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BOR - Papers in Press, published online ahead of print October 5, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.046409
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BIOLOGY OF REPRODUCTION 74, 169–176 (2006)
DOI: 10.1095/biolreprod.105.046409
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

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

Sara Matson 34 , Styliani Markoulaki 35 , and Tom Ducibella 2 45 

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

INTRODUCTION

In many organisms, the release of intracellular calcium (Ca2+) at fertilization is necessary and sufficient to initiate several events of egg activation (for review, see [1]). Mammalian fertilization stimulates oscillatory intracellular Ca2+ elevations [2, 3] that incrementally stimulate many events, including cortical granule (CG) exocytosis, cell-cycle resumption, and recruitment of maternal messenger RNAs [4]. Inhibition of Ca2+ oscillations in fertilized mouse eggs prevents CG exocytosis and cell-cycle resumption [5].

Events of egg activation are not stimulated directly by Ca2+ but, rather, through the actions of Ca2+-dependent regulatory proteins. An important master Ca2+ transducer is calmodulin (CAM), which has many potential downstream targets in all types of cells [6, 7]. Such a master transducer could, in theory, coordinate many of the known Ca2+-dependent events of fertilization. In fact, one CAM target, Ca2+/CAM-dependent protein kinase II (CAMK2), appears to regulate several events of egg activation [8–15]. Antagonists of CAMK2 inhibit cell-cycle resumption and CG exocytosis in artificially activated or fertilized mouse eggs. Activity of CAMK2 is oscillatory and parallels the transient elevations of Ca2+ in fertilized eggs [14, 15], suggesting that CAM becomes an active complex with Ca2+ in an oscillatory manner.

Other targets of Ca2+/CAM signaling, however, have not been investigated at fertilization and represent potentially important pathways that regulate events of egg activation. In this regard, another relevant Ca2+/CAM-dependent protein is myosin light chain kinase (MYLK2), which has the important function of regulating the cytoplasmic myosin isoform, myosin II [16, 17], an important cytoskeletal protein in eggs [18–20] and many other cell types [15, 21]. In immunofluorescence studies of preimplantation mouse embyros, MYLK2 has been reported in the 2-cell stage embryo [22]. Moreover, the MYLK2 inhibitor, ML-7, blocks chromatin-induced cortical actin localization and lateral CG movements in the mouse egg cortex [23]. Myosin II is reported to participate in eccentric meiotic spindle positioning as well as in the dynamics of the sperm incorporation cone in the mouse egg [18]. In that study, myosin II was found in the cortex and in the second polar body (PB) cleavage furrow.

Because the aforementioned studies indicate that MYLK2 and myosin have the potential to regulate cytoskeletal-mediated events in the egg cortex, the present study was undertaken to investigate possible roles for MYLK2 and myosin II in the regulation of CG exocytosis and second PB formation. These events are associated with the egg cortex, a region that is rich in actin and microfilaments and that undergoes changes at fertilization [24–28]. Little is known about how the egg cytoskeleton regulates CG translocation during exocytosis and spindle rotation during meiosis. Because they are commonly used in other cell types to investigate vesicle translocation and cytokinesis, two inhibitors (ML-7 and blebbistatin) were employed to antagonize MYLK2 activity and myosin II function, respectively. The results indicate new functions for MYLK2 and myosin II during fertilization-induced egg activation.

MATERIALS AND METHODS

Egg Collection

Ovulated metaphase II-arrested eggs were obtained from 6- to 14-wk-old female CF-1 mice (Harlan) following standard injection of 7.5 IU of eCG (Calbiochem) and, 48 h later, 7.5 IU of hCG (Sigma). Cumulus masses were collected 13–14 h post-hCG in Earle balanced salt solution (EBSS; Sigma), 0.3% bovine serum albumin (Sigma), and 25 mM Hepes buffer (pH 7.3) at 37°C. Cumulus cells were removed by treatment with 0.015% hyaluronidase (Calbiochem) in EBSS at 37°C for 2–4 min. Zonae were dissolved with Tyrode solution (pH 2.7; exposure time, <1 min) [29], and eggs were allowed to recover in minimal essential medium (MEM) [30] containing 0.1% polyvinyl alcohol (PVA; average molecular weight, ~10000; Sigma) at 37°C and under an atmosphere of 5% CO2 until use. Animals were used in accordance with the regulations of the National Institutes of Health and the Institutional Animal Care and Use Committee of Tufts-New England Medical Center.

