Background and Significance

    Significance to human health.

    Female meiosis is critical for the development of human embryos.  Down Syndrome and a high proportion of miscarriages are caused by non-disjunction during female meiosis (Koehler and Hassold, 1998). In addition, current methods for mammalian cloning involve removing the meiotic spindle from the metaphase II-arrested egg and introducing a G1 diploid nucleus which is converted into a diploid pseudo-meiosis II spindle.  If human cloning is to be used for generating therapeutic stem cells that are immunologically matched to a patient, the donor nucleus must come from adult somatic cells.  These somatic cell nuclei, however, assemble abnormal meiosis II spindles (Miyara et al., 2006) that may lead to aneuploidy and developmental defects that currently limit production of therapeutic stem cells.  Thus the mechanisms that mediate assembly and function of female meiotic spindles have direct medical relevance.  The significance of female meiosis is also supported by the absolute conservation of cortical meiotic spindle positioning in animal eggs.

    Cortical positioning of female meiotic spindles is highly conserved in animals.

    During meiosis in most species, chromosome number is reduced four-fold by first segregating pairs of homologous chromosomes and subsequently segregating pairs of sister chromatids.  In male animals, these sequential chromosome segregation events result in four haploid sperm.  In contrast, only one of four meiotic products is inherited during female meiosis.  This asymmetric inheritance is mediated by meiotic spindles that are attached by one pole to the oocyte cortex at anaphase (Albertson and Thompson, 1993; Endow and Komma, 1997 and 1998; Maro et al., 1984; Gard et al., 1995).  Cortical meiotic spindle positioning is followed by asymmetric cell divisions that leave one haploid set of chromosomes in the female pronucleus of a large egg and the "extra" chromosome in tiny cells called polar bodies (Fig. 1).  This type of meiotic division has led to the common belief that asymmetric spindle positioning has been conserved in evolution because it maximizes the volume of the resulting zygote.  Many species of insects, however, do not extrude polar bodies (Endow and Komma, 1998; Riparbelli et al., 2005; Tram and Sullivan, 2000) but instead generate a "tetrad" of 4 haploid nuclei.  One nucleus is inherited and the others are killed (Fig. 1).  In these species, there is no asymmetric cytokinesis and yet meiotic spindles are still attached to the egg cortex by one pole during anaphase.  At least in Nasonia, where these events have been directly filmed (Tram and Sullivan, 2000), the nucleus farthest from the cortex is always inherited.  Thus the cortical position of insect meiotic spindles may still mediate the selective "killing" of ¾ of the meiotic products that were pushed toward the cortex, just as in species that do extrude polar bodies.

Figure 1

Another group of animals that illustrate the conservation of female meiotic spindle positioning are those that exhibit "mitotic parthenogenesis".  In mitotic parthenogenesis, there are no cross-overs or other attachments between homologous chromosomes and Mendelian segregation of markers on homologous chromosomes does not occur.  Thus a Strongyloides ratti mother that is heterozygous for a molecular marker has only heterozygous progeny (Viney, 1994).  In "mitotic" strains of Meloidogyne hapla, a single mitosis-like division occurs in which half the sister chromatids of a diploid oocyte are expelled in a polar body (Triantaphyllou, 1966).  In the triploid fish, Poecilliopsis, an extra S-phase occurs and the transiently hexaploid oocyte goes through two consecutive asymmetric divisions expelling two polar bodies but only sister chromatids are expelled at each division (Cimino, 1972).  Thus asymmetric spindle positioning and asymmetric cell division during female meiosis appear to be more conserved than recombination or even Mendelian segregation.

    There is also conservation in the spindle movements that lead up to the perpendicular cortical attachment of the spindle at anaphase.  In mouse (Verlhac et al., 2000), Xenopus (Gard et al., 1995) and C. elegans (Yang et al., 2003), the meiosis I spindle or its precursor assembles at a distance from the cortex and translocates to the cortex after nuclear envelope breakdown.  In C. elegans (Albertson and Thompson, 1993; Yang et al., 2003), Drosophila (Endow and Komma, 1997 and 1998), Xenopus (Gard et al., 1995) and at least meiosis II of mouse (Maro et al., 1984), the spindle is initially oriented parallel to the cortex and then undergoes a discrete rotation to the perpendicular orientation.  C. elegans, however, is the only genetically-tractable species where the complete sequence of spindle movements during meiosis I and meiosis II can be continuously tracked by time-lapse imaging (Fig. 2, Yang et al., 2003 and 2005). 

figure 2

    Spindle positioning mechanisms.

