Human male meiotic sex chromosome inactivation

In mammalian male gametogenesis the sex chromosomes are distinctive in both gene activity and epigenetic strategy. At first meiotic prophase the heteromorphic X and Y chromosomes are placed in a separate chromatin domain called the XY body. In this process, X,Y chromatin becomes highly phosphorylated at S139 of H2AX leading to the repression of gonosomal genes, a process known as meiotic sex chromosome inactivation (MSCI), which has been studied best in mice. Post-meiotically this repression is largely maintained. Disturbance of MSCI in mice leads to harmful X,Y gene expression, eventuating in spermatocyte death and sperm heterogeneity. Sperm heterogeneity is a characteristic of the human male. For this reason we were interested in the efficiency of MSCI in human primary spermatocytes. We investigated MSCI in pachytene spermatocytes of seven probands: four infertile men and three fertile controls, using direct and indirect in situ methods. A considerable degree of variation in the degree of MSCI was detected, both between and within probands. Moreover, in post-meiotic stages this variation was observed as well, indicating survival of spermatocytes with incompletely inactivated sex chromosomes. Furthermore, we investigated the presence of H3K9me3 posttranslational modifications on the X and Y chromatin. Contrary to constitutive centromeric heterochromatin, this heterochromatin marker did not specifically accumulate on the XY body, with the exception of the heterochromatic part of the Y chromosome. This may reflect the lower degree of MSCI in man compared to mouse. These results point at relaxation of MSCI, which can be explained by genetic changes in sex chromosome composition during evolution and candidates as a mechanism behind human sperm heterogeneity.     

Eukaryotic initiation factor 4E-3 is essential for meiotic chromosome segregation, cytokinesis and male fertility in Drosophila

Gene expression is translationally regulated during many cellular and developmental processes. Translation can be modulated by affecting the recruitment of mRNAs to the ribosome, which involves recognition of the 5' cap structure by the cap-binding protein eIF4E. Drosophila has several genes encoding eIF4E-related proteins, but the biological role of most of them remains unknown. Here, we report that Drosophila eIF4E-3 is required specifically during spermatogenesis. Males lacking eIF4E-3 are sterile, showing defects in meiotic chromosome segregation, cytokinesis, nuclear shaping and individualization. We show that eIF4E-3 physically interacts with both eIF4G and eIF4G-2, the latter being a factor crucial for spermatocyte meiosis. In eIF4E-3 mutant testes, many proteins are present at different levels than in wild type, suggesting widespread effects on translation. Our results imply that eIF4E-3 forms specific eIF4F complexes that are essential for spermatogenesis.     

Evidence that sex chromosome asynapsis, rather than excess Y gene dosage, is responsible for the meiotic impairment of XYY mice

There is extensive evidence for the existence of a meiotic checkpoint that acts to eliminate spermatocytes that fail to achieve full sex chromosome synapsis at the pachytene stage of the first meiotic prophase. XYY mice are nearly always sterile, with clear signs of meiotic impairment, and sex chromosome asynapsis has been proposed to underlie this impairment. However, a study of XYY*(X) mice (mice having three sex chromosomes but only a single dose of Y genes) revealed that these mice are fertile, and thus implicated Y gene dosage as a major factor in the sterility of XYY mice. To address this question further, sex chromosome synapsis and spermatogenic proficiency were compared between XYY*(X) and XYY mice generated in the same litters. This established that differences in spermatogenic proficiency within and between the two genotypes correlated with the frequency of radial trivalent formation (full sex chromosome synapsis); XYY*(X) males, as a group, had double the radial trivalent frequency of XYY males. This observation provides strong support for the view that sex chromosome asynapsis (or some consequence thereof), rather than Y gene dosage, is the major factor leading to the meiotic impairment of XYY mice.     

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.     

CENP-B is not critical for meiotic chromosome segregation in male mice

Centromere protein B (CENP-B) is a constitutive protein that binds to a highly conserved 17 bp motif located at most mammalian centromeres. To determine whether disruption of this gene affects chromosome segregation in male germ cells, we evaluated the frequencies of disomic and diploid sperm in CENP-B heterozygous and homozygous null mice using the mouse epididymal sperm aneuploidy (m-ESA) assay, a multicolor FISH method with probes for chromosomes X, Y and 8. The specificity and sensitivity of the m-ESA assay was demonstrated using Robertsonian (2.8) translocation heterozygotes as positive controls for sperm aneuploidy. Our results show that the frequencies of disomic and diploid sperm did not differ significantly between CENP-B heterozygous and homozygous null mice (P≥0.5) or from 129/Swiss isogenic mice (P≥0.5) and B6C3F1 mice (P≥0.2). These findings indicate that CENP-B does not have an essential role during chromosome segregation in male meiosis.     

Cul4A is essential for spermatogenesis and male fertility

The mammalian Cul4 genes, Cul4A and Cul4B, encode the scaffold components of the cullin-based E3 ubiquitin ligases. The two Cul4 genes are functionally redundant. Recent study indicated that mice expressing a truncated CUL4A that fails to interact with its functional partner ROC1 exhibit no developmental phenotype. We generated a Cul4A−/− strain lacking exons 4–8 that does not express any detectable truncated protein. In this strain, the male mice are infertile and exhibit severe deficiencies in spermatogenesis. The primary spermatocytes are deficient in progression through late prophase I, a time point when expression of the X-linked Cul4B gene is silenced due to meiotic sex chromosome inactivation. Testes of the Cul4A−/− mice exhibit extensive apoptosis. Interestingly, the pachytene spermatocytes exhibit persistent double stranded breaks, suggesting a deficiency in homologous recombination. Also, we find that CUL4A localizes to the double stranded breaks generated in pre-pachytene spermatocytes. The observations identify a novel function of CUL4A in meiotic recombination and demonstrate an essential role of CUL4A in spermatogenesis.     

