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Human Molecular Genetics Advance Access originally published online on August 8, 2005
Human Molecular Genetics 2005 14(18):2705-2715; doi:10.1093/hmg/ddi304
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

Deletions on mouse Yq lead to upregulation of multiple X- and Y-linked transcripts in spermatids

Peter J.I. Ellis1, Emily J. Clemente1, Penny Ball2, Aminata Touré3, Lydia Ferguson1, James M.A. Turner3, Kate L. Loveland2, Nabeel A. Affara1,* and Paul S. Burgoyne3

1Department of Pathology, Mammalian Molecular Genetics Group, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK, 2RC Centre of Excellence in Biotechnology and Development, Monash Institute for Medical Research, Monash University, Clayton, Melbourne, Victoria 3168, Australia and 3Division of Stem Cell Research and Developmental Genetics, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

* To whom correspondence should be addressed. Tel: +44 01223 333700; Fax: +44 01223 333346; Email: na{at}mole.bio.cam.ac.uk

Received June 24, 2005; Revised July 22, 2005; Accepted August 2, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 AK005922 and XM_358268 are...
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Deletions on the mouse Y-chromosome long arm (MSYq) lead to teratozoospermia and in severe cases to infertility. We find that the downstream transcriptional changes in the testis resulting from the loss of MSYq-encoded transcripts involve upregulation of multiple X- and Y-linked spermatid-expressed genes, but not related autosomal genes. Therefore, this indicates that in normal males, there is a specific repression of X and Y (gonosomal) transcription in post-meiotic cells, which depends on MSYq-encoded transcripts. Together with the known sex ratio skew in favour of females in the offspring of fertile MSYqdel males, this strongly suggests the existence of an intragenomic conflict between X- and Y-linked genes. Two potential antagonists in this conflict are the X-linked multicopy gene Xmr and its multicopy MSYq-linked relative Sly, which are upregulated and downregulated, respectively, in the testes of MSYqdel males. Xmr is also expressed during meiotic sex chromosome inactivation (MSCI), indicating a link between the MSCI and the MSYq-dependent gonosomal repression in spermatids. We therefore propose that this repression and MSCI itself are evolutionary adaptations to maintain a normal sex ratio in the face of X/Y antagonism.


   INTRODUCTION
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 ABSTRACT
 INTRODUCTION
RESULTS
 AK005922 and XM_358268 are...
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Several naturally occurring deletion models exist with losses of part or all of the male-specific region of the Y-chromosome long arm (MSYq) in mouse. These models show a range of teratozoospermia and infertility phenotypes with severities correlated to the extent of the deletion (1Go–7Go). Intriguingly, the models with less severe deletions, which retain fertility, show a sex ratio skew in favour of females in the offspring of mutant males (2Go). The gonosomes are of great importance in spermatogenesis and are pre-disposed to accumulate genes with roles in this process. Enrichment for spermatogenic genes on the X is likely to be due to hemizygous uncovering of male-benefit recessive alleles (8Go), whereas enrichment on the Y is due to recruitment and retention of male-benefit genes against a background of ongoing genetic degeneration resulting from its non-recombining status (9Go–13Go). However, the X gene complement is also affected by the consequences of meiotic sex chromosome inactivation (MSCI), leading to a deficiency of meiosis-expressed genes on the X (14Go).

Previous work has identified some of the morphological and hormonal processes affected in the various MSYq-deleted phenotypes, notably abnormal vesicle formation in the developing acrosome (15Go), a flattened sperm head shape (1Go–4Go,6Go) and overexpression of aromatase in spermatids and Leydig cells (16Go). Progress has also been made in identifying the MSYq-linked transcripts, deficiency of which is likely to be the primary cause of the spermiogenic abnormalities (2Go,17Go–19Go). In the course of our microarray analysis that identified reduction or loss of MSYq-encoded testis transcripts in MSYq-deficient mice (Touré et al., submitted for publication), we also identified a number of downstream transcriptional changes, which we have verified by northern blotting and real-time RT–PCR. Surprisingly, as we report here, the majority of these changes involve an increase rather than a decrease in transcript levels, the cellular and chromosomal distribution of these transcripts indicates a specific derepression of X- and Y-chromosome transcription in post-meiotic germ cells of the mutant mice.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
AK005922 and XM_358268 are...
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Our initial analysis of the data from the microarray screen of three different MSYqdel models (Fig. 1) served to provide a more comprehensive picture of the reduction in MSYq-encoded testis transcripts resulting from the deletions. Our further analysis of these models has focussed on the other (downstream) transcriptional changes. In order to distinguish the primary consequences of MSYq deletion from secondary degenerative changes in the mutant testes, we extended the study to include an earlier time point for the XSxraY*X model, which has the most severe deletion phenotype. Microarray hybridizations and analysis were carried out as described previously (20Go), except that microarray data normalization was based on the global median signal for Cy3 and Cy5 channels rather than on a panel of control genes. This form of normalization is valid because the majority of genes do not vary between the mutant and control samples for any of the models (Fig. 2). Array data were then filtered to select genes showing a 1.5-fold change and 1% t-test relative to control in both the 1 month and 2 month XSxraY*X samples.



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  		Figure 1. Diagrams illustrating the sex chromosome complements and sperm phenotypes of normal male mice and the mice with MSYq deficiencies. The short arm of the normal Y-chromosome (shown expanded) carries seven single copy genes, one duplicated gene (Zfy) and multiple copies of Rbmy. MSYq carries multiple copies of the Ssty, Sly and Asty gene families. Y– denotes the fact that the Y in this MSYq deletion mutant also carries a Yp deletion removing the testis determinant Sry. This is complemented by an autosomally located Sry transgene. XSxra denotes an X-chromosome bearing the Y short arm-derived factor Tp(Y)1CtSxr-a (hereafter Sxra) attached distal to the PAR. Further details of the genotype and phenotype of the deleted models are given in Materials and Methods. TEL, telomere; CEN, centromere and PAR, pseudoautosomal region.