Considerable research efforts are currently directed at mechanisms controlling mitotic spindle positioning and asymmetric mitotic cell divisions in budding yeast (Schuyler and Pellman, 2001), C. elegans mitosis, Drosophila neuroblasts (Gonczy, 2002) and mammalian epithelial cells (Lechler and Fuchs, 2005).  These spindle positioning mechanisms have conserved molecular components, such as cytoplasmic dynein, and these mechanisms are essential for differentiation of many tissues. These studies all focus on "astral" microtubules which are attached by their minus ends to mitotic spindle poles and which extend outward so that their plus ends interact with the cell cortex (Gonczy, 2002; Sheeman et al., 2003). The uniform polarity of astral microtubules allows a minus-end directed microtubule motor on the cortex to pull on astral microtubules and thus pull the spindle toward the cortex.  This type of mechanism could be applicable to female meiotic spindles that have astral microtubules like those of Chaetopterus (Lutz et al., 1988) or Xenopus, which assembles a transient microtubule array consisting of parallel microtubules extending to the cortex (Becker et al., 2003).  However, female meiotic spindles in mouse, humans (Sathananthan et al., 1991) and C. elegans (Albertson and Thompson, 1993), are considered "anastral" because they do not have visible astral microtubules or the centriole-containing centrosomes that organize astral microtubules.  Thus novel mechanisms are likely to mediate meiotic spindle positioning in C. elegans and human oocytes.  In fact, mouse meiotic chromosomes translocate to the cortex in the complete absence of spindle microtubules (Verlhac et al., 2000).


Utility of C. elegans for analyzing female meiosis.

Adult C. elegans hermaphrodites have two gonad arms, each containing a linear array of oocytes (Fig. 3A).  The oldest oocyte is adjacent to a sperm-filled structure called the spermatheca.  Every 23 min (McCarter et al., 1999), the oldest oocyte undergoes nuclear envelope breakdown and squeezes into the spermatheca in response to a secreted signal from the sperm (Miller et al., 2001; Kosinski et al., 2005; Fig. 3B).  The oocyte is fertilized in the spermatheca (Samuel et al., 2001), then the oocyte again squeezes out of the spermatheca into the uterus where meiosis I and II are completed.  The complete meiotic cycle from nuclear envelope breakdown through pronucleus formation can be filmed in an anesthetized worm in 35 - 45 min (Yang et al., 2003).  Because the immature oocytes are arrested in G2, maternal proteins can be depleted by RNAi for up to two days before filming meiotic divisions.  The ability to film all meiotic spindle movements in a short period of time, in combination with the feasibility of whole-genome RNAi (Simmer et al., 2003), make C. elegans an ideal organism in which to study spindle movements.

Figure 3

    Wild-type spindle movements during C. elegans meiosis.

 To determine how the C. elegans female meiotic spindle ends up attached to the cortex by one pole at anaphase, we tracked spindle movements directly by time-lapse imaging of GFP-tubulin or GFP-histone in living, anesthetized worms (Yang et al., 2003). During meiotic maturation, the nuclear envelope fenestrates, GFP-tubulin invades the nuclear space and meiotic spindle assembly initiates within the fenestrated nucleus.  The oocyte then squeezes into the spermatheca, where meiotic spindle assembly is completed.  The spindle then translocates to the cortex and is initially oriented parallel to the cortex (Fig. 2).  The spindle then begins to shorten and then rotates to a perpendicular orientation.  Homologous chromosomes segregate after spindle rotation while the spindle continues to shorten (Fig. 2).  After formation of the first polar body, a meiosis II spindle forms and undergoes the same translocation, shortening and rotation cycle.  At the end of meiosis II, the chromosomes segregated into the embryo form a pronucleus, indicating entry into interphase of the first mitosis.  The combination of spindle translocation and shortening generates a very small spindle that is contacting the cortex at the time that cell division is induced by the spindle (Fig. 2; Yang et al., 2003).  Our working hypothesis is that the purpose of both translocation and shortening is to minimize the size of the polar body and thereby maximize the volume of the embryo. 


The role of Kinesin-1 in the earlier of two redundant spindle translocation pathways.

The microtubule-dependence of meiotic spindle translocation to the cortex (Yang et al., 2003) suggested that a microtubule motor protein might transport the spindle to the cortex on the cytoplasmic microtubule meshwork.  To address this possibility, we initiated an RNAi screen of all 20 kinesin related genes and cytoplasmic dynein in search of a spindle translocation motor.  Among the first 7 kinesins examined, only embryos depleted of the kinesin-1 heavy chain homolog,UNC-116, showed a meiotic spindle translocation defect.  Filming of GFP-tubulin revealed that 27/27 wild-type meiosis I spindles initiated movement toward the cortex while the embryo was still inside the spermatheca and contacted the cortex either before the embryo exited the spermatheca or within 2 minutes of spermatheca exit (Fig. 4A; Yang et al., 2005).  In striking contrast, meiosis I spindles in 15/19 unc-116(RNAi) worms were stationary until 7 min after spermatheca exit when they suddenly moved to the cortex (Fig. 4B; Yang et al., 2005).  Wild-type spindles translocated to the cortex 7 min before the initiation of spindle shortening (Fig.4C) whereas unc-116(RNAi) spindles did not initiate translocation until spindle shortening initiated (Fig. 4D). 

