The DNA mismatch repair protein Msh4 is essential for meiosis of male but not for female in zebrafish.

A Role for SUMO in Meiotic Chromosome Synapsis

Accurate segregation of homologous chromosomes during meiosis I (MI) is essential for haploid gamete production. This review systematically summarizes the dynamic regulatory mechanisms governing chromosome segregation in male mammalian meiosis. During prophase I, crossover-dependent connections are established through DNA double-strand break repair, synaptonemal complex assembly, and sister chromatid cohesion—prerequisites for stable bivalent formation. At metaphase I, the spindle assembly checkpoint monitors kinetochore-microtubule attachments, while Aurora B kinase and the chromosomal passenger complex regulate attachment stability and error correction. Polo-like kinase 1 (PLK1) coordinates spindle assembly, whereas the anaphase-promoting complex/cyclosome drives the metaphase-to-anaphase transition, through targeted degradation of securin and cyclin B1. The Rec8-containing cohesin complex resists separase-mediated cleavage until anaphase I, thereby preserving sister chromatid cohesion until meiosis II. However, current knowledge of the regulatory mechanisms governing homologous chromosome segregation during male meiosis remains fragmented and has not been systematically integrated. Therefore, this review compares the mechanisms of chromosome segregation in male meiosis with those in mitosis and female meiosis in order to highlight both shared features and male-specific molecular mechanisms, and provides a systematic framework for understanding the separation of homologous chromosomes during male meiosis I.

The mechanism of action of the topoisomerase II inhibitor etoposide (VP-16) was investigated in male mouse meiosis using the spermatid micronucleus (MN) test and two molecular cytogenetic approaches: (i) fluorescence in situ hybridization (FISH) with a mouse centromere specific minor satellite DNA probe; and (ii) immunolabelling of kinetochore proteins with CREST autoimmune serum. VP-16 caused significant increases in the frequencies of MN at all meiotic stages studied. VP-16 induced MN showed significantly elevated frequencies of centromeric hybridization signals compared to the controls. Similarly, after CREST immunostaining the majority of MN induced by the drug showed kinetochore signals when meiotic S phase and diplotene-diakinesis were treated. This would suggest that most induced MN were due to lagging of whole chromosomes. However, more than 80% of the small MN observed were signal-positive and a large pool of minute MN almost exclusively (92%) contained a kinetochore or centromere-DNA signal. This indicates that VP-16 causes chromosome fragmentation at centromeres. In addition, arrested first division (MI) anaphase figures with stretched bivalent(s) at the spindle equator were observed when diplotene-diakinesis and MI were targeted. Moreover, many small and medium size MN had two centromere or kinetochore signals at opposite sides, suggesting that inhibition of topo II at MI causes lagging of whole bivalents. Together, these results indicate that VP-16 acts by several genotoxic mechanisms at male meiosis: (i) fragmentation of centromeres possibly as a result of inhibition of the DNA strand religation reaction in a topoisomerase II mediated decatenation process of sister centromeres; and (ii) the induction of aneuploidy as a result of failures in separation of homologous chromosome arms possibly due to disturbances of chiasma resolution and decatenation processes during MI. Our results indirectly suggest that topoisomerase II plays an important role in male meiosis and its activity is needed at the metaphase-anaphase transition of both meiotic divisions for proper chromosome disjunction.

