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Male Reproductive Tract |
a Institute of Medical Biochemistry and Molecular Biology
b Institute of Anatomy, University Hospital Hamburg-Eppendorf, D-20246 Hamburg, Germany
c Institute for Hormone and Fertility Research, University of Hamburg, D-22529 Hamburg, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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and protein kinase A in human hepatocarcinoma HepG2 cells activated a promoter C-driven luciferase construct in transient transfection experiments. Furthermore, mGPDH expression was undetectable in testis of CREM-deficient mice. The cellular localization of mGPDH expression and translation in adult rat testis was determined by in situ hybridization and immunohistochemistry techniques. The mGPDH transcripts were detected solely in postmeiotic germ cells. Expression of mGPDH was restricted from round spermatids to early elongating spermatids. The mGPDH protein was delayed in postmeiotic germ cells, restricted from late elongating spermatids to mature spermatids. Our results indicate that rat mGPDH is expressed by a testis-specific promoter from haploid male germ cells in a stage-specific manner.
gene regulation, male reproductive tract, spermatid, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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The expression of rat mGPDH is regulated by multiple promoters in a tissue-specific manner. The 5'-most upstream promoter A sequence is used predominantly in brain, brown adipose tissue, and pancreas. In contrast, promoter B activities are ubiquitously detected, and the 3'-most downstream promoter C is used exclusively in testis [4, 9]. The use of alternate mGPDH promoters results in variant exon 1-containing transcripts in different tissues. In particular, exon 1a-containing transcripts have been detected from brain, brown adipose tissue, and pancreas RNA, exon 1b-containing transcripts have been detected ubiquitously, and exon 1c-containing transcripts have been detected exclusively from testis RNA. Beside the variants at the 5' end of mGPDH transcripts, variations have been described at the 3' end. In testis, a 2.4-kilobase (kb) transcript has been described with a shortened 3' untranslated region, compared with the 6.5-kb transcript detected in most tissues [4–6]. In contrast to promoters A and B, which have been characterized in detail [9–12], very little is known about the regulation of the testis-specific promoter C.
Within the testis, spermatogenesis is a complex developmental process that includes the mitotic proliferation of spermatogonial stem cells, meiotic prophase, division of spermatocytes, and morphological changes of haploid spermatids to highly specialized spermatozoa (reviewed in [13]). The developmental program of spermatogenesis is regulated by several testis-specific transcription factors, e.g., the cAMP-response element (CRE) modulator (CREM) tau (CREM). CREM, a testis-specific transcriptional activator, is an alternative splice product of the CREM gene [14]. CREM, CRE binding protein (CREB), and activating transcription factor (ATF) belong to a family of proteins regulated by cAMP and binding to CREs [15–17]. Targeted gene interruption of the CREM gene (including CREM) leads to infertility in transgenic mice [18, 19]. In agreement with this observation, CRE sites have been described in several testicularly expressed genes involved in spermatogenesis and fertility [20–22].
The aim of the following study was to analyze the cellular localization of mGPDH within rat testis and to characterize the regulation of the testis-specific promoter C of the mGPDH gene. In situ hybridization and immunohistochemistry techniques localized mGPDH in a stage-specific manner to postmeiotic haploid germ cells. Furthermore, we analyzed the genomic region upstream of the transcription start site by comparative electrophoretic mobility shift experiments, using both nuclear extracts from entire testis and in vitro-translated CREM, and ectopic expression of CREM in heterologous transient transfection assays.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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Sequences of the 5' flanking exon 1c of the rat mGPDH gene were identified by a polymerase chain reaction (PCR)-based technique using commercially prepared, anchor-ligated genomic DNA from the rat (Genome Walker; Clontech, Palo Alto, CA) according to the instructions of the manufacturer. PCR was performed using exon 1c-specific primer WL24 (5'-ccatatagacagaagcctgg-3') and AP1 primer (directed against the anchor sequence) with Tth DNA polymerase (Clontech) on an Omnigene thermocycler (Hybaid, Heidelberg, Germany). After a nested PCR with exon 1c-specific primer WL23 (5'-ggtggtccattggatgtgac-3') and AP2 primer (Clontech), a 3063-base pair (bp) fragment was amplified from SspI-digested anchor-ligated genomic DNA, subcloned into pT7 (Novagen, Madison, WI), and sequenced.
