Alberto Viera

The cohesin subunit RAD21L functions in meiotic synapsis and exhibits sexual dimorphism in fertility.

The cohesin complex is a ring‐shaped proteinaceous structure that entraps the two sister chromatids after replication until the onset of anaphase when the ring is opened by proteolytic cleavage of its α‐kleisin subunit (RAD21 at mitosis and REC8 at meiosis) by separase. RAD21L is a recently identified α‐kleisin that is present from fish to mammals and biochemically interacts with the cohesin subunits SMC1, SMC3 and STAG3. RAD21L localizes along the axial elements of the synaptonemal complex of mouse meiocytes. However, its existence as a bona fide cohesin and its functional role awaits in vivo validation. Here, we show that male mice lacking RAD21L are defective in full synapsis of homologous chromosomes at meiotic prophase I, which provokes an arrest at zygotene and leads to total azoospermia and consequently infertility. In contrast, RAD21L‐deficient females are fertile but develop an age‐dependent sterility. Thus, our results provide in vivo evidence that RAD21L is essential for male fertility and in females for the maintenance of fertility during natural aging.

Mammalian SGO2 appears at the inner centromere domain and redistributes depending on tension across centromeres during meiosis II and mitosis

Shugoshin (SGO) is a family of proteins that protect centromeric cohesin complexes from release during mitotic prophase and from degradation during meiosis I. Two mammalian SGO paralogues—SGO1 and SGO2—have been identified, but their distribution and function during mammalian meiosis have not been reported. Here, we analysed the expression of SGO2 during male mouse meiosis and mitosis. During meiosis I, SGO2 accumulates at centromeres during diplotene, and colocalizes differentially with the cohesin subunits RAD21 and REC8 at metaphase I centromeres. However, SGO2 and RAD21 change their relative distributions during telophase I when sister‐kinetochore association is lost. During meiosis II, SGO2 shows a striking tension‐dependent redistribution within centromeres throughout chromosome congression during prometaphase II, as it does during mitosis. We propose a model by which the redistribution of SGO2 would unmask cohesive centromere proteins, which would be then released or cleaved by separase, to trigger chromatid segregation to opposite poles.

A high incidence of meiotic silencing of unsynapsed chromatin is not associated with substantial pachytene loss in heterozygous male mice carrying multiple simple Robertsonian translocations

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.

CDK2 is required for proper homologous pairing, recombination and sex-body formation during male mouse meiosis

Cyclin-dependent kinase 2 (CDK2) was assumed to be essential in the mammalian cell cycle both at the G1-S transition and throughout the S phase. Interestingly, ablation of Cdk2 in mice does not have substantial consequences for embryonic or postnatal development, but both males and females are infertile. In the present study, we have analysed the meiotic alterations leading to infertility in Cdk2–/– male mice. We have studied the distribution and dynamics of several proteins related to meiosis progression, such as synaptonemal complex proteins, cohesin complexes, and centromere-, telomere- and recombination-related proteins. Cdk2–/– spermatocytes show an incomplete chromosome pairing, an extensive non-homologous synapsis and arrest at a pachytene-like stage with unrepaired programmed double-strand breaks. In these spermatocytes, some telomeres do not attach to the nuclear envelope, and sex chromosomes do not form a sex body. Our data demonstrate an unpredicted participation of CDK2 in the accurate pairing and recombination between homologues during mammalian meiosis.

Dynamic relocalization of the chromosomal passenger complex proteins inner centromere protein (INCENP) and aurora-B kinase during male mouse meiosis

INCENP and aurora-B kinase are two chromosomal passenger proteins that are thought to play key roles in coordinating chromosome segregation with cytokinesis in somatic cells. Here we have analyzed their subcellular distribution, and that of phosphorylated histone H3, and the timing of their relative appearance in mouse spermatocytes during both meiotic divisions. Our results show that in mitotic spermatogonial cells, INCENP and aurora-B show the same pattern of distribution as they do in cultured somatic cells. INCENP labels the synaptonemal complex central element from zygotene up to late pachytene when it begins to relocalize to heterochromatic chromocentres. Aurora-B first appears at chromocentres in late diplotene before the initial phosphorylation of histone H3. INCENP and aurora-B concentrate at centromeres during diakinesis and appear during metaphase I as T-shaped signals at their inner domains, just below associated sister kinetochores. During late anaphase I both proteins relocalize to the spindle midzone. Both proteins colocalize at a connecting strand traversing the centromere region and joining sister kinetochores, in metaphase II centromeres. This strand disappears at the metaphase II/anaphase II transition and relocalizes to the spindle midzone. We discuss the complex dynamic relocalization of the chromosomal passenger complex during prophase I. Additionally, we suggest that this complex may regulate sister-chromatid centromere cohesion during both meiotic divisions.

