On-line version ISSN 1678-4685
Genet. Mol. Biol. vol. 21 n. 4 São Paulo Dec. 1998
Use of lymphocyte cultures for BrdU replication banding patterns in anuran species (Amphibia)
Sanae Kasahara, Ana Paula Zampieri Silva and Simone Lilian Gruber
Departamento de Biologia, Instituto de Biociências, Universidade Estadual Paulista (UNESP), Campus de Rio Claro, Caixa Postal 199, 13506-900, Rio Claro, SP, Brasil. Send correspondence to S.K. E-mail: firstname.lastname@example.org
We describe the standardization of lymphocyte culture procedures in order to improve cytological preparations of anuran species. This methodology permits the use of 5-bromodeoxyuridine (BrdU) treatment to obtain replication banding patterns in the chromosomes of these species.
The development of cytogenetic studies on amphibians is mainly due to improved procedures for obtaining cytological preparations as well as to new techniques for differential chromosome staining. Squashing- or spreading-fixed cells, two direct methods developed in early studies of amphibian cytogenetics, are commonly used in Brazil due to their easy and rapid execution and low cost. However, these methods have some limitations such as a low mitotic index and poor quality of metaphase chromosomes that are generally too condensed or overlapped. These characteristics prevent good resolution of banding patterns, which are indispensable in current cytogenetic studies.
Another important point is that, contrary to warm-blooded mammals and birds, amphibian chromosomes as well as those of fishes and most reptiles generally do not display multiple euchromatic bands when submitted to the usual G, Q or R banding procedures. A possible explanation is based on the high homogeneity of DNA base pair composition, which cannot be compartmentalized into AT-rich or GC-rich bands as in other vertebrates (Medrano et al., 1988; Schmid et al., 1990). Nevertheless, it has been shown that good longitudinal chromosome differentiation in cold-blooded vertebrates, including fish, amphibian and lizard groups, may be obtained with incorporation of 5-bromodeoxyuridine (BrdU) during DNA replication following in vivo or in vitro treatments (Schempp and Schmid, 1981; Kuro-o et al., 1986; Kasahara et al., 1987; Almeida-Toledo et al., 1988; Yonenaga-Yassuda et al., 1988). Although both procedures have been successfully used, in vitro treatment during cell culture is advantageous for better control of BrdU treatment.
In order to improve the cytogenetic analysis of anuran specimens, mainly regarding induction of BrdU replication banding, we standardized the lymphocyte culture in our laboratory as a relatively easy methodology for obtaining amphibian chromosome preparations.
MATERIAL AND METHODS
The lymphocyte culture technique was applied to representatives of the families Bufonidae, Hylidae and Leptodactylidae. Samples were collected from various sites in the State of São Paulo and other Brazilian states, as part of a project entitled "Cytogenetics of Anuran Amphibians".
The following solutions were used: heparin Liquémine Roche, 5000 IU/ml; 4 x 10-5 M colchicine solution (Cultilab); 5-bromodeoxyuridine (BrdU) and 5-fluorodeoxyuridine (FudR) stock solutions respectively containing 10 mg of BrdU and 0.5 mg of FudR in 2 ml of 0.9% NaCl solution, stored frozen in dark vials; 0.075 M KCl hypotonic solution; freshly prepared methanol:acetic acid fixative (3:1).
The following cell culture media were used: TC 199-Earle Salts (Cultilab): 100 ml supplemented with 20 ml fetal bovine serum (Cultilab) and 5 ml of phytohemagglutinin (Cultilab); RPMI 1640 (Cultilab): 100 ml supplemented with 20 ml fetal bovine serum (Cultilab) and 5 ml of phytohemagglutinin (Cultilab); MEM supplemented with fetal bovine serum and phytohemagglutinin, available in aliquots of 5 ml (Meio Cariótipo Cultilab); Amphibian Culture Medium (Gibco): 100 ml supplemented with 5 ml of phytohemagglutinin (Cultilab) and antibiotics (Cultilab).
All culture medium supplements, except MEM, were prepared in our laboratory. All were divided into aliquots of 2.5 ml or 5 ml and stored at -20°C.
Animals were anesthetized with chloroform or ether and fixed on a dissection plate. The thorax and abdominal surfaces were thoroughly disinfected with 70° alcohol before dissection. The blood sample was obtained with a heparinized syringe by puncturing the ventricle of the beating heart.
Blood sample volume varied according to animal size. In general 0.2 or 0.3 ml samples were obtained, but exceptionally up to 2.5 ml was collected. For example, larger blood sample volumes were obtained from adult specimens of Leptodactylus labyrinthicus, L. ocellatus, Phyllomedusa distincta, and Bufo paracnemis, who are generally medium or large sized.
