Mechanical stress combines with planar polarised patterning during metaphase to orient embryonic epithelial cell divisions.

The planar orientation of cell division (OCD) is important for epithelial morphogenesis and homeostasis. Here, we ask how mechanics and antero-posterior (AP) patterning combine to influence the first divisions after gastrulation in the Drosophila embryonic epithelium. We analyse hundreds of cell divisions and show that stress anisotropy, notably from compressive forces, can reorient division directly in metaphase. Stress anisotropy influences the OCD by imposing metaphase cell elongation, despite mitotic rounding, and overrides interphase cell elongation. In strongly elongated cells, the mitotic spindle adapts its length to, and hence its orientation is constrained by, the cell long axis. Alongside mechanical cues, we find a tissue-wide bias of the mitotic spindle orientation towards AP-patterned planar polarised Myosin-II. This spindle bias is lost in an AP-patterning mutant. Thus, a patterning-induced mitotic spindle orientation bias overrides mechanical cues in mildly elongated cells, whereas in strongly elongated cells the spindle is constrained close to the high stress axis.

The Spo13/Meikin pathway confines the onset of gamete differentiation to meiosis II in yeast.

Sexual reproduction requires genome haploidization by the two divisions of meiosis and a differentiation program to generate gametes. Here, we have investigated how sporulation, the yeast equivalent of gamete differentiation, is coordinated with progression through meiosis. Spore differentiation is initiated at metaphase II when a membrane-nucleating structure, called the meiotic plaque, is assembled at the centrosome. While all components of this structure accumulate already at entry into meiosis I, they cannot assemble because centrosomes are occupied by Spc72, the receptor of the γ-tubulin complex. Spc72 is removed from centrosomes by a pathway that depends on the polo-like kinase Cdc5 and the meiosis-specific kinase Ime2, which is unleashed by the degradation of Spo13/Meikin upon activation of the anaphase-promoting complex at anaphase I. Meiotic plaques are finally assembled upon reactivation of Cdk1 at entry into metaphase II. This unblocking-activation mechanism ensures that only single-copy genomes are packaged into spores and might serve as a paradigm for the regulation of other meiosis II-specific processes.

Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins.

Mitotic spindle microtubules (MTs) undergo continuous poleward flux, whose driving force and function in humans remain unclear. Here, we combined loss-of-function screenings with analysis of MT-dynamics in human cells to investigate the molecular mechanisms underlying MT-flux. We report that kinesin-7/CENP-E at kinetochores (KTs) is the predominant driver of MT-flux in early prometaphase, while kinesin-4/KIF4A on chromosome arms facilitates MT-flux during late prometaphase and metaphase. Both these activities work in coordination with kinesin-5/EG5 and kinesin-12/KIF15, and our data suggest that the MT-flux driving force is transmitted from non-KT-MTs to KT-MTs by the MT couplers HSET and NuMA. Additionally, we found that the MT-flux rate correlates with spindle length, and this correlation depends on the establishment of stable end-on KT-MT attachments. Strikingly, we find that MT-flux is required to regulate spindle length by counteracting kinesin 13/MCAK-dependent MT-depolymerization. Thus, our study unveils the long-sought mechanism of MT-flux in human cells as relying on the coordinated action of four kinesins to compensate for MT-depolymerization and regulate spindle length.

KINESIN-12E regulates metaphase spindle flux and helps control spindle size in Arabidopsis.

The bipolar mitotic spindle is a highly conserved structure among eukaryotes that mediates chromosome alignment and segregation. Spindle assembly and size control are facilitated by force-generating microtubule-dependent motor proteins known as kinesins. In animals, kinesin-12 cooperates with kinesin-5 to produce outward-directed forces necessary for spindle assembly. In plants, the relevant molecular mechanisms for spindle formation are poorly defined. While an Arabidopsis thaliana kinesin-5 ortholog has been identified, the kinesin-12 ortholog in plants remains elusive. In this study, we provide experimental evidence for the function of Arabidopsis KINESIN-12E in spindle assembly. In kinesin-12e mutants, a delay in spindle assembly is accompanied by the reduction of spindle size, demonstrating that KINESIN-12E contributes to mitotic spindle architecture. Kinesin-12E localization is mitosis-stage specific, beginning with its perinuclear accumulation during prophase. Upon nuclear envelope breakdown, KINESIN-12E decorates subpopulations of microtubules in the spindle and becomes progressively enriched in the spindle midzone. Furthermore, during cytokinesis, KINESIN-12E shares its localization at the phragmoplast midzone with several functionally diversified Arabidopsis KINESIN-12 members. Changes in the kinetochore and in prophase and metaphase spindle dynamics occur in the absence of KINESIN-12E, suggest it might play an evolutionarily conserved role during spindle formation similar to its spindle-localized animal kinesin-12 orthologs.

