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Regular Article |
a Laboratory of Zoology, Graduate School of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
b Department of Developmental Molecular Anatomy, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan
ABSTRACT
Postnatal development of mammalian seminiferous tubules can be divided into three phases: spermatogonial mitosis, spermatocyte meiosis, and a postmeiotic phase in which drastic morphological changes occur in spermatids (spermiogenesis). In an attempt to elucidate the molecular mechanisms involved in spermiogenesis, we have applied a differential display method to identify genes that are developmentally up-regulated during rat testis development. One of the cDNA fragments isolated by differential display turned out to be iba1, an ionized calcium binding adapter molecule-1, that contains two EF hand-like motifs. Expression of iba1 mRNA in the rat testis was detected first at 4 wk in postnatal development and then increased up to adulthood. Using the antibody against a synthetic peptide corresponding to the N-terminal Iba1 protein, we discovered that Iba1 protein was not detectable by immunohistochemistry in spermatogonia, spermatocytes, and round spermatids in adult rat testis but was specifically expressed in the cytoplasm of elongate spermatids (steps 10–19) as well as in residual bodies that are ultimately engulfed by Sertoli cells. In situ hybridization, on the other hand, revealed that iba1 mRNA is present in round spermatids as well as early elongate spermatids (steps 1–12) but not in late spermatids, suggesting that iba1 mRNA undergoes post-transcriptional regulation. Because Iba1 protein is specifically expressed in the cytoplasm of elongate spermatids, which is finally engulfed as residual bodies into Sertoli cells, we suggest that Iba1 may be involved in the final stage of spermiogenesis (i.e., in elimination of the residual cytoplasm from spermatids).
sperm, spermatid, spermatogenesis, testes
INTRODUCTION
Spermatogenesis is an excellent model system for studying regulation of gene expression during cell differentiation. Postnatal development of mammalian seminiferous tubules is a complicated process that can be subdivided into three phases: 1) a premeiotic phase characterized by an increase in cell number due to mitosis of diploid spermatogonia, 2) a meiotic phase that gives rise to haploid round spermatids, and 3) a post-meiotic phase in which round spermatids undergo the drastic morphological changes that lead to the production of elongate spermatozoa (spermiogenesis). The morphological changes in the postmeiotic phase include the formation of an acrosome and a flagellum, condensation of the nucleus, and elimination of extra spermatid cytoplasm as well as cell organelles, which are indispensable for spermatozoa formation [1, 2]. The precise regulation of such germ cell differentiation requires a strict program of stage- and cell-specific gene expression in germ cells as well as in Sertoli cells. To understand the molecular mechanisms of this differentiation, it is necessary to identify genes that are specifically expressed at each stages.
Differential display of mRNA is a method for studying differential gene expression in cultured cells [3]. In contrast to a subtraction method, the gel display allows many samples to be compared side-by-side, and individual bands that visually indicate differentially regulated mRNA can be isolated and subcloned [4]. Differential display also has a primary advantage over the subtraction method in that it is based on polymerase chain reaction (PCR), and therefore, relatively little starting material is required to compare gene expression from different sources. Using differential display, we have cloned and sequenced more than 100 cDNA fragments, which include several novel genes as well as already identified genes whose expression is developmentally up-regulated in rat testis. Reverse transcription (RT)-PCR analysis revealed that one of the known genes isolated by differential display was iba1, ionized calcium binding adapter molecule-1. In this study, we have examined the expression pattern and localization of iba1 in rat testes by immunohistochemical and in situ hybridization techniques.
MATERIALS AND METHODS
Differential Display
Investigations were conducted in accordance with the National Research Council publication, Guide for Care and Use of Laboratory Animals.
The mRNA differential display method [3] was carried out using an RNA map kit (GenHunter, Nashville, TN). Briefly, total testis RNAs were isolated from Wistar rats of age 1, 2, 3, 4, 5, 6, 7, and 8 wk, as described previously [5]. RNAs were reverse-transcribed with oligo(dT) primers anchored to the beginning of the poly(A) tail. The resulting cDNAs were amplified with oligo(dT) primers and arbitrary primers. The cycling parameters were as follows: 94°C for 30 sec, 40°C for 2 min, and 72°C for 30 sec for 40 cycles. The amplified cDNAs were then separated on 6% urea-polyacrylamide gels, fixed, and stained by the silver sequence system (Promega, Madison, WI). Complementary DNA fragments whose expression levels were developmentally increased were recovered directly by cutting out the gel slices. After elution by boiling the gel slices in distilled water for 15 min, cDNA fragments were reamplified. The cDNA fragments were then purified by electrophoresis, cloned into the pGEM easy T-vector (Promega), and sequenced using a DNA sequencer (Applied Biosystems, Foster City, CA).