To characterize the 5' end of the testicular mGPDH mRNA, we used a rapid amplification of cDNA ends (RACE) technique, which allows only amplification from full-length transcripts with an intact 5' cap structure (GeneRacer; Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA was prepared as described previously [23]. Four micrograms of total rat testis RNA was reverse transcribed using the mGPDH-specific reverse primer WL36 (5'-gggctctggatttactgagg-3') and 200 U of SuperScript II reverse transcriptase. The 5' end amplification was performed with the GeneRacer 5' primer and the mGPDH-specific primer WL35 (5'-tgccaccacttataaagtggc-3') using the PCR setup and cycling parameters suggested by the supplier's protocol. These first amplification products were subjected to a nested PCR using the GeneRacer 5' nested primer and the gene-specific nested primer WL177 (5'-agttggcacgttcatgaagg-3'). The amplification products were analyzed on 1% agarose gels, gel purified, ligated into pGEM-T Easy vector (Promega, Madison, WI), and sequenced.
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared from liver and testes of adult Wistar rats (
300 g body weight) by the method of Deryckere and Gannon [24]. In vitro-translated, FLAG-tagged CREM [25] was synthesized by a transcription/translation-coupled reticulocyte lysate system (Promega) according to the manufacturer's instructions. For electrophoretic mobility shift analyses (EMSA), seven overlapping DNA fragments encompassing the sequence from -787 to +28 of the rat mGPDH promoter C, were amplified using plasmid DNA as template and the following primers in a PCR: fragment 1 forward (F1 for): 5'-tttaaaaatcctcttggggg-3', F1 reverse (rev): 5'-atgagaccctgacccaaaag-3', F2 for: 5'-cttttgggtcagggtctcat-3', F2 rev: 5'-aatgccaaaacagaactggc-3', F3 for: 5'-gccagttctgttttggcatt-3', F3 rev: 5'-ccttgtataaggtcacctag-3', F4 for: 5'-ctaggtgaccttatacaagg-3', F4 rev: 5'-agcaataattttcactgtag-3', F5 for: 5'-ctacagtgaaaattattgct-3', F5 rev: 5'-atctcattgtaatctttcaa-3', F6 for: 5'-ttgaaagattacaatgagat-3', F6 rev: 5'-tgtggagtggtacacattag-3', F7 for: 5'-ctaatgtgtaccactccaca-3', F7 rev: 5'-ggtggtccattggatgtgac-3'. DNA amplification products (each 135 bp in length) were separated on 2% agarose gels, gel purified, and 32P end-labeled according to standard protocols. Seven hundred twenty nanograms of rat testis and 810 ng of rat liver nuclear extract or 2 µl in vitro-translated CREM were incubated with 7.2 fmol of radioactively labeled DNA fragment or double-stranded oligonucleotide probe and 1 µg poly (dA-dT) x (dA-dT) in band shift buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.1 EDTA, 1 mM dithiothreitol, 0.5 mM MgCl2, 100 µg/ml BSA, 5% glycerol) for 30 min at room temperature. For competition experiments, increasing amounts of unlabeled DNA fragment (100-, 200-, and 500-fold molar excess) or 150-fold molar excess of double-stranded competing oligonucleotides were added to the binding reaction. Competition oligonucleotides (CRE site wild type: 5'-gtttcctttgTGAGGTCAtgaatgatgtta-3', CRE site mutant: 5'-gtttcctttgTGAGATCAtgaatgatgtta-3') were synthesized as single-stranded oligonucleotides and annealed in 180 mM NaCl. For antibody interaction experiments, 2–8 µg rabbit polyclonal anti-CREM, anti-c-fos, and anti-c-jun antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) or mouse monoclonal anti-FLAG (M2) antibodies (Sigma, St. Louis, MO) were added to the binding reaction mixtures and preincubated for 30 min at room temperature. The reaction mixtures were loaded onto 5% polyacrylamide gels in electrophoresis buffer (45 mM Tris-borate, 1 mM EDTA) and electrophoresed for 2 h at 200 V. The shifted DNA bands were visualized by autoradiography on XAR films (Kodak, Rochester, NY).
