Meiotic Spindle Assembly Research Articles

A point mutation in the microtubule binding region of the Ncd motor protein reduces motor velocity.

Non-claret disjunctional (Ncd) is a kinesin-related microtubule motor protein in Drosophila that functions in meiotic spindle assembly in oocytes and spindle pole maintenance in early embryos. The partial loss-of-function mutant ncdD retains mitotic, but not meiotic, function. The predicted NcdD mutant protein contains a V556-->F mutation in the putative microtubule binding region of the Ncd motor domain. Here we report an analysis of the properties of recombinant Ncd and NcdD proteins. A GST-NcdD fusion protein translocated microtubules approximately 10-fold more slowly than the corresponding wild-type protein in gliding assays. The maximum microtubule-stimulated ATPase activity of an NcdD motor domain protein was reduced approximately 3-fold and an approximately 3-fold greater concentration of microtubules was required for half-maximal stimulation of ATPase activity, compared with the corresponding wild-type protein. The Km for ATP and basal rate of ATP turnover were, in contrast, similar for the NcdD mutant and wild-type Ncd motor domain proteins. Pelleting assays demonstrated that the binding of the mutant NcdD motor protein to microtubules was reduced in the absence of nucleotide, relative to wild-type. The reduced velocity of NcdD translocation on microtubules is therefore correlated with reductions in microtubule-stimulated ATPase activity and affinity of the mutant motor for microtubules. The characteristics of the NcdD motor explain its meiotic loss of function, and are consistent with partial motor activity of Ncd being sufficient for its mitotic, but not its meiotic, role.

Variation of mouse oocyte sensitivity to griseofulvin-induced aneuploidy and meiotic delay during the first meiotic division.

The effects of varying the time of chemical treatment on the induction of aneuploidy and meiotic delay in metaphase II (MII) oocytes were studied by administering 1,500 mg/kg griseofulvin (GF) at 0, 2, 4, 6, or 8 hr after an injection of human chorionic gonadotrophin (HCG). The results show that the oocytes have a different sensitivity to GF-induced aneuploidy and meiotic delay during the course of meiotic maturation. Although not restricted to a particular period of meiotic maturation, the frequency of aneuploidy was highest (P < 0.05) when GF was given at 2, 4, or 6 hr after HCG. The maximum frequency of hyperploidy (42.4%) occurred at the 4-hr treatment time. Also, GF treatment resulted in the induction of meiotic delay as demonstrated by ovulated metaphase I (MI) and polyploid MII oocytes. The meiotic delay data depict a period of relative resistance between two periods of sensitivity in that the percentages of ovulated MI oocytes were 53.3, 21.3, 3.5, 6.7, and 25.7 when GF was given at 0, 2, 4, 6, and 8 hr after HCG, respectively. Also, at these treatment times the percentages of polyploid oocytes were 0.6, 1.7, 7.7, 20.1, and 15.4, respectively. Therefore, the oocytes seem to be more sensitive to GF-induced meiotic delay during the periods preceding and following meiotic spindle assembly. In conclusion, the results demonstrate that the time of chemical treatment influences the frequency of aneuploidy and the degree of meiotic delay. Also, the results emphasize that to thoroughly characterize the aneugenic potential of a specific chemical several treatment times may be needed.

Ectopic Spindle Assembly during Maturation of Xenopus Oocytes: Evidence for Functional Polarization of the Oocyte Cortex

Inversion of stage VI Xenopus oocytes, when combined with cold-induced microtubule depolymerization, resulted in the displacement of the germinal vesicle (the oocyte nucleus) from its normal position in the animal hemisphere to a more equatorial or vegetal position. Confocal immunofluorescence microscopy with anti-tubulin antibodies revealed that the breakdown of ectopic germinal vesicles during progesterone-induced maturation of inverted oocytes was accompanied by the assembly of a transient MTOC-microtubule complex (MTOC-TMA complex) that migrated to the region of cortex nearest the site of GV breakdown. Formation of ectopic meiotic spindles, including spindle rotation and polar body formation, was observed with nearly equal frequency throughout the animal hemisphere. In contrast, the frequency of spindle formation in the vegetal hemisphere was substantially reduced. Many spindles that did form in more vegetal regions of the oocyte appeared unable to rotate and did not result in polar body formation. The results presented suggest that the assembly of meiotic spindles at the animal pole during maturation of Xenopus oocytes does not result from a specific targeting of the MTOC-TMA complex during oocyte maturation. However, the oocyte cortex does exhibit A-V polarization of its ability to support spindle rotation and polar body formation. Finally, the observed increase in the incidence of monaster formation in the vegetal hemisphere suggests that interactions between the nascent spindle and cortex may regulate spindle assembly and elongation.

