Mouse KL2 is a unique MTSE involved in chromosome-based spindle organization and regulated by multiple kinases during female meiosis

Microtubule-severing enzymes(MTSEs)play important roles in mitosis and meiosis of the primitive organisms.However,their roles in mammalian female meiosis,which accounts for over 80%of gamete-originated human reproductive diseases,remain unexplored.In the current study,we reported that katanin-like 2(KL2)was the only MTSE concentrating at chromosomes.Furthermore,the knockdown of KL2 significantly reduced the chromosome-based increase in the microtubule(MT)polymer,increased aberrant kinetochore-MT(K-MT)attachment,delayed meiosis,and severely affected normal fertility.We demonstrated that the inhibition of aurora B,a key kinase for correcting aberrant K-MT attachment,significantly eliminated KL2 expression from chromosomes.Additionally,KL2 interacted with phosphorylated eukaryotic elongation factor-2 kinase,and they competed for chromosome binding.Phosphorylated KL2 was also localized at spindle poles,with its phosphorylation regulated by extracellular signal-regulated kinase 1/2.In summary,the current study reveals a novel function of MTSEs in mammalian female meiosis and demonstrates that multiple kinases coordinate to regulate the levels of KL2 at chromosomes.

Localization of Phosphorylated Histone H3 at Mitosis and Meiosis in Wheat

One of the prominent cell cycle related modifications of histone proteins, whose function is correlated with chromosome condensation, is the phosphorylation of histone H3. Wheat (Triticum aestivum L.) mitotic and meiotic cells were analyzed with indirect immunoflurorescence labeling with an antibody recognizing histone H3 phosphorylated at Serine 10 to study the localization of phosphorylated histone H3 at mitosis and meiosis. Our results showed that, during mitotic division, the phosphoryiation of H3 started from early prophase and vanished at telophase, remaining mainly in the pericentromeric regions at metaphase and anaphase. During meiotic division, phosphorylation of H3 initiated at the transition from leptotene to zygotene and remained uniform, along the chromosomes from prophase I until telophase whereas it showed slightly stronger in the pericentromeric regions than along the chromosome arms from metaphase II until Lelophase II The different patterns of H3 phophorylation at mitosis and meiosis in wheat suggested that this evolutionarily conserved post-translational chromatin modification might be involved in more roles besides chromosome condensation.

Observation of Mitosis and Meiosis in Rice Cells by Simple Squash Method

[Objective] The aim of this study was to establish a feasible squashing technique for chromosome and obtain data of rice chromosome. [Method] With the materials of rice root tips and anther, the specimen was prepared by the modified squash method, and microscopic observation of mitosis and meiosis in rice cells was also carried out. [ Result] Mitosis in rice cells included interphase, prophase, metaphase, anaphase and telophase. Chromosome in metaphase shortened to the minmum, which was a good time for observing and investigating chromosome. However, meiosis in rice cells included meiosis Ⅰ and meiosis Ⅱ. Chromosome replication appeared in meiosis Ⅰ, while cell division only appeared in meiosis Ⅱ. [ Conclusion] The modified squashing technique for rice chromosome can obtain accurate data of rice chromosome, which provides evidence for genetic breeding.

Functional conservation of the meiotic genes SDS and RCK in male meiosis in the monocot rice

The Arabidopsis SDS （SOLO DANCERS） and RCK （ROCK-N-ROLLERS） genes are important for male meiosis, but it is still unknown whether they represent conserved functions in plants. We have performed phylogenetic analyses of SDS and RCK and their respective homologs, and identified their putative orthologs in poplar and rice. Quantitative real-time RT-PCR analysis indicated that rice SDS and RCK are expressed preferentially in young flowers, and transgenic RNAi rice lines with reduced expression of these genes exhibited normal vegetative development, but showed significantly reduced fertility with partially sterile flowers and defective pollens. SDS deficiency also caused a decrease in pollen amounts. Further cytological examination of male meiocytes revealed that the SDS deficiency led to defects in homolog interaction and bivalent formation in meiotic prophase I, and RCK deficiency resulted in defective meiotic crossover formation. These results indicate that rice SDS and RCK genes have similar functions to their Arabidopsis orthologs. Because rice and Arabidopsis, respectively, are members of monocots and eudicots, two largest groups of flowering plants, our results suggest that the functions of SDS and RCK are likely conserved in flowering plants.