Figure 4

Because spindle shortening is dependent on the anaphase promoting complex (APC) that drives the metaphase anaphase transition (Yang et al., 2003; Fig. 4E), we hypothesized that unc-116(RNAi) spindles are completely blocked in an early, APC-independent translocation mechanism normally used by wild-type spindles and that unc-116(RNAi) spindles move to the cortex through a late, APC-dependent mechanism.  This hypothesis predicts that simultaneous inhibition of UNC-116 and the APC will result in a complete block in translocation.  Indeed, a complete block to translocation was observed in worms lacking APC activity due to a temperature sensitive mutation in the MAT-2 subunit of the APC, mat-2(ts), that were also treated with unc-116 dsRNA (Fig. 4F).  Also as predicted by this hypothesis, spindle translocation was normal in worms that lacked APC activity but retained wild-type UNC-116 activity (Fig. 4E).  Further support for the hypothesis comes from the orientation of spindles during movement to the cortex.  82% of wild-type spindles moved to the cortex in a sideways orientation (Fig. 4A) whereas 83% of unc-116(RNAi) spindles moved to the cortex with one spindle pole leading (Fig. 4B).  Also consistent with the existence of two different translocation mechanisms, wild-type spindles moved at a velocity of 0.9 +/- 0.4 μm/min whereas unc-116(RNAi) spindles moved at 3.5 +/- 1.2 μm/min.

These results led us to the model shown in Fig.5.  In this model, kinesin 1 is attached to the meiotic spindle and moves the spindle along cytoplasmic microtubules toward the cortex immediately after spindle assembly.  (We have since shown that kinesin-1 is NOT attached to the meiotic spindle but the concept of 2 pathways is still valid.)  After the activation of the APC, we hypothesize that a small number of astral microtubules extend from the spindle poles with plus ends pointed toward the cortex.  We further proposed that a minus-end directed motor attached to the cortex then pulls on the astral microtubules and thereby pulls one pole into the cortex.  This mechanism would result in late spindle translocation in kinesin 1-depleted embryos and spindle rotation in wild-type embryos.  We propose this model because late translocation and wild-type rotation are both rapid, APC-dependent movements of one spindle pole toward the cortex and because this latter mechanism is thought to mediate movements of mitotic spindles in many species (see B3).  A caveat with this model is that C. elegans meiotic spindles are acentriolar (Albertson and Thomson, 1993) and no astral microtubules are visible extending from their poles. We suggest that a small number of astral microtubules extend from C. elegans meiotic spindle poles only after activation of the APC and that these microtubules are obscured by the large number of cytoplasmic microtubules.  The first mitotic spindle of a parthenogenetically-activated cow embryo lacks astral microtubules as would be expected for these acentriolar spindles.  During anaphase, however, these acentriolar spindles extend long astral microtubule arrays (Navara et al., 1994).  This anaphase-specific extension of astral microtubules may be due to a highly conserved mechanism since the astral microtubules of mitotic spindles become longer during anaphase in many species.

Figure 5

We have since shown that cytoplasmic dynein is the minus end directed motor required for spindle rotation and for late translocation in an unc-116 mutant (Ellefson and McNally, 2009) in agreement with the work of others (van der Voet     et al., 2009).

Kinesin light chains, KLC-1 and KLC-2, and the light chain binding protein, KCA-1, form a complex with kinesin-1 heavy chain and are required for the early spindle translocation pathway. 

Kinesin-1 from many species, including C. elegans, is a hetero-tetramer consisting of two heavy chain subunits and two light chain subunits (Vale, 2003; Signor et al., 1999).  Kinesin light chains are thought to interact with cargo through cargo-specific adaptor proteins (Kamal and Goldstein, 2002).  We have found that double RNAi-mediated depletion of both C. elegans kinesin light chains, KLC-1 and KLC-2, results in the same spindle translocation phenotype seen in unc-116(RNAi) (Yang et al., 2005; Fig.6).  To identify a cargo-adaptor used in meiotic spindle translocation, we used RNAi to deplete 21 different proteins identified as kinesin light chain interactors in a published, global yeast 2-hybrid screen (Li et al., 2004).  We found that depletion of C10H11.10 results in a phenotype identical to the unc-116(RNAi) or klc-1,2(RNAi)  phenotype (Yang et al., 2005; Fig. 6).  We also reconstituted a complex of UNC-116, KLC-2 and C10H11.10 using purified recombinant proteins (Yang et al., 2005).  These results indicate that meiotic spindle translocation is mediated by a complex of UNC-116, KLC's and C10H11.10.  We have therefore named the C10H11.10 predicted gene kca-1 (kinesin cargo adaptor).  KCA-1 has no sequence homology with known kinesin cargo adaptors.  A KCA-1 homolog is present in the C. briggsiae and C. remanei genomes but no homologs are present in other phyla, indicating that either it is a nematode-specific protein (and therefore a potential drug target) or its primary sequence has diverged beyond easy recognition in Caenorhabditis.  We have since found that UNC-116, KLC-1 and KCA-1 localize to the cytoplasm in the middle of the meiotic embryo and NOT on the meiotic spindle.  We are working on elucidating how kinesin-1 drives meiotic spindle translocation without being bound to the spindle.


Figure 6

    The role of the microtubule-severing ATPase, katanin, in meiotic spindle assembly and spindle length control.

