266
Mitotic analysis of the North American white
sturgeon, Acipenser transmontanus Richardson
(Pisces, Acipenseridae), a fish with a very high
chromosome number
A.L. Van Eenennaam, J.D. Murray, and J.F. Medrano
Abstract: The average chromosome number of the North American white sturgeon, Acipenser transmontanus Richardson,
was found to be 271 ± 2.5 (ranging from 265 to 276). This number is significantly higher than previous estimates for this
species. A representative karyotype was found to consist of 132 meta- and submeta-centric chromosomes, 44 acrocentric
chromosomes, and 98 microchromosomes. An improved C-banding technique revealed variation (2–7) between animals in the
number of entirely heterochromatic metacentric chromosomes. These heterochromatic chromosomes may represent
supernumerary chromosomes. There was no cytogenetic evidence of a heteromorphic sex chromosome pair or any sex-related
chromosomal polymorphism in either sex of this species.
Key words: C-banding, fish, karyotype, supernumerary chromosomes, white sturgeon.
Résumé : Le nombre moyen de chromosomes chez l’esturgeon blanc d’Amérique du Nord, Acipenser transmontanus
Richardson, s’élève à 271 ± 2,5 (varie de 265 à 276). Ce nombre est significativement plus élevé que les estimations
précédentes chez cette espèce. Un caryotype représentatif est constitué de 132 chromosomes méta- ou subméta-centriques, de
44 chromosomes acrocentriques et de 98 microchromosomes. Une technique améliorée de coloration des bandes C a permis
de révéler une variation (2–7) quant au nombre de chromosomes métacentriques entièrement hétérochromatiques d’un animal
à un autre. Ces chromosomes hétérochromatiques pourraient représenter des chromosomes surnuméraires. Aucune évidence
cytogénétique d’une paire de chromosomes sexuels hétéromorphes ou de tout autre polymorphisme chromosomique relié au
sexe n’a été observée chez l’un ou l’autre sexe de cette espèce.
Mots clés : coloration des bandes C, poisson, caryotype, chromosomes surnuméraires, esturgeon blanc.
[Traduit par la Rédaction]
Introduction
Some of the most ancient living ray-finned fish, sturgeon and
paddlefish, belong to the order Acipenseriformes. Karyotypes
of the sturgeon and paddlefish are characterized by a very large
number of chromosomes, about half of which are microchromosomes.2 The order can be divided into two groups: the first
group has a chromosome number of approximately 120 and the
second group has a chromosome number of 240–250 (Birstein
Corresponding Editor: C.B. Gillies.
Received August 29, 1997. Accepted January 14, 1998.
A.L. Van Eenennaam1 and J.F. Medrano. Department of
Animal Science, University of California, Davis,
CA 95616-8521, U.S.A.
J.D. Murray. Department of Animal Science and Department
of Population Health and Reproduction, University of
California, Davis, CA 95616-8521, U.S.A.
1
2
Author to whom all correspondence should be addressed
(email: [email protected]).
For an excellent summary of Acipenseriformes karyotypes see
the following URL on the internet:
http://dns.unife.it:/geneweb/sturgeon.html
Genome, 41: 266–271 (1998)
et al. 1993; Blacklidge and Bidwell 1993). Various authors
have claimed that the first group is of tetraploid origin and the
second group is of octoploid origin (Ohno et al. 1969; Burtzev
et al. 1976; Dingerkus and Howell 1976; Birstein and Vasiliev
1987). No extant Acipenseriformes species has been found to
have a diploid number of 60. Early Russian papers suggested
that certain sturgeon species have a chromosome number of 60
(Serebryakova 1969; Burtzev et al. 1976), but it seems that the
microchromosomes were not included in these counts. Cytogenetic studies have not revealed heteromorphic sex chromosomes in any sturgeon species (Fontana and Colombo 1974;
Holcík 1986; Van Eenennaam et al. 1998a), although genetic
evidence suggests that white sturgeon may have a genetic sex
determination system with female heterogamety (Van Eenennaam et al. 1998b).
