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Testis |
Institute of Molecular & Cell Biology,3 National Institute of Advanced Industrial Science & Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan
Institute of Applied Biochemistry,4 University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CXC domain, meiosis, spermatogenesis, tesmin, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A lambda FIXII library (9 x 105 clones) of the mouse 129 strain (Stratagene, La Jolla, CA) was screened using a 32P-labeled tesmin cDNA (accession no. NM010841) as a probe. After the first and second screenings, three clones that contained tesmin-30 cDNAs were chosen for further analyses. Restriction fragments were subcloned into the Bluescript SK (+) (Stratagene), and insert DNAs were sequenced from both ends by primer walking. Sequencing was performed using automatic sequencers (ABI PRISM 310 and ABI 373 DNA sequencers; Applied Biosystems, Foster City, CA) by the dideoxyterminator method. A total sequence of 22 856 nucleotides was determined (GenBank accession no. AB057422).
Screening of Full-Length cDNA
A mouse testis cDNA library (6 x 105 independent clones) of the Balb/c strain in 
gt11 (Clontech, Palo Alto, CA) was screened using a 32P-labeled probe, a 139-bp fragment (nucleotides 505–643 of tesmin cDNA, accession no. NM010841). Twelve isolated clones were subcloned into the Bluescript vector at an EcoRI site and were sequenced from both ends by primer walking. The three clones that had extended 5' upstream sequences (CT2, CT3, and CT9) were fully sequenced, and then after their comparison with genomic DNA sequences, the longest cDNA (2215 bp) was deduced (accession no. AB057423).
Northern Blot Analysis
A Northern membrane loaded with 2 µg of poly(A)+ RNA from a variety of mouse tissues was purchased from Ambion (Austin, TX). The membrane was hybridized with a random-primed 32P-labeled DNA fragment of the full-length coding region of mouse tesmin for 16 h at 45°C in a hybridization solution containing 50% (v/v) formamide. This was washed twice with 2x SSPE (1x SSPE: 150 mM NaCl, 10 mM NaH2PO4, and 1 mM Na/EDTA)/0.1% SDS for 15 min at room temperature and for 10 min at 50°C with 1x SSPE/0.1% SDS and 0.1x SSPE/0.1% SDS.
Promoter Assay
A green fluorescence protein reporter vector Promoter assay (Clontech) was used as the positive control. The cytomegalovirus (CMV) promoter region was removed from pEGFP-N1 by cutting with AseI and BglII and used as the negative control (pCMV--EGFP). The promoter region of tesmin (2.5-kilobase [kb] region between XhoI and BalI (no. 19 and no. 2564, respectively, of accession no. AB057422) was ligated to XhoI and SmaI sites of pCMV--EGFP (pTes-EGFP). GC2 cells (American Type Culture Collection no. CRL-2196) from spermatocytes of a BALB/c mouse were plated in a 12-well plate (4 x 104 cells/well) and transfected with plasmid DNA (1.5 µg) using SuperFect (Qiagen, Hilden, Germany) 24 h after the plating. Cells in PBS were examined 24 h after the transfection under fluorescence microscopy.
Preparation of 30-kDa Recombinant Tesmin Protein and Raising of Anti-Tesmin Antibody
Tesmin-30 was cloned into the pQE-30 bacterial expression vector (Qiagen). Tesmin-30 was isolated as a His6-tagged protein using Ni-NTA resin (Qiagen) according to the manufacturer's instructions. It was further purified by preparative SDS-PAGE and then by elution (HBS-Elutor E51; Biometra, Gottingen, Germany). Purified tesmin-30 was dissolved to 0.2 mg/ml in physiological saline. Two rabbits were immunized with tesmin-30 three times at an interval of 10 days. After two booster injections of the antigen, blood was collected for antiserum and examined for its reactivity to tesmin protein by Western blot analysis.
