Molecular Cloning and Characterization of a Novel Cytoplasmic Protein-tyrosine Phosphatase That Is Specifically Expressed in Spermatocytes*

We identified a novel gene encoding protein-tyrosine phosphatase using a polymerase chain reaction-based method. Northern blot hybridization of RNAs from various tissues with the polymerase chain reaction-amplified DNA fragment showed that this gene was expressed exclusively in the testis. Complementary DNAs for this gene, termed typ (testis-specific tyrosine phosphatase), were obtained from a mouse testis cDNA library. Nucleotide sequencing of the cDNAs revealed an open reading frame that encoded 426 amino acids. The predicted Typ protein contained a single catalytic domain at the carboxyl-terminal half. No hydrophobic stretch for a possible transmembrane sequence or signal sequence was found, suggesting that Typ is a cytoplasmic protein-tyrosine phosphatase. The amino-terminal half of Typ did not share significant homologies with the other known proteins but contained a region rich in PEST residues. Indirect immunofluorescence studies and in situ hybridization analysis showed that Typ was specifically expressed in testicular germ cells that underwent meiosis. Developmentally, Typ was detected between 2 and 3 weeks after birth, in parallel with the onset of meiosis. Thus, Typ is a new member of the cytoplasmic protein-tyrosine phosphatases that may play an important role(s) in spermatogenesis and/or meiosis.

Protein tyrosine phosphorylation is one of the important regulatory events in cell growth, activation, and differentiation (1). The levels of tyrosine phosphorylation of cellular proteins are regulated by the opposing actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs). 1 In contrast to the extensive information available on the protein-tyrosine kinases, that on the biological roles of PTPs is limited. PTPs have been classified into two subfamilies, receptor-type PTPs and nonreceptor-type PTPs, based on the presence or absence of the extracellular and transmembrane portions. Each nonreceptor-type PTP ordinarily comprises a single PTP domain and a variable length of noncatalytic segment. Some nonreceptortype PTPs show dual specificity for both phosphotyrosine and phosphoserine or phosphothreonine (2)(3)(4). The PTP domain consists of approximately 240 amino acids carrying several consensus motifs including a sequence (I/V)HCXAGXXR(S/T)G (X can be any amino acid). The cysteine residue within the motif is involved in the formation of the catalytic core of the enzyme (5). Based on the consensus sequences, a number of cDNAs encoding PTPs have been identified using both reverse transcription-polymerase chain reaction (RT-PCR) and low stringency hybridization methods.
In mammals, spermatogenesis takes place in the seminiferous tubule in which mitotic division of spermatogonia, meiosis of spermatocytes, and differentiation of spermatid to sperm occurs. Spermatogenesis is an apparently essential step in sexual reproduction. However, the molecular mechanisms by which differentiation of germ cells is regulated are not well understood. Recently, several genes have been reported to be expressed specifically before or at meiosis. These include the genes for protein-tyrosine kinases (c-Abl, c-Kit, and FerT) (6 -10), serine/threonine kinases (Mak, c-Mos, and TESK) (11)(12)(13), a dual-specific kinase (Nek-1) (14), a protein tyrosine phosphatase (OST-PTP) (15), a serine/threonine phosphatase (calcineurin B subunit isoform ␤ 1) (16), and a dual specific phosphatase (Twine) (17). Because of the putative regulatory function of the protein kinases and phosphatases, these proteins are thought to contribute to germ cell differentiation. Indeed, c-Kit is directly involved in the mitotic cell cycle of spermatogonia (9), and the dual-specific phosphatase Twine is expressed specifically in germ cells and is suggested to be important in male meiosis in Drosophila (17).
Here we report identification of the typ gene that encodes a novel cytoplasmic PTP. The typ gene is expressed specifically in spermatocytes that are under meiosis, suggesting an important role of the Typ protein in spermatogenesis.

