Evidence that meiotic sex chromosome inactivation is essential for male fertility

The mammalian X and Y chromosomes share little homology and are largely unsynapsed during normal meiosis. This asynapsis triggers inactivation of X- and Y-linked genes, or meiotic sex chromosome inactivation (MSCI). Whether MSCI is essential for male meiosis is unclear. Pachytene arrest and apoptosis is observed in mouse mutants in which MSCI fails, e.g., Brca1(-/-), H2afx(-/-), Sycp1(-/-), and Msh5(-/-). However, these also harbor defects in synapsis and/or recombination and as such may activate a putative pachytene checkpoint. Here we present evidence that MSCI failure is sufficient to cause pachytene arrest. XYY males exhibit Y-Y synapsis and Y chromosomal escape from MSCI without accompanying synapsis/recombination defects. We find that XYY males, like synapsis/recombination mutants, display pachytene arrest and that this can be circumvented by preventing Y-Y synapsis and associated Y gene expression. Pachytene expression of individual Y genes inserted as transgenes on autosomes shows that expression of the Zfy 1/2 paralogs in XY males is sufficient to phenocopy the pachytene arrest phenotype; insertion of Zfy 1/2 on the X chromosome where they are subject to MSCI prevents this response. Our findings show that MSCI is essential for male meiosis and, as such, provide insight into the differential severity of meiotic mutations' effects on male and female meiosis.     

Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation

Chromosome synapsis during zygotene is a prerequisite for the timely homologous recombinational repair of meiotic DNA double-strand breaks (DSBs). Unrepaired DSBs are thought to trigger apoptosis during midpachytene of male meiosis if synapsis fails. An early pachytene response to asynapsis is meiotic silencing of unsynapsed chromatin (MSUC), which, in normal males, silences the X and Y chromosomes (meiotic sex chromosome inactivation [MSCI]). In this study, we show that MSUC occurs in Spo11-null mouse spermatocytes with extensive asynapsis but lacking meiotic DSBs. In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response. We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs. All four mutants fail to silence the X and Y chromosomes (MSCI failure), which is sufficient to explain the midpachytene apoptosis. Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.     

Sex chromosome configurations in pachytene spermatocytes of an XYY mouse

Karyotypic investigation of a phenotypically normal but sterile male mouse showed the presence of an XYY sex chromosome constitution. The synaptic behaviour of the three sex chromosomes was examined in 65 pachytene cells. The sex chromosomes formed a variety of synaptic configurations: an XYY trivalent (40%); an XY bivalent and Y univalent (38.5%); an X univalent and YY bivalent (13.8%); or X, Y, Y univalence (7.7%). There was considerable variation in the extent of synapsis and some of the associations clearly involved nonhomologous pairing. These observations have been compared with previously published information on chromosome configurations at metaphase I from other XYY males.     

An analysis of meiotic impairment and of sex chromosome associations throughout meiosis in XYY mice

The existing XYY meiotic data for mice present a very heterogeneous picture with respect to the relative frequencies of different sex chromosome associations, both at pachytene and diakinesis/metaphase I. Furthermore, where both pachytene and diakinesis/MI data are available for the same males, the frequencies of the different configurations at the two stages are very different. In the present paper we utilise "XYY" and "XY/XYY" mosaic mice with cytologically distinguishable Y chromosomes to investigate the factors responsible for this heterogeneity between different males and between the two meiotic stages. It is concluded (1) that the initial pattern of synapsis is driven by the relatedness of the three pseudoautosomal regions (PARs); (2) that the order and extent of PAR synapsis within radial trivalents are also affected by PAR relatedness and that this leads to chiasmata being preferentially formed between closely related PARs; (3) that trivalents with a single chiasma resolve into a bivalent + univalent by the diakinesis stage; (4) that although many spermatocytes with asynapsed sex chromosomes are eliminated between pachytene and diakinesis, those that survive this phase of elimination progress to the first meiotic metaphase (MI) and accumulate in large numbers, leading to an over-representation of those with univalents as compared to radial trivalents; and (5) that the arrested MI cells are eventually eliminated, so that very few "XYY" cells contribute products to MII.     