There is often considerable variation (±8) in the chromosome number reported for each species. This has been variously attributed to chromosome loss or fragmentation during
slide preparation, owing to the strong hypotonic shock needed
to avoid superimposition, the inclusion of cellular fragments
or artifacts resembling microchromosomes in chromosome
counts, overlapping of chromosomes leading to undercounting, difficulty in resolving the numerous dot-like microchromosomes, and actual counting errors (Dingerkus and Howell
1976; Vasiliev et al. 1980; Fontana et al. 1996; Gorshkova
© 1998 NRC Canada
267
Van Eenennaam et al.
Table 1. Individual fish and average chromosome numbers (including various numbers of
entirely heterochromatic chromosomes) obtained from eight California white sturgeon
(Acipenser transmontanus Richardson).
Heterochromosomesb
Mean number
Sex
Na
of chromosomes
SD
Range
Na
Male
Malec
Male
Male
Female
Female
Female
Femalee
Total
6
14
11
2
13
12
3
14
75
270
271
271
273
267
271
272
274
271
2.3
1.6
1.7
0.7
1.1
2.0
1.2
1.2
2.5
267–272
268–273
268–273
272–273
265–269
268–275
271–273
272–276
265–276
20
20
20
20
20
20
20
20
160
No.
3–4
3
5
6–7
2d
2
5–6
6
2d–7
a
N, number of metaphase spreads counted to determine chromosome or heterochromosome number.
Entirely heterochromatic (C-band positive) metacentric chromosome.
c
Male shown in Figs. 2a and 2b.
d
One of the two heterochromosomes had only one heterochromatic arm.
e
Female shown in Figs. 1, 2c, and 2d.
b
et al. 1996). A few authors have postulated that some of the
chromosome-number variation may be attributed to the presence of supernumerary chromosomes (Vasiliev et al. 1980;
Birstein and Vasiliev 1987; Sola et al. 1994). C-banding has
been reported for only three sturgeon species (Sola et al. 1994;
Fontana et al. 1996; Rab et al. 1996a), including a preliminary
study on white sturgeon that found that a variable number
(1–7) of small macrochromosomes in each metaphase spread
were entirely heterochromatic.
The chromosome number of white sturgeon exported from
North America to Italy during the 1980s has been reported to
be 248 ± 8 (Fontana 1994), with the karyotype including
52 metacentric and submetacentric pairs of chromosomes and
74 pairs of acrocentric chromosomes and microchromosomes.
Hedrick et al. (1991) found a modal chromosome number of
219 in a spleen-cell line from this species and a bimodal distribution with modes of 237 and 243 in a heart-cell line. These
chromosome numbers contrast with that which might be predicted based on the meiotic synaptonemal complex (SC) count
of 139 ± 3.4 for a California white sturgeon population (Van
Eenennaam et al. 1998a). Here we report a mitotic chromosome study that was undertaken: (i) to determine the chromosome number of and develop a mitotic karyotype for a
California white sturgeon population, (ii) to examine for the
presence of entirely heterochromatic (C-band positive heterochromatin) chromosomes in metaphase spreads, and (iii) to
critically appraise metaphase spreads from both sexes for the
presence of a heteromorphic sex chromosome pair. An improved and rapid C-band positive heterochromatin staining
technique for this species is also described.
