THE JOURNAL OF EXPERIMENTAL ZOOLOGY 265:301-308 (1993)
Primitive Sex Chromosomes in Poeciliid Fishes Harbor Simple Repetitive DNA Sequences INDRAJIT NANDA, MANFRED SCHARTL, JÖRG T. EPPLEN, WOLFGANG FEICHTINGER, AND MICHAEL SCHMID Departments ofHuman Genetics (l.N., W.F., Mi.S.), andPhysiological Chemistry I (Ma.S.), Biocenter, University ofWürzburg, W-8700, Würzburg, Department of Molecular Human Genetics, Ruhr-University, Bochum (J.T.E.), Germany
ABSTRACT The demonstration ofthe chromosomal mode ofsex determinationvia genetic exper- iments as well as the absence of heteromorphic sex chromosomes affirm poeciliid fishes as a unique · group among vertebrates that are endowed with the mostprimitive form of sex chromosornes. In many different taxa the evolutionary process involved in the differentiation ofadvanced sex chromo somes is outlined through sex specifically organized repetitive sequences. In this investigation hydridization of synthetic probes specific to genomic simple repeat motifs uncovers a sex-specific hybrid ization pattern in certain viviparaus fishes ofthe family Poeciliidae. The hybridization pattern together with specific staining ofthe constitutive heterochromatin by C-banding reveals heterogamety in males (Poecilia reticulata) as weil as in females (P. sphenops). In P. velifera, however, C-banding alone fails to unravel the heterogametic status. The female specific W-chromosome can be detected by simple repetitive sequence probes. Therefore, the principal significance ofheterochromatization as a means of generating differentiated sex chromosomes is evident. © 1993 Wiley-Liss,lnc.
In comparison with other vertebrate classes, the apparently analogous. For example, the Y or W cytological observation of structurally distinct sex chromosomes in most vertebrates become reduced chromosomes in fishes is somewhat sporadic. Con in size and have retained few structural genes. They ventional microscopic analysis, in which sex chro also acquire a large proportion of heterochromatin mosomes are precisely heteromorphic, have succeeded and consequently confer to special staining prop in describing cases of heterogamety of the XX/XY erties in the interphase nucleus. In addition, not and XX/XO types (Ebeling and Chen, '70; Chen, only late replicating throughout the S-phase the '69), the ZZ/ZW type (Chen and Ebeling, '68) and Y and W chromosomes are genetically isolated from even multiple sex chromosomes (Uyeno and Miller, the rest of the genome by restricting the exchange '71) in several species offishes (for details see Ohno, of the genetic material with that oftheX and Z '7 4; Price, '84). However, prior to the onset of cyto chromosome during meiosis (for reviews see J ablonka genetic analyses, in several gonochoristic fishes and Lamb, '90; Charlesworth, '91). In contrast, the belanging to the family Poeciliidae (Cyprinodonti poeciliid sex chromosomes have not been found to formes) the existence of either male (Poecilia re display any of these properties and therefore are ticulata, Xiphophorus maculatus) or female (X. considered tobe in the mostprimitive stage of devel maculatus) heterogamety has been inferred from oping sex chromosomes as they still resemble func genetic crosses using suitable phenotypic markers tional autosomes carrying many functional genes. (Winge, '22; Kallman, '84). Previous cytogenetic A large part ofthe Y or W chromosomal DNA in investigations on a number of poeciliid fishes in most species as revealed by C-banding is constitu cluding that on P. reticulata have failed to document tively heterochromatic and is usually associated the heteromorphic sex chromosomes (Prehn and with the presence ofhighly repetitive DNA (Stefos Rasch, '69). Also the probable existence of sex chro and Arrighi, '71; t}"ohn and Miklos, '79). While the mosomes has not been convincingly demonstrated analysis ofrepetitive sequences is of general inter- from karyological sturlies on several species of X i phophorus (but see Foerster and Anders, '77). In spite ofits independent origin in different taxa the Received August 7, 1992; revision accepted September 30, 1992. Address reprint requests to Michael Schmid, Department ofHuman features of t}:le molecular mechanism underlying Genetics, University ofWürzburg Biozentrum, Am Hubland, W-8700, the evolution of chromosomal sex determination are Würzburg, Federal Republic ofGermany. © 1993 WILEY-LISS, INC. 302 I. NANDA ET AL. est in studies of genome evolution, the distribution aration. The tissues were repeatedly minced in a of a conserved repetitive sequence initially isolated small drop ofhypotonic solution with a finepair of from a snake, shows close linkage to the heteroga scissors. The released cells along with the small