ABSTRACTIn higher plants, male meiosis is a key process of microsporogenesis and is crucial for plant fertility. Male meiosis programs are prone to be influenced by altered temperature conditions. Studies have reported that an increased temperature (28°C) within a fertile threshold can affect the frequency of meiotic recombination in Arabidopsis. However, not much has been known how male meiosis responses to an extremely high temperature beyond the fertile threshold. To understand the impact of extremely high temperature on male meiosis in Arabidopsis, we treated flowering Arabidopsis plants with 36-38°C and found that the high-temperature condition significantly reduced pollen shed and plant fertility, and led to formation of pollen grains with varied sizes. The heat stress-induced unbalanced tetrads, polyad and meiotic restitution, suggesting that male meiosis was interfered. Fluorescence in situ hybridization (FISH) assay confirmed that both homologous chromosome separation and sister chromatids cohesion were influenced. Aniline blue staining of tetrad-stage pollen mother cells (PMCs) revealed that meiotic cytokinesis was severely disrupted by the heat stress. Supportively, immunolocalization of ɑ-tubulin showed that the construction of spindle and phragmoplast at both meiosis I and II were interfered. Overall, our findings demonstrate that an extremely high-temperature stress over the fertile threshold affects both chromosome segregation and cytokinesis during male meiosis by disturbing microtubular cytoskeleton in Arabidopsis.

Sister chromatid cohesion is maintained from DNA replication to metaphase-to-anaphase transition by multisubunit protein complexes called cohesin, which include at least four proteins, SMC1alpha, SMC3, Rad21 and either SA1 or SA2, in mammalian somatic cells. We report here the first evidence of the involvement of Rec8 protein, a mammalian homolog of yeast Rec8p, in meiosis-specific chromosome behavior in mammals. In immunoblotting and immunohistochemical analysis using specific antibodies against mouse Rec8, we found that Rec8 was expressed in the testis but not in the kidney or liver; more precisely, it was expressed in spermatocytes and spermatids but not in spermatogonia or other somatic cells. We also found that Rec8 is present in both phosphorylated and dephosphorylated states in vivo. Immunoprecipitation analyses revealed that Rec8 associates with other cohesin proteins, SMC1beta (meiosis-specific protein) and SMC3 and with a component of synaptonemal complexes, SCP3, but not with SMC1alpha. In meiotic chromosome spreads, Rec8 was localized along the axial/lateral elements of the synaptonemal complexes in meiotic prophase from the leptotene to diplotene stages. At later stages, diakinesis and metaphase I, Rec8 was localized along the interstitial axes of chromosomes, including both centromere and arm regions of chromosomes. However, concomitantly with separation of homologous chromosomes in anaphase I, Rec8 was no longer detected along the arm regions, while it persisted on centromere regions up to metaphase II. In anaphase II, the centromeric signals were diminished. We propose from these results that mammalian Rec8 protein, in association with SMC3 and SMC1beta but not SMC1alpha, is involved in meiosis-specific chromosome behavior, and that homologous chromosome separation is triggered by selective loss of Rec8 from chromosome arms in meiosis I, while sister chromatid cohesion is maintained until metaphase II/anaphase II transition by centromeric Rec8 during mammalian meiosis.