EXPERIMENTAL PROCEDURES
Amplification of PTP cDNAs-For the cDNA synthesis and PCR, oligonucleotide primers were designed from conserved regions within the PTP domain. A primer for first-strand cDNA synthesis corresponded to the amino acid sequence QT(E/D/A)Q (primer 1, 5Ј-(A/T) (A/G)(C/T)TG(C/G/T)(G/T)CX(G/T)(A/C/G)(C/G/T)GT(C/T)TG-3Ј), and primers for PCR corresponded to DYINA (primer 2 (sense), 5Ј-AT-GAAGCTTGA(C/T)TA(C/T)AT(C/T)AA(C/T)GC-3Ј) and to HCSAG (primer 3 (antisense), 5Ј-CATGAATTC(A/G/T)GCACTGCA (A/G)TG-3Ј), where X is a mixture of A, C, G, and T in oligonucleotide sequences. Primer 2 and primer 3 contained restriction sites for HindIII and EcoRI (underlined), respectively. Total RNA was isolated from human breast carcinoma cell line MDA-MB453 and human embryo fibroblast cell line TIG1 using the acid guanidinium-phenol-chloroform (AGPC) method (18). The cDNA was synthesized with Superscript reverse transcriptase (Life Technologies, Inc.) and primer 1. PCRs were performed for 50 cycles with denaturation at 94°C for 30 s, annealing at 53°C for 1 min, and extension at 72°C for 30 s. At the end of the reaction, the samples were incubated at 72°C for 5 min. PCR products of the expected size (ϳ420 base pairs) were purified through 6% polyacrylamide gel electrophoresis and cloned into the pUC119 vector for sequence analysis.
Colony Hybridization-The pUC119 vector DNAs were ligated with the RT-PCR products amplified using RNAs from MDA-MB453 cells, and the resulting plasmids were transfected into Escherichia coli JM109. Colonies were transferred onto nitrocellulose filters (Schleicher & Schuell), and colony hybridization of the filters was performed using a mixture of 32 P-labeled PCR fragments corresponding to B-PTP2 (19), PTP-MEG (20), DEP-1 (21), and LAR (22) as probes. Hybridization was performed in a stringent condition as described (23).
Northern Hybridization Analysis-A Human Multiple Tissue Northern Blot, Human Multiple Tissue Northern Blot II, and Human Fetal Multiple Tissue Northern blot (CLONTECH) were hybridized to the 32 P-labeled human typ PCR fragment using the hybridization and washing conditions recommended by the supplier. Samples of total RNAs isolated from adult mice tissues were loaded on each lane (20 g/lane) of 1.0% formaldehyde-agarose gels, electrophoresed, and blotted overnight onto Hybond-N membrane filters (Amersham). The filters were hybridized to the 32 P-labeled human typ PCR fragment or the cDNA insert of clone 5 (see Fig. 2A) using the previously described hybridization and washing procedures (24).
DNA Sequencing Analysis and Computer Analysis-DNA sequencing was carried out by the dideoxy chain termination method (46) using the BcaBEST dideoxy sequencing kit (Takara). The nucleotide sequence and the deduced amino acid sequence were subjected to a homology search with GenBank TM and Protein Information Resource data bases using BLAST and FASTA programs.
Molecular Cloning of Mouse typ cDNA-A mouse testis cDNA library was constructed with oligo(dT) [12][13][14][15][16][17][18] primer (TimeSaver cDNA synthesis kit, Pharmacia) in the LAMDA ZAP II cloning vector (Stratagene) using poly(A) ϩ RNA prepared from 8 -12-week-old mice testes. The library was screened with standard protocols of plaque hybridization using 32 P-labeled human typ PCR fragment as a probe. Hybridization of the filters representing 1 ϫ 10 6 plaques was performed at low stringency condition as described (23). Complementary DNA inserts of positive clones were excised and partially sequenced using an antisense oligonucleotide primer corresponding to amino acid sequence WPDHGT (5Ј-AGTGCCATGGTCTGGCCA-3Ј), a highly conserved sequence in the PTP domain.
