Male Meiotic Chromosomes Research Articles

BRCA1, Histone H2AX Phosphorylation, and Male Meiotic Sex Chromosome Inactivation

In mammalian spermatogenesis, the X and Y chromosomes are transcriptionally silenced during the pachytene stage of meiotic prophase (meiotic sex chromosome inactivation, MSCI), forming a condensed chromatin domain termed the sex or XY body. The nucleosomal core histone H2AX is phosphorylated within the XY chromatin domain just prior to MSCI, and it has been hypothesized that this triggers the chromatin condensation and transcriptional repression. Here, we show that the kinase ATR localizes to XY chromatin at the onset of MSCI and that this localization is disrupted in mice with a mutant form of the tumor suppressor protein BRCA1. In the mutant pachytene cells, ATR is usually present at nonsex chromosomal sites, where it colocalizes with aberrant sites of H2AX phosphorylation; in these cells, there is MSCI failure. In rare pachytene cells, ATR does locate to XY chromatin, H2AX is then phosphorylated, a sex body forms, and MSCI ensues. These observations highlight an important role for BRCA1 in recruiting the kinase ATR to XY chromatin at the onset of MSCI and provide compelling evidence that it is ATR that phosphorylates H2AX and triggers MSCI.

Etoposide exposure during male mouse pachytene has complex effects on crossing-over and causes nondisjunction

In experiments involving different germ-cell stages, we had previously found meiotic prophase of the male mouse to be vulnerable to the induction of several types of genetic damage by the topoisomerase-II inhibitor etoposide. The present study of etoposide effects involved two end points of meiotic events known to occur in primary spermatocytes—chromosomal crossing-over and segregation. By following assortment of 13 microsatellite markers in two chromosomes (Ch 7 and Ch 15) it was shown that etoposide significantly affected crossing-over, but did not do so in a uniform fashion. Treatment generally changed the pattern for each chromosome, leading to local decreases in recombination, a distal shift in locations of crossing-over, and an overall decrease in double crossovers; at least some of these results might be interpreted as evidence for increased interference. Two methods were used to explore etoposide effects on chromosome segregation: a genetic experiment capable of detecting sex-chromosome nondisjunction in living progeny; and the use of FISH (fluorescence in situ hybridization) technology to score numbers of Chromosomes X, Y, and 8 in spermatozoa. Taken together these two approaches indicated that etoposide exposure of pachytene spermatocytes induces malsegregation, and that the findings of the genetic experiment probably yielded a marked underestimate of nondisjunction. As indicated by certain segregants, at least part of the etoposide effect could be due to disrupted pairing of achiasmatic homologs, followed by precocious sister-centromere separation. It has been shown for several organisms that absent or reduced levels of recombination, as well as suboptimally positioned recombination events, may be associated with abnormal segregation. Etoposide is the only chemical tested to date for which living progeny indicates an effect on both male meiotic crossing-over and chromosome segregation. Whether, however, etoposide-induced changes in recombination patterns are direct causes of the observed malsegregation requires additional investigation.

Functional analysis of the Drosophila Rad51 gene ( spn-A) in repair of DNA damage and meiotic chromosome segregation

Rad51 is a crucial enzyme in DNA repair, mediating the strand invasion and strand exchange steps of homologous recombination (HR). Mutations in the Drosophila Rad51 gene ( spn-A) disrupt somatic as well as meiotic double-strand break (DSB) repair, similar to fungal Rad51 genes. However, the sterility of spn-A mutant females prevented a thorough analysis of the role of Rad51 in meiosis. In this study, we generated transgenic animals that express spn-A dsRNA under control of an inducible promoter, and examined the effects of inhibiting expression of spn-A on DNA repair, meiotic recombination and meiotic chromosome pairing and segregation. We found that depletion of spn-A mRNA had no effect on the viability of non-mutagen-treated transgenic animals but greatly reduced the survival of larvae that were exposed to the radiomimetic drug MMS, in agreement with the MMS and X-ray sensitivity of spn-A mutant animals. We also found that increases in dose of spn-A gene enhanced larval resistance to MMS exposure, suggesting that at high damage levels, Rad51 protein levels may be limiting for DNA repair. spn-A RNAi strongly stimulated X–X nondisjunction and decreased recombination along the X in female meiosis, consistent with a requirement of Rad51 in meiotic recombination. However, neither RNAi directed against the spn-A mRNA nor homozygosity for a spn-A null mutation had any effect on male fertility or on X–Y segregation in male meiosis, indicating that Rad51 likely plays no role in male meiotic chromosome pairing. Our results support a central role for Rad51 in HR in both somatic and meiotic DSB repair, but indicate that Rad51 in Drosophila is dispensable for meiotic chromosome pairing. Our results also provide the first demonstration that RNAi can be used to inhibit the functions of meiotic genes in Drosophila.