The metaphase spindle at steady state - Mechanism and functions of microtubule poleward flux.

The mitotic spindle is a bipolar cellular structure, built from tubulin polymers, called microtubules, and interacting proteins. This macromolecular machine orchestrates chromosome segregation, thereby ensuring accurate distribution of genetic material into the two daughter cells during cell division. Powered by GTP hydrolysis upon tubulin polymerization, the microtubule ends exhibit a metastable behavior known as the dynamic instability, during which they stochastically switch between the growth and shrinkage phases. In the context of the mitotic spindle, dynamic instability is furthermore regulated by microtubule-associated proteins and motor proteins, which enables the spindle to undergo profound changes during mitosis. This highly dynamic behavior is essential for chromosome capture and congression in prometaphase, as well as for chromosome alignment to the spindle equator in metaphase and their segregation in anaphase. In this review we focus on the mechanisms underlying microtubule dynamics and sliding and their importance for the maintenance of shape, structure and dynamics of the metaphase spindle. We discuss how these spindle properties are related to the phenomenon of microtubule poleward flux, highlighting its highly cooperative molecular basis and role in keeping the metaphase spindle at a steady state.

Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis.

In mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its co-activator Cdc20. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 (CycB3) is an activator of the Cyclin-Dependent Kinase 1 (Cdk1) that is required for meiotic anaphase in flies, worms and vertebrates. It has been hypothesized that CycB3-Cdk1 may be responsible for APC/C activation in meiosis but this remains to be determined. Using Drosophila, we found that mutations in CycB3 genetically enhance mutations in tws, which encodes the B55 regulatory subunit of Protein Phosphatase 2A (PP2A) known to promote mitotic exit. Females heterozygous for CycB3 and tws loss-of-function alleles lay embryos that arrest in mitotic metaphase in a maternal effect, indicating that CycB3 promotes anaphase in mitosis in addition to meiosis. This metaphase arrest is not due to the Spindle Assembly Checkpoint (SAC) because mutation of mad2 that inactivates the SAC does not rescue the development of embryos from CycB3-/+, tws-/+ females. Moreover, we found that CycB3 promotes APC/C activity and anaphase in cells in culture. We show that CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its Cdc20 co-activators Fizzy and Cortex. Our results strongly suggest that CycB3-Cdk1 directly activates the APC/C to promote anaphase in both meiosis and mitosis.

Active forces shape the metaphase spindle through a mechanical instability.

The metaphase spindle is a dynamic structure orchestrating chromosome segregation during cell division. Recently, soft matter approaches have shown that the spindle behaves as an active liquid crystal. Still, it remains unclear how active force generation contributes to its characteristic spindle-like shape. Here we combine theory and experiments to show that molecular motor-driven forces shape the structure through a barreling-type instability. We test our physical model by titrating dynein activity in Xenopus egg extract spindles and quantifying the shape and microtubule orientation. We conclude that spindles are shaped by the interplay between surface tension, nematic elasticity, and motor-driven active forces. Our study reveals how motor proteins can mold liquid crystalline droplets and has implications for the design of active soft materials.

WDR62 is involved in spindle assembly by interacting with CEP170 in spermatogenesis.

