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MRC National Institute for Medical Research,3 London NW7 1AA, United Kingdom
Département Génétique & Développement,4 Institut Cochin, Paris F-75014, France
Karolinska Institute,5 Stockholm, Sweden SE-17177
ABSTRACT
The mouse multi-copy X-linked gene Xlr-related, meiosis-regulated (Xmr/Slx) has previously been described as encoding a testis-specific nuclear protein expressed during male meiotic prophase, and during which it becomes concentrated in the inactive X and Y chromatin domain. These conclusions were based on Western blot and immunolocalization analysis using an antibody raised against a related lymphocyte protein, XLR; however, our recently published RNA in situ for Xmr revealed that transcripts are predominantly or exclusively postmeiotic, and this is supported by a growing body of microarray data. This led us to reanalyze the expression of Xmr, both at the RNA level by RT-PCR and by RNA fluorescence in situ hybridization, and at the protein level by using antibodies raised against XMR that do not recognize XLR. In agreement with our previous RNA in situ data, our further transcription analysis showed almost exclusive expression in spermatids, and Western blot and immunostaining with the XMR antibodies showed that the protein is cytoplasmic and restricted to spermatids. Furthermore, the previously used XLR antibody was shown not to cross-react with XMR, and it is suggested that the meiotically expressed nuclear protein recognized by this antibody is another member of the complex Xlr superfamily. As a result of these findings, the gene previously known as Xmr is now officially know as Slx, Sycp3-like, X-linked.
Cor1 domain,, gametogenesis,, meiosis,, spermatid,, spermatid-specific,, spermatogenesis,, testis,, XLR,, Xlr superfamily,, XMR
Spermatogenesis is a complex developmental process whereby diploid stem cells generate haploid spermatozoa; recent microarray analyses have begun to document the underlying complexity of spermatogenic gene expression [1–4]. One gene postulated to play an important role during the meiotic prophase stage of spermatogenesis is Xmr/Slx [5], a multi-copy gene located proximally on the murine X chromosome. Xmr is a member of the Xlr superfamily, with an estimated 50–70 loci on the X chromosome [6]. The majority of these loci were thought to be pseudogenes, but the new microarray data indicate that a substantial proportion are transcribed and maintain an open reading frame.
The foundational member of this superfamily is Xlr, which encodes a 30-kDa nuclear protein expressed during the late stages of maturation in B lymphocytes [7] and preceding the rearrangement of the T-cell receptor genes in T lymphocytes [8]. XLR has been shown to colocalize with SATB1, a nuclear matrix protein [8], and is also expressed in the fetal ovary where it is located in the oocyte nucleus during prophase of meiosis I [9]. Other members of the Xlr superfamily include the Xlr3, Xlr4, and Xlr5 subfamilies. Xlr3a and Xlr3b have been shown to be expressed in lymphoid cells and the testis; intriguingly, they also show imprinted expression in the brain [10–12]. Although little is known about the function of the Xlr superfamily, the encoded proteins contain a partially conserved ‘Cor1' domain that is also found in the meiotically expressed synaptonemal complex protein SYCP3 (also called COR1) [13, 14]. Although the function of the Cor1 domain is not defined, it has been hypothesized to play a role in the localization of Cor1 domain proteins to chromatin.
Xmr belongs to a subgroup of the Xlr superfamily that includes multiple genomic copies of Xmr as well as related loci encoding closely similar testis transcripts such as AK015913 [2, 3, 15]. Xmr was first identified by Northern blot using a full length Xlr cDNA probe, which hybridized to an abundant, testis-specific transcript of approximately 1 kb [5]. Through subsequent screening of a testis cDNA library, an 812-bp transcript was identified that shared 94% homology with Xlr over the latter two-thirds of the transcript and appeared to encode an XLR-related protein with a conserved Cor1 domain [5]. Northern analysis of RNA from first spermatogenic wave testis samples detected transcripts from 3 wk postpartum, suggesting spermatid-specific expression; however, RT-PCR suggested that low-level transcription initiates as early as 6 days postpartum (dpp), 2 to 3 days before the onset of meiosis [5].