Production and Purification of Bacterially Expressed Protein-Amino acid sequences of residues 51-164, 165-285, and 168 -427 of the predicted Typ protein were expressed in E. coli as glutathione S-transferase (GST) fusion proteins (GST-TypN1, GST-TypC1, and GST-TypPTP, respectively). For construction of plasmids encoding GST-TypN1, GST-TypC1, and GST-TypPTP, the blunted EcoRI-EcoRV fragment encompassing nucleotides 365-709, EcoRV-EcoRI fragment encompassing nucleotides 710 -1071 and blunted EcoRV-XhoI fragment encompassing nucleotides 710 -1525 were ligated to the SmaI-digested pGEX-3X vector, SmaI-and EcoRI-digested pGEX-3X vector, and SmaI-digested pGEX-5X vector (Pharmacia), respectively. For protein expression, these constructs and empty pGEX-5X vector were transformed into the E. coli JM109. The GST-Typ fusion proteins and GST protein made in E. coli were purified by using glutathione-Sepharose beads as described (25). The purified recombinant proteins were suspended in phosphate-buffered saline (PBS), an aliquot of each sample was electrophoresed on a 10% SDS-polyacrylamide gel, and the gel was stained with Coomassie Blue. The amount of protein in the gel was estimated by comparing the stained band with that of a molecular weight marker (SDS-PAGE standard low range; Bio-Rad).
Generation of anti-Typ Polyclonal Antibodies-Polyclonal antibodies were generated by immunizing New Zealand White rabbits with the GST-TypN1 or GST-TypC1 protein. Affinity-purified anti-Typ antibodies were prepared from the crude anti-GST-TypN1 or anti-GST-TypC1 antiserum by use of affinity columns and the purified antibodies termed anti-TypN1 and anti-TypC1, respectively.
Transfection of the typ cDNA into 293T Cells-To construct a typ expression plasmid, pME-Typ, the typ cDNA insert of clone 5 (HindIII-XhoI) was recloned into an eukaryotic expression vector, pME18S (Ref. 26; a gift from K Maruyama, Tokyo Medical and Dental University). An amino-terminal-deleted Typ expression plasmid, pME-Flag-4M, contains the typ cDNA corresponding to amino acid residues 98 -426 tagged with FLAG epitope (Eastman Kodak Co.) at the amino terminus. Expression plasmids with mutations at the ATG or CTG codon of typ (pME-Typ1MA, pME-Typ2MS, pME-Typ3MV, pME-Typ4ML, pMETyp1LV, and pME-Typ2LA) were made from pME-Typ by sitedirected mutagenesis (27). Introduction of the mutations were confirmed by DNA sequencing. Each plasmid DNA was transfected into human 293T kidney cells by the calcium phosphate method.
Western Blotting-293T cells were lysed in a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 1% Nonidet P-40, 0.1% SDS, 20 mM EDTA, 150 mM NaCl, 50 mM NaF, 0.1 mM Na 3 VO 4 , and aprotinine (50 units/ml) after 48 h of transfection. Cell lysates of the testis, ovary, and brain were prepared by using the same lysis buffer. Samples of 25 g of proteins from the lysate of 293T cells and 100 g of proteins from the lysate of mouse tissues were fractionated by 10% SDS-PAGE. The filters were subjected to immunoblot analysis as described (24).
In Vitro Translation-The sense strand of the insert of clone 5 was transcribed using T7 RNA polymerase (Stratagene). The proteins encoded by the insert were translated in vitro in the presence of L-[ 35 S]methionine by using the wheat germ cell extract (Promega).
Phosphatase Assay-The catalytic activity of GST-TypPTP was assayed using p-nitrophenyl phosphate (pNPP) as a substrate (24,28). The glutathione beads capturing 0.25-3 g of GST-TypPTP or GST were subjected to the reaction.
In Situ Hybridization-Three DNA fragments (fragments 1-3, see below) were prepared from the cDNA insert of clone 5 (see Fig. 2A) by PCR amplification and restriction enzyme digestion. The amplified fragments were subcloned into the pBluescript II sk(Ϫ) vector. The resulting plasmid DNAs were linearized with appropriate enzymes and transcribed in vitro with T7 RNA polymerase (Stratagene). The transcripts from fragment 1 (nucleotide position 21-709 of typ), fragment 2 (position 218 -709), and fragment 3 (position 710 -1071) were used as antisense probes (antisense (AS)-probe 1, AS-probe 2, and AS-probe 3, respectively). Fragment 1 was also subcloned into pBluescript II KS(ϩ), linearized, and transcribed with T7 RNA polymerase to generate a sense RNA probe. The RNA probes were labeled using the digoxigenin RNA Labeling Kit (Boehringer Mannheim) following manufacturer's protocol.
