Analysis of the kar3 Meiotic Arrest in Saccharomyces cerevisiae

The motor protein Kar3p and its associated protein Cik1p are essential for passage through meiosis I. In the absence of either protein, meiotic cells arrest in prophase I. Experiments were performed to determine whether the arrest was caused by a structural inability to proceed through meiosis, or by a regulatory mechanism. The data demonstrate that the meiotic arrest is not structural; kar3 and cik1 mutants are able to form normal looking bipolar spindles and divide their DNA into two masses in spo11 mutant backgrounds. To identify the regulatory system necessary for the kar3/cik1 meiotic arrest, we tested whether the arrest could be bypassed by eliminating the pachytene checkpoint or the spindle checkpoint. The arrest is not solely dependent upon the pachytene checkpoint that monitors recombination and aspects of chromosome synapsis. Elimination of the spindle checkpoint failed to allow kar3 mutants to undergo meiosis I nuclear division, but phenotypes of the kar3/spindle checkpoint double mutants suggest that the kar3 meiotic arrest may be mediated by the spindle checkpoint.     

Analysis of the kar3 meiotic arrest in Saccharomyces cerevisiae

The motor protein Kar3p and its associated protein Cik1p are essential for passage through meiosis I. In the absence of either protein, meiotic cells arrest in prophase I. Experiments were performed to determine whether the arrest was caused by a structural inability to proceed through meiosis, or by a regulatory mechanism. The data demonstrate that the meiotic arrest is not structural; kar3 and cik1 mutants are able to form normal looking bipolar spindles and divide their DNA into two masses in spo11 mutant backgrounds. To identify the regulatory system necessary for the kar3/cik1 meiotic arrest, we tested whether the arrest could be bypassed by eliminating the pachytene checkpoint or the spindle checkpoint. The arrest is not solely dependent upon the pachytene checkpoint that monitors recombination and aspects of chromosome synapsis. Elimination of the spindle checkpoint failed to allow kar3 mutants to undergo meiosis I nuclear division, but phenotypes of the kar3/spindle checkpoint double mutants suggest that the kar3 meiotic arrest may be mediated by the spindle checkpoint.     

Differential regulation of the Kar3p kinesin-related protein by two associated proteins, Cik1p and Vik1p

The mechanisms by which kinesin-related proteins interact with other proteins to carry out specific cellular processes is poorly understood. The kinesin-related protein, Kar3p, has been implicated in many microtubule functions in yeast. Some of these functions require interaction with the Cik1 protein (Page, B.D., L.L. Satterwhite, M.D. Rose, and M. Snyder. 1994. J. Cell Biol. 124:507-519). We have identified a Saccharomyces cerevisiae gene, named VIK1, encoding a protein with sequence and structural similarity to Cik1p. The Vik1 protein is detected in vegetatively growing cells but not in mating pheromone-treated cells. Vik1p physically associates with Kar3p in a complex separate from that of the Kar3p-Cik1p complex. Vik1p localizes to the spindle-pole body region in a Kar3p-dependent manner. Reciprocally, concentration of Kar3p at the spindle poles during vegetative growth requires the presence of Vik1p, but not Cik1p. Phenotypic analysis suggests that Cik1p and Vik1p are involved in different Kar3p functions. Disruption of VIK1 causes increased resistance to the microtubule depolymerizing drug benomyl and partially suppresses growth defects of cik1Delta mutants. The vik1Delta and kar3Delta mutations, but not cik1Delta, partially suppresses the temperature-sensitive growth defect of strains lacking the function of two other yeast kinesin-related proteins, Cin8p and Kip1p. Our results indicate that Kar3p forms functionally distinct complexes with Cik1p and Vik1p to participate in different microtubule-mediated events within the same cell.     

