XY Body Research Articles

DAZAP1, an hnRNP protein, is required for normal growth and spermatogenesis in mice

DAZAP1 (Deleted in Azoospermia Associated Protein 1) is a ubiquitous hnRNP protein that is expressed most abundantly in the testis. Its ability to shuttle between the nucleus and the cytoplasm and its exclusion from the transcriptionally inactive XY body in pachytene spermatocytes implicate it in mRNA transcription and transport. We generated Dazap1 mutant alleles to study the role of DAZAP1 in mouse development. Most mice homozygous for the null allele as well as a hypomorphic Fn allele died soon after birth. The few Dazap1(Fn/Fn) mice that survived could nonetheless live for more than a year. They appeared and behaved normally but were much smaller in size compared to their wild-type and heterozygous littermates. Both male and female Dazap1(Fn/Fn) mice were sterile. Males had small testes, and the seminiferous tubules were atrophic with increased numbers of apoptotic cells. The tubules contained many germ cells, including pachytene spermatocytes with visible XY-bodies and diplotene spermatocytes, but no post-meiotic cells. FACS analyses confirmed the absence of haploid germ cells, indicating spermatogenesis arrested right before the meiotic division. Female Dazap1(Fn/Fn) mice had small ovaries that contained normal-appearing follicles, yet their pregnancy produced no progeny due to failure in embryonic development. The phenotypes of Dazap1 mutant mice indicate that DAZAP1 is not only essential for spermatogenesis, but also required for the normal growth and development of mice.

In human pachytene spermatocytes, SUMO protein is restricted to the constitutive heterochromatin

SUMO-1, a ubiquitin-like protein, is covalently bound to many proteins, leading to chromatin inactivation and transcriptional repression. The high concentration of SUMO-1 on the XY body in rodents suggests that this protein has an important role in facultative heterochromatin organization. In human, the precise role of SUMO in chromatin/heterochromatin organization remains to be defined. Here we describe the SUMO-1 distribution, during human male meiosis, in relation to the formation of the different types of heterochromatin. We show that, during late pachynema, SUMO-1 appears on the constitutive heterochromatin, but is excluded from the XY body facultative heterochromatin. At the SUMO-1 labelled areas, the presence of HP1alpha protein, as well as of trimethylated H3-K9 and H4-K20 histone modifications, supports a role for SUMO-1 in constitutive heterochromatin organization. We also establish that, on the constitutive heterochromatin, H4-K20me3 staining progressively decreases as SUMO-1 staining increases, suggesting that core histone(s), and histone H4 in particular, are direct targets for sumoylation. Our results also suggest that, in the context of global histone H4 hyperacetylation that precedes the histone-to-protamine transition at postmeiotic stages of spermatogenesis, histone H4 sumoylation may represent an important epigenetic marker replacing methylation on the constitutive heterochromatin.

Developmental control of sumoylation pathway proteins in mouse male germ cells

Protein sumoylation regulates a variety of nuclear functions and has been postulated to be involved in meiotic chromosome dynamics as well as other processes of spermatogenesis. Here, the expression and distribution of sumoylation pathway genes and proteins were determined in mouse male germ cells, with a particular emphasis on prophase I of meiosis. Immunofluorescence microscopy revealed that SUMO1, SUMO2/3 and UBE2I (also known as UBC9) were localized to the XY body in pachytene and diplotene spermatocytes, while only SUMO2/3 and UBE2I were detected near centromeres in metaphase I spermatocytes. Quantitative RT-PCR and Western blotting were used to examine the expression of sumoylation pathway genes and proteins in enriched preparations of leptotene/zygotene spermatocytes, prepubertal and adult pachytene spermatocytes, as well as round spermatids. Two general expression profiles emerged from these data. The first profile, where expression was more prominent during meiosis, identified sumoylation pathway participants that could be involved in meiotic chromosome dynamics. The second profile, elevated expression in post-meiotic spermatids, suggested proteins that could be involved in spermiogenesis-related sumoylation events. In addition to revealing differential expression of protein sumoylation mediators, which suggests differential functioning, these data demonstrate the dynamic nature of SUMO metabolism during spermatogenesis.