In Vitro Fertilization

Sperm from the caudal region of the epididymides from 10- to 14-wk-old CF-1 male, proven-breeder mice (Harlan) were collected in MEM containing 1% bovine serum albumin (fatty-acid free; Sigma) and allowed to capacitate for 1 h at 37°C under an atmosphere of 5% CO2. Eggs were preincubated in MEM containing 0.1% PVA and inseminated in the same medium with 10–15 x 104 sperm/ml.

ML-7 and Blebbistatin

Both ML-7 and blebbistatin enantiomers (both from Calbiochem) were dissolved in dimethyl sulfoxide (DMSO), aliquoted, and stored at –80°C until use. The biologically active and inactive preparations of blebbistatin were used in a final concentration of 0.5% DMSO to minimize precipitation, which sometimes was evident at higher concentrations. Hence, 0.5% DMSO was added to all control groups to account for any effects from the DMSO. The day of the experiment, inhibitors (using stock solutions; see above) were added to MEM containing 0.1% PVA while gently vortexing to maximize solubility and minimize a change in pH. Unless otherwise specified, all eggs were preincubated for 15 min in inhibitors before insemination. Experiments were performed in four-well Nunclon {Delta}-treated plates (Fisher Scientific) at 37°C in a 5% CO2 incubator with the exception of eggs monitored for Ca2+ (see below).

Ca2+ Imaging and Ionomycin Treatment

Before ionomycin activation or insemination, eggs were first loaded with the fluorescent Ca2+ indicator, Fura-2 AM (0.75 µM), and then, if required, preincubated with 15 µM ML-7 in MEM containing 0.1% PVA. For ionomycin treatment, approximately 15 eggs were placed in a well of a Nunc Lab-Tek chambered cover glass containing 500 µL of Ca2+/Mg2+-free EBSS, and Ca2+ monitoring was begun as described previously [14, 15]. After 2 min at baseline, 1 µM ionomycin (final concentration; dissolved in DMSO) was added. The activated eggs were washed, frozen, and analyzed for CAMK2 activity as described previously [14, 15].

To determine if ML-7 altered the pattern of intracellular Ca2+ at fertilization, approximately 10 eggs, which previously had undergone Fura-2 loading and ML-7 preincubation, were placed in 500 µL of MEM containing 0.1% PVA in a well of a Cell-Tak (BD Biosciences)-treated chambered cover glass [14]. The eggs were inseminated, and Ca2+ monitoring was begun as described previously [14, 15].

CAMK2 Activity Assays

Autonomous CAMK2 activity, due to enzyme subunit autophosphorylation, was examined in the absence of added Ca2+/CAM as described previously [14]. The SignaTECT assay system (Promega), using a biotinylated peptide substrate and 2.5 µCi of [{gamma}32P]ATP (Perkin Elmer), was used.

Cortical Granule Experiments, Staining, and Quantification

At 1 h postinsemination, eggs were taken out of the incubator, fixed, and stained for CGs, chromatin, and sperm head decondensation as described previously [14]. Cortical granule density was examined using epifluorescence microscopy and computerized digital analysis, also as described previously [12].

Second PB Formation Experiments

Second PB formation was examined 1.5 h postinsemination and every 15 min thereafter until PB formation was approximately 90% complete in untreated control groups. Rescue experiments were performed to ensure that inhibitors were not used at toxic levels: Inhibitor-treated eggs were taken out of inhibitors at 2 h postinsemination and put into inhibitor-free MEM. These eggs were further cultured for 1 h and then examined for second PB formation.