Calenda et al. [5] also assayed for protein expression in the testis using an existing antibody raised against XLR [5]. Consistent with their RT-PCR data, staining was seen in meiotic cells, the protein being first detected throughout the nucleus during leptotene and zygotene, when meiotic recombination is initiated [16]. The protein then associates with the X and Y chromosomes at the zygotene-pachytene transition as they undergo the chromatin changes that result in formation of the transcriptionally repressed XY- or sex-body [17, 18]. However, no protein was detected in spermatids, hence the name Xlr-related, meiosis-regulated (Xmr). The authors suggested that because Xlr and Xmr encode nuclear proteins that are expressed at a time of genome rearrangement, they may play an important role in chromatin metabolism [5, 8]. Indeed, both proteins share distant homology to Saccharomyces cerevisiae MER2, which is essential for the formation of meiotic DNA double-strand breaks [19].
Recently, by microarray analysis of males with mouse Y long arm (MSYq) deficiencies, we identified an MSYq-encoded multi-copy spermatid-expressed gene, Sly, with substantial homology to Xmr [20]. In a linked microarray study, Ellis et al. [15] made the intriguing finding that deletion of the mouse Y-chromosome long arm leads to up-regulation of multiple spermatid-expressed transcripts originating from the X chromosome and from the Y-chromosome short arm; these up-regulated transcripts included Xmr and the related transcript AK015913. Surprisingly, RNA in situ hybridization revealed that Xmr was expressed in round and early elongating spermatids but was undetectable in meiotic cells. Furthermore, by real-time PCR, Xmr transcripts were seen to appear some time between 15 dpp and 23 dpp, consistent with spermatid-derived expression [4, 15]. Ellis et al. [15] concluded that in normal males, the X and Y chromosomes are specifically repressed in spermatids, and that this depends on a factor encoded by MSYq. They noted that the sex-ratio distortion of offspring from males with partial MSYq deficiencies was suggestive of intragenomic conflict between X- and Y-linked genes, and proposed that Xmr and Sly may be key players in this conflict [15].
Given the direct evidence from RNA in situ hybridization that Xmr is highly transcribed during spermiogenesis, we have carried out a detailed reexamination of Xmr expression in mouse spermatogenesis. Because any role of Xmr in intragenomic conflict could be RNA or protein mediated, we have paid particular attention to establishing if the abundant Xmr transcripts in spermatids are translated.
Animal procedures were in accordance with the United Kingdom Animal Scientific Procedures Act 1986 and were subject to local Ethical Review. All mice were produced on an MF1 random-bred background at the MRC National Institute for Medical Research. XSxraY*X males [21] were produced by mating XY*X females [22, 23] to XYSxra males [24, 25]. XYRIIIqdel males were derived from a stock originating from the mice described by Conway et al. [26].
Total testis RNA was isolated using TRIzol reagent (Invitrogen) in accordance with the manufacturer's instructions. Three micrograms of total RNA was incubated with 5 units RQ DNAse 1 (Promega) for 1 h at 37°C and then ethanol precipitated. Eight hundred nanograms of total RNA was reverse-transcribed with Oligo dT primers (Invitrogen) in a 15-µl reaction using Superscript II reverse transcriptase (150 U; Invitrogen) according to the manufacturer's instructions; a 1-µl aliquot was then added to a 25-µl PCR reaction. Amplification was carried out for 35 cycles, with an annealing temperature of 60°C for Xmr and AK015913 using primers from Ellis et al. [15] and 56°C for primers A/B and D/C from Calenda et al. [5] (Fig. 1A).
