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Regular Article |
a Department of Physiology & Pharmacology, University of Queensland, Brisbane, Queensland 4072, Australia
b North Shore Assisted Reproductive Technologies, Chatswood NSW 2067, Australia
ABSTRACT
Although mouse oocytes and cleavage-stage embryos prefer pyruvate and lactate for metabolic fuels, they do take up and metabolize glucose. Indeed, presentation of glucose during the cleavage stages is required for subsequent blastocyst formation, which normally relies on uptake and metabolism of large amounts of glucose. Expression of the facilitative glucose transporter GLUT1 was examined using immunohistochemistry and Western blotting, and in polyspermic oocytes, metabolism of glucose was measured and compared with that of pyruvate and glutamine. GLUT1 was observed in all oocytes and embryos, and membrane and vesicular staining was present. Additionally, however, in polyspermic oocytes, the most intense staining was in the pronuclei, and this nuclear staining persisted in cleaving normal embryos. Furthermore, GLUT1 expression appeared to be up-regulated both in nuclei and plasma membranes following culture of oocytes in the absence of glucose. In polyspermic oocytes, the metabolism of glucose, but not of pyruvate or glutamine, was directly proportional to the number of pronuclei formed. After compaction, nuclear staining diminished, and GLUT1 localized to basolateral membranes of the outer cells and trophectoderm. In blastocysts, a weak but uniform staining of inner-cell-mass plasma membranes was apparent. The results are discussed in terms of potential roles for GLUT1 in pronuclei of oocytes and zygotes, nuclei of cleavage-stage embryos, and a transepithelial transport function for GLUT1, probably coupled with GLUT3, in compacted embryos and blastocysts.
developmental biology
INTRODUCTION
Although glucose is a major source of metabolic energy for most mammalian cells, it cannot support embryonic development before compaction [1]. Mouse zygotes have an absolute requirement for pyruvate during the first cleavage division, whereas the second division is supported by pyruvate and lactate acting synergistically [2, 3]. Glucose becomes the preferred substrate following compaction at the eight-cell stage [4], when it is required to fuel the high-energy-requiring Na+, K+-ATPases that facilitate cavitation. In vivo, these requirements are supplied by epithelial secretions of the oviduct and uterus. Low levels of glucose in the uterus (1 mM [5]) necessitate the embryonic expression of a high-affinity glucose transport system. This system was identified as GLUT3, a high-affinity/high-capacity transporter expressed on the apical trophectoderm at compaction, which, together with a basolateral GLUT1, provides the compacted embryo and blastocyst with maternal glucose [6].
The requirement for glucose postcompaction and the high ATP turnover required for cavitation are well established, but the role of glucose in fertilized oocytes and cleaving embryos before compaction remains elusive. Glucose uptake and metabolism are essential for gamete fusion and fertilization [7]. Indeed, glucose utilization by glycolytic and pentose phosphate pathways is increased with oocyte-sperm fusion [8]. In vitro, if glucose is removed after fertilization, normal blastocyst formation does not occur. However, brief exposure to glucose before the morula stage is sufficient to permit subsequent blastocyst formation [9–12]. The requirement for this brief exposure is intriguing and has led to the speculation that glucose may be required for activating blastocyst formation [11]. However, two-cell embryos that have formed in vivo may develop into blastocysts in vitro without glucose by relying on pyruvate and lactate. In cleaving embryos, glucose is taken up and converted to glycogen [13], lactate, and CO2 [14–16], and as much as 16% is utilized via the pentose phosphate pathway [17].
Glucose uptake by cleaving mouse embryos occurs via a classic, saturable facilitative system [18, 19] that has been identified as the GLUT1 transporter isoform [20, 21]. As development proceeds, GLUT3 is expressed at the four- to six-cell stage [6] and is joined at the eight-cell stage by GLUT2 [22]. In the blastocyst, GLUT3 concentrates in the apical trophectodermal membranes, where it provides predominant entry for exogenous glucose [6], whereas GLUT2 is located on the basal trophectoderm membranes [22].
The unchanging Km values during development [18] indicate participation of a single mechanism for glucose uptake throughout preimplantation development, despite documented expression of at least three transporter isoforms [6, 20, 22]. Furthermore, the expression of a specific glucose transporter during the cleavage stages, when glucose is not required as an energy source but, nonetheless, is essential [9–12], is intriguing. The subcellular distribution of GLUT1 in postcompacted mouse embryos is established [6], but to our knowledge, no evidence of its localization during earlier stages in development has been found. We propose that an unusual location may also be found in oocytes, where glucose is essential for gamete fusion [7].