Blood samples from some L. labyrinthicus representatives were obtained without sacrificing the specimens by direct heart puncture according to the procedure described by Agostinho (1994). In these cases, a maximum of 1 ml of blood was collected.
The syringe with the blood sample was allowed to stand vertically just long enough to separate an adequate amount of lymphocyte-rich plasma. The needle was then bent, and this layer plus the interface layer between the plasma and the red blood cells were transferred to a sterilized flask. After gently shaking, about 3 to 5 drops of the mixture were inoculated into 2.5 to 5 ml complete medium.
When less than 0.5 ml of blood was obtained, the whole sample was immediately inoculated into the culture medium at a concentration of approximately 3 drops or less per 2.5 ml.
Culture tubes were incubated at 26 or 30°C, for 3 to 4 days.
Cell harvesting and slide preparations
Two drops of colchicine were added to each tube containing 5 ml cell culture 90 min before harvesting. For replication banding, the stock solution of BrdU plus FudR was added to each culture at a final concentration of 100 µg/ml, 6 to 15 h before completing the culture time. Tubes were incubated in the dark by wrapping with black paper or aluminum foil in order to prevent photodegradation of BrdU and colchicine treatment was applied for 60 min.
Following colchicine treatment, cell suspensions were centrifuged at 800 to 1000 rpm for 7 min, the supernatant discarded, and the pellet dissociated with a Pasteur pipette prior to the addition of 5 ml pre-warmed KCl hypotonic solution. Cells were resuspended in this solution and then incubated at 37°C for 45 min.
Pre-fixation was performed at room temperature by first adding 6 drops of ice-cold fresh fixative prepared with methanol plus acetic acid (3:1). Cells were then resuspended gently by tapping the tube, and after 5 min, another 0.5 ml of fixative was added. Cells were resuspended with a pipette, and after 5 min a new centrifugation was performed to remove the supernatant. Cells were subjected to two or more changes of fresh fixative.
Following the last fixative change, the pellet was resuspended in a small volume of fixative according to the amount of cells. Next, one or two drops of the cell suspension were deposited on a clean slide kept horizontally on a wire support, inside a water bath at 60°C. The slide was placed 0.5 cm above water level and removed from the water bath as soon as the cell drop covered its surface, in order to avoid excessive heating. Slides were air dried at room temperature and after 24 h were ready for conventional and differential staining techniques.
Fluorochrome plus Giemsa staining
BrdU replication bands were differentiated according to the fluorochrome plus Giemsa (FPG) technique of Perry and Wolff (1974), modified by Dutrillaux and Couturier (1981).
One- or two-day aged slides were stained with 10 µg/ml Hoechst 33258 solution for 20 min, in the dark, at room temperature. After briefly rinsing in distilled water and 2x SSC solution, the slides were mounted in this saline solution and incubated in a moist chamber prepared on a Petri dish with 2x SSC wet filter paper and covered with plastic wrap.
Slides were irradiated with black light at a distance of approximately 10 cm from the source for 2 h, rinsed with distilled water to remove the coverslips, and then incubated in 2x SSC at 60°C for 20 min.
After rinsing with distilled water, the slides were stained with 2% Giemsa prepared in Na+ and K+ phosphate-buffered solution, pH 6.8, for 7 min.
RESULTS AND DISCUSSION
The standardization of anuran species lymphocyte culture described here was based on amphibian cytogenetic reports (Doyle and Beckert, 1970; Griffin et al., 1970; Schmid, 1978; Beck and Mahan, 1979; Matsui et al., 1985; Miura, 1994; Nishioka et al., 1994) and basically follows the steps described in the technique of Moorhead et al. (1960) for obtaining human chromosomes. Four major questions investigated are discussed below.
The first concerns blood samples for cultivation. It was observed that better results were obtained regarding amount of cell suspension and mitotic index when lymphocyte-rich plasma was cultivated. Possibly, more lymphocytes are inoculated into the culture than when whole blood is used, allowing greater cell multiplication. However, the procedure of separating lymphocyte-rich plasma from red blood cells was not always applicable, mainly for small-sized animals, which generally provided only a few drops of blood.
The second question under investigation was the most suitable culture medium. Undoubtedly Amphibian Culture Medium (Gibco) produced the best results due to its specificity for amphibian cells. Good results were also obtained with MEM medium (Meio Cariótipo Cultilab) followed by TC199-Earle Salts (Cultilab) and RPMI (Cultilab).