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RNA Fluorescence In Situ Hybridization
RNA fluorescence in situ hybridization (FISH) was performed as previously described [27, 28] using an X-chromosomal BAC clone RP23-470D15 containing an Xmr locus (BACPAC Resources). Staging of spermatogenic cells was based on DAPI fluorescence morphology, together with immunolabeling for the synaptonemal complex protein SYCP3 (ab 15092; Abcam) and the phosphorylated histone H2AFX (
H2AX) detected by a mouse monoclonal antibody (05–636; Upstate), as previously described [27].
DNA Fluorescence In Situ Hybridization
DNA FISH was carried out after RNA FISH and immunofluorescence as previously published [28]. Digoxigenin-labeled probes were prepared using the Digoxigenin Nick Translation Kit (Roche), and hybridizations were carried out as described for RNA FISH (see previous section). After stringency washes were carried out as described previously [27], DNA FISH signals were developed by using anti-DIG-FITC (Chemicon) diluted 1:10 for 2 h at 37°C.
Antibody Production and Western Blot Analysis
The amino acid sequences of XMR, XLR, SLY, and the predicted sequence for AK015913 were aligned using ClustalW. Two peptides, (VSFSEEWQRFARS [amino acids 69–81] and QQVRNASELDL [amino acids 96–106]) were identified to be specific to XMR, but have one- and two-amino-acid differences from the AK015913-encoded predicted protein (AK015913), respectively. Two rabbits were each immunized with the two XMR peptides, followed by booster injections at 2, 4, and 8 wk as part of the AS-BOUB-LXP standard peptide immunization package offered by Eurogentec. A third anti-XMR rabbit antibody was raised against the first 93 amino acids of XMR as follows. First, the relevant region of the XMR cDNA was subcloned in frame with DHFR in the bacterial expression vector pQE-40 (Qiagen), and the XMR/DHFR fusion protein was expressed in E. coli M15pREP4 cells; the fusion protein was then purified in one step by affinity chromatography as described by the supplier (Qiagen). Female New England rabbits were injected with the bacterially expressed XMR polypeptide, followed by three separate booster injections. The sera from the rabbits were affinity purified on nitrocellulose membrane strips onto which the XMR polypeptide had been transferred. These three antibodies are subsequently designated XMR69–81, XMR96–106, and XMR1–93.
Testis protein lysates were obtained by homogenization on dry ice and resuspension in Laemmli buffer (Sigma) at 10% w/v. Lysates were then denatured for 10 min at 95°C, and 5-µl was electrophoresed through a 12% SDS/polyacrylamide minigel. Transfer to Hybond-C membrane (Amersham) was performed at 100 V for 1 h, and the membrane was then processed for immunodetection. The membrane was blocked (PBSA [PBS, autoclaved] containing 5% milk powder and 0.1% Tween) for 1h at room temperature and incubated with primary antibody diluted in blocking solution overnight at 4°C (rabbit anti-XMR antibodies XMR69–81 and XMR96–106, 1:2000; XMR1–93, 1:1000; mouse anti-XLR antibody, 1:1000 [5]; mouse anti-ACTIN, 1:200 [sc-8432; Santa Cruz]; mouse monoclonal anti-H2AX, 1:2000 [05–636; Upstate]). Membranes were washed three times in PBSA containing 0.1% Tween and incubated in the corresponding secondary antibody (anti-rabbit IgG or anti-mouse IgG) coupled to peroxidase as described by the manufacturer (DAKO). After three washes (PBS containing 0.1% Tween), the signal was revealed by chemiluminescence (SuperSignal) and recorded on x-ray film.