Materials and methods
Synchronized lymphocyte cultures were prepared using modifications of a protocol obtained from F. Fontana (personal communication). Blood from each fish was collected from the caudal vein in a
5-mL sodium heparin vacutainer. Plasma supernatant was removed
from the tube and the remaining blood was washed once by mixing
with 2 mL of phosphate buffered saline (PBS (pH 7.2); Gibco BRL,
Gaithersburg, Md.). The tubes were then centrifuged (500 rpm,
10 min), and 8 drops of the buffy coat were removed and gently
mixed with 8 mL of Dulbecco’s modified Eagle medium (DMEM;
Gibco BRL), 1.5 mL of heat-inactivated fetal bovine serum, 200 µL
of PSN antibiotic mixture (5 mg penicillin, 5 mg streptomycin, plus
10 mg neomycin/mL; Gibco BRL), 200 µL phytohemagglutin
(PHA-M; Gibco BRL), and 100 µL pokeweed (Gibco BRL) in a
25-mL tissue culture flask and incubated at 25°C with 8% CO2 for
5 days. On the afternoon of the fourth day, 100 µL of 10 µM methotrexate (10–7 M final concentration) was added to the culture to block
DNA replication and, after a further 16–18 h, 100 µL of 1 mM
thymidine (10–5 M final concentration) was added to release the
methotrexate block. Harvest was initiated 4 h later by exposing the
cells to 100 µL of colcemid solution (10 µg/mL; Gibco BRL) for 4 h.
Cells were then separated from the culture medium by centrifugation
(500 rpm, 10 min), hypotonized (30 min in 10 mL of 0.075 M KCl),
and fixed in freshly prepared fixative (3:1 (v/v) methanol – acetic acid).
Slides of metaphase chromosome spreads were prepared from 8
domestically reared white sturgeon derived from wild San Francisco
Bay (California) broodfish of known sex (4 % and 4 &), using standard procedures. For conventional karyotyping, the slides were stained
for 20–30 min with a 4% Giemsa solution (pH 6.8) (Bio/medical
Specialities Inc., Santa Monica, Calif.). Staining of constitutive heterochromatin (C-banding) was achieved following the method of
Sumner (1972), using slides that had been dessicated for at least
3 days and a 15–30 s Ba(OH)2 incubation. Slides were then either
incubated in 2× SSC (1× SSC: 0.15 M NaCl plus 0.015 M sodium
citrate) at 60°C for 1h, rinsed in distilled H2O, allowed to air-dry,
stained with a 4% Giemsa solution for 60–90 min, and examined with
a light microscope (Olympus BH–2) or they were stained for 10 min
in propidium iodide (400 ng/mL in 2× SSC), rinsed for 2 min in 2×
SSC, mounted in 2 drops of DAPCO antifade stock (2.3% 1,4diazabicyclo-(2,2,2-octane) in glycerol), and observed under the
same microscope equipped for epifluorescence microscopy. Chromosomes recorded in photographs of discrete well-spread cells were
counted to determine the chromosome number, and good quality
spreads were scanned and arranged according to length and morphology, using ADOBE PHOTOSHOP software (Adobe Systems Inc., Mountain
View, Calif.), to produce a mitotic karyotype.
Results
Photographs of 75 discrete spreads (2–14 per fish) were
counted to determine the chromosome number (Table 1). We
© 1998 NRC Canada
268
Genome, Vol. 41, 1998
Fig. 1. California white sturgeon (Acipenser transmontanus Richardson) karyotype (2n = 274). The meta- and submeta-centric chromosomes
are aligned in order of declining size, followed by the acrocentric chromosomes and microchromosomes (×2800).
found that the diploid chromosome number of white sturgeon
was 271 ± 2.5. Figure 1 shows a representative mitotic karyotype (2n = 274) from a female fish. This karyotype consists of
132 meta- and submeta-centric chromosomes, 44 acrocentric
chromosomes, and 98 microchromosomes. These numbers are
somewhat arbitrary, as it is difficult to differentiate between
small macrochromosomes and microchromosomes. The size
of the chromosomes decreases in a rather continuous pattern
and approaches the resolution limits of the light microscope.
Karyotypes of male and female fish did not appear to differ
from each other, and there was no evidence of a heteromorphic
sex chromosome pair.
