Peter B. Moens

The fine structure of meiotic chromosome polarization and pairing in Locusta migratoria spermatocytes

At the leptotene stage of meiotic prophase in Locusta spermatocytes (2n=22 telocentric autosomes + X-chromosome), each chromosome forms an axial core. The 44 ends of the autosomal cores are all attached to the nuclear membrane in a small region opposite the two pairs of centrioles of the juxtanuclear mitochondrial mass. At later stages of meiotic prophase, the cores of homologous chromosomes synapse into synaptinemal complexes. Synapsis is initiated near the nuclear membrane, in the centromeric and the non-centromeric ends of the chromosomes. Homologous cores have their attachment points close together and some cores are co-aligned prior to synapsis. At subsequent stages of zygotene, the number of synaptinemal complexes at the membrane increases, while the number of unpaired axial cores diminishes. At pachytene, all 11 bivalents are attached to the membrane at both ends, so that there are 22 synaptinemal complexes at the membrane near the centrioles. Because each bivalent makes a complete loop, the configuration of the classic Bouquet stage is produced. The X-chromosome has a poorly defined single core at pachytene which also attaches to the nuclear membrane. These observations are based on consecutive serial sections (50 to 100) through the centriolar zone of the spermatocytes. Labeling experiments demonstrated that tritiated thymidine was incorporated in the chromatin of young spermatocytes prior to the formation of the axial cores at leptotene. It is concluded that premeiotic DNA synthesis is completed well in advance of pairing of homologous chromosomes, as marked by the formation of synaptinemal complexes.

RAD51 IMMUNOCYTOLOGY IN RAT AND MOUSE SPERMATOCYTES AND OOCYTES

Abstract.On the assumption that Rad51 protein plays a role in early meiotic chromosomal events, we examine the location and time of appearance of immuno-reactive Rad51 protein in meiotic prophase chromosomes. The Rad51 foci in mouse spermatocytes appear after the emergence of, and attached to, short chromosomal core segments that we visualize with Cor1-specific antibody. These foci increase in number to about 250 per nucleus at the time when core formation is extensive. The numbers are higher in mouse oocytes and lower in rat spermatocytes, possibly correlating with recombination rates in those cases. In the male mouse, foci decrease in number to approximately 100 while chromosome synapsis is in progress. When synapsis is completed, the numbers of autosomal foci decline to near 0 while the X chromosome retains about 15 foci throughout this time. This stage coincides with the appearance of testis-specific histone H1t at mid- to late pachytene. Electron microscopy reveals that at first Rad51 immunogold-labeled 100 nm nodules are associated with single cores, and that they come to lie between the chromosome cores during synapsis. It appears that these nodules may be the homologs of the Rad51-positive early nodules that are well documented in plants. The reciprocal recombination-correlated late nodules appear after the Rad51 foci are no longer detectable. The absence of Rad51 foci in the chromatin loops suggests that in wild-type mice Rad51/DNA filaments are restricted to DNA at the cores/synaptonemal complexes. The expected association of Rad51 protein with Rad52 could not be verified immunocytologically.

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.

The structure and function of the synaptinemal complex inLilium longiflorum sporocytes

The development of meiotic prophase in pollen mother cells ofLilium longiflorum is presented through photomicrographs of squashes and sections and through electron micrographs of thick and thin sections. Emphasis is placed on the first appearance of axial cores, the participation of axial cores in the formation of synaptinemal complexes, the fine structure of the complex and the fate of the complex at the end of pachytene. It is shown that axial cores are formed in early meiotic prophase chromosomes and that the two axial cores of a set of homologous chromosomes participate in the formation of a synaptinemal complex. It is proposed that the transverse filaments of each axial core meet and interdigitate and so produce the transverse filaments of the complex. It is shown that the complex is axial to the pachytene bivalent and that the association of the complex with chromosomal material is terminated at the end of pachytene. The pairing affinity of the cores in homologous and non-homologous chromosome associations is discussed. The zygotene stage is defined in terms of the occurrence of synaptinemal complexes and the attachment of the nucleolus to the nuclear membrane during this stage is noted.

Impaired meiotic DNA-damage repair and lack of crossing-over during spermatogenesis in BRCA1 full-length isoform deficient mice

Breast tumor suppressor gene 1 (BRCA1) plays an essential role in maintaining genomic integrity. Here we show that mouse Brca1 is required for DNA-damage repair and crossing-over during spermatogenesis. Male Brca1Δ11/Δ11p53+/- mice that carried a homozygous deletion of Brca1 exon 11 and a p53 heterozygous mutation had significantly reduced testicular size and no spermatozoa in their seminiferous tubules. During spermatogenesis, homologous chromosomes from the mutant mice synapsed and advanced to the pachytene stage but failed to progress to the diplotene stage. Our analyses revealed that the Brca1 mutation affected cellular localization of several DNA damage-repair proteins. This included prolonged association of γH2AX with sites of DNA damage, reduced sex body formation, diminished Rad51 foci and absence of Mlh1 foci in the pachytene stage. Consequently, chromosomes from mutant mice did not form chiasmata, a point that connects exchanging homologous chromosomes. Brca1-mutant spermatocytes also exhibited decreased RNA expression levels of several genes that are involved in DNA-damage repair, including RuvB-like DNA helicase, XPB, p62 and TFIID. Of note, the premature termination of spermatogenesis at the pachytene stage was accompanied by increased apoptosis by both p53-dependent and p53-independent mechanisms. Thus, our study revealed an essential role of Brca1 in DNA-damage repair and crossing-over of homologous chromosomes during spermatogenesis.