The XX/XY sex chromosomal system of mammals, including human, challenges the chromosome pairing mechanism during male meiosis. Pairing and subsequent separation of homologous chromosomes generates haploid cells from diploid cells during the meiotic divisions. One of the basic requirements for recognition between homologous chromosomes is DNA sequence identity. Since the X and Y chromosome share little homology, their quest for each other is difficult, and has special characteristics. During the lengthy meiotic prophase, all autosomal chromosomes synapse, by forming a special protein structure called the synaptonemal complex, which connects the chromosomal axes. In contrast, the X and Y chromosome synapse only in the short homologous pseudoautosomal regions, and form the so-called XY body.1 In this specialized chromatin area, transcription is shut down by a mechanism named meiotic sex chromosome inactivation (MSCI) (reviewed by Refs. 2 and 3). The sex chromosomes remain silent throughout the rest of meiotic prophase, and only few sex-linked genes are (re)activated in postmeiotic spermatids.2 Since long, scientists have wondered how MSCI is achieved, and what its biological significance is. Recently, two papers4, 5 significantly increased our understanding of how and why MSCI works. So far, the only molecule that was known to be absolutely essential for initiation of MSCI was γH2AX.6 This phosphorylated form of histone H2AX marks the XY body from its formation until diplotene in mouse.7 In mice lacking H2AX, no XY body is formed, the sex chromosomes are not silenced and meiosis arrests at pachytene.6 The phosphorylation of H2AX at the XY body is mediated by the checkpoint kinase ATR that can be visualized along the unsynapsed axes of X and Y from late zygotene onwards.8 In their recent paper, Ichijima et al.4 reveal that the phosphorylation of H2AX occurs in two phases; first, it is restricted to the chromosomal axes, then the area extends, and H2AX also becomes phosphorylated in the surrounding chromatin loops. This second phase, when γH2AX spreads, depends on a protein named mediator of DNA damage checkpoint 1 (MDC1), and this function is required for transcriptional silencing of the sex chromosomes (Figure 1). MDC1 is a known binding partner of γH2AX,9, 10 and both proteins, as well as ATR, are well known for their role in the DNA damage response pathway in somatic cells. This pathway plays a pivotal role in sensing the presence of single-stranded DNA at stalled replication fork to allow repair as well as activation of cell cycle checkpoints. Interestingly, the authors also analyzed the role of MDC1 in somatic cells. In analogy to their observations in meiotic cells, they observed reduced amplification of ATR-dependent γH2AX signals after replicative stress in the absence of MDC1, compared to control cells. They also showed that RNA polymerase II is excluded from chromatin at replication-stalled sites that are marked by ATR- and MDC1-dependent γH2AX accumulation, thereby confirming and extending the observations described by Shanbag et al.,11 who discovered that DNA double-strand breaks (DSBs) elicit transcriptional silencing in cis. These data reveal an analogy between somatic and meiotic cells, with respect to the link between DNA repair and DNA damage response protein accumulation (at respectively DNA damage sites and unsynapsed axes), and transcriptional silencing. It should be noted that 200–400 enzymatically generated DSBs are induced at the onset of meiotic prophase, to mediate homology recognition and meiotic recombination. Meiotic DSBs are also induced in the non-homologous regions of X and Y, and DSB repair molecules are present on the unsynapsed X chromosomal axis.12 Together with the observation that meiotic silencing is impaired when repair of meiotic DSBs is hampered,13 this indicates that MSCI is also mechanistically related to DSB repair and the DNA damage response pathway in somatic cells. Figure 1 Zoom in on sex chromosomes during pachytene. Schematic representation of spermatogenesis, showing the different substages of meiotic prophase (leptotene, zygotene, pachytene and diplotene). During meiotic prophase, progression of chromosome pairing and ... In yeast, a pachytene checkpoint operates to block development when meiotic recombination or synapsis has failed. In mouse, there also is a very strong pachytene arrest during male meiosis whenever synapsis and DSB repair are affected.2 In addition, MSCI fails in such situations.13 A recent series of experiments by Royo et al.5 has shown that pachytene arrest in male mice can be induced by inappropriate expression of sex-linked genes, and they identified a single Y-linked gene, named Zfy, whose expression is sufficient to induce apoptosis of pachytene spermatocytes. Thus, in the Mdc1 knockout mice analyzed by Ichijima et al.,4 it is to be expected that the failure to achieve MSCI also explains the strict pachytene arrest that is observed in these mice (Figure 1). Whether the presence of autosomal asynapsed chromatin and/or persistent DNA damage can also trigger activation of a pachytene checkpoint is not clear. In mouse oocytes with an impaired meiotic recombination machinery, checkpoint activation is not very efficient, leading to the formation of many aneuploid oocytes and early embryo loss upon fertilization of such oocytes.14 Thus, although the presence of heteromorphic sex chromosomes could be viewed as a problem for the male, it seems to have evolved into a blessing in disguise by providing a very efficient means to avoid the generation of aberrant sperm. In this context, it is also relevant to mention that the same gene (Zfy2) that needs to be repressed by MSCI to prevent an apoptotic response during pachytene, is somehow required to mediate apoptotic removal of cells with misaligned sex chromosomes during the first meiotic metaphase.15 This provides another male advantage to the fidelity of meiosis. Meiosis is a tricky business; no other cell generates hundreds of DSBs on purpose, and their complete repair is required to generate gametes with an intact genome. As a back-up, unrepaired damage present in postmeiotic cells may be repaired during spermiogenesis or in the zygote,16 but such repair would be associated with an increased risk of generating mutations. Human spermatocytes also contain a silenced XY body, and meiotic arrest is a frequent cause of infertility. To be able to asses whether meiotic defects could be associated with an increased risk to transmit mutations or chromosome aberrations via intacytoplasmatic sperm injection, it will be important to determine the quality of the pachytene checkpoint/arrest in human males. In a case report of a patient with an impairment of meiotic DSB repair, cell degeneration was reported to occur both during meiotic prophase and during meiotic divisions.17 X-autosome or autosome-to-autosome translocations that might be associated with impaired MSCI are usually associated with infertility, but sperm cells for intacytoplasmatic sperm injection could be obtained in at least one case, leading to transmission of the translocation.18 Solari and Rey Valzacchi19 have analyzed spermatocytes in an XYY human male. Since YY synapsis is observed in almost half of these cells, it would be expected that this would lead to a failure to induce MSCI, which should be associated with cell death during pachytene, as was observed in mouse XYY cells. However, Solari and Rey Valzacchi19 observe more prominent loss of XYY germ cells during metaphase I, indicating that the pachytene arrest that should be elicited by defective MSCI may be weaker in man compared to mouse. This is in accordance with data from microarray analyses of testicular gene expression in man and chimpanzee, that indicate that human MSCI may be less efficient or complete.20 Future in-depth analyses of meiotic progression in human spermatocytes from infertile patients should reveal whether MSCI in man also acts to safeguard the male germline against transmitting a damaged or incomplete genome.