Protein synthesis requirements during resumption of meiosis in the hamster oocyte: early nuclear and microtubule configurations.

The organization of chromatin and cytoplasmic microtubules changes abruptly at M-phase entry in both mitotic and meiotic cell cycles. To determine whether the early nuclear and cytoplasmic events associated with meiotic resumption are dependent on protein synthesis, cumulus-enclosed hamster oocytes were cultured in the presence of 100 micrograms/ml puromycin or cycloheximide for 5 hr. Both control (untreated) and treated oocytes were analyzed by fluorescence microscopy after staining with Hoechst 33258 and tubulin antibodies. Freshly isolated oocytes exhibit prominent nucleoli and diffuse chromatin within the germinal vesicle as well as an interphase network of cytoplasmic microtubules. After 4-4.5 hr in culture, most oocytes were in prometaphase I of meiosis as characterized by a prominent spindle with fully condensed chromosomes and numerous cytoplasmic asters. After 5-5.5 hr in culture, microtubule asters are no longer detected in most cells, and the spindle is the only tubulin-positive structure. Incubation for 5 hr in the presence of inhibitors does not impair germinal vesicle breakdown, chromatin condensation, kinetochore microtubule assembly, or cytoplasmic aster formation in the majority of oocytes examined; however, under these conditions, a population of oocytes retains a germinal vesicle, exhibiting variable degrees of chromatin condensation and cytoplasmic aster formation. Meiotic spindle formation is inhibited in all oocytes. These effects are fully reversible upon culture of treated oocytes in drug-free medium for 5 hr. The data indicate that meiotic spindle assembly is dependent on ongoing protein synthesis in the cumulus-enclosed hamster oocyte; in contrast, chromatin condensation and aster formation are not as sensitive to protein synthesis inhibitors during meiotic resumption.

Centriole duplication in lysates of Spisula solidissima oocytes.

A cell-free system has been developed that executes centriole duplication. Surf clam (Spisula solidissima) oocytes, arrested at late prophase of meiosis I, do not contain centrioles, centrosomes, or asters. Serial section high-voltage electron microscopy (HVEM) of asters and spindles isolated from potassium chloride-activated oocytes indicates that within 4 minutes oocytes assemble a single centriole that is duplicated by 15 minutes when assembly of the first meiotic spindle is complete. A mixture of lysates from unactivated oocytes and potassium chloride-activated oocytes induces centriole formation and duplication. Astral microtubule content in these lysate mixtures increases with time.

Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein.

Mature Drosophila oocytes are arrested in metaphase of the first meiotic division. We have examined microtubule and chromatin reorganization as the meiosis I spindle assembles on maturation using indirect immunofluorescence and laser scanning confocal microscopy. The results suggest that chromatin captures or nucleates microtubules, and that these subsequently form a highly tapered spindle in which the majority of microtubules do not terminate at the poles. Nonexchange homologs separate from each other and move toward opposite poles during spindle assembly. By the time of metaphase arrest, these chromosomes are positioned on opposite half spindles, between the metaphase plate and the spindle poles, with the large nonexchange X chromosomes always closer to the metaphase plate than the smaller nonexchange fourth chromosomes. Nonexchange homologs are therefore oriented on the spindle in the absence of a direct physical linkage, and the spindle position of these chromosomes appears to be determined by size. Loss-of-function mutations at the nod locus, which encodes a kinesin-like protein, cause meiotic loss and nondisjunction of nonexchange chromosomes, but have little or no effect on exchange chromosome segregation. In oocytes lacking functional nod protein, most of the nonexchange chromosomes are ejected from the main chromosomal mass shortly after the nuclear envelope breaks down and microtubules interact with the chromatin. In addition, the nonexchange chromosomes that are associated with spindles in nod/nod oocytes show excessive poleward migration. Based on these observations, and the structural similarity of the nod protein and kinesin, we propose that nonexchange chromosomes are maintained on the half spindle by opposing poleward and anti-poleward forces, and that the nod protein provides the anti-poleward force.

