Human Reproduction Vol.18, No.3 pp. 576±582, 2003
DOI: 10.1093/humrep/deg114
Abnormal assembly of annulate lamellae and nuclear pore
complexes coincides with fertilization arrest at the
pronuclear stage of human zygotic development
V.Y.Rawe1,4, S.Brugo Olmedo1, F.N.Nodar1, R.Ponzio2 and P.Sutovsky3
1
Centro de Estudios en GinecologõÂa y ReproduccioÂn, CEGyR, Buenos Aires, 2Centro de Investigaciones en ReproduccioÂn, Facultad
de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina and 3Departments of Obstetrics and Gynecology, and
Animal Science, University of Missouri±Columbia, S141 ASRC, 920 E Campus Drive, Columbia, MO 65211±5300, USA
4
To whom correspondence should be addressed at: Pittsburgh Developmental Center of Magee±Women's Research Institute and
Departments of Obstetrics±Gynecology±Reproductive Sciences and Cell Biology and Physiology, University of Pittsburgh,
204 Craft Ave, Pittsburgh, PA 15213, USA. E-mail: [email protected]
BACKGROUND: The assembly of nuclear pore complexes (NPC) and their cytoplasmic stacks, annulate lamellae
(AL), promote normal nucleocytoplasmic traf®cking and accompany pronuclear development within the mammalian zygote. Previous studies showed that a percentage of human oocytes fertilized in vitro failed to develop normal
pronuclei and cleave within 40±48 h post insemination. We hypothesized that an aberrant recruitment of NPC
proteins, nucleoporins and/or NPC preassembled into AL, might accompany human fertilization arrest. METHODS
AND RESULTS: We explored NPC and AL assembly in unfertilized human oocytes, and fertilized and arrested
zygotes by immuno¯uorescence with an NPC- and AL-speci®c antibody, mAb 414, and by transmission electron
microscopy. Major NPC or AL assembly was not observed in the unfertilized human oocytes. Once fertilization
took place, the formation of AL was observed throughout the cytoplasm and near the developing pronuclei with
NPC. On the contrary, NPC assembly was disrupted in the arrested zygotes, whereas AL were clustered into large
sheaths. This was accompanied by the lack of NPC incorporation into the nuclear envelopes. CONCLUSIONS: We
conclude that the aberrant assembly of NPC and AL coincides with early developmental failure in humans.
Key words: fertilization failure/human zygotes/nuclear envelope/nuclear pore complex/pronuclear development
Introduction
One of the most striking features of the cell cycle is the stagespeci®c separation and blending of the nuclear and cytoplasmic
compartments. The nuclear envelope (NE) of mitotic cells
dissolves at the entry point of the M-phase (Newport and
Spann, 1987) and reforms at M-exit, or interphase entry
(Pfaller et al., 1991). Three steps are necessary for NE
assembly (Boman et al., 1992; Macaulay and Forbes, 1996).
First, membrane vesicles, which may contain receptors for the
proteins of nuclear lamina (Collas et al., 1996), assemble
around the chromatin and eventually fuse to form a continuous
NE around it (Burke and Gerace, 1986; Pfaller et al., 1991).
Second, the nuclear pore complexes (NPC), assembled from
the speci®c set of O-glycosilated glycoproteins called nucleoporins (Davis and Blobel, 1986, 1987; Hanover et al., 1987;
Snow et al., 1987), are incorporated into the NE and provide
the channels for bi-directional nucleocytoplasmic traf®cking,
between the newly established nuclear and cytoplasmic
compartments (Benavente et al., 1989; reviewed by PanteÂ
and Aebi, 1993; GoÈrlich and Mattaj, 1996). Third, nuclear
576
lamins are imported into the nucleus and form the scaffold of
nuclear lamina underneath the NE (Newport et al., 1990).
The assembly of the NE following mammalian fertilization
starts with membrane-free chromatin (SzoÈllosi et al., 1972;
Longo, 1973) represented by two distinct entities: a set of
maternal chromosomes and the sperm nucleus. Subsequently,
the assembly of the NE takes place in a fashion similar to that
seen in somatic cells, regardless of whether the sperm entered
the oocytes during IVF or ICSI (Sutovsky et al., 1997, 1998).
