Mechanisms Of Spindle Assembly Research Articles

P-558 Characterization of the morphology of the outer-kinetochore expandable module, aka fibrous corona, and its role in ensuring proper chromosome segregation in human and bovine oocytes

Abstract Study question How does the Fibrous Corona (FC) behave in human and bovine oocyte meiosis and what is its function in chromosome segregation during meiosis I? Summary answer In human and bovine oocytes, FC shows extreme plasticity expanding beyond the centromeric region of the chromosomes. Pharmacological inhibition of FC’s expansion induces chromosome mis-segregation. What is known already Embryonic aneuploidy of meiotic origin is one of the major causes of implantation failure and early pregnancy loss. Erroneous microtubule (MT)-kinetochore attachments in human meiosis are the main cause of meiotic chromosome segregation errors. In somatic cells, the FC, a multiprotein complex, assembles on kinetochores and expands around the centromere during prometaphase to facilitate MT interactions and stimulate correct MT-kinetochore attachment. We have shown that in bovine oocytes FC expand beyond the centrosomes and is a potent MTs nucleator (10.1016/j.cub.2023.01.010). However, it is still unknown how FC behaves and what are its functions in MT-kinetochore interactions in human oocytes. Study design, size, duration FC morphology was studied in immature human oocytes discarded during ICSI procedures and donated (informed consent) by the patients for research (Ethical permit TCBio.21-754) during the years 2021-2024. Because of ethical concerns and reduced availability of human oocytes, functional studies on FC were performed on bovine oocytes collected from slaughterhouse ovaries. Participants/materials, setting, methods 180 immature human oocytes were matured in vitro and either fixed at different meiosis I stages or treated 2h with colcemid, a MT depolymerizing drug, to induce maximum FC expansion and fixed. The oocytes were then immuno-stained for chromatin(DAPI), FC(ZW10), MT(alpha tubulin) and kinetochores(CREST) and imaged with super-resolution confocal microscopy. Bovine immature oocytes were matured in vitro with or without lonafarnib (inhibitor of FC expansion). After maturation chromosome spreads were performed to evaluate oocyte ploidy. Main results and the role of chance In human oocytes, after nuclear envelope breakdown, before microtubule appearance when the chromosomes appear compacted, FCs are visible on the top of the kinetochores. During MT nucleation, FCs form thick mesh in the center of the compacted chromosomes. During prometaphase I, FCs expand beyond the centromeric region of the chromosomes forming a cup around the sister kinetochores of the paired homologous chromosomes. When the chromosomes starts to align at the metaphase plate, FCs start to be stripped from the kinetochores and colocalize to the MTs. At metaphase I and II, only kinetochore localization is visible. When oocytes are treated with colcemid, FCs extend to partially cover chromosome arms. We observed a wide variability in FCs expansion between and within patients. Inhibition of FC expansion in bovine oocytes increase the incidence of oocyte aneuploidy (90% in lonafarnib treated vs 37% in control oocytes). Altogether, our data reveal that in human oocytes FCs expand more compared to human somatic cells. Considering that inhibition of FC expansion induces chromosome segregation errors, we propose that the large FC size in human oocytes may play a role in achieving correct chromosome-MT attachments within their extremely large cytoplasm thus ensuring proper chromosome segregation. Limitations, reasons for caution The limited availability of human oocytes requires the use of an animal model for in depth mechanistic studies. Although bovine oocytes have similar aneuploidy rate and spindle assembly mechanisms as human oocytes, they show morphological differences in FC expansion which could underlie intrinsic functional discrepancies between the two organisms. Wider implications of the findings A better understanding of the chromosome segregation mechanisms in human meiosis will help understanding what causes the high incidence of aneuploidies in human oocytes and possibly contribute to the development of interventions or of oocyte selection procedures to reduce the incidence of oocyte aneuploidy during in vitro maturation. Trial registration number Not applicable

Early onset of APC/C activity renders SAC inefficient in mouse embryos.