WDR62 is the second most common genetic alteration associated with microcephaly. It has been shown that Wdr62 is required for germ cell meiosis initiation in mice, and the majority of male germ cells are lost in the meiotic defect of first wave spermatogenesis in Wdr62 mutants. Strikingly, in this study, we found that the initiation of meiosis following spermatogenesis was not affected and the germ cells were gradually repopulated at later developmental stages. However, most germ cells were arrested at metaphase of meiosis I and no mature sperm were detected in epididymides. Further, this study demonstrated that metaphase I arrest of Wdr62-deficient spermatocytes was caused by asymmetric distribution of the centrosome and aberrant spindle assembly. Also, mechanistic studies demonstrated that WDR62 interacts with centrosome-associated protein CEP170, and deletion of Wdr62 causes downregulation of the CEP170 protein, which in turn leads to the aberrant spindle assembly. In summary, this study indicates that the meiosis of first wave spermatogenesis and the following spermatogenesis started from spermatogonium is probably regulated by different mechanisms. We also demonstrated a new function of WDR62 in germ cell meiosis, through its interaction with CEP170.

Checkpoint kinases are required for oocyte meiotic progression by the maintenance of normal spindle structure and chromosome condensation.

Checkpoint kinases (Chk) 1/2 are known for DNA damage checkpoint and cell cycle control in somatic cells. According to recent findings, the involvement of Chk1 in oocyte meiotic resumption and Chk2 is regarded as an essential regulator for progression at the post metaphase I stage (MI). In this study, AZD7762 (Chk1/2 inhibitor) and SB218078 (Chk1 inhibitor) were used to uncover the joint roles of Chk1/2 and differentiate the importance of Chk1 and Chk2 during oocyte meiotic maturation. Inhibition of Chk1/2 or Chk1 alone had no significant effect on germinal vesicle breakdown (GVBD) but significantly inhibited the first polar body (PB1). Interestingly, inhibition of Chk1 alone could not increase or completely block the extrusion of PB1 like Chk1/2 inhibition. Also, Chk1/2 inhibition resulted in defective meiotic spindle organization and chromosome condensation both in MI and metaphase II (MII) stages of oocytes. The location of γ-tubulin and Securin were abnormal or missing, while P38 MAPK was activated by Chk1/2 inhibition. Meanwhile, Chk1/2 inhibition reduced the percentage of the second polar body extrusion and pronuclear formation. In conclusion, our results further understand the functions and regulatory mechanism of Chk1/2 during oocyte meiotic maturation.

AIP1 and Cofilin control the actin dynamics to modulate the asymmetric division and cytokinesis in mouse oocytes.

Actin-interacting protein 1 (AIP1), also known as WD repeat-containing protein 1 (WDR1), is ubiquitous in eukaryotic organisms, and it plays critical roles in the dynamic reorganization of the actin cytoskeleton. However, the biological function and mechanism of AIP1 in mammalian oocyte maturation is still largely unclear. In this study, we demonstrated that AIP1 boosts ADF/Cofilin activity in mouse oocytes. AIP1 is primarily distributed around the spindle region during oocyte maturation, and its depletion impairs meiotic spindle migration and asymmetric division. The knockdown of AIP1 resulted in the gathering of a large number of actin-positive patches around the spindle region. This effect was reduced by human AIP1 (hAIP1) or Cofilin (S3A) expression. AIP1 knockdown also reduced the phosphorylation of Cofilin near the spindle, indicating that AIP1 interacts with ADF/Cofilin-decorated actin filaments and enhances filament disassembly. Moreover, the deletion of AIP1 disrupts Cofilin localization in metaphase I (MI) and induces cytokinesis defects in metaphase II (MII). Taken together, our results provide evidence that AIP1 promotes actin dynamics and cytokinesis via Cofilin in the gametes of female mice.

Femtosecond laser-induced blastomere fusion results in embryo tetraploidy by common metaphase plate formation.

The cell fusion is a widespread process, which takes place in many systems in vivo and in vitro. Fusion of cells is frequently related to tetraploidy, which can be found within natural physiological conditions, e.g., placentation, and in pathophysiological conditions, such as cancer and early pregnancy failure in humans. Here we investigate the mechanism of tetraploidization with help of femtosecond laser-induced mouse blastomere fusion by the means of Hoechst staining, GFP, BODIPY dyes and fluorescent species generated intracellularly by a femtosecond laser. We establish diffusive mixing of cytosol, whereas the large components of a cytoplasm (organelles, cytoskeleton) are poorly diffusible and are not completely mixed after cell fusion and a subsequent division. We show that mechanisms which are responsible for the formation of a common metaphase plate triggered tetraploidization in fused mouse embryos and could be a significant factor in polyploidy formation in vivo. Thus, our results suggest that microtubules play a critical role in tetraploidization.

