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a Department of Biological Sciences, Fordham University, Bronx, New York 10458
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PRESSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PRESSREFERENCES |
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Investigations of cell-cell communication mechanisms in the testis have mostly focused on paracrine and hormonal pathways. A large number of paracrine factors have been identified, but we have little understanding of their roles in spermatogenesis [1–3]. The roles of gap junctions in testis cell communication have received less attention, yet they are clearly part of the communication network in the seminiferous tubules. Gap junctions are clusters of intercellular channels that assemble between homologous and heterologous cell types in nearly all epithelia (reviewed in [6, 7]). The channels permit regulated flow between cells of ions, metabolites, and second messengers, thus mediating electrotonic coupling, metabolic coupling, and coordinated responses of coupled cells to hormones and growth factors.
Electron microscopy has produced morphological evidence for gap junctions between Sertoli cells and between Sertoli and germ cells (reviewed in [8, 9]). The gap junctions between mature Sertoli cells are seen predominantly within the basolateral Sertoli occluding junctions but are also found in the adluminal compartment [10–14]. The relative abundance of Sertoli-Sertoli gap junctions is regulated during postnatal development [10, 11] and during the cycle of the seminiferous epithelium [8, 14]. Sertoli cell-germ cell gap junctions frequently appear as "desmosome-gap" junctions [15, 16]. They are present on spermatogonial through early spermatid stages, but they appear most abundantly on pachytene spermatocytes [17–19]. Gap junctions have not been detected on elongate spermatids. The number of junctions (2–3 per spermatocyte [17–19]) and the number of channels per junction (5–55 [16]) are relatively small. Nevertheless, cytoplasmic bridges between germ cells of the same stage may permit the rapid amplification and spread of second messenger waves (e.g., calcium waves) from Sertoli cells throughout the germ cell clone. Germ cells usually do not form gap junctions with each other because they are separated by thin extensions of Sertoli cells. However, molecules may pass from one germ cell to another by passing through two Sertoli-germ cell gap junctions present on opposite sides of the Sertoli cell extensions, but this remains to be demonstrated. To this investigator's knowledge, morphological descriptions of gap junctions between peritubular cells have not been published. Nevertheless, Risley et al. [20] have demonstrated dye coupling between peritubular cells in situ.
Individual Sertoli cells in a specific epithelial stage establish contacts with about 5 other Sertoli cells and as many as 50 spermatogenic cells from up to five different stages of sperm development [17, 18]. This suggests that gap junctions may permit extensive coupling of diverse cell types in the seminiferous epithelium, thereby minimizing the number of communication-independent cell populations. However, each intercellular channel in a gap junction may be assembled from one or more of the 14 polypeptides coded by the connexin (Cx) multigene family. In mice and rats, the Cxs are Cx-26, -30, -30.3, -31, -31.1, -32, -33, -36, -37, -40, -43, -45, -46, and -50 [6, 7]. Some Cxs are widely expressed (e.g., Cx-43) and show overlapping tissue distributions with other Cxs, while others (Cx-31.1 in skin, and Cx-33 in testes [21]) show more restricted expression. When a cell expresses more than one Cx, a single gap junction may contain one or multiple Cxs. Maculae containing two Cxs may contain homomeric (single Cx) channels that are mixed or segregated into separate domains [22]. Two Cxs may also co-oligomerize into heteromeric channels, such as those formed by Cx-26 and Cx-32 in transfected insect cells [23] and Cx-46 and Cx-50 in the lens [24]. If two heterologous cells form gap junctions, with each cell expressing multiple Cxs, channels may be formed in which each hemichannel contains several different Cxs (heterotypic heteromeric channels). Thus, the expression of multiple Cxs in a tissue permits assembly of biochemically diverse gap junction channels.
