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Regular Article |
a Department of Anatomy and Physiology, Royal Veterinary and Agricultural University, 1870 Frederiksberg C, Denmark
b Konstantin the Philosopher University, Nitra, Slovak Republic
c Research Institute of Animal Production, Nitra, Slovak Republic
d Veterinary University, Vienna, Austria
e Department of Biotechnology, Institut für Tierzucht und Tierverhalten, Mariensee, (FAL), Neustadt, Germany
f Precision Therapeutics, Pittsburgh, Pennsylvania 15213
g Universität Bonn, Institut für Tierzuchtwissenschaft, Germany
ABSTRACT
Ribosomal RNA genes are transcribed in the nucleolus. The formation of this organelle after fertilization is essential for embryonic protein synthesis and viability. We have examined nucleolus formation in in vivo-derived porcine embryos by light microscopical autoradiography following 20 min of 3H-uridine incubation, transmission electron microscopy (TEM), and immunocytochemical localization by confocal laser scanning microscopy of key nucleolar proteins involved in rRNA transcription (nucleolin, upstream binding factor, topoisomerase I, and RNA polymerase I) and processing (fibrillarin, nucleophosmin). During the first two postfertilization cell cycles, TEM revealed fibrillar spheres as the most prominent intranuclear entity of the blastomeres. Fibrillogranular nucleoli were established during the third cell cycle. Initially, fibrillar centers, a dense fibrillar component, and a granular component were formed on the surface of the fibrillar spheres. At the same time, autoradiographic labeling over the nucleoplasm and in particular the nucleoli was detected for the first time. The nucleolar proteins were, in general, not immunocytochemically localized to the presumptive nucleolar compartment until late during the third or early during the fourth cell cycle.
early development, embryo, gamete biology, gene regulation, oviduct
INTRODUCTION
The initial development of mammalian preimplantation embryos is governed by gene transcripts and polypeptides produced by, and stored in, the oocyte during its development [1]. However, following one to three cleavage divisions, control of development is taken over by the expression of portions of the embryonic genome as the maternally derived transcripts and proteins are gradually degraded [2–4]. This transition from maternal to embryonic control of development is a gradual phenomenon. In the mouse and rabbit, it has been clearly demonstrated that a minor transcriptional activation occurring at the G2 stage of the first postfertilization cell cycle precedes a major activation taking place at the G2 stage of the second cell cycle in mouse and at the fourth to fifth cell cycle in rabbit [5]. Accordingly, using long-term incubation with 3H-uridine we have observed transcription during the first [6] and second cell cycle [7–9] in cattle, which is well before the major transcriptional activation that takes place during the fourth cell cycle in this species [10]. In swine, the major transcriptional activation is seen during the third cell cycle [11], but whether this activation is preceded by an earlier minor one has not been investigated.
The major transcriptional activation of the embryonic genome includes the formation of a fibrillogranular nucleolus signaling the activation of the ribosomal RNA (rRNA) genes [11–13]. The rRNA genes are located at the nucleolus organizer regions (NORs) of the chromosomes, and the transcription of these genes is associated with the formation of the nucleolus, which is the most prominent nuclear organelle and the site of formation of the ribosomal subunits. In the functionally active nucleolus three main ultrastructural components can be identified: The fibrillar components consisting of the fibrillar centers (FCs) and the dense fibrillar component (DFC), and the granular component (GC) [14]. These components of the so-called fibrillogranular nucleolus reflect the steps in the biosynthesis of ribosomes according to the following: The FCs house the enzymatic apparatus for the transcriptional process, the DFC carries the primary nascent transcripts, while the GC represents processed transcripts associated with proteins in the form of preribosomal particles.
In porcine embryos, the first fibrillogranular nucleoli develop during the third postfertilization cell cycle [11]. During the first and second cell cycle, the nuclei present large spherical masses of densely packed fibrillar material referred to by many authors as so-called nucleolus precursor bodies [15]. The nucleolus contains a number of proteins. Their roles are to control the transcription of the rRNA genes, to process the transcripts, to assemble the transcripts with other ribosomal proteins, and to transport the newly synthesized ribosomal subunits to the cytoplasm [14]. In in vitro-produced bovine embryos these proteins gradually become localized to the nucleolar compartment over several cell cycles [16]. So far, however, the development of the nucleolar protein compartment in the porcine embryo has not been investigated.