Western Blot Analysis
The mouse testis homogenates from 8-wk-old mice were fractionated and subjected to 12% SDS-PAGE. Proteins were electroblotted to polyvinylidene fluoride membranes (Sequi-Blot PVDF membrane; Bio-Rad, Hercules, CA) using a Trans-Blot Cell (Bio-Rad). Membranes were hybridized with an anti-tesmin-30 serum (1:4000 dilution) or a preimmune serum and then with an anti-rabbit antibody conjugated with peroxidase (POD-
Rabbit IgG, 1:2000; Amersham Pharmacia Biotech, Buckinghamshire, U.K.). Reactive protein bands were visualized by chemiluminescence (Western Blot Chemiluminescence Reagent Plus; NEN Life Science, Boston, MA).
Peptide Sequencing of Native Tesmin
Recombinant tesmin-30 was covalently bound to CNBr-Sepharose 4B (Amersham) according to the manufacturer's protocols and was then used to purify anti-tesmin-30 serum. Purified anti-tesmin-30 serum was used for immunoprecipitation of native tesmin protein from mouse testis as follows: Cytoplasmic and nuclear fractions from the mouse testes of 10 ICR mice (age, 8 wk old; Nihon Clea, Suita, Osaka, Japan) were separated by centrifugation. Cytosolic and microsomal fractions were further separated from the cytoplasm by centrifugation at 19 000 x g for 60 min. Nuclei were purified from the nuclear fraction by two-step sucrose density-gradient centrifugation in 2.3 M sucrose at 12 000 x g for 30 min and in 0.25 M sucrose at 2000 x g for 5 min. Tesmin-30-specific antibody bound to Sepharose 4B (100 µl; Amersham Biosciences, Piscataway, NJ) and the testis cytosolic fraction (total protein, 51 mg) were mixed, and unbound proteins were removed by centrifugation. The precipitate was washed with buffers and suspended in an SDS-PAGE sample buffer containing 2-mercaptoethanol. Native tesmin-bound Sepharose 4B was boiled and applied to a 10% SDS-PAGE gel. Marker edges of the gel were subjected to either Quick-CBB or silver staining (Silver Staining Kit; Wako Pure Chemicals Industries, Osaka, Japan). Bands close to 60 kDa were cut out and digested with lysylendopeptidase in a Tris-HCl buffer (pH 8.5) at 35°C for 20 h. The digested sample was subjected to reverse-phase high-performance liquid chromatography, and 81 fractions were obtained. From fractions 45, 52, 61, 68, 69, and 70, the following sequences consisting of 9, 11, 20, 10, 10, and 10 amino acids, respectively, were obtained using a Hewlett-Packard sequencer (model G1005A; Hewlett-Packard, Palo Alto, CA).
Immunohistochemisry
The testes of an 8-wk-old ICR mouse were fixed in formalin and embedded in paraffin, and then thin sections (thickness, 3 µm) were prepared. Specimens were blocked with 1% bovine serum albumin/5% skim milk in distilled water and then sequentially stained with the anti-tesmin rabbit antibody (1:2000 dilution), Cy3-labeled anti-rabbit goat serum (1:1000 dilution), and a mixture of 4',6'-diamidino-2-phenylindole and fluorescein isothiocyanate-labeled lectin PNA. The slides were covered with a glass slip using Cell-Mount (Biomeda Corp., Foster City, CA). Cryosections were also prepared using a microtome (HM 500 OM; Microm Laborgerate GmbH, Walldorf, Germany). The testes of an ICR mouse were fixed with ice-cold 4% paraformaldehyde (PFA) in PBS and embedded in an O.C.T. compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan). Staining was performed as described above. Imprinting specimens were prepared as follows: A testis was cut into halves, and the cut-edge was pressed onto the surface of a glass slide. Specimens were fixed with ice-cold 4% PFA and immunohistochemically stained as described above. These were then examined by fluorescent microscopy (Leica Microsystems, Wetzlar, Germany).
