Luiz A. C. Bertollo, Michel S. Fontes, Alberto S. Fenocchio & Jesus Cano

Transcription

1 Chromosome Research 1997, 5, 493±499 The X 1 X 2 Y sex chromosome system in the sh Hoplias malabaricus. I. G-, C- and chromosome replication banding Luiz A. C. Bertollo, Michel S. Fontes, Alberto S. Fenocchio & Jesus Cano Received 11 June 1997; received in revised form and accepted for publication by M. Schmid 29 July 1997 Hoplias malabaricus, a widely distributed neotropical sh (Central America to Argentina), may represent a group of distinct species showing diversi ed cytotypes with respect to chromosome number, morphology and sex systems. One of these karyotypic forms is characterized by an X 1 X 1 X 2 X 2 =X 1 X 2 Y sex chromosome system, with 2n = 40 and 39 chromosomes in females and males respectively. Analyses with G-, C- and chromosome replication banding permitted a better characterization of the sex chromosomes in this cytotype. The Y chromosome, unique in males, resulted from a translocation event between two biarmed chromosomes: one similar to chromosome 6 (X 1 ) and the other one similar to chromosome 20 (X 2 ), the latter corresponding to a probable identi- cation. On the basis of the observed banding patterns, the Y chromosome may represent a stable dicentric, with an inactive centromere interstitially located on its long arm. The results are also related to a speci c satellite DNA subfamily, previously characterized in Hoplias malabaricus, which appears to be associated with the X 1 chromosome. Key words: chromosome banding, Hoplias malabaricus, inactive centromere, sex chromosomes, translocation rearrangement Introduction Although morphologically differentiated sex chromosomes are not generally found in sh, several occurrences have been reported (reviewed in Kirpichnikov 1981, Price 1984, Bertollo et al. 1986, Oliveira et al. 1988, Moreira-Filho et al. 1993, among others). With respect to neotropical shes, more than 30 cases have been related over the last decades, approximately 50% showing female heterogamety, and representing ve different sex chromosome systems: ZZ=ZW, ZZ=ZW 1 W 2, XX=XY, X 1 X 1 X 2 X 2 =X 1 X 2 Y and XX=XY 1 Y 2 (Moreira-Filho et al. 1993). These occurrences are even more signi cant if we consider that, until recently, reports on sex chromosome heteromorphisms in this group were uncommon. Hoplias malabaricus represents one of the neotropical sh groups that are widely distributed in Latin America currently being extensively studied from the cytogenetic viewpoint. Populations with different karyotypes have been described, with differences in chromosome number, structure and sex chromosome system (Bertollo et al. 1979, 1983, 1997, Dergam & Bertollo 1990). These cytotypes may represent a group of distinct species with species-speci c karyotypes (Bertollo et al. 1997). An X 1 X 2 Y system has been reported for one of these populations (Bertollo et al. 1983), with 2n ˆ 40 metasubmetacentric chromosomes in females and 2n ˆ 39 meta-submetacentric chromosomes in males. This X 1 X 2 Y system was characterized by comparison of male and female karyotypes (males have an exclusive chromosome ± the Y ± not observed in females, as well as two other chromosomes ± X 1 and X 2 ) and by analysis of meiotic cells during spermatogenesis. Meiotic cells showed 18 bivalents and 1 trivalent at metaphase I and two modal numbers at metaphase II, corresponding to 18 and 19 chromosomes. A priori, metacentric chromosomes 6 and 19 were considered to be the X 1 and X 2, respectively, whereas another large metacentric with no equivalent in females was designated the Y. However, X 1 and X 2 are arbitrary denominations as they do not refer to the original X chromosome (X 1 ) or to the new X (X 2 ) resulting from the establishment of the system. Although later studies have con rmed the multiple sex chromosome system in the same cytotype of Hoplias malabaricus (Dergam & Bertollo 1990, Scavone et al. 1994), the identi cation of the sex chromosomes in the karyotype was not