Meiotic studies in an azoospermic boar carrying a Y;14 translocation

A reciprocal translocation between the q arm of the Y chromosome and the q arm of chromosome 14 was identified in a young, phenotypically normal boar presenting azoospermia. Testicular biopsies were analyzed by classical histological and immunolocalization techniques, and by fluorescence in situ hybridization. Meiotic pairing analysis of 85 pachytene spreads showed the presence of an open structure corresponding to a quadrivalent formed by chromosomes 14, X, and the derivative chromosomes 14 and Y in 84.7% of the cases. In the remaining cases (15.3%), a ‘trivalent plus univalent’ configuration was observed. Immunolocalization of γH2AX revealed the presence of this modified histone in the chromatin domains of unsynapsed segments (centromeric region of chromosome 14) and spreading of the γH2AX signal from the XY body throughout chromosome 14 in 7.05% of the cells analyzed. The potential causes of the observed infertility, i.e. activation of meiotic checkpoints and/or silencing of genes necessary for the progression of meiosis, are discussed.

Increased frequency of asynapsis and associated meiotic silencing of heterologous chromatin in the presence of irradiation-induced extra DNA double strand breaks

In meiotic prophase of male placental mammals, the heterologous X and Y chromosomes remain largely unsynapsed, which activates meiotic sex chromosome inactivation (MSCI), leading to formation of the transcriptionally silenced XY body. MSCI is most likely related to meiotic silencing of unsynapsed chromatin (MSUC), a mechanism that can silence autosomal unsynapsed chromatin. However, heterologous synapsis and escape from silencing also occur. In mammalian species, formation of DNA double strand breaks (DSBs) during leptotene precedes meiotic chromosome pairing. These DSBs are essential to achieve full synapsis of homologous chromosomes. We generated 25% extra meiotic DSBs by whole body irradiation of mice. This leads to a significant increase in meiotic recombination frequency. In mice carrying translocation chromosomes with synaptic problems, we observed an approximately 35% increase in asynapsis and MSUC of the nonhomologous region in the smallest chromosome pair following irradiation. However, the same nonhomologous region in the largest chromosome pair, shows complete synapsis and escape from MSUC in almost 100% of the nuclei, irrespective of exposure to irradiation. We propose that prevention of synapsis and associated activation of MSUC is linked to the presence of unrepaired meiotic DSBs in the nonhomologous region. Also, spreading of synaptonemal complex formation from regions of homology may act as an opposing force, and drive heterologous synapsis.

Affected Homologous Chromosome Pairing and Phosphorylation of Testis Specific Histone, H2AX, in Male Meiosis Under FKBP6 Deficiency

A gene for FK506 binding protein 6 (Fkbp6) expresses during a specific stage of male and female meiosis. Disruption of the gene influences male reproduction, i.e. arrests spermatogenesis, but not female reproduction. Using the mouse model (targeted disruption), the role of the gene in homologous chromosome pairing has been demonstrated in a previous study. For further understanding the function of Fkbp6 in chromosome synapsis, we evaluated chromosome pairings during male meiosis in the as/as rat, a spontaneous null mutation, and compared them with those of the mouse model. Electron microscopy of the pachytene nuclei unveiled several types of abnormal chromosome pairing in the rat model, as shown in the mouse previously. The frequencies of aberrant pairings in the knockout mice and mutant rats were 42 of 67 nuclei (62.7%) and 20 out of 74 nuclei (27.0%), respectively. In order to clarify the mechanism of male specific infertility in Fkbp6 deficiency, the localization of gammaH2AX, a marker protein of XY chromosome inactivation during male meiosis, was examined. Immunostaining of gammaH2AX unveiled normal localization of the molecule to XY chromosomes (XY body) in both models, showing the independency of FKBP6 in sex chromosome inactivation. Besides the XY body, focal localization of gammaH2AX was observed in accordance with the unsynapsed chromosomes in both types of null animal. These results indicate the fundamental role of Fkbp6 in homologous chromosome synapsis during male meiosis. In conclusion, male specific infertility under Fkbp6 deficiency remains unsolved.

