Determining the potency of primordial germ cells by injection into early mouse embryos.

Primordial germ cells (PGCs) are the earliest precursors of the gametes. During normal development, PGCs only give rise to oocytes or spermatozoa. However, PGCs can acquire pluripotency in vitro by forming embryonic germ (EG) cells and in vivo during teratocarcinogenesis. Classic embryological experiments directly assessed the potency of PGCs by injection into the pre-implantation embryo. As no contribution to embryos or adult mice was observed, PGCs have been described as unipotent. Here, we demonstrate that PGCs injected into 8-cell embryos can initially survive, divide, and contribute to the developing inner cell mass. Apoptosis-deficient PGCs exhibit improved survival in isolated epiblasts and can form naive pluripotent embryonic stem cell lines. However, contribution to the post-implantation embryo is limited, with no functional incorporation observed. In contrast, PGC-like cells show an extensive contribution to mid-gestation chimeras. We thus propose that PGC formation in vivo establishes a latent form of pluripotency that restricts chimera contribution.

CRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes.

Animals are essential genetic tools in scientific research and global resources in agriculture. In both arenas, a single sex is often required in surplus. The ethical and financial burden of producing and culling animals of the undesired sex is considerable. Using the mouse as a model, we develop a synthetic lethal, bicomponent CRISPR-Cas9 strategy that produces male- or female-only litters with one hundred percent efficiency. Strikingly, we observe a degree of litter size compensation relative to control matings, indicating that our system has the potential to increase the yield of the desired sex in comparison to standard breeding designs. The bicomponent system can also be repurposed to generate postnatal sex-specific phenotypes. Our approach, harnessing the technological applications of CRISPR-Cas9, may be applicable to other vertebrate species, and provides strides towards ethical improvements for laboratory research and agriculture.

The BCL-2 pathway preserves mammalian genome integrity by eliminating recombination-defective oocytes.

DNA double-strand breaks (DSBs) are toxic to mammalian cells. However, during meiosis, more than 200 DSBs are generated deliberately, to ensure reciprocal recombination and orderly segregation of homologous chromosomes. If left unrepaired, meiotic DSBs can cause aneuploidy in gametes and compromise viability in offspring. Oocytes in which DSBs persist are therefore eliminated by the DNA-damage checkpoint. Here we show that the DNA-damage checkpoint eliminates oocytes via the pro-apoptotic BCL-2 pathway members Puma, Noxa and Bax. Deletion of these factors prevents oocyte elimination in recombination-repair mutants, even when the abundance of unresolved DSBs is high. Remarkably, surviving oocytes can extrude a polar body and be fertilised, despite chaotic chromosome segregation at the first meiotic division. Our findings raise the possibility that allelic variants of the BCL-2 pathway could influence the risk of embryonic aneuploidy.

Correction to: Identification of novel Y chromosome encoded transcripts by testis transcriptome analysis of mice with deletions of the Y chromosome long arm.

Following publication of the original article [1], the following error was reported: The actin control panel in Fig. 3 of this paper is reproduced from Fig. 7 of Touré et al, 2004 [2] by kind permission of the Genetics Society of America. Touré et al, 2004 used Northern blotting to show that the Y-linked genes Ssty1 and Ssty2 have reduced expression in a range of mouse genotypes with deletions on the Y chromosome long arm. This paper shows that two novel genes, Sly and Asty are also present on mouse Yq and have reduced expression in these deleted genotypes. A further companion paper was published in Human Molecular Genetics (Ellis et al, 2005 [3]) showing that X-linked genes are upregulated in the various deleted genotypes. Since two of the genotypes concerned are sterile and very hard to generate, all the Northern blot experiments in these papers were performed on a single membrane that was stripped and re-probed with a range of different X- and Y-linked genes. The same beta-actin loading control image thus necessarily applies to all the data presented, and was shown in all three papers. We regret that this was not mentioned appropriately in the Methods and figure legends at the time of publication.