always certain. In the present investigation, complementary analyses with G-, C- and replication banding led us to better characterize the sex chromosomes and the process involved in the evolution of the sex chromosome system. Materials and methods Chromosome preparation Twelve adult animals (7 males and 5 females), with easily recognizable testes or ovaries, were obtained from the Monjolinho reservoir (Campus of the Federal University of SaÄo Carlos) L. A. C. Bertollo (corresponding author) and M. S. Fontes are at the Departamento de GeneÂtica e EvolucËaÄo, Universidade Federal de SaÄo Carlos, C.P SaÄo Carlos, SP, Brazil. Tel: ( 55) ; Fax: ( 55) A. S. Fenocchio is at the Departamento de GeneÂtica, Facultad de Ciencias Exactas, Quimicas y Naturales, Universidad Nacional de Misiones, Felix de Azara 1552, 3300 Posadas, Misiones, Argentina. J. Cano is at the Departamento de Biologia Celular y GeneÂtica, Facultad de Ciencias, Universidad de MaÂlaga, Campus Universitario de Teatinos, MaÂlaga, Spain. # 1997 Rapid Science Publishers Chromosome Research Vol

2 L. A. C. Bertollo et al. or from the Mogi GuacËu river, both belonging to the Upper Parana basin, State of SaÄo Paulo, Brazil. Metaphase preparations were directly obtained from cephalic kidney cells after conventional in vivo colchicine treatment or from short-term cell cultures (Fenocchio et al. 1991) lasting on average 30 h. In some cases the specimens were previously treated with a yeast suspension for the stimulation of mitoses (Lee & Elder 1980). C- and G- banding Besides conventional Giemsa staining, several chromosome banding techniques were also performed. C-banding followed the basic procedure of Sumner (1972), with small modi cations. G-banding was carried out using the method of Gold et al. (1990) with the following adaptations: slides aged for 2 days at room temperature were rst incubated in 2 3 standard saline citrate (SSC) at 608C for 2 h, washed in distilled water and air dried. Preparations were then treated with a trypsin/giemsa solution for 20 min at room temperature, washed in distilled water and air dried. The trypsin/giemsa solution contained 2.0 ml of a stock solution of trypsin 0.7 ml of Giemsa 45 ml of phosphate buffer, ph 7.2. The stock trypsin solution, freshly prepared, contained 1.0 ml of 2.5% trypsin 2.4 ml of distilled water. 5-BrdU incorporation and replication bands 5-BrdU incorporation in chromosomal segments was performed both in vivo and in vitro. For in vivo incorporation, animals were injected intraperitoneally with a BrdU solution (0:1 mg=ml) at the proportion of 1 ml=100 g body weight for 6±7 h (Almeida- Toledo et al. 1988). For in vitro incorporation, 0.1 ml of a BrdU solution (2:0 mg=ml) was added per 10 ml of culture medium 6± 7 h before the harvest of cells (Fenocchio et al. 1991). Differential staining of replication bands was performed using the uorescence-plus-giemsa (FPG) technique (Perry & Wolff 1974). For BrdU photodegradation, chromosomes were irradiated with UV light for 2 h in 2 3 SSC, at a distance of 10 cm from the source. Results and discussion Chromosomal analyses showed the standard karyotypic structure expected for Hoplias malabaricus populations studied here. All female specimens presented 2n ˆ 40 chromosomes and the males 2n ˆ 39, with a multiple sex chromosome system of the X 1 X 1 X 2 X 2 =X 1 X 2 Y type. The Y chromosome is among the largest in male karyotype, whereas X 1 is of medium size (no. 6) and X 2 is probably the smallest (no. 20) in the complement (Figure 1). Precise identi cation of these chromosomes is not always possible by standard Giemsa staining. However, banding techniques used in this study identi- ed several chromosomal pairs in the H. malabaricus karyotype, with particularly good results for sex chromosome characterization. Figure 1. Conventional Giemsa-stained karyotypes of Hoplias malabaricus female (2n ˆ 40) and male (2n ˆ 39) showing the X 1, X 2 and Y sex chromosomes. Arrowheads indicate the sex chromosomes. Bar ˆ 5 ìm. 494 Chromosome Research Vol

