Acentriolar spindle assembly in mammalian female meiosis and the consequences of its perturbations on human reproduction

The purpose of meiosis is to generate developmentally competent, haploid gametes with the correct number of chromosomes. For reasons not completely understood, female meiosis is more prone to chromosome segregation errors than meiosis in males, leading to an abnormal number of chromosomes, or aneuploidy, in gametes. Meiotic spindles are the cellular machinery essential for the proper segregation of chromosomes. One unique feature of spindle structures in female meiosis is spindles poles that lack centrioles. The process of building a meiotic spindle without centrioles is complex and requires precise coordination of different structural components, assembly factors, motor proteins, and signaling molecules at specific times and locations to regulate each step. In this review, we discuss the basics of spindle formation during oocyte meiotic maturation focusing on mouse and human studies. Finally, we review different factors that could alter the process of spindle formation and its stability. We conclude with a discussion of how different assisted reproductive technologies (ART) could affect spindles and the consequences these perturbations may have for subsequent embryo development.

M2 Muscarinic Receptor Activation Impairs Mitotic Progression and Bipolar Mitotic Spindle Formation in Human Glioblastoma Cell Lines

Background: Glioblastoma multiforme (GBM) is characterized by several genetic abnormalities, leading to cell cycle deregulation and abnormal mitosis caused by a defective checkpoint. We previously demonstrated that arecaidine propargyl ester (APE), an orthosteric agonist of M2 muscarinic acetylcholine receptors (mAChRs), arrests the cell cycle of glioblastoma (GB) cells, reducing their survival. The aim of this work was to better characterize the molecular mechanisms responsible for this cell cycle arrest. Methods: The arrest of cell proliferation was evaluated by flow cytometry analysis. Using immunocytochemistry and time-lapse analysis, the percentage of abnormal mitosis and aberrant mitotic spindles were assessed in both cell lines. Western blot analysis was used to evaluate the modulation of Sirtuin2 and acetylated tubulin—factors involved in the control of cell cycle progression. Results: APE treatment caused arrest in the M phase, as indicated by the increase in p-HH3 (ser10)-positive cells. By immunocytochemistry, we found a significant increase in abnormal mitoses and multipolar mitotic spindle formation after APE treatment. Time-lapse analysis confirmed that the APE-treated GB cells were unable to correctly complete the mitosis. The modulated expression of SIRT2 and acetylated tubulin in APE-treated cells provides new insights into the mechanisms of altered mitotic progression in both GB cell lines. Conclusions: Our data show that the M2 agonist increases aberrant mitosis in GB cell lines. These results strengthen the idea of considering M2 acetylcholine receptors a novel promising therapeutic target for the glioblastoma treatment.

O-154 First mitotic spindle formation led by sperm centrosome-dependent microtubule organising centres may cause high incidence of zygotic division errors in humans

Study question Why do multinucleated blastomeres appear at high frequency in two-cell-stage embryos in humans? Summary answer Failure in microtubule assembly during the first mitotic spindle body formation by sperm centrosome-dependent microtubule organising centres (MTOCs) may lead to chromosomal instability. What is known already Unlike that in mice, multinucleated blastomeres appear at high frequency in two-cell-stage embryos in humans. However, the underlying mechanism remains elusive. In mice, multiple acentriolar MTOCs appear around the male and female pronuclei after pronuclear disappearance and contribute to dual-spindle formation, engulfing each parental chromosome. This spindle formation may ensure an error-free division, keeping the chromosomes stable during the first cleavage, as observed in mice, but it is unclear whether a similar mechanism exists in humans. Study design, size, duration To examine how sperm centrosomes contribute to MTOC formation in humans, two types of 3PN zygotes derived fromeither conventional in vitro fertilization (c-IVF, n = 30) or intracytoplasmic sperm injection (ICSI, n = 10) were used. The zygotes were collected from October 2018 to January 2020. MTOC and mitotic spindle formation at consecutive stages of development during the first cleavage were analysed under static and dynamic conditions using immunofluorescence assay and fluorescent live-cell imaging. Participants/materials, setting, methods Under ethics approval, 3PN zygotes were donated by infertile couples undergoing c-IVF or ICSI cycles at the Yamashita Shonan Yume Clinic in Japan. All participants provided informed consent. Immunofluorescence assay was performed using antibodies against α-tubulin, pericentrin, and H3K9me3 after fixation with MTSB-XF solution. Fluorescent live-cell imaging was performed using TagGFP2-H2B mRNA (chromosome marker) and FusionRed-MAP4 mRNA (microtubule marker). Main results and the role of chance Immunofluorescence revealed that while 3PN zygotes derived from c-IVF showed four pericentrin dots, those derived from ICSI exhibited two pericentrin dots. In pro-metaphase, an independent group of chromosomes derived from each pronucleus and MTOCs were formed by the sperm centrosome at the core. Microtubules from each MTOC extended toward the chromosomes in the early metaphase; a quadrupolar spindle was formed in the c-IVF-derived zygotes, and a bipolar spindle was formed in the ICSI-derived zygotes by the MTOCs at the zygote apex after chromosome alignment. In pro-metaphase, the microtubules extended from the MTOCs to the nearest chromosome. Since microtubule assembly was found on oocyte-derived chromosomes, we hypothesised that whether a chromosome is surrounded by microtubules depends on the location of the MTOCs, irrespective of its origin. Live-cell imaging of histone H2B and MAP4 revealed that four MTOCs appeared around the three pronuclei just before the disappearance of the pronuclear membrane; microtubules then extended from the MTOCs toward the chromosomes, beginning to form a mitotic spindle as the chromosomes moved to the centre of the oocyte. Interestingly, one of the three assembled chromosome groups showed no microtubule assembly in the pro-metaphase. Similar results were obtained in all six 3PN zygotes subjected. Limitations, reasons for caution We demonstrated the high risk of developing bare chromosomes not surrounded by microtubules during the formation of the first mitotic spindle, using human tripronuclear zygotes. However, owing to unavailability of normal fertilized oocytes for this study because of the clinical use, we were unable to confirm this in normal zygotes. Wider implications of the findings Although two sperm centrosome-dependent MTOCs are expected to be formed in normal fertilized oocytes, these MTOCs are not sufficient to completely enclose physically separated female and male chromosomes with the microtubules. This explains the high frequency of zygotic division errors that lead to unstable human chromosomes. Trial registration number not applicable