To examine this problem in a topographical context, this study has identified the cellular localization of GLUT1 using confocal scanning laser microscopy and a specific anti-mouse GLUT1 antiserum. The aim was to determine precisely when the mRNA for GLUT1 was translated and where the protein was expressed in the oocyte and cleaving embryos. Perhaps the low uptake and metabolism of glucose are reflected in an unusual localization of GLUT1. Furthermore, the effect of the absence of glucose on the unusual cellular localization of GLUT1 was sought. The metabolism of glucose, pyruvate, and glutamine by unfertilized and polyspermic oocytes was also examined to investigate their unusual location of GLUT1.
MATERIALS AND METHODS
Ethics
All experiments on mice were approved by the Animal Ethics and Experimentation Committees of the University of Queensland and The Royal North Shore Hospital. These committees are approved by the National Health and Medical Research Council of Australia.
Materials
Radiochemicals were purchased from Amersham Pharmacia Biotech (Castle Hill, New South Wales, Australia). The affinity-purified rabbit anti-rat-GLUT1 antibody, which was kindly provided by D. James (Centre for Molecular and Cellular Biology, University of Queensland), was raised against a synthetic carboxyl-terminal dodecapeptide (amino acids 480–492) of the brain/HepG2 transporter, coupled to keyhole limpet hemocyanin through the N-terminal cysteine residue. The antiserum was affinity purified against the peptide [23]. All other reagents were of analytical grade unless stated otherwise.
Oocyte and Embryo Collection and Preparation
Oocytes and embryos were collected from superovulated Quackenbush mice at 8–10 wk of age. Polyspermic mouse oocytes were prepared as follows: Oocytes were collected at 15 h post-hCG. Cumulus cells were removed in Hepes-human tubal fluid (HTF) [24] medium, supplemented with 1500 IU/L of hyaluronidase. Zonae pellucidae were removed by brief exposure to acidified Tyrode solution (pH 2.5), and oocytes were incubated in HTF with 3 g/L of BSA (product no, A4697; Sigma, St. Louis, MO) in the presence of 107–108 motile sperm/L. The oocytes were assessed 6 h later under Hoffman optics (Modulation Optics, Greenvale, NY) for the presence of multiple pronuclei.
Embryos were collected at 24, 48, 72, and 96 h post-hCG in M2 medium [25] modified to contain 0.33 mM sodium pyruvate, 5.56 mM glucose, and 4 g/L of BSA [26]. For culture experiments, oocytes were collected 24 h post-hCG in M2 medium without glucose, and cumulus cells were removed with hyaluronidase as described above. Oocytes were then cultured under mineral oil at 37°C in 5% CO2 in air in KSOM medium [27] in both the presence and absence of glucose, and the effect of this treatment on GLUT1 immunoreactivity was determined.
Antisense Oligodeoxynucleotides
Oligonucleotide sequences used were GLUT1 antisense, 5'-CTT CTT GCT GCT GGG CTC GAT-3' (complementary to bases 207–228 and overlapping the starting codon of the rat brain GLUT1 cDNA sequence), and GLUT1 sense, 5'-ATG GAG GGG AGC AGC AAG AAG-3' (identical to bases 207–228). An 8-base pair sequence (5'-GCG AAA GC-3') that forms a stable, hairpin-like structure resistant to 3'-exonucleases was included at the 3'-end of both sequences as previously described [6]. Sense oligonucleotides were used to determine nonspecific effects of oligonucleotides in the embryo culture system.
Embryos were collected from superovulated mice at 48 h post-hCG in M2 medium. The two-cell embryos were then washed through two changes of BMOC2 culture medium [1] and cultured in that medium supplemented with 30 µM sense or antisense oligodeoxynucleotides in 84% N2, 5% O2, 5% CO2, and 6% H2O at 37°C until the four-cell stage, when they were fixed for immunolocalization.