In Vitro Expression of Tesmin-60
The EcoRI fragment of CT3/9, a combined cDNA clone of CT3 and CT9, was cloned into the pcDNA3 expression vector (Invitrogen, Carlsbad, CA), giving pcDNA/M3. Because CT3/9 had three possible translation initiation codons (ATGs) at the 5' upstream region and the first ATG was not found in the genomic sequence, a cDNA clone lacking this first ATG-containing region was also constructed (pcDNA/M2). Plasmid DNA was purified and transfected into CHO cells using SuperFect (Qiagen). Cells were cultured in Dulbecco modified minimal essential medium supplemented with 10% fetal bovine serum and harvested with a rubber policeman 2 days after transfection in cold PBS. The cells were lysed in an SDS-PAGE sample buffer, and the supernatant fraction was used for Western blot analysis. COS7 cells were also transfected with pcDNA/M2 as described above. Cells grown on a cover slip were also transfected and used for immunohistochemistry.
Oxidative Stress Experiments
Diethylmaleate (DEM; 5 mmol/kg; Aldrich Chemical Co., Inc., Milwaukee, WI) or cobalt chloride (10 µmol/kg; Wako) was administered to mice at 0.1 ml per 10 g body weight. Mice were killed by cervical dislocation at 4, 24, or 48 h after administration, and imprint specimens of the testes were prepared. Staining for apoptotic cells was carried out using In Situ Cell Death Detection Kit (Fluorescein; Roche Diagnostics GmbH, Mannheim, Germany) following the manufacturer's protocol. For a positive control, specimens were treated with DNase I (100 µg/ml) to introduce nicks into DNA for 10 min at room temperature. Other staining procedures are described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The four tesmin cDNAs reported so far encode a 30-kDa protein consisting of the same 295 amino acid residues. An anti-tesmin-30 antibody raised against this protein produced in an Escherichia coli system was confirmed to be specifically reactive (Fig. 1A, lane 8). When used against mouse testis and liver lysates, a single band of 60 kDa was identified only in the soluble fraction from the testis. In agreement with previous RT-PCR and Northern blot data [1], this suggested that tesmin is exclusively expressed in the testis. However, its size was double that expected from the cDNA clones reported earlier [1]. To confirm that the 60-kDa protein was not an artifact of cross-reactivity, we purified the immunoprecipitated protein (Fig. 1C). Trials to determine the N-terminal sequence were unsuccessful, but five fragments produced by digestion with lysylendopeptidase could be sequenced. One of them (with the sequence EAGGSVPGGSPEDAAFQAPL) matched exactly with the deduced amino acid sequence from the 5'-untranslated region (UTR) of cDNA encoding tesmin-30. The other four sequences were found in tesmin-30. This indicates that the native tesmin is a 60-kDa protein and that the tesmin cDNAs encoding tesmin-30 are not full-length and start translation from an ATG far downstream to give a truncated product consisting of the C-terminal half-region of tesmin-60.
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Features of the Tesmin Gene
Three phage DNAs including tesmin-30 cDNA were sequenced and combined to yield a total of 22 856 nucleotides (Fig. 2). This composite clone contained several possible transcription factor-binding sites in the 5' upstream region. These include SP1 (-15, -462, -122, and -163 bp from the first base of the longest cDNA CT3/9), AP1 (-33 bp), GATA-1 (-258 bp), p53 (-369 bp), and CRE-BP1 (-450 bp). CCAAT or TATA boxes were not identified. This region of the tesmin gene is guanine-cytosine (GC)-rich and contains CpG islands (Table 1).