The DUET gene is necessary for chromosome organization and progression during male meiosis in Arabidopsis and encodes a PHD finger protein.

Progression through the meiotic cell cycle is an essential part of the developmental program of sporogenesis in plants. The duet mutant of Arabidopsis was identified as a male sterile mutant that lacked pollen and underwent an aberrant male meiosis. Male meiocyte division resulted in the formation of two cells instead of a normal tetrad. In wild type, male meiosis extends across two successive bud positions in an inflorescence whereas in duet, meiotic stages covered three to five bud positions indicating defective progression. Normal microspores were absent in the mutant and the products of the aberrant meiosis were uni- to tri-nucleate cells that later degenerated, resulting in anthers containing largely empty locules. Defects in male meiotic chromosome organization were observed starting from diplotene and extending to subsequent stages of meiosis. There was an accumulation of meiotic structures at metaphase 1, suggesting an arrest in cell cycle progression. Double mutant analysis revealed interaction with dyad, a mutation causing chromosome cohesion during female meiosis. Cloning and molecular analysis of DUET indicated that it potentially encodes a PHD-finger protein and shows specific expression in male meiocytes. Taken together these data suggest that DUET is required for male meiotic chromosome organization and progression.

The meiotic chromosomes of maleDrosophila willistoni

The male meiotic chromosomes of Drosophila willistoni are described and compared to Giemsa stained mitotic chromosomes. Meiosis was examined in males from two wild type stocks: WIP-4, an almost monomorphic stock, and 17A2, a high-inversion polymorphic stock. Hybrids between these stocks were also examined. Stocks and hybrids were cultured at 25oC and at 29oC, a tem- perature condition thought to induce male recombination associated with trans- posable element(s) mobilisation. Chromatin in primary spermatocytes is seen as pachytene and early diplotene bivalents, representing the two autosomal bivalents and a steady association between X and Y. Bivalents remain relatively extended from diplotene through anaphase I, which is followed by the conventional stages of meiosis II. Neither chiasmata nor chromatid bridges and fragments were detected in both control and experimental males. Thread-like bubble structures on the distal region of the X chromosome were observed in early diplotene cells from F1 male offspring between WIP-4 and 17A2 reared at 29oC; their functional meaning is unknown.

Meiotic sex chromosome inactivation in male mice with targeted disruptions of Xist.

X chromosome inactivation occurs twice during the life cycle of placental mammals. In normal females, one X chromosome in each cell is inactivated early in embryogenesis, while in the male, the X chromosome is inactivated together with the Y chromosome in spermatogenic cells shortly before or during early meiotic prophase. Inactivation of one X chromosome in somatic cells of females serves to equalise X-linked gene dosage between males and females, but the role of male meiotic sex chromosome inactivation (MSCI) is unknown. The inactive X-chromosome of somatic cells and male meiotic cells share similar properties such as late replication and enrichment for histone macroH2A1.2, suggesting a common mechanism of inactivation. This possibility is supported by the fact that Xist RNA that mediates somatic X-inactivation is expressed in the testis of male mice and humans. In the present study we show that both Xist RNA and Tsix RNA, an antisense RNA that controls Xist function in the soma, are expressed in the testis in a germ-cell-dependent manner. However, our finding that MSCI and sex-body formation are unaltered in mice with targeted mutations of Xist that prevent somatic X inactivation suggests that somatic X-inactivation and MSCI occur by fundamentally different mechanisms.

Mitotic and meiotic chromosomes in Somatochlora metallica (Cordulidae, Odonata). The absence of localized centromeres and inverted meiosis.