RT-PCR and Preparation of GST-Fusion Proteins
Complementary DNA strands were synthesized from 2 µg of total RNA by using a first-strand synthesis kit (Amersham Pharmacia Biotech) with random primers. The reverse-transcribed cDNA was used as PCR templates to synthesize iba1, mrf1, bart1, rab3A, rab3d, and rab6 genes. The primers used to amplify these cDNAs were as follows (the same reverse primer was used for iba1, mrf1, and bart1): iba1: 5'-CCATGAAGCCTGAGGAAATTTCA-3' (forward) and 5'-TTATATCCACCTCCAATTAGGGCA-3' (reverse), mrf1: 5'-CTATGAGCCAGAGCAAGGATTTG-3' (forward), bart1: 5'-CCACAATGCAATTATACTCTCTG-3' (forward), rab3a: 5'-ATGGCCTCAGCCACAGACTCTCGATATGG-3' (forward) and 5'-TCAGCAGGCGCAATCCTGATGAGGTGGTGCCTGCTG-3' (reverse), rab3d: 5'-ATGGCATCCGCTAGTGAGCCCCCTGC-3' (forward) and 5'-CTAACAGCCGCAGCTGCTCGGCTG-3' (reverse), rab6: 5'-ATGTCCACGGGCGGAGACTT-3' (forward) and 5'-TTAGCAGGAACAGCCTCCTTCACT-3' (reverse). Primers for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were 5'-TGAAGGTCGGTGTCAACGGATTTGGC-3' (forward) and 5'-CATGTAGGCCATGAGGTCCACCAC-3' (reverse). The PCR-amplified DNAs were cloned into pGEM-T easy vector (Promega) and sequenced using a DNA sequencer (Applied Biosystems).
Full-length iba1, mrf1, rab3a, rab3d, and rab6 genes were cloned in-frame to the COOH terminus of glutathione S-transferase (GST) using the pGEX-4T-1 system (Amersham Pharmacia). Recombinant proteins were expressed in E. coli and purified onto glutathione-sepharose (Amersham Pharmacia), as previously described [6, 7].
Antibody
The peptide used for raising antibody is derived from the N-terminal region (MKPEEISRGKA) of Iba1 [8]. The peptide (1 mg) coupled to keyhole limpet hemocyanin (KLH; Pierce, Rockford, IL) was dissolved in 1 ml of saline, emulsified with 1 ml of Freund's complete adjuvant, and injected at multiple sites on the back of a rabbit as described previously [6]. The rabbit was bled from the common carotid artery 8 days after the final injection. The antiserum was separated by centrifugation and stored at -80°C. Affinity purification of the antibody was carried out over a matrix of the peptide coupled to 2-fluoro-1-methylpyridinium toluene-4-sulfonate-activated sephadex (Seikagaku Kogyo, Japan) according to the supplier's protocol, as described previously [9].
Immunoblot Analysis
Seminiferous tubules were taken from testes of ether-anesthetized adult Wistar rats and washed in PBS at 4°C for 20 min with gentle agitation. The tubules were solubilized directly in RIPA buffer (50 mM Tris pH 7.2, 1 mM EDTA, 0.1% SDS, 0.1% Na deoxycholate, 1% Nonidet P-40, protease inhibitors [aprotinin 1 mU/ml, leupeptin 0.1 mmol/L, phenylmethylsulfonyl fluoride 0.5 mmol/L]), and centrifuged at 15 000 x g for 20 min. Clarified supernatants were subjected to electrophoresis. An aliquot of the tubules was homogenized in PBS, and centrifuged twice at 1000 x g to remove the nucleus fraction. Supernatants were further centrifuged for 60 min at 100 000 x g to obtain crude membrane fractions. The sample was dissolved in SDS-PAGE buffer for electrophoresis. GST-fusion recombinant proteins were also dissolved in SDS-PAGE sample buffer before electrophoresis. Proteins prepared for SDS-PAGE were separated on 12% or 15% acrylamide gel, and separated proteins were either stained with Coomassie brilliant blue or transferred to nitrocellulose sheets. The sheets were incubated for 2 h with the anti-Iba1 antibody diluted 1:1000 with a blocking buffer (PBS containing 5% nonfat milk and 0.1% Tween-20), followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG; BioRad, Richmond, CA) diluted 1:3000 in the same buffer. Antigen-antibody complexes were visualized using an electrochemiluminesence detection kit (Amersham Pharmacia).