For immunohistochemistry, one testis of each mouse was fixed in 4% buffered paraformaldehyde for 4 h at room temperature and post fixed in a diluted Bouin solution (Sigma) (0.675% picric acid, 4.9% glacial acetic acid, 9.25% formaldehyde) at room temperature overnight. The second testis was fixed in Bouin solution overnight. Tissues were washed in 70% ethanol, dehydrated, and embedded in paraffin. Three-micrometer paraffin sections of testes were mounted on a TESPA (3-aminopropyltriethoxysilane)-coated glass slide and dried overnight at 37°C. Sections were dewaxed in xylene and hydrated in a graded series of alcohols. After washing in PBS, the sections were boiled for 10 min in 0.01 M sodium citrate solution (pH 6) using a microwave oven. Slides were then washed three times in PBS for 5 min each. For immunofluorescent staining, slides were blocked for an hour at room temperature in PBT (PBS, 0.1% Tween, 0.15% BSA). After blocking, slides were incubated overnight at 37°C with primary antibody diluted in PBT (rabbit XMR antibodies, 1:50; mouse XLR antibody [5], 1:100; mouse monoclonal H2AX antibody, 1:200 [05–636; Upstate]; rabbit H2AX antibody, 1:50 [07–164; Upstate]. Slides were washed in PBS, incubated in either chicken anti-rabbit Alexa 594 (1:500; Molecular Probes) or chicken anti-mouse Alexa 488 (1:100; Molecular Probes) diluted in PBS for 1 h at 37°C, washed in PBS, and mounted in DAPI (DABCO). For antibodies XMR69–81 and XMR96–106, incubation with preimmune rabbit serum was used as a control.
Xmr Transcripts Are Predominantly Expressed in Spermatids
The positions of the primers used for RT-PCR analysis are given in Figure 1A, and alignment of the Xmr transcript with the related AK015913 and Xlr transcripts is given in Supplemental Figure 1 (available online at www.biolreprod.org). First, we repeated the RT-PCR for Xmr using the primers designed by Calenda et al. [5] on testes harvested at different ages during the first wave of spermatogenesis. In prepubertal mice, the first wave of spermatogenesis is a relatively synchronized process with progressively more mature spermatogenic cell types appearing at defined time points after birth. This allows the correlation of expression patterns with the appearance of specific populations of germ cells. Consistent with the data of Calenda et al. [5], RT-PCR using primer pairs A/B and D/C gave a faint Xmr PCR product from 7.5 dpp; primer pair D/C showed a clear increase in Xmr transcripts from 18.5 dpp (Fig. 1B). We then analyzed expression of Xmr during embryonic gonad development and found that these primers detect Xmr from 14.5 days postcoitum (dpc) (Fig. 1C). Primer pair A/B also detected Xlr at all ages assayed (Fig. 1, B and C). Although an earlier study indicated that XLR was not expressed in the testis [9], we have previously detected this transcript and confirmed that it is Xlr by sequencing [29]. To separately assess Xmr and AK015913 transcription, we then carried out RT-PCR using another primer pair, D/B, designed by Calenda et al. [5] but not previously used by them for RT-PCR expression analysis. This primer pair gives distinct product sizes for Xmr (813 bp) and AK015913 (623 bp). We detected a faint product for Xmr at 18.5 dpp, but it was nevertheless clear that the predominant expression for Xmr occurred later, from 18.5 dpp; AK015913 was only weakly detected by these primers from 21.5 dpp. The identity of both products was subsequently confirmed by direct sequencing. However, these primers detect multiple bands which may derive from other Xmr-related sequences in addition to Xmr and AK015913, so we then repeated the RT-PCR using primers designed to be specific to Xmr or to AK015913 sequences [15]. RT-PCR using the Xmr-specific primers (Fig. 1A) gave a product from 21.5 dpp, indicating that expression is exclusively postmeiotic in spermatids. RT-PCR for the related AK015913 gave a product from 18.5 dpp, which, in our stock, corresponds to the meiosis-spermatid transition.
