Testis-specific protein, Y-encoded (TSPY) expression in testicular tissues
Testis-specific protein, Y-encoded (TSPY) expression in testicular tissuesFrank Schnieders+, Thilo Dörk, Joachim Arnemann1, Tanja Vogel, Martin Werner2 and Jörg Schmidtke*
Institut für Humangenetik, Medizinische Hochschule Hannover, 30623 Hannover, Germany, 1Institut für Humangenetik, Johann-Wolfgang-Goethe-Universität zu Frankfurt, 60590 Frankfurt, Germany and 2Abteilung für Experimentelle Pathologie, Medizinische Hochschule Hannover, 30623 Hannover, Germany
Received June 25, 1996;Revised and Accepted August 1, 1996DDBJ/EMBL/GenBank accession no M98525
TSPY, the `testis-specific protein, Y-encoded', is the product of a tandem gene cluster on human proximal Yp. In order to gain insight into the function of this locus, we have analysed (i) the diversity of RNAs transcribed from the cluster, (ii) the sequence homology of the deduced TSPY to other proteins, and (iii) its protein properties both in tissue extracts and in tissue sections, using a TSPY-specific antiserum. We have identified a set of distinct TSPY transcripts with diverse exon compositions. We show that TSPY has homology with other human and non-human proteins, including SET and NAP, factors that are suggested to play a role in DNA replication. Protein analysis revealed TSPY to occur mainly in a modified, putatively phosphorylated form. By immunostaining it was detected in distinct subsets of spermatogonia. TSPY was also strongly immunostained in early testicular carcinoma in situ (CIS), while seminomatous tumour cells stained less intensely. The spermatogonial cells of two XY-TFM-females gave a strong immune response. The data presented here point to a phosphorylation-dependent TSPY-function in early spermatogenesis, immediately prior to the spermatogonia-to-spermatocyte transition, and in early testicular tumorigenesis.
TSPY is a gene family located on the proximal part of human Yp with each of its 20-40 copies embedded in a single unit of the DYZ5 tandem repeat array (1 -3 ). This array maps to the region immediately adjacent to the centromere on human Yp, thus rendering it exempt from pseudoautosomal recombination. TSPY-homologous gene families have also been found on the Y-chromosome of the great apes (4 ,5 ), cattle and other large mammals [(6 ) and own unpublished data], whereas TSPY-homologous sequences have not so far been isolated from rodents. By RNA analyses, expression of TSPY sequences was found exclusively in the testis both in prenatal and adult stages (2 ,4 ,6 ,7 ).
The differentiation of germ cells and the production of mature spermatozoa is an intricately regulated process, which, if deregulated, ends in tumour formation and/or infertility. The understanding of how this complex machinery is genetically controlled is only at its very beginning (8 ), as reviewed in (1 ). A link between spermatogenic processes and testicular tumour development is suggested by the finding that germ cell-derived tumours, especially gonadoblastomas, often develop on the basis of gonadal dysgenesis (9 ,10 ). There is evidence to invoke one or more Y-chromosomal causative genes, because gonadoblastomas have almost exclusively been encountered in cases where Y-chromosomal material was detected (9 ).
The organisation of TSPY as a tandem repeat gene family renders its functional analysis more difficult than it would be for a single-copy gene. It is, for instance, necessary to determine to what extent sequence diversity at the genomic level is reflected in transcript complexity and which of the transcripts encode functional products. Human TSPY sequences are characterised by sequence divergence of up to 10% (2 ,7 ). So far, two TSPY transcripts, JA923 (7 ) and Y-231 (4 ), differing in exon composition have been published, the latter of which may be functional. In this study, the question of transcript functionality was readdressed. Properties of the protein, such as biochemical parameters, tissue specificity, and the cellular site of expression in the testis were studied using a TSPY-specific polyclonal antiserum generated against a recombinant polypeptide derived from the JA923 open reading frame, which encompasses the Y-231-type exon-1-encoded amino acid sequence. We also discuss the results of database searches for proteins with homologies to TSPY.