Fertile male mice with three sex chromosomes: Evidence that infertility in XYY male mice is an effect of two Y chromosomes

In the mouse XYY males are sterile, presumably because pairing abnormalities resulting from the presence of three sex chromosomes lead to meiotic breakdown. We have produced male mice, designated XYY^*X, that have three sex chromosome pairing regions but only one intact Y chromosome. Unexpectedly XYY^*X males are fertile, although they are no more efficient in sex chromosome pairing than previously reported XYY males. We conclude that the sterility of XYY males is caused by a combination of the deleterious effect of two Y chromosomes, presumably acting prior to meiosis, and pairing abnormalities resulting in significant meiotic disruption.by P.B. Moens     

Recombination in the pseudoautosomal region in a 47,XYY male

Males with a 47,XYY karyotype generally have chromosomally normal children, despite the high theoretical risk of aneuploidy. Studies of sperm karyotypes or FISH analysis of sperm have demonstrated that the majority of sperm are chromosomally normal in 47,XYY men. There have been a number of meiotic studies of XYY males attempting to determine whether the additional Y chromosome is eliminated during spermatogenesis, with conflicting results regarding the pairing of the sex chromosomes and the presence of an additional Y. We analyzed recombination in the pseudoautosomal region of the XY bivalent to determine whether this is perturbed in a 47,XYY male. A recombination frequency similar to normal 46,XY men would indicate normal pairing within the XY bivalent, whereas a significantly altered frequency would suggest other types of pairing such as a YY bivalent or an XYY trivalent. Two DNA markers, STS/STS pseudogene and DXYS15, were typed in sperm from a heterozygous 47,XYY male. Individual sperm (23,X or Y) were isolated into PCR tubes using a FACStarPlus flow cytometer. Hemi-nested PCR analysis of the two DNA markers was performed to determine the frequency of recombination. A total of 108 sperm was typed with a 38% recombination frequency between the two DNA markers. This is very similar to the frequency of 38.3% that we have observed in 329 sperm from a normal 46,XY male. Thus our results suggest that XY pairing and recombination occur normally in this 47,XYY male. This could occur by the production of an XY bivalent and Y univalent (which is then lost in most cells) or by loss of the additional Y chromosome in some primitive germ cells or spermatogonia and a proliferative advantage of the normal XY cells.     

Robertsonian translocations modify genomic distribution of γH2AFX and H3.3 in mouse germ cells

Heterozygosity for Robertsonian translocations hampers pairing and synapsis between the translocated chromosome and its normal homologs during meiotic prophase I. This causes meiotic silencing of unsynapsed chromatin in pericentromeric regions. Several lines of evidence suggest that autosomal asynapsis leads to meiotic arrest in males and two underlying mechanisms have been proposed: (1) reactivation of the X and Y chromosomes due to competition for silencing factors and (2) meiotic silencing of genes that are located in the unsynapsed regions and are essential for meiotic progression. The latter mechanism requires that asynapsis and meiotic silencing spread beyond the p-arms of the normal homologs into gene-rich regions. We used chromatin immunoprecipitation assays to determine whether histones γH2AFX and H3.3, both marks of asynapsis and meiotic silencing, are enriched in gene-rich regions of the translocated chromosomes and their homologs in the spermatocytes of heterozygous carriers of Robertsonian translocations. We also asked if γH2AFX and H3.3 enrichment was reduced at the X chromosome and if γH2AFX and H3.3 enrichment was higher on the normal homolog. Our data show that γH2AFX enrichment extends as far as 9–15 Mb of the annotated genomic sequence of the q-arms of the translocated chromosomal trivalents and that both γH2AFX and H3.3 levels are reduced over the X chromosome. Our data are also suggestive of an asymmetry in γH2AFX and H3.3 enrichment with a bias toward the non-translocated homolog.     

The XYY condition in a wild mammal: an XY/XYY mosaic common shrew (Sorex araneus)

XY/XYY sex-chromosome mosaicism was demonstrated in both bone marrow and germ cells of a wild adult common shrew. Secondary sexual characteristics were those of a normal male, but the testes were small, and the sperm count was only about 3% of normal. Most of the seminiferous tubule cross-sections examined revealed serious spermatogenic impairment and a reduced diameter. A range of sex-chromosome pairing configurations was observed in XYY primary spermatocytes, including configurations involving the X and both Y chromosomes in a linear or radial array. The presence of metaphase II (MII) spreads with an XY sex-chromosome complement indicated that XYY primary spermatocytes could contribute products to MII. Following Burgoyne (1979) and Burgoyne and Biddle (1980), a number of models of spermatocyte loss were tested. The data indicated that there was an association between the sex-chromosome complement of primary spermatocytes and their contribution to MII. The best fit to the observed MII frequency data was provided by a model which assumed that all XYY primary spermatocytes with a univalent Y chromosome and a high proportion of XYY primary spermatocytes with an unpaired X chromosome failed to contribute products to MII.     

XYY spermatogenesis in XO/XY/XYY mosaic mice

The relative frequencies of XYY and XY cells in XO/XY/XYY mosaic mice were compared between somatic cells (bone marrow) and spermatogonia, and between spermatogonia and pachytene or MI spermatocytes. The results indicated there was no selection either for or against XYY spermatogonia. There was, however, a strong selection against XYY spermatocytes during pachytene, with their almost total elimination by the first meiotic metaphase. At pachytene, most XYY cells had trivalent or X univalent/YY bivalent configurations. These findings are contrasted with previous studies of XYY spermatogenesis in mice and are discussed with respect to a model that invokes sex-chromosome univalence as the cause of XYY spermatogenic failure.     

