Kinesin-1 prevents capture of the oocyte meiotic spindle by the sperm aster.

Centrioles are lost during oogenesis and inherited from the sperm at fertilization. In the zygote, the centrioles recruit pericentriolar proteins from the egg to form a mature centrosome that nucleates a sperm aster. The sperm aster then captures the female pronucleus to join the maternal and paternal genomes. Because fertilization occurs before completion of female meiosis, some mechanism must prevent capture of the meiotic spindle by the sperm aster. Here we show that in wild-type Caenorhabditis elegans zygotes, maternal pericentriolar proteins are not recruited to the sperm centrioles until after completion of meiosis. Depletion of kinesin-1 heavy chain or its binding partner resulted in premature centrosome maturation during meiosis and growth of a sperm aster that could capture the oocyte meiotic spindle. Kinesin prevents recruitment of pericentriolar proteins by coating the sperm DNA and centrioles and thus prevents triploidy by a nonmotor mechanism.

CDK-1 inhibits meiotic spindle shortening and dynein-dependent spindle rotation in C. elegans.

In animals, the female meiotic spindle is positioned at the egg cortex in a perpendicular orientation to facilitate the disposal of half of the chromosomes into a polar body. In Caenorhabditis elegans, the metaphase spindle lies parallel to the cortex, dynein is dispersed on the spindle, and the dynein activators ASPM-1 and LIN-5 are concentrated at spindle poles. Anaphase-promoting complex (APC) activation results in dynein accumulation at spindle poles and dynein-dependent rotation of one spindle pole to the cortex, resulting in perpendicular orientation. To test whether the APC initiates spindle rotation through cyclin B-CDK-1 inactivation, separase activation, or degradation of an unknown dynein inhibitor, CDK-1 was inhibited with purvalanol A in metaphase-I-arrested, APC-depleted embryos. CDK-1 inhibition resulted in the accumulation of dynein at spindle poles and dynein-dependent spindle rotation without chromosome separation. These results suggest that CDK-1 blocks rotation by inhibiting dynein association with microtubules and with LIN-5-ASPM-1 at meiotic spindle poles and that the APC promotes spindle rotation by inhibiting CDK-1.

Nuclear and spindle positioning during oocyte meiosis.

Female meiosis is unique in that an asymmetrically positioned meiotic spindle expels chromosomes into tiny, non-developing polar bodies. The extrusion of chromosomes into polar bodies is always mediated by meiotic spindles that are attached to the oocyte cortex by one pole. The asymmetric, cortical positioning of the oocyte meiotic spindle preserves the volume and contents of the oocyte. Recent work in C. elegans and mouse has provided mechanistic details of spindle positioning in oocytes.

Kinesin-dependent transport results in polarized migration of the nucleus in oocytes and inward movement of yolk granules in meiotic embryos.

During female meiosis, meiotic spindles are positioned at the oocyte cortex to allow expulsion of chromosomes into polar bodies. In C. elegans, kinesin-dependent translocation of the entire spindle to the cortex precedes dynein-dependent rotation of one spindle pole toward the cortex. To elucidate the role of kinesin-1 in spindle translocation, we examined the localization of kinesin subunits in meiotic embryos. Surprisingly, kinesin-1 was not associated with the spindle and instead was restricted to the cytoplasm in the middle of the embryo. Yolk granules moved on linear tracks, in a kinesin-dependent manner, away from the cortex, resulting in their concentration in the middle of the embryo where the kinesin was concentrated. These results suggest that cytoplasmic microtubules might be arranged with plus ends extending inward, away from the cortex. This microtubule arrangement would not be consistent with direct transport of the meiotic spindle toward the cortex by kinesin-1. In maturing oocytes, the nucleus underwent kinesin-dependent migration to the future site of spindle attachment at the anterior cortex. Thus the spindle translocation defect observed in kinesin-1 mutants may be a result of failed nuclear migration, which places the spindle too far from the cortex for the spindle translocation mechanism to function.

Kinesin-1 and cytoplasmic dynein act sequentially to move the meiotic spindle to the oocyte cortex in Caenorhabditis elegans.

During female meiosis in animals, the meiotic spindle is attached to the egg cortex by one pole during anaphase to allow selective disposal of half the chromosomes in a polar body. In Caenorhabditis elegans, this anaphase spindle position is achieved sequentially through kinesin-1-dependent early translocation followed by anaphase-promoting complex (APC)-dependent spindle rotation. Partial depletion of cytoplasmic dynein heavy chain by RNA interference blocked spindle rotation without affecting early translocation. Dynein depletion also blocked the APC-dependent late translocation that occurs in kinesin-1-depleted embryos. Time-lapse imaging of green fluorescent protein-tagged dynein heavy chain as well as immunofluorescence with dynein-specific antibodies revealed that dynein starts to accumulate at spindle poles just before the initiation of rotation or late translocation. Accumulation of dynein at poles was kinesin-1 independent and APC dependent, just like dynein driven spindle movements. This represents a case of kinesin-1/dynein coordination in which these two motors of opposite polarity act sequentially and independently on a cargo to move it in the same direction.

Differential timing of S phases, X chromosome replication, and meiotic prophase in the C. elegans germ line.

The replication of chromosomes in meiosis is an important first step for subsequent chromosomal interactions that promote accurate disjunction in the first of two segregation events to generate haploid gametes. We have developed an assay to monitor DNA replication in vivo in mitotic and meiotic germline nuclei of the nematode Caenorhabditis elegans. Using mutants that affect the mitosis/meiosis switch, we show that meiotic S phase is at least twice as long as mitotic S phase in C. elegans germ cell nuclei. Furthermore, our assay reveals that different regions of the genome replicate at different times, with the heterochromatic-like X chromosomes replicating at a distinct time from the autosomes. Finally, we have exploited S-phase labeling to monitor the timing of progression through meiotic prophase. Meiotic prophase for oocyte production in hermaphrodites lasts 54-60 h. Further, we find that the duration of the pachytene sub-stage is modulated by the presence of sperm. On the other hand, meiotic prophase for sperm production in males is completed by 20-24 h. Possible sources for the sex-specific differences in meiotic prophase kinetics are discussed.