Cell Cycle and Spindle Staining

To examine the cell-cycle further, eggs were fixed and stained for chromatin and spindle proteins using 4',6'-diamidino-2-phenylindole/Hoechst 33258 (Polysciences, Inc.) and {alpha}- and ß-tubulin antibodies (Sigma), respectively [31]. Briefly, eggs were preincubated in inhibitors, inseminated, and fixed at 1.5 h postinsemination in microtubule-stabilization buffer extraction fix for 1 h at 37°C. The eggs were then transferred to blocking solution (2% powdered milk, 2% normal goat serum, 1% bovine serum albumin, 0.1 M glycine, 0.2% azide, and 0.1% Triton X-100) until staining (within 1 wk of fixing; stored at 4°C). Monoclonal primary antibodies to {alpha}- and ß-tubulin were used at a dilution of 1:200, whereas secondary antibodies (goat anti-mouse coupled to Rhodamine Red; Molecular Probes) were used at a dilution of 1:500.

Statistics

Cortical granule densities were compared using the unpaired Student t-test. Second PB formation and spindle rotation, in the presence and absence of inhibitors in inseminated and fertilized eggs, respectively, were compared using chi-square analysis.

RESULTS

Evaluation of ML-7

To determine if events of egg activation are dependent on the activity of MYLK2, eggs were treated with ML-7, which is a cell-permeable, naphthalene sulfonamide derivative that inhibits MYLK2 by competing at its ATP-binding site [32]. ML-7 has been widely used to study MYLK2-dependent processes that are similar to those in activated eggs (e.g., vesicle movements, cell-cycle progression, and myosin II-mediated cytoskeletal changes; for references, see Discussion).

First, the potential for ML-7 to affect other signaling targets at fertilization was addressed (Fig. 1). To determine if ML-7 inhibits the activity of a Ca2+-dependent protein kinase, which is known to be required for events of egg activation [15], CAMK2 activity was analyzed in ML-7-treated eggs. Artificial activation was used to synchronize the elevation of Ca2+ in groups of eggs [14]. Eggs were preincubated in 15 µM ML-7, Ca2+ monitoring begun, and activation induced with 1 µM ionomycin (during continuous ML-7 treatment). Activity of CAMK2 was analyzed in eggs when Ca2+ reached a maximal level during the first large rise in Ca2+ [14] after ionomycin treatment (Fig. 1B). No detectable effect on CAMK2 activity was observed in 15 µM ML-7-treated groups compared to untreated controls (Fig. 1A). Consistent with the absence of an effect on CAMK2 activity, 15 µM ML-7 had no detectable effect on the elevation of intracellular Ca2+ by fertilization (Fig. 1, C and D). In addition, 15 µM ML-7 did not delay the onset of Ca2+ oscillations, as measured from the point of insemination, compared to the onset in untreated, fertilized controls (Fig. 1, C and D).


Figure 1
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  		FIG. 1. ML-7 (15 µM) does not inhibit CAMK2 activity or the onset of Ca2+ oscillations at fertilization. A) CAMK2 activity of eggs activated with 1 µM ionomycin in the presence and absence of 15 µM ML-7. Activity of CAMK2 is expressed relative to the activity in untreated metaphase II eggs, which was set at a value of one (see Materials and Methods). Values are presented as the mean ± SEM, and the number of determinations (three eggs/determination) is shown at the base of each bar. B) Representative Ca2+ traces of the first Ca2+ rise from in vitro fertilization (IVF) or 1 µM ionomycin treatment (IONO). Arrows represent the time at which eggs were frozen for analysis of CAMK2 activity. C andD) Representative Ca2+ traces of fertilized eggs, inseminated at zero time, in the absence (C) and the presence (D) of 15 µM ML-7. The mean Ca2+ oscillation frequencies were not significantly different. These and experiments in all subsequent figures were repeated at least twice

Although ML-7 is a relatively specific inhibitor [33], high concentrations also can inhibit protein kinase C and protein kinase A. The inhibition constants of ML-7 for these kinases, however, are higher than the median inhibitory concentration (IC50) values found for ML-7 in the present study (see below), and evidence suggests that these two kinases are not required for CG exocytosis and meiotic events in mouse eggs (see, e.g., [9, 34, 35] and that an active cAMP analog does not cause egg activation at metaphase II [36].