Immunohistochemistry
Adult rat testes were fixed in 4% paraformaldehyde in PBS at 4°C for 16 h, washed three times in PBS, incubated in PBS containing NH4Cl for 30 min, then rinsed in PBS. After infiltration of 20% (w/v) sucrose in PBS, the testes were embedded in OCT compound (Tissue-Tek, Miles Inc., Elkhart, IN) by liquid nitrogen. Frozen sections of 8-µm thickness were cut by a cryostat (Leica CM1850, Nussloch, Germany). The sections were washed in PBS, incubated with the anti-Iba1 antibody diluted 1:100 with a blocking buffer, and followed by incubation with Goat anti-rabbit IgG conjugated either with HRP (BioRad, Richmond, CA) or with Cy3 (Amersham Pharmacia Biotech). The samples were then washed with PBS and the peroxidase signal was visualized by incubation with PBS containing 0.01% hydrogen peroxide and 0.1% diaminobenzidine (diaminobenzidine reaction solution). For controls, the primary antibody was replaced by preimmune serum. Endogenous peroxidase activity was examined by exposure of the specimens to the diaminobenzidine reaction solution without immunostaining. Stained samples were examined either in a light microscope for immunoperoxidase labeling, or by confocal laser scanning microscopy (Olympus LSM-GB 200, Tokyo, Japan) for immunofluorescence labeling.
Sperm Isolation
Spermatozoa were isolated from epididymides of ether-anesthetized adult Wistar rats by a Percoll density gradient method described previously [7]. Purified spermatozoa were fixed in 3% paraformaldehyde in PBS and attached to poly-L-lysine-coated glass slides, and air-dried. The samples were processed for immunohistochemistry with the anti-Iba1 antibody as described earlier. An aliquot of purified spermatozoa was dissolved in RIPA buffer and processed for immunoblot analysis as described earlier.
In Situ Hybridization
In situ hybridization was carried out according to the method of Meehan et al. [10] with slight modification. Sense and antisense RNA probes were transcribed in vitro from linealized plasmids containing the entire protein-coding region of iba1 using digoxigenin-labeled UTP and T7 or SP6 RNA polymerase, as described in the manufacture's manual (DIG RNA Labeling Kit SP6/T7; Boehringer-Mannheim, Mannheim, Germany). Frozen sections of adult rat testis prepared as for immunohistochemistry were treated successively with 0.3% Triton X-100 in PBS for 5 min, proteinase K (1 µg/ml) in PBS for 5 min at 37°C, 4% paraformaldehyde in PBS for 5 min, 0.1 M glycine in PBS for 10 min, and 2x SSC (1x SSC = 0.15 M NaCl, 0.015 M sodium citrate pH 7.0) for 15 min. The sections were then preincubated for 30 min at 42°C in a hybridization buffer (20 mM Tris-HCl pH 8.0, 0.3 M NaCl, 2 M EDTA, 50% formamide, 1 mg/ml BSA, 0.02% Ficoll, 0.02% polyvinylpyrolidone, 1 mg/ml herring sperm DNA), and hybridized for 4–5 h at 42°C in the hybridization buffer containing a digoxigenin-labeled probe. Hybridization was performed in a MicroProbe staining apparatus (Fisher Biotech, Pittsburgh, PA). After hybridization, the sections were washed for 1 h in 2x SSC with 50% formamide at 42°C, incubated for 30 min at 37°C with RNAse A (20 µg/ml) in NTE buffer (0.5 M NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA), and washed in 0.1x SSC for 20 min at 42°C. The sections were incubated for 30 min with DIG blocking buffer reagent (Boehringer-Mannheim), and bound cRNA was detected using anti-Dig alkaline phosphatase-conjugated antibody (1:500 dilution, Boehringer-Mannheim), and visualized with NTB-BCIP (Boehringer-Mannheim).