 


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  		Figure 2. Array analysis shows a bias in the chromosomal location of regulated genes. Scatter plots show gene expression levels in the testis of mutant relative to control genotypes as measured by microarray hybridization. Following normalization and averaging of technical replicates, the measured fluorescence (arbitrary units) for the control genotype is plotted on the x-axis and for the mutant genotype on the y-axis in each panel. (A) XYRIII versus XYRIIIqdel, (B) XY– versus XY–qdel, (C) XYRIII versus XSxraY*X and (D) XYRIII versus XSxraY*X. (A–C) analysed at 2 months post-partum and (D) at 1 month post-partum. {ddi30406}, autosomal clones; {ddi30407}, X-linked clones; {ddi30408}, Y-linked clones and {ddi30409}, mitochondrial clones.

 
Strikingly, these downstream changes predominantly comprise increases in transcript levels, 23 genes being upregulated compared with only two being downregulated. This thus signifies that in normal males, the main effect of MSYq-linked transcription is a repression in transcript levels of a set of downstream genes. Even more strikingly, most (15/23) of the upregulated transcripts are X- or Y-linked, whereas both downregulated transcripts are autosomal (Fig. 2; Table 1). Mapping to X, Y or autosome was established by southern blotting in the cases where a definitive mapping was not already assigned for a given transcript (Supplementary Material, Fig. S1). Comparison with previous array data (20Go) indicated that the upregulated genes were all likely to be expressed in spermatids in normal males on the basis of their profile across the first wave of spermatogenesis and level of expression in four germ cell depleted models. The expression distribution of several of the most highly affected genes was confirmed in normal testes by in situ hybridization (Fig. 3) (full staging diagrams available as Supplementary Material, Fig. S2), and their upregulation in the MSYqdel models was confirmed by northern blotting (Fig. 4). Interestingly, one of the X-linked genes examined by ISH (AK006251, Fig. 3E) proved to be expressed in late pachytene spermatocytes as well as in spermatids, indicating that this gene is not silenced during MSCI.


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  		Table 1. Transcripts found to be significantly altered in the initial microarray screen and their chromosomal location
 


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  		Figure 3. Expression of the upregulated X-linked transcripts is confirmed to be specific to spermatids and late stage spermatocytes. In situ localization of transcripts in testes of adult C57BL/6J mice. Roman numerals indicate tubule stage. (A) AK005922 localizes to round and early elongating spermatids. (B) mgclh localizes to round and early elongating spermatids. (C) AK015913 localizes to round spermatids. (D) Xmr localizes to round and early elongating spermatids. (E) AK006251 localizes to pachytene spermatocytes, round and early elongating spermatids. (F) Tm4sf6 localizes to round and early elongating spermatids.

 


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  		Figure 4. Upregulation of key X-linked genes in the MSYqdel models is confirmed. Northern blot of total testis RNA probed with clones for AK005922, mgclh and Xmr. An actin probe was used as a loading control, and a probe for Ssty1 was used to demonstrate the loss of MSYq sequence in the deleted genotypes. Hybridization to AK005922 and mgclh probes clearly shows a dose-dependent response with the degree of upregulation depending on the degree of MSYq deficiency. Xmr is more evenly upregulated in the MSYq-deleted models; however, the microarray and RT–PCR results again show a degree of dose dependency.

 
Further in situ analysis (Supplementary Material, Fig. S3) showed that the expression distribution was unchanged in testes of the nine-tenth MSYqdel and XSxraY*X models, indicating that the observed upregulation was due to elevated levels of transcription in spermatids (spermatocytes in the case of AK006251), rather than expression in different cell types. Analysis of the chromosomal distribution of the arrayed libraries in conjunction with our prior data (20Go) showed that the sex chromosome bias of the upregulated clones was not due to bias in the underlying libraries or due to bias in the haploid-expressed gene clusters (data not shown). Many of the genes identified by the microarray screen are members of highly homologous gene families, and thus hybridization-based assays, such as northern blotting, microarray expression profiling and ISH, are insufficient to unpick the complexity of changes in transcript abundance. Therefore, we performed an extensive series of real-time PCR experiments to precisely distinguish between near relatives. The panel of genes investigated comprised:
  • every gene found to be significantly upregulated in the microarray screen, along with all further known X-, Y- or autosomal homologues;
  • all known MSYq-linked genes and X-homologues, and four further sets of X–Y homologous genes mapping to the short arm of the Y-chromosome (thus outside the extent of the deletion);
  • Ckt1 and relatives, which gave inconsistent results in the microarray screen and thus were not counted in the 23 genes found as significant by the array screen;
  • a panel of control genes chosen to include autosomal housekeeping genes, X-linked non-spermatid genes and autosomal spermatid-specific genes;
  • aromatase and the androgen receptor. The former has been reported to be upregulated in spermatids of B10.BR-Ydel males bearing a two-third MSYq deletion (16Go), and the latter is an X-linked component of the androgen response pathway.
Primer pairs for all genes are listed in Supplementary Material, Table S1. The results of the RT–PCR survey are shown in Table 2 and are plotted in Figure 5A, demonstrating the upregulation of multiple X- and Y-linked transcripts as well as downregulation of MSYq-linked transcripts. The 10 control genes included in the survey showed slight or no significant change in expression levels in the three MSYqdel models relative to normal mice; this was also true for aromatase and the X-linked androgen receptor. Eight of the genes indicated as upregulated by the array screen (Testatin, AK086357 [GenBank] , NM_198677 [GenBank] , AK007033 [GenBank] , BY366152 [GenBank] , XM_146592, AK006132 [GenBank] and Tm4sf6) were not confirmed by RT–PCR. These false positives showed no significant chromosomal bias, seven being autosomal and only one being X-linked. Discounting these leaves an even higher degree (14/15 genes) of sex chromosome linkage among the genes confirmed as upregulated by both techniques. Figure 5B–G highlights further RT–PCR results of particular interest.