C-banded metaphase spreads from a male and a female
white sturgeon stained with either Giemsa or propidium iodide
are shown in Fig. 2. We observed that the C-banding pattern
was the same regardless of which stain was used, however it
was easier to identify the C-band positive heterochromatin using the propidium iodide staining method. Twenty C-banded
propidium iodide stained cells from each fish were examined
to determine the number of entirely heterochromatic chromosomes (Table 1). There were a variable number (2–7) of het-
erochromatic chromosomes in the eight fish examined in this
experiment. In three of the fish, one of the heterochromatic
chromosomes stained slightly less brightly than the others, and
it could most clearly be classified as heterochromatic in
spreads where the chromosomes were somewhat condensed.
Therefore a range of values is listed in Table 1 for the number
of heterochromatic chromosomes found in these three fish.
This should not be interpreted to mean that we observed withinanimal variation in the number of heterochromatic chromosomes. It merely reflects the fact that one of the heterochromosomes in these fish sometimes stained less intensely,
depending upon the degree of chromosome condensation.
Discussion
The white sturgeon chromosome number that we observed,
271 (±2.5), is significantly higher than the previously published value of 248 (±8) (Fontana 1994) reported for a population
derived from North American white sturgeon first imported
into Italy as juveniles in 1981. Fish originating from both
the San Francisco Bay (California) and Columbia River
© 1998 NRC Canada
269
Van Eenennaam et al.
Fig. 2. C-banded metaphase spreads from male (a and b) and female (c and d) white sturgeon (Acipenser transmontanus) stained with
Giemsa (a and c) or propidium iodide (b and d). This male had 3 entirely heterochromatic chromosomes (arrowheads) and the female had
6 (a, ×1700; b, ×1000; c, ×975; d, ×1250).
(Washington/Oregon) white sturgeon populations were included in this exported group.3 In the Italian study, there were
52 meta- and submeta-centric pairs of chromosomes and
3
Ken Beer, The Fishery, Galt, Calif., personal communication.
74 pairs of acrocentric chromosomes and microchromosomes
in the karyotype. Using the same categorization, the present
study found 66 meta- and submeta-centric pairs of chromosomes and 71 pairs of acrocentric chromosomes and microchromosomes. This difference can be partially attributed to the
© 1998 NRC Canada
270
subjective classification that is required to categorize small
meta-, submeta-, and acro-centric macrochromosomes and
microchromosomes. It was not possible to karyotypically identify the position of the additional pairs of chromosomes that
were found in the present study.
One very clear difference between the karyotypes presented
in the two studies is the size of the largest acrocentric chromosome pair. The largest acrocentric chromosome pair found in
the Italian study was approximately the 7th largest pair in the
karyotype, whereas in the present study it was approximately
the 20th largest pair. We also found that the karyotype of a
white sturgeon (2n = 271) derived from the Columbia River
population was similar to the one reported here for California
white sturgeon (preliminary unpublished data). Our data
agrees with the meiotic SC karyotype for California white sturgeon in which the first acrocentric SC was found to be approximately the 20th element (Van Eenennaam et al. 1998a). The
chromosome number found in this study also better agrees
with the chromosome number that might be predicted from the
average white sturgeon SC number, i.e., 139 × 2 = 278. A
variable number of the SCs included in the meiotic count (1–7)
were univalents, implying that this value of 278 is actually a
slight (1–7) overestimate of the true chromosome number of
the California white sturgeon. Overall, these data strongly suggest that there are chromosome number and karyotypic differences between the Californian and Italian white sturgeon
populations. These differences do not appear to be related to
the origin of the white sturgeon broodfish that founded the two
domestic populations.
Counting a large number of discrete cells from each individual allowed us to detect chromosome number variation between animals (Table 1). Part of this variation can be explained
by the variable number (2–7) of entirely heterochromatic chromosomes found in different individuals. Within each individual the number of heterochromatic chromosomes was
consistent, whereas there was clear between-animal variation.
The heterochromatic chromosomes were most commonly
small metacentric chromosomes (Fig. 2). Supernumerary
chromosomes tend to be entirely heterochromatic in most organisms (Jones and Rees 1982), and it is tempting to speculate
that the heterochromatic chromosomes seen in this study are
supernumerary chromosomes, as was suggested by Sola et al.