Meiotic sex chromosome inactivation

X chromosome inactivation is most commonly studied in the context of female mammalian development, where it performs an essential role in dosage compensation. However, another form of X-inactivation takes place in the male, during spermatogenesis, as germ cells enter meiosis. This second form of X-inactivation, called meiotic sex chromosome inactivation (MSCI) has emerged as a novel paradigm for studying the epigenetic regulation of gene expression. New studies have revealed that MSCI is a special example of a more general mechanism called meiotic silencing of unsynapsed chromatin (MSUC), which silences chromosomes that fail to pair with their homologous partners and, in doing so, may protect against aneuploidy in subsequent generations. Furthermore, failure in MSCI is emerging as an important etiological factor in meiotic sterility.     

CENP-B is not critical for meiotic chromosome segregation in male mice.

Centromere protein B (CENP-B) is a constitutive protein that binds to a highly conserved 17bp motif located at most mammalian centromeres. To determine whether disruption of this gene affects chromosome segregation in male germ cells, we evaluated the frequencies of disomic and diploid sperm in CENP-B heterozygous and homozygous null mice using the mouse epididymal sperm aneuploidy (m-ESA) assay, a multicolor FISH method with probes for chromosomes X, Y and 8. The specificity and sensitivity of the m-ESA assay was demonstrated using Robertsonian (2.8) translocation heterozygotes as positive controls for sperm aneuploidy. Our results show that the frequencies of disomic and diploid sperm did not differ significantly between CENP-B heterozygous and homozygous null mice (P> or = 0.5) or from 129/Swiss isogenic mice (P> or = 0.5) and B6C3F1 mice (P> or = 0.2). These findings indicate that CENP-B does not have an essential role during chromosome segregation in male meiosis.     

Non-muscle myosin IIB is essential for cytokinesis during male meiotic cell divisions

Cytokinesis, the final stage of cell division, bisects the cytoplasm into two daughter cells. In mitotic cells, this process depends on the activity of non-muscle myosin II (NMII), a family of actin-binding motor-proteins that participate in the formation of the cleavage furrow. The relevance of NMII for meiotic cell division, however, is poorly understood. The NMII family consists of three members, NMIIA, NMIIB, and NMIIC, containing different myosin heavy chains (MYH9, MYH10, and MYH14, respectively). We find that a single non-muscle myosin II, NMIIB, is required for meiotic cytokinesis in male but not female mice. Specifically, NMIIB-deficient spermatocytes exhibit cytokinetic failure in meiosis I, resulting in bi-nucleated secondary spermatocytes. Additionally, cytokinetic failure at meiosis II gives rise to bi-nucleated or even tetra-nucleated spermatids. These multi-nucleated spermatids fail to undergo normal differentiation, leading to male infertility. In spite of the presence of multiple non-muscle myosin II isoforms, we demonstrate that a single member, NMIIB, plays an essential and non-redundant role in cytokinesis during meiotic cell divisions of the male germline.     

SDX on the X chromosome is required for male sex determination

Dear Editor, Sex determination is one of the most fundamental develop-ment processes,as gender is the first and most important identity of human.In most mammals...     

An intact Pms2 ATPase domain is not essential for male fertility

The DNA mismatch repair (MMR) machinery in mammals plays critical roles in both mutation avoidance and spermatogenesis. Meiotic analysis of knockout mice of two different MMR genes, Mlh1 and Mlh3, revealed both male and female infertility associated with a defect in meiotic crossing over. In contrast, another MMR gene knockout, Pms2 (Pms2^ko/ko), which contained a deletion of a portion of the ATPase domain, produced animals that were male sterile but female fertile. However, the meiotic phenotype of Pms2^ko/ko males was less clear-cut than for Mlh1- or Mlh3-deficient meiosis. More recently, we generated a different Pms2 mutant allele (Pms2^cre), which results in deletion of the same portion of the ATPase domain. Surprisingly, Pms2^cre/cre male mice were completely fertile, suggesting that the ATPase domain of Pms2 is not required for male fertility. To explore the difference in male fertility, we examined the Pms2 RNA and found that alternative splicing of the Pms2^cre allele results in a predicted Pms2 containing the C-terminus, which contains the Mlh1-interaction domain, a possible candidate for stabilizing Mlh1 levels. To study further the basis of male fertility, we examined Mlh1 levels in testes and found that whereas Pms2 loss in Pms2^ko/ko mice results in severely reduced levels of Mlh1 expression in the testes, Mlh1 levels in Pms2^cre/cre testes were reduced to a lesser extent. Thus, we propose that a primary function of Pms2 during spermatogenesis is to stabilize Mlh1 levels prior to its critical crossing over function with Mlh3.     

Transcription-wide mapping of dihydrouridine reveals that mRNA dihydrouridylation is required for meiotic chromosome segregation

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