Direct measures of permeability through channels composed of different Cxs have demonstrated that individual Cxs show ion selectivity (e.g., Cx-26 channels favor cations, while Cx-32 favors anions [25]), as well as selectivities for tracer dyes less than 1000 daltons that depend on charge, charge density, and molecular shape [26–30]. Thus, in osteoblast cell lines, Cx-43, but not Cx-45, readily passes lucifer yellow [27]. In HeLa cells, propidium iodide readily permeates channels formed from Cx-26, -37, -40, -43, and -45, but not Cx-31 or Cx-32 channels, which are permeable to lucifer yellow [28]. Channels may also show asymmetric permeability as seen in heterotypic homomeric channels formed by Cx-26 and Cx-32 [25]. Asymmetry may be responsible for the unidirectional dye movements from astrocytes to neurons [31] and oligodendrocytes [32]. Finally, certain combinations of Cxs are incompatible and do not assemble into functional channels [28, 29, 33]. Thus, Cx-31 is self-compatible but is incompatible with Cx-26, -32, -37, -40, -43, and -45. Cx-40 is compatible with itself, but not Cx-26, -31, -32, -43, -46, or -50. In contrast, Cx-46 is compatible with Cx-26, -32, -43, -46, and -50, but not Cx-40 [29]. Cx incompatibility may permit the establishment of communication barriers that prevent coupling between contacting cells from adjacent cell clusters. Also, Cx-33 does not form channels in Xenopus oocyte assays and interferes with channel formation by Cx-37 [34]. Finally, the gating of channels is sensitive to transjunctional voltage, pH, intracellular calcium, lipophilic agents, and phosphorylation; and the response of channels to gating by these agents may be Cx dependent [6].
Differential Cx expression and assembly within the seminiferous tubules may facilitate the formation of multiple unique and independently regulated pathways of cell-cell communication even within a single epithelial stage. Multiple Cx mRNAs are expressed in testes. Northern blot analyses of total rat testes RNA have identified mRNAs for Cx-26 and -32 [35], -43 [20, 36], and -33 and -37 [21]. Cx-30 [37], -31 [38], -31.1 [39], and -43 [20] were detected by Northern analyses of mouse testis RNA. Many of the Cxs detected by Northern analysis may be from interstitial tissue rather than tubule cells, however. Only Cx-33 and Cx-43 proteins have been identified in tubule cell gap junctions, between Sertoli cells [40]. To obtain a more comprehensive and sensitive analysis of the complexity of Cx mRNA expression and translation in tubules, this study used reverse transcription-polymerase chain reaction (RT-PCR) to identify Cx mRNAs in purified seminiferous tubules and in isolated Sertoli, peritubular, and spermatogenic cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PRESSREFERENCES |
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Sprague-Dawley rats of various ages (Zivic-Miller, Pittsburgh, PA) were killed by CO2 asphyxiation. Heart, liver, and kidney were rapidly frozen and stored at -90°C. Testes were quickly removed and decapsulated in Hanks' Balanced Salts lacking calcium and magnesium (HBSS-CMF).
Seminiferous Tubule Preparations
Interstitial tissues were dissociated and tubules released from decapsulated testes by gentle agitation at 32°C in HBSS-CMF (or Dulbecco's modified Eagle's medium [DMEM]:F12) containing 0.05–0.1% collagenase Type 1A (Sigma Chemical Co., St. Louis, MO) for 10–20 min. The tubules were settled, washed three times with ice-cold HBSS-CMF, and placed in a Petri dish on ice with HBSS containing 5% heat-inactivated dialyzed fetal calf serum to reduce further enzyme digestion. With the aid of a dissection microscope, small knots of interstitial tissue and blood vessels were manually removed, and tubules were collected and washed again with HBSS-CMF by sedimentation.
To obtain spermatogenic cells, isolated tubules (not treated with serum) were digested and triturated in 0.1% collagenase and 0.1% hyaluronidase Type 1-S (Sigma) in HBSS-CMF also containing 10 µg/ml DNase I (Sigma), 5 mM L-lactate, and 1 mM pyruvate. Tubules were settled and supernatants discarded. This process was repeated until peritubular cells were removed. The digestions and trituration were then repeated until most tubules were dissociated. The cell suspension was treated with serum, as above, and filtered through 100-µm Nitex and 20-µm Nitex (Tetko, Depew, NY) to remove aggregates. The cells were then pelleted and resuspended in ice-cold 2.5% Percoll and HBSS and separated by a 1-h sedimentation at 4°C through a 5–12% Percoll gradient formed over 50 ml 40% Percoll in a Celsep unit (Dupont, Newtown, CT). All Percoll-HBSS solutions contained 5 mM lactate, 1 mM pyruvate, and 0.1% BSA. Gradient fractions (25 ml) were examined microscopically, and those enriched for pachytene spermatocytes and round spermatids were pooled. These fractions were routinely enriched to at least 90%.