The association of ATR protein with mouse meiotic chromosome cores

Abstract. The ATR (ataxia telangiectasia- and RAD3-related) protein is present on meiotic prophase chromosome cores and paired cores (synaptonemal complexes, SCs). Its striking characteristic is that the protein forms dense aggregates on the cores and SCs of the last chromosomes to pair at the zygotene-pachytene transition. It would appear that the ATR protein either signals delays in pairing or it is directly involved in the completion of the pairing phase. Atm-deficient spermatocytes, which are defective in the chromosome pairing phase, accumulate large amounts of ATR. The behaviour of ATR at meiotic prophase sets it apart from the distribution of the RAD51/DMC1 recombinase complex and our electron microscope observations confirm that they do not co-localize. We failed to detect ATM in association with cores/SCs and we have reported elsewhere that RAD1 protein does not co-localize with DMC1 foci. The expectation that putative DNA-damage checkpoint proteins, ATR, ATM and RAD1, are associated with RAD51/DMC1 recombination sites where DNA breaks are expected to be present, is therefore not supported by our observations.

miRNA and piRNA localization in the male mammalian meiotic nucleus

During mammalian meiosis, transcriptional silencing of the XY bivalent is a necessary event where defects may lead to infertility in males. While not well understood, the mechanism of meiotic gene silencing is believed to be RNA-dependent. In this study, we investigated the types and localization of non-coding RNAs in the meiotic nucleus of the male mouse using a microarray screen with different cell isolates as well as FISH. We report that the dense body, a component of the murine spermatocyte sex body similar to that of a dense body in Chinese hamster spermatocytes, is DNA-negative but rich in proteins and RNA including miRNAs (micro RNAs) and piRNAs (PIWI associated small RNAs), or their precursors. Selective miRNAs and piRNAs localize to chromosome cores, telomeres and the sex body of spermatocytes. These RNAs have not previously been detected in meiotic nuclei. These RNAs appear to associate with the nucleolus of the Sertoli cells as well as with the dense body. While in MIWI-null male mice the nucleolar signal from miRNA and piRNA probes in Sertoli cells is largely diminished, a differential regulation must exist in meiotic nuclei since the localization of these two components appears to be unaffected in the null animal.

Anti-topoisomerase II recognizes meiotic chromosome cores.

At meiotic prophase the chromatin becomes arranged in loops on newly formed chromosome cores. The cores of homologous chromosomes become aligned in parallel and thus form the synaptonemal complex (SC), a structure found in the meiocytes of nearly all recombinationally competent, sexually reproducing organisms. We report that two polyclonal antibodies against topoisomerase II (topo II), which recognize the mitotic metaphase chromosome scaffold give, at pachytene, a positive immunocytological reaction with the chromatin and, predominantly, with the cores and centromeric regions of the paired chromosomes. It therefore appears that during meiotic prophase, topo II — a DNA-binding enzyme implicated in transient double-strand breaks, chromosome condensation, and anaphase separation — is associated with the chromatin and SCs of the pachytene and diplotene chromosomes.

Immunocytology of chiasmata and chromosomal disjunction at mouse meiosis

Immunocytological and in situ hybridization evidence supports the hypothesis that at meiosis of chiasmate organisms, chromosomal disjunction and reductional segregation of sister centromeres are integrated with synaptonemal complex functions. The Mr 125,000 synaptic protein, Syn1, present between cores of paired homologous chromosomes during pachytene of meiotic prophase, is lost from synaptonemal complexes coordinately with homolog separation at diplotene. Separation is constrained by exchanges between non-sister chromatids, the chiasmata. We show that the Mr 30,000 chromosomal core protein, Cor1, associated with sister chromatid pairs, remains an axial component of post-pachytene chromosomes until metaphase I. We demonstrate that at this time the chromatin loops are still attached to their cores. A reciprocal exchange event between two homologous non-sister chromatids is therefore immobilized by anchorage of sister chromatids to their respective cores. Cores thus contribute to the sister chromatid cohesiveness required for maintenance of chiasmata and proper chromosomal disjunction. Cor1 protein accumulates in juxtaposition to pairs of sister centromeres during metaphase I. Presumably, independent movement of sister centromeres at anaphase I is restricted by Cor1 anchorage. That reductional separation of sister centromeres is mediated by Cor1, is supported by the dissociation of Cor1 from separating sister centromeres at anaphase II and by its absence from mitotic anaphases.

The development, structure and function of modified synaptonemal complexes in mosquito oocytes.

The nucleus of the maturing oocytes expands to a large thin body of 400×140×3 μm but the chromosomes remain together in a small sphere, 15 μm in diameter. In Aedes aegypti this sphere becomes surrounded by one to several layers of polycomplexes, annulated polycomplexes, and related annulated pseudomembranes. Just prior to egg laying the expanded nucleus disintegrates while the sphere of chromosomes is surrounded by several layers of membranes. In Culex pipiens the elements which normally connect the lateral elements of the synaptonemal complexes become extended so that all bivalents become interconnected by a framework of pseudomembranes. The continuity between the modified synaptonemal complexes and various membranes associated with the karyosphere suggest that a relationship exists, by origin or by specialization, between the synaptic structures and nuclear envelope.