Testis samples of 8-week-old mice were embedded in OCT compound (Tissue-Tek, Miles) and frozen. The frozen sections (5-8 m) were fixed for 20 min with 4% paraformaldehyde freshly prepared in 0.1 M sodium phosphate buffer, pH 7.4. They were then treated with 0.2 M HCl and acetylated with 0.1 M triethanolamine containing 0.25% acetic anhydride. After washes in PBS, sections were dehydrated in a series of ethanol washes. The hybridization solution contained 50% formamide, 10 mM Tris, pH 7.6, 1 ϫ Denhardt's solution, 10% dextran sulfate, 600 mM NaCl, 0.25% SDS, 1 mM EDTA, pH 8.0, 200 g/ml tRNA, and 5 g/ml digoxigenin-labeled RNA probes. Hybridization solution (80 l) was placed on each section and incubated at 50°C in a moist chamber. After a 16-h incubation, sections were washed in a solution containing 50% formamide and 2 ϫ SSC (20 ϫ SSC ϭ 3 M NaCl, 0.3 M sodium citrate) at 65°C for 30 min, treated with RNase-A (10 g/ml) at 37°C for 30 min, and washed again in 2 ϫ SSC, 0.1% SDS and in 0.2 ϫ SSC, 0.1% SDS at 50°C for 60 min. The hybridization signals were visualized with alkaline phosphatase-conjugated anti-digoxigenin antibody (Boehringer Mannheim) by adding the substrate for alkaline phosphatase TNBT (Sigma).
Immunofluorescence Microscopy-Testis sections were cut and fixed as described above and permeabilized by incubation at room temperature with 0.2% (v/v) Triton X-100 in PBS for 10 min. The permeabilized sections were treated with PBS containing 1% (w/v) bovine serum albumin (fraction V from Sigma) for 30 min at room temperature. Then the sections were incubated for 2.5 h with the anti-TypN1 and anti-TypC1 antibodies in PBS containing 0.02% (v/v) Triton X-100 at room temperature. As a control staining, anti-TypN1 antibody in PBS containing 0.02% (v/v) Triton X-100 were pre-incubated with GST-TypN1 (see Fig. 7B). After being washed three times with PBS for 15 min, the sections were incubated for 1 h with the second immune reagent, FITC-labeled goat anti-rabbit IgG. For localization of chromosomes, DNAs were stained with propidium iodide. Samples were observed using laser scanning microscopes (Zeiss and Bio-Rad).

Isolation of a Novel PTP cDNA Fragments-To identify novel
PTPs, we employed the RT-PCR-based cloning procedure. Complementary DNAs were synthesized by RT of mRNAs prepared from MDA-MB453 cells and TIG1 cells. The RT reaction was primed with a degenerate primer (primer 1) designed from amino acid sequence QRE(A/E/D)Q, which was conserved among PTPs. The PCRs were performed using the cDNA and degenerate primers corresponding to other conserved regions in PTPs (primers 2 and 3 corresponding to DYINA and HCSA, respectively). The PCR products were subcloned into the pUC119 vector. Nucleotide sequencing of the inserts of the resulting 28 clones revealed that most of them encoded amino acid sequences corresponding to previously reported sequences of B-PTP2 (19), PTP-MEG (20), DEP-1 (21), and LAR (22). Some cDNA clones did not contain PTP consensus sequences. To identify clones that represent minor PCR products encoding novel PTPs, we employed a "negative selection" screening method. About 1,200 E. coli clones carrying the PCR products were analyzed by colony hybridization with probes of a mixture of the 32 P-labeled PCR products containing sequences for B-PTP2, PTP-MEG, DEP-1, and LAR. Consequently, eight colonies gave negative or weak signals. DNA sequence analysis of the eight clones revealed that one clone had an insert encoding a novel PTP. The other clones contained sequences corresponding to known PTPs or sequences that did not share any homology to PTPs.
