorial segment), which are retained after the acrosome reaction, is discussed.     

Role of the cytoskeleton in sperm entry during fertilization in the freshwater bivalve Dreissena polymorpha

The present study examined the role of the cytoskeleton in sperm entry and migration through the egg cytoplasm during fertilization in the zebra mussel, Dreissena polymorpha (Bivalvia: Veneroida: Dreissenidae). Fertilization in this freshwater bivalve occurs outside the mantle cavity, permitting detailed observations of fertilization. After its initial binding to the egg surface, the sperm is incorporated in two stages: (1) a gradual incorporation of the sperm nucleus into the egg cortex, followed by (2) a more rapid incorporation of the sperm axoneme, and translocation of the sperm head through the egg cytoplasm. Initial incorporation into the egg cortex was shown to be microfilament dependent. Microfilaments were found in the sperm's preformed acrosomal filament, the microvilli on the egg surface, and in an actin-filled insemination cone surrounding the incorporating sperm. Treatment of eggs with cytochalasin B inhibited sperm entry in a dose- and time-dependent manner. Microtubule polymerization was not necessary for initial sperm entry. Following incorporation of the sperm head, the flagellar axoneme entered the egg cytoplasm and remained active for several minutes. Associated with the incorporated axoneme was a flow of cytoplasmic particles originating near the proximal end of the flagella. Inhibition of microtubule polymerization prevented entry of the sperm axoneme, and the subsequent cytoplasmic current was not observed. After sperm incorporation into the egg cortex, no appreciable microfilaments were associated with the sperm nucleus. A diminutive sperm aster was associated with the sperm nucleus during its decondensation, but no obvious extension toward the female pronucleus was observed. The sperm aster was significantly smaller than the spindle associated with the female pronucleus, suggesting a reduced role for the sperm aster in amphimixis.     

Wave of cortical actin polymerization in the sea urchin egg

The distribution of actin filaments in the cortical layer of sea urchin eggs during fertilization has been investigated by light microscopy using fluorescently labeled phallotoxins. The cortical layer of both whole eggs and cortices isolated on a glass surface was examined. In cortices of unfertilized eggs, numerous fluorescent spots were seen, which may correspond to short actin filament cores in microvilli. After insemination, one of the sperm-attaching points on the egg surface first became strongly fluorescent. This fluorescence grew around the point of sperm penetration with the growth of the fertilization cone. Then, the cortical layer of the egg around the fertilization cone became strongly fluorescent and the fluorescence propagated in a wavelike manner over the entire cortex. The mechanism of the propagation of actin polymerization is discussed.     

Microtubular and actin cytoskeletons and ultrastructural characteristics of the potentially pathogenic basidiomycetous yeast Malassezia pachydermatis

Microtubular and actin cytoskeletons were investigated in the lipophilic yeast Malassezia pachydermatis by fluorescence and electron microscopy. To detect microtubules by indirect immunofluorescence using monoclonal anti-tubulin antibody, a prolonged incubation with lysing enzymes was necessary due to its very thick cell wall. Cytoplasmic microtubules were detected in interphase and a spindle with astral microtubules was seen in M-phase. The disintegration of cytoplasmic microtubules and migration of the nucleus to the bud before mitosis were characteristic features of the basidiomycetous yeast Malassezia pachydermatis. The visualisation of F-actin structures (patches, cables and cytokinetic rings) by fluorescence microscopy using both monoclonal anti-actin antibody and rhodamine-phalloidin failed, but actin was detected by electron microscopy with immunogold labelling. Clusters of gold particles indicating actin structures were detected at the plasma membrane of cells with unique cortical ultrastructural features characteristic of the genus Malassezia. A possible association of these with the actin cytoskeleton is suggested.     

Microinjected anti-actin antibodies decrease gap junctional intercellular commmunication in cultured astrocytes

The purpose of the present study was to investigate the influence of the actin cytoskeleton on gap junctional intercellular communication (GJIC) in cultured astrocytes. This report describes an decrease of GJIC following microinjection of anti-actin antibodies or cytochalasin D treatment. Dye transfer of microinjected neurobiotin was used to assay gap junctional permeability in cultured astrocytes. Besides this translocation of connexins to the plasma membrane we investigated subsequent anti-actin antibody injection. While control cultures exhibited intensive dye spreading of microinjected neurobiotin, GJIC was impaired by microinjection of anti-actin antibodies. Additionally, impaired GJIC was observed after cytochalasin D treatment for 15 min. After the drug had been washed out, a recovery of GJIC was achieved. Cultured astrocytes exhibited a prominent actin cytoskeleton, with strong staining of actin filaments at the plasma membrane. Confocal laser scanning microscopy revealed an impaired translocation of Cx 43 from the Golgi apparatus to the cell membrane of cell processes following anti-actin antibody injection. These results suggest that the morphological integrity of microfilaments seems to be fundamental for GJIC, probably by means of associations among actin filaments, actin binding proteins, and Cx 43 at the plasma membrane or indirectly through the transport of connexins from the cytoplasm to the cell membrane.     

Microtubule and centrosome distribution during sheep fertilization

The distribution of microtubules and centrosomes was studied during sheep fertilization by electron and immunofluorescence microscopy. Tubulin and centrosomal material was identified with monoclonal anti-alpha-tubulin and MPM-2 antibodies, respectively. In ovulated eggs, microtubules were exclusively found in the meiotic spindle and centrosomal material at each of its poles. At fertilization, sperm centrosomes were incorporated into the egg and organized the sperm astral microtubules. During pronuclear development and migration, the sperm aster increased in size; microtubules of the sperm aster extended from the male pronucleus to the egg center and towards the female pronucleus. The position of the sperm aster during pronuclear migration suggests that it plays a role in this process. When the pronuclei were in apposition in the egg center, a dense array of microtubules and the centrosomal material were present between the two pronuclei. The proximal centriole of the sperm was identified by electron microscopy, between the apposed pronuclei. The centrosomal material extending around the centriole and the sperm neck and proximal mid-piece, apparently contained several foci from which microtubules radiated. These data suggest that in sheep unlike in mice, centrosomal material originating from the sperm is involved in the fertilization events.     

Small GTPase RhoA is required for ooplasmic segregation and spindle rotation, but not for spindle organization and chromosome separation during mouse oocyte maturation, fertilization, and early cleavage

RhoA, a small GTPase, plays versatile roles in many aspects of cell function such as stress fiber formation, cytokinesis, and cell polarization. In this study, we investigated the subcellular localization of RhoA and its possible roles during oocyte maturation and fertilization. RhoA was localized in the cytoplasm of eggs from the germinal vesicle (GV) stage to 2-cell stage, especially concentrating in the midbody of telophase spindle when oocyte extruded PB1 and PB2. The RhoA kinases (ROCKs) specific inhibitor Y-27632 blocked GV breakdown (GVBD) and first polar body extrusion, but did not affect apparatus formation and anaphase/telophase I entry. Anti-RhoA antibody microinjection into the oocytes showed similar results. RhoA inhibitor caused abnormal organization of microfilaments, failure of spindle rotation, PB2 extrusion as well as cleavage furrow formation, while sister chromatid separation was not affected. Microinjection of RhoA antibody also blocked PB2 emission. Our findings indicate that RhoA, by regulating microfilament organization, regulates several important events including GVBD, polar body emission, spindle rotation, and cleavage.