Immunolocalization
Oocytes and embryos were fixed in 2% paraformaldehyde in PBS (pH 7.4) for 30 min at 25°C and washed four times with PBS before placing on Cell-Tak (Collaborative Biomedical Products, Two Oak Park, Bedford, MA)-coated coverslips. Following application of the primary antibody (10 µg/ml), embryos were washed in PBS and exposed for 1 h at 25°C to Texas red-conjugated goat-anti-rabbit IgG (Calbiochem-Novachem, Alexandria, NSW, Australia) diluted 1:100 with PBS. Coverslips were mounted on cavity slides in glycerol following brief exposure to 2.5%, 5%, 10%, 20%, 50%, and 70% (v/v) glycerol in PBS and examined using a Bio-Rad (Hercules, CA) MRC-600 confocal laser scanning microscope mounted on a Zeiss Axioskop equipped with a Zeiss (Oberkochen, Germany) Plan-APOCHROMAT x63 oil immersion objective.
Western Immunoblotting
Embryos were collected in minimum M2 medium and 15 µl of 10% SDS containing 1 mM PMSF, 125 mM dithiothreitol, 20% glycerol, 0.002% bromophenol blue, and 125 mM Tris-HCl (pH 6.8). The mixture was frozen before polyacrylamide electrophoresis through a 10% resolving gel [28]. Proteins were transferred to 0.45-µm nitrocellulose membranes [29]. Membranes were incubated in a blocking solution of 5% skim milk powder in PBS for 1 h at room temperature before incubation for 1 h at 37°C with anti-GLUT1 antibody diluted in 1% skim milk in PBS to a final concentration of 5 µg/ml. They were then washed through three incubations of 10 min in PBS containing 0.1% Tween 20 (Sigma) before incubation at 25°C for 1 h with a horseradish peroxidase-labeled donkey anti-rabbit secondary antibody (Amersham) diluted 1:10 000 in PBS containing 0.2% BSA. After three further washes, labeled protein was visualized using the enhanced chemiluminescence detection method (Amersham).
Oocyte Metabolism
A random selection of oocytes was assessed individually during a 2-h incubation period at 6 h post-hCG for glycolytic activity by conversion of 1.0 mM D-5-3H-glucose to 3H2O and for glucose oxidation by conversion of 1.0 mM U-14C-glucose to 14CO2 simultaneously [17]. The amount of 0.4 mM 2-14C-pyruvate or L-14C-glutamine oxidized to 14CO2 during 2 h of incubation was also assessed. Zona-intact and zona-free oocytes not exposed to sperm served as controls. Following metabolic assay, oocytes were fixed, stained with orcein, and examined for sperm tails and pronuclei.
Statistical Analysis
The metabolic data were analyzed by ANOVA, and means were compared using the Neuman-Keul's post-hoc test for comparison of multiple means.
RESULTS
GLUT1 Immunolocalization
No staining was apparent when the primary antibody was omitted or replaced with nonimmune rabbit serum (Fig. 1, A and B). GLUT1 immunoreactivity was detected in cytoplasmic vesicles in oocytes and embryos of all stages (Fig. 1). However, other expression sites varied with development. In oocytes and cleavage-stage embryos, GLUT1 was associated with nuclear membranes and nucleoli and, additionally, with mitotic spindles in cleavage-stage embryos. Little staining of the plasma membranes or the cytocortex was found (Fig. 1, C–F). When two-cell embryos were incubated with GLUT1 antisense oligodeoxynucleotides, the GLUT1 staining of the resultant four-cell embryos was reduced dramatically (Fig. 2D) and uniformly compared with sense oligodeoxynucleotide-treated embryos (Fig. 2C) and embryos developing without supplementation (Fig. 2B).
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Compacted morulae and blastocysts also demonstrated positive cytoplasmic staining, but membranous/cytocortical GLUT1 was confined to basolateral membranes (Fig. 1, G and H). This basolateral staining of trophectoderm was also found in Day 6 pig blastocysts (Fig. 1I). No apical staining for GLUT1 was observed in any of these compacted embryos or blastocysts. Membranes of the inner cell mass (ICM) stained weakly compared with the trophectoderm and cleavage-stage embryos, but the staining was distributed uniformly on the plasma membranes.