SETDB1 Links the Meiotic DNA Damage Response to Sex Chromosome Silencing in Mice.

Meiotic synapsis and recombination ensure correct homologous segregation and genetic diversity. Asynapsed homologs are transcriptionally inactivated by meiotic silencing, which serves a surveillance function and in males drives meiotic sex chromosome inactivation. Silencing depends on the DNA damage response (DDR) network, but how DDR proteins engage repressive chromatin marks is unknown. We identify the histone H3-lysine-9 methyltransferase SETDB1 as the bridge linking the DDR to silencing in male mice. At the onset of silencing, X chromosome H3K9 trimethylation (H3K9me3) enrichment is downstream of DDR factors. Without Setdb1, the X chromosome accrues DDR proteins but not H3K9me3. Consequently, sex chromosome remodeling and silencing fail, causing germ cell apoptosis. Our data implicate TRIM28 in linking the DDR to SETDB1 and uncover additional factors with putative meiotic XY-silencing functions. Furthermore, we show that SETDB1 imposes timely expression of meiotic and post-meiotic genes. Setdb1 thus unites the DDR network, asynapsis, and meiotic chromosome silencing.

ATR is a multifunctional regulator of male mouse meiosis.

Meiotic cells undergo genetic exchange between homologs through programmed DNA double-strand break (DSB) formation, recombination and synapsis. In mice, the DNA damage-regulated phosphatidylinositol-3-kinase-like kinase (PIKK) ATM regulates all of these processes. However, the meiotic functions of the PIKK ATR have remained elusive, because germline-specific depletion of this kinase is challenging. Here we uncover roles for ATR in male mouse prophase I progression. ATR deletion causes chromosome axis fragmentation and germ cell elimination at mid pachynema. This elimination cannot be rescued by deletion of ATM and the third DNA damage-regulated PIKK, PRKDC, consistent with the existence of a PIKK-independent surveillance mechanism in the mammalian germline. ATR is required for synapsis, in a manner genetically dissociable from DSB formation. ATR also regulates loading of recombinases RAD51 and DMC1 to DSBs and recombination focus dynamics on synapsed and asynapsed chromosomes. Our studies reveal ATR as a critical regulator of mouse meiosis.

DNA damage response protein TOPBP1 regulates X chromosome silencing in the mammalian germ line.

Meiotic synapsis and recombination between homologs permits the formation of cross-overs that are essential for generating chromosomally balanced sperm and eggs. In mammals, surveillance mechanisms eliminate meiotic cells with defective synapsis, thereby minimizing transmission of aneuploidy. One such surveillance mechanism is meiotic silencing, the inactivation of genes located on asynapsed chromosomes, via ATR-dependent serine-139 phosphorylation of histone H2AFX (γH2AFX). Stimulation of ATR activity requires direct interaction with an ATR activation domain (AAD)-containing partner. However, which partner facilitates the meiotic silencing properties of ATR is unknown. Focusing on the best-characterized example of meiotic silencing, meiotic sex chromosome inactivation, we reveal this AAD-containing partner to be the DNA damage and checkpoint protein TOPBP1. Conditional TOPBP1 deletion during pachynema causes germ cell elimination associated with defective X chromosome gene silencing and sex chromosome condensation. TOPBP1 is essential for localization to the X chromosome of silencing "sensors," including BRCA1, and effectors, including ATR, γH2AFX, and canonical repressive histone marks. We present evidence that persistent DNA double-strand breaks act as silencing initiation sites. Our study identifies TOPBP1 as a critical factor in meiotic sex chromosome silencing.

Fertile offspring from sterile sex chromosome trisomic mice.

