Adela Calvente

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.

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.

DNA double-strand breaks, recombination and synapsis: the timing of meiosis differs in grasshoppers and flies

The temporal and functional relationships between DNA events of meiotic recombination and synaptonemal complex formation are a matter of discussion within the meiotic field. To analyse this subject in grasshoppers, organisms that have been considered as models for meiotic studies for many years, we have studied the localization of phosphorylated histone H2AX (γ‐H2AX), which marks the sites of double‐strand breaks (DSBs), in combination with localization of cohesin SMC3 and recombinase Rad51. We show that the loss of γ‐H2AX staining is spatially and temporally linked to synapsis, and that in grasshoppers the initiation of recombination, produced as a consequence of DSB formation, precedes synapsis. This result supports the idea that grasshoppers display a pairing pathway that is not present in other insects such as Drosophila melanogaster, but is similar to those reported in yeast, mouse and Arabidopsis. In addition, we have observed the presence of γ‐H2AX in the X chromosome from zygotene to late pachytene, indicating that the function of H2AX phosphorylation during grasshopper spermatogenesis is not restricted to the formation of γ‐H2AX foci at DNA DSBs.

DNA double-strand breaks and homology search: inferences from a species with incomplete pairing and synapsis

The relationship between meiotic recombination events and different patterns of pairing and synapsis has been analysed in prophase I spermatocytes of the grasshopper Stethophyma grossum, which exhibit very unusual meiotic characteristics, namely (1) the three shortest bivalents achieve full synapsis and do not show chiasma localisation; (2) the remaining eight bivalents show restricted synapsis and proximal chiasma localisation, and (3) the X chromosome remains unsynapsed. We have studied by means of immunofluorescence the localisation of the phosphorylated histone H2AX (γ-H2AX), which marks the sites of double-strand breaks; the SMC3 cohesin subunit, which is thought to have a close relationship to the development of the axial element (a synaptonemal complex component); and the recombinase RAD51. We observed a marked nuclear polarization of both the maturation of SMC3 cohesin axis and the ulterior appearance of γ-H2AX and RAD51 foci, these being exclusively restricted to those chromosomal regions that first form cohesin axis stretches. This polarised distribution of recombination events is maintained throughout prophase I over those autosomal regions that are undergoing, or about to undergo, synapsis. We propose that the restricted distribution of recombination events along the chromosomal axes in the spermatocytes is responsible for the incomplete presynaptic homologous alignment and, hence, for the partial synaptonemal complex formation displayed by most bivalents.

A perikinetochoric ring defined by MCAK and Aurora-B as a novel centromere domain

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.

Cohesin axis maturation and presence of RAD51 during first meiotic prophase in a true bug

We have analyzed in a true bug, Graphosoma italicum (Pentatomidae, Hemiptera), the temporal and functional relationships between recombination events, synapsis progression, and SMC1α and SMC3 cohesin axis maturation throughout the male first meiotic prophase. The localization of the histone variant histone H3 trimethylated at lysine 9 at chromosome ends has allowed us to determine the association of these heterochromatic domains through prophase I stages. Results highlighted that cohesins provide to be good markers for synapsis progression since the formation, morphology, and development of the SMC1α and SMC3 cohesin axes resemble the synaptonemal complex dynamics and, also, that in this species the initiation of recombination precedes synapsis. In addition, we have carried out an accurate cytological characterization of the diffuse stage, which takes place after pachytene, and also analyzed the presence of the cohesin subunits, SMC1α and SMC3, and the recombinase RAD51 at this stage. The mechanisms underlying the absence of SMC1α and SMC3 axes from the diffuse stage onwards are discussed.

Meiosis in Stethophyma (Mecostethus) Grossum (Orthoptera: Acrididae): An Exciting History

Abstract The present study is a sincere tribute from some of the cytogenetic laboratories of Madrid involved in the study of grasshopper meiosis, for the inestimable contribution of Professor Michael J.D. White to orthopteran cytogenetics, on the 100th anniversary of his birth. Here we review the knowledge accumulated over almost 90 y on the meiosis of the grasshopper Stethophyma grossum, especially on the male side, because of the very unusual meiotic characteristics shown by the spermatocytes, namely: whereas the three shortest bivalents achieve full synapsis and do not show chiasma localization and the single X chromosome remains asynapsed, the remaining eight bivalents show restricted synapsis and proximal chiasma localization. In addition, supernumerary segments and accessory chromosomes are present in some natural populations. Special attention is paid to the relationships among cohesin axis morphogenesis, initiation of recombination events and synapsis achievement.

Dynamics of cohesin subunits in grasshopper meiotic divisions.

The cohesin complex plays a key role for the maintenance of sister chromatid cohesion and faithful chromosome segregation in both mitosis and meiosis. This complex is formed by two structural maintenance of chromosomes protein family (SMC) subunits and two non-SMC subunits: an α-kleisin subunit SCC1/RAD21/REC8 and an SCC3-like protein. Several studies carried out in different species have revealed that the distribution of the cohesin subunits along the chromosomes during meiotic prophase I is not regular and that some subunits are distinctly incorporated at different cell stages. However, the accurate distribution of the different cohesin subunits in condensed meiotic chromosomes is still controversial. Here, we describe the dynamics of the cohesin subunits SMC1α, SMC3, RAD21 and SA1 during both meiotic divisions in grasshoppers. Although these subunits show a similar patched labelling at the interchromatid domain of metaphase I bivalents, SMCs and non-SMCs subunits do not always colocalise. Indeed, SA1 is the only cohesin subunit accumulated at the centromeric region of all metaphase I chromosomes. Additionally, non-SMC subunits do not appear at the interchromatid domain in either single X or B chromosomes. These data suggest the existence of several cohesin complexes during metaphase I. The cohesin subunits analysed are released from chromosomes at the beginning of anaphase I, with the exception of SA1 which can be detected at the centromeres until telophase II. These observations indicate that the cohesin components may be differentially loaded and released from meiotic chromosomes during the first and second meiotic divisions. The roles of these cohesin complexes for the maintenance of chromosome structure and their involvement in homologous segregation at first meiotic division are proposed and discussed.

Incomplete Synapsis and Chiasma Localization: The Chicken or the Egg?

In the present study, and as a sincere tribute from the Cytogenetics teams from Madrid to Professor Máximo Drets on his 80th birthday, we have analyzed and compared 3 different grasshopper species with different synaptic patterns, a standard pattern, a second pattern with synapsis restricted to the proximal regions, and a third pattern with synapsis restricted to the distal regions. In the 3 species we have thoroughly analyzed the relationships among cohesin axis morphogenesis, formation of double strand breaks (DSBs) and recombination initiation. Our results demonstrate that in every case recombination initiation precedes synapsis, and that there is a direct relationship between the absence of meiotic recombination and the existence of particular unsynapsed chromosomal regions during prophase I. Based on our results we propose and discuss the mechanisms underlying the existence of incomplete synapsis and the localization of chiasma in wild species.

Do Exogenous DNA Double-Strand Breaks Change Incomplete Synapsis and Chiasma Localization in the Grasshopper Stethophyma grossum?

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.