Analysis of the meiosis in the F_1 hybrids of Longiflorum × Asiatic(LA) of lilies(Lilium) using genomic in situ hybridization

Longiflorum and Asiatic lilies of the genus Lilium of the family Liliaceae are two important groups of modem lily cultivars. One of the main trends of lily breeding is to realize introgression between these groups. With cut style pollination and embryo rescue, distant hybrids between the two groups have been obtained. However, the FI hybrids are highly sterile or some of them could produce a small number of 2n gametes, and their BC1 progenies are usually triploids. Dutch lily breeders have selected many cultivars from these BC1 progenies based on their variation. It is presumably suggested that such variation could be caused by intergenomic recombination and abnormal meiosis during gamete formation in F1 hybrids of Longiflorum × Asiatic （LA） hybrids in Lilium. Therefore, the meiotic process of ten F1 LA hybrids was cytologically investigated using genomic in situ hybridization and traditional cytological methods in the present research. The results showed that： at metaphase I, the homoeologous chromosome pairing among different F1 hybrids ranged from 2.0 to 11.4 bivalents formed by homoeologous chromosomes per pollen mother cell （PMC）, and very few multivalents, and even very few bivalents were formed by two chromosomes within one genome rather than homoeologous chromosomes in some PMCs; at anaphase I, all biva- lents were disjoined and most univalents were divided. Both the disjoined bivalents （half-bivalents） and the divided univalents （sister chromatids） moved to the opposite poles, and then formed two groups of chromosomes; because the two resulting half-bivalents retained their axes in the cell undisturbed, many crossover types, including single crossovers, three strand double crossovers, four strand double crossovers, four strand triple crossovers, and four strand multiple crossovers between the non-sister chromatids in the tetrads of bivalents, were clearly inferred by analyzing the breakpoints on the disjoined bivalents. The present investigation not only explained the reason for sterility of the Fl LA hybrids and the variation of their BCx progenies, but also provided a new method to analyze crossover types in other F1 interspecific hybrids as well.

The Arabidopsis MutS homolog AtMSH5 is required for normal meiosis

MSH5, a member of the MutS homolog DNA mismatch repair protein family, has been shown to be required for proper homologous chromosome recombination in diverse organisms such as mouse, budding yeast and Caenorhabditis elegans. In this paper, we show that a mutant Arabidopsis plant carrying the putative disrupted AtMSH5 gene exhibits defects during meiotic division, producing a proportion of nonviable pollen grains and abnormal embryo sacs, and thereby leading to a decrease in fertility. AtMSH5 expression is confined to meiotic floral buds, which is consistent with a possible role during meiosis. Cytological analysis of male meiosis revealed the presence of numerous univalents from diplotene to metaphase I, which were associated with a great reduction in chiasma frequencies. The average number of residual chiasmata in the mutant is reduced to 2.54 per meiocyte, which accounts for ~25% of the amount in the wild type. Here, quantitative cytogenetical analysis reveals that the residual chiasmata in Afresh5 mutants are randomly distributed among meiocytes, suggesting that AtMSH5 has an essential role during interferencesensitive chiasma formation. Taken together, the evidence indicates that AtMSH5 promotes homologous recombination through facilitating chiasma formation during prophase Ⅰ in Arabidopsis.