Transient Transfection Assay
Human hepatocarcinoma HepG2 cells were cultured as described previously [9, 11]. Rat promoter C sequences from -2836 to +28 (plus five 5'-deletion mutant fragments) were ligated into pGL2 basic vectors (Promega) upstream of the luciferase reporter gene and verified by sequencing. Transient transfection analyses were conducted as previously described [9, 11] with minor modifications. For ectopic CREM expression, 1.4 µg of promoter C (-179/+28)-pGL2-basic luciferase reporter plasmid was mixed with 0.8 µg CREM expression construct in pRc/CMV (Invitrogen) and 0.6 µg of protein kinase A expression construct in pcDNA3.1(+) (Invitrogen) for each 8.7-cm2 dish. Expression plasmids were as follows: CREM, CREM-10/FLAG; CREM
, CREM-22/FLAG; protein kinase A subunit Cß wild type (PKAwt); and protein kinase A subunit Cß mutated (PKAmut) [25]. Luciferase activities were determined as described previously [9, 11] and normalized to the total protein concentration of the samples, which was determined by the Bradford method (BioRad, Hercules, CA). Luciferase assays were carried out in duplicate, and each construct was tested in at least three independent transfections with three culture dishes per experiment.
In Situ Hybridization and Northern Blot
Two DNA fragments representing 96 bp from -31 to +65 of the promoter C sequence (including the sequence of the testis-specific exon 1c) and 724 bp from the 5' end of rat mGPDH cDNA [26] were used as hybridization probes. The fragments were ligated into pGEM-7Zf(+) vectors (Promega) and linearized with an appropriate restriction endonuclease. Single-stranded sense and antisense probes were transcribed in vitro in the presence of digoxigenin-11-dUTP with T7 (forward) or SP6 (reverse) primers using the DIG RNA Labeling Kit (Roche, Indianapolis, IN) according to the manufacturer's instructions. Digoxigenin-labeled probes were purified using the High Pure PCR Product Purification Kit (Roche) and checked for labeling efficiency by control hybridization. RNA was prepared from tissues of adult Wistar rats, and prehybridization and hybridization were conducted as described previously [27] with a minor modification. Digoxigenin-labeled sense or antisense probes were applied to 10-µm rat testis sections before an overnight hybridization was performed at 40°C. Northern blot analyses were performed using 18 µg total RNA from rat testis as described previously [23].
Immunohistochemistry
A rabbit polyclonal anti-mGPDH antibody directed against a protein fragment from amino acids 42–206 of rat mGPDH [11] was used for immunohistochemistry. To increase the intensity of the reaction product, a combination of the peroxidase anti-peroxidase and the avidin-biotin peroxidase complex method was used [28]. Immunohistochemistry experiments were performed on 6-µm paraffin-embedded sections of adult rat testes fixed in Bouin solution. After dewaxing and hydrating in descending ethanol solutions (100%, 96%, 80%, and 70%), sections were washed in PBS (154 mM NaCl, 10 mM NaH2PO4, pH 7.5) and incubated for 30 min with 2% normal swine serum in PBS. Sections were then incubated overnight at 4°C with antibody at a 1:600 dilution in PBS, 0.2% BSA, and 0.1% NaN3, washed three times in PBS, and again incubated with biotinylated swine anti-rabbit secondary antibody (DAKO, Carpinteria, CA) at a 1:250 dilution in PBS for 1 h at room temperature. Sections were washed twice in PBS and incubated with a monoclonal anti-rabbit PAP complex (DAKO) at a 1:200 dilution for 30 min at room temperature. After washing three times in PBS, sections were finally incubated with an ABC (Vector, Burlingame, CA) at a 1:250 dilution for 30 min at room temperature and then washed in PBS followed by 100 mM sodium phosphate buffer (pH 7.4), and the peroxidase-enzyme reaction was developed for 10–18 min in 100 mM sodium phosphate (pH 7.4), 1 M diaminobenzidene substrate solution, 3.4 M ammonium chloride, 50 mM nickel sulfate, 10% glucose, and 1.2 mg/ml glucose oxidase. After final incubation in PBS and dehydrating in ascending ethanol solutions (70%, 80%, 96%, and twice in 100%), sections were mounted with Eukitt (Kindler, Freiburg, Germany). Control sections were treated in parallel but were incubated without anti-mGPDH antibody or with preimmune serum.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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Rat mGPDH is regulated by at least three promoters (Fig. 1A). To obtain sequence information for the testis-specific promoter C, we amplified 3000 bp of 5' upstream sequence of exon 1c from adapter-ligated genomic rat sequences by PCR. Sequence inspections identified several repetitive sequences, e.g., an Alu repeat from -667 to -528, a tRNA-Ala-GCG sequence from -761 to -700, and three low-complexity sequences (-2121/-2079, -2355/-2310, and -2533/-2356).