The aim of the present study was to reveal the chronology of the major transcriptional activation of the embryonic genome in relation to the ultrastructural development of the nucleolus and the allocation of key nucleolar proteins to this organelle in preimplantation porcine embryos developed in vivo.
MATERIALS AND METHODS
Animal Treatment and Collection of Presumptive Zygotes and Embryos
Cyclic gilts (n = 58) were housed in single pens and were fed daily over a period of 11 days with 5 ml Regumate (Altrenogest, Iffa Merieux, France). On the last day of treatment the animals received an injection of 1500 IU eCG (Pregnenolon; Dessau, Germany) followed by 500 IU hCG (Ovogest; Intervet, Tönisvorst, Germany) 72 h later. The gilts were inseminated twice at 24 h and 36 h after hCG injection with freshly collected semen diluted in Androhep extender (Minitüb; Tiefenbach, Germany) to 3 x 109 sperm/100 ml. Subsequently, the gilts were killed at the Institute's own abattoir (Mariensee) at 48–56 h (n = 12), 70–74 h (n = 14), 94–98 h (n = 10), 118–122 h (n = 12), and 142–146 h (n = 10) after hCG injection. Then the oviducts and uteri were flushed and presumptive zygotes and embryos recovered. Due to asynchrony between cleavage of blastomeres, the separation of the data on the fourth and fifth cell cycles is not complete. Thus, 8- and 16-cell embryos are in the following referred to as tentative developmental stages. Immediately after flushing, presumptive zygotes were processed for transmission electron microscopy (TEM, n = 4) or immunocytochemistry (IC, n = 62) and 2-cell embryos for 3H-uridine incubation, light microscopical autoradiography, and TEM (A-TEM, n = 2) and IC (n = 57). The remaining collected 2-cell embryos (n = 168) and all the collected 4- (n = 164) and tentative 8-cell embryos (n = 92) were then cultured in vitro in NCSU 23 [17] at 39°C in a humidified atmosphere with 5% CO2 in air into the subsequent cell cycle. The embryos were examined every second hour in order to detect the time of cleavage. The embryos that did not pass the cleavage to the subsequent cell cycle were excluded from further experimentation. Subsequently, 4-cell embryos were harvested at 10 (A-TEM, n = 2; IC, n = 48), 20 (A-TEM, n = 2; IC, n = 47), and 30 (A-TEM, n = 2; IC, n = 50) h postcleavage (hpc), tentative 8-cell embryos at 10 (A-TEM, n = 4; IC, n = 68) and 20 (A-TEM, n = 2; IC, 72) hpc and tentative 16-cell embryos according to morphological evaluation (A-TEM, n = 2; IC, n = 79) for further experimentation.
3H-Uridine Incubation
Embryos from different developmental stages as defined above were incubated with 3H-uridine (specific activity 962 GBq/mmol; Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany) at a final concentration of 4 Mbq/mmol [11]. The embryos were labeled for 20 min in gas-equilibrated culture medium. After incubation with precursor, the embryos were repeatedly washed in 3H-uridine-free culture medium.
Processing for Light Microscopical A-TEM
After labeling with radioactive precursor (or immediately after flushing for the presumptive zygotes), the ova were fixed in a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.2). Subsequently, the specimens were washed in buffer, postfixed in 1% OsO4 in 0.1 M cacodylate buffer, embedded in Epon, and serially sectioned into semithin sections (2 µm). Every second section was stained with basic toluidine blue and evaluated by brightfield light microscopy. For the zygotes, all sections were stained. Selected semithin sections were re-embedded according to Hyttel and Madsen [18] and processed for ultrathin sectioning (70 nm). The ultrathin sections were examined on a Philips CM100 transmission electron microscope.
Selected unstained semithin sections were processed for autoradiography for detection of total RNA synthesis and nucleolus-associated RNA synthesis. The sections were coated with Ilford K5 liquid nuclear emulsion (Ilford; Basildon, Essex, UK) and exposed for 6 wk at 4°C. Finally, the specimens were developed in Kodak D19 at 17°C, stained with toluidine blue, and evaluated by brightfield and epipolarized light microscopy.