Sertoli and peritubular cells RNAs were isolated (using Trizol, see below) from postnatal Day 20 rats and generously provided to us by Dr. Michael Skinner (Washington State University, Pullman, WA).
RNA Extraction from Cells and Polysomes
All solutions were prepared with diethyl pyrocarbonate-treated water. RNA was isolated from tubule and cell pellets using the Trizol reagent [41] as recommended by the manufacturer (Gibco-BRL, Grand Island, NY). It was then digested with RNase-free DNase I (10 U/ml, 37°C, 20 min). The DNase was removed by using Snap binding columns (Invitrogen, San Diego, CA) or by reextraction of the RNA with Trizol and reprecipitation. When small amounts of RNA were expected, Pellet Paint (Novagen, Madison, WI) was employed as a carrier to enhance RNA recovery.
Tubules dissociated in DMEM:F12 were collected and incubated in ice-cold HBSS containing 100 µg/ml cycloheximide for 10–15 min while they were manually sorted from blood vessels and interstitium. They were then quick frozen on dry ice and stored at -90°C or used immediately. Tubules were homogenized (1 ml packed tubules/1 ml buffer) in ice-cold 20 mM Hepes, pH 7.2, 0.1 M NaCl, 1.5 mM MgCl2, 500 U/ml RNasin (Promega, Madison, WI), 10 mM dithiothreitol (DTT), 1% Nonidet P-40, 0.4% sodium deoxycholate, and 10 µg/ml cycloheximide. The homogenates were centrifuged at 5000 rpm for 5 min. The supernatants were removed, adjusted to 0.5 M NaCl and 30 mM MgCl2, and centrifuged again. EDTA was added to 50 mM in some of the supernatants and incubated at room temperature for 15 min to disrupt polysomes. Supernatants (0.5 ml) were layered onto linear 10.5-ml gradients of 10–40% sucrose over 0.5 ml 60% sucrose. The sucrose solutions also contained 20 mM Hepes, pH 7.2, 0.1 M NaCl, 1.5 mM MgCl2, and 100 µg/ml heparin. Gradients were centrifuged in a SW41 rotor at 35 000 rpm for 2 h at 4°C. Tubes were punctured with a needle (27G1/2) and fractions collected. The bottom 6 ml (fraction A), middle 2 ml (fraction B), and top 4 ml (fraction C) were pooled, and RNA was isolated with Trizol. RNA yields were quantified by spectrophotometry (A260), and integrity was assessed by electrophoresis in denaturing 1% agarose-formaldehyde gels [42].
RT-PCR
Primers were synthesized in-house using an Applied Biosystems (Foster City, CA) Model 392 DNA synthesizer or were obtained commercially from Gibco-BRL. The primer sequences were mostly those defined by De Sousa et al. (Cx-43 [43]) and Davies et al. (Cx-26, -31.1, -32, -37, -45, -46, -50 [44]). Cx-40 sense primer was as described by Davies et al. [43], but the antisense primer was modified to 3'-CGATGGTGTCACTGTTCGCG-5'. Rat Cx-31 primers were 'AAGCATCGCCAGAAGCACG3' and 3'GAAGGTGTCCTAATACGCTCCG-5'; 342-bp (base pair) amplicon. Cx-33 primer sequences (5'-ATGAGTGATTGGAGTGCCTTACAC-3' and 3'-CATTTCTCTTGTCTTGGTACA-5'; 877-bp amplicon) were designed by D. Paul (Harvard Medical School, Boston, MA).
