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

Abstract

Background: The male-specific region of the mouse Y chromosome long arm (MSYq) is comprised largely of repeated DNA, including multiple copies of the spermatid-expressed Ssty gene family. Large deletions of MSYq are associated with sperm head defects for which Ssty deficiency has been presumed to be responsible.

Results: In a search for further candidate genes associated with these defects we analyzed changes in the testis transcriptome resulting from MSYq deletions, using testis cDNA microarrays. This approach, aided by accumulating mouse MSYq sequence information, identified transcripts derived from two further spermatid-expressed multicopy MSYq gene families; like Ssty, each of these new MSYq gene families has multicopy relatives on the X chromosome. The Sly family encodes a protein with homology to the chromatin-associated proteins XLR and XMR that are encoded by the X chromosomal relatives. The second MSYq gene family was identified because the transcripts hybridized to a microarrayed X chromosome-encoded testis cDNA. The X loci ('Astx') encoding this cDNA had 92-94% sequence identity to over 100 putative Y loci ('Asty') across exons and introns; only low level Asty transcription was detected. More strongly transcribed recombinant loci were identified that included Asty exons 2-4 preceded by Ssty1 exons 1, 2 and part of exon 3. Transcription from the Ssty1 promotor generated spermatid-specific transcripts that, in addition to the variable inclusion of Ssty1 and Asty exons, included additional exons because of the serendipitous presence of splice sites further downstream.

Conclusion: We identified further MSYq-encoded transcripts expressed in spermatids and deriving from multicopy Y genes, deficiency of which may underlie the defects in sperm development associated with MSYq deletions.

Figure 1

Sex chromosome complements of the mice with MSYq deficiencies and relevant control mice. (a) XY^RIII control, illustrating the previously documented male specific gene content of the mouse Y chromosome. The short arm (shown expanded) carries seven single copy genes, one duplicated gene (Zfy), and multiple copies of Rbmy. MSYq carries multiple copies of the Ssty gene family. (b) XY^Tdym1 control. This male has a normal Y gene complement except that a 11 kb deletion has removed the testis determinant Sry; the Sry deletion is complemented by an Sry transgene located on an autosome. (c) The variant Y^RIIIqdel has a deletion removing about two-thirds of MSYq. (d) The variant Y^Tdym1qdel has a large deletion removing about nine-tenths of MSYq, together with the small 11 kb deletion removing Sry (complemented by an Sry transgene). (e) XSxr^aY* ^X mice are male because of the presence of the Y^RIII short arm derived, sex reversal factor Sxr^a attached distal to the X pseudo-autosomal region (PAR). Sxr^a comprises most of the Y short arm except for a substantial reduction in copies of Rbmy. The Y* ^X chromosome is in effect an X chromosome with a deletion from just proximal to Amel (close to the X PAR boundary) to within the DXHXF34 sequence cluster adjacent to the X centromere. It provides a second PAR, which is essential in order to avoid meiotic arrest. CEN, centromere; kb, kilobase; TEL, telomere.

Figure 2

Microarray analysis of the testis transcriptomes of the three MSYq deficient models. (a) Scatter plots showing transcription levels for the testis transcripts of MSYq deficient models relative to their controls. Expression in the MSYq deficient mice (y-axis, Cy3 label, arbitrary units) for each clone is plotted versus expression in age- and strain-matched normal testis control (x-axis, Cy5 label, arbitrary units). Data from four technical replicates are combined. Data are normalized on the median signal for each channel and then filtered (as described in Materials and methods) to show only clones with data for two technical replicates from each model. The data points showing significant reduction are plotted as enlarged triangles (Y clones green or red, and one X clone black - for clone identities see b). (b) The 23 cDNA clones identifying transcripts that were significantly reduced in one or more of the MSYq deficient models. The clones deriving from the two Y families and the one X family are color coded.

Figure 3

Transcription of Sly is reduced or absent in the MSYq deficient males. (a) Northern blot of total testis RNA probed with the microarrayed Sly cDNA clone MTnH-K10 and with an actin probe as a loading control. Hybridization to the Sly probe is clearly reduced in 2/3MSYq^- males and is further markedly reduced in the two models with more severe MSYq deficiency. (b) Reverse transcriptase polymerase chain reaction analysis of testis cDNA with primers that distinguish between Sly and Xmr transcripts and with Hprt primers as an amplification control. Some Sly transcripts are retained in 9/10MSYq^- males but they are absent in MSYq^- males.