Mutants of the microtubule motor protein, nonclaret disjunctional, affect spindle structure and chromosome movement in meiosis and mitosis.

The Drosophila microtubule motor protein, nonclaret disjunctional (ncd), is required for proper chromosome distribution in meiosis and mitosis. We have examined the meiotic and mitotic divisions in wild-type Drosophila oocytes and early embryos, and the effects of three ncd mutants (cand, ncd and ncdD) on spindle structure and chromosome movement. The ncd mutants cause abnormalities in spindle structure early in meiosis I, and abnormal chromosome configurations throughout meiosis I and II. Defective divisions continue in early embryos of the motor null mutant, cand, with abnormal early mitotic spindles. The effects of mutants on spindle structure suggest that ncd is required for proper meiotic spindle assembly, and may play a role in forming or maintaining spindle poles in meiosis. The disruption of normal meiotic and mitotic chromosome distribution by ncd mutants can be attributed to its role as a spindle motor, although a role for ncd as a chromosome-associated motor protein is not excluded. The ncd motor protein functions not only in meiosis, but also performs an active role in the early mitotic divisions of the embryo.

Two Drosophila beta tubulin isoforms are not functionally equivalent.

We have tested the functional capacity of different beta tubulin isoforms in vivo by expressing beta 3-tubulin either in place of or in addition to beta 2-tubulin in the male germ line of Drosophila melanogaster. The testes-specific isoform, beta 2, is conserved relative to major metazoan beta tubulins, while the developmentally regulated isoform, beta 3, is considerably divergent in sequence. beta 3-tubulin is normally expressed in discrete subsets of cells at specific times during development, but is not expressed in the male germ line. beta 2-Tubulin is normally expressed only in the postmitotic germ cells of the testis, and is required for all microtubule-based functions in these cells. The normal functions of beta 2-tubulin include assembly of meiotic spindles, axonemes, and at least two classes of cytoplasmic microtubules, including those associated with the differentiating mitochondrial derivatives. A hybrid gene was constructed in which 5' sequences from the beta 2 gene were joined to protein coding and 3' sequences of the beta 3 gene. Drosophila transformed with the hybrid gene express beta 3-tubulin in the postmitotic male germ cells. When expressed in the absence of the normal testis isoform, beta 3-tubulin supports assembly of one class of functional cytoplasmic microtubules. In such males the microtubules associated with the membranes of the mitochondrial derivatives are assembled and normal mitochondrial derivative elongation occurs, but axoneme assembly and other microtubule-mediated processes, including meiosis and nuclear shaping, do not occur. These data show that beta 3 tubulin can support only a subset of the multiple functions normally performed by beta 2, and also suggest that the microtubules associated with the mitochondrial derivatives mediate their elongation. When beta 3 is coexpressed in the male germ line with beta 2, at any level, spindles and all classes of cytoplasmic microtubules are assembled and function normally. However, when beta 3-tubulin exceeds 20% of the total testis beta tubulin pool, it acts in a dominant way to disrupt normal axoneme assembly. In the axonemes assembled in such males, the doublet tubules acquire some of the morphological characteristics of the singlet microtubules of the central pair and accessory tubules. These data therefore unambiguously demonstrate that the Drosophila beta tubulin isoforms beta 2 and beta 3 are not equivalent in intrinsic functional capacity, and furthermore show that assembly of the doublet tubules of the axoneme imposes different constraints on beta tubulin function than does assembly of singlet microtubules.

Microtubules during germinal vesicle breakdown (GVBD) of Xenopus oocytes: effect of Ca2+ ionophore A-23187 and taxol.

During the first meiotic cell division of the Xenopus oocyte, a transient microtubule network appears at the basal part of the disintegrating nucleus. In order to know if this structure plays a role in the formation of the first meiotic spindle, we have studied the actions of Ca2+ ions and taxol during the whole maturation process. We now report that when the free cytosolic Ca2+ concentration is raised by ionophore A 23187, the perinuclear microtubule array is disrupted and the spindle formation is inhibited. When taxol is microinjected into maturing oocytes, the perinuclear microtubules become anarchically distributed and the normal organization of the metaphase spindle is blocked. MPF (Maturation promoting factor) activity is maintained in these experimental conditions (Germinal vesicle breakdown is induced). These results indicate that polymerization/depolymerization of perinuclear microtubules are involved in the nuclear envelope breakdown and the subsequent assembly of the first meiotic spindle.