Immunofluorescence staining with an antiserum raised against a presumptive meiotic histone, which has been shown to appear prior to male meiosis in liliaceous plants, preferentially stained the centromere (kinetochore) region of meiotic chromosomes in microsporocytes and megasporocytes. Using this antiserum, we were able clearly to visualize the centromeres at all important meiotic stages in microsporocytes, namely, the association and fusion of centromeres of homologous chromosomes at zygotene-pachytene in prophase I, the disjunction of the homologous centromeres at diplotene, the doubling of each centromere at metaphase I and nonseparation of the sister centromeres at anaphase I, by confocal laser scanning microscopy. Thus, this report provides a complete picture of the behavior of centromeres during meiosis in a eukaryote for the first time. This antiserum also decorated centromeres during female meiosis in cryo-sectioned megasporocytes, but did not stain the centromeres of mitotic chromosomes in root-tip meristem. From these observations, it is suggested that a meiosis-specific centromere protein is required for the meiosis-specific behavior of the centromere.

• Mutations in the breast cancer susceptibility gene 2 (BRCA2) are correlated with hereditary breast cancer in humans. Studies have revealed that mammalian BRCA2 plays crucial roles in DNA repair. Therefore, we wished to define the role of the BRCA2 homologs in Arabidopsis in detail. • As Arabidopsis contains two functional BRCA2 homologs, an Atbrca2 double mutant was generated and analyzed with respect to hypersensitivity to genotoxic agents and recombination frequencies. Cytological studies addressing male and female meiosis were also conducted, and immunolocalization was performed in male meiotic prophase I. • The Atbrca2 double mutant showed hypersensitivity to the cross-linking agent mitomycin C and displayed a dramatic reduction in somatic homologous recombination frequency, especially after double-strand break induction. The loss of AtBRCA2 also led to severe defects in male meiosis and development of the female gametophyte and impeded proper localization of the synaptonemal complex protein AtZYP1 and the recombinases AtRAD51 and AtDMC1. • The results demonstrate that AtBRCA2 is important for both somatic and meiotic homologous recombination. We further show that AtBRCA2 is required for proper meiotic synapsis and mediates the recruitment of AtRAD51 and AtDMC1. Our results suggest that BRCA2 controls single-strand invasion steps during homologous recombination in plants.