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To delineate the transcriptional start site(s) of exon 1c, we applied a PCR-based RACE technique. A total of 10 independent RACE-PCR fragments have been sequenced, and the main transcription start site (8 of 10 clones) (indicated by a bold bent arrow in Fig. 1B) and two alternative start sites (indicated by fine bent arrows in Fig. 1B) have been determined. The main transcription start site has been defined as +1. This site is located 27 bp downstream of the site approximately calculated previously [4]. We further observed two alternative 3' splice donor sites of exon 1c (indicated by closed points in Fig. 1B), which have also been described previously [4]. The 3' donor sites were used with similar efficiency: 4 of 10 clones used the upstream site, and 6 of 10 clones used the downstream site.
Functional Analysis by EMSA Experiments
To identify testis-specific DNA binding proteins able to interact with the rat promoter C sequence, we performed comparative EMSA experiments. The sequence of promoter C from -787 to +28 was divided into seven overlapping DNA fragments (schematically summarized in Fig. 1A). Incubation of fragments 2, 3, 4, and 6 with either liver or testis nuclear extracts revealed no specific DNA-protein interaction in the presence of unspecific competitor DNA poly (dA-dT) x (dA-dT) (data not shown). Incubation of fragment 1 (from -787 to -650) and of fragment 5 (-327/-200) with nuclear extracts from liver and testis, respectively, generated identical DNA-protein interaction patterns (data not shown). In contrast, incubation of fragment 7 (-107/+28) with nuclear extract from testis revealed a DNA-protein complex (Fig. 2A, arrow) that was not detected for the liver nuclear proteins (Fig. 2A, arrowheads). Increasing amounts of fragment 7 abolished the formation of the testis-specific band, whereas nonspecific bands remained almost unaffected (Fig. 2A, asterisks). A detailed sequence inspection of fragment 7 identified a putative CRE site at -57 to -50 (Fig. 1B). EMSA experiments, using the putative CRE motif as competitor DNA, abolished the formation of the testis-specific complex as efficiently as competition with the complete DNA fragment 7 (Fig. 2B). An anti-CREM antibody, but neither anti-c-fos nor anti-c-jun antibodies, interfered with the testis-specific complex (Fig. 2B, arrow). An in vitro-translated FLAG-tagged CREM protein was also able to bind the CRE site. This binding was competed by an oligonucleotide carrying the CRE wild-type sequence but not by a mutated CRE oligonucleotide bearing only a single point mutation. An anti-CREM antibody abolished the formation of the CREM-DNA complex, whereas an anti-FLAG antibody supershifted the complex to higher mobility (Fig. 2C).
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Transient Transfection
To prove the importance of CREM for testicular mGPDH expression, we performed a series of transient transfection experiments. Ectopic expression of CREM activator (CREM) plus PKA wild type activates promoter C (-179/+28) construct 5-fold (P < 0.05 versus CREM/PKAwt) after transient transfection in HepG2 cells (Fig. 3). A CREM repressor (CREM, lacking the activator domain of CREM) plus PKAwt cotransfection activates promoter C (-179/+28) 2.5-fold (P < 0.01 versus CREM/PKAmut), whereas an activation was not observed by cotransfection with a mutated PKA (Fig. 3). Moreover, a significant promoter C activity was undetectable in TM3 (not shown), HepG2, HEK293, Rat1, and primary rat hepatocytes without CREM/PKA cotransfection [9, 11].