    Mains et al. (1990) found that complete loss of function mutations in mei-1 or mei-2 result in highly disorganized meiotic spindles as assayed by immunofluorescence.  We independently purified a heterodimeric, microtubule-severing ATPase, called katanin, from sea urchin eggs (McNally and Vale, 1993) and demonstrated that katanin is responsible for the microtubule-severing activity present in meiotic metaphase-arrested Xenopus eggs (McNally and Thomas, 1998).  Molecular cloning and sequence analysis demonstrated that MEI-1 (Clark-Maguire and Mains, 1994a) shares limited sequence homology with the p60, ATPase subunit, of sea urchin katanin (Hartman et al., 1998) and that MEI-2 (Srayko et al., 2000) shares limited sequence homology with the C-terminal domain of p80 katanin.  The C-terminal domain of p80 katanin is sufficient for binding and activating p60 katanin through a conserved N-terminal sequence in p60 (McNally et al., 2000).  These results suggested that MEI-1 and MEI-2 dimerize to form a katanin-like microtubule-severing enzyme.  Consistent with this interpretation, we found that MEI-1 and MEI-2 co-purified with each other and induced microtubule disassembly in vivo when co-expressed in HeLa cells (Srayko et al., 2000).  More recently, we have expressed either MEI-1 alone or MEI-1 with MEI-2 in insect cells and purified the polypeptides.  As shown in Fig. 7, purified MEI-1/MEI-2 complexes (but not MEI-1 alone) sever microtubules assembled from purified tubulin in an ATP-dependent manner.  This experiment demonstrates that MEI-1/MEI-2 indeed assemble into a katanin-like microtubule-severing protein.

Figure 7
Figure 7. Rhodamine-labelled, taxol-stabilized microtubules were immobilized on a coverslip surface with a mutant kinesin and incubated with MEI-1 + MEI-2 + ADP (left panel), MEI-1 + ATP (center panel) or MEI-1 + MEI-2 + ATP (right panel).

    Loss of function mutations in MEI-1 or MEI-2 result in highly disorganized meiotic spindles (Mains et al., 1990) that completely lack any bipolar arrangement of microtubules (Yang et al., 2003).  These disorganized spindles exhibit abnormal translocation of the spindle to the cortex, a complete lack of chromosome congression or anaphase segregation and these spindles never shrink to a smaller diameter at the times that wild-type meiosis I and II spindles shorten (Yang et al., 2003).  The MEI-1/MEI-2 microtubule-severing protein might be directly involved in spindle translocation, spindle shortening and anaphase chromosome segregation or some of these defects might be the indirect result of the aberrant spindle structure in these mutants.  We are currently analyzing double mutant combinations that suppress some of the defects in a mei-1 null to address this question.

    The failure of mei-1 mutant spindles to shrink during the times that wild-type spindles shorten (Yang et al., 2003) along with the concentration of MEI-1 (Clark-Maguire and Mains, 1994b) and MEI-2 (Srayko et al., 2000) at the poles of wild-type spindles suggested a model in which spindle microtubules are severed near their minus ends to cause shortening of wild-type meiotic spindles.  To further investigate the role of MEI-1/MEI-2 in meiotic spindle length control, we initiated analysis of the mei-2 partial loss of function allele, ct98.  This is a misense allele that expresses a reduced amount of MEI-2 protein (Srayko et al., 2000) and results in a reduced amount of MEI-1 protein on meiotic spindles (Clark-Maguire and Mains, 1994b).  This allele causes a high frequency of viable embryos with abnormally large polar bodies (Mains et al., 1990).  The viability of embryos suggested that bipolar spindles form whereas the large polar bodies suggested that spindles might be abnormally long during polar body induction.  We captured complete time-lapse sequences of meiotic spindle movements in mei-2(ct98) worms and found that both the maximum spindle length at metaphase and the length at the time of spindle rotation was longer for mei-2(ct98) than for wild type (Table 1).  Surprisingly, the velocity of spindle shortening before the time of wild-type spindle rotation was the same for mei-2(ct98) and wild type (Table 1). 

Genotype Wild type mei-2(ct98)
Maximum spindle length (metaphase) 7.17 +/- 0.69 μm (n= 36) 9.45 +/- 0.8 μm (n= 19)
Spindle length at time of wt rotation 4.37 +/- 0.42 μm (n=36) 5.99 +/- 0.54 μm (n=20)
Shortening rate before time of wt rotation 1.08 +/- 0.36 μm/min (n=12) 0.95 +/- 0.3 μm/min (n=12)
Shortening rate after time of wt rotation 0.74 +/- .05 μm/min (n=11) 0.01 +/- 0.08 μm/min (n=9)

Table 1.  Analysis of average pole-pole spindle length and velocity of pole-pole spindle shortening from time-lapse sequences of GFP-tubulin fluorescence indicated that MEI-2 influences the length of spindles when they are first assembled and the rate of post-rotation shortening but does not play a role in pre-rotation shortening.