Effect of Glucose Deprivation on GLUT1 Expression
Fertilized oocytes cultured in the absence of glucose demonstrated a significant increase in GLUT1 immunoreactivity (Fig. 3). Again, no staining was apparent when the primary antibody was replaced with nonimmune IgG or omitted (Fig. 3, A and B). Two-cell and four-cell embryos derived from in vitro culture of oocytes in the presence of glucose (Fig. 3, C and E) showed a similar nuclear and cytoplasmic staining pattern to that of freshly collected embryos (Fig. 1). These embryos, however, exhibited a lower staining intensity than their fresh counterparts. Culture of oocytes in the absence of glucose led to increased immunoreactivity that was most predominant in the nuclei and plasma membranes of both two-cell (Fig. 3D) and four-cell embryos (Fig. 3F). An increase in diffuse cytoplasmic staining was also observed (Fig. 3D).
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Western Immunoblotting
Extracts of two-cell embryos and blastocysts were analyzed by Western blotting to confirm the identity of the embryonic antigen and to determine if the different cytoplasmic locations of GLUT1 during development were reflected in posttranslational modifications of the transporter. A broad, heterogeneous doublet was demonstrated in rat brain (Fig. 4, lane 1). The major band identified in brain (lane 1) was of mobility equivalent to approximately 47 kDa, with another prominent, broad, heterogeneous band of approximately 63 kDa. A similar pattern was apparent in both two-cell embryos and blastocysts (Fig. 4, lanes 2 and 3). The broad, 63-kDa band was consistent in embryos, but the more mobile band displayed mobility equivalent to 52 kDa in two-cell embryos and 47 kDa in blastocysts. Staining was almost equally distributed between these two forms in two-cell embryos, but in blastocysts, much more of the 63-kDa form was found compared with the 47-kDa form.
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Metabolic Studies of Oocytes and Zygotes
No significant differences between zona-intact and zona-free oocytes in the metabolism of glucose, pyruvate, or glutamine were found (P > 0.05; Fig. 5). Upon fertilization, glycolysis and glucose oxidation increased 53-fold and 14-fold, respectively (P < 0.05; Fig. 5A). In contrast, oxidation of both glutamine and pyruvate was unaffected by fertilization (Fig. 5B). When polypronuclear oocytes were assessed, both the rates of glycolytic and of oxidative glucose catabolism were linearly related to the number of pronuclei (P < 0.05). The slope of the line fitted by linear regression was 0.44 ± 0.06 pmol/h/pronucleus for glycolysis and 0.18 ± 0.023 pmol/h/pronucleus for glucose oxidation. These slopes were significantly different (P < 0.01). Again, no such relationship existed for oxidation of pyruvate or glutamine.
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DISCUSSION
This study examined if the unusual glucose metabolism of the cleaving mouse embryo was reflected in the topological expression pattern of GLUT1, the ubiquitous, facilitative glucose transporter. GLUT1 was expressed in oocytes and embryos of all stages, which is consistent with the findings of earlier studies that relied on mRNA expression or Western blotting [20–22]. However, more fascinating and relevant to our aim were the location of this expression and the changes in its pattern as development proceeded.
Whereas GLUT1 is expressed by embryos at all stages during development, it is absent from plasma membranes until compaction, which is consistent with the inability of the embryo to utilize glucose as an energy source during this stage. Intriguingly, in oocytes and cleaving embryos, the most conspicuous GLUT1 staining was pronuclear or nuclear. To our knowledge, a nuclear location for GLUT1 has not been reported previously. Moreover, this nuclear GLUT1 was also apparent when embryos were cultured both in the presence and the absence of glucose. It is difficult to quantitatively compare immunofluorescence between different experiments, but the apparent decrease in GLUT1 staining intensity between freshly collected and cultured embryos is consistent with the observation that GLUT1 expression in bovine embryos is down-regulated following in vitro culture [30]. The increase in GLUT1 expression observed in the absence of glucose is a well-characterized, chronic response of GLUT1 to cellular stress [31]. This response is also apparent following short periods of glucose deprivation in the mouse embryo [18, 32]. Increased nuclear immunoreactivity for GLUT1 under these conditions is further support for the unusual localization of this transporter at this stage in development.