metic sex chromosome (Singh et al., '81; Epplen et tissue pieces were collected in a tube and incubated al., '82). In previous reports, we analyzed different in a hypotonic solution ofKCl (0.046M) for 45 rnin evolutionary stages of sex chromosomes in poecil at room temperature. After hypotonic treatment, iid fishes by means of simple repetitive sequences the cell suspension was centrifuged and fixed with (Nanda et al., '92). Although, large restriction frag 6-8 ml of chilled acetic acid:methano} (1:3) Solu ments characterized by presence of simple repeti tion. After overnight fixation at 4°C, the fixed tive sequences served as specific heterogametic material was collected by centrifugation (400g), markers, cytological evidence for a primitive form resuspended in a small volume offresh fixative and of sexchromosomeswas only demonstrated in case then dropped onto precleaned slides and air-dried of P. reticulata (Nanda et al., '90). In this report at room temperature. Chromosome preparations detailed cytological analyses on several other poe were stained in a 5% Giemsa solution (pH 6.8) for ciliid fishes are presented. In retrospect, hybridiza 5-10 min, rinsed in distilled water, and perrnanently tion of informative simple repetitive sequences to mounted in Eukitt for conventional chromosome the genomic DNA digested with restriction enzymes analysis. Staining of constitutive heterochromatin allowed to correlate the sex-specific hybridization (C-banding) was performed according to the method signals to the cytological findings. Briefly, our obser described by Sumner ('72). - vations demoostrate that in P. reticulata and P. sphenops cytologically recognizable Y or W chro DNA preparation and oligonucleotide mosome correspond to the sex-specific hybridization bybridization as detected by simple repetitive sequence. In P. Genomic DNA isolation from brain, liver, spieen, velifera (sailfin molly) as weil as in two populations and gill was carried out according to Blin and Staf of Xiphophorus the hybridization patterns with sim ford ('76). DNA was digested with the restriction ple repeats do not concur with the cytological find enzymes Hinfl and Alu! according to the manufac ings. Hence, accumulation of simple sequences is turer's recommendation at a ratio of 1 f.Lg of DNA discussed here in the light of its critical bearing to 5 units enzyme overnight at 37°C and resolved on vertebrate sex chromosome differentiation. on 0. 7% horizontal agarase gels in TBE buffer (Maniatis et al., '82). The gels were stained with MATERIALSAND METHODS ethidium bromide, photographed, dry-blotted, and Animals then subjected to denaturation and neutralization Adult specimens from both sexes of ornamental for hybridization as described (Schäfer et al., '88). strains of Poecilia reticulata (guppy), black molly The oligonucleotide probes specific for simple repeats (Poecilia sphenops, var. melanistica; the entirely were synthesized on an automated DNA synthesizer black breed is known as the melanistica variant) (Applied Biosystems 381A, Weiterstadt, FRG) and 32 and sailfin molly (Poecilia velifera) were obtained were end-labelled with ['y-P ] ATP (Amersham, from local animal dealers. Fishes from the "Laguna Branschweig, FRG) in a standard kinase reaction. Catemaco" population of X iphophorus helleri and Hybridizations were carried out for 3-4 hr at 43°C specimens of X iphophorus maculatus from a pop for (GACA)4 , (CA)8 , (GT)8 in 5 x SSPE (20 x SSPE is ulation of the "Rio Papaloapan" system were ana 3MNaCl, 200mMNaH2P04 x H20, 20 mM EDTA), lysed. Founder fish for the stocks (Xiphophorus) were 0.1% sodium dodecyl sulphate (SDS), 10 JJ-g/ml son obtained from A. and F. Anders (Gießen, FRG) or icated and denatured Escherichia coli DNA, and K.D. Kaliman (New York, NY), respectively. The 1 x 106 cpm/ml oflabelled probes. After hybridiza Xiphophorus strains were bred in closed stocks derived from at least two or three brother-sister tion gels were washed three times for 30 min each matings to minimize intrastrain polymorphism of atroom temperature in6 x SSC (0.9M NaCl, 90mM the sex chromosomes analysed. trisodium citrate) followed by a 1min washat the hybridization temperature. Gels were then exposed Cbromosome preparation and to Kodak XAR-5 film at room temperature. In situ staining techniques hybridization with the biotinylated (GACA) 4 oligo After exposing the animals to a 0.03% solution nucleotide to the chromosome of guppy were per of colchicine for 6-10 hr, the gills, kidneys and formed according to the methods described previously spleen were carefully removed for chromosome prep- (Nanda et al., '90). FISH SEX CHROMOSOMES 303