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In Situ Hybridization
To investigate the expression and cellular localization of mGPDH in testis tissue, we performed in situ hybridization. To generate an exon 1c-specific probe (and therefore to analyze specifically promoter C-derived transcripts), we used a 96-bp fragment that includes the maximum 68-bp sequence portion of exon 1c as probe on adult rat testis. However, we failed to detect suitable in situ hybridization signals, even under reduced hybridization temperature and reduced washing stringency conditions (data not shown). To overcome this problem, we used a longer hybridization probe consisting of 724 bp from the 5' end of the rat cDNA [26]. To prove that the longer hybridization probe still detects the testis-specific transcript of mGPDH, we performed comparative Northern hybridizations with the short (96 bp) and the long (724 bp) hybridization probe on testis RNA. Both hybridization probes recognized the 2.4-kb testis-specific transcript of mGPDH with equal efficiency (Fig. 4). Furthermore, a quantitative PCR approach detected 18-fold more exon 1c-containing transcripts than exon 1b-containing transcripts in rat testicular RNA (data not shown). This finding indicates that the 724-bp hybridization probe is appropriate to detect promoter C-driven transcripts in testis by in situ hybridization analysis.
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The intensity of the mGPDH signal varies greatly between different seminiferous tubule cross sections, indicating stage-specific expression (Fig. 5). Hybridization signals appeared in only round spermatids (step 2; Fig. 5, arrow), and signal intensity increased to early elongating spermatids (step 11; Fig. 5, arrowhead) in seminiferous tubules. No hybridization signals were observed in any other testicular cell type.
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Control experiments, using a sense probe of the 724-bp mGPDH fragment, revealed no hybridization signal in any cell type under identical experimental conditions (Fig. 5D), supporting the specificity of the antisense probe. Furthermore, analysis of seminiferous tubule cross sections from CREM -/- mice, using the 724-bp mGPDH antisense fragment as probe, revealed no hybridization signal (data not shown).
Immunohistochemistry
To define the testicular cell type in which mGPDH is detectable at the protein level, we performed immunohistochemistry using an anti-mGPDH-specific antibody [11]. Within cross sections of adult rat testis, a germ cell-specific immunostaining pattern was observed using different anti-mGPDH antisera (Fig. 6). Immunostaining was observed from late elongating spermatids (step 16; Fig. 6, A and B, arrow) to mature spermatids (step 19; Fig. 6C, arrow) of postmeiotic germ cells. Spermatid-specific staining was absent when preimmune serum was used instead of the antibody (Fig. 6D). A weak nonspecific staining of Leydig cells was also observed using different anti-mGPDH antisera (Fig. 6, arrowheads). A similar staining of Leydig cells was seen with preimmune serum (Fig. 6D, arrowhead) and in controls after omitting the first antibody incubation (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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Specific competition of EMSA experiments and antibody interference experiments imply that CREM, a testis-specific activator of genes expressed in haploid cells, is a specific binding protein on the promoter C sequence of the mGPDH gene (Fig. 2, A–C). The specific complex identified in the testis extract migrates differently from weak complexes found with liver nuclear proteins and interacts specifically with anti-CREM antibodies. Although this complex might represent a CREM-like factor from somatic testicular cells, round spermatids (the site of expression of CREM) comprise >30% of all nuclei in rat testes at this age [29], more than any other individual cell type. The putative CRE site comprises the sequence 5'-tgaggtca-3', thus differing slightly from the sequence of a typical palindromic CRE site (5'-tgacgtca-3') [14] by one nucleotide. This site, however, is identical to the CRE site in the testis-specific promoter of angiotensin-converting enzyme (t-ACE) [21]. CRE sites are frequently observed close to the transcriptional start site of postmeiotically expressed target genes. For t-ACE, a CREM binding site has been characterized from -55 to -48 [21], and a small portion of the ACE promoter from -91 to +17 can efficiently target a reporter construct to those testicular cells in transgenic mice that normally produce t-ACE [30]. For Pdha-2, a testis-specific subunit of pyruvate dehydrogenase, a 187-bp promoter fragment is sufficient for the stage-specific localization of a reporter gene in testis of transgenic mice [31], and a CREM binding site has been characterized from -62 to -54 [32]. Here, we demonstrated that heterologous expression of CREM and a constitutively active protein kinase A stimulate promoter C of mGPDH after transient transfection in human hepatocarcinoma HepG2 cells (Fig. 3). Thus, mGPDH belongs to a group of postmeiotically expressed genes whose expression can be regulated by CREM in a very similar promoter context.
