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a Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
b Department of Urology, Osaka University Medical School, Osaka, Japan
c Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
ABSTRACT
We have isolated a cDNA clone encoding a mouse haploid germ cell-specific protein from a subtracted cDNA library. Sequence analysis of the cDNA revealed high homology with pig and human heart succinyl CoA:3-oxo acid CoA transferase (EC 2.8.3.5), which is a key enzyme for energy metabolism of ketone bodies. The deduced protein consists of 520 amino acid residues, including glutamate 344, known to be the catalytic residue in the active site of pig heart CoA transferase and the expected mitochondrial targeting sequence enriched with Arg, Leu, and Ser in the N-terminal region. Thus, we termed this gene scot-t (testis-specific succinyl CoA:3-oxo acid CoA transferase). Northern blot analysis, in situ hybridization, and Western blot analysis demonstrated a unique expression pattern of the mRNA with rapid translation exclusively in late spermatids. The scot-t protein was detected first in elongated spermatids at step 8 or 9 as faint signals and gradually accumulated during spermiogenesis. It was also detected in the midpiece of spermatozoa by immunohistochemistry. The results suggest that the scot-t protein plays important roles in the energy metabolism of spermatozoa.
sperm, sperm maturation, sperm motility and transport, spermatid, testis
INTRODUCTION
Spermatogenesis is a complex differentiation process that can be divided into three main phases: spermatogonial proliferation, meiosis, and spermiogenesis. During spermiogenesis, which lasts about 2 wk in the mouse, haploid round spermatids transform into morphologically and functionally differentiated spermatozoa. This striking change involves chromatin condensation, directed by the sequential replacement of the somatic and testicular histones with several highly basic proteins, the transition proteins or the protamines [1–7]. Concomitant with the unique chromatin alterations in round spermatids, an acrosome develops at the Golgi attached to the nucleus, and an axoneme and tail are assembled [8]. Following chromatin condensation, the nucleosomal structure disappears and transcriptional activity ceases. It was believed that little RNA was transcribed after meiosis because very little [3H]uridine was found to be incorporated in the postmeiotic spermatid by autoradiography [9]. However, further study revealed that a considerable number of genes are expressed in spermatids [5–7, 10–15]. Most of them are transcribed after postmeiotic spermatids. These haploid gene expressions could be supported by the TBP (TATA-binding protein) accumulated in early haploid germ cells at much higher levels than in other somatic cell types. In addition to TBP, TF IIB, and RNA polymerase II are also known to be overexpressed in the testis [16]. Recently, CREM (cyclic AMP-responsive element modulator) was shown to play an important role in the transcriptional regulation of spermiogenesis, because CREM-mutant mice were demonstrated to exhibit a deficiency in spermiogenesis [17, 18]. Thus, spermiogenesis includes the very interesting phenomena of morphological change, specific gene expression, and transcriptional regulation.
To understand the molecular mechanism of haploid germ cell differentiation, a subtracted mouse testicular cDNA library was prepared to concentrate cDNAs specifically expressed in haploid germ cells [12]. From this library, we have so far isolated 84 haploid germ cell-specific genes [14]. We report here a novel cDNA that is a mouse-specific homologue of scot (EC 2.8.3.5) expressed specifically in testicular haploid germ cells that we named scot-t. In this paper, the isolation and characterization of scot-t cDNA are described.
MATERIALS AND METHODS
Preparation of a Mouse Testis cDNA Library
Total RNA was extracted by the guanidine thiocyanate/Cs trifluoroacetic acid method followed by purification of poly(A)+ RNA from the testes of adult wild-type C57BL/6 mice. The cDNA library was prepared as described by Gubler and Hoffman [19] with some modifications [10]. Briefly, cDNAs were synthesized in a reaction mixture containing 5Me-dCTP with reverse transcriptase (Superscript II) from 2–5 µg of the mouse testis poly(A)+ RNAs and 1.6 µg of an oligo(dT) primer carrying a NotI site. The reaction mixture was treated with RNase H, followed by DNA polymerase I, blunt-ended with T4 DNA polymerase, and ligated to an unphosphorylated BglII-SmaI adaptor. After digestion with NotI, DNA fragments of less than 300 base pairs (bp) were removed by a CROMA spin-400 column (Clontech, Palo Alto, CA). The remaining cDNA fragments longer than 300 bp were directionally inserted between NotI (dephosphorylated) and BglII sites of pAP3neo vector (Takara, Shiga, Japan) [10]. The ligation mixture was electroporated into the MC1061 cells as described elsewhere [20]. The complexity of the cDNA library used here was 6.0 x 106 colony-forming units.