Having the correct number of chromosomes is vital for normal development and health. Sex chromosome trisomy affects 0.1% of the human population and is associated with infertility. We show that during reprogramming to induced pluripotent stem cells (iPSCs), fibroblasts from sterile trisomic XXY and XYY mice lose the extra sex chromosome through a phenomenon we term trisomy-biased chromosome loss (TCL). Resulting euploid XY iPSCs can be differentiated into the male germ cell lineage and functional sperm that can be used in intracytoplasmic sperm injection to produce chromosomally normal, fertile offspring. Sex chromosome loss is comparatively infrequent during mouse XX and XY iPSC generation. TCL also applies to other chromosomes, generating euploid iPSCs from cells of a Down syndrome mouse model. It can also create euploid iPSCs from human trisomic patient fibroblasts. The findings have relevance to overcoming infertility and other trisomic phenotypes.

Non-Canonical and Sexually Dimorphic X Dosage Compensation States in the Mouse and Human Germline.

Somatic X dosage compensation requires two mechanisms: X inactivation balances X gene output between males (XY) and females (XX), while X upregulation, hypothesized by Ohno and documented in vivo, balances X gene with autosomal gene output. Whether X dosage compensation occurs in germ cells is unclear. We show that mouse and human germ cells exhibit non-canonical X dosage states that differ from the soma and between the sexes. Prior to genome-wide reprogramming, X upregulation is present, consistent with Ohno's hypothesis. Subsequently, however, it is erased. In females, erasure follows loss of X inactivation, causing X dosage excess. Conversely, in males, erasure leads to permanent X dosage decompensation. Sex chromosomally abnormal models exhibit a "sex-reversed" X dosage state: XX males, like XX females, develop X dosage excess, while XO females, like XY males, develop X dosage decompensation. Thus, germline X dosage compensation states are determined by X chromosome number, not phenotypic sex. These unexpected differences in X dosage compensation states between germline and soma offer unique perspectives on sex chromosome infertility.

Recombination between the mouse Y chromosome short arm and an additional Y short arm-derived chromosomal segment attached distal to the X chromosome PAR.

In a male mouse, meiosis markers of processed DNA double strand breaks (DSBs) such as DMC1 and RAD51 are regularly seen in the non-PAR region of the X chromosome; these disappear late in prophase prior to entry into the first meiotic metaphase. Marker evidence for DSBs occurring in the non-PAR region of the Y chromosome is limited. Nevertheless, historically it has been documented that recombination can occur within the mouse Y short arm (Yp) when an additional Yp segment is attached distal to the X and/or the Y pseudoautosomal region (PAR). A number of recombinants identified among offsprings involved unequal exchanges involving repeated DNA segments; however, equal exchanges will have frequently been missed because of the paucity of markers to differentiate between the two Yp segments. Here, we discuss this historical data and present extensive additional data obtained for two mouse models with Yp additions to the X PAR. PCR genotyping enabled identification of a wider range of potential recombinants; the proportions of Yp exchanges identified among the recombinants were 9.7 and 22.4 %. The frequency of these exchanges suggests that the Yp segment attached to the X PAR is subject to the elevated level of recombinational DSBs that characterizes the PAR.

Preliminary Evidence for Aortopathy and an X-Linked Parent-of-Origin Effect on Aortic Valve Malformation in a Mouse Model of Turner Syndrome.

Turner syndrome (TS), most frequently caused by X-monosomy (45,X), is characterized in part by cardiovascular abnormalities, including aortopathy and bicuspid aortic valve (BAV). There is a need for animal models that recapitulate the cardiovascular manifestations of TS. Extracellular matrix (ECM) organization and morphometrics of the aortic valve and proximal aorta were examined in adult 39,XO mice (where the parental origin of the single X was paternal (39,XPO) or maternal (39,XMO)) and 40,XX controls. Aortic valve morphology was normal (tricuspid) in all of the 39,XPO and 40,XX mice studied, but abnormal (bicuspid or quadricuspid) in 15% of 39,XMO mice. Smooth muscle cell orientation in the ascending aorta was abnormal in all 39,XPO and 39,XMO mice examined, but smooth muscle actin was decreased in 39,XMO mice only. Aortic dilation was present with reduced penetrance in 39,XO mice. The 39,XO mouse demonstrates aortopathy and an X-linked parent-of-origin effect on aortic valve malformation, and the candidate gene FAM9B is polymorphically expressed in control and diseased human aortic valves. The 39,XO mouse model may be valuable for examining the mechanisms underlying the cardiovascular findings in TS, and suggest there are important genetic modifiers on the X chromosome that modulate risk for nonsyndromic BAV and aortopathy.