Northern hybridization of RNAs from adult human tissues was carried out with the 32 P-labeled PCR fragment encoding the novel PTP. A typ RNA transcript of about 3.2-kilobases was detected exclusively in the testis (Fig. 1A). The transcript of this gene was not detected in fetal human tissues (brain, lung, liver, and kidney) nor in human cell lines examined, including MDA-MB453. Thus, we designated this novel PTP gene typ (testis-specific tyrosine phosphatase). Since the typ clone was obtained by RT-PCR amplification using mRNAs from MDA-MB453 cells, we assumed that the typ gene was expressed at very low levels in the cells.
Isolation and Characterization of the typ cDNA-To clone the full-length cDNA of the typ gene, we constructed a cDNA library from mouse testis mRNAs. The library was screened with the human typ probe under conditions of reduced stringency.
By screening 1ϫ10 6 independent clones, nine positive clones were obtained. Nucleotide sequencing revealed that the inserts of eight clones were overlapping, suggesting that they were derived from the same mRNA species (Fig. 2A). One remaining clone had a sequence for a putative mouse homologue of PTP-MEG (23). This clone might have been selected by cross-hybridization because the screening was carried out under the low stringent condition. A composite sequence of 3,090 nucleotides constructed from the inserts of four clones, clones 2, 5, 6, and 8, contained the largest open reading frame of 426 amino acid residues. The coding frame began with a consensus-initiating methionine codon at position 218 that was preceded by five in-frame stop codons (Fig. 2B). We tentatively concluded that the open reading frame encoded the mouse Typ protein. Nucleotide sequence analysis also revealed that the cDNA insert of clone 2 contained internal 441-base pair deletions in the coding region compared with the insert of the other clones. This resulted in the precise deletion of 147 amino acid residues (amino acids 168 -314) in the predicted sequence. Therefore, the downstream coding frame was not affected. We then performed RT-PCR analysis of total RNAs extracted from mouse testis. Two pairs of oligonucleotide primers that flanked the deletion were utilized. Both sets of primers failed to amplify the fragment corresponding to the insert of clone 2 but was able to amplify the DNAs corresponding to the other cDNA clones. This suggested that the deletion was an artifact that occurred during the library construction. However, the possibility that the clone 2 cDNA was derived from an alternatively spliced mRNA species, which was expressed in very low abundance, cannot be excluded.
The nucleotide sequence of the mouse typ cDNA and the deduced amino acid sequence showed 74.8 and 84.1% identity, respectively, with the corresponding sequences of human typ. The similarities between human and mouse were relatively low when compared with those of the other PTPs. For example, predicted amino acid sequences of mouse SHP2 (29) and the fist PTP domain of mouse PTP␦ (30) are 99.6 and 96.3% identical with the corresponding sequences of their human homologues, respectively (31)(32)(33)(34)(35). The human and mouse Mos proteins, protein kinases involved in meiosis (12,36), are less homologous to each other (ϳ78% identity) even in their catalytic domains (37,38). We then examined the expression pattern of mouse typ and compared it with that of human typ. RNAs were extracted from adult mouse tissues and analyzed by Northern blot hybridization under stringent conditions. Using the 32 P-labeled cDNA insert of clone 5 as a probe, typ mRNA of 3.2 kilobases, the same size as that detected in human testis, was detected only in testis among seven tissues examined (Fig. 1B). The same result was obtained when the blot was probed with the human typ PCR fragment under a relaxed condition (data not shown). These data substantiated that we have cloned cDNAs for the mouse typ gene.