ML-7 and Cell-Cycle Progression

The only known target of MYLK2 is myosin II [17], which is known to be required for contractile ring function during cytokinesis. Because second PB formation also utilizes a contractile ring [18, 37], this event was monitored in eggs that were preincubated in various concentrations of ML-7, followed by insemination with continuous ML-7 treatment. Second PB formation was inhibited by ML-7 in a dose-dependent manner, with an approximate IC50 of 10 µM (Fig. 2A). At 2 h postinsemination, untreated eggs had undergone second PB formation, followed by basal constriction, whereas a very low percentage of eggs treated with 15 µM ML-7 had formed a second PB (Fig. 2, A and B).


Figure 2
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  		FIG. 2. Second PB formation is inhibited by ML-7 in a dose-dependent manner at 2 h postinsemination (A). The numbers of eggs analyzed are shown at the base of each bar, and values represent the mean of two experiments. Rescue refers to eggs that, after 2 h treatment in 15 µM ML-7, were transferred to medium without ML-7. A comparison of untreated and 15 µM ML-7-treated eggs indicated a significant difference (P < 0.005). Representative light micrographs of an untreated, uninseminated egg (B); an untreated egg at 2 h postinsemination (C); a 15 µM ML-7-treated egg at 2 h postinsemination (D); and an inseminated egg rescued for 1 h after 2 h of treatment with 15 µM ML-7 (E) also are shown. The first PB is lost during zona removal. Bar = 10 µm

To determine if failure of second PB formation was caused by an inhibition of meiotic resumption or subsequent cell-cycle events, eggs were preincubated and treated continuously with ML-7, inseminated, fixed, and stained for chromatin. In vitro fertilization was performed using zona-free eggs to optimize the synchronization of fertilization. Fertilized eggs were recognized by the presence of sperm head decondensation inside the egg; the mean percentage of eggs fertilized for untreated controls and 15 µM ML-7-treated eggs was 85% and 75%, respectively. Greater than 60% of fertilized, untreated control eggs had progressed to telophase II, as indicated by spindle rotation (i.e., the pole-to-pole axis of the spindle changed from being parallel to the egg surface [tangent] to being perpendicular) (Figs. 3 and 4, A and B). The chromosomes of the emerging second PB often were in a bulge on the cell surface, whereas the maternal zygotic chromatin was found below the egg cortex. In contrast, less than 10% of ML-7-treated eggs underwent these changes. Interestingly, ML-7-treated eggs underwent cell-cycle resumption, but approximately 90% appeared to be arrested in anaphase II, without spindle rotation (Figs. 3 and 4, A and C). Treatment for 2 h with 15 µM ML-7 alone (no sperm) did not cause cell-cycle resumption (Fig. 3). Also, 15 µM ML-7 treatment did not cause irreversible arrest of the cell cycle, because removal of ML-7 after 2 h resulted in second PB formation 1 h later (>90%) (Fig. 2, A and E).


Figure 3
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  		FIG. 3. ML-7 (15 µM) prevents cell-cycle completion. Cell-cycle analysis of fertilized eggs at 1 h postinsemination in the presence and absence of 15 µM ML-7 is shown. The numbers of eggs analyzed are indicated at the base of each bar


