No abstract

SCP3 is a meiosis-specific structural protein appearing at axial elements and lateral elements of the synaptonemal complex. We have analysed the behaviour of SCP3 and the cohesin subunit Rad21 in mouse spermatocytes by means of a squashing technique. Our results demonstrate that both proteins colocalize and are partially released from chromosome arms during late prophase I stages, although they persist at the interchromatid domain of metaphase I bivalents. Thus, Rad21 cannot be considered a `mitotic'-specific variant, but coexists with Rec8. During late prophase I SCP3 and Rad21 accumulate at centromeres, and together with the chromosomal passenger proteins INCENP and aurora-B kinase, show a complex `double cornet'-like distribution at the inner domain of metaphase I centromeres beneath the associated sister kinetochores. We have observed that Rad21 and SCP3 are displaced from centromeres during telophase I when sister kinetochores separate, and are not present at metaphase II centromeres. Thus, we hypothesise that Rad21, and the superimposed SCP3 and SCP2, are involved in the monopolar attachment of sister kinetochores during meiosis I, and are not responsible for the maintenance of sister-chromatid centromere cohesion during meiosis II as previously suggested.

Shugoshin-2 (SGOL2) is one of the two mammalian orthologs of the Shugoshin/Mei-S322 family of proteins that regulate sister chromatid cohesion by protecting the integrity of the multiprotein cohesin complexes. This protective system is essential for faithful chromosome segregation during mitosis and meiosis, which is the physical basis of Mendelian inheritance. Regardless of its evolutionary conservation from yeast to mammals, little is known about the in vivo relevance and specific role that SGOL2 plays in mammals. Here we show that disruption of the gene encoding mouse SGOL2 does not cause any alteration in sister chromatid cohesion in embryonic cultured fibroblasts and adult somatic tissues. Moreover, mutant mice develop normally and survive to adulthood without any apparent alteration. However, both male and female Sgol2-deficient mice are infertile. We demonstrate that SGOL2 is necessary for protecting centromeric cohesion during mammalian meiosis I. In vivo, the loss of SGOL2 promotes a premature release of the meiosis-specific REC8 cohesin complexes from anaphase I centromeres. This molecular alteration is manifested cytologically by the complete loss of centromere cohesion at metaphase II leading to single chromatids and physiologically with the formation of aneuploid gametes that give rise to infertility.[Keywords: Cohesion; chromosome segregation; Shugoshin-2; mouse; mitosis; meiosis] Supplemental material is available at http://www.genesdev.org.

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 sisterkinetochore 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.

CEP63 is a centrosomal protein that facilitates centriole duplication and is regulated by the DNA damage response. Mutations in CEP63 cause Seckel syndrome, a human disease characterized by microcephaly and dwarfism. Here we demonstrate that Cep63 deficient mice recapitulate Seckel syndrome pathology. The attrition of neural progenitor cells involves p53-dependent cell death and brain size is rescued by the deletion of p53. Cell death is not the result of an aberrant DNA damage response but is triggered by centrosome-based mitotic errors. In addition, Cep63 loss severely impairs meiotic recombination, leading to profound male infertility. Cep63 deficient spermatocytes display numerical and structural centrosome aberrations, chromosome entanglements and defective telomere clustering, suggesting that a reduction in centrosome-mediated chromosome movements underlies recombination failure. Our results provide novel insight into the molecular pathology of microcephaly and establish a role for the centrosome in meiotic recombination.

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.

In most organisms, telomeres attach to the nuclear envelope at the onset of meiosis to promote the crucial processes of pairing, recombination and synapsis during prophase I. This attachment of meiotic telomeres is mediated by the specific distribution of several nuclear envelope components that interact with the attachment plates of the synaptonemal complex. We have determined by immunofluorescence and electron microscopy that the ablation of the kinase CDK2 alters the nuclear envelope in mouse spermatocytes, and that the proteins SUN1, KASH5 (also known as CCDC155) and lamin C2 show an abnormal cap-like distribution facing the centrosome. Strikingly, some telomeres are not attached to the nuclear envelope but remain at the nuclear interior where they are associated with SUN1 and with nuclear-envelope-detached vesicles. We also demonstrate that mouse testis CDK2 phosphorylates SUN1 in vitro. We propose that during mammalian prophase I the kinase CDK2 is a key factor governing the structure of the nuclear envelope and the telomere-led chromosome movements essential for homolog pairing.

SummaryFour 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 IIa, and then accumulate at the inner domain of the centromeres during the first and second meiotic divisions. Interestingly, SMC6 and DNA Topoisomerase IIa 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 IIa 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 IIa, in regulating centromere cohesion during meiosis II.