Transcript diversity was assessed by RT-PCR and direct sequencing of cDNAs prepared from testicular tissues of seven individuals. RT-PCR was performed using cDNAs primed with TSPY-specific as well as poly-dT primers to avoid a possible product bias due to one or the other of the methods. Three major products amplified with the primers Mu and TD3 (indicated in Fig. 1 B) of 569, 257, and 170 bp in length, respectively, were isolated and sequenced. The RT-PCR products obtained by specific cDNA priming and, as a control, the product amplified with the same primers from genomic DNA, are shown in Figure 1 A. Products were of equal numbers and sizes in all seven cDNA samples analysed (not shown). Specific and oligo-dT-priming revealed no differences in product amounts and sizes, thus all variants appeared to be equally polyadenylated. The most abundant and longest product (11 ) was found to be nearly identical with Y-231, except for a major difference at the exon 4-5 border (Figure 1 B,C). The exon 5 of this product is 11 bp shorter than its Y-231 equivalent, most likely due to the use of a different splice acceptor site 11 bases downstream. Thus, the open reading frame is shifted and the deduced amino acid sequence is 14 amino acids longer. In none of the seven individuals tested was a transcript with the Y-231-type exon 4-5 border found. We assume that, in our samples, Y-231 is a very rare variant. Two further single bp differences between the Y-231 and TSPY cDNAs analysed here were detected in the cDNAs 5' region, which further support the view that the products analysed here are transcribed from genomic TSPYcopies that are distinct from the Y-231-type copy. On the basis of these data and in view of the sequence homologies to related proteins shown below, we suggest that the major transcript seen here is the most promising candidate for a functional TSPY (referred to as TSPYmajor). The TSPY variants A and B, schematically drawn in Figure 1 B, show altered splice patterns with respect to exon 1. Since no genomic products having a corresponding size were found, genomic deletions were excluded. Instead, the deletion breakpoints almost perfectly match the consensus sequences of donor and acceptor splice sites (12 ). This sequence context strongly suggests that both transcript variants have formed by the use of a cryptic splice donor site (TSPY-A and TSPY-B) and a cryptic splice acceptor site (TSPY-A) within exon 1 (Fig. 1 B). Whether these products are generated by alternative splicing of one and the same transcript or from different distinct transcripts with individual splice donor and acceptor sites is as yet unclear.
Detailed database and sequence analyses revealed that TSPY is a member of a protein super family including the proto-oncogene SET, the nucleosome-assembly factor NAP-1, and a not further characterised protein from plasmodium falciparum. The alignment of human TSPYmajor and bovine TSPY (unpublished data) with SET (Fig. 2 ) reveals stretches of conserved amino acids distributed over the entire length of the protein sequences. The major difference between TSPY and SET is the C-terminal stretch of acidic moieties found in SET, but not in TSPY. This acidic domain was shown in vitro to promote DNA replication, presumably by interacting with nucleosomes (13 -15 ). The most carboxy-terminal group of homologous amino acids shared by all three proteins in this alignment is only present in the TSPYmajor reading frame but not in the Y-231 frame, further supporting the idea that TSPYmajor is functional.
For the biochemical analysis of the TSPY gene product, we used a rabbit antiserum (837/3) raised against a fusion protein corresponding to the JA923-deduced amino acid sequence (JA923-TSPY, shown in Fig. 3 A), almost identical with the first 149 amino acids of the TSPYmajor gene product (shown in Fig. 3 A). In order to test for the specificity of the antiserum, competition experiments were done with the carrier of the fusion protein, TrpE, on its own, preimmune serum and with sequence-specific peptides using the full-length immunisation peptide or a deleted (shortened) peptide, which covers the region of identity between both types of transcripts, the JA923-TSPY-type and the TSPYmajor-type (shown in Fig. 3 A). Both the fusion protein corresponding to the immunisation peptide and the shortened peptide were able to block the antiserum from binding on western blots and in immunohistochemical stainings on tissue sections.
In order to elucidate the topology of TSPY expression in the testis, sections of testicular tissue with normal spermatogenesis were immunostained in situ using the antiserum 837/3. As in the western analysis, TSPY-specific immunostaining was assessed using antiserum blocked with the control polypeptide. The antiserum strongly reacts with groups of spermatogonia (Fig. 4 A,B). Immunostaining occurs predominantly in pairs of spermatogonia, with neighbouring cells showing decreased dye intensities. A small number of primary spermatocytes appeared weakly stained (not shown). Staining is mainly concentrated in the cytoplasm of the cells, but some spermatogonia also appear to carry grains of the dye on their nucleoplasm. It should be pointed out that an amino acid motif which could serve as a nuclear localisation signal (NLS) is present in the amino acid sequence of TSPYmajor (see Fig. 2 ). Carbonic anhydrase (p29, see above) is only expressed in Sertoli cells of the adult testis and was thus excluded to cause the signal on spermatogonia found (16 ). The antiserum with and without control polypeptide was also applied to sections of human liver, lung, stomach, pancreas, intestine, colon, brain, thyroid gland, carotic arteria, lymph node and skin, but none of the tissues reacted specifically, in accordance with the testis-specificity of TSPY expression seen at the mRNA level.