After compaction, the antibody targets an antigen distributed in the plasma membrane and cytoplasm in a pattern quite distinct from the nuclear localization in oocytes and cleaving embryos. Yet, this same antibody detects a protein of similar electrophoretic mobility in two-cell embryos, blastocysts, and brain, as evidenced by Western immunoblotting, which showed a diffuse pattern consistent with that of brain in cleavage-stage embryos and blastocysts. Further evidence for the specificity of nuclear GLUT1 staining comes from earlier antisense oligodeoxynucleotide studies that showed complete and specific abolition of GLUT1 immunoreactivity following GLUT1 antisense, but not sense, treatment [6]. To specifically identify the nuclear labeling as GLUT1, four-cell embryos treated with GLUT1 antisense oligodeoxynucleotides were also examined. The staining for GLUT1 was dramatically attenuated, which is consistent with selective ablation of GLUT1 expression, and this attenuation of staining was consistent across all staining locations, including the nuclear region. Together, these results support the conclusion that the unusual nuclear staining in oocytes and cleavage-stage embryos is caused by the previously unreported nuclear location of GLUT1.
The broad doublet displayed by GLUT1 on electrophoresis is characteristic, arising from differential N-glycosylation [33]. Expression of GLUT1 in two glycosylation states in adipocytes deprived of glucose [34, 35] and a change in N-linked glycosylation associated with the tumor-suppressor function of fibroblasts [36] suggest that GLUT1 expression responds to different stimuli. The changes in cell physiology induced by these stimuli appear as posttranslational glycosylation. Perhaps the different glycosylation states in embryos are reflected in the nuclear localization in oocytes and cleaving embryos and indicate a particular physiological state of the embryo. This concept is supported by observations of macrophages, where nuclear GLUT1 was translocated to the cell membrane upon activation with phorbol ester [37]. Alternatively, the altered glycosylation may reflect the reduced substrate affinity (i.e., increased Km) as mouse two-cell embryos develop to morulae and blastocysts [18], as has been observed in tumorigenic cells [36].
Functional Significance of Nuclear GLUT1 in Precompacted Embryos
Consistent with a nuclear association for GLUT1 is the positive correlation between the number of pronuclei in polyspermic polypronucleate oocytes and glycolytic activity. In contrast, these polypronucleate oocytes exhibited no differences in the metabolism of pyruvate or glutamine, regardless of the number of pronuclei present. Increased glucose catabolism occurs during fertilization but not after parthenogenetic activation (unpublished observations and [8]). An increase in glycolysis is associated with sperm entry [7], as observed in this study, but to our knowledge, no comparable data exist for a relationship with polypronucleation. Because polypronucleation was induced by polyspermy, the linear relationship may derive from sperm entry or pronuclear formation. Although parthenogenetic activation did not cause an increase in glucose metabolism [8], suggesting pronuclear formation is not the cause, resolution of this question would require metabolic analysis of oocytes microinjected with male pronuclei. The activity of hexokinase, which phosphorylates glucose to glucose-6-phosphate, is also reported to increase upon fertilization [38], which is consistent with the metabolic results reported here. Increased glycolysis must be reflected by increased flux through hexokinase from GLUT1, because it is the only means of entry by glucose into the oocyte. To our knowledge, no evidence exists for nuclear glycolysis. Glycogen-like material and glycogen synthase kinase are enriched in nuclear fractions prepared from rat liver and Xenopus oocytes [39], and glycogen is required for the formation of functional nuclei in Xenopus egg extracts [40]. Possibly, GLUT1 and other enzymes required for glycogen synthesis, such as hexokinase, phosphoglucomutase, and glycogen synthase, are required in or close to the nuclei.
Their function at this location is currently unknown. It may be that these are required for transcriptional glucose signaling. Glucokinase (i.e., hexokinase IV isozyme) and its regulatory protein have been detected in hepatocyte nuclei [41], and the shuttling of glucokinase between the nucleus and cytoplasm in response to glucose is believed to be essential for the regulation of glucose metabolism in hepatocytes [42]. More recently, GLUT2 was shown to have the capacity to translocate to the nucleus in response to glucose [43], suggesting that GLUT2 may be involved in transcriptional glucose signaling.
Interestingly, mitochondria appear to be distributed in a tight, perinuclear cluster within each blastomere in mouse and hamster cleavage-stage embryos [44, 45]. In mouse oocytes, mitochondria are also associated with mitotic spindle structures (unpublished results), which is consistent with the hamster, where intense mitochondrial staining has been reported in the second polar body as well as the mitotic spindles [45]. This close association between the energy-generating organelles and the mitotic apparatus may facilitate delivery of energy required for spindle formation and subsequent chromosomal movements [45]. If so, then the proportional increase in glucose metabolism with an increasing number of nuclei might simply reflect incrementally increasing requirements for energy.