Versatility of multivalent orientation, inverted meiosis, and rescued fitness in holocentric chromosomal hybrids.

Chromosomal rearrangements (e.g., fusions/fissions) have the potential to drive speciation. However, their accumulation in a population is generally viewed as unlikely, because chromosomal heterozygosity should lead to meiotic problems and aneuploid gametes. Canonical meiosis involves segregation of homologous chromosomes in meiosis I and sister chromatid segregation during meiosis II. In organisms with holocentric chromosomes, which are characterized by kinetic activity distributed along almost the entire chromosome length, this order may be inverted depending on their metaphase I orientation. Here we analyzed the evolutionary role of this intrinsic versatility of holocentric chromosomes, which is not available to monocentric ones, by studying F1 to F4 hybrids between two chromosomal races of the Wood White butterfly (Leptidea sinapis), separated by at least 24 chromosomal fusions/fissions. We found that these chromosomal rearrangements resulted in multiple meiotic multivalents, and, contrary to the theoretical prediction, the hybrids displayed relatively high reproductive fitness (42% of that of the control lines) and regular behavior of meiotic chromosomes. In the hybrids, we also discovered inverted meiosis, in which the first and critical stage of chromosome number reduction was replaced by the less risky stage of sister chromatid separation. We hypothesize that the ability to invert the order of the main meiotic events facilitates proper chromosome segregation and hence rescues fertility and viability in chromosomal hybrids, potentially promoting dynamic karyotype evolution and chromosomal speciation.

Impact of Maturation and Vitrification Time of Human GV Oocytes on the Metaphase Plate Configuration.

The combination of in vitro maturation (IVM) techniques and oocyte vitrification (OV) could increase the number of useful oocytes in different types of patients. IVM and subsequent OV is the most widely used clinical strategy. Would the results improve if we reverse the order of the techniques? Here, we evaluated survival, in vitro maturation, time to extrude the first polar body (PB), and the metaphase plate configuration of human prophase I (GV) oocytes before or after their vitrification. Specific, 195 GV oocytes from 104 patients subjected to controlled ovarian stimulation cycles were included. We stablished three experimental groups: GV oocytes vitrified and IVM (Group GV-Vit), GV oocytes IVM and vitrified at MII stage (Group MII-Vit), and GV oocytes IVM (Group not-Vit). All of them were in vitro matured for a maximum of 48 h and fixed to study the metaphase plate by confocal microscopy. According to our results, the vitrification of immature oocytes and their subsequent maturation presented similar survival, maturation, and metaphase plate conformation rates, but a significantly higher percentage of normal spindle than the standard strategy. Additionally, the extension of IVM time to 48 h did not seem to negatively affect the oocyte metaphase plate configuration.

l-Serine transport in growing and maturing mouse oocytes.

Serine has roles in cell metabolism besides protein synthesis including providing one-carbon units to the folate cycle. Since growing mouse oocytes undergo a burst of folate accumulation as they near full size, we have investigated whether oocytes transport serine. Substantial serine transport appeared in oocytes near the end of their growth. Serine transport continued when oocytes resumed meiosis but ceased partway through first meiotic metaphase, remaining quiescent in mature eggs in second meiotic metaphase. The serine transporter was sodium dependent and inhibited by alanine, cysteine, leucine, or histidine, and had a Michaelis-Menten constant (Km ) for serine of 200 µM. Unexpectedly, exposing cumulus cell-enclosed oocytes to the physiological mediator of meiotic arrest, natriuretic peptide precursor Type C, substantially stimulated serine transport by the enclosed oocyte. Finally, in addition to transport by the oocyte itself, cumulus cells also supply serine to the enclosed oocyte via gap junctions within intact cumulus-oocyte complexes.