Parallel occurrence of asynaptic sex chromosomes in gray voles (Microtus Schrank, 1798)

In many eutherian species, pairing and recombination of X and Y chromosomes are indispensable for normal meiotic progression and correct segregation of sex chromosomes. The rodent subfamily Arvicolinae provides an interesting exception. The majority of arvicoline species with asynaptic sex chromosomes belong to the genus Microtus sensu lato. However, some vole species of the genus Microtus and other genera display normal X-Y pairing in meiosis. These observations indicate that synaptic condition was typical for the common ancestor of all voles, but the gaps in taxonomic sampling makes impossible to identify a lineage or lineages, in which the asynapsis occurred. The methods of electron and fluorescent microscopy were used to study the synapsis of sex chromosomes in males of some additional species of the subfamily Arvicolinae. This extended taxonomic list allowed us to identify asynaptic species in every large lineage of the tribe Microtini. Apparently, the ability of sex chromosomes to pair and recombine in male meiosis was lost in arvicoline evolution for at least three times independently. Our results indirectly suggest the unnecessity of sex chromosome pairing in male meiosis of arvicoline rodents, and presence of alternate molecular mechanism of sex chromosome segregation in this large mammalian tribe.     

Two new X-autosome Robertsonian translocations in the mouse

The influence of X-autosome Robertsonian (Rb) translocation hemizygosity on meiotic chromosome behaviour was investigated in male mice. Two male fertile translocations [Rb(X.2)2Ad and Rb(X.9)6H] and a male sterile translocation [Rb(X.12)7H] were used. In males of all three Rb translocation types, the acrocentric homologue of the autosome involved in the rearrangement regularly failed at pachytene to pair completely with its partner in the Rb metacentric. The centric end of the acrocentric autosome was found regularly to associate either with the proximal end of the Y chromosome or with the ends of nonhomologous autosomal bivalents; the proportions of cells with such configurations varied between pachytene substages and genotypes. Various other categories of synaptic anomaly, such as nonhomologous synapsis, foldback pairing and interlocks, affected the sex chromosome multivalent in a substantial proportion of cells. In one of the Rb(X.12)7H males screened, an unusual, highly aneuploid spermatocyte that contained trivalent and bivalent configurations was found. Rb translocation hemizygosity did not appear to increase to a significant extent the incidence of X-Y pairing failure at pachytene, although the incidence was elevated at metaphase I in Rb(X.12)7H animals. Overall, a comparison of the frequencies and types of chromosome pairing anomalies did not suggest that these were important factors in the aetiology of infertility in males carrying the Rb(X.12)7H translocation.     

Deficiency of X and Y chromosomal pairing at meiotic prophase in spermatocytes of sterile interspecific hybrids between laboratory mice (Mus domesticus) and Mus spretus

The normal association between the X and Y chromosomes at metaphase I of meiosis, as seen in air-dried light microscope preparations of mouse spermatocytes, is frequently lacking in the spermatocytes of the sterile interspecific hybrid between the laboratory mouse strains C57BL/6 and Mus spretus. The purpose of this work is to determine whether the separate X and Y chromosomes in the hybrid are asynaptic, caused by failure to pair, or desynaptic, caused by precocious dissociation. Unpaired X-Y chromosomes were observed in air-dried preparations at diakinesis, just prior to metaphase I. Furthermore, immunocytology and electron microscopy studies of surface-spread pachytene spermatocytes indicate that the X and Y chromosomes frequently fail to initiate synapsis as judged by the failure to form a synaptonemal complex between the pairing regions of the X and Y Chromosomes. Several additional chromosomal abnormalities were observed in the hybrid. These include fold-backs of the unpaired X or Y cores, associations between the autosome and sex chromosome cores, and autosomal univalents. The occurrence of abnormal autosomal and XY-autosomal associations was also correlated with cell degeneration during meiotic prophase. The primary breakdown in hybrid spermatogenesis occurs at metaphase I (MI), with the appearance of degenerated cells at late MI. In those cells, the X and Y are decondensed rather than condensed as they are in normal mouse MI spermatocytes. These results, in combination with the previous genetic analysis of spermatogenesis in hybrids and backcrosses with fertile female hybrids, suggest that the spermatogenic breakdown in the interspecific hybrid is primarily correlated with the failure of XY pairing at meiotic prophase, asynapsis, followed by the degeneration of spermatocytes at metaphase I. Secondarily, the failure of XY pairing can be accompanied by failure of autosomal pairing, which appears to involve an abnormal sex vesicle and degeneration at pachytene or diplotene.by C. Heyting     

