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Sex chromosomes of eutherian mammals are highly different in size and gene content, and share only a small region of homology (pseudoautosomal region, PAR). They are thought to have evolved through an addition-attrition cycle involving the addition of autosomal segments to sex chromosomes and their subsequent differentiation. The events that drive this process are difficult to investigate because sex chromosomes in almost all mammals are at a very advanced stage of differentiation. Here, we have taken advantage of a recent translocation of an autosome to both sex chromosomes in the African pygmy mouse Mus minutoides, which has restored a large segment of homology (neo-PAR). By studying meiotic sex chromosome behavior and identifying fully sex-linked genetic markers in the neo-PAR, we demonstrate that this region shows unequivocal signs of early sex-differentiation. First, synapsis and resolution of DNA damage intermediates are delayed in the neo-PAR during meiosis. Second, recombination is suppressed or largely reduced in a large portion of the neo-PAR. However, the inactivation process that characterizes sex chromosomes during meiosis does not extend to this region. Finally, the sex chromosomes show a dual mechanism of association at metaphase-I that involves the formation of a chiasma in the neo-PAR and the preservation of an ancestral achiasmate mode of association in the non-homologous segments. We show that the study of meiosis is crucial to apprehend the onset of sex chromosome differentiation, as it introduces structural and functional constrains to sex chromosome evolution. Synapsis and DNA repair dynamics are the first processes affected in the incipient differentiation of X and Y chromosomes, and they may be involved in accelerating their evolution. This provides one of the very first reports of early steps in neo-sex chromosome differentiation in mammals, and for the first time a cellular framework for the addition-attrition model of sex chromosome evolution., Author summary Sex chromosomes seem to evolve and differentiate at different rates in different taxa. The reasons for this variability are still debated. It is well established that recombination suppression around the sex-determining region triggers differentiation, and several studies have investigated this process from a genetic point of view. However, the cellular context in which recombination arrest occurs has received little attention so far. In this report, we show that meiosis, the cellular division in which pairing and recombination between chromosomes takes place, can affect the incipient differentiation of X and Y chromosomes. Combining cytogenetic and genomic approaches, we found that in the African pygmy mouse Mus minutoides, which has recently undergone sex chromosome-autosome fusions, synapsis and DNA repair dynamics are disturbed along the newly added region of the sex chromosomes. We argue that these alterations are a by-product of the fusion itself, and cause recombination suppression across a large region of the neo-sex chromosome pair. Therefore, we propose that the meiotic context in which sex or neo-sex chromosomes arise is crucial to understand the very early stages of their differentiation, as it could promote or hinder recombination suppression, and therefore impact the rate at which these chromosomes differentiate.

Natural populations of the house mouse Mus musculus domesticus show great diversity in chromosomal number due to the presence of chromosomal rearrangements, mainly Robertsonian translocations. Breeding between two populations with different chromosomal configurations generates subfertile or sterile hybrid individuals due to impaired meiotic development. In this study, we have analyzed prophase-I spermatocytes of hybrids formed by crossing mice from Vulcano and Lipari island populations. Both populations have a 2n = 26 karyotype but different combinations of Robertsonian translocations. We studied the progress of synapsis, recombination, and meiotic silencing of unsynapsed chromosomes during prophase-I through the immunolocalization of the proteins SYCP3, SYCP1, γH2AX, RAD51, and MLH1. In these hybrids, a hexavalent is formed that, depending on the degree of synapsis between chromosomes, can adopt an open chain, a ring, or a closed configuration. The frequency of these configurations varies throughout meiosis, with the maximum degree of synapsis occurring at mid pachytene. In addition, we observed the appearance of heterologous synapsis between telocentric and metacentric chromosomes; however, this synapsis seems to be transient and unstable and unsynapsed regions are frequently observed in mid-late pachytene. Interestingly, we found that chiasmata are frequently located at the boundaries of unsynapsed chromosomal regions in the hexavalent during late pachytene. These results provide new clues about synapsis dynamics during meiosis. We propose that mechanical forces generated along chromosomes may induce premature desynapsis, which, in turn, might be counteracted by the location of chiasmata. Despite these and additional meiotic features, such as the accumulation of γH2AX on unsynapsed chromosome regions, we observed a large number of cells that progressed to late stages of prophase-I, indicating that synapsis defects may not trigger a meiotic crisis in these hybrids.