Asynchronous meiosis in Cucumis hystrix–cucumber synthetic tetraploids resulting in low male fertility

Interspecific hybridization and allopolyploidization contribute to the improvement of many important crops. Recently, we successfully developed an amphidiploid from an interspecific cross between cucumber(Cucumis sativus, 2n = 2x = 14) and its relative C. hystrix(2n = 2x = 24) followed by chemical induction of chromosome doubling. The resulting allotetraploid plant was self-pollinated for three generations. The fertility and seed set of the amphidiploid plants were very low. In this study, we investigated the meiotic chromosome behavior in pollen mother cells with the aid of fluorescence in situ hybridization, aiming to identify the reasons for the low fertility and seed set in the amphidiploid plants. Homologous chromosome pairing appeared normal, but chromosome laggards were common, owing primarily to asynchronous meiosis of chromosomes from the two donor genomes. We suggest that asynchronous meiotic rhythm between the two parental genomes is the main reason for the low fertility and low seed set of the C. hystrix–cucumber amphidiploid plants.

CENP-A Regulates Chromosome Segregation during the First Meiosis of Mouse Oocytes

Proper chromosome separation in both mitosis and meiosis depends on the correct connection between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy, thus kinetochores are critical for faithful segregation of chromosomes. Centromere protein A(CENP-A) is an important component of the inner kinetochore plate. Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores, leading to abnormal chromosome segregation, aneuploidy and apoptosis in cells. Here we report the expression and function of CENP-A during mouse oocyte meiosis. Our study found that microinjection of CENP-A blocking antibody resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs. Thus, our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis.

Double-stranded DNA breaks and gene functions in recombination and meiosis

Meiotic prophase I is a long and complex phase. Homologous recombination is an important process that occurs between homologous chromosomes during meiotic prophase I. Formation of chiasmata, which hold homologous chromosomes together until the metaphase I to anaphase I transition, is critical for proper chromosome segregation. Recent studies have suggested that the SPO 11 proteins have conserved functions in a number of organisms in generating sites of double-stranded DNA breaks （DSBs） that are thought to be the starting points of homologous recombination. Processing of these sites of DSBs requires the function of RecA homologs, such as RAD5 1, DMC 1, and others, as suggested by mutant studies; thus the failure to repair these meiotic DSBs results in abnormal chromosomal alternations, leading to disrupted meiosis. Recent discoveries on the functions of these RecA homologs have improved the understanding of the mechanisms underlying meiotic homologous recombination.

A Three-Dimensional (3D) Environment to Maintain the Integrity of Mouse Testicular Can Cause the Occurrence of Meiosis

Adhesions between different cells and extracellular matrix have been studied extensively in vitro, but little is known about their functions in testicular tissue counterparts. Spermatogonia and their companion somatic cells maintain a close association throughout spermatogenesis and this association is necessary for normal spermatogenesis. In order to keep the relative integrity of the testicular tissues, and to detect the development in vitro, culture testicular tissues in a three- dimensional （3D） agarose matrix was examined. Testicular tissues isolated from 6.5 d postpartum （dpp） mouse were cultured on the top of the matrix for 26 d with a medium height up to 4/5 of the 3D agarose matrix. The results showed that in this 3D culture environment, each type of testicular cells kept the same structure, localization and function as in vivo and might be more biologically relevant to living organisms. After culture, germ cell marker VASA and meiosis markers DAZL and SCP3 showed typical positive analysed by immunofluorescence staining and RT-PCR. It demonstrated that this 3D culture system was able to maintain the number of germ cells and promote the meiosis initiation of male germ cells.

Defective callose walls and cell plates during abnormal meiosis cause male-sterility in the oat mutant zbs1

During meiosis in flowering plants,degradation of the callose wall in tetrads releases newly produced microspores,which develop into mature pollen grains.In this study,we identified zbs1,a male-sterile mutant of naked oat（Avena nuda L.）that displayed complete spikelet sterility due to inviable mature pollen.The abnormal pollen grains originated from microspores with a defective callose wall and cell plate during meiosis.The defective callose wall and cell plate of the zbs1 mutant were detected by the labeling of cell wall epitopes（β-1,3-glucan） with immunogold during meiosis,and an abnormal chromosome configuration was observed by propidium iodide staining.The mature pollen grains of the zbs1 mutant were irregular in shape,and abnormal germination was observed by scanning electron microscopy.Together,our results indicate that the cause of male sterility in zbs1 is abnormal meiosis.