Control mechanisms of spindle assembly and chromosome segregation are vital for preventing aneuploidy during cell division. The mammalian germ cells and embryos are prone to chromosome segregation errors, and the resulting aneuploidy is a major cause of termination of development or severe developmental disorders. Here we focused on early mouse embryos, and using combination of methods involving microinjection, immunodetection and confocal live cell imaging, we concentrated on the Spindle Assembly Checkpoint (SAC) and Anaphase Promoting Complex/Cyclosome (APC/C). These are two important mechanisms cooperating during mitosis to ensure accurate chromosome segregation, and assessed their activity during the first two mitoses after fertilization. Our results showed, that in zygotes and 2-cell embryos, the SAC core protein Mad1 shows very low levels on kinetochores in comparison to oocytes and its interaction with chromosomes is restricted to a short time interval after nuclear membrane disassembly (NEBD). Exposure of 2-cell embryos to low levels of spindle poison does not prevent anaphase, despite the spindle damage induced by the drug. Lastly, the APC/C is activated coincidentally with NEBD before the spindle assembly completion. This early onset of APC/C activity, together with precocious relocalization of Mad1 from chromosomes, prevents proper surveillance of spindle assembly by SAC. The results contribute to the understanding of the origin of aneuploidy in early embryos.

Ran-GTP assembles a specialized spindle structure for accurate chromosome segregation in medaka early embryos

Despite drastic cellular changes during cleavage, a mitotic spindle assembles in each blastomere to accurately segregate duplicated chromosomes. Mechanisms of mitotic spindle assembly have been extensively studied using small somatic cells. However, mechanisms of spindle assembly in large vertebrate embryos remain little understood. Here, we establish functional assay systems in medaka (Oryzias latipes) embryos by combining CRISPR knock-in with auxin-inducible degron technology. Live imaging reveals several unexpected features of microtubule organization and centrosome positioning that achieve rapid, accurate cleavage. Importantly, Ran-GTP assembles a dense microtubule network at the metaphase spindle center that is essential for chromosome segregation in early embryos. This unique spindle structure is remodeled into a typical short, somatic-like spindle after blastula stages, when Ran-GTP becomes dispensable for chromosome segregation. We propose that despite the presence of centrosomes, the chromosome-derived Ran-GTP pathway has essential roles in functional spindle assembly in large, rapidly dividing vertebrate early embryos, similar to acentrosomal spindle assembly in oocytes.

Interview with Journal of Cell Science Editor Renata Basto

ABSTRACT Renata Basto is a Centre National de la Recherche Scientifique (CNRS) Team Leader at the Institut Curie and Deputy Director of the Cell Biology and Cancer Research Unit in Paris, France. Renata completed her undergraduate degree at the University of Lisbon, Portugal, before moving abroad to Gif-sur-Yvette, France for her doctoral studies on mitotic spindle assembly checkpoint proteins in Drosophila, mentored by Prof. Rui Gomes and Prof. Roger Karess. She then received a postdoctoral fellowship with Prof. Jordan Raff at the University of Cambridge, UK, where her work helped identify centrosome amplification as a tumour-initiating mechanism. In 2008, she joined the Department of Cell Biology at CNRS and the Institut Curie, where her laboratory studies the roles of centrosomes, and mechanisms of mitotic spindle assembly, polyploidy and genetic instability in development, health and disease. Renata joined Journal of Cell Science as an Editor in August 2023, bringing to the journal her significant expertise in the cell biology of centrosomes, spindle proteins, microtubules and mitosis. We spoke with Renata over Zoom to learn about her fascinating career, her advice for young scientists and the impact she hopes to make as an Editor.

Mechanism of spindle assembly regulated by the human oocyte microtubule organizing centre in human oocytes.

Mechanism of spindle assembly regulated by the human oocyte microtubule organizing centre in human oocytes.