Although the assembly and fusion of membrane vesicles
during NE formation have been studied extensively in somatic
cells and in a cell-free system (reviewed by Poccia and Collas,
1996), little is known about the pathways leading to the
formation of NPC on the zygotic NE, especially in mammalian
models. Previous studies in bovine have demonstrated that the
fertilizing sperm triggers the assembly of annulate lamellae
(AL), in parallel with NPC insertion into NE (Sutovsky et al.,
1998). AL, the cytoplasmic stacks of NPC (reviewed by
Kessel, 1992), cross-react with a nucleoporin-speci®c antibody
mAb 414 in Xenopus egg extracts (Meier et al., 1995) and in
ã European Society of Human Reproduction and Embryology
Nuclear pore complex in human zygotes
mammalian pronuclear zygotes (Sutovsky et al., 1998;
Ramalho-Santos et al., 2000).
Despite the continuous improvement of IVF techniques,
several levels at which fertilization can fail have been
described (Asch et al., 1995; Rawe et al., 2000). The arrest
at two-pronuclei (2PN) stage is one of frequently seen patterns.
Although AL have been found in human metaphase II oocytes
(Baca and Zamboni, 1967) and pronuclear zygotes (Hertig and
Adams, 1967; Zamboni et al., 1966; Van Blerkom et al., 1987),
it is not known how and when NPC are incorporated into NE of
developing human pronuclei, or if NPC and/or AL play any
role during human fertilization and zygotic development.
In the present study we used immuno¯uorescence (IF) with
an NPC- and AL-speci®c antibody, mAb 414, to visualize the
assembly of NPC and AL in the fertilized and arrested human
zygotes. Ultrastructural features of fertilized and arrested
human zygotes were characterized by electron microscopy
(EM).
Materials and methods
Patients and treatments
A total of 75 unfertilized, supernumerary, fertilized and arrested
human oocytes and zygotes were studied using IF and transmission
electron microscopy (TEM). The studied material was obtained from
couples that had signed an informed consent undergoing IVF and ICSI
in the Assisted Fertilization Programme of CEGyR, Buenos Aires,
Argentina, where all studies were performed. Fertilized zygotes
described as controls were supernumerary, discarded zygotes de®ned
by the development of two pronuclei (2PN) ~16 h after insemination
or sperm injection. At that time-point, these supernumerary zygotes
had to be discarded upon the request of consenting donors. In the
CEGyR programme, ~96% of the 2PN zygotes undergo mitosis.
Supernumerary, pronuclear embryos that came from cycles with 100%
cleavage rate were used as controls. Therefore, such embryos were
considered to be normally fertilized. Couples that donated such
zygotes indeed achieved pregnancy. Arrested oocytes/zygotes
developed two apparently normal PN and did not cleave after ~40 h
post insemination or sperm injection. Two of the studied arrested
zygotes appeared to be normally fertilized and were cryopreserved in a
previous attempt. These 2PN embryos did not cleave after 24 h and
remained at PN stage. All unfertilized oocytes and fertilized zygotes
used were discarded upon the request of consenting donors. Ethics
committees of participating groups in this work have approved all
procedures. Wives' ovarian stimulation and IVF or ICSI procedures
were performed as previously described (Rawe et al., 2000).
Nucleoporin and chromatin labelling
Nine prophase I (PI), 11 metaphase I (MI) and 10 metaphase II (MII)
human oocytes were screened after the labelling of nucleoporins and
chromatin by conventional epi¯uorescence microscopy. At the same
time, a total of 21 supernumerary fertilized zygotes (11 from IVF and
Figure 1. Nuclear pore complex (NPC) labelling in non-inseminated oocytes, visualized by immuno¯uorescence with nucleoporin-speci®c
antibody mAb 414. (A) Continuous ring of NPC on the nuclear envelope of a human germinal vesicle (GV) stage oocyte. Diffuse,
background labelling and some auto¯uorescent particles are seen in human oocytes after germinal vesicle breakdown (B; metaphase I) and at
the second metaphase arrest (C; metaphase II). (D) Negative control labelling of a GV stage oocyte by the omission of the ®rst antibody,
mAb 414. (E) Negative control of a metaphase II oocyte, DNA counterstaining is shown in E¢. Scale bars = 10 mm.
577
V.Y.Rawe et al.