Next, we examined the expression pattern of Xmr during spermatogenesis using RNA FISH. We observed Xmr-specific FISH signals (i.e., colocalizing with the Xmr DNA FISH signals) exclusively in round spermatid nuclei, identifiable by their small size and prominent chromocenters (Fig. 1D). Approximately one half (38/72) of all round spermatids had multiple RNA signals as expected from the multi-copy nature of Xmr. These signals originated from the edge of the X chromosome, which appeared as a characteristic DAPI-dense body located at the edge of the chromocenter [28]. Despite detailed examination, we did not detect Xmr-specific RNA signals in meiotic cells (n = 47; Fig. 1D) or in spermatogonia. We excluded the possibility that chromatin accessibility problems influenced our ability to detect Xmr RNA in these cell types by subsequently carrying out Xmr DNA FISH, which gave clear FISH signals. Importantly, the RNA and DNA FISH signals in round spermatids colocalized, confirming that the X-encoded transcripts identified originated from Xmr.
Together with published microarray studies and RNA in situ data [1–4], our results show that Xmr is transcribed in spermatids with little or no transcription detectable before this time.
XMR Protein Is Expressed in Spermatids
In their previous work, Calenda et al. [5] used an antibody raised against XLR to study XMR expression in the testis and concluded that XMR was meiosis-specific. We found this difficult to reconcile with our current finding that Xmr and AK015913 transcription is largely confined to spermatids. Here we have used three XMR antibodies that were designed to avoid recognizing XLR (see Materials and Methods).
To determine if our new antibodies detect both XMR and AK015913, COS-7 cells were transiently transfected with either the Xmr or the AK015913 ORF. Though the XMR69–81 and XMR1–93 antibodies detected both proteins, XMR96–106 only detected the XMR protein, demonstrating that it is specific for XMR (See Supplemental Fig. 2, available online at www.biolreprod.org).
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Next, we used the XMR69–81, XMR96–106, and XMR1–93 antibodies to investigate XMR/AK015913 expression in mouse tissues. Using all three antibodies, we detected a 34-kDa band on Western blots that was testis-specific (Fig. 2A). This 34-kDa protein was detected by all three antibodies after immunoprecipitation with the anti-XMR96–106 antibody, confirming these antibodies recognize the same protein (see Supplemental Fig. 3, available online at www.biolreprod.org). The specificity of the antibody for XMR was confirmed by Western blot analysis of a mouse model lacking all MSYq material that shows an increase in the X-linked transcripts Xmr and AK015913 [15] (Fig. 2B). As expected, we found an increase in intensity of the 34-kDa band in the MSYq- sample compared to wild type (Fig. 2B).
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To identify which testis cell types express XMR protein, we then performed Western blot analysis on first-wave testis samples. For all three antibodies, we detected XMR protein at low levels from 21.5 dpp and increasing until 28.5 dpp, with no detectable band at 18.5 dpp or earlier (Fig. 2C). The timing of expression was further refined by Western blot analysis of daily time points from 18.5 dpp to 22.5 dpp, which confirmed that XMR first appeared at 21.5 dpp (Fig. 2D). These results indicate that XMR is expressed in spermatids. More crucially, the absence of XMR prior to 21.5 dpp as shown with the XMR-specific antiserum XMR96–106 confirms that XMR is not a meiotic protein, as previously thought [5, 9, 30, 31]. It is not clear if AK015913 is translated, as we were unable to detect a protein of the predicted size, but if it is, it must have the same pattern of expression as XMR.
XMR Encodes a Spermatid-Specific Cytoplasmic Protein
To examine the localization of XMR during spermatogenesis, we performed immunostaining on testis sections using all three XMR antibodies. In each case, we observed labeling exclusively in spermatids, at high levels from stages 2 to 9 and at low levels from stages 10 to 11. To confirm that XMR is not present in meiotic cells, we then carried out dual labeling with either XMR69–81 or XMR96–106, or XMR1–93, together with an antibody to H2AX, which labels the whole nucleus of leptotene and zygotene cells and the sex-body of pachytene cells [16]. No colocalization was seen, confirming that XMR is not a meiotic protein (Fig. 3A, top panel). Furthermore, dual labeling with XMR69–81, XMR96–106, or XMR1–93, together with the anti-XLR antibody used by Calenda et al. [5], gave mutually exclusive staining patterns (Fig. 3A, middle panel). As expected, the anti-XLR antibody [5] and the anti-H2AX antibody colocalized in the sex-body (Fig. 3A, bottom panel).