Meiotic recombination occurs as a programmed event that initiates by the formation of DNA double-strand breaks (DSBs) that give rise to the formation of crossovers that are observed as chiasmata. Chiasmata are essential for the accurate chromosome segregation and the generation of new combinations of parental alleles. Some treatments that provoke exogenous DSBs also lead to alterations in the recombination pattern of some species in which full homologous synapsis is achieved at pachytene. We have carried out a similar approach in males of the grasshopper Stethophyma grossum, whose homologues show incomplete synapsis and proximal chiasma localization. After irradiating males with γ rays we have studied the distribution of both the histone variant γ-H2AX and the recombinase RAD51. These proteins are cytological markers of DSBs at early prophase I. We have inferred synaptonemal complex (SC) formation via identification of SMC3 and RAD 21 cohesin subunits. Whereas thick and thin SMC3 filaments would correspond to synapsed and unsynapsed regions, the presence of RAD21 is only restricted to synapsed regions. Results show that irradiated spermatocytes maintain restricted synapsis between homologues. However, the frequency and distribution of chiasmata in metaphase I bivalents is slightly changed and quadrivalents were also observed. These results could be related to the singular nuclear polarization displayed by the spermatocytes of this species.

Telomeric DNA repeats are key features of chromosomes that allow the maintenance of integrity and stability in the telomeres. However, interstitial telomere sites (ITSs) can also be found along the chromosomes, especially near the centromere, where they may appear following chromosomal rearrangements like Robertsonian translocations. There is no defined role for ITSs, but they are linked to DNA damage-prone sites. We were interested in studying the structural organization of ITSs during meiosis, a kind of cell division in which programmed DNA damage events and noticeable chromatin reorganizations occur. Here we describe the presence of highly amplified ITSs in the pericentromeric region of Mongolian gerbil (Meriones unguiculatus) chromosomes. During meiosis, ITSs show a different chromatin conformation than DNA repeats at telomeres, appearing more extended and accumulating heterochromatin markers. Interestingly, ITSs also recruit the telomeric proteins RAP1 and TRF1, but in a stage-dependent manner, appearing mainly at late prophase I stages. We did not find a specific accumulation of DNA repair factors to the ITSs, such as γH2AX or RAD51 at these stages, but we could detect the presence of MLH1, a marker for reciprocal recombination. However, contrary to previous reports, we did not find a specific accumulation of crossovers at ITSs. Intriguingly, some centromeric regions of metacentric chromosomes may bind the nuclear envelope through the association to SUN1 protein, a feature usually performed by telomeres. Therefore, ITSs present a particular and dynamic chromatin configuration in meiosis, which could be involved in maintaining their genetic stability, but they additionally retain some features of distal telomeres, provided by their capability to associate to telomere-binding proteins.