Spermatogonial metaphase chromosomes were examined in two dragonfly species, Somatochlora metallica (Cordulidae) and Aeshna grandis (Aeshnidae), and the behaviour of male meiotic chromosomes was studied in S. metallica. Both in S. metallica and A. grandis the male mitotic metaphase chromosomes from cells treated with colchicine consisted of two equidistantly aligned chromatids, showing no primary constriction. In meiosis the chromosomes of S. metallica males showed telokinetic activity during the first meiotic division, and kinetic activity was restricted in the middle parts of chromosomes during the second division. The kinetic behaviour of the chromosomes both in mitosis and meiosis showed that they were holocentric. One chiasma arises interstitially in each bivalent in S. metallica male meiosis. The chiasmata retain their interstitial position at metaphase I and do not terminalize. At metaphase I bivalents co-orient with homologous telomere regions towards the opposite poles. Thus genuine dyads segregate at the first anaphase. Meiosis in these male dragonflies is thus pre-reductional or conventional, not post-reductional or inverted, as has been previously proposed.

MITOTIC AND MEIOTIC CHROMOSOMES OF ARTEMIA (BRANCHIOPODA) FROM POPULATIONS OF LA PAMPA PROVINCE, ARGENTINA

Three populations of Artemia from La Pampa Province, Argentina, were cytogenetically analyzed: Salinas Grandes de Hidalgo, Laguna Callaqueo, and Laguna Colorada Chica. Both mitotic and male meiotic chromosomes were studied. All 3 populations share the same diploid (2n = 44) and/or haploid chromosome number (n = 22), and hence were determined as Artemia persimilis Piccinelli and Prosdocimi. Although bivalents decrease gradually in size, differences among the larger and the smaller ones were apparent. So far, no cytogenetic differences have been detected among the 3 populations. Chromosomes with metacentric, submetacentric, and telocentric morphology were detected in mitotic prometaphase cells, suggesting that chromosomes of Artemia may be monocentric. The anostracan genus Artemia is taxonomically regarded as a group of several reproductively isolated sibling species, distributed worldwide, except for the Antarctic continent. In the New World, the genus is represented by three endemic bisexual species: A. franciscana Kellogg, 1906, A. persimilis Piccinelli and Prosdocimi, 1968, and A. monica Verrill, 1869.

Study of mouse synaptonemal complex after injection of camptotecin

Total synaptonemal complexes of male mouse meiotic chromosomes were examined under electron microscope at different periods after a single injection of the antitumor drug camptotecin. At the stage of meiosis prophase I, the drug induced sharp decondensation of chromatin, distention of chromosomes and elements of synaptonemal complex, and blocked desynapsis of homologous chromosomes at the diplotene stage, thus “arresting” damaged cells. The effect of camptotecin lasted 34 days after injection and involved 15 to 90% cells in different periods.

Chromosomal Assignment of 311 Sequences Transcribed in Human Adult Testis

A total of 311 expressed sequence tags (ESTs) derived from human adult testis have been assigned to human chromosomes by Southern analysis of a monochromosome somatic cell hybrid panel. Over 70% of the ESTs show conservation to hamster and mouse DNA, and the overall distribution of transcripts correlates well with physical chromosome size and to a greater extent with male meiotic chromosome length. The notable exception is the X chromosome, for which the number of testis- derived ESTs is greatly underrepresented. This finding may reflect inactivation of the X chromosome during the meiotic phase of spermatogenesis and a consequent selection against large numbers of X-linked germ cell transcripts. Further analysis of the distribution of testis ESTs showed that the EST density remains significantly correlated with the recombination density of each autosome. Analysis of a comparable number (320) of brain EST autosome assignments showed no similar correlation. These data suggest a specific association between transcription in testis tissue and male meiotic recombination.

Generation of mouse chromosome painting probes by DOP-PCR amplification of microdissected meiotic chromosomes.

We describe here the development of four sets of painting probes for mouse chromosomes 1 and 13, 2 and 8, 6 and 15, and X and Y by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) amplification of 10-20 copies of microdissected male meiotic chromosomes. The X,Y probe was obtained from the X-Y bivalent of diakinesis/metaphase I complements of mice with a normal karyotype, whereas the other probes were derived from tri- or quadrivalents in diakinesis/metaphase I of two reciprocal translocations, T(1;13)70H and T(2;8)2Wa, and one inversion heterozygote carrying a small deletion, In(6;15)Rb1Ald del6(15)1Wa. The specificity of these probes was established by fluorescence in situ hybridization (FISH) to meiotic and mitotic metaphase complements. The chromosomes painted by these probes could be identified by single or multicolor FISH.

Mutagenic Potential of the Non-nodulous Nitrogen-fixing Bacterium, Xanthobacter flavus in Treated Male Parent Mice and Verified to Their F1 Progeny.

