Analysis of male meiotic "sex body" proteins during XY female meiosis provides new insights into their functions.

During male meiosis in mammals the X and Y chromosomes become condensed to form the sex body (XY body), which is the morphological manifestation of the process of meiotic sex chromosome inactivation (MSCI). An increasing number of sex body located proteins are being identified, but their functions in relation to MSCI are unclear. Here we demonstrate that assaying male sex body located proteins during XY female mouse meiosis, where MSCI does not take place, is one way in which to begin to discriminate between potential functions. We show that a newly identified protein, "Asynaptin" (ASY), detected in male meiosis exclusively in association with the X and Y chromatin of the sex body, is also expressed in pachytene oocytes of XY females where it coats the chromatin of the asynapsed X in the absence of MSCI. Furthermore, in pachytene oocytes of females carrying a reciprocal autosomal translocation, ASY associates with asynapsed autosomal chromatin. Thus the location of ASY to the sex body during male meiosis is likely to be a response to the asynapsis of the non-homologous regions [outside the pseudoautosomal region (PAR)] of the heteromorphic X-Y bivalent, rather than being related to MSCI. In contrast to ASY, the previously described sex body protein XY77 proved to be male sex body specific. Potential functions for MSCI and the sex body are discussed together with the possible roles of these two proteins.

Mre11 and Ku70 interact in somatic cells, but are differentially expressed in early meiosis.

Double-strand DNA breaks (DSBs) pose a major threat to living cells, and several mechanisms for repairing these lesions have evolved. Eukaryotes can process DSBs by homologous recombination (HR) or non-homologous end joining (NHEJ). NHEJ connects DNA ends irrespective of their sequence, and it predominates in mitotic cells, particularly during G1 (ref. 3). HR requires interaction of the broken DNA molecule with an intact homologous copy, and allows restoration of the original DNA sequence. HR is active during G2 of the mitotic cycle and predominates during meiosis, when the cell creates DSBs (ref. 4), which must be repaired by HR to ensure proper chromosome segregation. How the cell controls the choice between the two repair pathways is not understood. We demonstrate here a physical interaction between mammalian Ku70, which is essential for NHEJ (ref. 5), and Mre11, which functions both in NHEJ and meiotic HR (Refs 2,6). Moreover, we show that irradiated cells deficient for Ku70 are incapable of targeting Mre11 to subnuclear foci that may represent DNA-repair complexes. Nevertheless, Ku70 and Mre11 were differentially expressed during meiosis. In the mouse testis, Mre11 and Ku70 co-localized in nuclei of somatic cells and in the XY bivalent. In early meiotic prophase, however, when meiotic recombination is most probably initiated, Mre11 was abundant, whereas Ku70 was not detectable. We propose that Ku70 acts as a switch between the two DSB repair pathways. When present, Ku70 destines DSBs for NHEJ by binding to DNA ends and attracting other factors for NHEJ, including Mre11; when absent, it allows participation of DNA ends and Mre11 in the meiotic HR pathway.

SCP2: a major protein component of the axial elements of synaptonemal complexes of the rat.

In the axial elements of synaptonemal complexes (SCs) of the rat, major protein components have been identified, with relative electrophoretic mobilities (M rs) of 30 000-33 000 and 190 000. Using monoclonal anti-SC antibodies, we isolated cDNA fragments which encode the 190 000 M r component of rat SCs. The translation product predicted from the nucleotide sequence of the cDNA, called SCP2 (for synaptonemal complex protein 2), is a basic protein (pI = 8.0) with a molecular mass of 173 kDa. At the C-terminus, a stretch of approximately 50 amino acid residues is predicted to be capable of forming coiled-coil structures. SCP2 contains two clusters of S/T-P motifs, which are common in DNA-binding proteins. These clusters flank the central, most basic part of the protein (pI = 9.5). Three of the S/T-P motifs are potential target sites for p34(cdc2) protein kinase. In addition, SCP2 has eight potential cAMP/cGMP-dependent protein kinase target sites. The gene encoding SCP2 is transcribed specifically in the testis, in meiotic prophase cells. At the amino acid sequence and secondary structural level, SCP2 shows some similarity to the Red1 protein, which is involved in meiotic recombination and the assembly of axial elements of SCs in yeast. We speculate that SCP2 is a DNA-binding protein involved in the structural organization of meiotic prophase chromosomes.