Alternative Splicing of OsRAD1 Defines C-Terminal Domain Essential for Protein Function in Meiosis

Alternative splicing can generate multiple mRNAs that differ in their untranslated regions or coding sequences,and these differences might affect mRNA stability or result in different protein isoforms with diverse functions and/or localizations.In this study,we isolated a sterile mutant in rice with abnormal meiosis of microspore mother cells and megaspore mother cells that carried a point mutation in OsRAD1 gene.Cloning of OsRAD1 cDNAs revealed three transcript variants,named as OsRAD1.1,OsRAD1.2 and OsRAD1.3,respectively,which were derived from alternative splicing of the last intron.Proteins derived from the three transcripts were mostly identical except the difference in the very C-terminal domain.The three transcripts exhibited similar expression patterns in various tissues,but the expression level of OsRAD1.1 was the highest.Specific knockout of OsRAD1.1 led to sterility,while knockout of OsRAD1.2 and OsRAD1.3 together did not change the plant fertility.Overexpression of OsRAD1.2 and OsRAD1.3 cDNAs in OsRAD1.1-specific mutant did not complement the plant fertility.Yeast two-hybrid assay showed that OsRAD1.1,but not OsRAD1.2 and OsRAD1.3,interacted with the three other meiosis proteins OsHUS1,OsRAD9 and OsRAD17,suggesting that the C-terminal domain of OsRAD1.1 is critical for the protein function.

Dynamic Changes of β Tubulin during the Resumption of Meiosis of Mouse Oocyte

Morphological changes of tubulin during the resumption of meiosis in both mouse oocyte and fertilized egg were revealed by indirect immunofluorescent marking with monoclonal antibody against β tubulin. During germinal vesicle period (GV), tubulin was found to be distributed around the GV menibrane. With the disruption of GV membrane, microtubule complexes (MTCs) appeared in cytoplasm, first around GV membrane later to spread to other portions as well. Quantitative difference was noted among different oocytes. MTCs coexisted with spindlesformed by prometaphase tubulin, while metaphase tubulin polymerized into spindles and anaphase and telophase tubulin was concentrated in the two poles of the meiotic apparatus and the midbody. In egg arrested in the 2nd metaphase, whether maturing in vitro or vivo, all the tubulin went to form spindles with no MTCs left in the cytoplasm. After fertilization in vitro,MTCs reappeared in the egg cytoplasm activated by sperms while no MTCs could be revealed in cytoplasm after formation of pronucleus. As demonstrated by this experiment,cytoplasmic tubulin in eggs are polymerized chiefly into two forms: the star-shaped MTCs and the spindle.Cytoplasmic MTCs are the structure newly formed when the 1st and 2nd oocytes resumed meiosis. With colchicine disrupting the polymerization of tubulin, the maternal chromosomes,instead of orderly arrangement and orderly separation, either formed disordered mass or were divided into multiple chromatin masses. However, the penetration of sperm into egg, and decondensation and formation of pronuclei were not affected.

Caseinolytic mitochondrial matrix peptidase X is essential for homologous chromosome synapsis and recombination during meiosis of male mouse germ cells