3 Hoplias sex chromosome system C-banding results were similar to those obtained by Dergam & Bertollo (1990) and Haaf et al. (1993) for this same species. Small centromeric heterochromatic bands occur in practically all chromosomal pairs. In addition, telomeric bands are seen in some chromosomes in the complement (Figure 2); these may coincide with the location of the nucleolar organizer regions, which are telomeric and multiple in Hoplias malabaricus (Bertollo 1996). In standard analyses, identi cation of chromosomal pairs 5 and 6 (X 1 ) may be dif cult, despite the centromere of chromosome 5 being, in general, more median located. However, analysis of constitute heterochromatin clearly shows a small C-positive band interstitially located on the long arm of pair 5 both in males and in females (Figure 2). Similarly, the G- and replication banding patterns, together with C-banding, permit not only a good characterization of the X 1 and Y chromosomes but also identi cation of the rearrangements that occurred in the establishment of the X 1 X 2 Y sex chromosome system. The prominent C-positive block proximal to the centromere on the long arm of the X 1 chromosome is a G- negative band and is late replicating. This same band is also present in Y chromosome but occupies an inverse position, i.e. immediately above the centromere and extending along the short arm (Figures 2±4). This marker region demonstrates that the short arm of the Y chromosome corresponds to the long arm of the X 1 chromosome. The long arm of the Y comprises two segments, one related to the short arm of the X 1 chromosome and the other one related to the X 2 chromosome (Figures 2D, 3D & 4D). Thus, a translocation event is associated with the origin of the present sex chromosome system in which the neo-y retains a good similarity with the X 1 and the proposed X 2 chromosome. In some other sh species with a known X 1 X 2 Y sex system, the origin of the Y has been associated with a process of centric fusion involving Figure 2. C-banded karyotypes of Hoplias malabaricus female and male A, showing the sex chromosomes. Note the conspicuous heterochromatic band on the long arm of the X 1 chromosome and the corresponding band on the short arm of the Y chromosome. The interstitial C-positive band on the long arm of the latter may be related to the centromeric heterochromatin of the X 2 chromosome. Sex chromosomes from different female B and male C metaphases are included for comparison. Rearrangements proposed for the origin of the Y chromosome are presented in D. Bar ˆ 5 ìm. Chromosome Research Vol

4 L. A. C. Bertollo et al. acrocentric chromosomes of the karyotype, as in the cases of Eigenmannia sp. (Almeida-Toledo et al. 1984), one species of Cobitis (Saitoh 1989) and Brevoortia aurea (Brum et al. 1992), among others. A more precise identi cation of the X 2 chromosome proved to be harder, even with some banding techniques. However, G-banding data (Figure 3) in association with size and morphology of chromosome 20 suggest that this chromosome corresponds to the X 2, the smallest in the karyotype (Figures 1±4). This contrasts with the hypothesis of Bertollo et al. (1983) that chromosome 19 is the second X chromosome. Good results with serial chromosome banding, such as G-banding, are not often obtained in lower vertebrates. In the present study, use of the technique of Gold et al. (1990), with some modi cations, led to resolutive results. Factors associated with banding processes in higher vertebrates, such as the degree of compartmentalization genome and the presence of isocores (Medrano et al. 1988), have been considered as limiting factors in obtaining multiple structural bands in sh. Gold et al. (op. cit.) suggested that serial bands, although present in sh chromosomes, may not be rich in AT or GC bases compared with adjacent heterochromatin, a fact that would prevent their detection by basespeci c uorochromes, as is the case for higher vertebrates. In this sense, the