Characterization of the Typ Protein-The deduced amino acid sequence of the mouse typ protein revealed the presence of the PTP domain at the carboxyl-terminal half. The Typ protein had neither a signal peptide nor a membrane-spanning segment, indicating that Typ is a cytoplasmic PTP. The putative PTP domain contained the consensus sequences such as the catalytic core sequence (I/V)HCXAGXXR(S/T)G. Comparison of amino acid sequences between Typ and other PTPs at the catalytic domain revealed that the identity was about 45% at most. A computer-assisted search of the GenBank TM data base revealed no significant homologies of the amino-terminal half of the Typ protein to the other known proteins. However, there was a region rich in proline, glutamate, serine, and threonine residues that was flanked by positively charged amino acids ies were termed anti-TypN1 and anti-TypC1, respectively. In the extract of adult mouse testis, about 45-and 40-kDa proteins were detected by both of the affinity-purified anti-TypN1 and anti-TypC1 antibodies (Fig. 3A). Two proteins were not detected in the brain extracts nor in the ovary extracts (Fig.  3A). The 40-kDa protein could be a degradation product of the 45-kDa protein. Alternatively, a downstream ATG codon might be utilized for an initiation methionine. Then the typ cDNA containing the entire open reading frame that initiates from the ATG-218 codon was translated in vitro. As shown in Fig.  3A, the 45-and 40-kDa proteins were synthesized (lane 6). In addition, in the lysates of 293T cells transfected with the Typ expression plasmid, pME-Typ, proteins of the same size as detected in the testis lysate were detected by the anti-TypN1 antibodies (Fig. 3C, lanes 1 and 2). On the other hand, when the ATG-218 codon that encodes the first methionine was mutated to GCG, the resulting expression plasmid pME-Typ1MA gave rise to only the 40-kDa form of Typ upon transfection into 293T cells (Fig. 3C, lane 3). These results indicated that the 45-kDa Typ protein was initiated from the ATG-218 and that the 40-kDa protein was not produced by post-translational modification or degradation of the 45-kDa Typ. We next tested the possibility that the 40-kDa protein resulted from the alterna-tive use of internal translation initiation sites. When an expression plasmid (pME-Flag-4M) for the FLAG-tagged and amino-terminal-deleted Typ (corresponding to amino acid residues 98 -426) (Fig. 3B) was transfected into 293T, the cells expressed a protein smaller than the 40-kDa form of Typ (Fig.  3C, lane 9). The data suggested that the translation initiation site for the 40-kDa Typ protein was located between the TCT-221 codon next to the ATG-218 codon and the ATG-665 codon for Met-97. There are three ATG codons (Met-38, Met-49, and Met-97) and two CTG codons (Leu-25 and Leu-60) in this region. Since not only the ATG codon but also the CTG codon, in some cases, can serve as a translation initiation codon, we mutated these codons in the Typ expression plasmid to identify the initiation site for the 40-kDa protein (Fig. 3B). The resulting plasmid encoding mutant Typs pME-Typ2MS, pME-Typ3MV, pME-Typ4ML, pMETyp1LV, and pME-Typ2LA was transfected into 293T cells. As shown in Fig. 3C, both the 45and 40-kDa proteins were detected by anti-TypN1 antibodies in the lysate of the cells transfected with the mutant DNAs (lanes 4 -8). Virtually identical results were obtained using the anti-TypC1 antibodies (data not shown). Taken together, it was suggested that in addition to the first methionine encoded by the ATG-218 codon, an internal codon other than ATG nor CTG was utilized in vitro and in vivo as a translation initiation site.
Demonstration of Phosphatase Activity-To demonstrate the catalytic activity of the PTP domain of Typ, the carboxyl-terminal half of Typ that contained the entire PTP domain was expressed in E. coli as a GST fusion protein (GST-TypPTP). Purified GST and GST-TypPTP proteins were assayed for PTP activity by incubating with pNPP (20). As shown in Fig. 4, GST-TypPTP but not GST alone was able to dephosphorylate pNPP. Hydrolysis of pNPP was inhibited by a specific PTP inhibitor sodium orthovanadate.