The Mechanism of Acentrosomal Spindle Assembly in Human Oocytes

ABSTRACT During somatic cell mitosis and meiosis, spindle assembly is an essential step for ensuring accurate chromosome transmission and is mediated via duplicated centrosomes. In nonsomatic cells, canonical centrosomes are absent in the oocytes of many species, suggesting the mechanism of female meiotic spindle assembly is not conserved between species. Human oocytes lack detectable acentriolar microtubule organizing centers at the meiotic spindle poles and the exact mechanism of acentrosomal spindle assembly in human oocyte remains unclear. This study aimed to utilize 3-dimensional (3D) high-resolution imaging of more than 2000 human oocytes to identify and characterize key structures in the spindle assembly process. First, human oocytes were stained to visualize chromosomes, kinetochores, and microtubules before maturation. Approximately 2 to 4 hours following nuclear envelope breakdown (NEBD), the nucleation of spindle microtubules was initiated from kinetochores. To identify factors driving this spindle microtubule nucleation from kinetochores in human oocytes, 86 human centrosome and microtubule-related proteins were localized by immunofluorescence in more than 1000 fixed human oocytes. Of these, 43 proteins localized in the meiotic spindle, among which 4 proteins (centriolar coiled-coil protein 110 [CCP110], cytoskeleton-associated protein 5 [CKAP5], disrupted in schizophrenia 1 [DISC1], and transforming acidic coiled-coil-containing protein 3 [TACC3]) showed kinetochore and spindle microtubule localization. It was observed that these 4 proteins belong to the same structure (human oocyte microtubule organizing center [huoMTOC]), and a dense microtubule cluster was observed around this structure, which caused asymmetrical microtubule distribution around the nuclear envelope before NEBD. Next, it was observed that TACC3 had over 29-fold greater expression than CCP110, CKAP5, and DISC1, and after being depleted using siRNA, there was complete disruption of the huoMTOC, and the distribution of microtubules was drastically diminished. The other 3 proteins showed similar but less severe diminishment of the huoMTOC when depleted. Next, to visualize the dynamic process of huoMTOC-regulated spindle assembly, 3D time-lapse imaging was used following NEBD. This showed the huoMTOC fragments and relocates on the kinetochores to initiate microtubule nucleation and spindle assembly. Again, each huoMTOC component was down-regulated in human oocytes and brought through this stage, showing that with each component there was significant impairment of spindle microtubule nucleation and spindle assembly compared with the control group (P < 0.001), and spindle microtubules were greatly decreased. It was hypothesized that given the key role of the huoMTOC in human spindle assembly, a disrupted huoMTOC resulting from mutations in CCP110m CKAP5, DISC1, or TACC3 may cause abnormal oocyte maturation in patients. A cohort of 1394 infertile female patients characterized by oocyte maturation arrest was screened for mutations through whole-exome sequencing data sets. Two patients with loss-of-function mutations in TACC3 were identified, and analysis of their retrieved oocytes found a missing huoMTOC, suggesting the disruption of the huoMTOC impaired the nucleation of microtubules in the oocytes of patients. This study identified and characterized a structure, which was named the huoMTOC, which serves as a major site of microtubule nucleation and is required for spindle assembly in human oocytes.

An egg-ceptional mechanism of acentrosomal spindle assembly.

An egg-ceptional mechanism of acentrosomal spindle assembly.

Phosphorylation of adducin-1 by TPX2 promotes interpolar microtubule homeostasis and precise chromosome segregation in mouse oocytes

BackgroundADD1 (adducin-1) and TPX2 (targeting protein for Xklp2) are centrosomal proteins and regulate mitotic spindle assembly. Mammalian oocytes that segregate homologous chromosomes in Meiosis I and sister chromatids in Meiosis II with a spindle lacking centrosomes are more prone to chromosome segregation errors than in mitosis. However, the regulatory mechanisms of oocyte spindle assembly and the functions of ADD1 and TPX2 in this process remain elusive.ResultWe found that the expression levels and localization of ADD1, S726 phosphorylated ADD1 (p-ADD1), and TPX2 proteins exhibited spindle assembly-dependent dynamic changes during mouse oocyte meiosis. Taxol treatment, which stabilizes the microtubule polymer and protects it from disassembly, made the signals of ADD1, p-ADD1, and TPX2 present in the microtubule organizing centers of small asters and spindles. Knockdown of approximately 60% of ADD1 protein levels destabilized interpolar microtubules in the meiotic spindle, resulting in aberrant chromosome alignment, reduced first polar body extrusion, and increased aneuploidy in metaphase II oocytes, but did not affect K-fiber homeostasis and the expression and localization of TPX2. Strikingly, TPX2 deficiency caused increased protein content of ADD1, but decreased expression and detachment of p-ADD1 from the spindle, thereby arresting mouse oocytes at the metaphase I stage with collapsed spindles.ConclusionPhosphorylation of ADD1 at S726 by TPX2 mediates acentriolar spindle assembly and precise chromosome segregation in mouse oocytes.