Meiotic Pairing and Segregation of Achiasmate Sex Chromosomes in Eutherian Mammals: The Role of SYCP3 Protein

In most eutherian mammals, sex chromosomes synapse and recombine during male meiosis in a small region called pseudoautosomal region. However in some species sex chromosomes do not synapse, and how these chromosomes manage to ensure their proper segregation is under discussion. Here we present a study of the meiotic structure and behavior of sex chromosomes in one of these species, the Mongolian gerbil (Meriones unguiculatus). We have analyzed the location of synaptonemal complex (SC) proteins SYCP1 and SYCP3, as well as three proteins involved in the process of meiotic recombination (RAD51, MLH1, and γ-H2AX). Our results show that although X and Y chromosomes are associated at pachytene and form a sex body, their axial elements (AEs) do not contact, and they never assemble a SC central element. Furthermore, MLH1 is not detected on the AEs of the sex chromosomes, indicating the absence of reciprocal recombination. At diplotene the organization of sex chromosomes changes strikingly, their AEs associate end to end, and SYCP3 forms an intricate network that occupies the Y chromosome and the distal region of the X chromosome long arm. Both the association of sex chromosomes and the SYCP3 structure are maintained until metaphase I. In anaphase I sex chromosomes migrate to opposite poles, but SYCP3 filaments connecting both chromosomes are observed. Hence, one can assume that SYCP3 modifications detected from diplotene onwards are correlated with the maintenance of sex chromosome association. These results demonstrate that some components of the SC may participate in the segregation of achiasmate sex chromosomes in eutherian mammals.

Inactivation or non-reactivation: what accounts better for the silence of sex chromosomes during mammalian male meiosis?

During the first meiotic prophase in male mammals, sex chromosomes undergo a program of transcriptional silencing called meiotic sex chromosome inactivation (MSCI). MSCI is triggered by accumulation of proteins like BRCA1, ATR, and γH2AX on unsynapsed chromosomes, followed by local changes on the sex chromatin, including histone modifications, incorporation of specific histone variants, non-histone proteins, and RNAs. It is generally thought that MSCI represents the transition of unsynapsed chromatin from a transcriptionally active state to a repressed state. However, transcription is generally low in the whole nucleus during the early stages of the first meiotic prophase, when markers of MSCI first appear, and is then reactivated globally during pachytene. Thus, an alternative possibility is that MSCI represents the targeted maintenance and/or reinforcement of a prior repressed state, i.e., a failure to reactivate. Here, we present an analysis of the temporal and spatial appearance of transcriptional and MSCI markers, as well as chromatin modifications related to transcriptional regulation. We show that levels of RNA pol II and histone H3 acetylated at lysine 9 (H3K9ac) are low during leptotene, zygotene, and early pachytene, but increase strongly in mid-pachytene, indicating that reactivation occurs with some delay after synapsis. However, while transcription markers appear abundantly on the autosomes at mid-pachytene, they are not directed to the sex chromosomes. Interestingly, we found that chromatin modifications related to transcriptional silencing and/or MSCI, namely, histone H3 trimethylated at lysine 9 (H3K9me3), histone H3 monomethylated at lysine 4 (H3K4me1), γH2AX, SUMO1, and XMR, appear on the sex chromosomes before autosomes become reactivated. These results suggest that the onset of MSCI during late zygotene and early pachytene may prevent sex chromosome reactivation during mid-pachytene instead of promoting inactivation de novo. Additionally, we found temporal differences between the X and Y chromosomes in the recruitment of DNA repair and MSCI markers, indicating a differential regulation of these processes. We propose that many of the meiotic defects attributed to failure to silence sex chromosomes could be interpreted as a more general process of transcriptional misregulation that occurs under certain pathological circumstances in zygotene and early pachytene.

Involvement of synaptonemal complex proteins in sex chromosome segregation during marsupial male meiosis

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.

Sex chromosomes, synapsis, and cohesins: a complex affair

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.

Dynamic localization of SMC5/6 complex proteins during mammalian meiosis and mitosis suggests functions in distinct chromosome processes

Summary Four members of the structural maintenance of chromosome (SMC) protein family have essential functions in chromosome condensation (SMC2/4) and sister-chromatid cohesion (SMC1/3). The SMC5/6 complex has been implicated in chromosome replication, DNA repair and chromosome segregation in somatic cells, but its possible functions during mammalian meiosis are unknown. Here, we show in mouse spermatocytes that SMC5 and SMC6 are located at the central region of the synaptonemal complex from zygotene until diplotene. During late diplotene both proteins load to the chromocenters, where they colocalize with DNA Topoisomerase II&agr;, and then accumulate at the inner domain of the centromeres during the first and second meiotic divisions. Interestingly, SMC6 and DNA Topoisomerase II&agr; colocalize at stretched strands that join kinetochores during the metaphase II to anaphase II transition, and both are observed on stretched lagging chromosomes at anaphase II following treatment with Etoposide. During mitosis, SMC6 and DNA Topoisomerase II&agr; colocalize at the centromeres and chromatid axes. Our results are consistent with the participation of SMC5 and SMC6 in homologous chromosome synapsis during prophase I, chromosome and centromere structure during meiosis I and mitosis and, with DNA Topoisomerase II&agr;, in regulating centromere cohesion during meiosis II.