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Cloning of the cDNA for Tesmin-60
To obtain a full-length cDNA encoding tesmin-60, several attempts were made. These included 5' rapid amplification of cDNA ends and colony hybridization screenings of commercially available (Nippon Gene, Toyama, Japan; Takara, Tokyo, Japan; and Clontech) and homemade mouse testis cDNA libraries. We obtained cDNAs encoding 475 amino acid residues from the Clontech cDNA library. Three of these cDNAs, CT2, CT93, and CT9, out of 12 were fully sequenced (Fig. 3). CT3 (Fig. 3, nos. 1–1066) and CT9 (Fig. 3, nos. 60–2243) were combined to obtain the longest CT3/9 cDNA sequence. Genomic DNA and CT3/9 cDNA had nine common regions (exons) sharing 2185 bp excluding poly(A)n (Fig. 2). The GT-AG rule was applied to most introns except for intron 7, where GC-AG was used for splicing.
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The first ATG of CT3/9 was included in the 5' flanking sequence that was not found in the genomic DNA (Fig. 3, first 28 bp). This sequence mostly consists of a part of 28S ribosomal RNA (21 base matches out of 22 from nos. 7–28) (Fig. 3). The origin of this is unknown. However, it is thought to be inserted into the 5' end during the preparation of the cDNA library, because the first ATG was not necessary for the production of tesmin-60 (Fig. 1) and a promoter activity was found in the region just upstream of the second ATG (see below).
One cDNA clone had a 3'-UTR that was shorter by 406 nucleotides; this may explain the two splice isoforms of mRNA, 2.2 and 1.8 kb. This shorter mRNA used a noncanonical poly(A)n-addition signal ATAAAA (Fig. 2B, no. 1787) instead of the canonical AATAAA (Fig. 2B, no. 2193). The shorter mRNA (1.8 kb) was the major transcript (Fig. 4) as revealed by Northern blot analysis. Figure 4 also shows that tesmin is specifically expressed in the testis.
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The genomic DNA clone was isolated from the 129 strain of mice, whereas the cDNA clones were isolated from the BALB/c strain. Some differences exist between the two cDNAs shown in Figure 3: an insertion of a nucleotide (G) between numbers 1021 and 1022, and a deletion of a nucleotide (G) at number 1030 in the 129 strain. As a result, alanine at residue 305 was changed to glycine, and isoleucine at residue 306 was changed to histidine. Nucleotide G at positions 1108 and 1126 were replaced by T in 129, and accordingly, it resulted in the replacement of glutamine at residues 433 and 439 by histidine. In the 3'-UTR, nucleotide G and T at positions 1991 and 2039, respectively, were deleted, and nucleotide C was replaced by T at position 2089 in the 129 strain.
The molecular weight of tesmin-60 (475 amino acid residues), as deduced from the cloned cDNA, was 50.6 kDa. This was still shorter by 10 kDa from the apparent size of the protein detected from mouse testis lysates and suggested that CT3/9 may not still represent a full-length cDNA. By database search, we found a 580-bp EST sequence (accession no. AI875749) (Fig. 2, nos. 1018–1597) identical to our genomic DNA. Northern blot analysis using a probe including these 580 nucleotides and a further upstream probe (5 kb HincII-XhoI fragment from the first nucleotide in Fig. 2) did not detect any transcripts, indicating that no exons occur in at least the upper 7.5-kb region from the first exon of CT3/9. Indeed, the promoter activity was found in the 2.5-kb upstream region of CT3/9 (see below).
CT3/9 cDNA Encodes Tesmin-60
The cDNA encoding tesmin-60 had three in-frame ATGs in the 5' upstream region (Fig. 3). The first ATG occurred in the sequence not found in the genomic DNA that we have sequenced so far. Expression vectors having either all three (pcDNA/M3) or only the second and third ATGs (pcDNA/M2) were constructed and transfected into CHO cells. Because pcDNA/M3 produced the same-size protein as those produced by pcDNA/M2 (Fig. 1A, lanes 6 and 7), the first ATG in pcDNA/M3 was not involved in translation initiation.