Localization of SCP2 and SCP3 protein molecules within synaptonemal complexes of the rat.

SCP2 and SCP3 are major protein components of the lateral elements (LEs) of synaptonemal complexes (SCs) of the rat, with Mrs of 173, 000 and 30,000. We performed a detailed immunocytochemical comparison of the localization of SCP2 and SCP3 within SCs at the electron microscopic level. The ultrastructural localization of SCP2 and SCP3 was analyzed by immunogold labeling of two types of preparations, namely surface-spread spermatocytes and ultrathin sections of Lowicryl-embedded testicular tissue of the rat. For each of the antisera used, the distribution of immunogold label over SCs in surface-spread spermatocytes differed significantly from the distribution of label on sections. We attributed this difference to artifacts caused by the surface-spreading technique, and therefore we relied on sections for the precise localization of epitopes. On sections, the distribution of label obtained with two antisera against nonoverlapping, widely separated fragments of SCP2 did not differ significantly. There was a small but significant difference between the labeling pattern obtained with an anti-SCP3 serum and the pattern obtained with either of the two antisera against fragments of SCP2; although for all three antisera the peak of the immunogold label coincided with the center of the LE, the distributions of label obtained with the antisera against fragments of SCP2 were asymmetrical, with a shoulder at the inner side of the LE, whereas the distribution of label obtained with anti-SCP3 serum was symmetrical. Furthermore, we observed fuzzy connections between the LEs that were labeled by anti-SCP2 but not anti-SCP3 antibodies. It is possible that labeling of these fuzzy bridges caused the shoulder in the gold label distributions obtained with anti-SCP2 antibodies.

RecA-like proteins are components of early meiotic nodules in lily.

Early meiotic nodules (also called recombination nodules) are proteinaceous structures about 100 nm in diameter that are associated with forming synaptonemal complexes (SCs) during early prophase I of meiosis. Although their function is unknown, early nodules may be involved in searching for DNA homology before synaptic initiation. Two potential components of early nodules are Rad51 and Dmc1 proteins. These proteins are important for meiotic recombination in eukaryotes and are homologous to RecA, the major protein that catalyzes homologous pairing and DNA strand exchange in prokaryotes. In addition, Rad51 has been localized by immunofluorescence in abundant foci that may correspond to early nodules in yeast, lily, and mouse. In yeast and lily, Dmc1 and Lim15, the lily homolog of Dmc1, colocalize with Rad51. Here, using electron microscopic immunogold localization to spreads of zygotene and early pachytene SCs from lily, we confirm that RecA-like proteins are components of early nodules. The antibody used was generated to full-length tomato Rad51 protein and binds to both Rad51 and Lim15 in immunoblots of lily primary microsporocyte proteins. The labeled early nodules are heterogeneous in size and are associated with both axial elements and SCs. There are two classes of early nodules, those that are densely labeled with gold and those that are not labeled at all. This result may be due to technical limitations associated with using spread preparations or to differences in the nodules themselves. The presence of Rad51 and/or Lim15 proteins in early nodules supports the hypothesis that early nodules are involved in recombination-related events during meiosis.

Cloning, sequencing, disruption and phenotypic analysis of uvsC, an Aspergillus nidulans homologue of yeast RAD51.

We have cloned the uvsC gene of Aspergillus nidulans by complementation of the A. nidulans uvsC114 mutant. The predicted protein UVSC shows 67.4% sequence identity to the Saccharomyces cerevisiae Rad51 protein and 27.4% sequence identity to the Escherichia coli RecA protein. Transcription of uvsC is induced by methyl-methane sulphonate (MMS), as is transcription of RAD51 of yeast. Similar levels of uvsC transcription were observed after MMS induction in a uvsC+ strain and the uvsC114 mutant. The coding sequence of the uvsC114 allele has a deletion of 6 bp, which results in deletion of two amino acids and replacement of one amino acid in the translation product. In order to gain more insight into the biological function of the uvsC gene, a uvsC null mutant was constructed, in which the entire uvsC coding sequence was replaced by a selectable marker gene. Meiotic and mitotic phenotypes of a uvsC+ strain, the uvsC114 mutant and the uvsC null mutant were compared. The uvsC null mutant was more sensitive to both UV and MMS than the uvsC114 mutant. The uvsC114 mutant arrested in meiotic prophase-I. The uvsC null mutant arrested at an earlier stage, before the onset of meiosis. One possible interpretation of these meiotic phenotypes is that the A. nidulans homologue of Rad51 of yeast has a role both in the specialized processes preceding meiosis and in meiotic prophase I.

