Interrelaciones del complejo de cohesina, la recombinación y la sinapsis en la meiosis de Ortópteros

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología . Fecha de lectura: 21-12-0

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

Sex chromosomes, synapsis, and cohesins: a complex affair

The final publication is available at http://link.springer.co

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

Do Exogenous DNA Double-Strand Breaks Change Incomplete Synapsis and Chiasma Localization in the Grasshopper <i>Stethophyma grossum</i>? - Fig 1

<p><b>Acetic orcein and silver staining of metaphase I spermatocytes (A, B). (C-N)</b> γ<b>-H2AX and RAD51 double immunolocalization in non-irradiated squashed spermatocytesof <i>S</i>.<i>grossum</i>.</b> (A) The X chromosome is marked. Most bivalents show a single chiasma located near the pericentromeric regions. (B) Kinetochores of chromosomes are marked by asterisks. See text for the nomenclature applied to different bivalents. (C,G,H) γ-H2AX (green); (D,H,L) RAD51 (red); (E,I,M) DAPI; (F,J,N) merging of γ-H2AX and RAD51. (C-F) Leptotene. (G-J) Zygotene. (K-N) Pachytene. γ-H2AX and RAD51 signals are polarized in the same nuclear region. (C-N) images correspond to the sum of different focal planes. Bars represent 10 μm.</p

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

<div><p>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 <i>Stethophyma grossum</i>, 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.</p></div

Possible origin of quadrivalents.

<p>Diagramatic representation of two long bivalents, partially synapsed, of <i>S</i>.<i>grossum</i>, depicted in green and red, respectively. Whereas chiasmata normally occur in the pericentromeric regions of homologous chromosomes, they could also take place between homologous subdistal regions of non-homologous chromosomes as a consequence of reciprocal translocations produced by exogenous DSBs and /or COs between homologous DNA sequences of non-homologous chromosomes (non-allelic homologous recombination) (blue arrow).</p

Acetic orcein staining of squashed spermatocytes of <i>S</i>. <i>grossum</i> at anaphase I and second meiotic division, 8–12 days after irradiation.

<p>(A, B) Anaphase I. (C) Telophase I. (D) Metaphase II. (E,F) Anaphase II. (G) Haploid spermatids. (H-J) Aberrant spermatids. Chromatin bridges (double white arrowheads), chromosomal fragments (black arrows), chromatin constrictions (black arrowheads), heteromorphic chromosomes (blue arrows) and lagging chromosomes (blue arrowhead) are indicated. Bars represent 10 μm.</p