10 from ICSI) and 13 arrested fertilized zygotes (PN formation
without further cleavage after ~40 h of insemination or injection, six
from IVF and seven from ICSI) were screened for nucleoporins and
chromatin labelling using a modi®ed protocol of Messinger and
Albertini (1991). For this purpose, zona pellucida was removed by a
brief incubation with acid Tyrode's medium, and denuded oocytes/
zygotes were ®xed and permeabilized for 20 min at 37°C in a
microtubule stabilizing buffer (0.1 mol/l PIPES, pH 6.9, 5 mmol/l
MgCl2´6H2O, 2.5 mmol/l EGTA containing 2.0% formaldehyde, 0.5%
Triton X-100, and 1 mmol/l taxol). Fixed oocytes/zygotes were
blocked for 1 h at 37°C with 2% bovine serum albumin (BSA), 2%
powdered milk, 2% normal goat serum, 0.1 mol/l glycine and 0.01%
Triton X-100 in PBS. If necessary, oocytes/zygotes were stored for up
to 3 days at 4°C in this solution. To identify NPC, oocytes were
incubated for 40 min at room temperature with 1:250 dilution of
antibody mAb414 (BabCo; Berkeley, CA, USA) (Davis and Blobel,
1986, 1987; Meier et al., 1995; Sutovsky et al., 1998) in PBS
containing 0.1% BSA and 0.02% sodium azide (PBS + BSA). After a
short wash in blocking solution, oocytes were incubated in 1:500
dilution of ¯uorescein-conjugated goat anti-mouse immunoglobulin G
(IgG), for 1 h at room temperature. Finally, treated oocytes were
washed three times in PBS + BSA, counterstained with Hoechst 33258
(1 mg/ml) for 30 min at room temperature, washed again in PBS +
BSA and mounted between a microscopy slide and a coverslip for
examination using Olympus BX-40 epi¯uorescent microscope.
Images were photographed using Kodak Ektachrome ®lm
(1600ASA) and processed using Adobe Photoshop 5.0 software
(Adobe System Inc.). For control staining, PBS + BSA alone replaced
the speci®c antibody solution. Secondary antibodies and reagents used
were purchased from Sigma (St Louis, MO, USA).
Transmission electron microscopy
Six arrested zygotes (2PN stage) and ®ve control zygotes were ®xed
for 1 h in a ®xative composed of 2.5% glutaraldehyde and 0.6%
paraformaldehyde in 0.25 mol/l cacodylate buffer (pH 7.2), then
washed in 0.1 mol/l cacodylate buffer containing 0.2 mol/l sucrose and
post-®xed for 1 h in 1% osmium tetroxide. Following dehydration by
ascending ethanol series (30±100%), oocytes were in®ltrated by a
series of washes in a mixture of propylene oxide and Polybed 812
(Polyscience, Warrington, PA, USA) EM resin and embedded in
PolyBed 812. Ultrathin sections were cut on a Porter±Blum Sorvall
MT2B ultramicrotome and stained in two steps with uranyl acetate
and lead citrate. Thin sections were examined and photographed in a
Zeiss EM 9A electron microscope (Zeiss, Oberkochen, Germany).
Negatives were scanned and printed using Adobe Photoshop 5.0
software.
Results
Nucleoporin and DNA patterns in the non-inseminated
oocytes
All studied oocytes were informative after processing the
material. The nuclei (germinal vesicles, GV) of human
prophase I oocytes displayed a continuous ring of NPC
labelling on their NE, while some auto¯uorescence and
occasional punctate-speci®c ¯uorescence were found in the
cytoplasm (Figure 1A). After the resumption of meiosis (GV
breakdown) the nuclear envelope labelling disappeared and a
diffuse background-like labelling with occasional punctate
auto¯uorescence in the cytoplasm was seen in the MI and MII
oocytes (Figure 1B, C respectively). The numbers of oocytes
578
and zygotes studied as well as the labelling patterns are
summarized in Table I.
Distribution of nucleoporins and DNA in fertilized zygotes
Normal fertilization
A distinct punctate pattern of labelling with mAb 414 appeared
throughout the zygotic cytoplasm after 16±18 h of insemination and/or sperm injection (Figure 2A), suggestive of the
assembly of cytoplasmic AL. In most cases, this cytoplasmic
labelling was accompanied by the presence of NPC in the
nuclear envelope of the fertilized zygotes (Figure 2A, Table I).