differentiation of G-bands (rich in GC) and R-bands (rich in GC) was probably a more recent step in vertebrate evolution than the differentiation of replication units (Holmquist et al. 1982; Drouin et al. 1994). This seems to be supported by banding data available for sh in which replication bands by 5-BrdU incorporation have been obtained in a more generalized manner in different groups, being more reproducible than structural bands (Gold et al. 1990; Amores et al. 1995; VeÃnere & Galetti Jr 1995). The few sh species presenting reproducible G-banding patterns, as is the case for Hoplias malabaricus, may re ect differences in chromatin organization along the chromosomes, as proposed by VinÄ as et al. (1994) for Anguilla anguilla. If Figure 3. G-banded karyotypes of Hoplias malabaricus female and male A, showing the sex chromosomes. Note the conspicuous G-negative band on the long arm of the X 1 chromosome and the corresponding band on the short arm of the Y chromosome. The G-positive band on the distal third of the Y long arm corresponds with the centromeric G- positive band of the X 2 chromosome. Details of the sex chromosomes from different female B and male C metaphases are included for comparison. Rearrangements proposed for the origin of the Y chromosome are presented in D. Bar ˆ 5 ìm. 496 Chromosome Research Vol

5 Hoplias sex chromosome system Figure 4. Karyotypes of Hoplias malabaricus female and male with BrdU replication banding A, showing the sex chromosomes. Note the conspicuous late-replicating band on the long arm of the X 1 chromosome and the corresponding band on the short arm of the Y chromosome. Details of the sex chromosomes from different female B and male C metaphases are included. Rearrangements proposed for the origin of the Y chromosome are presented in D. Bar ˆ 5 ìm. so, this would represent a heterogenity with respect to this feature in the sh group (Amores et al. 1995). Alternatively, AbuõÂn et al. (1996) proposed that the dif culty in demonstrating bands in some groups of organisms may be solely due to technical problems. Clearly, detection of multiple structural bands in sh chromosomes continues to be a eld requiring further studies. Inferences about the structure of the Y chromosome can be made based on the observed banding patterns. The C-positive block proximal to the centromere is G- negative and late replicating, similar to that observed for the corresponding segment on the long arm of the X 1 chromosome. However, the small interstitial C-positive band on the distal third of the Y long arm is also G- positive, as is the case for the centromeric region of several other chromosomes, including the hypothesized X 2 (Figures 2 & 3). These facts support the proposition that the interstitial C-positive band in the Y chromosome represents the centromeric region of a chromosome similar to no. 20 (X 2 ) associated with the short arm of another one similar to no. 6 (X 1 ), giving rise to a stable dicentric neo-y in the X 1 X 2 Y sex chromosome system of Hoplias malabaricus. On this basis, this small C- and G-positive band of the Y chromosome represents an inactive centromere, which would permit a normal migration of the chromosome during cell division. Centromere inactivation has been proposed by Hedin et al. (1990) for Scincella lateralis (Sauria), presenting an X 1 X 2 Y system similar to that of H. malabaricus. In S. lateralis the origin of the Y may be due to fusion of a submetacentric chromosome with a metacentric by their telomeric ends. In insects of the genus Myrmecia, occurrence of an inactive centromere after telomeric fusion is also hypothesized, an event that appears to be similarly viable in several mammalian species (Imai & Taylor 1989). However, other possibilities associated with the centromeres of stable dicentric chromosomes have been analysed by Wandall (1994) in humans, including the binding of the two centromeres to the spindle but with different functions, without the need to be both related to chromosome mobility. Chromosome Research Vol