Immunocytochemistry and Confocal Laser Scanning Microscopy
The following primary antibodies against key nucleolar proteins were used: Mouse monoclonal antinucleolin (C23; 1:1000 [19]), mouse monoclonal antinucleophosmin (B23; 1:1000 [19]), human antifibrillarin (1:1000 [20]), human antitopoisomerase I (1:100 [21]), human anti-RNA polymerase I (1:500 [22]), and human anti-upstream binding factor (UBF; 1:500 [23]).
For indirect immunofluorescence the presumptive zygotes and embryos were fixed in a mixture of 4% paraformaldehyde and 0.1% Triton X-100 for 3 h at 4°C. Subsequently, the specimens were washed in 1% Triton X-100 in PBS and preincubated for 2 h with 5% rabbit serum (Dako, Glostrup, Denmark) in PBS at room temperature. Thereafter, they were incubated with the primary antibodies diluted in PBS containing 5% rabbit serum overnight at 4°C. Excess primary antibodies were removed by extensive washing in PBS prior to a 4-h (at 4°C) and 1-h (at room temperature) incubation in rabbit antihuman-biotin (Dako; for antitopoisomerase I, anti-RNA-polymerase I, anti-UBF, and antifibrillarin) or rabbit antimouse-biotin (Dako; for antinucleolin and antinucleophosmin), diluted in PBS containing 5% rabbit serum. The secondary antibodies were visualized by streptavidin-fluorescein isothiocyanate (Dako) in PBS. Finally, the ova were mounted on glass slides using Dako fluorescent mounting medium (Dako) and examined on a Leica confocal laser scanning microscope. Control immunostaining of unspecific labeling by the secondary antibody was performed by omitting the primary antibodies.
RESULTS
Autoradiography and Ultrastructure
First cell cycle, i.e., the zygote In all four zygotes the most prominent entities observed in the pronuclei were large electron-dense fibrillar spheres (Fig. 1).
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Second cell cycle, i.e., the 2-cell embryo None of the nuclei in the two 2-cell embryos presented autoradiographic labeling (Fig. 2). In all the embryos the most prominent intranuclear entities were the fibrillar spheres. Heterochromatic areas of slightly condensed chromatin were dispersed throughout the nuclei.
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Third cell cycle, i.e., the 4-cell embryo None of the nuclei in the two 4-cell embryos fixed early during the third cell cycle, i.e., 10 hpc, presented autoradiographic labeling (Fig. 3). The most prominent intranuclear entities in all evaluated embryos were still the fibrillar spheres that, however, had become associated with a halo of condensed chromatin that was connected to other heterochromatic areas.
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From the two 4-cell embryos fixed at the midpoint of the third cell cycle, i.e., 20 hpc, one lacked autoradiographic labeling. In the other specimens, all nuclei displayed autoradiographic labeling dispersed over the nucleoplasm and the nucleoli (Fig. 4). At the ultrastructural level, nuclei displayed different stages of nucleolus formation, ranging from the fibrillar spheres to fibrillogranular nucleoli presenting semilunar formations of FCs, DFC, and GC on the surface of the fibrillar spheres that were more or less encapsulated. Different stages of nucleolus development were observed within the same nucleus.
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Both the two 4-cell embryos fixed late during the third cell cycle, i.e., 30 hpc, displayed autoradiographic labeling dispersed over the nucleoplasm and concentrated over the nucleoli in all nuclei (Fig. 5). The nucleus ultrastructure resembled that in the previous group, and again, nucleoli at different developmental stages were observed within the same nucleus.
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Fourth cell cycle, i.e., the tentative 8-cell embryos All four tentative 8-cell embryos fixed early during the fourth cell cycle, i.e., 10 hpc, displayed intensive autoradiographic labeling over both the nucleoplasm and the nucleoli in all nuclei (Fig. 6). The nucleus ultrastructure resembled that in the previous group except for the fibrillogranular portions of the nucleoli that had become more reticulated. Spherical fibrillar masses and fibrillogranular nucleoli were still observed within the same nuclei.
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Both the two tentative 8-cell embryos fixed late during the fourth cell cycle, i.e., 20 hpc, presented the same autoradiographic and ultrastructural findings as the previous group.
Fifth cell cycle, i.e., the tentative 16-cell stage In two tentative 16-cell stages, pronounced autoradiographic labeling over nucleoplasm and nucleoli was observed. The nucleoli were reticulated fibrillogranular. Another four degenerated embryos were encountered in this group.