The presence of a bright signal on the surfaces of both
pronuclei can be seen in Figure 2A.
Arrested fertilization
Fertilization arrest at 1-cell stage coincided with an abnormal
distribution of NPC and AL (Figure 2B, Table I). A differential
pattern of AL assembly was seen in the cytoplasm of these
arrested zygotes compared with controls. AL formed large
patches assembled near the pronuclei (Figure 2C and C¢),
which in some cases became fragmented into multiple small
karyomeres (Figure 2B). No differences were seen in the
abnormal distribution pattern observed between IVF and ICSI
zygotes. It is noteworthy that two of the studied fertilized
zygotes were cryopreserved in a previous attempt. These
zygotes were normally fertilized, i.e. formed two pronuclei of
normal appearance. Once thawed, these 2PN zygotes did not
cleave after 24 h and remained at PN stage. The patterns
observed in this material were identical to the previously
observed clustering of AL near both pronuclei (Figure 2D
and D¢). Surprisingly, while the arrested zygotes showed
two pronuclei in light microscopy (Figure 2 E), and their
overall appearance was similar to control 2PN zygotes
(Figure 2F), epi¯uorescence revealed DNA condensation and
fragmentation.
Transmission electron microscopy of control and arrested
zygotes
To provide more support for the observations of fertilized
oocytes using IF, we explored the distribution of NPC and AL
by EM. At the ultrastructural level (Figure 3), the presumed
normally fertilized zygotes displayed small stacks of AL
Table I. Fluorescence patterns of unfertilized and fertilized oocytes studied
after nucleoporin labelling
Category
Number
NPC labelling pattern
GV oocytes
MI oocytes
MII oocytes
Normal zygotes
9
11
10
21
Arrested zygotes
13
Regular ring of NPC on NE
Diffuse cytoplasmic/background
Diffuse cytoplasmic/background
Mostly peri-pronuclear/punctate
pattern of AL in cytoplasm
Large cytoplasmic clumps
Normal fertilized zygotes were de®ned by the development of two pronuclei
(2PN) ~16 h after IVF and ICSI. Arrested fertilized zygotes developed two
apparently normal PN and did not cleave within 40 h post IVF or ICSI.
NPC = nuclear pores complexes; NE = nuclear envelope; GV = prophase
I/germinal vesicle-stage oocytes; MI = metaphase I oocytes; MII =
metaphase II oocytes; PN = pronuclei; AL = annulate lamellae.
Nuclear pore complex in human zygotes
Figure 2. Nuclear pore complex (mAb 414/NPC) and DNA con®guration in control and arrested oocytes. (A) Normally fertilized zygote at
the time of pronuclear apposition. Note the punctate pattern of annulate lamellae labelling throughout the cytoplasm, and a distinct labelling
of the nuclear envelope of both male and female pronuclei. (B) Large assemblies of NPC near the decondensed pronuclei after fertilization
arrest (B, C and C¢). (D and D¢). Note that the nuclear envelopes are not labelled. A similar pattern of aberrant NPC and annulate lamellae
distribution is seen in a fertilized zygote arrested at the pronuclear apposition stage after cryopreservation and thawing. Scale bars = 10 mm in
A, B, C and D, and 5 mm in C¢ and D¢. (E) Phase contrast micrograph of an arrested human zygote 40 h after insemination. (F) A control
zygote 16 h after insemination.
distributed through the cytoplasm and adjacent to the NE
(Figure 3A). In contrast, large assemblies of AL were
predominantly found in the vicinity of the male and female
pronuclei in the arrested zygotes (Figure 3B, C). An interesting
feature of arrested zygotes was the presence of numerous
mitochondria with accumulation of the electron-dense material
in mitochondrial matrix (Figure 3C). On serial ultrathin
sections, such mitochondria were found to be distributed
evenly throughout the zygotic cytoplasm, but were not detected
in the control zygotes. In the supernumerary fertilized zygotes
(controls), the EM study revealed an apparently normal
pronuclear development (Figure 3D). While the NE of such
zygotes contained numerous NPC (Figure 3E), the number of
NPC was greatly diminished in the arrested zygotes (Figure 3F,
579
V.Y.Rawe et al.