Meiosis is the process of producing haploid gametes through a series of complex chromosomal events and the coordinated action of various proteins.The mitochondrial protease complex(ClpXP),which consists of caseinolytic mitochondrial matrix peptidase X(ClpX)and caseinolytic protease P(ClpP)and mediates the degradation of misfolded,damaged,and oxidized proteins,is essential for maintaining mitochondrial homeostasis.ClpXP has been implicated in meiosis regulation,but its precise role is currently unknown.In this study,we engineered an inducible male germ cell-specific knockout caseinolytic mitochondrial matrix peptidase X(Clpx^(cKO))mouse model to investigate the function of ClpX in meiosis.We found that disrupting Clpx in male mice induced germ cell apoptosis and led to an absence of sperm in the epididymis.Specifically,it caused asynapsis of homologous chromosomes and impaired meiotic recombination,resulting in meiotic arrest in the zygotene-to-pachytene transition phase.The loss of ClpX compromised the double-strand break(DSB)repair machinery by markedly reducing the recruitment of DNA repair protein RAD51 homolog 1(RAD51)to DSB sites.This dysfunction may be due to an insufficient supply of energy from the aberrant mitochondria in Clpx^(cKO) spermatocytes,as discerned by electron microscopy.Furthermore,ubiquitination signals on chromosomes and the expression of oxidative phosphorylation subunits were both significantly attenuated in Clpx^(cKO) spermatocytes.Taken together,we propose that ClpX is essential for maintaining mitochondrial protein homeostasis and ensuring homologous chromosome pairing,synapsis,and recombination in spermatocytes during meiotic prophase I.

RETINOBLASTOMA RELATED 1 switches mitosis to meiosis in rice

The transition from mitosis to meiosis is a critical event in the reproductive development of all sexually reproducing species.However,the mechanisms that regulate this process in plants remain largely unknown.Here,we find that the rice(Oryza sativa L.)protein RETINOBLASTOMA RELATED 1(RBR1)is essential to the transition from mitosis to meiosis.Loss of RBR1 function results in hyper-proliferative sporogenous-celllike cells(SCLs)in the anther locules during early stages of reproductive development.These hyperproliferative SCLs are unable to initiate meiosis,eventually stagnating and degrading at late developmental stages to form pollen-free anthers.These results suggest that RBR1 acts as a gatekeeper of entry into meiosis.Furthermore,cytokinin content is significantly increased in rbr1 mutants,whereas the expression of type-B response factors,particularly LEPTO1,is significantly reduced.Given the known close association of cytokinins with cell proliferation,these findings imply that hyper-proliferative germ cells in the anther locules may be attributed to elevated cytokinin concentrations and disruptions in the cytokinin pathway.Using a genetic strategy,the association between germ cell hyper-proliferation and disturbed cytokinin signaling in rbr1 has been confirmed.In summary,we reveal a unique role of RBR1 in the initiation of meiosis;our results clearly demonstrate that the RBR1 regulatory module is connected to the cytokinin signaling pathway and switches mitosis to meiosis in rice.

foxl2l is a germ cell-intrinsic gatekeeper of oogenesis in zebrafish

Zebrafish serve as a valuable model organism for studying germ cell biology and reproductive processes.The AB strain of zebrafish is proposed to exhibit a polygenic sex determination system,where most males initially develop juvenile ovaries before committing to male fate.In species with chromosomal sex determination,gonadal somatic cells are recognized as key determinants of germ cell fate.Notably,the loss of germ cells in zebrafish leads to masculinization,implying that germ cells harbor an intrinsic feminization signal.However,the specific signal triggering oogenesis in zebrafish remains unclear.In the present study,we identified foxl2l as an oocyte progenitor-specific gene essential for initiating oogenesis in germ cells.Results showed that foxl2l-knockout zebrafish bypassed the juvenile ovary stage and exclusively developed into fertile males.Further analysis revealed that loss of foxl2l hindered the initiation of oocyte-specific meiosis and prevented entry into oogenesis,leading to premature spermatogenesis during early gonadal development.Furthermore,while mutation of the pro-male gene dmrt1 led to fertile female differentiation,simultaneous disruption of foxl2l in dmrt1 mutants completely blocked oogenesis,with a large proportion of germ cells arrested as germline stem cells,highlighting the crucial role of foxl2l in oogenesis.Overall,this study highlights the unique function of foxl2l as a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.