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  		Table 2. {Delta}{Delta}Ct values for control relative to mutant genotypes for all genes tested in the RT–PCR study
 


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  		Figure 5. RT–PCR analysis shows specific upregulation of X- and Y-linked members of multigene families. Values plotted are 2–{Delta}{Delta}Ct. (A) Line plot showing all genes surveyed. A log axis is used to demonstrate the dramatic drop in expression level of MSYq-encoded transcripts. Blue, Y-linked transcripts; pink, X-linked transcripts and black, autosomal transcripts. Remaining panels are bar charts showing {Delta}{Delta}Ct±SEM for (B) AK005922 and relatives, (C) mgclh and mgcl-1, (D) Xmr and relatives, (E) Ckt1 and relatives, (F) AK005630 and relatives and (G) further X–Y homologous genes and the new Y-linked transcript AK006152. *Dunnett post hoc test, significant at 95% CI (**99%). {ddi30410}, XYRIII expression (control for XYRIIIqdel and XSxraY*X); {ddi30411}, XY–Sry expression control for XY–qdel, Sry); {ddi30412}, XYRIIIqdel expression; {ddi30413}, XY–qdel, Sry expression; {ddi30414}, XSxraY*X expression.

 

   AK005922 and XM_358268 are upregulated, whereas an autosomal homologue is not (Fig. 5B)
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 AK005922 and XM_358268 are...
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The most highly upregulated X-linked gene in both the initial microarray screen and the RT–PCR follow-up was AK005922, an intronless gene containing histone-fold TAFII domains, present in multiple copies mapping close to the X-centromere. BLAST searches revealed a single-copy autosomal relative (AK018839) and several Y-linked copies (e.g. XM_358268), all further family members also being intronless. According to contig NT_078925 [GenBank] .3 from mouse genome build 34.1, the Y-members are located on Yp, 200 kb distal to the Rbmy cluster, between Rbmy1a1 and Sry. Importantly, transcripts from the X- and Y-linked copies were upregulated, whereas those from the autosomal copy were not.

Mgclh is upregulated, whereas the autosomal mgcl-1 is not (Fig. 5C)
The second most highly upregulated X-linked gene in both the microarray and RT–PCR studies was mgclh, an intronless, multicopy, centromeric, homologue of Drosophila gcl (germ cell-less). gcl is a maternal-effect transcriptional repressor necessary for specifying germ plasm in the developing embryo (21Go–23Go). There is a second, autosomal homologue of gcl in the mouse, mgcl-1 (24Go–27Go), an intron-containing gene located on chromosome 2, which shares 45% amino acid identity and 62% similarity with mgclh over the entire length of the coding region, suggesting that the two genes have a similar function. Once again, the autosomal homologue is not upregulated.

Xmr and AK015913 are upregulated, whereas Sly is downregulated (Fig. 5D)
Xmr and relatives are a highly multicopy gene family with an estimated 50–75 copies on each of the X- and Y-chromosomes (28Go). The founder member of the family, Xlr, was identified as encoding a lymphocyte antigen (29Go) and another member of the family, Xmr, was subsequently shown to encode a germ cell antigen which binds to the nucleolus and the asynapsed arms of the X- and Y-chromosomes during MSCI (30Go–32Go). The Y-linked copies, founder member Sly [Mouse Chromosome Y Mapping Project (Jessica E. Alfoldi, Helen Skaletsky, Steve Rozen and David C. Page at the Whitehead Institute for Biomedical Research, Cambridge MA, USA and the Washington University Genome Sequencing Center, St Louis, MO, USA)], map to MSYq and produce a transcript expressed specifically in spermatids; this transcript is consequently reduced or absent in the MSYqdel models. We find that Xmr and the closely related X-linked AK015913 are significantly upregulated in the MSYqdel models. Xlr was included in the panel for the sake of completeness, but is not thought to be expressed in any of the germ or supporting cell lineages. Absolute Ct values for this gene were high, and none of the ratios to control was significant. The high Ct values indicate very low transcript abundance, which may be derived from lymphocytes in testicular blood vessels.

Surprisingly, our ISH results indicate that Xmr, AK015913 [GenBank] and Sly are strongly expressed in spermatids in a tubule stage-specific manner (Fig. 3). The primers used to assay Xmr expression in our study show a marked increase in transcript abundance between testes at day 15 and day 23 post-partum (data not shown), again consistent with spermatid expression. This runs counter to immunofluorescence-based reports (30Go,32Go) that Xmr expression is specific to meiotic stages. It may be that the Xmr-related transcripts in spermatids are not translated or that the epitope recognized by the antibody used in these studies is absent or inaccessible in the family members expressed in spermatids. It is clear from the northern blot data in the original article of Calenda et al (30Go) that a great increase in Xmr-related transcript abundance occurs in the testis between post-natal weeks 2 and 3 and 5, consistent with the majority of these transcripts being spermatid derived.

Ckt1 and X-linked relatives are upregulated, whereas the autosomal Ckt1r2 is not (Fig. 5E)
Ckt1 and Cktr1-3 are a family of closely related genes identified as differentially phosphorylated spermatid-specific targets of Ck2 (X. Xu et al., unpublished data, direct submission to GenBank) and have since been shown to be a component of the post-acrosomal perinuclear theca (33Go). Ckt1r2 is the only autosomal member of the family and is intronless (thus most likely a retroposed copy), whereas the other X-linked family members all contain a single intron. Again, it is X-linked members of the family that are specifically upregulated.