Surprisingly, all three XMR antibodies labeled the cytoplasm, and not the nucleus, as would have been expected based on published data on the Xlr superfamily (Fig. 3B). In order to see whether epitope masking might have prevented us from detecting any nuclear XMR protein in sections, we then analyzed cytoplasmic versus nuclear testis fractions by Western blot analysis. This confirmed that XMR is cytoplasmic and not nuclear as previously predicted (Fig. 3C).
The Anti-XLR Antibody Does Not Detect XMR
The costaining experiments described above showed that the mouse monoclonal anti-XLR antibody used previously [5] fails to detect a spermatid-encoded protein. Formally speaking, however, this may have also been due to masking of the XMR/AK015913 epitope recognized by the anti-XLR antibody in testis sections. Therefore, we characterized the anti-XLR antibody in greater detail on multi-tissue Western blots and Western blots of first wave testis samples. Although many bands were seen, no testis-specific band of 34 kDa was detected on the multi-tissue Western blot (Fig. 2A, far right), and no product appeared either at the onset of meiosis or at the onset of spermatid differentiation on the first wave Western blot (data not shown). Furthermore, we were unable to detect any change in banding pattern intensity between control males and the MSYq- model (Fig. 2B, far right), in which both Xmr and AK015913 are known to be up-regulated [15]. The XLR antibody did not detect the XMR protein immunoprecipitated with XMR96–106 antibody, which was detected by XMR69–81 and XMR1–93 antibodies (Supplemental Fig. 3). Finally, no immunostaining of COS-7 cells transfected with the Xmr ORF (which we had earlier used to characterize our XMR69–81, XMR96–106, and XMR1–93 antibodies) was seen with the XLR antibody (Supplemental Fig. 2). We conclude that the anti-XLR antibody cannot detect XMR, and therefore that the meiotic epitope detected by Calenda et al. [5] results from cross-reaction of the anti-XLR antibody with another unidentified protein(s).
In this study, we have used RT-PCR and RNA FISH to further substantiate the growing evidence that the multiple-copy Xmr gene is predominantly or exclusively transcribed in spermatids. In agreement with earlier data [5] by RT-PCR, we do detect low-level transcription of some closely related transcripts in prepubertal testes prior to 18.5 dpp, when spermatids are not present. We have then used Western blot and immunostaining analysis with antibodies raised specifically to XMR in an attempt to demonstrate that it is a cytoplasmic protein present in stage 2–11 spermatids. These findings are inconsistent with the study of Calenda et al. [5] that concluded, using an antibody raised to the related XLR, that XMR is a nuclear protein expressed during meiosis. To explain this discrepancy, we have also demonstrated that this anti-XLR antibody is unable to recognize either XMR or the related protein AK015913. In light of these findings, the name Xmr (Xlr-related, meiosis regulated) is misleading, and the gene has been renamed Slx (Sycp3-like, X-linked) to reflect its homology to the Y-encoded multi-copy Sly gene, which we identified previously [20]. AK015913 is now named Slxl1 (Slx-like 1).
The XMR protein contains a Cor1 domain, which is hypothesized to facilitate chromatin binding, so our finding that XMR is cytoplasmic is surprising. The cellular location of XMR may be explained by loss of the putative nuclear localization signal motif KRKR found in the Cor1 domain of XLR and SYCP3, and by the lack of other identifiable nuclear localization signal motifs. Whereas the Cor1 domains of XLR and SYCP3 share 72.1% similarity, XMR only shares 45.8% similarity with SYCP3, and this loss of homology may allow the Cor1 domain to have different functions within these proteins. XLR has been shown to colocalize with the AT-rich DNA binding protein SATB1 [8], but there is no direct evidence that SYCP3 and XLR bind to chromatin, although SYCP3 contains a nucleotide binding motif A [13] that is not found in other Cor1 proteins. Thus it is possible that the Cor1 domain of XMR allows XMR to bind to cytoplasmic RNAs rather than DNA.