The mutagenic potential of log culture (20×107 cells per ml) of the non-nodulous nitrogenfixing free living bacterium, Xanthobacter flavus in treated male parent mice after mating with different sets of virgin untreated normal females for 7 consecutive weeks and in their F1 progeny verified by lethal test in vivisected mothers and in living ones by various cytogenetic assays was found positive in each test as compared to parallel controls. In male parents as well as in both sexes of F1 embryos and adults, as the cases might be, the frequencies of chromosome aberration and micronucleated erythrocytes in somatic cells, male meiotic chromosome aberration and sperm head abnormality were strikingly high in treated series than in respective controls, indicating the same trends of cytogenetical effects in parent and F1 progeny of treated series. The results have been explained with the hypothesis that the treatment of log culture of X. flavus to male parent mice induced mutation to some genetic locus/loci which in normal state maintained the structural integrity of chromosome, but when mutated lost the control. This might have led to various cytogenetic effects in F1 progeny, in some cases caused lethality while in others (living ones) produced visible cytogenetic anomalies of various forms assessed by different testing protocols.

The DNA content of Chinese hamster meiotic metaphase chromosomes.

DNA values of Chinese hamster male meiotic metaphase chromosomes were measured by slide-based Feulgen cytometry. All the autosomes were distinguishable on the basis of their DNA content. No significant differences were found between the autosomes of the two male animals studied, but a significant difference was found in the DNA content of the sex-chromosome pair between these two animals.

Regions of active chromatin conformation in 'inactive' male meiotic sex chromosomes of the mouse.

The sensitivity to DNase I of the meiotic sex chromosomes of the male mouse was determined by in situ nick translation. At pachytene and diakinesis-metaphase I, six segments, four at the ends of the X and Y chromosomes and two at internal sites on the X chromosome, were found to be more sensitive than the other parts of these chromosomes. The sensitive segments presumably reflect an active or potentially active chromatin conformation which is maintained in the sex chromosomes despite the earlier reported, almost complete cessation of uridine incorporation. The distribution of regions which are sensitive to DNase I corresponds to that of early DNA replication bands. Active conformation patterns like those figured here, probably exist in the sex chromosomes of other mammals as well.

Cytotaxonomy: II: Standard chromosomes of the Simulium (Edwardsellum) damnosum Theobold (Dipkra: Simuliidae)

Male meiotic chromosomes and larval salivary gland polytene chromosomes were studied in Feulgen squash preparations of Simulium (Edwardsellum) damnosum Theobald. The position of the primary constrictions of the three meiotic bivalents approximate the position of the centromeres in the three giant salivary gland polytene chromosomes. A description of the standard chromosomes of the subgenus and the micromophological landmarks associated with each chromosome is given and illustrated with microphotographs. The value of these landmarks, the uniqueness of the banding pattern and the use of both in the diagnosis of species complexes are discussed.

Analyse des caryotypes métaphasiques méiotiques chez le mâle dePleurodeles waltlii (Amphibien, Urodèle) après coloration des chromosomes par l'argent ammoniacal

Analysis of metaphasic meiotic karyotypes in male of Pleurode les waltlii(Amphibia, Urodela) after silver ammoniacal staining of chromosomes. In the newtPleurodeles waltlii, the ammoniacal silver staining technique was applied to the male meiotic chromosomes at metaphase I and II. A specific staining of paracentromeric heterochromatin and of the centromere is observed on each chromosome of the complements. The two karyotypes are analysed and the homology between meiotic and mitotic chromosomes is established.

XX/XY-Sex Chromosomes in Gekko gecko (Sauria, Reptilia)

The mitotic karyotype, chromosomal banding patterns and male meiotic chromosomes of Gekko gecko were studied in differentially stained preparations obtained from bone marrow and testes. A chromosome number of 38 was found with a heteromorphic chromosome pair in the male sex, which indicates a XX/XY-mechanism of chromosomal sex determination. The Y-chromosome was found to be larger than the X-chromosome.