Redistribution of centrosomal proteins by centromeres and Polo kinase controls partial nuclear envelope breakdown in fission yeast

Proper mitotic progression in Schizosaccharomyces pombe requires partial nuclear envelope breakdown (NEBD) and insertion of the spindle pole body (SPB – yeast centrosome) to build the mitotic spindle. Linkage of the centromere to the SPB is vital to this process, but why that linkage is important is not well understood. Utilizing high-resolution structured illumination microscopy (SIM), we show that the conserved SUN-domain protein Sad1 and other SPB proteins redistribute during mitosis to form a ring complex around SPBs, which is a precursor for localized NEBD and spindle formation. Although the Polo kinase Plo1 is not necessary for Sad1 redistribution, it localizes to the SPB region connected to the centromere, and its activity is vital for redistribution of other SPB ring proteins and for complete NEBD at the SPB to allow for SPB insertion. Our results lead to a model in which centromere linkage to the SPB drives redistribution of Sad1 and Plo1 activation that in turn facilitate partial NEBD and spindle formation through building of a SPB ring structure.

First mitotic spindle formation is led by sperm centrosome-dependent MTOCs in humans

Unlike in mice, multinucleated blastomeres appear at a high frequency in the two-cell-stage embryos in humans. In this Point of View article, we demonstrate that the first mitotic spindle formation led by sperm centrosome-dependent microtubule organizing centers may cause a high incidence of zygotic division errors using human tripronuclear zygotes.

Clathrin Heavy Chain 1 Plays Essential Roles During Oocyte Meiotic Spindle Formation and Early Embryonic Development in Sheep

As a major protein of the polyhedral coat of coated pits and vesicles, clathrin molecules have been shown to play a stabilization role for kinetochore fibers of the mitotic spindle by acting as inter-microtubule bridges. Clathrin heavy chain 1 (CLTC), the basic subunit of the clathrin coat, plays vital roles in both spindle assembly and chromosome congression during somatic-cell mitosis. However, its function in oocyte meiotic maturation and early embryo development in mammals, especially in domesticated animals, has not been fully investigated. In this study, the expression profiles and functional roles of CLTC in sheep oocytes were investigated. Our results showed that the expression of CLTC was maintained at a high level from the germinal vesicle (GV) stage to metaphase II stage and that CLTC was distributed diffusely in the cytoplasm of cells at interphase, from the GV stage to the blastocyst stage. After GV breakdown (GVBD), CLTC co-localized with beta-tubulin during metaphase. Oocyte treatments with taxol, nocodazole, or cold did not affect CLTC expression levels but led to disorders of its distribution. Functional impairment of CLTC by specific morpholino injections in GV-stage oocytes led to disruptions in spindle assembly and chromosomal alignment, accompanied by impaired first polar body (PB1) emissions. In addition, knockdown of CLTC before parthenogenetic activation disrupted spindle formation and impaired early embryo development. Taken together, the results demonstrate that CLTC plays a vital role in sheep oocyte maturation via the regulation of spindle dynamics and an essential role during early embryo development.

Tdrd3 regulates the progression of meiosis II through translational control of Emi2 mRNA in mouse oocytes

ABSTRACTAfter completion of meiosis I, the oocyte immediately enters meiosis II and forms a metaphase II (MII) spindle without an interphase, which is fundamental for generating a haploid gamete. Here, we identify tudor domain-containing protein 3 (Tdrd3) as a novel regulator of oocyte meiosis. Although early mitotic inhibitor 2 (Emi2) protein has been shown to ensure the meiosis I to II transition and the subsequent MII spindle formation by inhibiting the anaphase-promoting complex/cyclosome (APC/C), how it accumulates after meiosis I has remained unresolved. We isolated Tdrd3 as a protein directly binding to Emi2 mRNA. In GV-stage mouse oocytes, Emi2 mRNA assembled into RNA granules containing Tdrd3, while cyclin B1 mRNA, which was translated in early meiosis I, formed different granules. Knockdown of Tdrd3 attenuated Emi2 synthesis in meiosis II without affecting cyclin B1 synthesis in meiosis I. Moreover, Tdrd3-deficient oocytes entered interphase and failed to form an MII spindle after completion of meiosis I. Taken together, our results indicate the importance of Tdrd3-mediated translational control of Emi2 mRNA, which promotes Emi2 synthesis in meiosis II, for the progression of meiosis.

Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.