Preparation of a Haploid Germ Cell-Specific Subtracted cDNA Library
A haploid germ cell-specific cDNA library in pAP3neo vector was generated by subtracting mRNAs of 17-day-old testes from a cDNA library of 35-day-old testes as described previously [12, 14]. All clones from this subtracted library were further examined to confirm their haploid germ cell-specific expression by Northern blot analysis using total RNA from testes of 17- and 35-day-old mice.
Isolation and Sequencing of the MT-71 cDNA Clone
To obtain a full-length cDNA of the isolated clone, the pAP3neo cDNA library of mouse testis was screened under conditions of high stringency hybridization. A 32P-labeled probe was prepared with a BcaBest random primer kit (Takara) using a 700-bp EcoRI-NotI fragment of the partial MT-71 cDNA fragment (Fig. 1). Six independently isolated clones were identified, subcloned into pBluescript SK II(-) (Stratagene, La Jolla, CA), and used for sequence analysis. Nucleotide sequences were determined on both strands by the dideoxy chain termination method using fluorescent dye-labeled primers and a thermal cycle sequencing kit (Amersham Pharmacia Biotech, Tokyo, Japan). The reaction products were analyzed by model 4000 (Li-COR, Lincoln, NE). The nonredundant database at NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/BLAST/) was searched for sequences with homology to the nucleotide and deduced amino acid sequence of MT-71.
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Preparation of Fractionated Testicular Cells
Testicular germ cells and somatic cells were fractionated as described previously [21]. Briefly, four testes were collected from two adult wild-type C57BL/6 mice. The tunica albuginea was removed from each testis. Seminiferous tubules were placed in PBS containing 1 mM EDTA and dispersed by gentle pipetting a few times to remove interstitial cells. Then, tubules were transferred to a plastic petri dish, cut into small fragments with a knife, transferred to a 50-ml conical tube, and washed by pipetting in PBS (pH 7.0) containing 1 mM EDTA. The conical tube was left to stand for 5 min. Subsequently, the supernatant was filtered through a nylon mesh (NBC Industries Co., Ltd., Tokyo, Japan), centrifuged at 600 x g for 10 min, and used as a germ cell fraction. Leydig cell and Sertoli cell fractions were obtained from jsd/jsd mice that had no differentiated germ cells [22]. Seminiferous tubules from jsd/jsd mice were placed in 10 mM PBS containing 1 mM EDTA, gently unraveled with forceps, and transferred to a 50-ml conical tube. The tube was left to stand for 5 min to precipitate tubule fragments. The supernatant containing separated cells was filtered through a Nylon mesh and centrifuged at 600 x g for 10 min. The precipitant was used as a Leydig cell fraction. Remaining tubules were cut into small fragments with a knife in the petri dish, transferred to a 50-ml conical tube, and vigorously pipetted to remove germ cells. Then, the sample was left standing for 5 min. The supernatant fraction that mainly contained the remaining germ cells was discarded. The sedimented sample was used as a Sertoli cell fraction.
The germ cells were incubated with lysis buffer (10 mM Tris-HCl, pH.7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40) on ice for 5 min. After centrifugation (9000 x g), supernatant and precipitate were used as cytoplasmic and nuclear fractions, respectively [23].