Mechanisms of kinesin-7 CENP-E in kinetochore-microtubule capture and chromosome alignment during cell division.

Chromosome congression is essential for faithful chromosome segregation and genomic stability in cell division. Centromere-associated protein E (CENP-E), a plus-end-directed kinesin motor, is required for congression of pole-proximal chromosomes in metaphase. CENP-E accumulates at the outer plate of kinetochores and mediates the kinetochore-microtubule capture. CENP-E also transports the chromosomes along spindle microtubules towards the equatorial plate. CENP-E interacts with Bub1-related kinase, Aurora B and core kinetochore components during kinetochore-microtubule attachment. In this review, we introduce the structures and mechanochemistry of kinesin-7 CENP-E. We highlight the complicated interactions between CENP-E and partner proteins during chromosome congression. We summarise the detailed roles and mechanisms of CENP-E in mitosis and meiosis, including the kinetochore-microtubule capture, chromosome congression/alignment in metaphase and the regulation of spindle assembly checkpoint. We also shed a light on the roles of CENP-E in tumourigenesis and CENP-E's specific inhibitors.

Regulation of mitotic spindle orientation by phosphorylation of end binding protein 1.

End binding protein 1 (EB1) is a key regulator of microtubule dynamics that orchestrates hierarchical interaction networks at microtubule plus ends to control proper cell division. EB1 activity is known to be regulated by serine/threonine phosphorylation; however, how tyrosine phosphorylation affects EB1 activity remains poorly understood. In this study, we mapped the tyrosine phosphorylation pattern of EB1 in synchronized cells and identified two tyrosine phosphorylation sites (Y217 and Y247) in mitotic cells. Using phospho-deficient (Y/F) and phospho-mimic (Y/D) mutants, we revealed that Y247, but not Y217, is critical for astral microtubule stability. The Y247D mutant contributed to increased spindle angle, indicative of defects in spindle orientation. Time-lapse microscopy revealed that the Y247D mutant significantly delayed mitotic progression by increasing the duration times of prometaphase and metaphase. Structural analysis suggests that Y247 mutants lead to instability of the hydrophobic cavity in the EB homology (EBH) domain, thereby affecting its interactions with p150glued, a protein essential for Gαi/LGN/NuMA complex capture. These findings uncover a crucial role for EB1 phosphorylation in the regulation of mitotic spindle orientation and cell division.

Prediction of metaphase II oocytes according to different levels of serum AMH in poor responders using the antagonist protocol during ICSI: a cohort study.

The aim of our study was to assess the value of serum AMH in prediction of metaphase II oocytes in poor responders. We performed a prospective cohort study included 206 poor responders candidate for ICSI using antagonist protocol. They were classified into 3 groups. Group I included 50 women with AMH < 0.3 ng/ml, group II included 85 women with AMH 0.3-0.7 ng/ml and group III included 71 women with AMH > 0.7-1.0 ng/ml. The primary outcome parameter was the number of MII oocytes. There was a highly significant difference between the study groups regarding E2 at triggering (481.41 ± 222.653, 648.17 ± 264.353 and 728.74 ± 305.412 respectively, number of oocyte retrieved (2.37 ± 1.178, 3.38 ± 1.622 and 3.80 ± 1.427 respectively), number of MII oocytes (1.66 ± 1.039, 2.35 ± 1.171 and 2.61 ± 1.080 respectively), number of fertilized oocytes (1.39 ± 0.919, 1.91 ± 0.983 and 2.21 ± 0.937 respectively), , total number of embryos (1.34 ± 0.938, 1.76 ± 0.956 and 2.09 ± 0.907 respectively), clinical pregnancy rates (4.9 vs. 7.7 and 19.7% respectively). We concluded that AMH is a good predictor for number of MII oocytes in poor responders undergoing ICSI.

The key long non-coding RNA screening and validation between germinal vesicle and metaphase II of porcine oocyte in vitro maturation.