RT-PCR amplifications were standard two-step reactions. DNase I-treated RNA (up to 1 µg) was mixed with primer (50 pmol oligo dT), denatured at 70°C for 10 min, and quick chilled on ice. RNA was added to a 20-µl reaction mix (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 1 mM of each dNTP [Promega], 20 U RNasin [Promega], and 200 U murine Moloney leukemia virus [MMLV] reverse transcriptase [Promega]) and incubated at 42°C for 60 min, followed by 94°C for 5 min and a quick chill on ice. PCR was conducted in 25 µl containing 10 mM Tris, pH 9, 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl2, 0.2 mM each dNTP, 0.2 µM of each primer, 1 µl of cDNA, and 2.5 U Taq polymerase (Promega). PCR was also conducted using HotStarTaq DNA polymerase as recommended by the manufacturer (Qiagen, Valencia, CA). After hot-start incubation (95°C for 2.5 min for Taq, 95°C for 15 min for HotStarTaq), amplification was performed for 30–35 cycles as follows: denaturing 94°, 40 sec; annealing at 55° (Cx-45), 60°C (Cx-31, -31.1, -33, -40, -43, -46, -50) or 65°C (Cx-26, -32, -37) for 40 sec; extension at 72° for 1.5 min. A final 72°C extension was conducted for 10 min followed by quick chill. PCR was conducted in a PTC-200 Peltier thermal cycler from MJ Research (Watertown, MA). The cycling conditions were not optimized for efficiency but were chosen to provide maximum specificity of PCR with Taq polymerase. Indeed, recent results have shown that all of the Cxs amplified at 65°C with Taq may be more efficiently amplified at 60°C with HotStarTaq without loss of specificity.
RT-PCR reactions were also conducted without MMLV to control for potential DNA contamination. Further controls for genomic contamination and variations in RNA integrity included conducting RT-PCR with primers to rat ß-actin (5'-CGTGGGCCGCCCT-3' and 3'-CCGGGGAGACTTGGGATTCCGGTT-5') that span a small 87-bp intron in DNA, thus generating a 243-bp product from cDNA and a 330-bp product from genomic DNA [45]. PCR products were electrophoresed in 2% agarose-TAE gels [42] containing 1:5000 dilutions of SYBR Green I (Molecular Probes, Eugene, OR) and photographed with Polaroid (Cambridge, MA) 667 film using a 300-nm transilluminator. All photographs were digitized (Microtek ScanMaker 5; Microtek, Redondo Beach, CA), arranged, and annotated with Adobe Photoshop 5.0 (Adobe Systems, San Jose, CA).
PCR Amplicon Sequencing
Specific PCR products were isolated by excision from 2% agarose gels, followed by purification using a Qiaex II kit, as recommended by the manufacturer (Qiagen, Valencia, CA). Sense PCR primers (15 pmol) were 5'-labeled with [
-32P]ATP using recombinant T4 polynucleotide kinase according to supplier instructions (United States Biochemicals, Cleveland, OH). Unincorporated nucleotides were removed by gel filtration through Sephadex G-25 (Pharmacia, Kalamazoo, MI). Dideoxynucleotide-terminated PCR products were then generated by 45 cycles of asymmetric amplification of 20 ng of each Cx PCR amplicon using 1.5 pmol of 32P-labeled sense primers, appropriate dideoxynucleotides, and Thermosequenase (Amersham, Piscataway, NJ). Amplified products were electrophoresed on 6% polyacrylamide-7.9 M urea gels at 80 watts for 1.5 h and autoradiographed. At least 100 bases of nucleotide sequence were determined for each PCR product and compared (BLAST) to sequences for mouse and rat Cx DNAs present in GenBank.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PRESSREFERENCES |
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When RT-PCR was used to amplify Cx transcripts from other organs, it was found that the expression profile for each organ was specific and of lower complexity than that for seminiferous tubules (Fig. 2). Kidney, an organ with many cell types, showed the most complexity of the three somatic tissues, containing Cx-26, -31, -32, -37, -40, -43, and -45. Adult liver contained the same Cx transcripts as kidney, except that Cx-31 and Cx-45 were very weak and variable after 35 cycles. Cx-37, -40, -43, and -45 were routinely found in heart. Seminiferous tubules contained transcripts from four Cx genes (Cx-31.1, -33, -46, and -50, Fig. 1) undetected after 35 PCR cycles of heart or kidney RNA. These results suggest that the large number of Cx transcripts found in seminiferous tubules is not a technical artifact and that seminiferous tubules show a complex Cx gene expression profile.