Meiosis is a complex type of cell division that involves homologous chromosome pairing, synapsis, recombination, and segregation. When any of these processes is altered, cellular checkpoints arrest meiosis progression and induce cell elimination. Meiotic impairment is particularly frequent in organisms bearing chromosomal translocations. When chromosomal translocations appear in heterozygosis, the chromosomes involved may not correctly complete synapsis, recombination, and/or segregation, thus promoting the activation of checkpoints that lead to the death of the meiocytes. In mammals and other organisms, the unsynapsed chromosomal regions are subject to a process called meiotic silencing of unsynapsed chromatin (MSUC). Different degrees of asynapsis could contribute to disturb the normal loading of MSUC proteins, interfering with autosome and sex chromosome gene expression and triggering a massive pachytene cell death. We report that in mice that are heterozygous for eight multiple simple Robertsonian translocations, most pachytene spermatocytes bear trivalents with unsynapsed regions that incorporate, in a stage-dependent manner, proteins involved in MSUC (e.g., γH2AX, ATR, ubiquitinated-H2A, SUMO-1, and XMR). These spermatocytes have a correct MSUC response and are not eliminated during pachytene and most of them proceed into diplotene. However, we found a high incidence of apoptotic spermatocytes at the metaphase stage. These results suggest that in Robertsonian heterozygous mice synapsis defects on most pachytene cells do not trigger a prophase-I checkpoint. Instead, meiotic impairment seems to mainly rely on the action of a checkpoint acting at the metaphase stage. We propose that a low stringency of the pachytene checkpoint could help to increase the chances that spermatocytes with synaptic defects will complete meiotic divisions and differentiate into viable gametes. This scenario, despite a reduction of fertility, allows the spreading of Robertsonian translocations, explaining the multitude of natural Robertsonian populations described in the mouse., Author Summary Cells have different mechanisms to assess the proper occurrence of cellular events. These mechanisms are called checkpoints and are involved in the surveillance of processes such as DNA replication and cell division. A checkpoint at the pachytene stage arrests meiosis when defects in the process of homologous chromosome synapsis and recombination are detected. In mammals, both transcriptional inactivation of chromosomal regions that are not correctly synapsed at pachytene and activation of sex chromosome genes that are normally silent during this stage could contribute to meiotic arrest. We found that when Robertsonian translocations appear in heterozygosis, many synapsis defects occur, and mechanisms that trigger transcriptional silencing of the unsynapsed chromatin are activated. However, meiotic prophase-I progression is not greatly compromised. This questions the ability of the meiotic checkpoints to halt meiosis progression when synapsis is not completed, allowing cells with synapsis defects to reach the first meiotic division. The fertility reduction of Robertsonian heterozygous mice seems to be mainly caused by errors detected by the metaphase-I checkpoint, when most of the spermatocytes die, rather than by synapsis defects. In an evolutionary context, a permissive pachytene checkpoint could contribute to increasing the chances of Robertsonian translocations to spread into natural populations.

10 páginas, 6 figuras.-- et al., The assembly of the mitotic centromere has been extensively studied in recent years, revealing the sequence and regulation of protein loading to this chromosome domain. However, few studies have analyzed centromere assembly during mammalian meiosis. This study specifically targets this approach on mouse spermatocytes. We have found that during prophase I, the proteins of the chromosomal passenger complex Borealin, INCENP, and Aurora-B load sequentially to the inner centromere before Shugoshin 2 and MCAK. The last proteins to be assembled are the outer kinetochore proteins BubR1 and CENP-E. All these proteins are not detected at the centromere during anaphase/telophase I and are then reloaded during interkinesis. The loading sequence of the analyzed proteins is similar during prophase I and interkinesis. These findings demonstrate that the interkinesis stage, regularly overlooked, is essential for centromere and kinetochore maturation and reorganization previous to the second meiotic division. We also demonstrate that Shugoshin 2 is necessary for the loading of MCAK at the inner centromere, but is dispensable for the loading of the outer kinetochore proteins BubR1 and CENP-E., This work was supported by grants BFU2008-00300, BFU2006-06655 and SAF-2008-03172 from Ministerio de Ciencia e Innovacion. RG has been supported by a Fundación Francisco Cobos and Ministerio de Ciencia e Innovación predoctoral fellowships.