Localization of the Kar3 kinesin heavy chain-related protein requires the Cik1 interacting protein

The Kar3 protein (Kar3p), a protein related to kinesin heavy chain, and the Cik1 protein (Cik1p) appear to participate in the same cellular processes in S. cerevisiae. Phenotypic analysis of mutants indicates that both CIK1 and KAR3 participate in spindle formation and karyogamy. In addition, the expression of both genes is induced by pheromone treatment. In vegetatively growing cells, both Cik1::beta-gal and Kar3::beta-gal fusions localize to the spindle pole body (SPB), and after pheromone treatment both fusion proteins localize to the spindle pole body and cytoplasmic microtubules. The dependence of Cik1p and Kar3p localization upon one another was investigated by indirect immunofluorescence of fusion proteins in pheromone-treated cells. The Cik1p::beta-gal fusion does not localize to the SPB or microtubules in a kar3 delta strain, and the Kar3p::beta-gal fusion protein does not localize to microtubule-associated structures in a cik1 delta strain. Thus, these proteins appear to be interdependent for localization to the SPB and microtubules. Analysis by both the two-hybrid system and co- immunoprecipitation experiments indicates that Cik1p and kar3p interact, suggesting that they are part of the same protein complex. These data indicate that interaction between a putative kinesin heavy chain-related protein and another protein can determine the localization of motor activity and thereby affect the functional specificity of the motor complex.     

The Yeast Motor Protein, Kar3p, Is Essential for Meiosis I

The recognition and alignment of homologous chromosomes early in meiosis is essential for their subsequent segregation at anaphase I; however, the mechanism by which this occurs is unknown. We demonstrate here that, in the absence of the molecular motor, Kar3p, meiotic cells are blocked with prophase monopolar microtubule arrays and incomplete synaptonemal complex (SC) formation. kar3 mutants exhibit very low levels of heteroallelic recombination. kar3 mutants do produce double-strand breaks that act as initiation sites for meiotic recombination in yeast, but at levels severalfold reduced from wild-type. These data are consistent with a meiotic role for Kar3p in the events that culminate in synapsis of homologues.     

The Cik1/Kar3 motor complex is required for the proper kinetochore-microtubule interaction after stressful DNA replication

In budding yeast Saccharomyces cerevisiae, kinetochores are attached by microtubules during most of the cell cycle, but the duplication of centromeric DNA disassembles kinetochores, which results in a brief dissociation of chromosomes from microtubules. Kinetochore assembly is delayed in the presence of hydroxyurea, a DNA synthesis inhibitor, presumably due to the longer time required for centromeric DNA duplication. Some kinetochore mutants are sensitive to stressful DNA replication as these kinetochore proteins become essential for the establishment of the kinetochore-microtubule interaction after treatment with hydroxyurea. To identify more genes required for the efficient kinetochore-microtubule interaction under stressful DNA replication conditions, we carried out a genome-wide screen for yeast mutants sensitive to hydroxyurea. From this screen, cik1 and kar3 mutants were isolated. Kar3 is the minus-end-directed motor protein; Cik1 binds to Kar3 and is required for its motor function. After exposure to hydroxyurea, cik1 and kar3 mutant cells exhibit normal DNA synthesis kinetics, but they display a significant anaphase entry delay. Our results indicate that cik1 cells exhibit a defect in the establishment of chromosome bipolar attachment in the presence of hydroxyurea. Since Kar3 has been shown to drive the poleward chromosome movement along microtubules, our data support the possibility that this chromosome movement promotes chromosome bipolar attachment after stressful DNA replication.     

Cik1 targets the minus-end kinesin depolymerase kar3 to microtubule plus ends

Kar3, a Saccharomyces cerevisiae Kinesin-14, is essential for karyogamy and meiosis I but also has specific functions during vegetative growth. For its various roles, Kar3 forms a heterodimer with either Cik1 or Vik1, both of which are noncatalytic polypeptides. Here, we present the first biochemical characterization of Kar3Cik1, the kinesin motor that is essential for karyogamy. Kar3Cik1 depolymerizes microtubules from the plus end and promotes robust minus-end-directed microtubule gliding. Immunolocalization studies show that Kar3Cik1 binds preferentially to one end of the microtubule, whereas the Kar3 motor domain, in the absence of Cik1, exhibits significantly higher microtubule lattice binding. Kar3Cik1-promoted microtubule depolymerization requires ATP turnover, and the kinetics fit a single exponential function. The disassembly mechanism is not microtubule catastrophe like that induced by the MCAK Kinesin-13s. Soluble tubulin does not activate the ATPase activity of Kar3Cik1, and there is no evidence of Kar3Cik1(.)tubulin complex formation as observed for MCAK. These results reveal a novel mechanism to regulate microtubule depolymerization. We propose that Cik1 targets Kar3 to the microtubule plus end. Kar3Cik1 then uses its minus-end-directed force to depolymerize microtubules from the plus end, with each tubulin-subunit release event tightly coupled to one ATP turnover.     