X-linked members of a novel multigene family are selectively upregulated (Fig. 5F)
Expressed sequence tags, AK005630, AK006132 [GenBank] , AK005817 [GenBank] and XM_141928, together comprise a multigene family with two subfamilies. AK006132 is intronless, appears to derive from a retroposed copy of AK005630 and maps to chromosome 12. The other three family members are X-linked and may be present in multiple copies. All three X-linked transcripts are significantly upregulated in the MSYqdel models, whereas the autosomal relative is not. All four transcripts potentially encode proteins, but there were no significant motifs found when the putative protein sequences were searched against the PROSITE database. It may be significant that all family members except AK005630 contain potential phosphorylation sites for Ck2.

Other X- and Y-linked genes show varying degrees of upregulation (Fig. 5G)
Upregulation of the Y-linked AK006152 was confirmed. A further four Yp-linked genes were assayed along with their X- and autosomal homologues. Of these, Rbmy1a1 showed significant downregulation in XSxraY*X, confirming the partial deletion of this multicopy gene in this genotype. Rbmx and Usp9x/y showed no clear pattern of variation between genotypes; however, both Ube1x and Ube1y were upregulated, the extent of the upregulation increasing with deletion size and being significant in the XSxraY*X model. None of Zfy1, Zfy2, Zfx or Zfa showed significant upregulation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 AK005922 and XM_358268 are...
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The aim of this study was to investigate the downstream transcriptional consequences of deletions on mouse MSYq. In addition to the loss of MSYq-linked transcripts, we found a moderate upregulation of several genes in the spermatids of MSYqdel males. These upregulated genes may contribute to the testicular phenotypes seen in the MSYq deletion models. In particular, mgclh is closely homologous to genes thought to function in sperm head development in mouse and in human (34Go,35Go), and the upregulated genes also include several known or potential substrates of the kinase Ck2, which is known to regulate sperm head morphogenesis (36Go).

We further observed that the upregulated genes were predominantly X- and Y-linked and confirmed this in three separate instances where there are closely homologous X and autosomal genes and in one instance where X, Y and autosomal copies exist. In every case, the X- and Y-genes were significantly upregulated, whereas the autosomal homologues were not. Therefore, this indicates that there is a specific derepression of gonosomal genes in mutant spermatids associated with a loss of MSYq-encoded transcripts. Among the genes known to lie within MSYq, the best candidate for a role in XY regulation in spermatids is Sly. Sly is the most highly transcribed of the known MSYq genes, all of which are spermatid specific. The putative SLY protein contains a COR1 chromatin-binding domain, and the X-linked relative Xmr produces a protein which associates with the transcriptionally repressed X- and Y-chromosomes in pachytene spermatocytes during MSCI (30Go,31Go). This suggests that Sly may be targeted to the gonosomes in spermatids and may regulate gonosomal chromatin conformation and expression.

Although Sly is downregulated in the MSYqdel models, its X-linked homologues, Xmr and AK015913, are upregulated in these models. Together with the highly expanded copy numbers of these genes and the sex ratio skew in the offspring of XYRIIIqdel males, this suggests that there is an intragenomic conflict between the X- and Y-members of this gene family. Further evidence in favour of this ‘Sly/Xmr conflict theory’ is given by a recent study of hybrid breakdown in Mus musculusxM. molossinus crosses (37Go). Haldane's rule suggests that hybrid sterility in males of interspecific crosses is due to unbalancing of sex ratio distorter genes and their suppressors (38Go). The hybrid breakdown in the musculus/molossinus cross leads to male-specific infertility with sperm head abnormalities highly similar to those seen in the nine-tenth MSYq deletion model addressed in this article. The sperm head phenotype was mapped to three QTL regions on the mouse X-chromosome, named Spha1–3 (37Go). Of these, Spha1 and Spha2 map closely to the two largest complexes of Xmr sequences, whereas Spha3 may also contain Xmr copies.

The potential for conflict between the X and the Y has long been advanced; however, strong selective constraints on offspring sex ratio dictate that any such conflict must inevitably be concealed by the evolution of suppressor alleles (39Go). The effects of the distorter/suppressor complexes are thus only seen when the suppressor is deleted (e.g. the MSYqdel models) or when the ‘wrong’ sets of drivers and suppressors are combined, as in the inter-subspecific cross discussed earlier. Under our Sly/Xmr conflict hypothesis, one or more of the X-linked transcripts upregulated in the mutant models are responsible for the sex ratio distortion in XYRIIIqdel, and the gene complement of MSYq has evolved to oppose the effect of the X-transcript and maintain a normal sex ratio. One candidate gene for the sex ratio distorter is Xmr itself; however, an alternative possibility is that the distorter gene is among the other upregulated X-linked loci, with the competitors Xmr and Sly being opposing regulators of this gene via an antagonistic action on sex chromatin.

The involvement of Xmr homologues also indicates that there may be a link between the MSCI and the gonosomal derepression seen in the mutant models, for example, a persistence of the meiotically silenced state into spermatids, which is modulated by the action of Xmr, Sly and other family members. Such a persistence has been suggested by Huynh and Lee (40Go) as a means of providing a ‘pre-inactivated’ X for dosage compensation in the early embryo. More recently, it has been shown that the X- and Y-chromosomes and all non-pairing DNA are silenced during meiosis in mouse, and MSCI may be a special case or elaboration of this more general phenomenon (41Go–43Go). Many of the upregulated genes discussed in this article are present in multiple copy ampliconic regions (e.g. AK005922, mgclh, Xmr and AK015913) or multigene families (Ckt and relatives and AK005630 and relatives), and some of those that are not multicopy are nevertheless contained within palindromes (e.g. Tgifx1) (44Go). Both the X- and Y-chromosomes have been shown to be enriched for such palindromic repeat regions (44Go–46Go). It is possible that these repetitive regions can self-synapse during pachytene, thus escaping recognition as unpaired DNA. DNase I hypersensitive regions (indicative of active chromatin conformation) have been observed on the otherwise silenced X and Y during pachytene, and this hypersensitivity persists through to at least metaphase 1 (47Go). The recent observation that Xmr also binds to the tandem repeat arrays of ribosomal genes in the nucleolus during MSCI may also have a bearing on this issue (32Go). If repetitive regions (whether palindromic or direct repeats) are processed differently during pachytene, this may explain why some X-genes (AK006251, Xmr) are expressed in spermatocytes, and also why some X-genes are able to activate expression in spermatids, whereas others remain silent following MSCI.