Immunocytochemistry and Confocal Laser Scanning Microscopy
The patterns of immunocytochemical labeling found within each developmental sampling point were very homogenous and are described in the following.
First cell cycle Labeling of nucleophosmin was localized to shell-like intrapronuclear bodies resembling the presumptive fibrillar spheres (Fig. 7). In addition, a dispersed labeling of the pronuclei of fibrillarin, nucleolin, UBF, topoisomerase I, and RNA polymerase I, excluding the presumptive fibrillar spheres, was noted. The labeling of UBF was weak.
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Second cell cycle A weak nucleoplasmic labeling of fibrillarin, nucleolin, and nucleophosmin excluding the presumptive fibrillar spheres was noted. No immunocytochemical labeling was detected with the remaining antibodies.
Third cell cycle A weak nucleoplasmic labeling of fibrillarin, nucleolin, nucleophosmin, and UBF excluding the presumptive fibrillar spheres was noted at 10, 20, and 30 hpc. In particular, the labeling of fibrillarin was weak. A weak nucleoplasmic labeling of RNA polymerase I excluding the presumptive fibrillar spheres was noted at 20 hpc and 30 hpc. At the latter time point, additional labeling of RNA polymerase I was localized to small foci surrounding the presumptive fibrillar spheres. No labeling of topoisomerase I was detected.
Fourth cell cycle Labeling of fibrillarin, UBF, topoisomerase I, and RNA polymerase I was localized to small foci surrounding the presumptive fibrillar spheres at both 10 and 20 hpc. The labeling of topoisomerase I was weak. At the same time points, labeling for nucleolin and nucleophosmin was localized to more or less shell-like bodies, probably outlining the fibrillar spheres and the attached fibrillogranular nucleoli. A weak nucleoplasmic labeling was observed at both sampling points with all antibodies except antifibrillarin.
Fifth cell cycle Similar immunocytochemical observations as described for the previous group were done.
Control No immunocytochemical labeling was noted in the control specimens in which the primary antibodies had been omitted.
DISCUSSION
In mammals, the restoration of rRNA transcription after fertilization is accompanied by a gradual differentiation over several cell cycles of the nucleolus through a process referred to as embryonic nucleologenesis. As described earlier, fibrillogranular nucleoli were absent in porcine embryos during the first and second postfertilization cell cycles when large electron-dense fibrillar spheres were observed [11, 24–26]. However, during the third cell cycle the nuclei displayed different stages of transformation of the fibrillar spheres into fibrillogranular nucleoli. Later, during the fourth cell cycle, the fibrillogranular portions of the nucleoli became more extensive and reticulated. Similar to previous findings, different stages of nucleolus development were observed within the same nucleus [11]. This rate of nucleolus development is in accordance with the onset of autoradiographic labeling of the blastomere nucleoplasm and nucleoli observed during the third cell cycle [11] (present study).
Embryonic nucleologenesis shows a species-specific pattern. In the porcine embryo, the fibrillogranular nucleolus emerges by the formation of FCs, DFC, and GC on the surface of the fibrillar sphere that can be considered as a nucleolar anlage. In the bovine embryo, where the formation of fibrillogranular nucleoli occurs during the fourth cell cycle, the FCs and DFC develop in the periphery of the fibrillar sphere and along the rim of vacuoles formed inside the sphere [12, 13, 16]. With respect to the morphological aspects of nucleologenesis, the porcine embryo is more similar to the murine embryo [27, 28] than to the bovine.
In order to drive transcription and subsequent processing of the rRNA, the nucleolus requires a panel of proteins to accommodate a number of specific functions [14]. Information is scarce on the protein composition of the nucleolus in the preimplantation mammalian embryo. In the blastocyst, in which fully functional nucleoli are present, it is reasonable to believe that the protein compartment is in accordance with that known from somatic cell nucleoli. How this critical protein compartment is established during the process of embryonic nucleologenesis is, however, to a great extent unknown.
Basically, the present experiment resulted in two different patterns of immunocytochemical labeling: A homogeneous labeling of the nucleoplasm and a labeling confined to the presumptive nucleolar compartment. The latter labeling pattern can directly be related to the nucleolar tasks of the different proteins in rRNA transcription and processing, whereas the significance of the nucleoplasmic labeling remains an enigma. Due to the fact that the nucleoplasmic labeling was never observed in the control specimens where the primary antibody was omitted, notes on this labeling pattern are included in this paper in a descriptive form.