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Polysomes were isolated from seminiferous tubules to determine which Cx transcripts may be translated. Figure 3 shows that the three fractions (A,B,C) pooled from sucrose gradients each contained 28 and 18S rRNAs. If tubule homogenates were treated with 50 mM EDTA prior to centrifugation, however, little rRNA was present in fractions A and B, suggesting that these fractions were enriched for polysomes. As expected, fraction C rRNA content was not affected by EDTA treatment, indicating that this light fraction contained ribosomal subunits and other ribonucleoproteins.
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RT-PCR showed that all of the Cx transcripts except Cx-46 were detected in the polysome fraction A, and EDTA treatment eliminated the Cx transcripts from this polysome fraction but not from C (Fig. 4). None of the Cx transcripts were found completely loaded on polysomes. These data provide further evidence that the RT-PCR procedure amplified Cx mRNAs, and suggest that at least 10 of the 11 Cx mRNAs examined are translated in seminiferous tubules.
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Cx transcripts were also examined in seminiferous tubules from postnatal rats of different ages. Figure 5 shows that tubules from postnatal Day 6 rats as compared to adult rat tubules (Fig. 1) had fewer Cx transcripts detectable after 30 cycles of PCR. Cx-43 and Cx-45 were readily amplified, but only faint bands were obtained for Cx-26, -31, -32, and -37. Cx-31.1, -33, -40, -46, and -50 were not reproducibly detected. When 35 cycles of PCR were used, however, only Cx-33 and Cx-50 were undetected. Cx transcripts from postnatal Day 15 rat tubules were amplified more robustly than those from postnatal Day 6 tubules. All of the Cx transcripts studied were detected in tubule RNA from postnatal Day 15 rats after 35 cycles of PCR, although Cx-50 was usually amplified weakly. These observations suggest that the complexity and abundance of Cx transcripts increase in tubules during the first wave of spermatogenesis.
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Pachytene spermatocytes, round spermatids, Sertoli cells, and peritubular cells were isolated in order to compare their Cx mRNA expression patterns. All of the Cx transcripts typical of adult tubules, except Cx-31.1 and -46, were detected in RNAs from pachytene spermatocytes and round spermatids (Fig. 6). Cx-33 and Cx-50 both appeared as strong bands in these samples in contrast to RNAs from postnatal Day 6 tubules. Sertoli cells from postnatal Day 20 rat testes contained all of the Cx transcripts present in the germ cells except Cx-50. Peritubular cells from postnatal Day 20 rats contained predominantly Cx-31, -37, -40, -43, and -45 mRNAs. Occasionally, Cx-26 and -32 transcripts were also weakly detected by RT-PCR of peritubular cell RNAs, suggesting that these mRNAs may also be present in relatively low abundance. Cx-50 was never detected in Sertoli or peritubular cells; thus, Cx-50 expression seemed to be germ cell specific in tubules. The differences in Cx transcripts between cell types also attests to the relatively high purities of the different cell isolates.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PRESSREFERENCES |
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The comparisons of the Cx mRNAs in postnatal rat tubules and isolated cell types suggest that Cx gene expression becomes more complex with the development of spermatogenic cells, especially meiotic cells. Postnatal Day 6 tubules contain mostly immature peritubular cells, proliferating, immature Sertoli cells, and spermatogonia [5]. In postnatal Day 15 tubules, Sertoli cells are not proliferating [5], and they begin forming tight junctions [10]. The germ cell population is larger than that of the Sertoli cells, and many have developed to early meiotic prophase [5]. Postnatal Day 15 tubules contained all of the Cx mRNAs in adult testes. In contrast, the Cx mRNA population in postnatal Day 6 tubules appeared less complex and less abundant on the basis of the relative number of PCR cycles required to achieve detection of each Cx mRNA from the various cell and tissue sources. Thus, the development of spermatocytes from spermatogonia may result in increased Cx gene expression. This conclusion is further supported by the observation that all of the Cx mRNAs present in adult tubules and postnatal Day 15 tubules, except Cx-31.1 and -46, were present in pachytene spermatocytes and round spermatids. Cx-33 and -50 transcripts appear to accumulate coincident with the development of meiotic cells.