Irrespective of the precise mechanism involved, our observations demonstrate a link between breakdown of gonosomal silencing and sex ratio abnormalities, indicating that one function of this silencing is to prevent transmission distortion by selfish elements. We therefore propose that meiotic silencing of unpaired DNA is a general genomic defence mechanism against transmission distorters and other selfish elements, which acts in the form of MSCI to maintain a normal sex ratio in the face of X/Y antagonism. MSCI thus has the effect of ‘brokering a truce’ between warring X- and Y-genomes. However, the proposed involvement of the mouse-specific Xmr gene (and relatives) with gonosomal regulation means that caution must be exercised in generalizing observations in the mouse to other species, especially concerning gonosomal regulation in spermatids.

If silencing of unpaired DNA is indeed an adaptation for genomic defence, it is tempting to see the largely heterochromatic nature of the mouse Y-chromosome long arm as a more extreme manifestation of the same type of process. Recruitment of heterochromatin may provide a longer term means of achieving the silencing reiterated in each generation by MSCI, or it may be a secondary process occurring at sites where selfish elements and/or their repressors have become amplified. In this regard, the extensive heterochromatic blocks on other Y-chromosomes (including the human Y) may be viewed as representing ‘genomic scars’ left at the site of past intragenomic battles, the mouse Y long arm revealing an intermediate stage in the process of Y degeneration and heterochromatinization.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 AK005922 and XM_358268 are...
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Mice
All mice were produced on a random-bred albino MF1 strain (NIMR colony) background. The mice used to provide RNA for microarray analysis were the three MSYq-deficient genotypes that we have previously analysed with respect to Ssty, Sly and Asty expression and sperm abnormalities, together with appropriate age- and strain-matched controls.

  1. XYRIIIqdel males (approximately two-third MSYq deletion). These mice have an RIII strain Y-chromosome with a deletion removing approximately two-thirds of MSYq. The sperm has mild distortions of head shape; the mice are nevertheless fertile with an intriguing distortion of the sex ratio in favour of females (2Go). XYRIII males are the appropriate controls.
  2. XYTdym1qdelSry males (approximately nine-tenth MSYq deletion). These mice have a 129 strain Y-chromosome with a deletion removing approximately nine-tenths of MSYq and also a small deletion (Tdym1) removing the testis determinant Sry from the short arm, this deficiency being complemented by an Sry transgene. These males are sterile with virtually all the sperm having grossly distorted heads (6Go). XYTdym1Sry males are the appropriate controls. YTdym1 is referred earlier as Y–.
  3. XSxraY*X males (no MSYq). In these mice, the only Y-specific material is provided by the Y short arm-derived factor Sxra; the Y*X-chromosome provides a second PAR, thus allowing fulfilment of the requirement for PAR synapsis. These males lack the entire Y-specific (non-PAR) gene content of MSYq; they also have a 7.5-fold reduction in copies of the Rbmy gene family located on the short arm adjacent to the centromere. The males are sterile and have even more severe sperm head defects than XY–qdelSry males (1Go,6Go). As Sxra orginated from a YRIII-chromosome, the appropriate controls are again XYRIII.

Sample collection
Testes were obtained from two 2-month-old XYRIIIqdel, XY–qdelSry and XSxraY*X males, from three 1-month-old XSxraY*X males and from equal numbers of age- and strain-matched controls. Total RNA was isolated using TRI reagent (Sigma) and cleaned using RNEasy columns (Qiagen), according to the manufacturers' protocols.

Array analysis
Microarray hybridizations and analysis were carried out as described (20Go), except that microarray data normalization was based on the global median signal for Cy3 and Cy5 channels, rather than on a panel of control genes, and intensity-dependent Lowess correction was applied. This form of normalization is valid because the majority of genes do not vary between the mutant and control samples for any of the models (Fig. 2). Normalization, t-tests and fold-change filtering were performed using GeneSpring version 4.2.1. Genes were designated as significant if they showed a 1.5-fold change and a 1% t-test relative to control in both the 1 month and 2 month XSxraY*X samples (Table 1). Full experimental protocols and raw data have been deposited in the public access ArrayExpress database, accession no. E-MEXP-251.

Real-time RT–PCR
Real-time RT–PCR was performed in 96-well plates using the QuantiTect SYBR Green RT–PCR kit (Qiagen) according to the manufacturer's protocols, quantifying the resulting fluorescence using an iCycler system (BioRad, UK). Thirty nanograms of total RNA was used for each RT–PCR reaction. RNA was DNase-treated before RT–PCR, and primer pairs (Supplementary Material, Table S1) were designed to span an intron boundary and to include at least one intron wherever possible. The identity of all RT–PCR products was confirmed by sequencing.