(1994). It may be that these heterochromatic chromosomes
were the source of the variable number (1–7) of univalents
seen in meiotic SC spreads derived from a different group of
California white sturgeon males (Van Eenennaam et al. 1998a).
Supernumerary chromosomes have been reported in other animals (Switonsky et al. 1987; Fletcher and Hewitt 1988; Del
Cerro et al. 1994), including fish (Salvador and Moreira-Filho
1992; Andreata et al. 1993). It would be interesting to examine
the metaphase I behavior of these heterochromatic chromosomes.
We consistently obtained good quality spreads using the
lymphocyte culture protocol outlined in Materials and methods.
We had difficulty staining C-band positive heterochromatin
with Giemsa, and found that using propidium iodide as the
stain allowed us to better visualize the C-banding patterns
(Fig. 2). In addition, we found that when using propidium iodide as the stain, the hot salt solution incubation following
denaturation (2× SSC, 1h, 60°C) could be omitted, allowing
the length of time it took to complete the C-banding procedure
Genome, Vol. 41, 1998
to be reduced. Aside from the entirely heterochromatic chromosomes, C-band positive heterochromatin was located
mainly in the centromeric region of small to medium macrochromosomes and microchromosomes. Large metacentric
chromosomes exhibited weak or no C-banding in the centromeric region.
Gold et al. (1990) state that fluorochromes may be used to
resolve C-bands, provided the heterochromatic regions are differentially rich in AT or CG base pairs relative to the remainder
of the chromatin. In this study, we resolved C-bands with
propidium iodide, which does not preferentially bind to either
GC- or AT-rich DNA (Saitoh and Laemmli 1994). It seems
likely that the propidium iodide was fluorescing more brightly
in regions of DNA that had not been fully denatured by treatment with barium hydroxide, regardless of the base-pair content. A similar use of propidium iodide staining to detect
nucleolus organizer regions in fish chromosomes has recently
been reported (Rab et al. 1996b).
C-banding and karyotypic analyses did not reveal a heteromorphic sex chromosome pair or any sex-related chromosomal polymorphism in white sturgeon. An analysis of
spermatocyte SCs also showed no evidence of a heteromorphic
sex chromosome pair in males (Van Eenennaam et al. 1998a).
Similarly, in a series of fluorescence in situ hybridization
(FISH) experiments, where labeled male and female white
sturgeon genomic DNA was hybridized to metaphase chromosome spreads of each sex, no chromosome or chromosome arm
was seen to be specifically hybridizing only to genomic DNA
from the same sex (Van Eenennaam 1997). Overall, these results suggest that the sex chromosomes of white sturgeon are
at a very early stage of differentiation, so that they appear
homomorphic and cannot be detected by cytogenetic analyses.
Recent genetic evidence suggests that white sturgeon may
have a female heterogametic genetic sex determination system
(Van Eenennaam et al. 1988b). It would therefore be of considerable interest to examine SCs from oocytes of this species,
to determine if there is evidence of a ZW bivalent exhibiting
the atypical pairing behavior characteristically associated with
heteromorphic sex chromosomes.
Acknowledgments
The authors thank L.V. Millon and F. Fontana for helpful suggestions regarding sturgeon lymphocyte culture and
J.P. Van Eenennaam for technical assistance with sample collection. This research was funded by a grant from the National
Sea Grant College Program, National Oceanic and Atmospheric Administration (NOAA), United States Department of
Commerce, under grant NA36RG0537, project R/A–99,
through the California Sea Grant College System, and in part
by the California State Resources Agency. The views expressed herein are those of the authors and do not necessarily
reflect the views of NOAA or any of its subagencies. The
United States Government is authorized to reproduce and distribute this work for governmental purposes. A.L. Van Eenennaam was supported by a Sea Grant traineeship.
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