Figure 4
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  		FIG. 4. Spindle rotation is inhibited by ML-7 (15 µM) or blebbistatin (100 µM) in fertilized eggs analyzed at 1.5 h postinsemination (A). The numbers of eggs analyzed are shown at the base of each bar, and values represent the mean of two experiments. A comparison of untreated and 15 µM ML-7-treated eggs indicated a significant difference (P < 0.005); similarly, 100 µM blebbistatin (Blebb)-treated eggs were significantly different from controls (P < 0.005). Also shown (B–E) are representative fluorescence and light (inset) micrographs of the same fertilized egg at 1.5 h postinsemination, costained for chromatin (blue) and tubulin (red): untreated egg forming a second PB (B); yellow arrowhead, PB chromatin; white arrow, egg chromatin), 15 µM ML-7-treated egg (C), 100 µM blebbistatin-treated egg (D), and 100 µM inactive blebbistatin analog-treated egg (E). Rectangular box indicates decondensed sperm head. The spindle, maternal chromatin, and decondensed sperm chromatin were photographed at their respective focal planes, and the three images were merged. Other irregular blue-staining is primarily from single or multiple, superficially bound sperm chromatin, some of which is not in focus. Bar = 20 µm

ML-7 and CG Exocytosis

Because MYLK2 and myosin regulate secretion in other cells and ML-7 affects secretory vesicle recruitment (see Discussion), the effect of ML-7 on CG exocytosis was investigated. A treatment of 15 µM was chosen, because this concentration inhibits recruitment of the reserve pool of synaptic vesicles to the cell surface [38], a process that is analogous to CG translocation from the cortex to the plasma membrane during fertilization [13]. Eggs were preincubated in 15 µM ML-7, inseminated, and received continuous ML-7 treatment. In monospermically fertilized eggs (identified by chromatin staining), approximately 50% inhibition of CG exocytosis was observed in ML-7-treated eggs compared to untreated controls at 1 h postinsemination (Fig. 5). Polyspermically fertilized eggs appeared to be less inhibited, likely because of an increase in the frequency of Ca2+ oscillations [39]. Treatment with 15 µM ML-7 also inhibited ionomycin-induced CG exocytosis (data not shown). Uninseminated control eggs treated with 15 µM ML-7 had no detectable CG exocytosis compared to uninseminated, untreated control eggs (Fig. 5).


Figure 5
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  		FIG. 5. ML-7 (15 µM) inhibits CG exocytosis. At 1 h postinsemination, the percentage CG exocytosis was determined in monospermic and polyspermic fertilized eggs treated with 15 µM ML-7 and untreated fertilized eggs (calculated from the percentage of the mean CG density of uninseminated, untreated control eggs). The numbers of eggs analyzed are shown at the base of each bar. Bars represent the mean + SEM. Asterisks denote a significant difference (P < 0.001)

Blebbistatin and Events of Egg Activation

Blebbistatin is a recently discovered, cell-permeable, myosin inhibitor that has high affinity and selectivity for inhibiting the ATPase activity of myosin isoform II compared to other isoforms [40–42]. In living cells, the inhibitory concentration for preventing cytokinesis is 50–100 µM, and the inactive analog of blebbistatin is used as a control [40, 43].

In the inseminated eggs preincubated and maintained in blebbistatin, inhibition of second PB formation occurred in a dose-dependent manner (Fig. 6A). The IC50 was 6–12 µM, and 90% inhibition was observed using 100 µM. In contrast, at 2 h postinsemination, untreated eggs, as well as eggs cultured in 100 µM of the inactive analog, had undergone second PB formation (Fig. 6, A, C, and D). After chromatin staining, these two groups were found to have undergone similar extents of cell-cycle progress to telophase II at 1 h, whereas fertilized eggs treated with 100 µM active blebbistatin were in anaphase II (~90%) but had failed to enter telophase II, which is consistent with the absence of second PB formation (Figs. 6 and 7). Failure of entry into telophase II was associated with the absence of spindle rotation in blebbistatin-treated eggs but not in those treated with the inactive analog (Fig. 4, D and E). Blebbistatin also resulted in prolonged elevation of intracellular Ca2+ in fertilized eggs (unlike ML-7; a detailed report of these findings will appear elsewhere). Although this renders the analysis of CG exocytosis difficult to interpret (data not shown), significant inhibition of exocytosis was observed despite prolonged elevated Ca2+.