Kar3Vik1, a member of the kinesin-14 superfamily, shows a novel kinesin microtubule binding pattern

Kinesin-14 motors generate microtubule minus-end-directed force used in mitosis and meiosis. These motors are dimeric and operate with a nonprocessive powerstroke mechanism, but the role of the second head in motility has been unclear. In Saccharomyces cerevisiae, the Kinesin-14 Kar3 forms a heterodimer with either Vik1 or Cik1. Vik1 contains a motor homology domain that retains microtubule binding properties but lacks a nucleotide binding site. In this case, both heads are implicated in motility. Here, we show through structural determination of a C-terminal heterodimeric Kar3Vik1, electron microscopy, equilibrium binding, and motility that at the start of the cycle, Kar3Vik1 binds to or occludes two αβ-tubulin subunits on adjacent protofilaments. The cycle begins as Vik1 collides with the microtubule followed by Kar3 microtubule association and ADP release, thereby destabilizing the Vik1-microtubule interaction and positioning the motor for the start of the powerstroke. The results indicate that head-head communication is mediated through the adjoining coiled coil.     

Neck Rotation and Neck Mimic Docking in the Noncatalytic Kar3-associated Protein Vik1

It is widely accepted that movement of kinesin motor proteins is accomplished by coupling ATP binding, hydrolysis, and product release to conformational changes in the microtubule-binding and force-generating elements of their motor domain. Therefore, understanding how the Saccharomyces cerevisiae proteins Cik1 and Vik1 are able to function as direct participants in movement of Kar3Cik1 and Kar3Vik1 kinesin complexes presents an interesting challenge given that their motor homology domain (MHD) cannot bind ATP. Our crystal structures of the Vik1 ortholog from Candida glabrata may provide insight into this mechanism by showing that its neck and neck mimic-like element can adopt several different conformations reminiscent of those observed in catalytic kinesins. We found that when the neck is α-helical and interacting with the MHD core, the C terminus of CgVik1 docks onto the central β-sheet similarly to the ATP-bound form of Ncd. Alternatively, when neck-core interactions are broken, the C terminus is disordered. Mutations designed to impair neck rotation, or some of the neck-MHD interactions, decreased microtubule gliding velocity and steady state ATPase rate of CgKar3Vik1 complexes significantly. These results strongly suggest that neck rotation and neck mimic docking in Vik1 and Cik1 may be a structural mechanism for communication with Kar3.     

The ATPase Pathway That Drives the Kinesin-14 Kar3Vik1 Powerstroke

Kar3, a Saccharomyces cerevisiae microtubule minus-end-directed kinesin-14, dimerizes with either Vik1 or Cik1. The C-terminal globular domain of Vik1 exhibits the structure of a kinesin motor domain and binds microtubules independently of Kar3 but lacks a nucleotide binding site. The only known function of Kar3Vik1 is to cross-link parallel microtubules at the spindle poles during mitosis. In contrast, Kar3Cik1 depolymerizes microtubules during mating but cross-links antiparallel microtubules in the spindle overlap zone during mitosis. A recent study showed that Kar3Vik1 binds across adjacent microtubule protofilaments and uses a minus-end-directed powerstroke to drive ATP-dependent motility. The presteady-state experiments presented here extend this study and establish an ATPase model for the powerstroke mechanism. The results incorporated into the model indicate that Kar3Vik1 collides with the microtubule at 2.4 μm⁻¹ s⁻¹ through Vik1, promoting microtubule binding by Kar3 followed by ADP release at 14 s⁻¹. The tight binding of Kar3 to the microtubule destabilizes the Vik1 interaction with the microtubule, positioning Kar3Vik1 for the start of the powerstroke. Rapid ATP binding to Kar3 is associated with rotation of the coiled-coil stalk, and the postpowerstroke ATP hydrolysis at 26 s⁻¹ is independent of Vik1, providing further evidence that Vik1 rotates with the coiled coil during the powerstroke. Detachment of Kar3Vik1 from the microtubule at 6 s⁻¹ completes the cycle and allows the motor to return to its initial conformation. The results also reveal key differences in the ATPase cycles of Kar3Vik1 and Kar3Cik1, supporting the fact that these two motors have distinctive biological functions.     