6 L. A. C. Bertollo et al. In a study on the organization and molecular cytogenetics of Hoplias malabaricus DNA, Haaf et al. (1993) cloned and sequenced a family of satellite DNA characterized by two evolutionarily related subfamilies, A and B. In situ hybridization showed that subfamily A is associated with the centromeres of various chromosomes, including the smallest in the karyotype (chromosome 20). Subfamily B is highly speci c for a given chromosome, with a paracentromeric location on the pair 6. Comparison of the female karyotype reported by Haaf et al. (op. cit.) with the present results suggests that this last chromosome is the same as that considered and identi ed by us as the X 1 chromosome. If so, the B subfamily of satellite DNA would then be speci c for this chromosome and should also occur on the Y chromosome. Unfortunately, Haaf et al. (op. cit.) did not obtain results from male specimens. The chromosomal data in the Hoplias malabaricus group are still not resolutive for an appropriate comparison between the different cytotypes, with respect to the evolutionary history of the X 1 X 1 X 2 X 2 =X 1 X 2 Y sex chromosome system. Additional banding studies are needed in other systems already proposed for H. malabaricus, as in the XX=XY 1 Y 2 system (Bertollo et al 1983). Moreover, the suggested XX=XY sex chromosome system for some populations (Bertollo et al. 1979) requires reanalysis and con rmation. On the other hand, if we consider the different cytotypes already described in the H. malabaricus populations, the one that is more strictly related to the cytotype studied here appears to show no heteromorphic sex chromosomes, with males and females carrying a similar standard karyotype, both in number (2n ˆ 40) and morphology. This nding reinforces the similarities between the compound Y and the X 1 and X 2 chromosomes observed in the present study. At present, two pieces of evidence seem to support chromosome 6 (X 1 ) as being the original X chromosome. First is the fact that the active centromere of the Y chromosome is homologous to that of X 1. Second, this chromosome has an unique repetitive DNA `signature' (Haaf et al. 1993). Repetitive DNAs on sex chromosomes tend to evolve at a different rate to those on autosomes because of altered recombination frequency. Meanwhile, further banding and molecular cytogenetic studies of related sh are needed and these should allow de nitive identi cation of the original X chromosome in this species. Acknowledgements We wish to thank Dr Lurdes F. Almeida-Toledo for help in re ning the replication banding technique and Dr Pedro Manoel Galetti Jr for comments on the manuscript. We are also grateful to MSc Paulo Cesar Venere, Roberto Ferreira Artoni and Vladimir Pavan Margarido for photographic assistance. This work was supported by Conselho Nacional de Desenvolvimento Cientõ co e TecnoloÂgico (CNPq - Brazil) and Proyecto de CooperacioÂn Cientõ ca con IberoameÂrica (Agencia EspanÄ ola de CooperacioÂn Internacional). References AbuõÂn M, MartõÂnez P, SaÂnches L (1996) G-like banding pattern in two salmonid species: Oncorhynchus mykiss and Oncorhynchus kisutch. Chrom Res 4: 471±473. Almeida-Toledo LF, Foresti F, Toledo-Filho SA (1984) Complex sex chromosome system in Eigenmannia sp (Pisces, Gymnotiformes). Genetica 64: 165±169. Almeida-Toledo LF, Viegas-PeÂquinot E, Foresti F, Toledo-Filho SA, Dutrillaux B (1988) BrdU replication patterns demonstrating chromosome homeologies in two sh species, genus Eigenmannia. Cytogenet Cell Genet 48: 117±120. Amores A, Bejar J, Alvarez MC (1995) BrdU replication bands in the anguilliform sh Echelus myrus. Chrom Res 3: 423±426. Bertollo LAC (1996) The nucleolar organizer regions of Erythrinidae sh. An uncommon situation in the genus Hoplias. Cytologia 61: 75±81. Bertollo LAC, Takahashi CS, Moreira-Filho O (1979) Karyotypic studies of two allopatric populations of the genus Hoplias (Pisces, Erythrinidae). Brazil J Genet 2: 17±37. Bertollo LAC, Takahashi