Repetitive DNA and meiotic behavior of sex chromosomes in Gymnotus pantanal (Gymnotiformes, Gymnotidae)

Neotropical fishes have a low rate of chromosome differentiation between sexes. The present study characterizes the first meiotic analysis of sex chromosomes in the order Gymnotiformes. Gymnotus pantanal - females had 40 chromosomes (14m/sm, 26st/a) and males had 39 chromosomes (15m/sm, 24st/a), with a fundamental number of 54 - showed a multiple sexual determination chromosome system of the type X(1)X(1)X(2)X(2)/X(1)X(2)Y. The heterochromatin is restricted to centromeres of all chromosomes of the karyotype. The meiotic behavior of sex chromosomes involved in this system in males is from a trivalent totally pared in the pachytene stage, with a high degree of similarity. The cells of metaphase II exhibit 19 and 20 chromosomes, normal disjunction of sex chromosomes and the formation of balanced gametes with 18 + Y and 18 + X(1)X(2) chromosomes, respectively. The small amount of heterochromatin and repetitive DNA involved in this system and the high degree of chromosome similarity indicated a recent origin of the X(1)X(1)X(2)X(2)/X(1)X(2)Y system in G. pantanal and suggests the existence of a simple ancestral system with morphologically undifferentiated chromosomes.     

An asymmetric chromosome pair undergoes synaptic adjustment and crossover redistribution during Caenorhabditis elegans meiosis: implications for sex chromosome evolution

Heteromorphic sex chromosomes, such as the X/Y pair in mammals, differ in size and DNA sequence yet function as homologs during meiosis; this bivalent asymmetry presents special challenges for meiotic completion. In Caenorhabditis elegans males carrying mnT12, an X;IV fusion chromosome, mnT12 and IV form an asymmetric bivalent: chromosome IV sequences are capable of pairing and synapsis, while the contiguous X portion of mnT12 lacks a homologous pairing partner. Here, we investigate the meiotic behavior of this asymmetric neo-X/Y chromosome pair in C. elegans. Through immunolocalization of the axis component HIM-3, we demonstrate that the unpaired X axis has a distinct, coiled morphology while synapsed axes are linear and extended. By showing that loci at the fusion-proximal end of IV become unpaired while remaining synapsed as pachytene progresses, we directly demonstrate the occurrence of synaptic adjustment in this organism. We further demonstrate that meiotic crossover distribution is markedly altered in males with the asymmetric mnT12/+ bivalent relative to controls, resulting in greatly reduced crossover formation near the X;IV fusion point and elevated crossovers at the distal end of the bivalent. In effect, the distal end of the bivalent acts as a neo-pseudoautosomal region in these males. We discuss implications of these findings for mechanisms that ensure crossover formation during meiosis. Furthermore, we propose that redistribution of crossovers triggered by bivalent asymmetry may be an important driving force in sex chromosome evolution.     

哺乳动物X染色体失活机制

哺乳动物X染色体连锁基因的剂量平衡,是通过雌性胚胎发育早期随机或印记失活一条X染色体来实现的,这是一个复杂的过程,包括：启动、计数、选择、维持等一系列的步骤。X染色体失活中心是X染色体失活的主控开关座位,调节X失活的早期事件,失活发生后,X染色体的失活状态可稳定地存在并传递给后代,这一过程涉及基因组印记的形成。此外,在雄性动物,精原细胞减数分裂早期也存在着短暂的X染色体失活现象。现对哺乳动物X染色体失活机制的最新进展进行综述。     

Taurodontism and the presence of an extra Y chromosome: study of 47,XYY males and analytical review

A sample of 47,XYY males was examined for taurodontism to provide further information on the effects of chromosome aneuploidies on the trait. The etiology of taurodontism is reviewed in light of recent findings. Two models have been put forward to explain the association of taurodontism with chromosome abnormalities: (1) Taurodontism results from a generalized disruption of developmental homeostasis, and (2) the development of taurodontism reflects a more specific action of the genes. The recent findings in 45,X females indicate that this chromosome aneuploidy does not have any effect on the development of taurodontism, in contrast to the findings of increased frequency of the trait in individuals with extra X chromosomes. The present results in 47,XYY males suggest that the presence of an extra Y chromosome does not cause an increase in the expression of taurodontism. It is concluded that the observed variation in the occurrence of taurodontism in individuals with sex chromosomes aneuploidies does not corroborate the hypothesis of disrupted homeostasis. Instead, the findings indicate that more specific action of gene(s) on the X chromosome is involved. We suggest that the effect of the Y chromosome on growth of both enamel and dentin, possibly in a regulative way, could be involved in the balanced growth of dental structures in 47,XYY males.