RESULTS probes. The informative (GGAT)4 signal is restricted to the 23 kb region in females whereas the dimeric DNA analysis repeat (CA)8 displays several bands ranging from DNA from both sexes ofthe species surveyed was 8 kb to extremely high molecular weight range (Fig. Hinfl digested and challenged with several simple 1b). Correspondingly two less prominent but con repeat probes in the gel. In most cases the infor sistent female specific signals were noted with (GT)s mative probe revealed a fingerprint pattern compris in P. velifera (Fig. 1c). In comparison with P. reticulata ing some 15-20 fragments in each digest. Principally and the two other Poecilia species, hybridization of two categories offragments were generated on DNA simple repeat probes to theXiphophorus model sys fingerprint: 1) fragments that appeared tobe invar tem could not alloca te specific bands to Y, W, or X iably at the high molecular weight region were chromosomes. In particular, a population of X. macu always present in one ofthe sexes but never in the latus as weil as X. helleri carrying a definite Y chro other sex; 2) polymorphic fragments (independent mosome (genetic marker) exhibited an intense smear ofthe sex) in which the nurober ofrepeats appeared signal in males with (GATA)4 (data not shown). to differ extensively in different individuals. While After a prolonged exposure ofthe X-ray film, sim the first category ofhybridization fragment served ilar smear signal could not be marked in fernales. as marker for evaluating the heterogametic status, Probably the large Y-chromosomal DNA lacking the latter pattern of hybridization with the same restriction sites are just randomly broken and their probeisnot at all surprising as sex specifically con separation results in the smear signal. served sequences are known to cross-hybridize simul taneously with several hypervariable autosümalloci Cytogenetic analyses (Ali et al., '86; Traut, '87). Table 1 Blustrates The evolution of differentiated sex chromosomes the details of the hybridization pattern with sev often correlates with a significant accumulation of eral simple repeats in the context ofheterogametic simple repetitive sequences. In some species this sexes. Most conspicuously, in the Poecilia reticulata consistent occurrence can be visualized by conven a singlemale specific (GACA)4 signalwas observed tional C-banding (Schmid, '83). Accordingly, C in all individuals (Fig. 1a) at the high molecular banding analysis of the somatic chromosomes was weight region which indicates the existence of a performed to correlate the sex specific hybridization long stretch of simple sequences on the Y chromo observed in poeciliid fishes. Figure 2 displays the some. On the other hand, the probe (GATA)4 reveals C-banded karyotypes for all the five species inves a Y-chromosome specific signal in certain individ tigated. The chromosome lengths decrease only uals (data not shown). Thus a stable (monomorphic) slightly frornonepair to another. Therefore, in the as weil as a polymorphic locus on the Y chromo karyotypes the chromosomes form a continuous some could be detected in P. reticulata. With respect series of decreasing size, and their numeration is to the heterogamety, the GACA and GATA simple arbitrary. In accordance to the previous reports, all sequences appeared less informative in P. sphenops 23 chromosome pairs in Poecilia species as weil as and P. velifera. In females of P. sphenops prominent 24 chromosome pairs in X iphophorus are telocen sex-specific hybridization in the high. molecular tric. The C-band positive heterochromatin is localized weight range predicting the presence of a W chro mainly in the pericentromeric region of the chro mosome was obtained with the (GGAT)4 and (CA)s mosomes. Additionally, a very distinct heteromor-
. TABLE 1. Summary ofsex-specific hybridization patterns as detected by simple repetitive sequences Informativeenzymes Heterogametic Approx. range ofsex Species andprobes status specific fragments (kb)
P. reticulata HinfliAlul (GACA)4 ·xY/XX >23.0 '
b c
Fl'IOu'· ·.·,t :< ;I·· .. ····>''f:\f:(·e· A·' '···. '· .. t. . . ,, .o:8. Hinf I ... ·(GT)8
Fig. 1. In-gel hybridization of genomic DNA from Poecilia reticulata (a), Poecilia sphenops
(b), and Poecilia uelifera (c) using the simple repetitive oligonucleotide probes (GACA) 4 , (CA) 8 and (GT)8• Note the sex-specific signals at high molecular weight region. Size markers (Hindlll digested Lambda DNA) are indicated in kilobases. FISH SEX CHROMOSOMES 305
1·1: II II II II 1'1 '·.·2: 3 8 9 10 12 II II BI II • 111 15· '•t9 20 21 22 ec23 II II II II 7 8 10 1'1 12
20 .22 II 3 6 6 .8: 9 10 1l 12 II 15 t7 II II 6· 6 9 10 11 12 II•a,-··.· );,,· '15 16 19 e II II II II •1 2.. 4 6 9 1(). 11 •12 II II 14 21 22 f 111 II 1 '9 lf 1.2 lll &" .~ 111 13 i6 19 •'20 22 :23
