"I n vivo" Occurrence of Premature Chromosomes Condensation in
_??_ 1992 The Japan
Cytologia 57: 377-381
n vivo" Occurrence of Premature ChromosomesCondensation
in Kidney Cells of Eigenmannia virescens
de Morfologia, Instituto de Biociencias , Universidade
Paulista "Julio de Mesquita Filho", Botucatu, Sao Paulo, Brasil
, Instituto de Biociencias, Universidade
de Sao Paulo, Sao Paulo, Brasil
of Cell Biology, The University of Texas,
M. D. Anderson Hospital and Tumor Institute Houston, TX, 77030, U. S. A.
Accepted March 5, 1992
The technique of premature chromosome condensation (PCC) allows the visualization
of chromosomes in G1, S and G2 interphase nuclei when such cells are fused with a mitotic
metaphase cell. Johnston and Rao (1970) demonstrated that the fusion of a mitotic metaphase
with an interphase cell resulted in the breakdown of the nuclear membrane and the subsequent
premature condensation of the interphase chromatin.
PCC has been used to study the chro
mosome constitution of nondividing cells and in this way, to visualize insults on genetic material
induced by Xray-radiation of mammalian spermatids that otherwise do not divide (Drwinga
et al. 1979). The phenomenon of PCC has been extensivelly studied in virally and/or chemi
cally-induced multinucleate cells "in vitro" (Johnston et al. 1970, Ikeuchi 1973) and "in vivo"
(Kurten and Obe 1975, Yamamoto et al. 1986). Most of these studies were only restricted
to mammalian cells. Spontaneous cell fusions were first described between tumoral cells (Barski
et al. 1961, Atkin 1979) and also in cultured lynphocytes of two sibs with Bloom's syndrome
(Otto and Therman 1982), which is the first description of spontaneous fusion between non
malignant somatic human cells.
The present communication reports recent observations on the spontaneous occurrence
of PCC in metaphase spreads from the kidney cells of two out of five specimens of a fish,
Eigenmannia virescens (2n=38) manteined for fifteen days before sacrifice in aquaria which
was verified to be strongly infested by a ciliate protozoan, Ichthyophthirius multifiliis.
The chromosomal analyses were made on cytological preparations from five specimens of
a fish, Eigenmannia virescens belonging to the family Sternopygidae, collected in the Mogi
Guacu river, at Pirassununga (SP), Brasil. The specimens were maintained for fifteen days in
well aerated aquaria in the laboratory till sacrifice for chromosome preparations.
time two specimens presented a very strong infestation of a ciliate protozoan, Ichthyophthirius
multifiliis, distributed over fish bodies like white velvet spots.
Chromosome preparations were made from kidney cells according to the procedure de
scribed by Foresti et al. (1981). In order to obtain appropriate chromosome condensation
for karyotypic analysis, the concentration of colchicine solution was reduced to 0.05% (1ml/
100g body weight) and the animals were killed 50 to 60 min after intraperitonealy drug
and Sen Pathak
Typical diploid and tetraploid metaphase plates are shown in Figs. la and 1b, respectively.
The diploid chromosome number in this species is 2n=38 as described by Foresti et al. (1981).
Fig. 1. Diploid (a) and tetraploid (b) metaphase spreads of Eigenmannia virescens, 2n=38, show
ing typical chromosome morphology. Autosomes carrying secondary constrictions in their short
arms are marked by arrows.
Table 1. Frequency of normal and PCC type cells found in Eigenmannia virescens. Two
out of five specimens were infested by the ciliate protozoan, Ichthyophthirius multifiliis
Each diploid metaphase spread is characterized by a single pair of autosomes with secondary
constriction in the short arms (arrows in Fig. la). In 266 metaphase spreads examined in two
infested specimens of E. virescens, polyploid like mitosis were encountered in 8 cells (3.01%)
and 41 (15.41%) metaphase cells showed a typical PCC chromosome configuration (Fig. 2).
"In vivo" Occurrence
In such cases, two types of chromosome morphology , one condensed with double chromatids
and other elongated with double and single elements were observed. Most of these cells
showed G1 and G2 types of PCC morphology . PCC of the S type cells were not so frequently
seen in the sample of cells analysed (Table 1). Otherwise, the tetraploid cells found in the
Fig. 2. Mitotic spreads depicting G1-PCC (a and b), late S-phase PCC (c), and possible G2-PCC
(d and e) configurations. Note the presence of secondary-constrictions in well condensed chro
mosomes of mitotic morphology.
be the result
of a fusion
Other than PCC, no additional
though the exact mechanism
by which PCC and tetraploid
cells were formed is unknow,
could possibly throw some light on it.
and Sen Pathak
The fishes in whose chromosome preparations PCC were observed, were invariably infest
ed with a ciliate protozoan, Ichthyophthirius multifiliis. Not a single PCC type cell morphol
ogy was observed in preparations from healthy and noninfected specimens of the same fish
species maintained at the same laboratory conditions. Could it be possible that this protozoan
is acting as a fusogen in the host cells? If so, then in addition to causing disease, it might be able
to fuse cells situated at different points of cell cycle. Also, the frequency of tetraploid cells
in non infested specimens was very small, i.e. 1 tetraploid cell (0.25%) in 396 normal diploid
cells anlysed, suggesting that such cell could not be the result of a cell fusion process but the
effect of colchicine injected in healthy fishes during the treatment for developing chromosome
preparations. We have not ruled out the possibility that these fishes also could be infected
with some kind of virus which might be acting as a fusogen in their host cell as pointed by
Johnston and Rao (1970). One might also speculate that the species of protozoa which is
infesting these fishes or even the virus might be producing some kind of chemical into fish
bodies which could be acting similarly to polyethilenoglicol (PEC) and inducing cell fusions
and PCC (Drwinga et al. 1979). At least it could be possible that these two specimens that
presented PCC might be old fishes in which the occurrence of tetraploidy seems to be a pre
visible event. Besides these speculations, the real answer to this "in vivo" PCC occurrence
in fishes should await for further research.
The occurrence of premature chromosome condensation (PPC) was demonstrated in
cytological preparations developed in two out of five specimens of a fish, Eigenmannia virescens,
2n=38, analysed after a period they were maintained in laboratory conditions.
mens in which PCC occurred and tetraploid cells had come about by cell fusions were invariab
ly infested with a ciliate protozoan, Ichthyophthirius multifiliis. It is speculated that in some
way the infestation of fish with this organism may cause cell fusions in the body of the hosts
resulting into PCC and tetraploidy.
The author are grateful to Drs. Priscila G. Otto and Yatyio Y. Yassuda (Universidade
de Sao Paulo, SP, Brasil) for their suggestions.
Funds supporting this study were provided
by CNPq, FAPESP and FINER
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