Developmentally Regulated typ Gene Expression in Testicular Germ Cells-To understand the role of Typ, we analyzed its expression during spermatogenesis. We first examined the developmental expression of the typ products in mouse testis by Western blotting. As shown in Fig. 5, the 45-kDa Typ protein was detected in the lysate of a 3-week-old mouse testis, whereas the 40-kDa typ product was not detected until 4 weeks after birth (Fig. 3A). This indicates that the Typ protein becomes detectable between 2 and 3 weeks after birth, in parallel with the appearance of the pachytene spermatocyte (41). Then we examined typ mRNA expression by in situ hybridization.  6). B, schematic representation of FLAG-tagged N-terminal-deleted Typ protein (Flag-4M) and the mutant constructs. C, proteins in the lysate of testis (100 g) (lane 1) and 293T cells (25 g) transfected with the pME-Typ (pME-Typ1MA (1MA), pME-Typ2MS (2MS), pME-Typ3MV (3MV), pME-Typ4ML (4ML), pMETyp1LV (1LV), pME-Typ2LA (2LA), pME-Flag-4M, and empty pME18S vector (mock) (lanes 2-10)) were fractionated by 10% SDS-PAGE and analyzed by immunoblotting with anti-TypN1 antibodies. Cryostat sections of the 8-week-old mouse testis were hybridized with digoxigenin-labeled typ antisense RNA probe, which was complementary to nucleotides 21-709 (AS-probe 1). Expression of the typ mRNA was confined to rings of cells adjacent to the spermatogonial layer of the tubule circumference (Fig.  6A). Both the expression pattern and the morphology of typexpressing cells strongly suggested that typ was expressed in primary spermatocytes. Under lower magnification, typ mRNA was not detectable in all seminiferous tubules (Fig. 6C). The process of spermatogenesis in mature testis proceeds asynchronously and can be divided into 12 steps according to the composition and distribution of spermatocytes, spermatids, and sperm (42). The failure to detect the typ mRNA in some tubules indicated that expression of the typ gene is restricted to particular stages of the spermatogenetic cycle. The other two antisense probes (AS-probe 2 and 3, see "Experimental Procedures") gave essentially identical results (data not shown), and the sense control probe did not give any specific signals (Fig.  6B).
The distribution of Typ proteins during spermatogenesis was also determined immunocytochemically by employing an indirect immunofluorescence technique. Consistent with the result of the in situ hybridization analysis, strong signals were detected in the primary spermatocytes by both anti-TypN1 and anti-TypC1 antibodies. No significant signals were detected in spermatogonia, early round spermatids, sperm, or in testicular somatic cells such as Sertoli cells (Fig. 7, panels D, E, G, and  H). Signals were blocked by preincubation with the immunizing antigen (Fig. 7B). Together these results strongly suggest that expression of the typ gene is temporarily regulated during spermatogenesis and is restricted to germ cells at a particular meiotic stage, possibly at the pachytene stage.

DISCUSSION
In this study to identify novel PTPs, a modified RT-PCRbased method was employed for identifying mRNAs expressed in low abundance within the cell. To eliminate the previously known and/or abundantly amplified sequences from the PCR products, we first generated a plasmid library of amplified PCR products. Then the library was subjected to colony hybridization with probes of a mixture of the 32 P-labeled PCR amplified products. Using the negative selection screening method, we cloned a DNA fragment encoding a novel PTP named Typ. Northern blot analysis revealed that the typ gene is expressed exclusively in the testis of both human and mouse. The tran-script of the typ gene was not detected in any human cell lines examined, including MDA-MB453, from which the template cDNA for PCR amplification was prepared. Thus, the negative selection screening method allowed identification of the typ mRNA present in low abundance in MDA-MB453 cells.
The predicted Typ protein does not contain a signal peptide or a membrane-spanning region but has a single catalytic domain located in the carboxyl terminus-proximal half. The PTP domain of Typ shows an overall homology with that of other PTPs, although the shared identity is about 45% at most. This indicates that Typ is a new member of the cytoplasmic PTPs. The amino terminus-proximal nonenzymatic region of Typ does not share significant homologies with the other known proteins. The noncatalytic domain contains PEST-like sequences, characteristic of proteins that display very short half-lives (39). Therefore, Typ might be an unstable protein.