Genetic interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during nuclear fusion in Saccharomyces cerevisiae

During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.     

Novel roles for saccharomyces cerevisiae mitotic spindle motors.

The single cytoplasmic dynein and five of the six kinesin-related proteins encoded by Saccharomyces cerevisiae participate in mitotic spindle function. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle. Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell. This study reveals that kinesin-related Kar3p and Kip3p are unique in that they perform roles both inside and outside the nucleus. Kar3p, like Kip3p, was found to be required for spindle positioning in the absence of dynein. The spindle positioning role of Kar3p is performed in concert with the Cik1p accessory factor, but not the homologous Vik1p. Kar3p and Kip3p were also found to overlap for a function essential for the structural integrity of the bipolar spindle. The cytoplasmic and nuclear roles of both these motors could be partially substituted for by the microtubule-destabilizing agent benomyl, suggesting that these motors perform an essential microtubule-destabilizing function. In addition, we found that yeast cell viability could be supported by as few as two microtubule-based motors: the BimC-type kinesin Cin8p, required for spindle structure, paired with either Kar3p or Kip3p, required for both spindle structure and positioning.     

Loss of function of the Cik1/Kar3 motor complex results in chromosomes with syntelic attachment that are sensed by the tension checkpoint

The attachment of sister kinetochores by microtubules emanating from opposite spindle poles establishes chromosome bipolar attachment, which generates tension on chromosomes and is essential for sister-chromatid segregation. Syntelic attachment occurs when both sister kinetochores are attached by microtubules from the same spindle pole and this attachment is unable to generate tension on chromosomes, but a reliable method to induce syntelic attachments is not available in budding yeast. The spindle checkpoint can sense the lack of tension on chromosomes as well as detached kinetochores to prevent anaphase onset. In budding yeast Saccharomyces cerevisiae, tension checkpoint proteins Aurora/Ipl1 kinase and centromere-localized Sgo1 are required to sense the absence of tension but are dispensable for the checkpoint response to detached kinetochores. We have found that the loss of function of a motor protein complex Cik1/Kar3 in budding yeast leads to syntelic attachments. Inactivation of either the spindle or tension checkpoint enables premature anaphase entry in cells with dysfunctional Cik1/Kar3, resulting in co-segregation of sister chromatids. Moreover, the abolished Kar3-kinetochore interaction in cik1 mutants suggests that the Cik1/Kar3 complex mediates chromosome movement along microtubules, which could facilitate bipolar attachment. Therefore, we can induce syntelic attachments in budding yeast by inactivating the Cik1/Kar3 complex, and this approach will be very useful to study the checkpoint response to syntelic attachments.     