We analysed XYRIIIqdel, XY–qdel, Sry, XSxraY*X, XYRIII and XY–, Sry genotypes, all at 2 months of age. Duplicate RT–PCR reactions for each gene were performed for each genotype, and Ubb was included on every plate as a loading control, along with appropriate negative controls. A ‘crossing-point’ Ct was obtained for each well, being the fractional cycle number at which the measured fluorescence crossed the threshold of 100 U, a value chosen such that all reactions in all plates were in log phase. Differential loading was controlled on a per-genotype per-plate basis by subtracting the mean Ct for the loading control Ubb to give {Delta}Ct. {Delta}{Delta}Ct values were then calculated as the change in {Delta}Ct for the MSYqdel genotypes relative to the appropriate control genotype. Finally, the complete expression profiles for each genotype were normalized to the mean {Delta}{Delta}Ct of the 10 control genes included in the panel. This last step was necessary as the fold changes in transcript abundance are small, and a panel of control genes forms a more robust basis for comparison than the single gene Ubb. Raw Ct values and all stages of normalization are shown in Supplementary Material, Table S2. Significance testing was performed using Analyse-It software to perform one-way analysis of variance of the five genotypes (three mutants and two controls) within Microsoft Excel, followed by Dunnett post hoc testing of the mutant/control comparisons.

RNA in situ analysis
In situ hybridization using digoxigenin-labelled cRNAs was used to localize each mRNA on Bouin's fixed paraffin-embedded mouse testis sections using procedures previously described (48Go) with hybridization and washing temperatures up to 55°C. Both antisense and sense (negative control) cRNAs were used on each sample, in every experiment, for each set of conditions tested.

Northern blot analysis
Clones for AK005922, mgclh and Xmr were used to perform northern blot analysis. An actin probe that recognizes {alpha}- and ß-actin transcripts (49Go) served as a loading control. Total RNA (20 µg) was electrophoresed in a 1.4% formaldehyde/agarose gel and transferred to Hybond-N membrane (Amersham) using 20x SSC buffer. The RNA was cross-linked to the membrane with ultraviolet (StrataLinkerTM), the membrane fixed for 1 h at 80°C and hybridized overnight at 60°C with 32P-labelled probes in hybridization buffer (6x SSC, 5x Denharts, 0.1% SDS, 50 mM sodium phosphate, 100 µg/ml salmon sperm DNA). After two 60°C washes (30 min 0.5x SSC, 0.1% SDS and 30 min 0.1x SSC, 0.1% SDS), the membrane was exposed to X-ray film for 5–6 h except for the actin filter which was exposed overnight.

Southern blot analysis
Southern blot analysis was performed for the unmapped transcripts AK006152, XM_146592, NM_198677 [GenBank] and AK007033. Genomic DNA was extracted from normal XX and XY mouse livers and 12 µg aliquots of each sample digested to completion with three different restriction enzymes, EcoR1, Taq{alpha}1 and Sst1 (NEB). Results for digestion with Taq{alpha}1 are shown in Supplementary Material, Figure S1. Digested samples were electrophoresed through 0.8% TAE agarose gel, depurinated, denatured and transferred to Hybond-N nylon membranes (Amersham). Membranes were baked at 80°C for 2 h to fix the DNA, followed by overnight hybridization of 32P-labelled probes at 65°C in Church buffer. Wash step: 15 min at 65°C in 2x SSC, 0.1% SDS. Membranes were exposed to X-ray film at –80°C for times ranging from 3 h to 2 weeks.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 AK005922 and XM_358268 are...
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank Aine Rattigan for PCR genotyping and Anthony Brown, David Carter and Rob Furlong at the Department of Pathology Centre for Microarray Resources for printing and QC of microarray slides. J.M.A.T. is an MRC Career Development Fellow. The study was funded partly by BBSRC and the Wellcome Trust (E.J.C. and P.J.I.E.), the NHMRC of Australia (K.L.L. no. 143792) and the ARC (Paul S. Burgoyne and Kate L. Loveland).

Conflict of Interest statement. There are no conflicts of interest in regard to this manuscript for any of the authors.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 AK005922 and XM_358268 are...
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

  1. Burgoyne, P.S., Mahadevaiah, S.K., Sutcliffe, M.J. and Palmer, S.J. (1992) Fertility in mice requires X–Y pairing and a Y-chromosomal ‘spermiogenesis’ gene mapping to the long arm. Cell, 71, 391–398.[CrossRef][ISI][Medline]

  2. Conway, S.J., Mahadevaiah, S.K., Darling, S.M., Capel, B., Rattigan, A.M. and Burgoyne, P.S. (1994) Y353/B: a candidate multiple-copy spermiogenesis gene on the mouse Y chromosome. Mamm. Genome, 5, 203–210.[CrossRef][ISI][Medline]

  3. Styrna, J., Imai, H.T. and Moriwaki, K. (1991) An increased level of sperm abnormalities in mice with a partial deletion of the Y chromosome. Genet. Res., 57, 195–199.[ISI][Medline]

  4. Styrna, J., Klag, J. and Moriwaki, K. (1991) Influence of partial deletion of the Y chromosome on mouse sperm phenotype. J. Reprod. Fertil., 92, 187–195.[Abstract]

  5. Suh, D.S., Styrna, J. and Moriwaki, K. (1989) Effect of Y chromosome and H-2 complex derived from Japanese wild mouse on sperm morphology. Genet. Res., 53, 17–19.[ISI][Medline]

  6. Toure, A., Szot, M., Mahadevaiah, S.K., Rattigan, A., Ojarikre, O.A. and Burgoyne, P.S. (2004) A new deletion of the mouse Y chromosome long arm associated with the loss of Ssty expression, abnormal sperm development and sterility. Genetics, 166, 901–912.[Abstract/Free Full Text]

  7. Xian, M., Azuma, S., Naito, K., Kunieda, T., Moriwaki, K. and Toyoda, Y. (1992) Effect of a partial deletion of Y chromosome on in vitro fertilizing ability of mouse spermatozoa. Biol. Reprod., 47, 549–553.[Abstract]

  8. Vallender, E.J. and Lahn, B.T. (2004) How mammalian sex chromosomes acquired their peculiar gene content. Bioessays, 26, 159–169.[CrossRef][ISI][Medline]