Figure 3. Transmission electron microscopy of normal and arrested 2-pronuclear oocytes. (A) Small stacks of cytoplasmic annulate lamellae
(al) are seen near the nuclear envelope (ne) and distributed through the cytoplasm of a control, fertilized zygote. (B) Annulate lamellae in the
cytoplasm near the pronucleus (pn) of a zygote that failed to divide within 40 h after insemination. Large clumps of annulate lamellae are
found in the vicinity of the developing pronuclei inside an arrested zygote (C). Note the presence of numerous mitochondria with
accumulation of electron-dense material (arrows) not seen in control zygotes (see A). Control fertilized zygotes show an apparently normal
pronuclear development (D) and regions of nuclear envelope containing nuclear pore complex (NPC) (E). Large areas of continuous nuclear
envelope without NPC were observed in the arrested zygotes (F, inset). Internalization of annulate lamellae is seen in the pronucleus (F,
arrows). Scale bars = 20 nm in A, B, D and E and 1 mm in C.
Table II. Electron microscopic evaluation of normal and arrested zygotes
Category
Number
Findings
Normal zygotes
5
Arrested zygotes
6
AL homogeneously distributed in cytoplasm
NPC-incorporated NE
AL clustered in cytoplasm
Absence of NPC from the NE
AL internalization into both pronuclei
AL = annulate lamellae; NPC = nuclear pores complexes; NE = nuclear
envelope.
Table II). Internalization of AL into both pronuclei, described
previously (Zamboni et al., 1966; Van Blerkom and Motta,
1989) in presumably normal zygotes, and the presence of NPCfree stretches of NE were recorded in the arrested zygotes
(Figure 3F), but not in the control ones.
580
Discussion
The present results indicate that the assembly of cytoplasmic
AL in humans is triggered by fertilization and accompanies
pronuclear development. Although seldom observed in GV
stage, MI and MII oocytes, the AL were assembled in the
oocyte cytoplasm after sperm penetration. This event was
evidenced by the presence of punctate labelling of cytosolic
AL, recognized by mAb 414 (Meier et al., 1995; Sutovsky
et al., 1998) after fertilization. Accordingly, AL have been
described in human oocytes at all stages ranging from GV
oocytes to pronuclear zygotes (Adams and Hertig, 1965;
Zamboni et al., 1966; Baca and Zamboni, 1967; Hertig and
Adams, 1967; Van Blerkom and Motta, 1989). Based solely on
ultrastructural observations, these studies did not allow for a
comparison of AL density in the fertilized versus non-fertilized
oocytes. An abnormal male and/or female PN development,
observed after ICSI in rhesus monkey oocytes (Sutovsky et al.,
Nuclear pore complex in human zygotes
1996), is also accompanied by accumulation of AL and
reduced recruitment of NPC to the male pronuclear NE
(Ramalho-Santos et al., 2000). Related to the deviant turnover
of AL in the arrested zygotes could be the absence of NPC from
large areas of pronuclear NE. This irregular NPC insertion
pattern was not seen in the control, fertilized zygotes.
While the arrested zygotes showed 2PN in light microscopy,
and their overall appearance was similar to control 2PN
zygotes (Figure 2E, F), epi¯uorescence revealed DNA condensation and fragmentation in the arrested zygotes. It is
possible that DNA within the pronuclei of arrested zygotes
became fragmented and condensed while the NE remained
intact, thus maintaining `normal' appearance in light microscopy. This is supported by the observation that all arrested
zygotes screened by immuno¯uorescence showed 2PN con®gurations indistinguishable from normal zygotes when examined by light microscopy. DNA fragmentation and
disappearance of NPC from NE of the arrested zygotes could
be signs of abortive apoptotic process.
Previous studies suggested that NPC are involved in
selective nucleocytoplasmic transport in the pronuclear bovine
zygotes (Sutovsky et al., 1998). The anomalies of AL assembly
were shown to accompany fertilization arrests after ICSI in
rhesus monkey (Ramalho-Santos et al., 2000). NPC are
removed from the sperm NE during spermatid elongation in
the testis (Sutovsky et al., 1999) and the NE of mature sperm is
removed completely during fertilization in order to make the
sperm chromatin accessible to oocyte cytoplasmic factors
necessary for the remodelling of the sperm nucleus (Perreault
et al., 1984; Poccia and Collas, 1996; Sutovsky et al., 1996,
1997; Sutovsky and Schatten, 1997, 2000; Usui et al., 1997;
Wright, 1999).