Mechanistic analysis of the Saccharomyces cerevisiae kinesin Kar3

Kar3 is a minus-end-directed microtubule motor that is implicated in meiotic and mitotic spindle function in Saccharomyces cerevisiae. To date, the only truncated protein of Kar3 that has been reported to promote unidirectional movement in vitro is GSTKar3. This motor contains an NH2-terminal glutathione S-transferase (GST) tag followed by the Kar3 sequence that is predicted to form an extended alpha-helical coiled-coil. The alpha-helical domain leads into the neck linker and COOH-terminal motor domain. Kar3 does not homodimerize with itself but forms a heterodimer with either Cik1 or Vik1, both of which are non-motor polypeptides. We evaluated the microtubule-GSTKar3 complex in comparison to the microtubule-Kar3 motor domain complex to determine the distinctive mechanistic features required for GSTKar3 motility. Our results indicate that ATP binding was significantly faster for GSTKar3 than that observed previously for the Kar3 motor domain. In addition, microtubule-activated ADP release resulted in an intermediate that bound ADP weakly in contrast to the Kar3 motor domain, suggesting that after ADP release, the microtubule-GSTKar3 motor binds ATP in preference to ADP. The kinetics also showed that GST-Kar3 readily detached from the microtubule rather than remaining bound for multiple ATP turnovers. These results indicate that the extended alpha-helical domain NH2-terminal to the catalytic core provides the structural transitions in response to the ATPase cycle that are critical for motility and that dimerization is not specifically required. This study provides the foundation to define the mechanistic contributions of Cik1 and Vik1 for Kar3 force generation and function in vivo.     

Microtubule motor protein Kar3 is required for normal mitotic division and morphogenesis in Candida albicans

The kinesin-related protein Kar3 is a minus end-directed molecular motor that plays a multifunctional role in microtubule-directed nuclear movement. Previously, it was shown that Candida albicans Kar3p is critical for nuclear fusion during mating as kar3 mutants were defective in karyogamy. In this study, we confirm that Kar3p is required for nuclear congression in mating but that neither Kar3p nor the dynein motor protein Dyn1p is required for nuclear migration in the mating projection prior to cell fusion. In addition, we show that C. albicans Kar3p plays an important role in the cell and colony morphology of mitotically dividing cells, as evidenced by diminished filamentation of kar3 cells on Spider medium and an increased tendency of mutant cells to form pseudohyphal cells in liquid culture. Loss of Kar3p also led to defects in nuclear division, causing cells to grow slowly and exhibit reduced viability compared to wild-type cells. Slow growth was due, at least in part, to delayed cell cycle progression, as cells lacking Kar3p accumulated in anaphase of the cell cycle. Consistent with a role in mitotic division, Kar3 protein was shown to localize to the spindle pole bodies. Finally, kar3 cells exhibited unstable or aberrant mitotic spindles, a finding that accounts for the delay in cell cycle progression and decreased viability of these cells. We suggest that the altered morphology of kar3 cells is a direct consequence of the delay in anaphase, and this leads to increased polarized growth and pseudohypha formation.     

Genetic control of recombination partner preference in yeast meiosis. Isolation and characterization of mutants elevated for meiotic unequal sister-chromatid recombination.

Meiotic exchange occurs preferentially between homologous chromatids, in contrast to mitotic recombination, which occurs primarily between sister chromatids. To identify functions that direct meiotic recombination events to homologues, we screened for mutants exhibiting an increase in meiotic unequal sister-chromatid recombination (SCR). The msc (meiotic sister-chromatid recombination) mutants were quantified in spo13 meiosis with respect to meiotic unequal SCR frequency, disome segregation pattern, sporulation frequency, and spore viability. Analysis of the msc mutants according to these criteria defines three classes. Mutants with a class I phenotype identified new alleles of the meiosis-specific genes RED1 and MEK1, the DNA damage checkpoint genes RAD24 and MEC3, and a previously unknown gene, MSC6. The genes RED1, MEK1, RAD24, RAD17, and MEC1 are required for meiotic prophase arrest induced by a dmc1 mutation, which defines a meiotic recombination checkpoint. Meiotic unequal SCR was also elevated in a rad17 mutant. Our observation that meiotic unequal SCR is elevated in meiotic recombination checkpoint mutants suggests that, in addition to their proposed monitoring function, these checkpoint genes function to direct meiotic recombination events to homologues. The mutants in class II, including a dmc1 mutant, confer a dominant meiotic lethal phenotype in diploid SPO13 meiosis in our strain background, and they identify alleles of UBR1, INP52, BUD3, PET122, ELA1, and MSC1-MSC3. These results suggest that DMC1 functions to bias the repair of meiosis-specific double-strand breaks to homologues. We hypothesize that the genes identified by the class II mutants function in or are regulators of the DMC1-promoted interhomologue recombination pathway. Class III mutants may be elevated for rates of both SCR and homologue exchange.     

