Christer Höög

The Murine SCP3 Gene Is Required for Synaptonemal Complex Assembly, Chromosome Synapsis, and Male Fertility

During meiosis, the homologous chromosomes pair and recombine. An evolutionarily conserved protein structure, the synaptonemal complex (SC), is located along the paired meiotic chromosomes. We have studied the function of a structural component in the axial/lateral element of the SC, the synaptonemal complex protein 3 (SCP3). A null mutation in the SCP3 gene was generated, and we noted that homozygous mutant males were sterile due to massive apoptotic cell death during meiotic prophase. The SCP3-deficient male mice failed to form axial/lateral elements and SCs, and the chromosomes in the mutant spermatocytes did not synapse. While the absence of SCP3 affected the nuclear distribution of DNA repair and recombination proteins (Rad51 and RPA), as well as synaptonemal complex protein 1 (SCP1), a residual chromatin organization remained in the mutant meiotic cells.

PKA and MPF-Activated Polo-like Kinase Regulate Anaphase-Promoting Complex Activity and Mitosis Progression

Ubiquitin-mediated proteolysis is the key to cell cycle control. Anaphase-promoting complex/cyclosome (APC) is a ubiquitin ligase that targets cyclin B and factors regulating sister chromatid separation for proteolysis by the proteasome and, consequently, regulates metaphase-anaphase transition and exit from mitosis. Here we report that Cdc2-cyclin B-activated Polo-like kinase (Plk) specifically phosphorylates at least three components of APC and activates APC to ubiquitinate cyclin B in the in vitro-reconstituted system. Conversely, protein kinase A (PKA) phosphorylates two subunits of APC but suppresses APC activity. PKA is superior to Plk in its regulation of APC, and Plk activity peaks whereas PKA activity is falling at metaphase. These results indicate that Plk and PKA regulate mitosis progression by controlling APC activity.

Age-Related Meiotic Segregation Errors in Mammalian Oocytes Are Preceded by Depletion of Cohesin and Sgo2

BACKGROUND The growing trend for women to postpone childbearing has resulted in a dramatic increase in the incidence of trisomic pregnancies. Maternal age-related miscarriage and birth defects are predominantly a consequence of chromosome segregation errors during the first meiotic division (MI), which involves the segregation of replicated recombined homologous chromosomes. Despite the importance to human reproductive health, the events precipitating female age-related meiotic errors are poorly understood. RESULTS Here we use a long-lived wild-type mouse strain to show that the ability to segregate chromosomes synchronously during anaphase of MI declines dramatically during female aging. This is preceded by depletion of chromosome-associated cohesin in association with destabilization of chiasmata, the physical linkages between homologous chromosomes, and loss of the tight association between sister centromeres. Loss of cohesin is not due to an age-related decline in the ability of the spindle checkpoint to delay separase-mediated cleavage of cohesin until entry into anaphase I. However, we find that reduced cohesin is accompanied by depletion of Sgo2, which protects centromeric cohesin during MI. CONCLUSIONS The data indicate that cohesin declines gradually during the long prophase arrest that precedes MI in female mammals. In aged oocytes, cohesin levels fall below the level required to stabilize chiasmata and to hold sister centromeres tightly together, leading to chromosome missegregation during MI. Cohesin loss may be amplified by a concomitant decline in the levels of the centromeric cohesin protector Sgo2. These findings indicate that cohesin is a key molecular link between female aging and chromosome missegregation during MI.

Identification of BIME as a Subunit of the Anaphase-Promoting Complex

The initiation of anaphase and exit from mitosis require the activation of a proteolytic system that ubiquitinates and degrades cyclin B. The regulated component of this system is a large ubiquitin ligase complex, termed the anaphase-promoting complex (APC) or cyclosome. Purified Xenopus laevis APC was found to be composed of eight major subunits, at least four of which became phosphorylated in mitosis. In addition to CDC27, CDC16, and CDC23, APC contained a homolog of Aspergillus nidulans BIME, a protein essential for anaphase. Because mutation of bimE can bypass the interphase arrest induced by either nimA mutation or unreplicated DNA, it appears that ubiquitination catalyzed by APC may also negatively regulate entry into mitosis.

A Meiotic Chromosomal Core Consisting of Cohesin Complex Proteins Recruits DNA Recombination Proteins and Promotes Synapsis in the Absence of an Axial Element in Mammalian Meiotic Cells

ABSTRACT The behavior of meiotic chromosomes differs in several respects from that of their mitotic counterparts, resulting in the generation of genetically distinct haploid cells. This has been attributed in part to a meiosis-specific chromatin-associated protein structure, the synaptonemal complex. This complex consist of two parallel axial elements, each one associated with a pair of sister chromatids, and a transverse filament located between the synapsed homologous chromosomes. Recently, a different protein structure, the cohesin complex, was shown to be associated with meiotic chromosomes and to be required for chromosome segregation. To explore the functions of the two different protein structures, the synaptonemal complex and the cohesin complex, in mammalian male meiotic cells, we have analyzed how absence of the axial element affects early meiotic chromosome behavior. We find that the synaptonemal complex protein 3 (SCP3) is a main determinant of axial-element assembly and is required for attachment of this structure to meiotic chromosomes, whereas SCP2 helps shape the in vivo structure of the axial element. We also show that formation of a cohesin-containing chromosomal core in meiotic nuclei does not require SCP3 or SCP2. Our results also suggest that the cohesin core recruits recombination proteins and promotes synapsis between homologous chromosomes in the absence of an axial element. A model for early meiotic chromosome pairing and synapsis is proposed.