Anti-Typ antibodies recognized 45-and 40-kDa proteins in testicular lysates. In vitro translation of the typ open reading frame resulted in the production of the same sizes of proteins. The results demonstrate that the two proteins represent the typ gene products and arise from a single unprocessed typ mRNA species. As shown in Fig. 3C, even when the 45-kDa protein was not synthesized due to the mutation in the ATG-218 codon, the 40-kDa Typ protein was detected. Therefore, that 40-kDa Typ protein was not produced by the proteolytic cleavage of the 45-kDa Typ protein. In addition, mutations in the other three internal ATG codons and two CTG codons, which might be putative translation initiator, did not affect the production of the two proteins. These results suggest that the 40-kDa form of Typ is produced by utilizing non-ATG nor CTG codon as a translation initiator. Since pME-Flag-4M directs the synthesis of a protein slightly smaller than 40 kDa, the translation initiation site for this protein is confined within a se- quence between TCT-221 and ATG-665. We note by passing that some triplets that differ from ATG and CTG have the ability to direct initiation of the protein synthesis in mammalian cells (43).
In the mouse seminiferous tubule, spermatogenetic cells of the same developmental stage form a concentric layer of cells. Mitotic proliferation of stem cells, which is the initial phase of spermatogenesis, occurs in the basal compartment. Successively, meiosis and spermiogenesis occur toward the inside of the seminiferous tubule. Therefore, it is generally possible to specify the developmental stage of germ cells on the basis of their sizes, shapes, and spatial distribution. The present data reveal that both the typ mRNA and the protein products are specifically expressed in testicular germ cells adjacent to the spermatogonial layer. Both their position relative to the spermatogonial layer and their morphology (large round cells) strongly suggest that the typ-expressing cells are primary spermatocytes. Consistently, expression of the Typ protein became detectable around 3 weeks after birth, at which meiosis for spermatogenesis begins. Moreover, since the typ mRNA was not detectable in all seminiferous tubules, we concluded that expression of the typ gene is temporarily regulated during spermatogenesis. Similar patterns of expression in testis have been reported for several genes such as nek1 (14), ferT (10), CTfin51 (44), and OST-PTP (15).
Although expression of typ is not detectable in the adult ovary ( Figs. 1 and 4), it does not necessarily indicate that Typ is not involved in meiosis of ovarian germ cells. Adult ovaries contain a much smaller number of germ cells than adult testes. Moreover, after migration into prospective ovary, female germ cells cease to proliferate and enter meiosis about 13.0 days postcoitum. All oocytes are in the diplotene stage of the prophase of the first meiotic division by 5 days after birth. Thus, oocytes in adult ovary are in a phase later than the equivalent stage of the testicular germ cells in which typ is expressed. Further analysis of typ expression in the ovaries of female mouse embryo will demonstrate whether the expression of the typ gene is restricted to testicular germ cells or commonly found in both male and female germ cells.
In conclusion, we have reported the isolation and characterization of a novel cytoplasmic PTP Typ. Typ expresses exclusively in the testis. The highest level of the typ mRNA and Typ proteins were detected in the primary spermatocyte of the adult mouse testis. Accumulating evidence suggests that protein tyrosine phosphorylation is a key reaction in various biological systems. Protein-tyrosine kinases such as c-Kit (9) and Fer-T (45) are implicated in spermatogenesis. However, the molecular mechanisms of spermatogenesis that involve protein tyrosine phosphorylation remain to be clarified. Less understood is the relevance of tyrosine phosphatases in spermatogenesis, though receptor-type PTPs termed OST-PTP (osteotesticular protein-tyrosine phosphatase) is reported to be expressed within the seminiferous tubule as well as in bone (15). Further investigation of the function of Typ will provide an important insight into the signal transduction pathway that regulates spermatogenesis or the maturation of mammalian germ cells. FIG. 7. Immunohistochemical analysis of Typ expression in testis. Testis sections (5-8 m) of the 8-week-old mouse were stained with anti-TypN1 antibodies (D), anti-TypC1 antibodies (G), or anti-TypN1 antibodies pre-incubated with the immunizing antigen (B). Panels A, C, and F are the staining of chromosome DNA by propidium iodide. The image recorded in panels C and F were electronically overlapped with that in panels D and G, respectively (E and H). The red signal shows distribution of the Typ protein, and the green signal shows chromosome DNA stained with propidium iodide.