A previous version of this article appeared as an Early Online Release on January 2, 2007 (doi:10.1371/journal.pgen.0030028.eor)., The cohesin complexes play a key role in chromosome segregation during both mitosis and meiosis. They establish sister chromatid cohesion between duplicating DNA molecules during S-phase, but they also have an important role during postreplicative double-strand break repair in mitosis, as well as during recombination between homologous chromosomes in meiosis. An additional function in meiosis is related to the sister kinetochore cohesion, so they can be pulled by microtubules to the same pole at anaphase I. Data about the dynamics of cohesin subunits during meiosis are scarce; therefore, it is of great interest to characterize how the formation of the cohesin complexes is achieved in order to understand the roles of the different subunits within them. We have investigated the spatio-temporal distribution of three different cohesin subunits in prophase I grasshopper spermatocytes. We found that structural maintenance of chromosome protein 3 (SMC3) appears as early as preleptotene, and its localization resembles the location of the unsynapsed axial elements, whereas radiation-sensitive mutant 21 (RAD21) (sister chromatid cohesion protein 1, SCC1) and stromal antigen protein 1 (SA1) (sister chromatid cohesion protein 3, SCC3) are not visualized until zygotene, since they are located in the synapsed regions of the bivalents. During pachytene, the distribution of the three cohesin subunits is very similar and all appear along the trajectories of the lateral elements of the autosomal synaptonemal complexes. However, whereas SMC3 also appears over the single and unsynapsed X chromosome, RAD21 and SA1 do not. We conclude that the loading of SMC3 and the non-SMC subunits, RAD21 and SA1, occurs in different steps throughout prophase I grasshopper meiosis. These results strongly suggest the participation of SMC3 in the initial cohesin axis formation as early as preleptotene, thus contributing to sister chromatid cohesion, with a later association of both RAD21 and SA1 subunits at zygotene to reinforce and stabilize the bivalent structure. Therefore, we speculate that more than one cohesin complex participates in the sister chromatid cohesion at prophase I., This work was supported by grants BFU2005–05668-C03–01, BFU2006–06655, BFU2005–01266, BFU2005–02431, and BFU2006–04406 from Ministerio de Educación y Ciencia, España, and grants 1001160016 and 11/BCB/013 from Universidad Autónoma de Madrid and Comunidad de Madrid. The Department of Immunology and Oncology was founded and is supported by the Spanish Council for Scientific Research (CSIC).

Marsupial sex chromosomes break the rule that recombination during first meiotic prophase is necessary to ensure reductional segregation during first meiotic division. It is widely accepted that in marsupials X and Y chromosomes do not share homologous regions, and during male first meiotic prophase the synaptonemal complex is absent between them. Although these sex chromosomes do not recombine, they segregate reductionally in anaphase I. We have investigated the nature of sex chromosome association in spermatocytes of the marsupial Thylamys elegans, in order to discern the mechanisms involved in ensuring their proper segregation. We focused on the localization of the axial/lateral element protein SCP3 and the cohesin subunit STAG3. Our results show that X and Y chromosomes never appear as univalents in metaphase I, but they remain associated until they orientate and segregate to opposite poles. However, they must not be tied by a chiasma since their separation precedes the release of the sister chromatid cohesion. Instead, we show they are associated by the dense plate, a SCP3-rich structure that is organized during the first meiotic prophase and that is still present at metaphase I. Surprisingly, the dense plate incorporates SCP1, the main protein of the central element of the synaptonemal complex, from diplotene until telophase I. Once sex chromosomes are under spindle tension, they move to opposite poles losing contact with the dense plate and undergoing early segregation. Thus, the segregation of the achiasmatic T. elegans sex chromosomes seems to be ensured by the presence in metaphase I of a synaptonemal complex-derived structure. This feature, unique among vertebrates, indicates that synaptonemal complex elements may play a role in chromosome segregation., Synopsis Meiosis is the fascinating kind of cell division that leads to the formation of gametes, and thus is essential for sexual reproduction. During meiosis, the part of our genome coming from our father and that coming from our mother play the last act of a love story initiated at the very moment of fertilization. This performance includes the search and close pairing of each chromosome with its homolog, the interchange of their genetic information, and their ultimate segregation into different cells. Everything occurs in an ordered and proper fashion because each chromosome is able to recognize its homolog and recombine with it. This ensures they are correctly transmitted to the next generation. But exceptions exist to this general rule. The authors show in this report that in the meiosis of the South American marsupial, Thylamys elegans, the sex chromosomes have the ability to segregate without undergoing meiotic recombination. The sex chromosomes take profit of the organization of a structure only present in marsupials, the dense plate, which serves the purpose of maintaining X and Y chromosomes associated until they segregate to different daughter cells. Surprisingly, this dense plate is composed of two proteins of the synaptonemal complex not meant to be at that place at that time. This finding teaches us that wildlife has plenty of way-out species, most of them unexplored, which challenge our accepted paradigms.