Protein immunolocalization supports the presence of identical mechanisms of XY body formation in eutherians and marsupials

The meiotic sex chromosomes of the American marsupials Monodelphis dimidiata and Didelphis albiventris were studied with electron microscopy (EM) and with immunofluorescence localization of meiotic proteins SYCP1 and SYCP3, and proteins essential for meiotic sex chromosome inactivation (MSCI), gamma-H2AX and BRCA1. The chromatin of the non-synaptic X and Y chromosomes contains gamma-H2AX, first as foci and then as homogeneous staining at late stages. The thick and split X and Y axes are labelled with BRCA1 except at one terminus. The bulgings of the axes contain SYCP1 as well as the inner side of the dense plate. The evenly spaced and highly packed chromatin fibres of the conjoined XY body in these species have the same behaviour and the same components (gamma-H2AX in the chromatin, BRCA1 in the axes) as in the XY body of eutherian species. These observations and recent data from the literature suggest that XY body formation is ancestral to the metatherian-eutherian divergence.

Chromosomal rearrangement interferes with meiotic X chromosome inactivation

Heterozygosity for certain mouse and human chromosomal rearrangements is characterized by the incomplete meiotic synapsis of rearranged chromosomes, by their colocalization with the XY body in primary spermatocytes, and by male-limited sterility. Previously, we argued that such X-autosomal associations could interfere with meiotic sex chromosome inactivation. Recently, supporting evidence has reported modifications of histones in rearranged chromosomes by a process called the meiotic silencing of unsynapsed chromatin (MSUC). Here, we report on the transcriptional down-regulation of genes within the unsynapsed region of the rearranged mouse chromosome 17, and on the subsequent disturbance of X chromosome inactivation. The partial transcriptional suppression of genes in the unsynapsed chromatin was most prominent prior to the mid-pachytene stage of primary spermatocytes. Later, during the mid-late pachytene, the rearranged autosomes colocalized with the XY body, and the X chromosome failed to undergo proper transcriptional silencing. Our findings provide direct evidence on the MSUC acting at the mRNA level, and implicate that autosomal asynapsis in meiosis may cause male sterility by interfering with meiotic sex chromosome inactivation.

Increased phosphorylation and dimethylation of XY body histones in theHr6b-knockout mouse is associated with derepression of the X chromosome

Mono-ubiquitylated H2A marks the transcriptionally silenced XY body during male meiotic prophase. Concomitant with H2A(K119ub1), the ubiquitin-conjugating enzyme HR6B is also enriched on the XY body. We analyzed H2A and H2B ubiquitylation in Hr6b-knockout mouse spermatocytes, but no global changes were detected. Next, we analyzed phosphorylation of the threonine residues T120 and T119 that are adjacent to the K119 and K120 target sites for ubiquitylation in H2A and H2B, respectively. In wild-type cells, H2A(T120ph) and H2B(T119ph) mark meiotically unpaired and silenced chromatin, including the XY body. In Hr6b-knockout spermatocytes, the H2B(T119ph) signal was unchanged, but H2A(T120ph) was enhanced from late pachytene until metaphase I. Furthermore, we found increased H3(K4) dimethylation on the X and Y chromosomes of diplotene Hr6b-knockout spermatocytes, persisting into postmeiotic round spermatids. In these cells, the X and Y chromosomes maintained an unchanged H3(K9m2) level, even when this modification was lost from centromeric heterochromatin. Analysis of gene expression showed derepression of X chromosome genes in postmeiotic Hr6b-knockout spermatids. We conclude that HR6B exerts control over different histone modifications in spermatocytes and spermatids, and that this function contributes to the postmeiotic maintenance of X chromosome silencing.

Trimethylation of Histone H3 Lysine 4 is an Epigenetic Mark at Regions Escaping Mammalian X Inactivation

It is now estimated that 150-200 genes clustered in several discrete regions escape X inactivation in somatic cells of human females by unknown mechanisms. Here, we show that although the human female inactive X chromosome is largely devoid of histone 3 lysine 4 trimethylation (H3K4me3), regions that are known to escape X inactivation, including the pseudoautosomal regions, are enriched with this modification. Also, H3K4me3, unlike H3K4me2 and H4 and H3 acetylation, is restricted to discrete regions on metaphase chromosomes. In contrast to humans, there are only a few genes that are known to escape X inactivation in the mouse. Therefore, we examined mouse female somatic cells with H3K4me3 to identify candidate regions with genes that escape X inactivation. We found the mouse female inactive X in somatic cells and the male inactive X in meiosis to have seven discrete regions that are enriched with H3K4me3. Furthermore, RNA polymerase II is largely excluded from the XY body at male pachytene except for several discrete regions on the X and Y suggesting the presence of regions that also escape sex chromosome inactivation during male meiosis.