Time of cultivation, the third question investigated, may be highly variable. Good results were obtained with 3- or 4-day cultures, but there were no time restrictions, and culture could be continued up to 7 days.
The fourth question concerns optimal culture temperature. Two temperatures were tested based on reports in the literature (Doyle and Beckert, 1970; Griffin et al., 1970; Schmid, 1978; Beck and Mahan, 1979; Matsui et al., 1985; Miura, 1994; Nishioha et al., 1994), in which amphibian cultures were incubated at 25, 26, 27 and 30°C. Both 26 and 30°C seem to be equally satisfactory for cell growth since good cell division was observed in aliquots cultivated at either temperature.
It is important to note that, in spite of working under very simple laboratory conditions (laminar flow not available), a low index of bacterial contamination was observed.
Lymphocyte culture procedures yielded good chromosome preparations when applied to some representatives of Bufonidae, Hylidae and Leptodactylidae regarding mitotic index and quality of metaphase spreads. This was very important for improving data on routine differential staining techniques, such as C-banding or Ag-NOR impregnation, because of the larger number of suitable metaphases produced when compared to that obtained by direct methods.
The standardization of lymphocyte culture methodology in our laboratory represented, above all, an important step in overcoming difficulties in obtaining conventional euchromatic G, Q or R banding patterns in anuran species. More refined banding techniques using BrdU incorporation could be successfully applied to induce reproducible replication bands in several amphibian species.
Figures 1, 2, and 3 show BrdU-banded karyotypes of Phyllomedusa distincta (Hylidae), Bufo paracnemis and a specimen of Bufo with an intermediate phenotype between B. paracnemis and B. ictericus (Bufonidae). Multiple euchromatic bands were demonstrated on large or medium-sized chromosomes allowing the unequivocal pairing of homologues. Small-sized chromosomes also presented some longitudinal differentiation but this was not always sufficient for a precise identification of each pair. The results obtained here for two representatives of the family Bufonidae are of higher quality than those reported by Kasahara et al. (1996) for B. crucifer. Chromosomes of this animal, which was submitted to in vivo BrdU treatment, exhibited only vestiges of banding.
Figure 1 - Banded karyotype of a male Phyllomedusa distincta (2n = 26) after BrdU treatment.
Figure 2 - Banded karyotype of a female Bufo paracnemis (2n = 22) after BrdU treatment.
Figure 3 - Banded karyotype of a male specimen of Bufo (2n = 22) with an intermediate phenotype between Bufo paracnemis and B. ictericus after BrdU treatment.
Comparison of BrdU banding patterns of the two Bufo specimens in the present study shows that apparently there are no differences in the major bands between the two karyotypes, which are also indistinguishable when preparations are submitted to conventional staining.
Figure 4 A, B, C, and D shows results obtained for Leptodactylus labyrinthicus and L. ocellatus (family Leptodactylidae), Aparasphenodon brunoi and Phyllomedusa distincta (family Hylidae), with different levels of resolution. A number of metaphases of these species exhibited chromosomes with medium-resolution banding along their lengths. Rare prometaphases observed in the cytological preparations occasionally presented high-resolution banded chromosomes. Undoubtedly, the introduction of a lymphocyte culture methodology in our laboratory and its use for obtaining BrdU replication bands represent an important step toward better chromosome characterization and more reliable comparative cytogenetic studies of anuran species.
Figure 4 - BrdU replication banding with different levels of resolution. A, Leptodactylus labyrinthicus (2n = 22); B, L. ocellatus (2n = 22); C, Aparasphenodon brunoi (2n = 24); D, Phyllomedusa distincta (2n = 26).
Lymphocyte culture methodologies are more accessible than those that use fibroblast cultures, which require equipment or installations not always available in laboratories. Another significant advantage is the possibility of collecting blood samples directly in the field, thereby preserving the life of wild specimens.
The authors are grateful to Dr. Célio F.B. Haddad, Dr. Rita Maria Ladeira Pires and Luciano M. Castanho for providing some of the specimens used in this study. The collaboration of Miss Carmem Silvia Mengardo was greatly appreciated.
This work was supported by CNPq, CAPES and FAPESP. Publication supported by FAPESP.
Descreve-se a padronização da cultura de linfócitos com a finalidade de melhorar as preparações citológicas de espécies de anfíbios anuros. Esta metodologia permite o uso do tratamento pelo BrdU para se obter padrões de bandas de replicação nos cromossomos dessas espécies.