RESULTS
Iba1 Is a Developmentally Up-Regulated Gene in Rat Testis
To identify developmentally up-regulated genes in the rat testis, transcripts derived from the testes of 2- to 7-wk-old rats were examined by differential display screening using 40 different combinations of primer pairs. During this screening, we identified a cDNA of approximately 430 base pairs (bp) in length, which became detectable at 4 wk and was still expressed at 7 wk (Fig. 1). Sequence analysis of the cDNA fragment followed by a database search revealed a high homology with three different genes: ionized calcium binding adapter molecule-1 (iba1) [8], microglia response factor-1 (mrf1) [11], and balloon angioplasty responsive transcript-1 (bart1) [12]. Mrf1 gene is identical to the iba1 gene except for a short N-terminal region corresponding to a 9 amino acid sequence. Mrf1 and iba1 are produced from the same gene by alternative splicing (personal communication with Dr. Y. Imai). Expression of these genes in 7-wk-old rat testis was investigated by RT-PCR. We found that approximately 430 bp PCR products of both iba1 and mrf1 were amplified, whereas amplification of bart1 was not observed (Fig. 2). PCR for mrf1 yielded an extra band migrating at 650 bp, which seems to be an artificial band because such a spurious band was undetectable after nested PCR (not shown). We next examined by RT-PCR the expression of the iba1 gene in various organs of adult rats. Iba1 was strongly expressed in testis, moderately in epididymis and lung, weekly in kidney and brain, and was undetectable in heart and liver (Fig. 3), data that agree well with that of previous Northern blot analyses [8]. When the developmental expression of iba1 and mrf1 in rat testes was examined by RT-PCR, iba1 was first detectable at 4 wk in postnatal development and its expression increased thereafter, whereas the expression level of mrf1 was constant throughout the developmental period (Fig. 4). Because of a similarity in the expression pattern of iba1 on differential display and RT-PCR, the cDNA fragment isolated by differential display appeared to be Iba1.
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Specificity of Anti-Iba1 Antibody
To examine the expression and localization of Iba1 protein in rat testis, a polyclonal antibody was raised against the synthetic peptide (MKPEEISRGKA) corresponding to the N-terminal of Iba1, which was chosen to distinguish Iba1 protein from a cognate protein, Mrf1. The anti-Iba1 antibody was used after affinity purification. Specificity of the anti-Iba1 antibody was examined on the blots to which several GST-fusion recombinant proteins were transferred. As shown in Figure 5, A and B, the anti-Iba1 antibody specifically recognized GST-Iba1 protein but did not react with GST-Mrf1 or other GST-fusion proteins, indicating that the antibody is specific for Iba1 protein. On the blots to which whole seminiferous tubule extract and crude membrane proteins of the seminiferous tubules were transferred, the anti-Iba1 antibody recognized a protein migrating at approximately 16.5 kDa (Fig. 5, C and D), close to 16 893 Da calculated from the Iba1 amino acid sequence deduced from the cDNA sequence [8].
