Mechanism of spindle pole organization and instability in human oocytes

Human oocytes are prone to assembling meiotic spindles with unstable poles, which can favor aneuploidy in human eggs. The underlying causes of spindle instability are unknown. We found that NUMA (nuclear mitotic apparatus protein)–mediated clustering of microtubule minus ends focused the spindle poles in human, bovine, and porcine oocytes and in mouse oocytes depleted of acentriolar microtubule-organizing centers (aMTOCs). However, unlike human oocytes, bovine, porcine, and aMTOC-free mouse oocytes have stable spindles. We identified the molecular motor KIFC1 (kinesin superfamily protein C1) as a spindle-stabilizing protein that is deficient in human oocytes. Depletion of KIFC1 recapitulated spindle instability in bovine and aMTOC-free mouse oocytes, and the introduction of exogenous KIFC1 rescued spindle instability in human oocytes. Thus, the deficiency of KIFC1 contributes to spindle instability in human oocytes. Description A missing motor in human meiosis Chromosomal errors in human eggs are a leading cause of miscarriages and infertility. These errors result from chromosome missegregations during the maturation of oocytes into eggs. Chromosome segregation is driven by the spindle, a macromolecular machinery that pulls chromosomes apart. However, human oocytes often assemble unstable spindles, favoring chromosome missegregations. So et al. discovered that human oocyte spindles are unstable because they are deficient in the molecular motor KIFC1. KIFC1 stabilizes spindles in other mammalian oocytes and in cancer cells. By introducing exogenous KIFC1, the authors were able to increase the fidelity of spindle assembly and chromosome segregation in human oocytes. —SMH Human oocytes have unstable meiotic spindles compared with oocytes from other mammals because of the lack of the molecular motor KIFC1. INTRODUCTION Many human eggs carry an incorrect number of chromosomes, a condition known as aneuploidy. Aneuploidy in human eggs is the leading cause of aberrant embryonic development, resulting in pregnancy loss and genetic disorders such as Down syndrome. Most aneuploidy results from chromosome segregation errors during the maturation of oocytes into fertilizable eggs. Unlike somatic cells, human oocytes segregate chromosomes with a specialized microtubule spindle that lacks centrosomes. Previous live-imaging studies revealed that human oocytes often assemble spindles with unstable poles, favoring chromosome segregation errors. The causes of high spindle instability in human oocytes were unknown. RATIONALE Identifying the causes of spindle instability may lead to therapeutic strategies that reduce chromosome segregation errors in human eggs and improve outcomes of assisted reproductive technology. We thus set out to investigate how spindle poles are organized in the absence of centrosomes and why spindles are unstable in human oocytes. To this end, we systematically studied the localization and function of proteins that are required for spindle pole assembly or spindle stability in oocytes of different mammalian species. In stark contrast to human oocytes, the spindles of other mammalian oocytes were stable. We thus carried out a comparative analysis to investigate whether differences in molecular composition can explain the high degree of spindle instability in human oocytes. RESULTS Spindle pole assembly requires the bundling of parallel microtubules by microtubule cross-linking proteins as well as stabilization and/or anchoring of microtubule minus ends in the spindle pole region by minus end–binding proteins. We found that the microtubule cross-linking protein NUMA (nuclear mitotic apparatus protein) localized to microtubule minus ends, where it recruited the molecular motor dynein for spindle pole focusing. Depletion of NUMA or inhibition of dynein splayed microtubule minus ends, demonstrating that NUMA and dynein organize the spindle poles in human oocytes. NUMA was similarly enriched at the spindle poles in bovine and porcine oocytes, which naturally lack centrosomes, as well as in mouse oocytes that we artificially depleted of acentriolar microtubule organizing centers (aMTOCs). We thus asked whether spindle instability is a general feature of mammalian oocytes that use NUMA for spindle pole organization. Live imaging, however, revealed that bovine, porcine, and aMTOC-free mouse oocytes did not assemble unstable spindles, indicating that additional mechanisms stabilize spindles in these oocytes. Using an RNA interference screen of proteins with diverse functions in spindle organization, we identified the molecular motor KIFC1 (kinesin superfamily protein C1) as a spindle-stabilizing factor that is present in other mammalian oocytes but deficient in human oocytes. Depletion of KIFC1 in other mammalian oocytes recapitulated the spindle instability of human oocytes, resulting in spindles with unstable poles and an increase in aneuploidy. To investigate further if the spindle instability in human oocytes was a result of KIFC1 deficiency, we injected recombinant KIFC1 protein into human oocytes and performed live imaging of spindle assembly. Introduction of exogenous KIFC1 stabilized the spindles and reduced chromosome segregation errors, confirming that KIFC1 deficiency contributes to spindle instability in human oocytes. CONCLUSION Our data reveal notable differences in spindle pole organization in different systems. In somatic cells, two centrosomes act as the main MTOCs and promote bipolar spindle assembly. In mouse oocytes, centrosomes are functionally replaced by aMTOCs. In other mammalian oocytes, including humans, NUMA is enriched at microtubule minus ends. NUMA primarily engages the motor activity of dynein but can also cross-link microtubules itself. These activities allow NUMA to cluster microtubule minus ends, and to thereby organize the spindle poles in the absence of centrosomes or aMTOCs. Our data also elucidate a cause of spindle instability in human oocytes: Human oocytes are deficient in KIFC1, a key spindle-stabilizing protein in other mammalian oocytes and in cancer cells. KIFC1 stabilizes the spindle poles and prevents their fragmentation. This is likely achieved through the formation of static cross-links along parallel microtubules at the poles and the alignment of antiparallel microtubules in the central region of the spindle. Because human oocytes are deficient in KIFC1, we propose that the deficiency of these activities renders their spindles unstable. By delivering a defined amount of KIFC1 protein into human oocytes, we were able to reduce spindle instability and the risk of aneuploidy in human oocytes. Thus, our data also reveal a potential method for increasing the fidelity of spindle assembly and chromosome segregation in human oocytes. Spindle instability in mammalian oocytes. Depletion of the molecular motor KIFC1 in aMTOC-free mouse oocytes (left) and bovine oocytes (middle) resulted in multipolar spindles (gray) and chromosome (magenta) segregation errors, recapitulating the spindle instability of human oocytes (right), as shown in these immunofluorescence images. Conversely, introduction of exogenous KIFC1 stabilized the spindles in human oocytes, which are naturally deficient in KIFC1. Yellow arrowheads highlight multiple spindle poles.