Germ cell tumours are believed to arise from the carcinoma in situ (CIS) stage of the testis, where tumour cells are located at the basal membrane of the tubulus (17 ). These cells share a number of morphological features and cell surface markers with embryonic germ cells (18 ,19 ) supporting the hypothesis that they are persisting primordial germ cells (19 ). Up to a point where a CIS begins to develop into a rapidly growing germ cell tumour, proliferation of CIS cells is thought to proceed slowly (17 ). We have stained sections of early and later stages of testicular tumours employing the antiserum 837/3. As can be seen in Figure 5 A,B, CIS cells show strong immunostaining. The concentration of the dye mostly on and around the cell nucleus is probably artificial, because CIS cells contain large cytoplasmic stocks of glycogen that are washed out during the tissue fixation process, causing the cytoplasm to stack around the nucleus and at the plasma membrane. Figure 5 B shows a CIS stage together with both tumour cells on their way out of the seminiferous tubule and cells forming an interstitially located solid seminoma. Also, the migrating cells and groups of cells forming a solid tumour are specifically immunostained, the latter to a much lesser extent than CIS cells.
We have shown that at least three major transcripts, differing in size, abundance, and splice pattern, are expressed from the human TSPY locus, a tandem repeat gene family with some 20-40 elements on proximal Yp. The transcript heterogeneity could, in principle, be caused either by the transcription of structurally different genes within the cluster, or by one or several structurally identical copies permitting alternative splice patterns. In contrast to observations made at the genomic level (1 ,2 ,4 ), no sequence heterogeneity due to single base-pair exchanges or small deletions/insertions was seen among these transcripts. It is not possible, however, to assess the number of expressed TSPY copies from these data.
On the basis of structural comparisons with the SET/NAP protein, the product of the largest cDNA, termed above as TSPYmajor, is also likely to represent the functional transcript. In comparison with the cY-231 reading frame, its region of homology with SET has significantly extended at the C-terminus. The evolutionary conservation of amino acid sequences of TSPY proteins in man and cattle as well as between TSPY and SET is suggested to reflect some important, though still unknown physiological function. SET and the more distantly related NAP have been described as activating factors of the replication process (13 -15 ) and as binding-partners of cyclin B with a cyclin/CDK complex (15 ,22 ). More recently, SET has also been identified as a potent inhibitor of protein phosphatase 2A, a major mammalian protein serine threonine phosphatase that regulates diverse cellular processes (23 ). Although it is presently unclear how these observations fit together in a cellular network of proliferation and differentiation processes, further advances in the characterisation of SET function may provide some important clues also to the biological role of the TSPY gene family.
Our data indicate that TSPY occurs in two alternative forms of apparent relative molecular masses of 33 and 38 kDa. Alkaline phosphatase treatment experiments suggest p38 to be the phosphorylated p33. SET with its predicted molecular weight of 33 kDa is likewise predominantly found in a phosphorylated form of apparent 38 kDa and was shown by phosphopeptide mapping to be phosphorylated at two serine residues (25 ). This suggests that functional regulation by phosphorylation may be a common feature of the SET/TSPY protein family.
A TSPY function specific for the proliferation of germ cells is suggested by the immunohistochemical data of TSPY expression. The marked variation in immunostaining of spermatogonia with pairs of spermatogonia showing equivalent staining indicates that TSPY may be involved in spermatogonial proliferation. If the rare staining in spermatocytes labels TSPY originating from mother spermatogonia, then TSPY protein expression may mark the branch point between mitotic proliferation and meiotic differentiation.
That TSPY expression is somehow tied to the mitotic division of early germ cells is also indicated by immunostaining in CIS and early seminoma cells. CIS cells are thought to originate from embryonic germ cells persisting in the testis, as suggested by the presence of embryonic antigens detected with specific antibodies (18 ,19 ). Although the anti-TSPY-antiserum stained both the CIS cells and normal spermatogonia, together with the evaluation of morphological parameters (for review see 17 ), TSPY immunostaining may add to the recognition of early germ cell tumorigenesis. In this context, it is of interest to note that in the dysgenetic testicular tissue of the two XY-TFM patients, TSPY is expressed in patches of spermatogonia-like cells while the majority of tubules remain negative. It is tempting to speculate that the TSPY-negative cells are resting, while the TSPY-positive cells are actively proliferating, with a potential to transform to a malignant state. Interestingly, Tsuchiya et al. (27 ) have recently shown that TSPY maps into the candidate region for the Y-linked gonadoblastoma locus.