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Localization of Iba1 Protein in Rat Testis
Using the anti-Iba1 antibody, we intended to determine the cell typesexpressing Iba1 protein immunohistochemically in frozen sections of adultrat testis. Both light microscopy with an immunoperoxidase probe andconfocal laser scanning microscopy with an immunofluorescence probe (notshown) produced an identical result. In a cross-section of the testisviewed at low power of the light microscope, Iba1 immunoperoxidase reactionproduct was detected at the luminal border of the seminiferous tubules,although the staining pattern varied between the tubules due to the stagesof the spermatogenetic cycle in the seminiferous epithelium (Fig. 6A). Replacement of the anti-Iba1antibody with preimmune serum gave no specific staining (Fig. 6B). Endogenous peroxidase activitywas only detected in red blood cells in the specimens that were notimmunostained (not shown). At higher magnification, Iba1 immunoproduct wasobserved as diffuse staining in the cytoplasm of step 10–12spermatids (Fig. 6, C and D). Instep 18 spermatids, Iba1 immunostaining was detected as diffuse staining inthe cytoplasm of the middle piece of the developing tail, which oftencontained more densely immunolabeled globular structures (Fig. 6, E and F). These globularstructures might represent RNA-containing chromatoid bodies [13]. In step 19 spermatids at stage VII(near spermiation), Iba1 immunoperoxidase reaction product appeared assmall spheres located in the vicinity of sperm heads, whereas elongatespermatids themselves were devoid of immunolabeling (Fig. 6, G and H). The immunostained smallspheres appeared to be residual bodies in the lobes of spermatid cytoplasmengulfed by Sertoli cells. By contrast, Iba1 immunostaining was virtuallyundetectable in round spermatids, spermatocytes, spermatogonia, and Sertolicells (Fig. 6, A–H). In order to examine whether mature epididymal spermatozoa express Iba1 protein, confocal laser scanning microscopy was performed on the spermatozoa immunostained for Iba1. As shown in Figure 7, there was no specific immunolabeling on mature spermatozoa. We also failed to detect Iba1 protein on the blot to which total proteins of mature spermatozoa had been transferred (not shown).
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In Situ Localization of iba1 Messenger RNA
The immunohistochemical procedure described above revealed that Iba1 protein is not detectable in round spermatids but is specifically expressed in elongate spermatids in which synthesis of mRNA has almost finished. This raised the possibility that iba1 mRNA is synthesized before the elongation phase of spermatids and is translated later into protein. To address this question, we performed in situ hybridization to determine the cell types expressing iba1 mRNA. Frozen sections of adult rat testis were hybridized either with an RNA probe having the antisense sequence of iba1 mRNA or with a sense probe as control. Hybridization with the antisense probe created strong signals in the inner half layer of the seminiferous epithelium of adult rat testis (Fig. 8A), whereas hybridization with the sense probe for iba1 gave no signal (Fig. 8B), indicating its specificity for the iba1 sequence. At higher magnification, iba1 mRNA was found to be present in round spermatids located in the inner half layer of the seminiferous epithelium (Fig. 8C) as well as in early spermatids (steps 10–12) whose cytoplasm protruded into the tubular lumen (Fig. 8D). Both less-mature germ cells (spermatogonia, primary spermatocytes) located in the outer half layer of the seminiferous epithelium and Sertoli cells were negative (Fig. 8, C and D). In contrast to Iba1 protein, iba1 mRNA was not detectable in more advanced spermatids (steps 15–19) nor in residual bodies (Fig. 8C). Thus, these data indicate that iba1 mRNA is synthesized and stored in round spermatids and early elongate spermatids.
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We also performed in situ hybridization to study the developmental expression of iba1 mRNA in the seminiferous epithelium of 2- to 7 wk-old rats. We found that iba1 mRNA was detected first at 4 wk in postnatal development and continued to be expressed up to 7 wk (not shown). The data were highly consistent with those of differential display (Fig. 1) and RT-PCR (Fig. 4).
DISCUSSION
The process of spermatid differentiation can be subdivided into three successive processes of equal duration: a round phase, an elongation phase, and a maturation phase [2]. During the maturation phase, future spermatozoa discard the cytoplasm and organelles as residual bodies, which are engulfed and digested by Sertoli cells [1, 14–18]. Elimination of excessive cytoplasm, which leads to cell volume reduction, is essential for making spermatozoa smaller and more slender to facilitate their smooth movement toward eggs. The reduction in spermatid volume may be also achieved by an efflux of water through a water channel Aquaporin 7 [19].
In this study, a cDNA fragment developmentally up-regulated in rat testis was isolated by differential display. The encoded protein turned out to be Iba1, ionized calcium binding adapter molecule-1. Iba1 is a small protein of 146 amino acids containing two EF hand-like motifs and no transmembrane region [8]. The protein is expressed in microglia, but not in neurons, astroglia, or oligodendroglia. Because of up-regulation of Iba1 protein in activated microglia in response to nerve injury, it has been speculated that Iba1 may play a role in recovery from brain injuries [20]. Expression of Mrf1, a cognate protein expressed in microglia, is also markedly enhanced upon apoptosis of neuron and necrotic neuronal death [11].