Figure 6
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  		FIG. 6. Second PB formation is inhibited by blebbistatin in a dose-dependent manner by 2 h post-insemination (A). The numbers of eggs analyzed are shown at the base of each bar, and values represent the mean of at least two experiments. Rescue refers to eggs that, after 2 h treatment in 100 µM blebbistatin, were transferred to medium without blebbistatin. Untreated and 25 µM blebbistatin-treated eggs were significantly different (P < 0.005). Representative light micrographs (at 2 h) of an untreated, uninseminated egg (B) or eggs postinsemination (C–G). C) Egg without blebbistatin treatment. D) 100 µM inactive blebbistatin analog-treated egg. E) 100 µM blebbistatin-treated egg, preincubated for 15 min prior to insemination. F) 100 µM blebbistatin-treated egg, without preincubation. G) Egg rescued for 1 h after 2 h of treatment with 100 µM blebbistatin. The first PB is lost during zone removal. Bar = 10 µm


Figure 7
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  		FIG. 7. Blebbistatin (100 µM) prevents cell-cycle completion. Uninseminated and fertilized eggs were fixed at 1 h postinsemination in the groups indicated and scored for cell-cycle stage after chromatin staining. The numbers of eggs analyzed are shown at the base of each bar

DISCUSSION

To our knowledge, this is the first report providing evidence that second PB formation and CG exocytosis are regulated by an MYLK2-myosin II pathway. This pathway appears to regulate spindle rotation and the meiotic transition from anaphase II to telophase II but not progress from metaphase II to anaphase II. Thus, cell-cycle progression in fertilized mammalian eggs relies on a temporal series of protein kinase activities, some of which regulate different steps required for the completion of meiosis, such as CAMK2 activity for the transition from metaphase II to anaphase II (see Introduction) and MYLK2 activity for progress from anaphase II to telophase II.

These findings extend earlier important studies of myosin II in mouse eggs by Simerly et al. [18]. For example, an important step in CG exocytosis is the translocation of CGs from the mammalian egg cortex to the plasma membrane, especially because full-grown, preovulatory mouse oocytes are incompetent to undergo this translocation [12, 13]. Because the basis for this maturation-associated change in the ability to undergo CG exocytosis is unknown, identification of the proteins required for translocation in mature eggs is needed, and MYLK2 may be an important regulator. For example, MYLK2 activity recently was found to be necessary for lateral movements of CGs in the mouse egg cortex [23], and other cell types require MYLK2 activity for secretory vesicle translocation and secretion [38, 44, 45]. Our finding that 15 µM ML-7 did not result in complete inhibition of CG exocytosis may be caused by the concentration used (increased ML-7 concentrations could lead to nonspecific effects) or other explanations, such as the involvement of MYLK2-independent myosin isoforms in secretion in other cells [21, 46, 47]. For example, it remains to be established whether CGs closest to the plasma membrane use precisely the same translocation proteins as CGs located in the deep cortex.

Because the myosin II light chain is the only known target of MYLK2 activity [17], we propose that MYLK2 activity is stimulated at fertilization by the increase in Ca2+ (see below), resulting in the activation of a myosin motor involved in CG translocation. In support of this, many studies have demonstrated that CG translocation to the cortex during oocyte maturation and to the plasma membrane during egg activation use microfilaments and actin, whereas microtubules are not required [48–52]. In addition, an RHO-signaling pathway, which has the potential to regulate egg cortical actin [53] and myosin function (see below), mediates CG translocation in sea urchin oocytes [54]. Thus, a myosin-actin system, not a kinesin-microtubule system, becomes the more likely CG translocation mechanism, which also is supported by the results of the present study. This model is consistent with studies in other cell types that have demonstrated a role for myosin II in secretion [55–58].