    These results suggested that wild-type MEI-1/MEI-2 causes initial assembly of a short metaphase spindle, possibly because microtubule severing during spindle assembly results in a shorter average microtubule length.  This assembly is then followed by a MEI-1/MEI-2-independent spindle shortening phase that occurs before spindle rotation.  Wild-type spindles continue to shorten after rotation (Table 1) whereas mei-2(ct98) spindles essentially stop shortening at the time of wild-type rotation (Table 2). 
Figure 8
These results indicate that a second, MEI-1/MEI-2-dependent shortening mechanism is activated at the time of rotation.  We hypothesized that the pre-rotation, MEI-1/MEI-2-independent shortening might be due to inward sliding of anti-parallel microtubules within the spindle by motor proteins that form cross-bridges between anti-parallel microtubules.  This model is based on the proposed roles of ncd as an inward sliding motor in Drosophila mitotic spindles (Sharp et al., 1999 and 2000) and of the ncd homolog, HSET, as an inward sliding motor in human mitotic spindles (Mountain et al., 1999).  Inward sliding of microtubules should result in an increase in microtubule density within the spindle, which should result in an increase in the fluorescence intensity of GFP-tubulin.  As shown in Fig. 8, the average intensity of GFP-tubulin fluorescence within a wild-type spindle is constant during the period of constant metaphase spindle length, increases during pre-rotation shortening and decreases during post-rotation shortening. These observations are consistent with a pre-rotation shortening mechanism driven by inward microtubule sliding and a post-rotation shortening mechanism that is accompanied by net microtubule disassembly.  mei-2(ct98) spindles, which do not show obvious rotation, exhibit an increases in GFP-tubulin fluorescence intensity during spindle shortening and no second phase of shortening while the spindle decreases in fluorescence intensity (Fig. 8).  These results indicate that MEI-1/MEI-2 is involved in post-rotation spindle shortening but not in pre-rotation shortening.
    The role of MEI-1/MEI-2 in post-rotation spindle shortening is more dramatically shown in Fig. 9.

Figure 9
Figure 9. The second phase of spindle shortening is accompanied by katanin-dependent disassembly of spindle pole microtubules and assembly of midzone microtubules.  Time-lapse images of a wild-type embryo and a mei-2(ct98) embryo expressing GFP::tubulin and mCherry::histone beginning at rotation were captured with a spinning disk confocal microscope.  Fluorescence intensity was plotted as a function of distance down the pole-pole axis of a single microtubule bundle for each image.  Images are shown adjacent to their corresponding plots.  Images are in the same orientation as the graphs.  In the wild-type spindle, GFP-tubulin fluorescence intensity (green) of the spindle poles decreased while the intensity of the spindle in between the separating chromosomes increased as the midzone formed.  The mei-2(ct98) spindle, which did not shorten  during this period, showed no change in the relative intensity of GFP::tubulin fluorescence at poles vs the midzone but did exhibit a decrease in microtubule density that progressed uniformly throughout the spindle.  Bar = 3.5 μm.

We are actively pursuing the mechanisms by which katanin promotes spindle length control and bipolar spindle assembly.

The role of fertilization and the SPE-11 protein in meiotic progression and polar body formation. 

The female meiotic cell cycle differs from that of males in being coordinated with fertilization. Elucidating the mechanisms that drive sequential spindle translocation, shortening, rotation and polar body extrusion in C. elegans will require an understanding of the species-specific role of fertilization in the female meiotic cell cycle.  Oocytes in all animal species enter a G2, prophase arrest during which homologous chromosomes become physically attached, typically through meiotic recombination. This first arrest is broken by a maturation hormone that induces nuclear envelope breakdown and assembly of the meiosis I spindle (Abrieu et al., 2001; Kishimoto, 1998).  Most species have a second meiotic cell cycle arrest point that is broken near the time of fertilization (Tunquist and Maller, 2003; Nixon et al., 2002; Page and Orr-Weaver, 1997; Harada et al., 2003; Heifetz et al., 2001).  In C. elegans, the maturation hormone, MSP, is secreted from sperm (Miller et al., 2001, Kosinski et al., 2005), which are stored in the spermatheca adjacent to the oldest oocyte (Fig. 3A).  In the absence of sperm, eg. in a fem-1 mutant, oocytes arrest in G2/diakinesis.  To observe matured but unfertilized oocytes requires the use of paternal-effect mutants such as fer-1 and spe-9 (L'Hernault et al., 1988) that produce sperm that secrete MSP but that cannot fertilize eggs (McCarter et al., 1999).

    To elucidate the role of fertilization in female meiosis, we generated worms that were homozygous for ts alleles of fer-1 or spe-9 that also expressed GFP-tubulin or GFP-histone H2B in their oocytes.  Time-lapse imaging revealed that spindle assembly, translocation, shortening and rotation were quantitatively identical with fertilized embryos.  Anaphase I chromosome separation was also identical between fertilized and unfertilized embryos (Fig. 10).  Unfertilized embryos, however, did not disassemble the meiosis I midbody, extrude a first polar body or assemble a meiosis II spindle.  Chromosomes that were segregated into the embryo at anaphase I formed a female pronucleus at the same time that anaphase II chromosomes formed a pronucleus in fertilized embryos, indicating that C. elegans has no second cell-cycle arrest point in meiosis (McNally and McNally, 2005).  Although we did not continue filming these embryos after pronucleus formation, one-celled, unfertilized embryos with no eggshells and a single large nucleus accumulated in a linear array in the uterus.  Older unfertilized embryos had higher DNA content as assayed by GFP-histone imaging and embryos with no nuclear envelope and condensed chromatin were occasionally observed.  These observations indicate that unfertilized C. elegans embryos cycle between S and M phases without ever dividing.  GFP-tubulin imaging did not reveal any centrosomes or astral microtubule arrays, consistent with a lack of sperm-derived centrioles.  A lack of centriole-containing centrosomes and their associated astral microtubule arrays might be sufficient to explain why unfertilized embryos do not exhibit mitotic cytokinesis but a centriole deficiency cannot readily explain why the first polar body is not formed nor why a meiosis II spindle does not form.