Kinetochore Orientation during Meiosis Is Controlled by Aurora B and the Monopolin Complex

Detailed cytological investigations performed in Anemone tetrasepala Royle from the Pangi Valley revealed the presence of various irregularities (spindle irregularities, chromatin transfer, chromatin stickiness, and nonsynchronous condensation of chromatin material) for the first time in the species that existed at diploid level (2n = 14). Spindle irregularities resulted in dyads, triads, polyads, and micronuclei in sporads during microsporogenesis and variable sized pollen grains and reduced fertility in pollen grains (85.38%). Direct fusion among proximate meiocytes resulted in the formation of syncyte pollen mother cells. Such syncytes yielded jumbo-sized pollen grains that were surely of unreduced nature in their genetic constitution. Due to severe chromosome stickiness, chromosome separation during anaphases was also affected. Another interesting observation was the nonsynchronous condensation of 1-2 chromosomes during anaphase-I. This paper discusses the consequences of chromosomal and spindle irregularities on the course of meiosis and microsporogenesis and ultimately on the end products.

Changes in environmental temperature affect multiple meiotic processes in flowering plants. Polyploid plants derived from whole-genome duplication (WGD) have enhanced genetic plasticity and tolerance to environmental stress but face challenges in organizing and segregating doubled chromosome sets. In this study, we investigated the impact of increased environmental temperature on male meiosis in autotetraploid Arabidopsis (Arabidopsis thaliana). Under low to mildly increased temperatures (5°C-28°C), irregular chromosome segregation universally occurred in synthetic autotetraploid Columbia-0 (Col-0). Similar meiotic lesions occurred in autotetraploid rice (Oryza sativa L.) and allotetraploid canola (Brassica napus cv Westar), but not in evolutionarily derived hexaploid wheat (Triticum aestivum). At extremely high temperatures, chromosome separation and tetrad formation became severely disordered due to univalent formation caused by the suppression of crossing-over. We found a strong correlation between tetravalent formation and successful chromosome pairing, both of which were negatively correlated with temperature elevation, suggesting that increased temperature interferes with crossing-over predominantly by impacting homolog pairing. We also showed that loading irregularities of axis proteins ASY1 and ASY4 co-localize on the chromosomes of the syn1 mutant and the heat-stressed diploid and autotetraploid Col-0, revealing that heat stress affects the lateral region of synaptonemal complex (SC) by impacting the stability of the chromosome axis. Moreover, we showed that chromosome axis and SC in autotetraploid Col-0 are more sensitive to increased temperature than those in diploid Arabidopsis. Taken together, our data provide evidence suggesting that WGD negatively affects the stability and thermal tolerance of meiotic recombination in newly synthetic autotetraploid Arabidopsis.

Accurate positioning of spindles is a critical aspect of cell division as it ensures that each daughter cell contains a single nucleus. In many flowering plants, two meiotic chromosome separations occur without intervening cytokinesis, resulting in two spindles in one cell during the second division. Here we report a detailed examination of two mutants, jason (jas) and parallel spindle1 (ps1), in which disturbed spindle position during male meiosis II results in the incorporation of previously separated chromosome groups into a single cell. Our study reveals that an organelle band provides a physical barrier between the two spindles. The loss of a single protein, JAS, from this organelle band leads to its disruption and a random movement of the spindles. JAS is largely associated with vesicles in the organelle band, revealing a role for vesicles in plant meiosis and that cytoplasmic events maintain spindle position during the chromosome division.