RNA Extraction and Northern Blot Analysis
Various organs and fractionated testicular cells were homogenized in RNA zol B (Tel-Test, Inc., Friendswood, TX). Total RNA was extracted according to the manufacturer's recommendation, quantified by optical density measurement, separated by electrophoresis on a 1% formaldehyde gel, and transferred to a nylon transfer membrane (Amersham). After baking for 2 h at 80°C, for prehybridization, the nylon membrane was incubated for 6 h at 42°C in a solution containing 50% formamide, 4x saline-sodium citrate (SSC, 0.15 M NaCl and 0.015 M sodium citrate), 5x Denhardt solution, 0.2% SDS, and 100 µg/ml denatured sonicated salmon sperm DNA and was then hybridized with 32P-labeled scot-t cDNA under the same conditions for 10 h. The membrane was washed twice in a solution of 0.2x SSC, and 0.1% SDS at 55°C for 30 min. The filters were exposed to an imaging plate (Fuji Photo Film Co., Ltd., Tokyo, Japan) for analysis by Bio Imaging analyzer BAS-1000 (Fuji Photo Film) and then exposed to x-ray films.
In Situ Hybridization
Antisense digoxygenin (DIG)-labeled RNA was used for in situ hybridization. Testes of adult C57BL/6 mice were frozen in OTC embedding compound (Tissue-Tek, Sakura Finetechnical Co., Ltd., Tokyo, Japan), and cryosections (10 µm) were collected on a Superfrost microslide glass with APS coat (Matsunami Glass Ind., Ltd., Osaka, Japan). The sections were dried and fixed in a solution of 4% paraformaldehyde (PFA), 0.5% glutaraldehyde, and 0.5 M sodium phosphate buffer (pH 7.4). A scot-t probe was generated from a 420-bp NcoI-NotI fragment containing 3'-UTR cloned into pBluescript II SK (-). An antisense probe was generated by transcription of an EcoRI digest with T3 RNA polymerase and a sense probe by transcription of a NotI digest with T7 RNA polymerase. Probes were labeled with DIG-labeled UTP (Boehringer Mannheim, Indianapolis, IN). In situ hybridization was performed as described previously [21]. After hybridization, the bound probe was detected by incubating with anti-DIG-Fab fragments conjugated with alkaline phosphatase (Boehringer Mannheim), followed by a color reaction involving NBT (4-nitroblue tetrazolium chloride; Boehringer Mannheim) and X-phosphate (5-bromo-4-chloro-3-indolyl-phosphate; Boehringer Mannheim). Sections were contrasted with 1% methyl green stain solution (Muto Pure Chemicals, Ltd., Tokyo, Japan), and examined under a microscope.
Preparation of Antiserum to Scot-t Protein
To obtain the specificity for immunohistochemistry, we prepared anti-scot-t antiserum against amino acids 439–520 that contained 66.7% identity with the deduced amino acid sequence from the EST of mouse kidney (accession no. AA117593). NcoI-NotI (1344–1763 nucleotides) cDNA fragment (Fig. 1A), corresponding to amino acids 439–520 (Fig. 1, A and B), was subcloned into a pET 30a expression vector (Novagen, Madison, WI) and transformed into Escherichia coli (BL-21 strain). The histidine-tagged fusion protein was induced with isopropyl-ß-D-thiogalactopyranoside (IPTG) under native conditions, then purified by affinity chromatography on Ni2+ cations that were immobilized on His Bind resin (Novagen), and recovered by elution with imidazole according to the manufacturer's instructions. For the preparation of polyclonal antiserum, 1 ml of PBS containing approximately 100 µg of this protein was mixed with Gerbu adjuvant 100 [24] and used as an antigen. New Zealand white rabbits were immunized with this antigen and boosted every 2 wk thereafter. Antiserum was collected 2 wk after the fifth immunization.
Antigen Extraction and Immunoblotting
Various organs from adult C57BL/6 mice were mechanically dissected, washed twice in cold PBS, and suspended in 3 volumes of RIPA buffer (10 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1% NP-40, 0.1% SDS, 1 mM EDTA, 1 mM PMSF). Sperm were collected from the distal portion of the vas deferens and washed several times in cold PBS. After homogenization, samples were centrifuged at 10 000 x g for 15 min at 4°C. Supernatant was resuspended in Laemmli sample buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 1 mM dithiothreitol, 1% SDS, 0.002% bromphenol blue) and separated by 10% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA) and blocked with 5% nonfat dry milk in TBST (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 50 mM KCl, 0.05% Tween 20). The filters were incubated overnight at 4°C in TBST with anti-scot-t polyclonal antibody, washed with TBST three times for 10 min each, and probed with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham). Immunoreactive bands were visualized by development with the POD staining kit (Wako Pure Chemicals, Ltd., Osaka, Japan).