Figure 10

To begin dissecting the roles of fertilization in first polar body extrusion and meiosis II spindle assembly, we conducted time-lapse imaging of GFP-histone in spe-11 mutant worms.  In a large collection of paternal effect mutants, spe-11 mutants were found to be the only ones where embryos were fertilized (Hill et al., 1989).  All the other fer and spe genes, like spe-9, are required for fertilization itself and thus dead embryos produced by these mutants do not form a male pronucleus nor do they contain centriole-containing centrosomes.  Work described in two consecutive papers from Susan Strome's lab (Hill et al., 1989; Browning and Strome, 1996) revealed that SPE-11 is a sperm protein introduced into the egg at fertilization. Paternally contributed SPE-11 is required for embryonic polarization and cleavage even though a male pronucleus and associated centrosomes are present in zygotes fertilized by spe-11 mutant sperm.  We reasoned that embryos fertilized by sperm lacking SPE-11 should exhibit a subset of the defects seen in unfertilized spe-9 embryos. Indeed, our time-lapse imaging of GFP-histone in embryos fertilized by spe-11 mutant sperm revealed that both anaphase I and anaphase II occur with normal timing and orientation relative to the cortex.  However, extrusion of both polar bodies fails in these embryos.  Because formation of the first polar body fails, the metaphase II spindle has 12 sister chromatid pairs instead of the usual 6 (Fig. 10; McNally and McNally, 2005), just as we previously reported for latrunculin-treated worms (Yang et al., 2003).  We propose that sperm-derived SPE-11 protein interacts with maternal cytokinesis regulators to mediate polar body extrusion and that sperm provide one or more additional factors that are required to assemble a meiosis II spindle. 

Polar body extrusion.

We are actively pursuing the mechanism of contractile ring positioning during meiosis.
Stay tuned for details.