Genetic and pharmacological modulation of the steroid sulfatase axis improves response control; comparison with drugs used in ADHD.

Maladaptive response control is a feature of many neuropsychiatric conditions, including attention deficit hyperactivity disorder (ADHD). As ADHD is more commonly diagnosed in males than females, a pathogenic role for sex-linked genes has been suggested. Deletion or point mutation of the X-linked STS gene, encoding the enzyme steroid sulfatase (STS) influences risk for ADHD. We examined whether deletion of the Sts gene in the 39,X(Y*)O mouse model, or pharmacological manipulation of the STS axis, via administration of the enzyme substrate dehydroepiandrosterone sulfate or the enzyme inhibitor COUMATE, influenced behavior in a novel murine analog of the stop-signal reaction time task used to detect inhibitory deficits in individuals with ADHD. Unexpectedly, both the genetic and pharmacological treatments resulted in enhanced response control, manifest as highly specific effects in the ability to cancel a prepotent action. For all three manipulations, the effect size was comparable to that seen with the commonly used ADHD therapeutics methylphenidate and atomoxetine. Hence, converging genetic and pharmacological evidence indicates that the STS axis is involved in inhibitory processes and can be manipulated to give rise to improvements in response control. While the precise neurobiological mechanism(s) underlying the effects remain to be established, there is the potential for exploiting this pathway in the treatment of disorders where failures in behavioral inhibition are prominent.

Altered brain gene expression but not steroid biochemistry in a genetic mouse model of neurodevelopmental disorder.

BACKGROUND: The 39,XY*O mouse, which lacks the orthologues of the ADHD and autism candidate genes STS (steroid sulphatase) and ASMT (acetylserotonin O-methyltransferase), exhibits behavioural phenotypes relevant to developmental disorders. The neurobiology underlying these phenotypes is unclear, although there is evidence for serotonergic abnormalities in the striatum and hippocampus. METHODS: Using microarray and quantitative gene expression analyses, and gas chromatography-mass spectrometry, we compared brain gene expression and steroid biochemistry in wildtype (40,XY) and 39,XY*O adult mice to identify non-obvious genetic and endocrine candidates for between-group differences in behaviour and neurochemistry. We also tested whether acute STS inhibition by COUMATE in wildtype (40,XY) adult male mice recapitulated any significant gene expression or biochemical findings from the genetic comparison. Data were analysed by unpaired t-test or Mann Whitney U-test depending on normality, with a single factor of KARYOTYPE. RESULTS: Microarray analysis indicated seven robust gene expression differences between the two groups (Vmn2r86, Sfi1, Pisd-ps1, Tagap1, C1qc, Metap1d, Erdr1); Erdr1 and C1qc expression was significantly reduced in the 39,XY*O striatum and hippocampus, whilst the expression of Dhcr7 (encoding 7-dehydrocholesterol reductase, a modulator of serotonin system development), was only reduced in the 39,XY*O hippocampus. None of the confirmed gene expression changes could be recapitulated by COUMATE administration. We detected ten free, and two sulphated steroids in 40,XY and 39,XY*O brain; surprisingly, the concentrations of all of these were equivalent between groups. CONCLUSIONS: Our data demonstrate that the mutation in 39,XY*O mice: i) directly disrupts expression of the adjacent Erdr1 gene, ii) induces a remarkably limited suite of downstream gene expression changes developmentally, with several of relevance to associated neurobehavioural phenotypes and iii) does not elicit large changes in brain steroid biochemistry. It is possible that individuals with STS/ASMT deficiency exhibit a similarly specific pattern of gene expression changes to the 39,XY*O mouse, and that these contribute towards their abnormal neurobiology. Future work may focus on whether complement pathway function, mitochondrial metabolism and cholesterol biosynthesis pathways are perturbed in such subjects.