A Mammal-Specific Doublesex Homolog Associates with Male Sex Chromatin and Is Required for Male Meiosis

Gametogenesis is a sexually dimorphic process requiring profound differences in germ cell differentiation between the sexes. In mammals, the presence of heteromorphic sex chromosomes in males creates additional sex-specific challenges, including incomplete X and Y pairing during meiotic prophase. This triggers formation of a heterochromatin domain, the XY body. The XY body disassembles after prophase, but specialized sex chromatin persists, with further modification, through meiosis. Here, we investigate the function of DMRT7, a mammal-specific protein related to the invertebrate sexual regulators Doublesex and MAB-3. We find that DMRT7 preferentially localizes to the XY body in the pachytene stage of meiotic prophase and is required for male meiosis. In Dmrt7 mutants, meiotic pairing and recombination appear normal, and a transcriptionally silenced XY body with appropriate chromatin marks is formed, but most germ cells undergo apoptosis during pachynema. A minority of mutant cells can progress to diplonema, but many of these escaping cells have abnormal sex chromatin lacking histone H3K9 di- and trimethylation and heterochromatin protein 1β accumulation, modifications that normally occur between pachynema and diplonema. Based on the localization of DMRT7 to the XY body and the sex chromatin defects observed in Dmrt7 mutants, we conclude that DMRT7 plays a role in the sex chromatin transformation that occurs between pachynema and diplonema. We suggest that DMRT7 may help control the transition from meiotic sex chromosome inactivation to postmeiotic sex chromatin in males. In addition, because it is found in all branches of mammals, but not in other vertebrates, Dmrt7 may shed light on evolution of meiosis and of sex chromatin.

Mammalian Polycomb Scmh1 mediates exclusion of Polycomb complexes from the XY body in the pachytene spermatocytes

The product of the Scmh1 gene, a mammalian homolog of Drosophila Sex comb on midleg, is a constituent of the mammalian Polycomb repressive complexes 1 (Prc1). We have identified Scmh1 as an indispensable component of the Prc1. During progression through pachytene, Scmh1 was shown to be excluded from the XY body at late pachytene, together with other Prc1 components such as Phc1, Phc2, Rnf110 (Pcgf2), Bmi1 and Cbx2. We have identified the role of Scmh1 in mediating the survival of late pachytene spermatocytes. Apoptotic elimination of Scmh1(-/-) spermatocytes is accompanied by the preceding failure of several specific chromatin modifications at the XY body, whereas synapsis of homologous autosomes is not affected. It is therefore suggested that Scmh1 is involved in regulating the sequential changes in chromatin modifications at the XY chromatin domain of the pachytene spermatocytes. Restoration of defects in Scmh1(-/-) spermatocytes by Phc2 mutation indicates that Scmh1 exerts its molecular functions via its interaction with Prc1. Therefore, for the first time, we are able to indicate a functional involvement of Prc1 during the meiotic prophase of male germ cells and a regulatory role of Scmh1 for Prc1, which involves sex chromosomes.