Oocyte maturation plays a vitally important role in the reproduction of pigs. However, the roles of messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) in the developmental process of porcine oocyte maturation are still largely unclear. In this study, a transcriptome analysis of germinal vesicle (GV) and metaphase II (MII) of oocytes from Chinese Duroc pigs was performed. A total of 1,753,030 and 2,486 differentially expressed (DE) mRNAs, 22,811 and 9,868 DE lncRNAs were identified between GV and MII stages, respectively. Furthermore, functional enrichment analysis showed that the common DE mRNAs and DE lncRNAs during the process of maturation were mainly involved in biological process and cellular components. Our study provides new insights of the expression changes of mRNAs and lncRNAs during GV and MII stages, which might contribute to the maturation of oocytes. These results greatly improve our understanding of the molecular mechanisms regulating the maturation of oocytes in pigs.

Embryonic poly(A)-binding protein is differently expressed and interacts with the messenger RNAs in the mouse oocytes and early embryos.

The embryonic poly(A)-binding protein (EPAB) functions in the translational regulation of the maternal messenger RNAs (mRNAs) required during oocyte maturation, fertilization, and early embryo development. Since there is no antibody specific to mammalian EPAB protein, all studies related to the Epab gene could be performed at the mRNA levels except for the investigations in the Xenopus. In this study, we have produced an EPAB-specific antibody. When we examined its expressional distribution in the mouse gonadal and somatic tissues, the EPAB protein was found to be expressed only in the mouse ovary and testis tissues, but it is undetectable level in the somatic tissues including stomach, liver, heart, small intestine, and kidney. Additionally, the spatial and temporal expression patterns of the EPAB and poly(A)-binding protein cytoplasmic 1 (PABPC1) proteins were analyzed in the mouse germinal vesicle (GV) and metaphase II (MII) oocytes, one-cell, and two-cell embryos. While EPAB expression gradually decreased from GV oocytes to two-cell embryos, the PABPC1 protein level progressively increased from GV oocytes to one-cell embryos and remarkably declined in the two-cell embryos ( P < 0.05). We have also described herein that the EPAB protein interacted with Epab, Pabpc1, Ccnb1, Gdf9, and Bmp15 mRNAs dependent upon the developmental stages of the mouse oocytes and early embryos. As a result, we have first produced an EPAB-specific antibody and characterized its expression patterns and interacting mRNAs in the mouse oocytes and early embryos. The findings suggest that EPAB in cooperation with PABPC1 implicate in the translational control of maternal mRNAs during oogenesis and early embryo development.

High proportion of immature oocytes in a cohort reduces fertilization, embryo development, pregnancy and live birth rates following ICSI.

RESEARCH QUESTION: Ooplasmic maturity has been studied for some time, but remains poorly defined. This study aimed to evaluate metaphase II (MII) oocyte competence in terms of fertilization, embryo development and cycle outcomes, according to the oocyte maturity ratio. DESIGN: Couples treated by intracytoplasmic sperm injection (ICSI) between 1993 and 2017 with female partners ≤35 years old were included. Cycles were divided into four groups according to proportion of MII oocytes at the time of retrieval: optimal (76-100%), adequate (51-75%), partial (26-50%) and minimal (1-25%). RESULTS: A total of 7672 ICSI cycles (optimal: 4838; adequate: 2252; partial: 518; minimal oocyte maturity: 64) were included, in which 95,667 MII oocytes were injected using ejaculated spermatozoa. The decreasing proportion of MII significantly reduced normal fertilization (two pronuclei) (78.9% to 71.3%; P < 0.0001) with a corresponding increase in digynic three-pronuclei that rose from 2.6% in the optimal group to 4.7% in the minimal group (P = 0.003). Implantation (33% to 17%; P < 0.0001), clinical pregnancy (63.6% to 37.5%; P < 0.0001) and live birth rates (49.2% to 26.6%; P < 0.0001) were affected by the decreasing proportion of MII oocytes. CONCLUSIONS: A high proportion of immature sibling oocytes in the retrieved cohort affects the fertilization rate and embryo developmental competence of MII inseminated oocytes, clinical pregnancy and live birth rates, suggesting that, in addition to nuclear maturity, ooplasmic and membrane maturity are required for developmental competence of MII oocytes. These findings may provide guidance toward ovarian stimulation protocols aimed at achieving a greater proportion of MII oocytes, leading to higher fertilization rates and better pregnancy outcomes.