In summary, TSPY gives rise to a pool of heterogeneously composed transcripts, at least one of which yields a protein that is a member of a superprotein family together with SET and NAP. Immunohistologically, TSPY was detected to be concentrated in the cytoplasm of spermatogonia in normal as well as pathological tissue. TSPY was found in restricted areas of the dysgenetic testes of two XY-female siblings with TFM. TSPY was also found in early testicular tumour cells at the CIS-stage and in the early forms of seminomatous testicular tumours. On the basis of the data presented here, its structural relationship to SET and NAP, TSPY is suggested to serve a function related to spermatogonial proliferation.
For the preparation of rabbit antisera, an EcoRI/HindIII fragment of cDNA clone pJA923, spanning the entire open reading frame (7 ), was subcloned into the pATH3 expression vector to yield the TrpE/JA923 fusion construct pJA10EX. After indoleacrylic acid-induced expression in RRI-cells (28 ) the fusion protein was separated from a whole cell lysate by SDS-PAGE and purified from the gel by band excision and electroelution. Rabbits were immunized according to standard methods(29 ). The resulting sera were preabsorbed with Sepharose-bound proteins extracted from TrpE-expressing RRI cells.
For western analysis, 50 [mu]g protein (per lane) of total tissue (for preparation see below) were separated on 12% gels in SDS-PAGE (30 ), and transferred onto nitrocellulose membrane (Hybond C extra) by semi-dry blotting (Biometra). Signals were immunodetected with antiserum 837/3 diluted 1/2000, using the ECL-chemiluminescence system (Amersham), and autoradio- graphy on Kodak Biomax films.
Poly(A)+ RNAs were isolated using the Fast Track RNA Isolation kit (Invitrogen) following the manufacturer's protocols. Total cellular RNA was extracted using the method reported in (31 ).
RT-PCR analyses were carried out with cDNAs reversely transcribed from total cellular RNA using the First Strand Synthesis kit (Pharmacia) following the manufacturer's instructions. cDNA synthesis was primed either with a sequence-specific primer or with the poly-(dT) primer supplied with the kit. RT-PCR was carried out with the TSPY primers Mu (5'-GGCCCTTCGCGCGCAGTCCCTTAG-3', forward, position -45 to -22 of the TSPYmajor-cDNA) and TD3 (5'-GTCAGTGATCAGGGCTGACATCTG-3', reverse, position 521 to 545 of the TSPYmajor cDNA), located in exons 1 and 2 of the Y-231-type TSPY cDNA, respectively.
Testicular tissues used in the western analyses and dephosphorylation assays were from patients with orchidectomy due to prostate cancer (patients 1 and 2 in Fig. 3 D) or a Leydig cell tumour (patient 3, Fig. 3 D), respectively. Approximately 100 mg of tissue were homogenized in 0.25 M sucrose, 20 mM Hepes, 1 mM EDTA, 1 mM DTT, pH 7.4, in a Dounce homogenizer, cell debris removed by short centrifugation at 4oC and 12 000 g. Supernatants of this tissue extract were subjected to protein analysis using a Lowry assay (DC Protein Assay, Biorad) and stored at -70oC prior to western blotting.
Total testis protein (16 [mu]g) was incubated in a 20 [mu]l volume either with or without the addition of 10 U calf intestine alkaline phosphatase (Boehringer Mannheim) for 2 h at 30oC. The reaction was stopped by mixing the components with 6.6 [mu]l 4 * Laemmli sample buffer and boiling the sample after reaction prior to western analysis.
Tissue sections were prepared after fixation of the tissues in Bouin's fixative and embedding in paraffin. Sections of 5-6 [mu]m were used for immunohistochemical analyses using a modified peroxidase ABC detection protocol (32 ).
Multialignment of the sequences given in Figure 2 was performed combining data of the program MULTALIGN with the data from pairwise alignments of the indicated sequences with the program GAP. MULTALIGN and GAP are included in the HUSAR software provided by the EMBL.
We would like to thank A. F. Holstein and M. Davidoff (Hamburg), for contributing histological specimens and giving advice on specific immunohistochemistry and the interpretation of the immunohistological results. We further thank Anthony Magee from NIMR, Mill Hill, UK, for his help in raising the antisera, T. Schroeder-Kurth (Heidelberg), J. Hundrieser, U. Jonas, H. Schmidt, and J. Serth (Hannover) for human tissues, S. Hoyer-Fender (Göttingen), B. Lüscher (Hannover) and E. Manz (Berlin) for helpful discussions, and S. Nedel and S. Horn (Hannover) for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft and by the Madeleine Bühler Kinderkrebs-Stiftung through grants to J.S.
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*To whom correspondence should be addressed
+Present address: Max-Planck-Gesellschaft und Humboldt Universität, Max Delbrück-Center für Molekulare Medizin, 13125 Berlin-Buch, Germany
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