Fig. 2. Karyotypes of(a) Poecilia velifera <.?, (b,c) P. reticulata ö (d), P. sphenops <.?, (e) Xipho phorus helleri ö, and (f) X. maculatus d. a,b,d-f: C-banding. (c) In situ hybridization with a biotinylated (GACA)4 oligonucleotide probe. The sex chromosomes are in brackets. Note the prominent telomeric heterochromatin in the Y and W chromosomes of (b) P. reticulata, (d) P. sphenops, and (c) the specific in situ hybridization signal in the Y chromosome of P. reticulata. phism in the telomeric heterochromatin between homologous chromosomes ofNo. 1 pair could be dif two homologaus chromosome allows the identifica ferentiated on the basis of occurrence of telomeric tion of sex chromosomes in P. reticulata and P. heterochromatin in only one of the homologues in sphenops. However, the extent ofC-band heteromor females (Fig. 2d). In the C-banded karyotypes of phism is manifested in a different pattem in both the male animals these two homologous chromo species. somes (No. 1) are identical and possess no telomeric In most of the metaphases of P. sphenops the heterochromatin (Haaf and Schmid, '84). Therefore 306 I. NANDA ET AL. such aC-band difference Ieads to the conclusion P. sphenops and in parallel by the presence of sim that these are sex-specific chromosomes of the ZW ple repetitive sequences visualized in these chro g /ZZ o type. Cytologically the only intrinsic differ mosomes. Yet, in contrast to the situation in most ence between the Z and W chromosomes is the higher vertebrates the sex chromosomes here are telomeric heterochromatin. In P. reticulata, although morphologically identical. It can be assumed that both the homologaus chromosomes No. 1 carry pro the situation in these species may reflect an inter minent telomeric heterochromatin, in one of the mediate stage between truly undifferentiated homo homologaus chromosomes the heterochromatin is morphic sex chromosomes and highly heteromorphic apparently more ~xtensive than on the other homol differentiated sex chromosomes. However, the pro ogaus chromosome in males. Consequently the cess which involves the transition ofa euchromatic length of the euchromatic region (C-band negative) segment on one ofthe functional homologaus chro · is relatively reduced in this particular chromosome mosomes to cytologically demonstrable, genetically (Fig. 2b). By comparing the C-banded preparation silent heterochromatin in P. sphenops cannot be between the sexes, there is always distinctly more explained satisfactorily. N evertheless this process telomeric heterochromatin on the heterogametic might have initiated an effective form of meiotic specific Y chromosome than in the X chromosome. isolation of the primeval Z and W chromosomes. Furthermore, the sex specific Y chromosome could The analysis of Banded krait minor (Bkm) sat be unequivocally identified by hybridizing (GACA)n ellite DNA in the snake has allowed us to recover simple repeat probes in-situ. The hybridization sig sex-specific sequences which are quantitatively nal always originates from the Y-chromosome het associated with the W chromosome (Singh et al., erochromatin, but not from the X chromosome (Fig. '81). Moreover, distribution of this sequence indi 2c). In contrast to the above observed phenomenon cates that accumulation of such genomic components similar heteromorphism could be observed neither coincides with the morphological differentiation of in Poecilia velifera nor in the two species of Xipho the W chromosome among snakes. However, in sev phorus (Fig. 2a,e,f). Hence the Z(or X) and W(or eral fishes and in particular amphibians with or Y) chromosomes are still structurally identical. without heteromorphic sex chromosomes, the Bkm Only in fish from the "Laguna Catemaco" popula sequences which are enriched with GATA/GACA tion of X. helleri, where sex-specific hybridization simple repeats (Epplen et al., '82) failed to reveal pattern was marked with (GATA) 4 inconsistent sex-specific hybridization pattems (Lloyd et al., '89; heterochromatin heteromorphism indicated a small Schmid et al., '91). Conspicuously, these Observa difference between a pair of chromosomes. Larger tions suggest that the GATAJGACA simple repeats samples have to be analysed to clarify this phe are probably not the exclusive sequences associated nomenon. with sex chromosome evolution. In this context, the present investigation on the basis of sex-specific DISCUSSION hybrdization patterns as observed in the primitive Among most lower vertebrates, simple karyolog sex-chromosomes ofpoeciliid fishes with additional ical analysis fails to detect the sex chromosomes, simple repetitive sequences would indicate that sev as the X(orZ) and Y(orW) chromosomes exhibit eral different simple repetitive