  9. Fisher, R. (1931) The evolution of dominance. Biol. Rev., 6, 345–368.

  10. Charlesworth, B. (1996) The evolution of chromosomal sex determination and dosage compensation. Curr. Biol., 6, 149–162.[CrossRef][ISI][Medline]

  11. Graves, J.A. (1995) The origin and function of the mammalian Y chromosome and Y-borne genes—an evolving understanding. Bioessays, 17, 311–320.[CrossRef][ISI][Medline]

  12. Lahn, B.T. and Page, D.C. (1997) Functional coherence of the human Y chromosome. Science, 278, 675–680.[Abstract/Free Full Text]

  13. Lahn, B.T., Pearson, N.M. and Jegalian, K. (2001) The human Y chromosome, in the light of evolution. Nat. Rev. Genet., 2, 207–216.[CrossRef][ISI][Medline]

  14. Khil, P.P., Smirnova, N.A., Romanienko, P.J. and Camerini-Otero, R.D. (2004) The mouse X chromosome is enriched for sex-biased genes not subject to selection by meiotic sex chromosome inactivation. Nat. Genet., 36, 642–646.[CrossRef][ISI][Medline]

  15. Siruntawineti, J., Yamagata, K., Nakanishi, T. and Baba T. (2002) Occurrence of small, round vesicles in the acrosome of elongating spermatids from a mouse mutant line with partial deletion of the Y chromosome. J. Reprod. Develop., 48, 513–521.[CrossRef]

  16. Kotula-Balak, M., Grzmil, P., Styrna, J. and Bilinska, B. (2004) Immunodetection of aromatase in mice with a partial deletion in the long arm of the Y chromosome. Acta Histochem., 106, 55–64.[CrossRef][ISI][Medline]

  17. Bishop, C.E. and Hatat, D. (1987) Molecular cloning and sequence analysis of a mouse Y chromosome RNA transcript expressed in the testis. Nucleic Acids Res., 15, 2959–2969.[Abstract/Free Full Text]

  18. Prado, V.F., Lee, C.H., Zahed, L., Vekemans, M. and Nishioka, Y. (1992) Molecular characterization of a mouse Y chromosomal repetitive sequence that detects transcripts in the testis. Cytogenet. Cell Genet., 61, 87–90.[ISI][Medline]

  19. Toure, A., Grigoriev, V., Mahadevaiah, S.K., Rattigan, A., Ojarikre, O.A. and Burgoyne, P.S. (2004) A protein encoded by a member of the multicopy Ssty gene family located on the long arm of the mouse Y chromosome is expressed during sperm development. Genomics, 83, 140–147.[CrossRef][ISI][Medline]

  20. Ellis, P.J., Furlong, R.A., Wilson, A., Morris, S., Carter, D., Oliver, G., Print, C., Burgoyne, P.S., Loveland, K.L. and Affara, N.A. (2004) Modulation of the mouse testis transcriptome during postnatal development and in selected models of male infertility. Mol. Hum. Reprod., 10, 271–281.[Abstract/Free Full Text]

  21. Leatherman, J.L., Levin, L., Boero, J. and Jongens, T.A. (2002) Germ cell-less acts to repress transcription during the establishment of the Drosophila germ cell lineage. Curr. Biol., 12, 1681–1685.[CrossRef][ISI][Medline]

  22. Robertson, S.E., Dockendorff, T.C., Leatherman, J.L., Faulkner, D.L. and Jongens, T.A. (1999) Germ cell-less is required only during the establishment of the germ cell lineage of Drosophila and has activities which are dependent and independent of its localization to the nuclear envelope. Dev. Biol., 215, 288–297.[CrossRef][ISI][Medline]

  23. Jongens, T.A., Ackerman, L.D., Swedlow, J.R., Jan, L.Y. and Jan, Y.N. (1994) Germ cell-less encodes a cell type-specific nuclear pore-associated protein and functions early in the germ-cell specification pathway of Drosophila. Genes Dev., 8, 2123–2136.[Abstract/Free Full Text]

  24. de la Luna, S., Allen, K.E., Mason, S.L. and La Thangue, N.B. (1999) Integration of a growth-suppressing BTB/POZ domain protein with the DP component of the E2F transcription factor. EMBO J., 18, 212–228.[CrossRef][ISI][Medline]

  25. Kimura, T., Yomogida, K., Iwai, N., Kato, Y. and Nakano, T. (1999) Molecular cloning and genomic organization of mouse homologue of Drosophila germ cell-less and its expression in germ lineage cells. Biochem. Biophys. Res. Commun., 262, 223–230.[CrossRef][ISI][Medline]

  26. Leatherman, J.L., Kaestner, K.H. and Jongens, T.A. (2000) Identification of a mouse germ cell-less homologue with conserved activity in Drosophila. Mech. Dev., 92, 145–153.

  27. Nili, E., Cojocaru, G.S., Kalma, Y., Ginsberg, D., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Berger, R., Shaklai, S., Amariglio, N. et al. (2001) Nuclear membrane protein LAP2beta mediates transcriptional repression alone and together with its binding partner GCL (germ-cell-less). J. Cell Sci., 114, 3297–3307.[Medline]

  28. Garchon, H.J., Loh, E., Ho, W.Y., Amar, L., Avner, P. and Davis, M.M. (1989) The XLR sequence family: dispersion on the X and Y chromosomes of a large set of closely related sequences, most of which are pseudogenes. Nucleic Acids Res., 17, 9871–9888.[Abstract/Free Full Text]

  29. Cohen, D.I., Hedrick, S.M., Nielsen, E.A., D'Eustachio, P., Ruddle, F., Steinberg, A.D., Paul, W.E. and Davis, M.M. (1985) Isolation of a cDNA clone corresponding to an X-linked gene family (XLR) closely linked to the murine immunodeficiency disorder xid. Nature, 314, 369–372.[CrossRef][Medline]