Tesmin-60 encoded by pcDNA/M2 was detected as a 60-kDa protein in the soluble fraction of cells transfected with pcDNA/M2 (Fig. 1A, lane 3). Its mobility was identical to that of native tesmin-60 (Fig. 1A, lane 9). The calculated molecular weight of the protein from this cDNA, however, was 50.6 kDa; some posttranslational modifications are expected because of the higher apparent molecular weight seen on SDS-PAGE. The sequence around the second ATG is GTGGCCATGG and matches 8 out of 10 bases in the Kozak consensus sequence GCCG/ACCAT [3]. The translation initiation site remains to be identified.
Promoter Activity of the 2.5-kb 5' Upstream Region
GC-2 cells did not show fluorescence transfected with pCMV--EGFP (Fig. 5) as did those without treatment (not shown). Many cells with bright and faint fluorescence were detected when cells were transfected with pEGFP-N1 or pTes-EGFP, indicating that the 2.5-kb genomic upstream region contains the promoter for tesmin. This region starts from the XhoI site 2.5-kb upstream and ends two bases upstream from the second ATG (Fig. 3, no. 110). The promoter activity of this region (Fig. 5D) was weaker than that of CMV (Fig. 5F) under the present experimental conditions.
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CHC Gene Family
The literature and database survey indicated that tesmin is highly homologous to genes such as TSO1 of Arabidopsis thaliana [4, 5], CPP1 of Glycine max [6], and a putative protein with 429 amino acid residues of Caenorhabditis elegans (accession no. Z82274) (Fig. 6). For simplicity, these genes with the phylogenetically conserved structure of CXC-hinge-CXC domains are identified here as the CHC family. When the amino acid sequence of MT-3 was compared with those of the CHC family, the sequence deviated quite far from those of the CHC family members. The hinge region is lacking in MT-3. No metal-responsive element (MRE), the consensus sequence of which is 5'-TGCAC-3' [7], was found in the 5' upstream region of tesmin gene. Tesmin-60, therefore, must not be a member of the MTs.
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Localization of Tesmin
When pcDNA/M2 was transfected into COS7 cells, tesmin was found in the cytoplasm (Fig. 7, A–C). In the testicular tubules embedded in paraffin, tesmin seemed to localize mainly in the cytoplasm of spermatocytes. Round spermatids were stained more faintly than elongated spermatids (Fig. 7, D–F).
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Cell fractionation and Western blot analyses indicated that tesmin occurs in the soluble fraction of the testis (Fig. 1B). Tesmin was therefore thought to be located in the cytoplasm of spermatocytes. Analyses of stage-specific distribution of tesmin in cryosections, however, revealed that it translocated from the nucleus and the cytoplasm just before meiotic division at stages X–IX (Fig. 7, H–J). These stages also indicated that it was localized in the cytoplasm of leptotene-zygotene spermatocytes (Fig. 7J, short arrow) and at the caudal ends of late spermatids (Fig. 7, L and R). Tesmin was also localized at the outer surface of the nuclear membrane of elongated spermatids, as revealed by the cut-edge of a cryosection (Fig. 7, H, arrow, and K). When tesmin was seen in the nucleus, it did not colocalize with condensing chromosomes (Fig. 7L, long arrow) or meiotic chromosomes (Fig. 7L, short arrows), indicating that tesmin is not a component of chromosomes at meiotic division.
Even though one spermatocyte gives rise to four spermatids at meiotic division, the relative intensity of signal per spermatid is expected to remain constant. However, tesmin was hardly detectable in early round spermatids (Fig. 7R). When spermiogenesis started and the acrosome vesicle began to appear on the nuclear surface of spermatids, tesmin began to localize on the nuclear membrane just under the acrosome vesicle (Fig. 7, M–O). Starting from the acrosome vesicle, the nuclear surface was covered with tesmin in the midstage of round spermatids (Fig. 7P). As spermatids elongated, tesmin began to move toward the caudal end (Fig. 7Q) and, finally, was relocated into the cytoplasm of elongated spermatids (Fig. 7R). When tesmin was on the nuclear surface, it stained faintly (Fig. 7R, short arrow). When tesmin was detached from the nuclear membrane, tesmin stained brightly (Fig. 7, R, long arrow, and L).