Recently, a GLUT1 C-terminal binding protein (GLUT1CBP) has been identified. This protein interacts with GLUT1 and cytoskeletal proteins via PDZ-domains and is implicated in targeting GLUT1 to specific subcellular sites, either by tethering the transporter to cytoskeletal motor proteins or by anchoring the transporter to the actin cytoskeleton [46]. Expression of GLUT1CBP by mouse embryos would provide a mechanism to enable GLUT1 to translocate to its unusual locale.
GLUT1 Function in the Compacted Embryo: Transepithelial Transport of Maternal Glucose
After compaction and polarization, GLUT1 was restricted to the basolateral membrane domains of outer morula cells and trophectoderm, with weak apolar staining of ICM cells. This is consistent with the pig blastocyst and replicates the arrangement in rabbit blastocysts [47], but it differs from the observations of mouse blastocysts by immunoelectron microscopy, in which "GLUT1 was found on cells of the trophectoderm and ICM and was randomly distributed along apical, basolateral, and intercellular portions of the plasma membrane of these cells" [22]. The latter study corroborates considerable vesicular staining for GLUT1 in blastocysts. The localization of GLUT1 to basolateral membranes of the epithelial trophectoderm in at least three different species suggests a typical transepithelial glucose transport function for this transporter by coupling with the apically expressed, high-capacity GLUT3 [6]. This arrangement is analogous to that in epithelia of the proximal convoluted tubule of the nephron and of the small intestine, where basolateral GLUT1 [48, 49] provides efflux of glucose actively absorbed across the apical membrane by the sodium-linked glucose transporter SGLT1 [48]. Interestingly, GLUT1 and its high-affinity partner, SGLT1, mediate glucose efflux in regions of the nephron and intestine with low luminal glucose concentration, but GLUT2 and SGLT2 (both low-affinity carriers) perform this function in areas of high luminal glucose levels further upstream in both the nephron and the gut. Evidence for the expression of SGLT in mouse embryos is conflicting [50], but GLUT3 provides the major route of entry for glucose into the blastocyst [6]. Its high facilitative capacity might serve a similar role to SGLT1, especially if the concentration of glucose in the blastocyst is kept low by rapid metabolism, as is likely during blastocyst formation and expansion [50].
The uniform distribution of GLUT1 in ICM cells suggests that this transporter is responsible for glucose uptake from the blastocoele cavity for utilization by these cells. The ability of GLUT1 to operate for both influx and efflux derives from its asymmetric kinetics. The Km for influx is relatively low (1–2 mM), but that for efflux is an order of magnitude higher (20–30 mM), thus providing an efficient efflux mechanism [49]. Together, these results suggest a model for glucose supply in blastocysts based on apical influx of maternal glucose via GLUT3, followed by basolateral efflux via GLUT1 into the blastocoele and interstitial spaces for subsequent uptake by ICM cells via GLUT1 [50]. This model, however, does not account for GLUT2, which we and others [21] have been unable to detect, despite the observations of mRNA [20] and protein [22, 51] expression in compacted embryos.
In conclusion, developmentally differentiated, targeted expression of GLUT1 has been demonstrated in mouse preimplantation embryos. A basolateral disposition in the trophectoderm and ICM is consistent with a function in the efflux of maternal glucose from the trophectodermal cells and subsequent uptake by the glycolytic ICM cells by virtue of its asymmetric, bidirectional transport characteristics. A predominantly nuclear location in cleaving embryos reflects the low glucose uptake exhibited by these embryos, and it suggests an alternate role for GLUT1, possibly related to the physiological state of these cells. A direct reliance of glycolytic metabolism on the degree of polyspermy, but not parthenogenetic activation, suggests that glucose metabolism is related to sperm penetration or pronuclear formation.
ACKNOWLEDGMENTS
We thank Kathleen Waite and Kathryn Markham for their technical assistance and Chris Corcoran for management of our mouse colony.
FOOTNOTES
1 Supported by National Health and Medical Research Council grants to P.L.K. and J.P.R. 
2 Correspondence. FAX: 617 3365 1766; kaye{at}plpk.uq.edu.au
Accepted: November 28, 2000.
Received: June 15, 2000.
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