Interpretation of the significance of the Cx mRNA expression in spermatogenesis is difficult, since some of the Cx mRNAs detected may be rare sequences and yield functionally insignificant amounts of protein if translated. For example, Kren et al. [46] have found Cx-43 transcripts in liver, but hepatocytes lack Cx-43 protein. However, Pozzi et al. [47] have shown that low levels of Cx mRNAs for Cx-26, -32, and -43 in mammary glands, detectable only by RT-PCR, are sufficient to produce the proteins for gap junctions in this tissue.
The RT-PCR results for total RNAs from liver and seminiferous tubules were unexpectedly complex. Interestingly, all organs and isolated cells studied contained transcripts for Cx-37, -40, -43, and -45, while others varied. The presence of blood vessels in all whole organs may account for the common expression of Cx-37, -40, and -43. Some of the Cx transcripts in total RNAs may also be rare nuclear RNAs that fail to become processed. Nevertheless, most of the Cx mRNAs in seminiferous tubules were found on polysomes; thus, they are functionally identified as mRNAs. In preliminary experiments, this laboratory has also immunolocalized Cx proteins-26, -32, -33, -37, -40, -43, and -45 in germ cell intracellular compartments (unpublished results). This suggests translation and intracellular transport of multiple Cx proteins in developing sperm. Cytoplasmic localization is inadequate evidence, however, for the role of a specific Cx protein in gap junction assembly. For example, rat osteoblastic cell lines express Cx-43 and Cx-46, but accumulate Cx-46 in the Golgi and use Cx-43 to assemble gap junctions [48]. Since there are many Cx transcripts in male germ cells, control of gap junction formation probably relies on translational and posttranslational regulation of Cx protein assembly.
Interpretation of the functional significance of the multiple Cx transcripts in germ cells is also confounded by the "transcriptional promiscuity" of spermatogenic cells that results in the production of unexpected gene products [49, 50]. The Cx genes may be broadly expressed in male germ cells because of this robust transcriptional activity rather than a need for each Cx protein. Consistent with this notion, null mutants of Cx genes (Cx-31, -32, -37, -40, -46, and -50) in mice have not produced male infertility (reviewed in [51]). Cx-43 null mutants in mice have lower germ cell numbers in immature testes, but this may be an indirect effect on primordial germ cell migration or coupling between Sertoli cells [52]. The apparent lack of effect of Cx knockouts on mouse spermatogenesis may be due, however, to the reliance on fertility as an endpoint. Knockouts of specific Cxs may cause quantitative changes in sperm numbers without affecting fertility. In addition, the large number of Cxs expressed in spermatogenic and Sertoli cells may allow compensatory assembly of compatible Cxs to minimize the negative effects of null mutations. Indeed, this mechanism may have evolved to minimize the effects of Cx mutations on male fertility.
The presence of gap junctions between Sertoli cells and spermatogonial through round spermatid stages argues for a role for these structures in Sertoli-germ cell communication. Although the number of channels between germ cells and Sertoli cells is relatively low, data from this laboratory have shown that there are sufficient channels to support dye coupling between rat Sertoli and germ cells at all epithelial stages, and coupling can be dye-selective [53]. Dye-selective coupling and stage-dependent expression of multiple Cx genes is consistent with the hypothesis that Sertoli cells and germ cells communicate via structurally diverse gap junctions [20, 40]. Current studies are directed at correlating the coupling status of germ cells with the ultrastructural localization of specific Cxs in Sertoli-germ cell gap junctions.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PRESSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This material is based upon work supported by the National Science Foundation under Grant No. IBN-9722987. 
2 Correspondence: Michael S. Risley, Department of Biological Sciences, Fordham University, Fordham Rd., Bronx, NY 10458. FAX: 718 817 3645; risley{at}fordham.edu
Accepted: October 29, 1999.
Received: August 26, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PRESSREFERENCES |
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