Regulation of APC/C Activity in Oocytes by a Bub1-Dependent Spindle Assembly Checkpoint

BACKGROUND Missegregation of chromosomes during meiosis in human females causes aneuploidy, including trisomy 21, and is thought also to be the major cause of age-related infertility. Most errors are thought to occur at the first meiotic division. The high frequency of errors raises questions as to whether the surveillance mechanism known as the spindle assembly checkpoint (SAC) that controls the anaphase-promoting complex or cyclosome (APC/C) operates effectively in oocytes. Experimental approaches hitherto used to inactivate the SAC in oocytes suffer from a number of drawbacks. RESULTS Bub1 protein was depleted specifically in oocytes with a Zp3-Cre transgene to delete exons 7 and 8 from a floxed BUB1(F) allele. Loss of Bub1 greatly accelerates resolution of chiasmata and extrusion of polar bodies. It also causes defective biorientation of bivalents, massive chromosome missegregation at meiosis I, and precocious loss of cohesion between sister centromeres. By using a quantitative assay for APC/C-mediated securin destruction, we show that the APC/C is activated in an exponential fashion, with activity peaking 12-13 hr after GVBD, and that this process is advanced by 5 hr in oocytes lacking Bub1. Importantly, premature chiasmata resolution does not occur in Bub1-deficient oocytes also lacking either the APC/Cs Apc2 subunit or separase. Finally, we show that Bub1s kinase domain is not required to delay APC/C activation. CONCLUSIONS We conclude that far from being absent or ineffective, the SAC largely determines the timing of APC/C and hence separase activation in oocytes, delaying it for about 5 hr.

Two novel proteins recruited by synaptonemal complex protein 1 (SYCP1) are at the centre of meiosis

Completion of meiosis in mammals depends on the formation of the synaptonemal complex, a tripartite structure that physically links homologous chromosomes during prophase I. Several components of the synaptonemal complex are known, including constituents of the cohesin core, the axial/lateral element and the transverse filaments. No protein has previously been identified as an exclusive component of the central element. Mutations in some synaptonemal-complex proteins results in impaired meiosis. In humans, cases of male infertility have been associated with failure to build the synaptonemal complex. To search for new components of the meiotic machinery, we have used data from microarray expression profiling and found two proteins localising solely to the central element of the mammalian synaptonemal complex. These new proteins, SYCE1 and CESC1, interact with the transverse filament protein SYCP1, and their localisation to the central element appears to depend on recruitment by SYCP1. This suggests a role for SYCE1 and CESC1 in synaptonemal-complex assembly, and perhaps also stability and recombination.

Transmembrane protein Sun2 is involved in tethering mammalian meiotic telomeres to the nuclear envelope

Dynamic repositioning of telomeres is a unique feature of meiotic prophase I that is highly conserved among eukaryotes. At least in fission yeast it was shown to be required for proper alignment and recombination of homologous chromosomes. On entry into meiosis telomeres attach to the nuclear envelope and transiently cluster at a limited area to form a chromosomal bouquet. Telomere clustering is thought to promote chromosome recognition and stable pairing of the homologs. However, the molecular basis of telomere attachment and movement is largely unknown. Here we report that mammalian SUN-domain protein Sun2 specifically localizes to the nuclear envelope attachment sites of meiotic telomeres. Sun2–telomere association is maintained throughout the dynamic movement of telomeres. This association does not require the assembly of chromosomal axial elements or the presence of A-type lamins. Detailed EM analysis revealed that Sun2 is part of a membrane-spanning fibrillar complex that interconnects attached telomeres with cytoplasmic structures. Together with recent findings in fission yeast, our study indicates that the molecular mechanisms required for tethering meiotic telomeres and their dynamic movements during bouquet formation are conserved among eukaryotes.

Mouse embryonic stem cells form follicle-like ovarian structures but do not progress through meiosis

Several recent studies have suggested that mouse embryonic stem cells (ESCs) can differentiate into female and male germ cells in vitro. The meiotic process in germ cell‐like cells derived from ESCs has not been studied in detail, but it has been reported that synaptonemal complex protein‐3 (SYCP3) is expressed in these cells. Here, we have carefully evaluated the meiotic process in germ cell‐like cells derived from ESCs, using a panel of meiosis‐specific markers that identify distinct meiotic signatures unique to meiotic prophase I development in vivo. We find that whereas SYCP3 is expressed in germ cell‐like cells, other meiotic proteins, such as SYCP1, SYCP2, STAG3 (stromal antigen 3), REC8 (meiotic protein similar to the rad21 cohesins), and SMC1 (structural maintenance of chromosomes‐1)‐β, are not expressed. The nuclear distribution of SYCP3 in the germ cell‐like cells is highly abnormal and not associated with the chromosomes of these cells. Fluorescence in situ hybridization analysis shows that the SYCP3‐positive germ cell‐like cells do not contain synapsed homologous chromosomes but instead display a chromosomal organization normally found in somatic cells. The absence of expression of essential meiotic proteins and a normal meiotic chromosomal organization strongly suggests that the germ cell‐like cells formed from ESCs fail to progress through meiosis.

Affinity Proteomics for Systematic Protein Profiling of Chromosome 21 Gene Products in Human Tissues

Here we show that an affinity proteomics strategy using affinity-purified antibodies raised against recombinant human protein fragments can be used for chromosome-wide protein profiling. The approach is based on affinity reagents raised toward bioinformatics-designed protein epitope signature tags corresponding to unique regions of individual gene loci. The genes of human chromosome 21 identified by the genome efforts were investigated, and the success rates for de novo cloning, protein production, and antibody generation were 85, 76, and 56%, respectively. Using human tissue arrays, a systematic profiling of protein expression and subcellular localization was undertaken for the putative gene products. The results suggest that this affinity proteomics strategy can be used to produce a proteome atlas, describing distribution and expression of proteins in normal tissues as well as in common cancers and other forms of diseased tissues.