CS, Moreira-Filho O (1983) Multiple sex chromosomes in the genus Hoplias (Pisces, Erythrinidae). Cytologia 48: 1±12. Bertollo LAC, Moreira-Filho O, Galetti Jr PM (1986) Cytogenetics and taxonomy: considerations based on chromosome studies of freshwater sh. J Fish Biol 28: 153±159. Bertollo LAC, Moreira-Filho O, Fontes MS (1997) Karyotypic diversity and distribution in Hoplias malabaricus (Pisces, Erythrinidae). Cytotypes with 2n ˆ 40 chromosomes. Brazil J Genet 20: 237±242. Brum MJI, Galetti Jr PM, CorreÃa MMO, Aguilar CT (1992) Multiple sex chromosomes in South Atlantic sh, Brevoortia aurea, Clupeidae. Brazil J Genet 15: 547±553. Dergam JA, Bertollo LAC (1990) Karyotypic diversi cation in Hoplias malabaricus (Osteichthyes, Erythrinidae) of the SaÄo Francisco and Alto Parana basins, Brazil. Brazil J Genet 13: 755±766. Drouin R, Holmquist GP, Richer CL (1994) High resolution replication bands compared with morphologic C- and R- bands. In: Harris H, Hirschhorn K, ed. Advances in Human Genetics. New York: Plenum Press, pp 47±115. Fenocchio AS, VeÃnere PC, Cesar ACG, Dias AL, Bertollo LAC (1991) Short term culture from solid tissues of shes. Caryologia 44: 161±166. Gold JR, Li YC, Shipley NS, Powers PK (1990) Improved methods for working with sh chromosomes with a review of metaphase chromosome banding. J Fish Biol 37: 563±575. Haaf T, Schmid M, Steinlein C, Galetti Jr PM, Willard HF (1993) Organization and molecular cytogenetics of a satellite DNA family from Hoplias malabaricus (Pisces, Erythrinidae). Chrom Res 1: 77±86. Hedin MC, Sudman PH, Greenbaum IF, Sites Jr JW (1990) Synaptonemal complex analysis of sex chromosome pairing in the common ground skink, Scincella lateralis (Sauria, Scincidae). Copeia 1990: 1114±1122. Holmquist G, Gray M, Porter T, Jordan J (1982) Characterization of Giemsa dark- and light-band DNA. Cell 31: 121±129. Imai HT, Taylor RW (1989) Chromosomal polymorphisms involving telomere fusion, centromeric inactivation and centromere shift in the ant Myrmecia (pilosula) n ˆ 1. Chromosoma 98: 456± Chromosome Research Vol

7 Hoplias sex chromosome system Kirpichnikov VS (1981) Genetic Bases of Fish Selection. Berlin: Springer. Lee MR, Elder FFB (1980) Yeast stimulation of bone marrow mitosis for cytogenetic investigations. Cytogenet Cell Genet 26: 36±40. Medrano L, Bernardi G, Couturier J, Dutrillaux B, Bernardi G (1988) Chromosome banding and genome compartmentalization in shes. Chromosoma 96: 178±183. Moreira-Filho O, Bertollo LAC, Galetti Jr PM (1993) Distribution of sex chromosome mechamisms in neotropical sh and description of a ZZ=ZW system in Parodon hilarii (Parodontidae). Caryologia 46: 115±125. Oliveira C, Almeida-Toledo LF, Foresti F, Britski HA, Toledo- Filho SA (1988) Chromosome formulae of neotropical freshwater shes. Brazil J Genet 11: 577±624. Perry P, Wolff S (1974) New Giemsa method for the differential staining of sister chromatids. Nature 251: 156±158. Price DJ (1984) Genetics of sex determination in shes: a brief review. In: Potts GW, ed. Fish Reproduction: Strategies and Tactics. London: Academic Press, pp 77±89. Saitoh K (1989) Multiple sex chromosome system in a loach sh. Cytogenet Cell Genet 52: 62±64. Scavone MD, Bertollo LAC, Cavallini MM (1994) Simpatric occurrence of two karyotypic forms of Hoplias malabaricus (Pisces, Erythrinidae). Cytobios 80: 223±227. Sumner AT (1972) A simple technique for demonstrating centromeric heterochromatin. Exp Cell Res 75: 304±306. VeÃnere PC, Galetti Jr PM (1995) Multiple longitudinal bands in sh chromosomes: comparison of structural G-banding and replication R-bands among curimatids. Cytobios 84: 71±78. VinÄas A, GoÂmez C, MartõÂnez P, SaÂnchez L (1994) Induction of G-bands on Anguilla anguilla chromosomes by the restriction endonucleases HaeIII, HinfI, and MseI. Cytogenet Cell Genet 65: 79±81. Wandall A (1994) A stable dicentric chromosome: both centromeres develop kinetochores and attach to the spindle in monocentric and dicentric con guration. Chromosoma 103: 56±62. Chromosome Research Vol