Meiotic transcriptional reprogramming mediated by cell-cell communications in humans and mice revealed by scATACseq and scRNA-seq

Meiosis is a highly complex process significantly influenced by transcriptional regulation.However,studies on the mechanisms that govern transcriptomic changes during meiosis,especially in prophase I,are limited.Here,we performed single-cell ATAC-seq of human testis tissues and observed reprogramming during the transition from zygotene to pachytene spermatocytes.This event,conserved in mice,involved the deactivation of genes associated with meiosis after reprogramming and the activation of those related to spermatogenesis before their functional onset.Furthermore,we identified 282 transcriptional regulators(TRs)that underwent activation or deactivation subsequent to this process.Evidence suggested that physical contact signals from Sertoli cells may regulate these TRs in spermatocytes,while secreted ENHO signals may alter metabolic patterns in these cells.Our results further indicated that defective transcriptional reprogramming may be associated with non-obstructive azoospermia(NOA).This study revealed the importance of both physical contact and secreted signals between Sertoli cells and germ cells in meiotic progression.

High temperature treatment induced production of unreduced 2n pollen in Camellia oleifera

Unreduced gametes through chromosome doubling play a major role in the process of plant polyploidization.Our previous work confirmed that Camellia oleifera can produce natural 2n pollen,and it is possible to induce the 2n pollen formation by high temperature treatment.This study focused on the optimization of the 2n pollen induction technique and the mechanisms of high temperature-induced2n pollen formation in C.oleifera.We found that the optimal protocol for inducing 2n pollen via high temperature was to perform 45℃with4 h at the prophaseⅠstage of the pollen mother cells(PMCs).Meanwhile,high temperature significantly decreased the yield and fertility of2n pollen.Through the observation of meiosis,abnormal chromosome and cytological behaviour was discovered under high-temperature treatment,and we confirmed that the formation of 2n pollen is caused by abnormal cell plate.Based on weighted gene co-expression network analysis,fifteen hub genes related to cell cycle control were identified.After male flower buds were exposed to heat shock,polygalacturonase gene(CoPGX3)was significantly upregulated.We inferred that high temperature causes the CoPGX3 gene to be overexpressed and that CoPGX3 is redistributed into the cytosol where it degrades cytoplasmic pectin,which leads to an abnormal cell plate.Furthermore,abnormal cytokinesis resulted in the formation of dyads and triads,and PMCs divided to produce 2n pollen.Our findings provide new insights into the mechanism of 2n pollen induced by high temperature in a woody plant and lay a foundation for further ploidy breeding of C.oleifera.

Cell Biological Characterization of Male Meiosis and Pollen Development in Rice

Abstract: Little systematic analysis has been undertaken in rice (Oryza sativa L.) on the stages of male meiosis from leptotene to telophase II or of pollen development from microspores to mature pollen grains. The present study describes multiple stages in detail from analysis of rice chromosome spreading with staining of 4′,6-diamidino-2-phenylindole. The description of normal wild-type male meiosis provides an important morphological reference for analyses of meiotic mutants. Meiosis in rice is largely similar to those of the well characterizing model plants Arabidopsis thaliana L. and Zea mays L. However, rice meiosis differs from that in Arabidopsis in that rice meiosis I is followed by the formation of a cell plate, instead of an organelle band that forms between the two nuclei and persist through meiosis II. This suggests a difference in the control of organelle biogenesis and distribution and cytokinesis. Our results should facilitate studies of rice meiosis and pollen development using molecular genetic and cell biological approaches.

Roles of MAP kinase signaling pathway in oocyte meiosis

Mitogen-activated protein kinase (MAPK) is a family of Ser/Thr protein kinases expressed widely in eu-karyotic cells. MAPK is activated by a cascade of protein kinase phosphorylation and plays pivotal roles in regulating meiosis process in oocytes. As an important physical substrate of MAPK, p90rsk mediates numerous MAPK functions. MAPK was activated at G2/M transition during meiosis. Its activity reached the peak at M I stage and maintained at this level until the time before the pronuclear formation after fertilization. There is complex interplay between MAPK and MPF in the meiosis regulation. Furthermore, other intracel-lular signal transducers, such as cAMP, protein kinase C and protein phosphotase, ect., also regulated the activity of MAPK at different stages during meiosis in oocytes. In the present article, the roles of MAPK signaling pathway in oo-cyte meiosis are reviewed and discussed.