Germ line polysomy in the grasshopper Atractomorpha similis

Nineteen Eastern Australian populations of the grasshopper Atractomorpha similis (Acridoidea, Pyrgomorphidae) were sampled and male meiotic chromosomes, as well as some male and female somatic mitoses, were examined. In fourteen of these populations, a proportion of the males were found to carry between one and ten extra copies of a particular autosome, the megameric chromosome (A9). Numbers of extra chromosomes varied between but not within the individual follicles of the testis. The extra chromosomes were not found in somatic tissue. In all, 20% of males from the field were germ-line polysomic and within these males, 91% of germ cells were polysomic. In meiosis, extra copies of A9 present as univalents lagged at anaphase I or II and subsequently formed micronuclei which degenerated early in spermiogenesis. As one extra univalent is the most common polysomic condition in natural populations, this elimination of univalents suggests that most polysomic males produce a large proportion of normal haploid sperm. In laboratory cultures, selection for increased frequency of germ-line polysomy, conducted over four generations, raised the proportion of polysomic males from 23% to 71%. Selection against polysomy reduced its frequency to 5%. These breeding experiments also showed that germ-line polysomy is equally transmissible through both the male and the female parent. Transmission data also suggested that these extra chromosomes can arise de novo, presumably by unequal disjunction in previously diploid lines. A computer model was devised, simulating the effects of repeated non-disjunction over a series of mitotic divisions. The behaviour of this model suggested that the distributions of extra chromosome numbers observed in the laboratory generations most probably resulted from such a series of non-disjunctions, occurring in an initially diploid cell population. It seems, therefore, that the transmission of polysomy occurs through the agency of heritable factors which determine the probability of non-disjunction and thus the accumulation of a particular autosome during a specific series of mitotic divisions in the embryonic germ-line.

Meiotic crossing over exchange in the female mouse visualised by BUdR substitution

ALTHOUGH chiasmata have long been thought to be sites of crossing over exchange between chromosomes during meiosis, in keeping with the partial chiasmatype hypothesis1,2, there has been no convincing visual evidence of this despite many attempts (summarised in refs 3–6). Taylor7 confirmed by autoradiography that meiotic crossing over occurs by exchange involving breakage and reunion of chromatids. However, because of the poor resolution of autoradiography, requiring study of meiotic chromosomes after 1st metaphase, and because of interference by sister chromatid exchanges, a one-to-one correspondence between chiasmata and exchanges of labelled chromatid segments could not be established8,9. The difficulty was alleviated by correlating chiasmata and exchanges with temperature variation10, or by selecting a species with localised chiasmata and thus with predictable points of exchange11. 5-Bromodeoxyuridine (BUdR) substitution with differential labelling and consequent differential staining of mitotic chromosomes12–15 offer the potential for studying chromosomes at 1st meiotic metaphase, giving an unequivocal picture and separating sister chromatid exchanges from chiasma exchange points between homologues. Tease16 has reported success with male grasshopper meiotic chromosomes, but an attempt with the male mouse showed only sister chromatid exchanges in the sex chromosomes, and the autosomal bivalents at 1st meiotic metaphase were not differentially stained17. In the male Armenian hamster the sex chromosomes showed differential staining and an apparent exchange of segments but again the autosomes were not critically differentially stained18. This may be partly due to the make-up of the male meiotic chromosomes. We have overcome these problems by using an in vitro/in vivo technique19 which enables us to expose the differentiating germ cells of embryonic ovaries to BUdR in vitro and to collect and display the chromosomes20 of the mature oocytes some time after transplantation into young spayed adults. We report our results here.

Cytological studies of heterochromatin function in the Drosophila melanogaster male: autosomal meiotic paring.

In Drosophila melanogaster it is now documented that the different satellite DNA sequences make up the majority of the centromeric heterochromatin of all chromosomes. The most popular hypothesis on this class of DNA is that satellite DNA itself is important to the pairing processes of chromosomes. Evidence in support of such a hypothesis is, however, circumstantial. This hypothesis has been evaluated by direct cytological examination of the meiotic behaviour of heterochromatically and/or euchromatically rear-ranged autosomes in the male. It was found that neither substantial deletions nor rearrangements of the autosomal heterochromatin cause any disruption of meiotic pairing. Autosomal pairing depends on homologs retaining sufficient euchromatic homology. This is the first clear demonstration that the highly repeated satellite DNA sequences in the heterochromatin of the second, third and fourth chromosomes are not important in meiotic pairing, but rather than some euchromatic homology in the autosome is essential to ensure a regular meiotic process. These results on the autosomes, when taken in conjunction with our previous studies on sex chromosome pairing, clearly indicate that satellite DNA is not crucial for male meiotic chromosome pairing of any member of the D. melanogaster genome.