Immunohistochemistry
Frozen sections (10 µm) of adult C57BL/6 mouse testis were dried, fixed in a solution of 4% PFA, treated with 0.3% H2O2 solution, and blocked with 5% nonfat dry milk for 1 h at room temperature. Then, sections were incubated with scot-t antiserum (1/100), followed by horseradish peroxidase-conjugated anti-rabbit IgG (Amersham). Immunocomplexes were detected using diaminobenzidine in 50 mM Tris-HCl (pH 7.5) plus 0.3% H2O2. Slides were counterstained with hematoxylin and examined under a microscope.
Sperm suspensions obtained from the cauda epididymis were placed onto a Superfrost microslide glass with silane coating (Matsunami) and fixed with 80% methanol on ice for 5 min. For indirect immunofluorescent staining, the slides were incubated with scot-t antiserum diluted 1:500 in PBS for 12 h at 4°C. Then the slides were treated with fluorescein isothiocyanate-labeled anti-rabbit Igs antibody (Amersham) for 2 h at room temperature, washed with PBS, and observed under a fluorescent microscope (Olympus BX50, Tokyo, Japan).
RESULTS
Isolation and Sequencing of Mouse Testicular Haploid Germ Cell-Specific Succinyl CoA:3-Oxo Acid CoA Transferase (Scot-t) cDNA
From the subtracted cDNA library concentrated with various haploid germ cell-specific clones, one clone operationally named MT-71 was isolated. Northern blotting and sequencing analysis revealed that MT-71 is expressed in a haploid germ cell-specific manner and is a partial clone in length. To obtain the full-length cDNA, 3 x 105 colonies of a pAP3neo mouse testicular cDNA library were rescreened by the radiolabeled cDNA insert of MT-71 under high stringency conditions. More than 60 positive clones were independently isolated. Among them, six clones having the longest insert of about 1.7 kb were sequenced. The complete nucleotide sequences that were almost the same in all six clones and the same deduced amino acid sequences are shown in Figure 1A. The longest cDNA contained 1763 nucleotides and a single open reading frame coding 520 deduced amino acids with a predicted molecular weight of 56 531 and pI of 9.24 calculated by Genetics version 8.0. The accession number of the cDNA sequence in the DDBJ/EMBL/GenBank is AB022180. The nucleotide and deduced amino acid sequences were used as a query to search the nonredundant database at NCBI using the BLAST network service. The nucleotide sequence analysis revealed a high homology with scot in pig (63.4%) and human (62.7%) [25, 26], and the deduced amino acid sequence showed high identities of 68.0% and 67.4%, respectively (Fig. 1, B and C). We named this gene scot-t. The initiation ATG codon was thought to be located at nucleotides 32–34 from the comparison with pig and human heart scot (Fig. 1A), although we could not find any nonsense codons in the 5' upstream region. The sequence ATTAAA located 18 nucleotides upstream of the poly(A) tract, deviated 1 nucleotide from the most common consensus polyadenylation signal AATAAA (Fig. 1A). This A-to-T substitution of the consensus sequence is well tolerated and the most frequent variant of the polyadenylation signal [27, 28]. The hydrophilic bridge region, 271–284 amino acids of the predicted helix structure, that was presumably nicked by proteolytic digestion during purification, was conserved as pig heart scot [25] (Fig. 1B).
Expression of Succinyl CoA:3-Oxo Acid CoA Transferase mRNA of Testis Type (Scot-t) in Various Organs and in Developmental Stages of the Testes
To investigate the expression of scot-t mRNA, total RNA was prepared from various mouse organs and analyzed by Northern blotting with the full length of scot-t cDNA as a probe. The scot-t mRNA was exclusively detected in the testis as a major transcript of 1.7 kb and a minor hybridizing band of 4.3 kb (Fig. 2A). During male germ cell development, scot-t transcript was not detected until 23 days of age. Significant signal was then detected by 29 days of age and increased at 35 days of age (Fig. 2B). Furthermore, the expression of scot-t mRNA was ascribed to the germ cells in the testis defined by cell separation (Fig. 2C). These results indicated that scot-t was exclusively expressed in haploid germ cells.