Figure 4

Identification of the novel MSYq encoded transcripts Asty and Asty(rec). (a) Testis transcripts identified from a BLAST (basic local alignment search tool) with the arrayed X encoded Astx cDNA clone 8832_f_22. BF019211, CF198098 and AK076884 are X encoded transcripts, whereas the rest are Y encoded transcripts. The Y encoded transcripts AK016790 and BY716467 include exons that do not derive from Asty (including two exons matching Ssty1). We refer to these transcripts as Asty(rec) because they derive from novel 'recombinant' loci on MSYq. For all the transcripts the percentage sequence identity is given for those regions that match the microarrayed clone. (b) The structure of the Asty(rec) locus encoding AK016790. The exons included in AK016790 are indicated by filled color coded rectangles and the inter-exonic distances are given in base pairs. The position of other Ssty1 and Asty exons not included in the AK016790 transcript are also indicated. (Note that the Ssty1 exon 3 is truncated in the Asty(rec) locus.) The two exons colored red are those that match the previously described transcript AK015935. (c) Reverse transcriptase polymerase chain reaction of testis cDNA with primers designed to specifically amplify Asty/Asty(rec) or Astx transcripts. It is clear that it is the Asty/Asty(rec) transcripts that are preferentially reduced in the MSYq deficient males. (d) Northern blot of total testis RNA probed with an Asty exon 4 probe and with an actin probe as a loading control. Transcripts ranging in size from about 1 kilobase (kb) to more than 9.5 kb are detected with the Asty exon 4 probe; the approximately 7.5 kb and larger transcripts definitely derive from the Asty/Asty(rec) loci (see text) and (in contrast to AK016790) must include Asty exon 4.

Figure 5

Multiple copies of Sly and Asty/Asty(rec) map to MSYq. (a, b) Southern of EcoRI digested DNAs showing hybridization of Sly probe MTnH-K10 and Asty exon 4 probe, respectively, to multiple male specific bands, with reduced hybridization to all bands in 2/3MSYq^- males. (c, d) Southern of EcoRI-digested DNAs from 2/3MSYq^-, 9/10MSYq^-, and MSYq^- males together with control XY^RIII, showing reduced hybridization in 2/3MSYq^- males and an apparent absence of hybridization to Y specific bands in the males with more extensive MSYq deficiency. (e) Two week exposure of a Southern blot of EcoRI digested DNAs from 9/10MSYq^- and MSYq^- males together with XO and XX females, and an underloaded XY^RIII control. X derived fragments now cross-hybridize with the Sly probe (dosing with the number of X chromosomes), but in addition there are at least three male specific bands (outlined) retained in the 9/10MSYq^- males that are absent in the MSYq^- males.

Figure 6

Sly and Asty/Asty(rec) are expressed in spermatids. (a) A multi-tissue northern blot of polyA⁺ RNA probed with Sly clone MTnH-K10 and Asty exon 4 probe. Sly transcripts are restricted to the testis, but Asty/Asty(rec) transcripts are also seen in the heart. (b) A northern blot of total testis RNA from testes of mice aged 12.5-30.5 days postpartum (dpp) hybridized with the same Sly and Asty probes. No transcripts are detectable before 20.5 dpp, suggesting that the transcripts are restricted to spermatid stages. (c) RNA in situ analysis confirming that Sly transcription in the testis is predominantly if not exclusively in spermatids. (d) Diagram summarizing the expression of Sly in spermatids throughout the spermatogenic cycle. The spermatogenic stages starting from the basal layer and reading left to right are: spermatogonia: A1 through to B; meiotic prophase spermatocytes: preleptotene (PL), leptotene (LE), zygotene (Z), pachytene (P), diplotene (D); meiotic divisions (mm); postmeiotic spermatid stages: 1-13. (e) RNA in situ analysis with the Asty exon 4 probe that should detect Asty and Asty(rec) transcripts. The most convincing signal is in round spermatid nuclei, but this is seen with the antisense and sense (control) probes. However, the previously reported cDNA clone BU936708 (Additional data file 3) contains Asty exon 4 sequence in the antisense oritentation and which would hybridize with the sense probe. (f) Exon 1-4 Asty reverse transcriptase polymerase chain reaction from 9.5 dpp to adult testes. Sequencing of the cloned amplification products confirmed the presence of full-length Asty transcripts at 14.5 dpp, and a shorter transcript lacking exon 3 from 22.5 dpp.

Figure 7

The protein encoding potential of Sly. (a) Sly encodes a putative protein with a COR1 region. This COR 1 region is shared with two closely related proteins, namely XMR and XLR, and with the less closely related synaptonemal complex protein SYCP3. (b) A comparison of the predicted protein sequence for SLY with the predicted proteins for other Sly related Y loci that are each represented by more than one copy (information collated from sequence information available in December 2004).

Figure 8

A comparison of the protein encoding potentials of Astx and Asty. (a) The predicted protein for the conserved open reading frame in exon 4 of the arrayed Astx clone (f22) and for the four protein variants predicted from the eight putative X chromosomal loci. (b) The two predicted ASTY protein variants encoded by two overlapping open reading frames in exon 1 present in six Asty loci.

Figure 9

Dendrogram showing the relationship between the SLY protein and other identified or predicted mammalian Cor1 domain proteins. The autosomally encoded SYCP3 is the presumed ancestral gene because it has been identified in a wide range of vertebrate species. The remaining proteins other than SLY are X encoded. For the rat, the putative X-encoded proteins have not yet been named and the labels given are simply a reflection of their position within the dendrogram. The massive expansion in copy number is only seen in the branch containing XMR, XMR-rel., XLR and SLY. This amplification therefore occurred subsequent to the divergence of mouse and rat lineages, concurrent with the appearance of Sly on the Y chromosome.