cDNA cloning and characterization of Npap60: a novel rat nuclear pore-associated protein with an unusual subcellular localization during male germ cell differentiation.

We have cloned and characterized a cDNA, Npap60, encoding a rat nuclear pore-associated protein. The 3-kb cDNA was obtained by antibody screening of a rat testis expression library. The predicted NPAP60 contains 381 amino acids with a composition of 25.6% charged residues and is highly hydrophilic. The Npap60 gene appears to be conserved in mouse, rat, and human. Immunofluorescence studies with anti-NPAP60 fusion protein antibody show that the NPAP60 protein colocalizes with nuclear pore complexes in RAT1A cells. The expression of Npap60 is about 10-20 times higher in rat testis than in somatic tissues. The subcellular localization of NPAP60 protein changes dramatically during male germ cell differentiation, from nuclear pore complex-like staining in spermatocytes to whole nucleus staining in spermatids and finally to a nuclear surface staining in mature spermatozoa. These changes are temporally and spatially related to nuclear reorganization during male germ cell differentiation.

Isolation and characterization of rad51 orthologs from Coprinus cinereus and Lycopersicon esculentum, and phylogenetic analysis of eukaryotic recA homologs.

In eubacteria, the recA gene has long been recognized as essential for homologous recombination and DNA repair. Recent work has identified recA homologs in archaebacteria and eukaryotes, thus emphasizing the universal role this gene plays in DNA metabolism. We have isolated and characterized two new recA homologs, one from the basidiomycete Coprinus cinereus and the other from the angiosperm Lycopersicon esculentum. Like the RAD51 gene of Saccharomyces cerevisiae, the Coprinus gene is highly induced by gamma irradiation and during meiosis. Phylogenetic analyses of eukarotic recA homologs reveal a gene duplication early in eukaryotic evolution which gave rise to two putatively monophyletic groups of recA-like genes. One group of 11 characterized genes, designated the rad51 group, is orthologous to the Saccharomyces RAD51 gene and also contains the Coprinus and Lycopersicon genes. The other group of seven genes, designated the dmc1 group, is orthologous to the Saccharomyces DMC1 gene. Sequence comparisons and phylogenetic analysis reveal extensive lineage- and gene-specific differences in rates of RecA protein evolution. Dmc1 consistently evolves faster than Rad51, and fungal proteins of both types, especially those of Saccharomyces, change rapidly, particularly in comparison to the slowly evolving vertebrate proteins. The Drosophila Rad51 protein has undergone remarkably rapid sequence divergence.

A change in the phosphorylation pattern of the 30000-33000 Mr synaptonemal complex proteins of the rat between early and mid-pachytene.

The lateral elements (LEs) of synaptonemal complexes (SCs) of the rat contain major components with relative electrophoretic mobilities (Mr s) of 30000-33000, which are the products of a single gene. After one-dimensional separation of SC proteins on polyacrylamide-SDS gels, these components show up as two major bands, whereas upon two-dimensional electrophoresis they are resolved in at least 24 spots, which focus at pH 6.5 to 9.5. In this paper we show that these spots represent phosphorylation variants. For the analysis of the phosphorylation of the 30000- to 33000-Mr SC components during progression through meiotic prophase, we developed a procedure for isolation of fractions of testicular cells of the rat that are enriched in separate stages of meiotic prophase. Analysis of the 30000- to 33000-Mr SC components in these fractions by two-dimensional electrophoresis and immunoblotting showed that phosphorylated variants of the 30000- to 33000-Mr SC proteins occur throughout meiotic prophase. However, the extent of phosphorylation changes between early and mid-pachytene, when one phosphate group is probably added to each of the variants.