Localization of mGPDH in Testis
To address the cellular localization of mGPDH in testis, we performed in situ hybridization and immunohistochemistry analysis. The mGPDH transcripts were detected in round spermatids (step 2) to early elongating spermatids (step 11; Fig. 5), whereas mGPDH protein was seen in late elongating spermatids (step 16) to mature spermatids (step 19; Fig. 6). The cellular localization of mGPDH mRNA and protein in male germ cells is schematically summarized in Figure 7.
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Because CREM appears to be involved in testicular mGPDH expression, we also analyzed seminiferous tubule cross sections from CREM -/- mice for the presence of mGPDH transcripts. Histological analysis of CREM-deficient mice revealed arrest of spermatogenesis at the round spermatid level (step 1), resulting in infertility [18, 19]. The lack of detectable mGPDH expression in the testes of these animals (data not shown) suggests either that CREM is directly involved in mGPDH gene expression or at least that its expression is a downstream event dependent upon CREM activity.
The detection of mGPDH protein in elongating spermatids also correlates well with data on mGPDH enzyme activity, with high mGPDH enzyme activity having been detected in late stages of spermatid differentiation (steps 18 and 19) in the rat testis [7, 8].
No mGPDH transcripts were detected later than step 11 nor were mGPDH proteins seen earlier than step 16 spermatids. Transcription of most germ cell-specific genes appears to cease at the remodeling of the spermatid nucleus, and mRNA molecules are then stored as translationally repressed messenger ribonucleoprotein particles to be translated at later stages of spermiogenesis. This phenomenon of uncoupled transcription and translation has frequently been observed in haploid spermatids for a number of different genes [33]. An alternative explanation would be the storage of the protein in a form not detectable by immunohistochemistry. However, the gap between step 11 and step 16 spermatids is surprisingly long, corresponding to 9 days of spermatid development. Further research is necessary to explore these possibilities.
Function of mGPDH in Testis
In the 1960s, several agents were identified as potent antifertility drugs inducing sperm abnormalities and impaired sperm motility, finally leading to infertility in male laboratory animals [34]. The application of some of these drugs (e.g., -chlorohydrin, procarbazine, 7,12-dimethylbenz()-anthracene) was correlated with reduced mGPDH enzyme activities in the testes of animal models [7, 35–37], suggesting that mGPDH may play a critical role in fertility. However, the enzyme activity levels of mGPDH in the testis differ greatly among rat (high activity), cat and bull (modest activity), and human, dog, and horse (low activity) [7]. These findings have been confirmed (in rodents and humans) by mGPDH expression data [4, 5, 26]. Further studies on mGPDH knockout mice [38] specifically addressing the testis-specific promoter C would help to clarify the functional importance of mGPDH in spermatozoa and its possible implications for male infertility.
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NOTE ADDED IN PROOF |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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40% decreased fertility of mGPDH KO mice compared to wild-type littermates, underscoring the importance of mGPDH for proper fertility of mice.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Joachim M. Weitzel, Institute of Medical Biochemistry and Molecular Biology, University Hospital Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany. FAX: 49 40 42803 4862; weitzel{at}uke.uni-hamburg.de
Received: 18 June 2002.
First decision: 30 June 2002.
Accepted: 28 August 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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