The expression of Y-linked Zfy2 in XY mouse oocytes leads to frequent meiosis 2 defects, a high incidence of subsequent early cleavage stage arrest and infertility.

Outbred XY(Sry-) female mice that lack Sry due to the 11 kb deletion Sry(dl1Rlb) have very limited fertility. However, five lines of outbred XY(d) females with Y chromosome deletions Y(Del(Y)1Ct)-Y(Del(Y)5Ct) that deplete the Rbmy gene cluster and repress Sry transcription were found to be of good fertility. Here we tested our expectation that the difference in fertility between XO, XY(d-1) and XY(Sry-) females would be reflected in different degrees of oocyte depletion, but this was not the case. Transgenic addition of Yp genes to XO females implicated Zfy2 as being responsible for the deleterious Y chromosomal effect on fertility. Zfy2 transcript levels were reduced in ovaries of XY(d-1) compared with XY(Sry-) females in keeping with their differing fertility. In seeking the biological basis of the impaired fertility we found that XY(Sry-), XY(d-1) and XO,Zfy2 females produce equivalent numbers of 2-cell embryos. However, in XY(Sry-) and XO,Zfy2 females the majority of embryos arrested with 2-4 cells and almost no blastocysts were produced; by contrast, XY(d-1) females produced substantially more blastocysts but fewer than XO controls. As previously documented for C57BL/6 inbred XY females, outbred XY(Sry-) and XO,Zfy2 females showed frequent failure of the second meiotic division, although this did not prevent the first cleavage. Oocyte transcriptome analysis revealed major transcriptional changes resulting from the Zfy2 transgene addition. We conclude that Zfy2-induced transcriptional changes in oocytes are sufficient to explain the more severe fertility impairment of XY as compared with XO females.

Biological mechanisms associated with increased perseveration and hyperactivity in a genetic mouse model of neurodevelopmental disorder.

Chromosomal deletions at Xp22.3 appear to influence vulnerability to the neurodevelopmental disorders attention deficit hyperactivity disorder (ADHD) and autism. 39,X(Y*)O mice, which lack the murine orthologue of the Xp22.3 ADHD candidate gene STS (encoding steroid sulfatase), exhibit behavioural phenotypes relevant to such disorders (e.g. hyperactivity), elevated hippocampal serotonin (5-HT) levels, and reduced serum levels of dehydroepiandrosterone (DHEA). Here we initially show that 39,X(Y*)O mice are also deficient for the recently-characterised murine orthologue of the Xp22.3 autism candidate gene ASMT (encoding acetylserotonin-O-methyltransferase). Subsequently, to specify potential behavioural correlates of elevated hippocampal 5-HT arising due to the genetic lesion, we compared 39,X(Y*)O MF1 mice to 40,XY MF1 mice on behavioural tasks taxing hippocampal and/or 5-HT function (a 'foraging' task, an object-location task, and the 1-choice serial reaction time task of impulsivity). Although Sts/Asmt deficiency did not influence foraging behaviour, reactivity to familiar objects in novel locations, or 'ability to wait', it did result in markedly increased response rates; these rates correlated with hippocampal 5-HT levels and are likely to index behavioural perseveration, a frequent feature of neurodevelopmental disorders. Additionally, we show that whilst there was no systematic relationship between serum DHEA levels and hippocampal 5-HT levels across 39,X(Y*)O and 40,XY mice, there was a significant inverse linear correlation between serum DHEA levels and activity. Our data suggest that deficiency for genes within Xp22.3 could influence core behavioural features of neurodevelopmental disorders via dissociable effects on hippocampal neurochemistry and steroid hormone levels, and that the mediating neurobiological mechanisms may be investigated in the 39,X(Y*)O model.