The mechanism of acentrosomal spindle assembly in human oocytes

Meiotic spindle assembly ensures proper chromosome segregation in oocytes. However, the mechanisms behind spindle assembly in human oocytes remain largely unknown. We used three-dimensional high-resolution imaging of more than 2000 human oocytes to identify a structure that we named the human oocyte microtubule organizing center (huoMTOC). The proteins TACC3, CCP110, CKAP5, and DISC1 were found to be essential components of the huoMTOC. The huoMTOC arises beneath the oocyte cortex and migrates adjacent to the nuclear envelope before nuclear envelope breakdown (NEBD). After NEBD, the huoMTOC fragments and relocates on the kinetochores to initiate microtubule nucleation and spindle assembly. Disrupting the huoMTOC led to spindle assembly defects and oocyte maturation arrest. These results reveal a physiological mechanism of huoMTOC-regulated spindle assembly in human oocytes.

Directly Measuring Forces within Reconstituted Active Microtubule Bundles.

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.

Mechanistic insights into central spindle assembly mediated by the centralspindlin complex

The central spindle spatially and temporally regulates the formation of division plane during cytokinesis in animal cells. The heterotetrameric centralspindlin complex bundles microtubules to assemble the central spindle, the mechanism of which is poorly understood. Here, we determined the crystal structures of the molecular backbone of ZEN-4/CYK-4 centralspindlin from Caenorhabditis elegans, which revealed the detailed mechanism of complex formation. The molecular backbone of centralspindlin has the intrinsic propensity to undergo liquid-liquid phase separation. The condensation of centralspindlin requires two patches of basic residues at ZEN-4 and multiple acidic residues at the intrinsically disordered region of CYK-4, explaining the synergy of the two subunits for the function. These complementary charged residues were critical for the microtubule bundling activity of centralspindlin in vitro and for the assembly of the central spindle in vivo. Together, our findings provide insights into the mechanism of central spindle assembly mediated by centralspindlin through charge-driven macromolecular condensation.

Aneuploidy in human eggs: contributions of the meiotic spindle.

Human eggs frequently contain an incorrect number of chromosomes, a condition termed aneuploidy. Aneuploidy affects ∼10–25% of eggs in women in their early 30s, and more than 50% of eggs from women over 40. Most aneuploid eggs cannot develop to term upon fertilization, making aneuploidy in eggs a leading cause of miscarriages and infertility. The cellular origins of aneuploidy in human eggs are incompletely understood. Aneuploidy arises from chromosome segregation errors during the two meiotic divisions of the oocyte, the progenitor cell of the egg. Chromosome segregation is driven by a microtubule spindle, which captures and separates the paired chromosomes during meiosis I, and sister chromatids during meiosis II. Recent studies reveal that defects in the organization of the acentrosomal meiotic spindle contribute to human egg aneuploidy. The microtubules of the human oocyte spindle are very frequently incorrectly attached to meiotic kinetochores, the multi-protein complexes on chromosomes to which microtubules bind. Multiple features of human oocyte spindles favour incorrect attachments. These include spindle instability and many age-related changes in chromosome and kinetochore architecture. Here, we review how the unusual spindle assembly mechanism in human oocytes contributes to the remarkably high levels of aneuploidy in young human eggs, and how age-related changes in chromosome and kinetochore architecture cause aneuploidy levels to rise even higher as women approach their forties.

Centriole-independent mitotic spindle assembly relies on the PCNT-CDK5RAP2 pericentriolar matrix.