The gene encoding a major component of the lateral elements of synaptonemal complexes of the rat is related to X-linked lymphocyte-regulated genes.

The lateral elements of synaptonemal complexes (SCs) of the rat contain major components with relative electrophoretic mobilities (M(r)S) of 30,000 and 33,000. After one-dimensional separation of SC proteins on polyacrylamide-sodium dodecyl sulfate gels, these components show up as two broad bands. These bands contain closely related proteins, as judged from their peptide maps and immunological reactivity. Using affinity-purified polyclonal anti-30,000- and anti-33,000-M(r) component antibodies, we isolated a cDNA encoding at least one of the 30,000- or 33,000-M(r) SC components. The protein predicted from the nucleotide sequence of the cDNA, called SCP3 (for synaptonemal complex protein 3), has a molecular mass of 29.7 kDa and a pI value of 9.4. It has a potential nucleotide binding site and contains stretches that are predicted to be capable of forming coiled-coil structures. In the male rat, the gene encoding SCP3 is transcribed exclusively in the testis. SCP3 has significant amino acid similarity to the pM1 protein, which is one of the predicted products of an X-linked lymphocyte-regulated gene family of the mouse: there are 63% amino acid sequence similarity and 35% amino acid identity between the SCP3 and pM1 proteins. However, SCP3 differs from pM1 in several respects, and whether the proteins fulfill related functions is still an open question.

A coiled-coil related protein specific for synapsed regions of meiotic prophase chromosomes.

Synaptonemal complexes (SCs) are structures that are formed between homologous chromosomes during meiotic prophase. They are probably involved in chromosome pairing and recombination. Using a monoclonal anti-SC antibody we isolated cDNAs encoding a major component of SCs which is localized specifically in synapsed segments of meiotic prophase chromosomes. The protein predicted from the nucleotide sequence of a full-length cDNA, named SCP1, consists of 946 amino acid residues and has a molecular weight of 111 kDa. It shares several features with nuclear lamins and some recently identified nuclear matrix proteins. The major part of SCP1 consists of long stretches capable of forming amphipathic alpha-helices. This region shows amino acid sequence similarity to the coiled-coil region of myosin heavy chain. A leucine zipper is included in this region. The carboxy-terminus has two small basic domains and several S/T-P-X-X motifs, which are characteristic of DNA-binding proteins. One of these motifs is a potential target site for p34cdc2 protein kinase. The amino-terminus is acidic and relatively proline-rich, but does not contain the S/T-P-X-X motif. The transcription of the gene encoding SCP1 is restricted to zygotene-diplotene spermatocytes. A polyclonal antiserum raised against the fusion protein of one of the cDNA clones recognizes a single protein on Western blots of isolated SCs, with an electrophoretic mobility identical to that of the antigen recognized by the original monoclonal antibody (mAb), IX5B2. From a detailed comparison of the immunogold labelling of rat SCs by mAb IX5B2 and the polyclonal anti-fusion protein antiserum respectively, we tentatively infer that the carboxy-terminus of SCP1 is orientated towards the lateral elements and that the other domains of the protein extend towards the central region between the lateral elements. We conclude that SCP1 is the major component of the transverse filaments of SCs, and speculate that it has evolved by specialization of a nuclear matrix protein.

The sequential appearance of components of the synaptonemal complex during meiosis of the female rat.