A prerequisite for transcription of the rRNA genes is that the supercoiled DNA must be uncoiled. This process is mediated by topoisomerase I [29] that has been localized to the FCs and DFC [14]. In the porcine embryo this protein was observed in the presumptive nucleoli for the first time early during the fourth cell cycle. The actual transcription of the rRNA genes is driven by RNA polymerase I, which as well has been localized to the FCs and the DFC [14]. A dispersed nucleoplasmic labeling for RNA polymerase I was observed in the blastomeres at the midpoint of the third cell cycle coinciding with the formation of the fibrillogranular nucleoli. Toward the end of the third cell cycle, however, the labeling was concentrated to foci that associated to form a shell-like structure, probably representing the FCs and DFC developing on the surface of the nucleolar anlage. During the subsequent cell cycles, the presumptive fibrillar components of the nucleoli displayed a pronounced labeling for RNA polymerase I in accordance with the establishment of functional fibrillogranular nucleoli. The chronological and spatial correlation between the ultrastructural detection of the FCs and the DFC, and the labeling of RNA polymerase I is in accordance with observations in cattle in which RNA polymerase I labeling is chronologically and spatially closely related to the formation of fibrillogranular nucleoli during the fourth cell cycle [16]. In contrast in the mouse, RNA polymerase I was not immunocytochemically localized until the third cell cycle, whereas the rRNA transcription in this species apparently is initiated during the second cycle [30].
Upstream binding factor is one of several transcription factors required for the binding of RNA polymerase I to the DNA [31]. Upstream binding factor is thought to bind to the promoter and to recruit another transcription factor, the promoter selectivity factor (SL1) [32], thus forming a preinitiation complex to which RNA polymerase I can bind and initiate transcription [33]. In porcine embryos, dispersed nucleoplasmic labeling excluding the presumptive fibrillar spheres was noted during the first and third cell cycle. Subsequently, during the fourth cycle, however, the labeling shifted to small foci that associated to form a shell-like structure compatible with the localization to the presumptive FCs and DFC as reported earlier for other cell types [14]. The detection of UBF prior to that of RNA polymerase I is in agreement with the sequential function of the two proteins during the transcription processes. Accordingly, in bovine embryos, UBF was localized to solitary foci presumably identical with the spherical nucleolar anlages already from the first cell cycle and during the fourth cycle, when fibrillogranular nucleoli are formed in this species, UBF relocalized to more complex structures compatible with a localization to FCs and DFC [16].
Fibrillarin was first identified by human autoimmune sera from patients with scleroderma and was localized to the FCs and DFC [20, 34]. Fibrillarin is a small nucleolar ribonucleoprotein (snoRNP) associated with U3 small nucleolar RNA (snoRNA) [35, 36] and with U8 and U13 snoRNAs [37, 38]. The snoRNAs are involved in the processing of the primary rRNA transcript [39–42]. In porcine embryos a dispersed nucleoplasmic labeling of fibrillarin excluding the presumptive fibrillar spheres was immunocytochemically detected during the first, second, and third cell cycles. From the fourth cell cycle and onward, however, the labeling shifted to small foci that associated to form a shell-like structure compatible with a localization to the presumptive FCs and DFC. In bovine embryos fibrillarin was immunocytochemically localized to solitary foci presumably identical with the spherical nucleolar anlages already from the first cell and during the fourth cycle, when fibrillogranular nucleoli are formed in this species, fibrillarin relocalized to more complex structures compatible with a localization to FCs and DFC [26].