Homeostasis of meiotic DNA double strand breaks (DSB) is critical for germline genome integrity and homologous recombination. Here we demonstrate an essential role for SKP1, a constitutive subunit of the SCF (SKP1-Cullin-F-box) ubiquitin E3 ligase, in early meiotic processes. SKP1 restrains accumulation of HORMAD1 and the pre-DSB complex (IHO1-REC114-MEI4) on the chromosome axis in meiotic germ cells. Loss of SKP1 prior to meiosis leads to aberrant localization of DSB repair proteins and a failure in synapsis initiation in meiosis of both males and females. Furthermore, SKP1 is crucial for sister chromatid cohesion during the pre-meiotic S-phase. Mechanistically, FBXO47, a meiosis-specific F-box protein, interacts with SKP1 and HORMAD1 and targets HORMAD1 for polyubiquitination and degradation in HEK293T cells. Our results support a model wherein the SCF ubiquitin E3 ligase prevents hyperactive DSB formation through proteasome-mediated degradation of HORMAD1 and subsequent modulation of the pre-DSB complex during meiosis.

Alterations in the DNA mismatch repair (MMR) proteins have been associated with an increased resistance of many cancer cell lines to cisplatin. The aim of this work was to investigate whether defects in DNA MMR proteins are involved in the survival of human colorectal cancer cells in the presence of high concentrations of cisplatin and oxaliplatin, a diaminocyclohexane (DACH) platinum compound whose adducts are not recognized by the MMR system. Six unselected human colon cancer cell lines (HT29, HCT15, HCT116, Caco2, SW480 and SW620) were treated with a single 3-h exposure to cisplatin or oxaliplatin at suprapharmacological concentrations, ranging from 50 to 200 microg/ml. The microsatellite stability and the expression of MMR proteins in the parental cell lines and in the drug-selected subpopulations were studied. Most cells underwent apoptosis in the days following the cisplatin or oxaliplatin treatment, but some colonies expanded 3 to 4 weeks after, suggesting the presence of innately resistant cells in the six parental cell lines. Microsatellite instability (MIN), which reflects genetic defects in the DNA MMR system, was detected only in the HCT116 parental cell line and its drug-selected counterparts, due to a known mutation in the hMLH1 gene. No acquired MIN was observed in the other cisplatin-selected sublines derived from the HT29, HCT15, Caco2, SW480 or SW620 parental cells. In the same way, Western blot analysis showed that expression of the DNA MMR proteins hMLH1, hPMS1, hPMS2, hMSH2 and hMSH6 did not differ between the parental and the drug-surviving cells. These results indicate that high-level resistance of human colon cancer cells to high doses of cisplatin and oxaliplatin does not seem to be related to acquired defects in the DNA MMR proteins.

Proper segregation of homologous chromosomes in meiosis I is ensured by pairing of homologs and maintenance of sister chromatid cohesion. In male Drosophila melanogaster, meiosis is achiasmatic and homologs pair at limited chromosome regions called pairing sites. We screened for male meiotic mutants to identify genes required for normal pairing and disjunction of homologs. Nondisjunction of the sex and the fourth chromosomes in male meiosis was scored as a mutant phenotype. We screened 2306 mutagenized and 226 natural population-derived second and third chromosomes and obtained seven mutants representing different loci on the second chromosome and one on the third. Five mutants showed relatively mild effects (<10% nondisjunction). mei(2)yh149 and mei(2)yoh7134 affected both the sex and the fourth chromosomes, mei(2)yh217 produced possible sex chromosome-specific nondisjunction, and mei(2)yh15 and mei(2)yh137 produced fourth chromosome-specific nondisjunction. mei(2)yh137 was allelic to the teflon gene required for autosomal pairing. Three mutants exhibited severe defects, producing >10% nondisjunction of the sex and/or the fourth chromosomes. mei(2)ys91 (a new allele of the orientation disruptor gene) and mei(3)M20 induced precocious separation of sister chromatids as early as prometa-phase I. mei(2)yh92 predominantly induced nondisjunction at meiosis I that appeared to be the consequence of failure of the separation of paired homologous chromosomes.