Using the antibody specific to Iba1 protein, we discovered that it was specifically expressed in the cytoplasm of elongate spermatids. Neither spermatogonia, spermatocytes, round spermatids, nor epididymal mature spermatozoa expressed the protein. We also clarified the diversiform distribution of Iba1 protein in elongated spermatids: the protein is diffusely distributed in the cytoplasm of step 10–12 spermatids, concentrated to some degree in globular structures in step 18 spermatids, and is finally discarded from step 19 spermatids as residual bodies.
Throughout the course of mammalian spermatogenesis, differentiating spermatogenic cells remain in close contact with somatic Sertoli cells until spermiation, at which mature spermatozoa are separated from their excess cytoplasm and released into the lumen of the seminiferous tubules. One important question to be answered is how Sertoli cells discriminate between the maturing spermatozoa and the residual spermatid cytoplasm, both of which are in contact with Sertoli cells [15, 16]. It is assumed that the extra cytoplasm of elongate spermatids has some "signal" that enables Sertoli cells to recognize and phagocytose only the extra cytoplasm. An example of this type of communication between Sertoli cells and spermatogenic cells is found in the disposal of apoptotic spermatogenic cells by Sertoli cells, in which phosphatidylserine exposed on the surface of spermatogenic apoptotic cells might serve as a signal for phagocytosis by Sertoli cells [21]. Because Iba1 protein is specifically expressed in the cytoplasm of elongate spermatids, and because the protein as well as the extra cytoplasm is finally engulfed as residual bodies into Sertoli cells, it is speculated that Iba1 may be involved in the signal for elimination of the extra spermatid cytoplasm.
In addition to the precise regulation of stringent stage-specific gene expression, post-transcriptional control is especially important toward the end of spermatogenesis because global transcription ceases several days before the completion of spermatogenesis (see review by Sassone-Corsi [22]). This means that mRNA storage and translational activation play prominent roles in the expression of spermatid and spermatozoon proteins that are synthesized in late stages of germ cell maturation. In early spermatids, mRNAs of spermatid-specific basic proteins, such as protamines and transition proteins, are translationally suppressed with long poly(A) tails for up to a week. A double-stranded RNA binding protein, Prbp, encoded by the Tarbp 2 gene, is required for proper translational activation of the suppressed mRNAs [23]. We provided the evidence showing the different localization of iba1 mRNA and Iba1 protein in rat seminiferous tubules. Iba1 mRNA appeared to be expressed in spermatids at steps 1–12, and Iba1 protein in step 10–19 spermatids, although it is difficult to determine the precise steps of spermatid differentiation in the samples used for immunohistochemistry and in situ hybridization. These results might suggest that iba1 mRNA is translationally suppressed until the early elongate spermatid stage. We hypothesize that iba1 mRNA undergoes post-transcriptional regulation in spermatids and that the protein is specifically expressed and functions in elongate spermatids.
In conclusion, we discovered that iba1 mRNA, which is developmentally up-regulated in rat testis, is expressed in round spermatids and early elongate spermatids. Iba1 mRNA is translated into protein as spermatids elongate, and the protein is specifically localized in the excess cytoplasm of elongate spermatids that is eventually engulfed as residual bodies into Sertoli cells. A recent study by Ohsawa et al. [24] demonstrated colocalization of Iba1 with F-actin and small GTPase Rac in membrane ruffles and phagocytic cups in macrophages. It is therefore likely that Iba1 might be linked to reorganization of actin cytoskeleton during spermiogenesis and residual body extrusion. Further studies of Iba1 may shed more light on the molecular mechanisms regulating the final step of spermiogenesis (i.e., spermiation).
ACKNOWLEDGMENTS
We are grateful to Dr. J. M. Bedford of the Cornell University Medical College and K. Toshimori of Miyazaki Medical College for critical review of the manuscript.
FOOTNOTES
First decision: 20 October 2000.
1 Supported by a Grant-in-Aid for Scientific Research by the Japan Society for the Promotion of Science. 
2 Correspondence: Hiroshi Iida, Laboratory of Zoology, Graduate School of Agriculture, Kyushu University, Higashiku Hakozaki 6-10-1, Fukuoka 812-8581, Japan. FAX: 92 642 2804; iidahiro{at}agr.kyushu-u.ac.jp
Accepted: November 15, 2000.
Received: August 31, 2000.
REFERENCES
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