Ellis et al. [15] have reported that mice with deletions of the Y long arm up-regulate a number of X and Yp linked transcripts, including Xmr. Our present results are important because they are the first to show that, at least for Xmr, the increase in transcription seen in MSYq- mice leads to a corresponding increase in protein levels. Aside from an increase in sperm head defects seen in MSYq- mice models, those that are fertile—such as the XYqdelRIII 2/3 MSYq deletion mutant—generate offspring with a distorted sex ratio in favor of females [26]. Ellis et al. [15] proposed that this is because of an underlying postmeiotic X-versus-Y intragenomic conflict that has been uncovered by the reduction in MSYq-encoded transcripts resulting from the deletion. It has further been suggested that Xmr and Sly may be important players in this genomic conflict [15, 20], perhaps due to a mutually antagonistic action of their encoded proteins on the transcriptionally repressed X and Y chromatin domains of spermatids [2, 28, 32]. However, our data indicate that XMR does not localize to the nucleus, so any potential genomic conflict between XMR and SLY is unlikely to occur by effects on sex chromatin. It is more likely that this potential interaction is mediated at the posttranscriptional level, perhaps in a manner similar to that described for the multiple-copy sex-linked Ste/Su(ste) genes in Drosophila [33, 34]. Alternatively, the reduction of other MSYq-encoded transcripts such as Ssty may be responsible for the distorted sex ratio in offspring of the XYqdelRIII 2/3 MSYq deletion mutant by control at either the transcriptional or posttranslational level.
The identity of the protein recognized by the monoclonal anti-XLR antibody [5] is at present unknown. An obvious candidate would be XLR, which we detected in the testis from 11.5dpc, despite previous data suggesting that it was not expressed in the testis [9, 31]. Alternatively, the antibody could recognize other members of the Xlr-superfamily. Microarray analyses suggest that Xlr3, Xlr4, and Xlr5 subfamily members are transcribed in spermatogenic cells, and for Xlr3 this has been confirmed by Northern blot analysis [10]. Transcription of all three subfamilies begins before MSCI takes place [1–3] and could therefore account for the appearance of the protein detected by the anti-XLR antibody [5]. The putative XLR3, XLR4, and XLR5 proteins all contain classical nuclear localization signal motifs [35], and are predicted to be nuclear using Psort II (http://psort.nibb.ac.jp/form2.html). The anti-XLR antibody also recognizes an ovarian meiotic protein reported to be XLR [9]. However, it is possible that the anti-XLR antibody detects the same protein in male and female meiosis, and microarray analysis indicates that the Xlr3, Xlr4, and Xlr5 genes are also transcribed in the ovary during meiosis [36]. Finally, it is conceivable that the antibody cross-reacts with an unrelated meiotic protein.
These findings add novel insights into our understanding of the role of Xlr-family members in germ line development. In light of the multi-copy nature of Xmr, RNA-mediated knockdown may be the best approach to elucidate the specific function of this gene in spermatid maturation.
ACKNOWLEDGMENTS
We thank O. A. Ojarike and Á. Rattigan for help with the mouse breeding and genotyping, P. Laskey and R. Sekido for invaluable advice, and H.-J. Garchon for providing the XLR antibody.
FOOTNOTES
1Supported by an MRC research studentship to L.N.R, a Marie Curie Fellowship to A.T., an EMBO Fellowship to J.C., and a grant from the Swedish Research Council to C.H. 
Correspondence: 2FAX: 44 20 8816 2009; pburgoy{at}nimr.mrc.ac.uk
Received: 23 February 2007.
First decision: 12 March 2007.
Accepted: 25 April 2007.
REFERENCES
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