Kar3Vik1 Mechanochemistry Is Inhibited by Mutation or Deletion of the C Terminus of the Vik1 Subunit

Force production by kinesins has been linked to structural rearrangements of the N and C termini of their motor domain upon nucleotide binding. In recent crystal structures, the Kar3-associated protein Vik1 shows unexpected homology to these conformational states even though it lacks a nucleotide-binding site. This conservation infers a degree of commonality in the function of the N- and C-terminal regions during the mechanochemical cycle of all kinesins and kinesin-related proteins. We tested this inference by examining the functional effects on Kar3Vik1 of mutating or deleting residues in Vik1 that are involved in stabilizing the C terminus against the core and N terminus of the Vik1 motor homology domain (MHD). Point mutations at two moderately conserved residues near the Vik1 C terminus impaired microtubule gliding and microtubule-stimulated ATP turnover by Kar3Vik1. Deletion of the seven C-terminal residues inhibited Kar3Vik1 motility much more drastically. Interestingly, none of the point mutants seemed to perturb the ability of Kar3Vik1 to bind microtubules, whereas the C-terminal truncation mutant did. Molecular dynamics simulations of these C-terminal mutants showed distinct root mean square fluctuations in the N-terminal region of the Vik1 MHD that connects it to Kar3. Here, the degree of motion in the N-terminal portion of Vik1 highly correlated with that in the C terminus. These observations suggest that the N and C termini of the Vik1 MHD form a discrete folding motif that is part of a communication pathway to the nucleotide-binding site of Kar3.     

Developmental control of oocyte maturation and egg activation in metazoan models

Production of functional eggs requires meiosis to be coordinated with developmental signals. Oocytes arrest in prophase I to permit oocyte differentiation, and in most animals, a second meiotic arrest links completion of meiosis to fertilization. Comparison of oocyte maturation and egg activation between mammals, Caenorhabditis elegans, and Drosophila reveal conserved signaling pathways and regulatory mechanisms as well as unique adaptations for reproductive strategies. Recent studies in mammals and C. elegans show the role of signaling between surrounding somatic cells and the oocyte in maintaining the prophase I arrest and controlling maturation. Proteins that regulate levels of active Cdk1/cyclin B during prophase I arrest have been identified in Drosophila. Protein kinases play crucial roles in the transition from meiosis in the oocyte to mitotic embryonic divisions in C. elegans and Drosophila. Here we will contrast the regulation of key meiotic events in oocytes.     

Zinc maintains prophase I arrest in mouse oocytes through regulation of the MOS-MAPK pathway

Meiosis in mammalian females is marked by two arrest points, at prophase I and metaphase II, which must be tightly regulated in order to produce a haploid gamete at the time of fertilization. The transition metal zinc has emerged as a necessary and dynamic regulator of the establishment, maintenance, and exit from metaphase II arrest, but the roles of zinc during prophase I arrest are largely unknown. In this study, we investigate the mechanisms of zinc regulation during the first meiotic arrest. Disrupting zinc availability in the prophase I arrested oocyte by treatment with the heavy metal chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) causes meiotic resumption even in the presence of pharmacological inhibitors of meiosis. We further show that the MOS-MAPK pathway mediates zinc-dependent prophase I arrest, as the pathway prematurely activates during TPEN-induced meiotic resumption. Conversely, inhibition of the MOS-MAPK pathway maintains prophase I arrest. While prolonged zinc insufficiency ultimately results in telophase I arrest, early and transient exposure of oocytes to TPEN is sufficient to induce meiotic resumption and bypass the telophase I block, allowing the formation of developmentally competent eggs upon parthenogenetic activation. These results establish zinc as a crucial regulator of meiosis throughout the entirety of oocyte maturation, including the maintenance of and release from the first and second meiotic arrest points.