Human and mouse ZFY genes produce a conserved testis-specific transcript encoding a zinc finger protein with a short acidic domain and modified transactivation potential.

Mammalian ZFY genes are located on the Y chromosome, and code putative transcription factors with 12-13 zinc fingers preceded by a large acidic (activating) domain. In mice, there are two genes, Zfy1 and Zfy2, which are expressed mainly in the testis. Their transcription increases in germ cells as they enter meiosis, both are silenced by meiotic sex chromosome inactivation (MSCI) during pachytene, and Zfy2 is strongly reactivated later in spermatids. Recently, we have shown that mouse Zfy2, but not Zfy1, is involved in triggering the apoptotic elimination of specific types of sex chromosomally aberrant spermatocytes. In humans, there is a single widely transcribed ZFY gene, and there is no evidence for a specific role in the testis. Here, we characterize ZFY transcription during spermatogenesis in mice and humans. In mice, we define a variety of Zfy transcripts, among which is a Zfy2 transcript that predominates in spermatids, and a Zfy1 transcript, lacking an exon encoding approximately half of the acidic domain, which predominates prior to MSCI. In humans, we have identified a major testis-specific ZFY transcript that encodes a protein with the same short acidic domain. This represents the first evidence that ZFY has a conserved function during human spermatogenesis. We further show that, in contrast to the full acidic domain, the short domain does not activate transcription in yeast, and we hypothesize that this explains the functional difference observed between Zfy1 and Zfy2 during mouse meiosis.

Steroid sulfatase-deficient mice exhibit endophenotypes relevant to attention deficit hyperactivity disorder.

Attention Deficit Hyperactivity Disorder (ADHD) is a common neurodevelopmental condition characterised by inattention, impulsivity and hyperactivity; it is frequently co-morbid with anxiety and conduct disorders, sleep perturbation and abnormal consummatory behaviours. Recent studies have implicated the neurosteroid-modulating enzyme steroid sulfatase (STS) as a modulator of ADHD-related endophenotypes. The effects of steroid sulfatase deficiency on homecage activity, feeding/drinking behaviours, anxiety-related behaviours (assayed in light-dark box and open field paradigms), social dominance and serum steroid hormone levels were determined by comparing 40,XY and 39,X(Y*)O mice. Subsequently, mice administered the steroid sulfatase inhibitor COUMATE acutely were compared to vehicle-treated mice on behavioural tasks sensitive to enzyme deficiency to dissociate between its developmental and ongoing effects. 39,X(Y*)O mice exhibited heightened reactivity to a novel environment, hyperactivity in the active phase, and increased water (but not food) consumption relative to 40,XY mice during a 24h period; the former group also demonstrated evidence for heightened emotional reactivity. There was no difference in social dominance between the 40,XY and 39,X(Y*)O mice. COUMATE administration had no effect on homecage activity, water consumption or anxiety measures in the open field. 39,X(Y*)O mice exhibited significantly lower dehydroepiandrosterone (DHEA) serum levels than 40,XY mice, but equivalent corticosterone levels. Together with previous data, the present results suggest that steroid sulfatase may influence core and associated ADHD behavioural endophenotypes via both developmental and ongoing mechanisms, and that the 39,X(Y*)O model may represent a useful tool for elucidating the neurobiological basis of these endophenotypes.

Altered serotonergic function may partially account for behavioral endophenotypes in steroid sulfatase-deficient mice.