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In Situ Hybridization of Scot-t Transcript
To confirm and get more detailed information on the results of Northern blotting, in situ hybridization of scot-t mRNA was performed with antisense and sense cRNA probes generated from a 420-bp NcoI-NotI (poly A-NotI linker [10]) fragment containing a 3'-untranslated region (UTR) (Fig. 1A). Specific staining with the antisense probe was observed first in the step 8–9 spermatids occupying the middle layer of stage VIII–IX seminiferous tubules. The signal could be detected at the same level until step 12 elongated spermatids, then gradually decreased at step 13 to 14 in stage I–III seminiferous tubules and disappeared. No detectable positive signal was observed in stage IV–VI tubules (Fig. 3). These results were consistent with the data of Northern blotting of the testes at different ages, which indicated that scot-t was expressed in haploid spermatids at the middle to late steps of spermiogenesis.
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Western Blot Analysis of Scot-t Protein
Western blot analysis with anti-scot-t antiserum yielded a specific band of Mr 50 000 in the extracts of the testis and sperm (Fig. 4A). Some signals in the extracts of other organs such as heart, liver, and kidney were thought to be cross-reactive materials, because no scot-t transcript was detected (Fig. 2A). Expression of scot-t in the mouse testis was detected at 29 days and became stronger at 35 days of age (Fig. 4B). To determine the localization of this protein, the testicular cells were divided into two fractions, the cytoplasmic portion and the nucleus. Specific staining was detected in the cytoplasmic portion of germ cells but not in the nucleus (Fig. 4C).
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Immunohistochemical Examination with Anti-Scot-t Antiserum
Immunohistochemical staining of the frozen section of an adult mouse testis with the anti-scot-t antiserum revealed that the positive signal was present in germ cells but not in Sertoli or Leydig cells. The scot-t protein was detected first in elongated spermatids at step 8 or 9 as faint signals, while round spermatids at step 1 to 7 had no staining. The intensity of the staining increased gradually during spermiogenesis. Flagella of condensing spermatids at the center of the lumen of the seminiferous tubules were stained and residual bodies remaining in the tubules of stage VII to IX were also positively stained (Fig. 5). These observations were in good agreement with the results of Western blot analysis, indicating that the scot-t protein was synthesized during spermiogenesis exclusively at the late stages of spermiogenesis, concomitant with the transcription of the mRNA. Furthermore, the localization of this protein in fixed sperm from cauda epididymis was examined by indirect immunofluorescent staining. Positive staining was restricted to the midpiece of sperm flagellum containing a large amount of mitochondria (Fig. 6).
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DISCUSSION
We have so far been able to isolate 84 haploid germ cell-specific cDNA clones from a subtracted cDNA library of mouse testis to study the molecular mechanism of haploid germ cell differentiation [12, 14]. In the present study, we isolated and characterized a novel gene that we named scot-t. It has high homology in amino acid sequence with heart scot in pig (68.0%) and human (67.4%) [25, 26]. Northern blot analysis showed that the scot-t transcript was exclusively expressed in the testis as a major transcript of 1.7 kb. Developmental analysis showed scot-t transcripts were not detected until 23 days of age, clearly detected by 29 days, and increased at 35 days of age. In situ hybridization demonstrated the specific staining in spermatids from step 8 to 9 that increased until step 12, then gradually decreased at step 13 to 14, and disappeared. Thus, the expression of this gene was restricted to the testis, especially in late spermatids. Recently, Penttila et al. [15] demonstrated that all of their 13 novel spermatid-specific cDNA tags were expressed in round spermatids, and the expression pattern resembled those previously described for the nuclear proteins protamine (Prm) 1, Prm 2, and transition protein 1 (TP1) [29, 30]. They also suggested that haploid gene expression was regulated in a similar manner, i.e., the transcription occurs in early round spermatids and then becomes inactive in elongated spermatids. The storage of transcripts and translational control would be common phenomena in the late stage of spermiogenesis in transcriptionally inactive elongating spermatids [15]. However, in the present study, the expression of scot-t was not observed before 29 days of age on Northern blot analysis (Fig. 2B) or earlier than step 7 in in situ hybridization of mRNA (Fig. 3). De novo synthesis of scot-t mRNA in the late stages of spermiogenesis concomitant with protein expression would be unique to condensed spermatids.