This paper describes the light microscopy (LM) and electron microscopy (EM) localization of synaptonemal complex (SC) antigens in oocytes of rats. For this purpose, we used monoclonal antibodies (Mabs) that recognize components of 30 + 33, 125, and 190 kDa antigens of SCs of rat spermatocytes. The LM localization was performed by immunofluorescence and the EM localization by immunogold staining. The reaction of the Mabs with oocytes was similar to the reaction with spermatocytes, but weaker. The 30 + 33 kDa as well as the 190 kDa antigens could always be demonstrated if axial elements of the SC were present, irrespective of whether these were paired or unpaired. Thus, these antigens could be detected from leptotene--early zygotene until diplotene. The 190-kDa antigen appeared in a diffuse manner just before the appearance of the 30 + 33 kDa antigens. The 30 + 33 kDa antigens were not only detected in the axial elements of SCs but also in characteristic aggregates, which appeared in zygotene and persisted until after the SCs had disappeared. Such aggregates had rarely been observed in spermatocytes. The 125 kDa antigen was only present in the tripartite segments of SCs, at the inner edge of the lateral elements. Thus, the reaction of the Mab against the 125 kDa antigen was detectable in zygotene, pachytene, and very early diplotene. It appeared later than 30 + 33 kDa and 190 kDa antigens and it disappeared earlier. We found that several steps of the immunostaining procedure could cause variation in the intensity of the Mab reaction.

Tissue distribution of two major components of synaptonemal complexes of the rat.

In this paper we describe an analysis of the tissue distribution of two recently identified components of synaptonemal complexes (SCs), an Mr 125,000 and an Mr 190,000 protein, in the male rat by immunoblot analysis and immunocytochemical techniques. We compared the tissue distribution of these antigens with that of two earlier identified SC components, an Mr 30,000 and an Mr 33,000 polypeptide. For this purpose we used monoclonal antibodies (Mabs) that react exclusively with SCs in lysed spermatocytes, and that recognize the above mentioned antigens specifically in immunoblots of SC proteins or of nuclear proteins from spermatocytes; these were Mab IX9D5 (anti-190,000), Mab IX5B2 (anti-125,000), Mab II52F10 (anti-30,000 + 33,000), and Mab IX8G9 (anti-30,000 + 33,000). In the immunoblot experiments, we could detect the Mr 190,000 and 125,000 antigens exclusively in blots of SC proteins or nuclear proteins from spermatocytes; these antigens were not detectable in blots of nuclear proteins from liver, brain, spermatogonia or spermatids or in blots of proteins from mitotic chromosomes or nuclear laminae. With the anti- 30,000 + 33,000 Mabs we obtained essentially the same result, except that Mab IX8G9, but not II52F10, recognizes a small amount of Mr 30,000 antigen in blots of nuclear proteins from spermatids and spermatogonia. Although this might be ascribed to contamination of the isolated spermatids and spermatogonia, we cannot exclude that a small amount of Mr 30,000 antigen is present in these cells. In the immunofluorescence analysis, the testis was the only tissue that reacted detectably with the above antibodies. Within the testis, spermatocytes and some early spermatids were the only cell types that contained detectable amounts of antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Synaptonemal complex proteins.

Synaptonemal complexes were isolated from rate spermatocytes for the purpose of biochemical and morphological analysis. Several monoclonal antibodies were elicited against purified synaptonemal complexes to study the composition and assembly of these structures. Four classes of antibodies could be discriminated according to the polypeptides that they recognize on Western blots of purified synaptonemal complexes, namely antibodies recognizing (i) a 190-kDa polypeptide; (ii) a 30- and a 33-kDa polypeptide; (iii) two polypeptides with molecular weights of about 120 kDa; and (iv) polypeptides with molecular weights of 66-55 kDa. The localization of these antigens within spermatocytes was analyzed light microscopically, by means of the immunoperoxidase technique and ultrastructurally, by immunogold labelling of surface-spread spermatocytes. The 66- to 55-kDa polypeptides are not confined to synaptonemal complexes; rather, these polypeptides appear to be chromosomal components. The 190-, 30-, and 33-kDa polypeptides make part of the lateral elements of paired as well as unpaired segments of synaptonemal complexes. The 120-kDa polypeptides were localized on the inner edge of the lateral elements, specifically in paired segments of synaptonemal complexes. The distribution of the 190-, 120-, 30-, and 33-kDa polypeptides within the testis was analyzed by immunofluorescence staining of cryostat sections. All these polypeptides turned out to be specific for nuclei of zygotene up to and including diplotene spermatocytes. Only in some early spermatids could the 190-, 30-, and 33-kDa polypeptides be detected, presumably in remnants of synaptonemal complexes.(ABSTRACT TRUNCATED AT 250 WORDS)