Premature Translocation of Tesmin from Cytoplasm to Nucleus by Oxidative Stress
Whereas tesmin appeared to play a role in spermiogenesis (Fig. 7, M–R), it was expressed in the cytoplasm of leptotene-zygotene spermatocytes (Fig. 7J, short arrow). There must be some reason why tesmin is expressed so early in spermatogenesis. Because spermatocytes are vulnerable to exogenous stresses such as DNA damage and oxidative conditions, we examined the effects of cobalt chloride, which mimics hypoxia, and DEM, an oxidative agent, on the localization of tesmin. Tesmin was localized in the cytoplasm during the early stages of spermatocytes. When cobalt chloride was administered to induce oxidative stress, the apoptotic cells were observed sporadically on TUNEL staining (Fig. 8, A–I). At the same time, multinucleated giant cells, a morphological feature of apoptosis [8, 9], appeared (Fig. 8, J–L). Cells with a few nuclei could be sporadically observed in the control specimens, but cells with more than 10 nuclei were limited to chemically treated cells. Tesmin was present in both the cytoplasm and nucleus in giant cells (Fig. 8, K and L). In pachytene spermatocytes, tesmin is normally localized in cytoplasm, and in the case of round and elongated spermatids, it is on the nuclear surface. However, after cobalt chloride treatment, it was found to be evenly distributed in both the cytoplasm and nucleus (Fig. 8, J–L). When DEM was administered to mice, tesmin translocated from the cytoplasm to the nucleus within 4 h (Fig. 8, M–P). The localization changes of tesmin is illustrated in Figure 9.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Multiple Transcription Initiation Sites Downstream of a GC-Rich Promoter
The tesmin promoter lacks CCAAT and TATA boxes but contains several SP1 sequences, with a consensus of CCGCCC. The region around the promoter as well as the first exon is GC-rich. Sequencing of this region was difficult, and the denaturation process was carried out at 98°C in the presence of dimethyl sulfoxide. The GC-rich promoters frequently occur in testis-specific genes [10, 11]. Multiple transcription initiations are seen in GC-rich, TATA-less genes [12–14]. Thus, the multiple transcripts may be ascribed, in part, to the high GC content and cause the difficulty in obtaining full-length cDNA; reported cDNAs (accession nos. U67176, U77383, AK015724, and AK015732), all of which encode 295 amino acid residues, were transcribed from the ATG far downstream of our CT9 cDNA encoding 475 amino acid residues (Figs. 2 and 3).
Major mRNA with an Atypical Polyadenylation Signal
In trials to isolate full-length cDNA, Northern blot analyses were performed. The results indicated that the major transcript was the 1.8-kb mRNA and that the minor transcript was the 2.2-kb mRNA. Our tesmin cDNA clone had an atypical ATAAAA polyadenylation signal located 406-bp upstream of a typical AATAAA signal (Fig. 3). In testis, non-AATAAA polyadenylation signals are frequently used [15]. A longer 3'-UTR may be effective in prolonging the life of mRNAs; however, when tesmin is translated soon after transcription during the early stages of spermatocytes (Fig. 7, H–J), a long 3'-UTR may not be needed.
Tesmin Fits Better in the CHC Family
Tesmin was originally reported to be a testis-specific, MT-like protein and an early marker of male germ cell differentiation [1]. Its status was reexamined within the context of the MT family of proteins. Mammals contain four MTs, the ubiquitous MT-1 and MT-2, the brain- and reproductive organ-predominant MT-3, and the squamous epithelium-specific MT-4. All are located on a single chromosome (i.e., no. 8 in mice). The 5'-UTR contains regulatory elements, including one or more copies of the MRE, the consensus sequence of which is 5'-TGCAC-3' [4]. This sequence acts as a binding target for the transcription-activating protein factor MTF-1, which regulates MT gene expression [16]. The ubiquitous MT-1 and MT-2 are expressed in spermatocytes and spermatids [17]. Because tesmin is also metal-responsive [18], metal-sensitivity may be shared by MTs and tesmin.