  • Abrieu, A., Doree, M., Fisher, D.,  2001.  The interplay between cyclin-B-Cdc2 kinase (MPF) and MAP kinase during maturation of oocytes.  J. Cell Sci. 114, 257 - 267.
  • Albertson, D.G. and J.N. Thomson.  1993.  Segregation of holocentric chromosomes at meiosis in the nematode, Caenorhabditis elegans.  Chromosome Res.  1:  15-26.
  • Becker, B.E., Romney, S.J., and D.L. Gard. 2003.  XMAP215, XKCM1, NuMA, and cytoplasmic dynein are required for the assembly and organization of the transient microtubule array during the maturation of Xenopus oocytes.  Dev. Biol. 261: 488-505.
  • Browning, H., and S. Strome.  1996.  A sperm-supplied factor required for embryogenesis in C. elegans.  Development.  122: 391 - 404.
  • Cimino, M.C.     1972. Meiosis in triploid all-female fish (Poeciliopsis, Poeciliidae).  Science. 175: 1484 - 1486.
  • Clark-Maguire, S., and P.E. Mains. 1994a. mei-1, a gene required for meiotic spindle formation in Caenorhabditis elegans, is a member of a family of ATPases. Genetics. 136: 533-546.
  • Clark-Maguire, S., and P.E. Mains.  1994b. Localization of the mei-1 gene product of Caenorhaditis elegans, a meiotic-specific spindle component. J. Cell Biol. 126: 199-209.
  • Ellefson, M.L., and McNally, F.J.  (2009).  Kinesin-1 and Cytoplasmic Dynein Act Sequentially to Move the Meiotic Spindle to the Oocyte Cortex in C. elegans.  Mol. Biol. Cell. 20, 2722-2730
  • Endow, S.A. and D.J. Komma. 1997.  Spindle dynamics during meiosis in Drosophila oocytes. J. Cell Biol. 137: 1321-1336.
  • Endow ,S.A., and D.J. Komma. 1998. Assembly and dynamics of an anastral:astral spindle: the meiosis II spindle of Drosophila oocytes. J. Cell Sci. 111: 2487-2495.
  • Gard, D.L., Cha, B.J., and M.M. Schroeder.  1995.  Confocal immunofluorescence microscopy of microtubules, microtubule-associated proteins, and microtubule-organizing centers during amphibian oogenesis and early development.  Curr. Top. in Dev. Biol.  31: 383-431.
  • Gard, D.L., Cha. B.J., and A.D. Roeder.  1995.  F-actin is required for spindle anchoring and rotation in Xenopus oocytes: a re-examination of the effects of cytochalasin B on oocyte maturation. Zygote 3: 17-26.
  • Gonczy, P.  2002.  Mechanisms of spindle positioning: focus on flies and worms. Trends Cell Biol. 12: 332-339.
  • Harada, K., Oita, E., Chiba, K.,  2003.  Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH.  Development. 130, 4581 - 4586.
  • Heifetz, Y., Yu, J., Wolfner, M.F.,  2001.  Ovulation triggers activation of Drosophila oocytes.  Dev. Biol.  234, 416 - 424.
  • Hill, D.P., Shakes, D.C., Ward, S., Strome, S.,  1989.  A sperm-supplied product essential for initiation of normal embryogenesis in Caenorhabditis elegans is encoded by the paternal-effect embryonic-lethal gene, spe-11.  Dev. Biol. 136, 154 - 166. 
  • Kamal, A., Almenar-Queralt, A., LeBlanc, J.F., Roberts, E.A., and L.S. Goldstein LS.     2001. Kinesin-mediated axonal transport of a membrane compartment containing beta-secretase and presenilin-1 requires APP.  Nature. 414: 643-648.
  • Kamal, A. and Goldstein, L.S. 2002.  Principles of cargo attachment to cytoplasmic motor proteins. Curr. Opin. Cell Biol. 14: 63-68.
  • Kishimoto, T.,  1998.  Cell cycle arrest and release in starfish oocytes and eggs.  Semin. Cell Dev. Biol. 9, 549 - 557.
  • Koehler, K.E. and T.J. Hassold.  1998.  Human aneuploidy: lessons from achiasmate segregation in Drosophila melanogaster.  Ann. Hum. Genet.  62: 467-479.
  •     Kosinski, M., McDonald, K., Schwartz, J., Yamamoto, I., and D. Greenstein.  2005.  C. elegans sperm bud vesicles to deliver a meiotic maturation signal to distant oocytes. Development. 132: 3357-3369.
  • Lechler, T., and E. Fuchs.  2005.  Asymmetric cell divisions promote stratification and differentiation of mammalian skin.  Nature. 437: 275-280.
  • L'Hernault, S.W., Shakes, D.C., Ward, S.,  1988.  Developmental genetics of chromosome I spermatogenesis-defective mutants in the nematode Caenorhabditis elegans.  Genetics.  120, 435 - 452.
  • Li, S., Armstrong, C.M., Bertin, N., Ge, H., Milstein, S., Boxem, M., Vidalain, P.-O., Han, J.-D. J., Chesneau, A., Hao, T., Goldberg, D.S., Li, N., Martinez, M., Rual, J.-F., Lamesch, P., Xu, L., Tewari, M., Wong, S.L., Zhang, L.V., Berriz, G.F., Jacotot, L., Vaglio, P., Reboul, J., Hirozane-Kishikawa, T., Li, Q., Gabel H. W., Elewa, A., Baumgartner, B., Rose, D.J., Yu, H., Bosak, S., Sequerra, R., Fraser, A., Mango, S.E., Saxton, W.M., Strome, S., van den Heuvel, S., Piano, F., Vandenhaute, J., Sardet, C., Gerstein, M., Doucette-Stamm, L., Gunsalus, K.C., Harper, J.W., Cusick, M.E., Roth, F.P., Hill, D.E., and M. Vidal.  2004.  A map of the interactome network of the metazoan C. elegans. Science. 303: 540-543.
  • Lutz, D.A., Hamaguchi, Y., and S. Inoue. 1988.  Micromanipulation studies of the asymmetric positioning of the maturation spindle in Chaetopterus sp. oocytes: I. Anchorage of the spindle to the cortex and migration of a displaced spindle. Cell Motil. Cytoskeleton. 11: 83-96.
  • Mains, P.E., Kemphues, K.J., Sprunger, S.A., Sulston, I.A., and W.B. Wood.  1990.  Mutations affecting         meiotic and mitotic divisions of the early Caenorhabditis elegans embryo.  Genetics  126: 593-605.