Nucleolin is a phosphorylated protein present in large amounts in nucleoli with active ribosomal biogenesis [43]. It has RNA binding properties [44], and furthermore, it is identical to the human DNA helicase IV that unwinds RNA-RNA, DNA-DNA, and DNA-RNA duplexes [45]. The protein is associated with the primary rRNA transcripts, in particular the 18S and 28S sequences. Nucleolin may act in promoting the functional secondary structures of the 18S and 28S RNA that are necessary for the assembly of the preribosomal particles [46]. However, it is not part of the final product [47]. Nucleolin has been localized to the DFC and the GC [48]. Nucleophosmin may be involved in shuttling other proteins, such as nucleolin [49] and the nucleolar protein p120 [50], to the nucleolus. However, nucleophosmin also has DNA and RNA binding properties [51], ribonuclease activity [52], and associates with the most mature nucleolar preribosomal RNP [53]. Therefore, it has been proposed that nucleophosmin, together with nucleolin, functions in the assembly of preribosomal particles. Nucleophosmin has been localized to the DFC and the GC [48]. In porcine embryos, a dispersed nucleoplasmic labeling of nucleolin excluding the presumptive fibrillar spheres was observed during the initial three cell cycles. The same applied for nucleophosmin except for the labeling of the periphery of the presumptive fibrillar spheres during the first cycle. During the fourth cycle, when fibrillogranular nucleoli had been established, however, the labeling shifted to a shell-like pattern compatible with labeling of all components of the nucleolus. In bovine embryos the two proteins were localized to solitary nuclear foci at least two cell cycles before the formation of fibrillogranular nucleoli at which time the pattern shifted to a large shell-like structure compatible with labeling of GC preferentially [16].
The nucleolar proteins investigated in the present study may be divided into two categories: Proteins such as RNA polymerase I, UBF, and topoisomerase I that are characterized by a well-defined action during transcription, while others such as fibrillarin, nucleolin, and nucleophosmin are mainly characterized by their spatial localization within the nucleolus. It was demonstrated that the immunocytochemical detection of RNA polymerase I chronologically and spatially coincided with the formation of the structural elements of the functional fibrillogranular nucleolus and the onset of RNA transcription in porcine embryos during the third cell cycle. However, the absence of labeling for topoisomerase I during this cell cycle remains an enigma and may be related to the limitation of the immunocytochemical sensitivity.
In addition to the differences in the ultrastructural transformation of the nucleolar anlage to a fibrillogranular nucleolus between porcine and bovine embryos, there are also marked differences with respect to the localization of some of the nucleolar proteins. Thus, in bovine embryos fibrillarin, UBF, nucleolin, and nucleophosmin were localized to distinct nuclear foci several cell cycles before fibrillogranular nucleoli were established [16]. The same proteins were also found in porcine embryos in the cell cycles preceding the formation of fibrillogranular nucleoli, but at a dispersed nucleoplasmic distribution the significance of which remains an enigma. On the other hand, the detection of RNA polymerase I and topoisomerase I appears to be closely linked chronologically and spatially with the formation of the fibrillar components of the nucleoli in both species. Similar to our observations in bovine embryos [16], some proteins were immunocytochemically detected during the first cell cycle but were undetectable during the second and in certain instances the initial phase of the third cycle before they were redetected during the third or fourth cell cycle. The proteins detected during the first cell cycle are believed to be of maternal origin. The proteins detected later during development may be of embryonic origin on the background of translation of either maternal or embryonic mRNAs. On the other hand, the redetection may also result from relocalization of existing pools of maternal proteins to local higher concentrations.
In conclusion, a major activation of the transcription of the embryonic genome including the activity of the rRNA genes as visualized by the formation of a fibrillogranular nucleolus occurs during the third postfertilization cell cycle. Concomitantly, or at least from the onset of the fourth cell cycle a number of specific nucleolar proteins can be localized to the nucleolar compartment. This line of research is worth developing, both in order to understand mammalian embryonic development as well as for establishing an alternative model to somatic cell nucleologenesis in basic research on nuclear and nucleolar biology.
ACKNOWLEDGMENTS
The authors are grateful to Ms. Jytte Nielsen, Birgit Sieg, and Antje Frenzel for excellent technical assistance.
FOOTNOTES
1 This project was supported by the Bundesministerium für Wissenschaft und Verkehr der Republik Österreich GZ 45.023/1-III/B/8/98, Danish Agricultural and Veterinary Research Council, and through an Alexander von Humboldt fellowship. 
2 Correspondence: Poul Hyttel, Department of Anatomy and Physiology, Royal Veterinary and Agricultural University Groennegaardsvej 7, DK-1870 Frederiksberg C, Denmark. FAX: 45 3528 2547; poh{at}kvl.dk
Accepted: August 4, 2000.
Received: April 12, 2000.
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