  30. Calenda, A., Allenet, B., Escalier, D., Bach, J.F. and Garchon, H.J. (1994) The meiosis-specific Xmr gene product is homologous to the lymphocyte Xlr protein and is a component of the XY body. EMBO J., 13, 100–109.[ISI][Medline]

  31. Escalier, D. and Garchon, H.J. (2000) XMR is associated with the asynapsed segments of sex chromosomes in the XY body of mouse primary spermatocytes. Chromosoma, 109, 259–265.[CrossRef][ISI][Medline]

  32. Escalier, D. and Garchon, H.J. (2005) XMR, a dual location protein in the XY pair and in its associated nucleolus in mouse spermatocytes. Mol. Reprod. Dev., 72, 105–112.[CrossRef][ISI][Medline]

  33. Kitamura, K., Iguchi, N., Kaneko, Y., Tanaka, H. and Nishimune, Y. (2004) Characterization of a novel postacrosomal perinuclear theca-specific protein, CYPT1. Biol. Reprod., 71, 1927–1935.[Abstract/Free Full Text]

  34. Kimura, T., Ito, C., Watanabe, S., Takahashi, T., Ikawa, M., Yomogida, K., Fujita, Y., Ikeuchi, M., Asada, N., Matsumiya, K. et al. (2003) Mouse germ cell-less as an essential component for nuclear integrity. Mol. Cell Biol., 23, 1304–1315.[Abstract/Free Full Text]

  35. Kleiman, S.E., Yogev, L., Gal-Yam, E.N., Hauser, R., Gamzu, R., Botchan, A., Paz, G., Yavetz, H., Maymon, B.B., Schreiber, L. et al. (2003) Reduced human germ cell-less (HGCL) expression in azoospermic men with severe germinal cell impairment. J. Androl., 24, 670–675.[Abstract/Free Full Text]

  36. Xu, X., Toselli, P.A., Russell, L.D. and Seldin, D.C. (1999) Globozoospermia in mice lacking the casein kinase II alpha' catalytic subunit. Nat. Genet., 23, 118–121.[CrossRef][ISI][Medline]

  37. Oka, A., Mita, A., Sakurai-Yamatani, N., Yamamoto, H., Takagi, N., Takano-Shimizu, T., Toshimori, K., Moriwaki, K. and Shiroishi, T. (2004) Hybrid breakdown caused by substitution of the X chromosome between two mouse subspecies. Genetics, 166, 913–924.[Abstract/Free Full Text]

  38. Haldane, J.B.S. (1922) Sex ratio and unisexual sterility in hybrid animals. J. Genet., 12, 101–109.[ISI]

  39. Partridge, L. and Hurst, L.D. (1998) Sex and conflict. Science, 281, 2003–2008.[Abstract/Free Full Text]

  40. Huynh, K.D. and Lee, J.T. (2003) Inheritance of a pre-inactivated paternal X chromosome in early mouse embryos. Nature, 426, 857–862.[CrossRef][Medline]

  41. Turner, J.M., Mahadevaiah, S.K., Fernandez-Capetillo, O., Nussenzweig, A., Xu, X., Deng, C.X. and Burgoyne, P.S. (2005) Silencing of unsynapsed meiotic chromosomes in the mouse. Nat. Genet., 37, 41–47.[ISI][Medline]

  42. Baarends, W.M., Wassenaar, E., van der Laan, R., Hoogerbrugge, J., Sleddens-Linkels, E., Hoeijmakers, J.H., de Boer, P. and Grootegoed, J.A. (2005) Silencing of unpaired chromatin and histone H2A ubiquitination in mammalian meiosis. Mol. Cell Biol., 25, 1041–1053.[Abstract/Free Full Text]

  43. Huynh, K.D. and Lee, J.T. (2005) X-chromosome inactivation: a hypothesis linking ontogeny and phylogeny. Nat. Rev. Genet., 6, 410–418.[CrossRef][ISI][Medline]

  44. Warburton, P.E., Giordano, J., Cheung, F., Gelfand, Y. and Benson, G. (2004) Inverted repeat structure of the human genome: the X-chromosome contains a preponderance of large, highly homologous inverted repeats that contain testes genes. Genome Res., 14, 1861–1869.[Abstract/Free Full Text]

  45. Rozen, S., Skaletsky, H., Marszalek, J.D., Minx, P.J., Cordum, H.S., Waterston, R.H., Wilson, R.K. and Page, D.C. (2003) Abundant gene conversion between arms of palindromes in human and ape Y chromosomes. Nature, 423, 873–876.[CrossRef][Medline]

  46. Kuroda-Kawaguchi, T., Skaletsky, H., Brown, L.G., Minx, P.J., Cordum, H.S., Waterston, R.H., Wilson, R.K., Silber, S., Oates, R., Rozen, S. et al. (2001) The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men. Nat. Genet., 29, 279–286.[CrossRef][ISI][Medline]

  47. Richler, C., Uliel, E., Kerem, B.S. and Wahrman, J. (1987) Regions of active chromatin conformation in ‘inactive’ male meiotic sex chromosomes of the mouse. Chromosoma, 95, 167–170.[CrossRef][ISI][Medline]

  48. Meinhardt, A., O'Bryan, M.K., McFarlane, J.R., Loveland, K.L., Mallidis, C., Foulds, L.M., Phillips, D.J. and de Kretser, D.M. (1998) Localization of follistatin in the rat testis. J. Reprod. Fertil., 112, 233–241.[Abstract]

  49. Minty, A.J., Caravatti, M., Robert, B., Cohen, A., Daubas, P., Weydert, A., Gros, F. and Buckingham, M.E. (1981) Mouse actin messenger RNAs. Construction and characterization of a recombinant plasmid molecule containing a complementary DNA transcript of mouse alpha-actin mRNA. J. Biol. Chem., 256, 1008–1014.[Free Full Text]


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