Some differences between MTs and tesmin are their molecular sizes (6–7 and 60 kDa, respectively), chromosomal location (no. 8 vs. no. 19 in mice), number of exons (three vs. nine), composition of amino acids (without vs. with aromatic amino acids), cysteine contents (20% vs. 5.5%), different spacing of cysteine residues (see Fig. 6), localization (mainly in the cytoplasm vs. in the cytoplasm, nucleus, and nuclear membrane), and promoter regions (with metal-responsive elements and TATA box vs. without these elements). In light of these and other known characteristics of MTs, the tesmin protein characterized in the present study deviates far from the MT family. As shown in Figure 6, tesmin is highly homologous to the CHC family genes that occur in worms, insects, plants, and animals, suggesting the indispensable roles it has played during the evolution of eukaryotes from before the separation of plants and animals 160 million years ago. It is postulated that TSO1 is involved in cytokinesis at flower development [4, 5] and that CPP1 is involved in the expression of the soybean leghemoglobin c3 gene [6]. Because tesmin is the first mammalian CHC family protein, a new paradigm for tesmin-60 as a novel gene product could be established.
Dynamic Changes of Tesmin Localization
One of the most prominent characteristics of tesmin is its persistent occurrence from early to late stages during spermatogenesis (Fig. 9). Because the long course of spermatogenesis proceeds under the nursery of Sertoli cells, the functions of tesmin could be achieved through a direct or indirect interaction with Sertoli cells. First, this explains the long life of tesmin. Second, multinucleated giant cells may have been formed by disruption of Sertoli-germ cell junctions under oxidative stresses (Fig. 8, J–L). Third, translocation of tesmin from cytoplasm to nucleus just before meiotic division (Fig. 7, H–J) could be explained by a temporal dissolution of Sertoli-germ cell junctions. Fourth, the movement of tesmin from head to tail in the nucleus of elongated spermatids (Fig. 7R) is reminiscent of the maturation process of spermatozoa and of independence from the nursery of Sertoli cells. Indeed, when yeast two-hybrid experiments were conducted using tesmin as a bait, testin was detected. Testin is a testosterone-responsive, Sertoli cell-secretory protein and a key factor of Sertoli-germ cell junctions [19, 20]. Both tesmin and testin might be associated with Sertoli-germ cell junctions.
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FOOTNOTES |
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2 Current address: Department of Genetics, Osaka University Medical School, Osaka 565-0871, Japan
Received: 3 April 2002.
First decision: 30 April 2002.
Accepted: 11 December 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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diplotene spermatocyte. K) Tesmin at the cut-edge of an elongated spermatid (Cy3-staining, cryosection, see arrow in H). L) Part of the seminiferous tubule at stage XII (merge of Cy3-, DAPI-, and PNA-staining; cryosection). Short arrow: meiotic dividing cell. Long arrow: spermatocyte with condensing chromosomes. M) Round spermatids at steps 2–3 (PNA-staining, imprinting). Arrow: acrosome vesicle. N) Cy3-staining of M. Arrow: acrosome vesicle. O) Round spermatids at steps 2–3 (merge of Cy3-, DAPI-, and PNA-staining; imprinting). P) Round spermatids at step 8 (merge of Cy3-, DAPI-, and PNA-staining; imprinting). Q) Round spermatids at step 9 (merge of Cy3-, DAPI-, and PNA-staining; imprinting). R) Round spermatids at step 1 and elongated spermatids at step 13 (merge of Cy3-, DAPI-, and PNA-staining; imprinting). Short arrow: tesmin on the nuclear surface. Long arrow: tesmin detached from the nuclear surface. Bar = 10 µm (A, H, M–O, and Q) and 50 µm (D)