Impact of trisomy on fertility and meiosis in male mice

Chromosomal abnormalities frequently are associated with impairment or arrest of spermatogenesis in mammals but are compatible with fertility in female carriers of the same anomaly. In the case of trisomy, mice have extra genomic DNA as well as the chromosomal abnormality, usually present as an extra, unpaired chromosome. Thus, impairment of spermatogenesis in trisomic males could be due to the presence of extra genomic material (i.e. triplicated genes) or due to the chromosomal abnormality and presence of an unpaired chromosome in meiosis. In this study, fertility and chromosomal pairing configurations during meiotic prophase were analysed in male mice trisomic for different segments of the genome. Four have an extra segmental or tertiary trisomic chromosome--Ts(17(16))65Dn, Ts(10(16))232Dn, Ts(12(17))4Rk and Ts(4(17))2Lws--and one has the triplicated segment attached to another chromosome--Ts(16C-tel)1Cje. Ts(17(16))65Dn and Ts(16C-tel)1Cje have similar gene content triplication and differ primarily in whether the extra DNA is in an extra chromosome or not. The presence of an intact extra chromosome, rather than trisomy per se, is associated with male sterility. Additionally, sterility is correlated with a high frequency of association of the unpaired chromosome with the XY body, which contains the largely unpaired X and Y chromosomes. Intact extra chromosomes disrupt spermatogenesis, and unpaired chromosomes establish a unique chromatin territory within meiotic nuclei.

Whole-arm translocations between chromosome 1 and acrocentric G chromosomes are associated with a poor prognosis for spermatogenesis: two new cases and review of the literature

To analyze unusual translocations involving a chromosome 1 whole arm and an acrocentric G chromosome p arm found in two men with azoospermia. Case report with review of the scientific literature. Cytogenetics department. Two men with azoospermia and normal hormonal levels. Peripheral blood lymphocytes were obtained for karyotype, and metaphases were studied by standard GBG, RBG, and CBG banding procedures. Karyotype GBG, RBG, and CBG banding. Karyotype revealed balanced translocation involving a chromosome 1 whole arm and an acrocentric G chromosome p arm: 46,XY,t(1;21)(q11;p13) (patient 1) and 46,XY,t(1;22)(q11;p11) (patient 2). With regard to published cases of whole-arm translocation of human chromosome 1 with an acrocentric p arm and a maternal origin of these abnormalities, we argue for an impairment of meiosis resulting in a high probability of quadrivalent-XY-body interaction. Male factor infertility might be due to two poor prognostic factors, first the involvement of human chromosome 1 (and its heterochromatic region) and second the involvement of an acrocentric chromosome p-arm breakpoint. This probable interaction between the pachytene quadrivalent and XY body might explain azoospermia.

The asynaptic chromatin in spermatocytes of translocation carriers contains the histone variant γ-H2AX and associates with the XY body

The close apposition of multivalents with the XY body has been repeatedly described in heterozygous carriers of chromosomal rearrangements. Because in many of these carriers spermatogenesis is deeply disturbed at the spermatocyte level, the association of autosomal chromatin with the XY body may impair the spermatocyte life. Testicular biopsies from three men carriers of three different chromosomal rearrangements have been analysed by electron microscopy (EM) and immunolocalization of meiotic proteins. There is an ordered transition from isolated multivalents at early pachytene to XY body association in late pachytene, as shown in a carrier of a rob t(13;14) translocation by EM and in a reciprocal translocation t(9;14) carrier by immunofluorescence. The non-synapsed ends of the quadrivalent show BRCA1 located on the axes and the variant histone gamma-H2AX located on the chromatin. The area covered by gamma-H2AX increases with the association of the asynaptic ends with the XY body in the t(9;14) carrier, and the area covered with gamma-H2AX in the t(Y;15) carrier is larger than that of the XY body of controls. The affinity between the inactive XY body and asynaptic regions of multivalents is given a material basis, and transcriptional inactivation is probably shared by these two chromatin types.

WITHDRAWN: DErratum to “Phosphorylated extracellular signal-regulated kinase 1/2 is localized to the XY body of the meiotic prophase spermatocytes” [Biochem. Biophys. Res. Commun. 346 (2006) 1261–1266

We found that phosphorylated extracellular signal-regulated kinase 1/2 (phospho-ERK1/2) is localized to the XY body of meiotic prophase spermatocytes. A more detailed surface spread analysis showed that phospho-ERK1/2 is localized to the synaptonemal complex of the XY pair of pachytene spermatocytes or the entire XY body of zygotene spermatocytes. In the XY body of meiotic prophase spermatocytes, both transcription and homologous recombination are inactivated. These results suggest a novel function of ERK1/2 in meiotic sex chromosome inactivation .