Centrosomes, composed of centrioles that recruit a pericentriolar material (PCM) matrix assembled from PCNT and CDK5RAP2, catalyze mitotic spindle assembly. Here, we inhibit centriole formation and/or remove PCNT-CDK5RAP2 in RPE1 cells to address their relative contributions to spindle formation. While CDK5RAP2 and PCNT are normally dispensable for spindle formation, they become essential when centrioles are absent. Acentriolar spindle assembly is accompanied by the formation of foci containing PCNT and CDK5RAP2 via a microtubule and Polo-like kinase 1-dependent process. Foci formation and spindle assembly require PCNT-CDK5RAP2-dependent matrix assembly and the ability of CDK5RAP2 to recruit γ-tubulin complexes. Thus, the PCM matrix can self-organize independently of centrioles to generate microtubules for spindle assembly; conversely, an alternative centriole-anchored mechanism supports spindle assembly when the PCM matrix is absent. Extension to three cancer cell lines revealed similar results in HeLa cells, whereas DLD1 and U2OS cells could assemble spindles in the absence of centrioles and PCNT-CDK5RAP2, suggesting cell type variation in spindle assembly mechanisms.

Mechanisms of spindle assembly and size control.

The spindle is crucial for cell division by allowing the faithful segregation of replicated chromosomes to daughter cells. Proper segregation is ensured only if microtubules (MTs) and hundreds of other associated factors interact to assemble this complex structure with the appropriate architecture and size. In this review, we describe the latest view of spindle organisation as well as the molecular gradients and mechanisms underlying MT nucleation and spindle assembly. We then discuss the overlapping physical and molecular constraints that dictate spindle morphology, concluding with a focus on spindle size regulation.

Mechanisms of chromosome biorientation and bipolar spindle assembly analyzed by computational modeling.

The essential functions required for mitotic spindle assembly and chromosome biorientation and segregation are not fully understood, despite extensive study. To illuminate the combinations of ingredients most important to align and segregate chromosomes and simultaneously assemble a bipolar spindle, we developed a computational model of fission-yeast mitosis. Robust chromosome biorientation requires progressive restriction of attachment geometry, destabilization of misaligned attachments, and attachment force dependence. Large spindle length fluctuations can occur when the kinetochore-microtubule attachment lifetime is long. The primary spindle force generators are kinesin-5 motors and crosslinkers in early mitosis, while interkinetochore stretch becomes important after biorientation. The same mechanisms that contribute to persistent biorientation lead to segregation of chromosomes to the poles after anaphase onset. This model therefore provides a framework to interrogate key requirements for robust chromosome biorientation, spindle length regulation, and force generation in the spindle.

Kif2a Scales Meiotic Spindle Size in Hymenochirus boettgeri.

Size is a fundamental feature of biological systems that affects physiology at all levels. For example, the dynamic, microtubule-based spindle that mediates chromosome segregation scales to a wide range of cell sizes across different organisms and cell types. Xenopus frog species possess a variety of egg and meiotic spindle sizes, and differences in activities or levels of microtubule-associated proteins in the egg cytoplasm between Xenopus laevis and Xenopus tropicalis have been shown to account for spindle scaling [1]. Increased activity of the microtubule severing protein katanin scales the X.tropicalis spindle smaller compared to X.laevis [2], as do elevated levels of TPX2, a protein that enriches the cross-linking kinesin-5 motor Eg5 at spindle poles [3]. To examine the conservation of spindle scaling mechanisms more broadly across frog species, we have utilized the tiny, distantly related Pipid frog Hymenochirus boettgeri. We find that egg extracts from H.boettgeri form meiotic spindles similar in size to X.tropicalis but that TPX2 and katanin-mediated scaling is not conserved. Instead, the microtubule depolymerizing motor protein kif2a functions to modulate spindle size. H.boettgeri kif2a possesses an activating phosphorylation site that is absent from X.laevis. Comparison of katanin and kif2a phosphorylation sites across a variety of species revealed strong evolutionary conservation, with X.laevis and X.tropicalis possessing distinct and unique alterations. Our study highlights the diversity and complexity of spindle assembly and scaling mechanisms, indicating that there is more than one way to assemble a spindle of a particular size.

The balance between KIFC3 and EG5 tetrameric kinesins controls the onset of mitotic spindle assembly.