The X-linked gene STS encodes the steroid hormone-modulating enzyme steroid sulfatase. Loss-of-function of STS, and variation within the gene, have been associated with vulnerability to developing attention deficit hyperactivity disorder (ADHD), a neurodevelopmental condition characterized by inattention, severe impulsivity, hyperactivity, and motivational deficits. ADHD is commonly comorbid with a variety of disorders, including obsessive-compulsive disorder. The neurobiological role of steroid sulfatase, and therefore its potential role in ADHD and associated comorbidities, is currently poorly understood. The 39,X(Y)*O mouse, which lacks the Sts gene, exhibits several behavioral abnormalities relevant to ADHD including inattention and hyperactivity. Here, we show that, unexpectedly, 39,X(Y)*O mice achieve higher ratios than wild-type mice on a progressive ratio (PR) task thought to index motivation, but that there is no difference between the two groups on a behavioral task thought to index compulsivity (marble burying). High performance liquid chromatography analysis of monoamine levels in wild type and 39,X(Y)*O brain tissue regions (the frontal cortex, striatum, thalamus, hippocampus, and cerebellum) revealed significantly higher levels of 5-hydroxytryptamine (5-HT) in the striatum and hippocampus of 39,X(Y)*O mice. Significant correlations between hippocampal 5-HT levels and PR performance, and between striatal 5-HT levels and locomotor activity strongly implicate regionally-specific perturbations of the 5-HT system as a neurobiological candidate for behavioral differences between 40,XY and 39,X(Y)*O mice. These data suggest that inactivating mutations and functional variants within STS might exert their influence on ADHD vulnerability, and disorder endophenotypes through modulation of the serotonergic system.

The Y-encoded gene zfy2 acts to remove cells with unpaired chromosomes at the first meiotic metaphase in male mice.

During male but not female mammalian meiosis, there is efficient apoptotic elimination of cells with unpaired (univalent) chromosomes at the first meiotic metaphase (MI) [1]. Apoptotic elimination of MI spermatocytes is seen in response to the univalent X chromosome of XSxr(a)O male mice [2], in which the X chromosome carries Sxr(a) [3, 4], the Y-chromosome-derived sex-reversal factor that includes the testis determinant Sry. Sxr(b) is an Sxr(a)-derived variant in which a deletion has removed six Y short-arm genes and created a Zfy2/Zfy1 fusion gene spanning the deletion breakpoint [4, 5]. XSxr(b)O males have spermatogonial arrest that can be overcome by the re-addition of Eif2s3y from the deletion as a transgene; however, XSxr(b)OEif2s3y transgenic males do not show the expected elimination of MI spermatocytes in response to the univalent [6]. Here we show that these XSxr(b)OEif2s3y males have an impaired apoptotic response with completion of the first meiotic division, but there is no second meiotic division. We then show that Zfy2 (but not the closely related Zfy1) is sufficient to reinstate the apoptotic response to the X univalent. These findings provide further insight into the basis for the much lower transmission of chromosomal errors originating at the first meiotic division in men than in women [7].

Deficiency in the multicopy Sycp3-like X-linked genes Slx and Slxl1 causes major defects in spermatid differentiation.

The human and mouse sex chromosomes are enriched in multicopy genes required for postmeiotic differentiation of round spermatids into sperm. The gene Sly is present in multiple copies on the mouse Y chromosome and encodes a protein that is required for the epigenetic regulation of postmeiotic sex chromosome expression. The X chromosome carries two multicopy genes related to Sly: Slx and Slxl1. Here we investigate the role of Slx/Slxl1 using transgenically-delivered small interfering RNAs to disrupt their function. We show that Slx and Slxl1 are important for normal sperm differentiation and male fertility. Slx/Slxl1 deficiency leads to delay in spermatid elongation and sperm release. A high proportion of delayed spermatids are eliminated via apoptosis, with a consequent reduced sperm count. The remaining spermatozoa are abnormal with impaired motility and fertilizing abilities. Microarray analyses reveal that Slx/Slxl1 deficiency affects the metabolic processes occurring in the spermatid cytoplasm but does not lead to a global perturbation of sex chromosome expression; this is in contrast with the effect of Sly deficiency which leads to an up-regulation of X and Y chromosome genes. This difference may be due to the fact that SLX/SLXL1 are cytoplasmic while SLY is found in the nucleus and cytoplasm of spermatids.