After fertilization arrest at 1-cell stage, the AL- and NPCspeci®c antibody mAb 414 (Meier et al., 1995; Sutovsky et al.,
1998) revealed the accumulation of AL in the cytoplasm of
arrested zygotes. AL formed large patches assembled near the
fragmented pronuclei, as previously observed in some rhesus
monkey ICSI oocytes (Sutovsky et al., 1996; Ramalho-Santos
et al., 2000). It is possible that the pronuclear development and
nucleocytoplasmic communication were impaired in these
zygotes. Consequently, the pronuclear development was not
completed and the mitotic division had not taken place. This is
consistent with the observation that the microinjection of an
NPC antagonist, wheat germ agglutinin, prevented embryo
development in bovine (Sutovsky et al., 1998). Similarly, the
S-phase entry was blocked in rhesus monkey embryos with
deviant PN and AL appearance after ICSI (Ramalho-Santos
et al., 2000). Studies of Xenopus oocytes suggest that one of the
NPC components, RanBP2 (also called Nup358), is a key
factor in the interphase nucleocytoplasmic transport, but also in
the control of mitotic events, including spindle assembly
during metaphase and the reformation of the NE during
telophase (Greber and Carafoli, 2002).
Alternatively, the abnormal biogenesis or function of the
zygotic centrosome, documented previously in some cases of
human fertilization failure (Asch et al., 1995; Van Blerkom,
1996), could affect the recruitment of NPC and cytoplasmic
traf®cking of AL in some of the arrested, fertilized zygotes,
mainly in those not showing pronuclear apposition. The
nocodazole-induced disruption of the microtubules within
fertilized bovine oocytes was indeed shown to derail the
pronuclear recruitment of NPC, PN development and cytoplasmic movement of AL in fertilized bovine oocytes
(Sutovsky et al., 1998).
EM studies con®rmed the pathological reorganization of
NPC and AL in arrested zygotes, previously observed by IF.
Large assemblies of AL have been found in the cytoplasm of
arrested 2PN zygotes, while smaller stacks of AL were seen in
the control ones. A similar pattern of AL assembly was
observed by IF in normally fertilized zygotes that did not
cleave after cryopreservation/thaw. Cytoskeletal damage
caused by cryopreservation (Sathananthan et al., 1988; Van
der Elst et al., 1992; Park et al., 1997; Wei-Hua et al., 2001;
Boiso et al., 2002) may affect the pattern of AL distribution.
The association of AL with microtubules has been demonstrated previously (reviewed by Kessel, 1992; Sutovsky et al.,
1998). The electron-dense deposits observed in the mitochondria throughout the cytoplasm of arrested zygotes were
reminiscent of calcium phosphate deposits in preimplantation
embryos of mouse t-mutant (Hillman and Tasca, 1973). In our
studies, such mitochondria were distributed randomly throughout the cytoplasm of arrested zygotes, and could be suggestive
of apoptotic process, known to start with alterations of the
mitochondrial membrane and matrix. Furthermore, it might be
interesting to speculate that the release of activated caspases by
these abnormal mitochondria could lead to the digestion of
many cellular proteins responsible for cell cycle regulation,
DNA damage recognition and repair and regulation of the
cellular structure (for review see Nalepa and ZukowskaSzczechowska, 2002).
The results presented in this study document that the patterns
of NPC and AL distribution in human oocytes that failed to
fertilize do not mimic those seen in normal zygotes. Such
observations suggest a differential distribution of NPC and AL
in the normal and arrested pronuclear zygotes. These ®ndings
have implications for understanding the aetiology and cellular
basis of early developmental failure in humans.
Acknowledgements
Authors would like to thank to Dr ArquõÂmedes Bolondi, GermaÂn
Galaverna, Isabel Farias, Blanca LoÂpez, Alberto Valcarcel and
Gustavo MartõÂnez for their technical assistance and dedication in
collecting the selected material. We gratefully acknowledge Miriam
Sutovsky and Angela George for clerical assistance. CEGyR
Foundation has supported this work.
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Submitted on May 17, 2002; resubmitted on August 30, 2002; accepted on
November 19, 2002