One of the first steps in mitotic spindle assembly is the dissolution of the centrosome linker followed by centrosome separation driven by EG5, a tetrameric plus-end-directed member of the kinesin-5 family. However, even in the absence of the centrosome linker, the two centrosomes are kept together by an ill-defined microtubule-dependent mechanism. Here we show that KIFC3, a minus-end-directed kinesin-14, provides microtubule-based centrosome cohesion. KIFC3 forms a homotetramer that pulls the two centrosomes together via a specific microtubule network. At mitotic onset, KIFC3 activity becomes the main driving force of centrosome cohesion to prevent premature spindle formation after linker dissolution as it counteracts the increasing EG5-driven pushing forces. KIFC3 is eventually inactivated by NEver in mitosis-related Kinase 2 (NEK2) to enable EG5-driven bipolar spindle assembly. We further show that persistent centrosome cohesion in mitosis leads to chromosome mis-segregation. Our findings reveal a mechanism of spindle assembly that is evolutionary conserved from yeast to humans.

OsMTOPVIB is required for meiotic bipolar spindle assembly

The organization of microtubules into a bipolar spindle is essential for chromosome segregation. Both centrosome and chromatin-dependent spindle assembly mechanisms are well studied in mouse, Drosophila melanogaster, and Xenopus oocytes; however, the mechanism of bipolar spindle assembly in plant meiosis remains elusive. According to our observations of microtubule assembly in Oryza sativa, Zea mays, Arabidopsis thaliana, and Solanum lycopersicum, we propose that a key step of plant bipolar spindle assembly is the correction of the multipolar spindle into a bipolar spindle at metaphase I. The multipolar spindles failed to transition into bipolar ones in OsmtopVIB with the defect in double-strand break (DSB) formation. However, bipolar spindles were normally assembled in several other mutants lacking DSB formation, such as Osspo11-1, pair2, and crc1, indicating that bipolar spindle assembly is independent of DSB formation. We further revealed that the mono-orientation of sister kinetochores was prevalent in OsmtopVIB, whereas biorientation of sister kinetochores was frequently observed in Osspo11-1, pair2, and crc1 In addition, mutations of the cohesion subunit OsREC8 resulted in biorientation of sister kinetochores as well as bipolar spindles even in the background of OsmtopVIB Therefore, we propose that biorientation of the kinetochore is required for bipolar spindle assembly in the absence of homologous recombination.

Acentriolar microtubule organization centers and Ran-mediated microtubule formation pathways are both required in porcine oocytes.

Mammalian oocytes lack centrioles but can generate bipolar spindles using several different mechanisms. For example, mouse oocytes have acentriolar microtubule organization centers (MTOCs) that contain many components of the centrosome, and which initiate microtubule polymerization. On the contrary, human oocytes lack MTOCs and the Ran-mediated mechanisms may be responsible for spindle assembly. Complete knowledge of the different mechanisms of spindle assembly is lacking in various mammalian oocytes. In this study, we demonstrate that both MTOC- and Ran-mediated microtubule nucleation are required for functional meiotic metaphase I spindle generation in porcine oocytes. Acentriolar MTOC components, including Cep192 and pericentrin, were absent in the germinal vesicle and germinal vesicle breakdown stages. However, they start to colocalize to the spindle microtubules, but are absent in the meiotic spindle poles. Knockdown of Cep192 or inhibition of Polo-like kinase 1 activity impaired the recruitment of Cep192 and pericentrin to the spindles, impaired microtubule assembly, and decreased the polar body extrusion rate. When the RanGTP gradient was perturbed by the expression of dominant negative or constitutively active Ran mutants, severe defects in microtubule nucleation and cytokinesis were observed, and the localization of MTOC materials in the spindles was abolished. These results demonstrate that the stepwise involvement of MTOC- and Ran-mediated microtubule assembly is crucial for the formation of meiotic spindles in porcine oocytes, indicating the diversity of spindle formation mechanisms among mammalian oocytes.

Dual-spindle formation in zygotes keeps parental genomes apart in early mammalian embryos.

At the beginning of mammalian life, the genetic material from each parent meets when the fertilized egg divides. It was previously thought that a single microtubule spindle is responsible for spatially combining the two genomes and then segregating them to create the two-cell embryo. We used light-sheet microscopy to show that two bipolar spindles form in the zygote and then independently congress the maternal and paternal genomes. These two spindles aligned their poles before anaphase but kept the parental genomes apart during the first cleavage. This spindle assembly mechanism provides a potential rationale for erroneous divisions into more than two blastomeric nuclei observed in mammalian zygotes and reveals the mechanism behind the observation that parental genomes occupy separate nuclear compartments in the two-cell embryo.