  • Maro, B., Johnson, M.H., Pickering, S.J., and G. Flach.  1984.  Changes in actin distribution during fertilization of the mouse egg.  J. Embryol. Exp. Morph. 81: 211-237.
  • McCarter, J., Bartlett, B., Dang, T., and T. Schedl.  1999.  On the control of oocyte meiotic maturation and ovulation in Caenorhabditis elegans.  Dev. Biol.  205, 111-128.
  • McNally, K.L., and F.J. McNally. 2005.  Fertilization initiates the transition from anaphase I to metaphase II during C. elegans meiosis. Dev. Biol. 282: 218-230.
  • McNally, K.P., Bazirgan, O.A., and F.J. McNally.  2000.  Two domains of p80 katanin regulate microtubule severing and spindle pole targeting by p60 katanin.  J. Cell Science.  113:  1623-1633.
  • McNally, F. and R. Vale. 1993. Identification of katanin, an ATPase that severs and disassembles stable microtubules. Cell 75: 419-429.
  • Miller, M.A., Nguyen, V.Q., Lee, M.H., Kosinski, M., Schedl, T., Caprioli, R.M. and D. Greenstein.  2001.  A sperm cytoskeletal protein that signals oocyte meiotic maturation and ovulation.  Science.  291: 2144-2147.
  • Miyara, F., Han, Z., Gao, S., Vassena, R., and K.E. Latham.  2006.  Non-equivalence of embryonic and somatic cell nuclei affecting spindle composition in clones.  Dev. Biol.  289: 206.
  • Mountain, V., Simerly, C., Howard, L., Ando, A., Schatten, G., and D.A. Compton.  1999. The kinesin-related protein, HSET, opposes the activity of Eg5 and cross-links microtubules in the mammalian mitotic spindle. J. Cell Biol. 147: 351-366.
  • Navara, C.S., First, N.L., and G. Schatten.  1994.  Microtubule organization in the cow d uring fertilization, polyspermy, parthenogenesis, and nuclear transfer: the role of the sperm aster.  Dev. Biol. 162: 29-40.
  • Nixon, V.L., Levasseur, M., McDougall, A., Jones, K.T.,  2002.  Ca(2+) oscillations promote APC/C-dependent cyclin B1 degradation during metaphase arrest and completion of meiosis in fertilizing mouse eggs. Curr. Biol. 12, 746 - 750.
  • Page, A.W., Orr-Weaver, T.L.,  1997.  Activation of the meiotic divisions in Drosophila oocytes.  Dev. Biol. 183, 195 - 207.
  • Samuel, A.D., Murthy, V.N. and M.O. Hengartner.  2001.  Calcium dynamics during fertilization in C. elegans.  BMC Dev Biol. 1: 8.
  • Sathananthan, A.H., Kola, I., Osborne, J., Trounson, A., NG, S.C., Bongso, A., and S.S. Ratnam.  1991. Centrioles in the beginning of human development.  Proc. Natl. Acad. Sci. USA 88: 4806-4810.
  • Schuyler, S.C., and D. Pellman.  2001.  Search, capture and signal: games microtubules and centrosomes play. J.  Cell. Sci. 114: 247-255.
  • Sharp, D.J., Brown, H.M., Kwon, M., Rogers, G.C., Holland, G., and J.M. Scholey.  2000. Functional coordination of three mitotic motors in Drosophila embryos. Mol. Biol. Cell. 11: 241-253.
  • Sharp, D.J., Yu, K.R., Sisson, J.C., Sullivan, W., and J.M. Scholey JM. 1999.  Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos. Nat. Cell Biol. 1: 51-54.
  • Sheeman, B., Carvalho, P., Sagot, I., Geiser, J., Kho, D., Hoyt, M.A., and D. Pellman.  2003.  Determinants of S. cerevisiae dynein localization and activation: implications for the mechanism of spindle positioning.  Curr. Biol. 13: 364-372.
  • Signor, D., Wedaman, K.P., Rose, L.S., and J.M. Scholey.  1999.  Two heteromeric kinesin complexes in chemosensory neurons and sensory cilia of Caenorhabditis elegans. Mol. Biol. Cell. 10: 345 - 360.
  •     Simmer, F., Moorman, C., van der Linden, A.M., Kuijk, E., van den Berghe, P.V., Kamath, R.S., Fraser, A.G., Ahringer, J., and R.H. Plasterk. 2003.  Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions. PLoS Biol. 1: E12.
  • Srayko, M., Buster, D.W., Bazirgan, O.A., McNally, F.J., and P.E. Mains.  2000.  MEI-1/MEI-2 katanin-like microtubule severing activity is required for Caenorhabditis elegans meiosis.  Genes and Dev.  14: 1072-1084.
  • Tram, U., and W. Sullivan. 2000. Reciprocal inheritance of centrosomes in the parthenogenetic hymenopteran Nasonia vitripennis. Curr. Biol. 10: 1413-1419.
  • Triantaphyllou, A.C. 1966. Polyploidy and reproductive patterns in the root-knot nematode Meloidogyne hapla. J. Morphol. 118: 403-13.
  • Tunquist, B.J., Maller, J.L.,  2003.  Under arrest: cytostatic factor (CSF)-mediated metaphase arrest in vertebrate eggs.  Genes Dev.  17, 683 - 710.
  • Vale, R.D. 2003.  The molecular motor toolbox for intracellular transport. Cell. 112: 467-480.
  • van der Voet, M., Berends, C.W., Perreault, A., Nguyen-Ngoc, T., Gönczy, P., Vidal, M., Boxem, M., and van den Heuvel, S.  (2009).  NuMA-related LIN-5, ASPM-1, calmodulin and dynein promote meiotic spindle rotation independently of cortical LIN-5/GPR/Galpha.  Nat. Cell Biol. 11, 269-277.
  • Verlhac, M.H., Lefebvre, C., Guillaud, P., Rassinier, P., and B. Maro.  2000.  Asymmetric division in mouse oocytes: with or without Mos.  Curr. Biol.  10: 1303-1306.
  • Viney, M.E. 1994. A genetic analysis of reproduction in Strongyloides ratti. Parasitology. 109: 511 - 515.
  • Yang, H., McNally, K., and F.J. McNally.  2003.  MEI-1/katanin is required for translocation of the meiosis I spindle to the oocyte cortex in C. elegans.  Dev. Biol. 260:  245-259.
  • Yang, H., Mains, P.E., and F.J. McNally. 2005.  Kinesin-1 mediates translocation of the meiotic spindle to the oocyte cortex through KCA-1, a novel cargo adapter.  J. Cell Biol. 169: 447-457.