The inhibition of second PB formation by both ML-7 and blebbistatin is consistent with reports that myosin II is required for cytokinesis [59, 60]. Our finding that spindle rotation is prevented by both inhibitors, however, raises the possibility that an earlier step, just before cytokinesis, also may require MYLK2 and myosin II in eggs. Mouse eggs must undergo rotation of the axis of the second meiotic spindle [37, 61] before telophase II, the stage at which two putative daughter cells—the zygote and the second PB—are forming. The axis of rotation changes from parallel to perpendicular relative to the egg surface, and experiments with cytochalasin D indicate a requirement for actin and microfilaments in this process [37, 62]. In the presence of inhibitors, the failure of rotation prevents second PB protrusion from the egg surface; thus, the normal, small cytokinesis furrow cannot form at the base of the protrusion. Before egg activation, myosin IIA and IIB (like actin) are found in the cortex, with polarization over the spindle region [18]. Interestingly, in that study, only myosin IIA was observed in the spindle, whereas both isoforms normally are present in the cleavage furrow of the second PB. Thus, the localization of myosin is consistent with the inhibitor results in the present study.

The mechanism by which MYLK2 is regulated during spindle rotation is not clear at this time. After fertilization, Ca2+ oscillations continue during second PB formation in mammalian eggs [63], so MYLK2 may be activated in an oscillatory manner, as has been reported for CAMK2 [14, 15]. The fact that CAMK2 can negatively regulate MYLK2 [17] leads to the prediction that they are not colocalized. Interestingly, Ca2+ does not appear to be required at the time of second PB formation when artificial activation agents are used, especially in postovulatory, aged eggs. Such agents cause a single, early Ca2+ rise [64, 65] or act downstream of Ca2+ [35, 66, 67], and they are able to stimulate progress to telophase II of meiosis and second PB formation. Activity of MYLK2 appears to be necessary for spindle rotation based on inhibition by ML-7 and the normal dependence of myosin II activity on MYLK2. However, because a coincident increase in Ca2+ may not be required, it cannot be ruled out that MYLK2 also is regulated through a combination of both MAPK [23, 68] and another factor, the activity of which is fertilization-dependent. Regulation of MAPK has been implicated in normal second PB formation [67]. Alternatively, myosin II may be regulated by another mechanism, such as one involving RHO kinase [17]. In fact, RHOA is localized to the cleavage furrow of the second PB of mouse eggs, and RHOA kinase inhibitors prevent spindle rotation, cleavage furrow formation, and PB formation [69, 70].

Several comments about the approaches used are relevant. Similar effects with two different inhibitors in the same pathway (i.e., MYLK2/myosin II pathway) provide corroborating evidence for the role of this pathway in egg activation; further support comes from other studies of actin and RHO in second PB formation and CG translocation (see above). The antagonists used herein have the benefits of cell permeability, constant exposure during events of egg activation, dose-response effects, acceptable specificity, and extensive background information (see Results). In addition, multiple useful inhibitors for both MYLK2 and myosin II are not readily available. For example, microinjection of inhibitory antibodies to myosin II in eggs does not prevent second PB formation [18], which is unexpected, because myosin II provides the motor for contractile ring closure in cells [40, 71]. One potential explanation is that the usefulness of a single microinjection of protein is dependent on the maintenance of its activity over the considerable period of time required for the completion of the events of egg activation, and some proteins are actively targeted for degradation in activated eggs. Regarding immunofluorescence, the locations of myosin IIA and IIB have been reported during mouse fertilization [18], and currently available MYLK2 antibodies are unsuitable for immunofluorescence in mouse eggs [23]. Of the three isoforms of myosin II, myosin IIC is not required for cytokinesis in COS-7 cells [72].

In conclusion, the present study provides evidence for a new signaling pathway, which has important functions during egg activation and may act in concert with CAMK2. In addition, the proposed roles of MYLK2 and myosin II provide the basis for future studies to determine the development of competence to undergo CG translocation just before ovulation [73] and the mechanisms regulating the control of meiotic telophase II, spindle rotation, and second PB formation.

ACKNOWLEDGMENTS

The authors would like to thank the NICHD for financial support and Tim Ryan at Cornell University for his insights.

FOOTNOTES

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

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 Back

3 These authors contributed equally to this work. Back

Received: 5 August 2005.

First decision: 18 August 2005.

Accepted: 27 September 2005.

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