The X and Y Chromosomes Assemble into H2A.Z, Containing Facultative Heterochromatin, following Meiosis

Spermatogenesis is a complex sequential process that converts mitotically dividing spermatogonia stem cells into differentiated haploid spermatozoa. Not surprisingly, this process involves dramatic nuclear and chromatin restructuring events, but the nature of these changes are poorly understood. Here, we linked the appearance and nuclear localization of the essential histone variant H2A.Z with key steps during mouse spermatogenesis. H2A.Z cannot be detected during the early stages of spermatogenesis, when the bulk of X-linked genes are transcribed, but its expression begins to increase at pachytene, when meiotic sex chromosome inactivation (MSCI) occurs, peaking at the round spermatid stage. Strikingly, when H2A.Z is present, there is a dynamic nuclear relocalization of heterochromatic marks (HP1beta and H3 di- and tri-methyl K9), which become concentrated at chromocenters and the inactive XY body, implying that H2A.Z may substitute for the function of these marks in euchromatin. We also show that the X and the Y chromosome are assembled into facultative heterochromatic structures postmeiotically that are enriched with H2A.Z, thereby replacing macroH2A. This indicates that XY silencing continues following MSCI. These results provide new insights into the large-scale changes in the composition and organization of chromatin associated with spermatogenesis and argue that H2A.Z has a unique role in maintaining sex chromosomes in a repressed state.

Mouse MAELSTROM: the link between meiotic silencing of unsynapsed chromatin and microRNA pathway?

Meiotic silencing of unsynapsed chromatin (MSUC) is a key mechanism in spermatogenesis and a model system to study the dynamics of gene silencing. Here we show that MAEL, the ortholog of Drosophila's high mobility group box protein Maelstrom, is associated not only with the silenced XY body, but also with unsynapsed autosomes. Characterization of MAEL revealed that it interacts directly with the chromatin remodeler SNF5/INI1 and chromatin-associated protein SIN3B, which we also find localized to the XY body. This is the first time that a chromatin remodeler has been shown to associate with whole chromosomes. In addition, we show that MAEL is a component of the mouse meiotic nuage and its haploid cell counterpart, the chromatoid body. This is a site of accumulation of RNA and RNA processing enzymes, including proteins involved in the microRNA (miRNA) pathway. Furthermore, in the nuage, MAEL is present in a complex with germ cell specific MVH, an RNA helicase and Argonaute family members, MILI and MIWI. The presence of MAEL in these critical compartments of male germ cells and its interactions provide a link suggesting the involvement of the miRNA pathway in MSUC.

Phosphorylated extracellular signal-regulated kinase 1/2 is localized to the XY body of meiotic prophase spermatocytes

We found that phosphorylated extracellular signal-regulated kinase 1/2 (phospho-ERK1/2) is localized to the XY body of meiotic prophase spermatocytes. A more detailed surface spread analysis showed that phospho-ERK1/2 is localized to the synaptonemal complex of the XY pair of pachytene spermatocytes or the entire XY body of zygotene spermatocytes. In the XY body of meiotic prophase spermatocytes, both transcription and homologous recombination are inactivated. These results suggest a novel function of ERK1/2 in meiotic sex chromosome inactivation.

Postmeiotic Sex Chromatin in the Male Germline of Mice

In mammals, the X and Y chromosomes are subject to meiotic sex chromosome inactivation (MSCI) during prophase I in the male germline, but their status thereafter is currently unclear. An abundance of X-linked spermatogenesis genes has spawned the view that the X must be active . On the other hand, the idea that the imprinted paternal X of the early embryo may be preinactivated by MSCI suggests that silencing may persist longer . To clarify this issue, we establish a comprehensive X-expression profile during mouse spermatogenesis. Here, we discover that the X and Y occupy a novel compartment in the postmeiotic spermatid and adopt a non-Rabl configuration. We demonstrate that this postmeiotic sex chromatin (PMSC) persists throughout spermiogenesis into mature sperm and exhibits epigenetic similarity to the XY body. In the spermatid, 87% of X-linked genes remain suppressed postmeiotically, while autosomes are largely active. We conclude that chromosome-wide X silencing continues from meiosis to the end of spermiogenesis, and we discuss implications for proposed mechanisms of imprinted X-inactivation.