Recently, male germ cells were reported to have numerous isozymic variants commonly expressed in the somatic tissues. Among them, several isozymes involved in energy metabolism were also reported to be specifically expressed in male germ cells, i.e., phosphoglycerate kinase-2 [31], lactate dehydrogenase-C [32, 33], spermatogenic cell-specific glyceraldehyde 3-phosphate dehydrogenase (GAPD-S) [34, 35], cytochrome C [36], type 1 hexokinase isozyme [37, 38], and pyruvate dehydrogenase-2 [39]. These genes are speculated to have some metabolic advantage in male germ cells [40]. Although the somatic cell-type isozyme of scot-t has not yet been isolated in mouse, and conversely, no testicular type of scot has been reported in pig and human, an EST having high homology with scot-t has been isolated from a mouse kidney cDNA library (accession no. AA117593). These results indicate that some isozymic variants of scot should also exist in these animals.
In the mammalian mitochondrion, the enzyme scot is essential for the energy metabolism of ketone bodies such as acetoacetate and 3-hydroxybutyrate in the extrahepatic tissues, because this enzyme catalyzes the formation of acetoacetyl CoA by transfer of a CoA moiety from succinyl CoA to 3-oxo acid, which is further broken down to two acetyl CoA molecules capable of entering the tricarboxylic acid cycle. Its catalytic mechanism is well known to involve the transient thioesterification of an active-site glutamate residue by CoA [41]. Because the deduced amino acid sequence of scot-t also has a glutamate residue (342) corresponding to glutamate 344 known to be conserved in all sequenced CoA transferases [42] (Fig. 1B), the gene product of scot-t should have a similar important function in energy metabolism.
In pig heart scot, the N-terminal 1–39 residues have an abundance of Arg, Leu, and Ser, an absence of acidic residues, and the potential to form an amphiphilic 
-helix suggested to be a mitochondrial targeting sequence [25, 43]. The N-terminal sequence of scot-t was also enriched with Arg, Leu, and Ser and expected to be a mitochondrial targeting sequence. The finding was supported by immunocytochemistry that showed that scot-t protein was localized in the midpiece of sperm flagellum containing a large amount of mitochondria (Fig. 6). Western blot analysis revealed one positive band of approximately Mr 50 000 in the testis and sperm, although the deduced amino acid sequence gave a predicted molecular weight of 56 531. The discrepancy in molecular weight may come from the N-terminal mitochondrial targeting sequence processed in mitochondria.
It has been reported that this enzyme is most active in the heart and kidney in various mammals [44, 45] and that the synthesis of ketone bodies is accelerated to meet the energy requirements during starvation and diabetes. Recently, a patient with hereditary scot deficiency was reported to have sustained hyperketonemia and episodes of severe ketoacidosis due to a nonsense mutation in the scot gene [26]. Furthermore, a bacterial scot gene of Helicobacter pylori was isolated recently as a dimeric protein in contrast to the monomeric mammalian proteins [46]. Thus, scot plays a crucial role in ketone body metabolism and is evolutionarily conserved.
It is generally believed that sperm motility needs energy derived from glycolysis, the tricarboxylic acid cycle, and mitochondrial respiration, although various mechanisms have been demonstrated to be species-specific [47]. In mammals, spermatozoa have a high glycolytic capacity [48], sugar seems to be used for energy metabolism but not glycogen storage, and consequently motility depends to some extent on exogenous fuels. However, if the uterovaginal tract is starved of energy substrates, mammalian spermatozoa may use endogenous unsaturated fatty acids or ketone bodies, although there are no reports about ketone body metabolism in spermatozoa. Further study is required to elucidate the physiological function of scot-t products in spermatozoa.
FOOTNOTES
1 Correspondence: Y. Nishimune, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. FAX: 81 6 6879 8339; nishimun{at}biken.osaka-u.ac.jp 
Accepted: July 11, 2000.
Received: April 11, 2000.
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