Mitotic Centromere-Associated Kinesin (MCAK) is a member of the kinesin-13 subfamily of kinesin-related proteins. In mitosis, this microtubule-depolymerising kinesin seems to be implicated in chromosome segregation and in the correction of improper kinetochore-microtubule interactions, and its activity is regulated by the Aurora-B kinase. However, there are no published data on its behaviour and function during mammalian meiosis. We have analysed by immunofluorescence in squashed mouse spermatocytes, the distribution and possible function of MCAK, together with Aurora-B, during both meiotic divisions. Our results demonstrate that MCAK and Aurora-B colocalise at the inner domain of metaphase I centromeres. Thus, MCAK shows a “cone”-like three-dimensional distribution beneath and surrounding the closely associated sister kinetochores. During the second meiotic division, MCAK and Aurora-B also colocalise at the inner centromere domain as a band that joins sister kinetochores, but only during prometaphase II in unattached chromosomes. During chromosome congression to the metaphase II plate, MCAK relocalises and appears as a ring below each sister kinetochore. Aurora-B also relocalises to appear as a ring surrounding and beneath kinetochores but during late metaphase II. Our results demonstrate that the redistribution of MCAK at prometaphase II/metaphase II centromeres depends on tension across the centromere and/or on the interaction of microtubules with kinetochores. We propose that the perikinetochoric rings of MCAK and Aurora-B define a novel transient centromere domain at least in mouse chromosomes during meiosis. We discuss the possible functions of MCAK at the inner centromere domain and at the perikinetochoric ring during both meiotic divisions., Synopsis The centromere is a chromosome domain essential for the correct partitioning of chromosomes during mitotic and meiotic cell divisions. MCAK is a centromeric protein that depolymerises microtubules, and seems to be implicated in chromosome segregation, and in the correction of improper microtubule interactions with the chromosome. However, there are no published data on its behaviour and function during meiotic divisions. Here, Parra et al. analyse the pattern of distribution of MCAK during male mouse meiosis in relation to Aurora-B, a kinase that regulates its activity. They show that MCAK and Aurora-B appear at the inner domain of metaphase I bivalents and unaligned metaphase II chromosomes. Most importantly, the authors discovered that these proteins relocalise to a novel perikinetochoric ring in aligned metaphase II chromosomes. The discovery of this novel structure adds a new dimension to the understanding of kinetochore structure and biology. The authors propose that, at least for mouse centromeres, the perikinetochoric ring represents a transient centromere domain whose appearance depends on tension across centromeres once microtubules interact with both sister kinetochores. This study shows that the analysis of the behaviour of different centromere proteins during meiosis can offer new insights concerning the centromere functionality.

During first meiotic prophase, homologous chromosomes are held together by the synaptonemal complex, a tripartite proteinaceous structure that extends along the entire length of meiotic bivalents. While this feature is applicable for autosomes, sex chromosomes often escape from this rule. Many species present sex chromosomes that differ between them in their morphology, length, and gene content. Moreover, in some species, sex chromosomes appear in a single dose in one of the sexes. In all of these cases, the behavior of sex chromosomes during meiosis is conspicuously affected, and this includes the assembly and dynamics of the synaptonemal complex. We review in this study the structure of the synaptonemal complex in the sex chromosomes of three groups of organisms, namely: mammals, orthopterans, and hemipterans, which present different patterns of sex chromosome structure and behavior. Of special interest is the analysis of the organization of the axial/lateral elements of the synaptonemal complex in relation to other axial structures organized along meiotic chromosomes, mainly the cohesin axis. The differences found in the behavior of both axial structures reveal that while the organization of a cohesin axis along sex chromosomes is a conserved feature in most organisms and it shows very little morphological variations, the axial/lateral elements of the synaptonemal complex present a wide range of structural modifications on these chromosomes.