Agostinho, C.A. (1994). Caracterização de populações de rã-pimenta Leptodactylus labyrinthicus (Spix, 1824) e avaliação de seu desempenho em cativeiro. Doctoral thesis, Centro de Ciências Biológicas e da Saúde/Universidade de São Carlos, São Carlos, SP. [ Links ]
Almeida-Toledo, L.F., Viegas-Péquignot, E., Foresti, F., Toledo Filho, S.A. and Dutrillaux, B. (1988). BrdU replication patterns demonstrating chromosome homoeologies in two fish species, genus Eigenmannia. Cytogenet. Cell Genet. 48: 117-120. [ Links ]
Beck, M.L. and Mahan, J.T. (1979). Ammoniacal silver staining of nucleolar organizer regions in four species of Bufo. Copeia 2: 341-345. [ Links ]
Doyle, B.W. and Beckert, W.H. (1970). Chromosome characteristics of the Bufonidae among species and within population. Caryologia 23: 143-154. [ Links ]
Dutrillaux, B. and Couturier, J. (1981). La Pratique de L'Analyse Chromosomique. Masson, Paris, pp. 86. [ Links ]
Griffin, C.S., Scott, D. and Papworth, D.G. (1970). The influence of DNA content and nuclear volume on the frequency of radiation-induced chromosome aberration in Bufo species. Chromosoma 30: 228-249. [ Links ]
Kasahara, S., Yonenaga-Yassuda, Y. and Rodrigues, M.T. (1987). Geographical karyotypic variations and chromosome banding patterns in Tropidurus hispidus (Sauria, Iguanidae). Caryologia 40: 43-57. [ Links ]
Kasahara, S., Silva, A.P.Z. and Haddad, C.F.B. (1996). Chromosome banding in three species of Brazilian toads (Amphibia-Bufonidae). Braz. J. Genet. 19: 237-242. [ Links ]
Kuro-o, M., Ikebe, C. and Kohno, S. (1986). Cytogenetic studies of Hynobiinae (Urodela). IV. DNA replication bands (R-banding) in the genus Hynobius and the banding karyotype of Hynobius nigrescens Stejneger. Cytogenet. Cell Genet. 43: 14-18. [ Links ]
Matsui, M., Seto, T., Kohsaka, Y. and Borkin, L.J. (1985). Bearing of chromosome C-banding patterns on the classification of Eurasian toads of the Bufo bufo complex. Amphibia-Reptilia 6: 23-33. [ Links ]
Medrano, L., Bernardi, G., Couturier, J., Dutrillaux, B. and Bernardi, G. (1988). Chromosome banding and genome compartmentalization in fishes. Chromosoma 96: 178-183. [ Links ]
Miura, I. (1994). Sex chromosome differentiation in the Japanese brown frog, Rana japonica. I. Sex related heteromorphism of the distribution pattern of constitutive heterochromatin in chromosome No. 4 of the Wakuya population. Zool. Sci. 11: 797-806. [ Links ]
Moorhead, P.S., Nowell, P.C., Mellman, W.J., Battips, D.M. and Hungerford, D.A. (1960). Chromosome preparations of leukocytes cultured from human peripheral blood. Exptl. Cell Res. 20: 613-616. [ Links ]
Nishioka, M., Hanada, H., Miura, I. and Ryuzaki, M. (1994). Four kinds of sex chromosomes in Rana rugosa. Sci. Rep. Lab. Amphib. Biol. Hiroshima Univ. 13: 1-34. [ Links ]
Perry, P. and Wolff, S. (1974). New Giemsa method for the differential staining of sister chromatids. Nature 251: 156-158. [ Links ]
Schempp, W. and Schmid, M. (1981). Chromosome banding in Amphibia. VI. BrdU replication patterns in Anura and demonstration of XX/XY sex chromosomes in Rana esculenta. Chromosoma 83: 697-710. [ Links ]
Schmid, M. (1978). Chromosome banding in Amphibia. I. Constitutive heterochromatin and nucleolus organizer regions in Bufo and Hyla. Chromosoma 66: 361-388. [ Links ]
Schmid, M., Steinlein, C., Nanda, I. and Epplen, J.T. (1990). Chromosome banding in Amphibia. In: Cytogenetics of Amphibians and Reptiles (Olmo, E., ed.). Birkhäuser Verlag, Basel, pp. 21-45. [ Links ]
Yonenaga-Yassuda, Y., Kasahara, S., Chu, T.H. and Rodrigues, M.T. (1988). High-resolution RBG-banding pattern in the genus Tropidurus (Sauria, Iguanidae). Cytogenet. Cell Genet. 48: 68-71. [ Links ]
(Received March 3, 1998)