elements may have equallengths, the same staining behavior and degree accumulated independently on the sex chromo of condensation in conventionally stained prepa somes. Most notable is the sex-specific hybridization rations. In certain vertebrates, particularly in the in Xiphophorus and P. velifera where cytologically colubrid snake Ptyas mucosus or in the hylid frog sex chromosomes can not be detected. Therefore, · Rana clamintas, the homomorphic sex chromo it is conceivable that accumulation of simple se somes differ from one another by their C-band quences could have preceded the morphological dif character (Singh et al., '76; King, '80). Therefore, ferentiation of sex chromosomes. heterochromatin staining by C-banding is frequently The theory pertaining to the evolution ofhetero used to assign the heterogametic status in primi morphic sex chromosomes from an ancestral pair tive vertebrates. In fish, heterochromatin analysis of homologous chromosome (Ohno, '67) that were in most instances has been performed on already morphologically identical is convincingly explained morphologically differentiated sex chromosomes · by two cytological means: (1) primary heterochro (Galetti and Foresti, '86). Using the C-banding tech matization where differentiation of the sex-pair nique alone, the sex chromosomes could be distin actually depends on the heterochromatization event guished here on the basis of a higher accumulation to impede recombination and (2) structural alter of telomeric heterochromatin in P. reticulata and ation through pericentric inversion (Ohno, '67, Bull, FISH SEX CHROMOSOMES 307 '83). The observation on Poeciliid fishes here favors the beginning of a process mimicking "hitch-hiking" the heterochromatization concept for sex chromo phenomena can be predicted. some differentiation. Whether this initial hetero chromatization is a cytological manifestation of a ACKNOWLEDGMENTS process supporting Muller's "ratchet" hypothesis This work has been supported by the Deutsche (Muller, '64) or a phenomenon of "hitch-hiking" Forschungsgemeinschaft (EP 7/6-1, SCHM 484/4-2). needs tobe discussed. The functional degeneration The oligonucleotide probes are subjected to patent ofthe Y(or W) chromosome through several muta applications. Commercial enquiries should be di tional events ("ratchet") has been conjectured to rected to Fresenius AG, Oberursel, Germany. draw a hypothetical scheme for the evol ution of Sex chromosomes. In the absence of recombination LITERATURE CITED between the proto-type sex chromosomes the muta Ali, S., C.R. Müller, and J.T. Epplen (1986) DNA fingerprint tional event may have affected the Y chromosome ing by oligonucleotide probes specific for simple repeats. Hum. exclusively (Charlesworth, '78). Therefore it is nec Genet., 74:239-243. Blin, N., and D.W. Stafford (1976) A general method for isola essary to analyze whether the accumulation of sim tion ofhigh rnolecular weight DNA from eukaryotes. Nucleic ple repetitive sequences per se might have functioned Acids Res., 3:2303-2308. as mechanism for suppressing the recombination. Bull, J.J. (1983) Evolution of Sex Determining Mechanisms. Although, classical observations clearly demoo Benjamin/Cumming, Menlo Park, California. strate that the recombination frequency is extremely Charlesworth, B. (1978) Model for evolution of Y chromosome and dosage compensation. Proc. Natl. Acad. Sei. USA., 75: low in the heterochromatic region, certain simple 5618-5622. - sequences are said tobe the hot-spot for recombi Charlesworth, B. (1991) The evolution of sex chromosomes. Sci nation (Stallings et al., '91). However, it must be ence, 251:1030-1033. mentioned that in most cases intimately inter Chen, T.R., and A.W. Ebeling (1968) Karyological evidence of spersed simple sequences are distributed simulta female heterogamety in the mosquitofish Gambusia af{inis (Baird and Girad). Copeia, 1:70-75. neously on both homologaus chromosomes (both Chen, T.R. (1969) Karyological heterogamety of deep-sea fishes. alleles). This distribution pattern may itselfbe the Postilla, 130:1-29. cause ofpotentiation ofrecombination. On the other Ebeling, A.W., and T.R. Chen (1970) Heterogamety in teleos band the presence of a simple sequence stretch tean fishes. Trans. Am. Fish, 99:131-138. observed here on a single chromosome (Y or W) Epplen, J.T., J.R. Mccarrey, S. Sutou, and S. Ohno (1982) Base sequence of a cloned snake W-chromosome DNA fragment would have imposed a qualitative barrier at the and identification of a male specific putative mRNA in the sequence level for the recombination to occur and mause. Proc. Natl. Acad. Sei. USA., 79:3798-3802. consequently would allow the "ratcheting process" Foerster, W., and F. Anders (1977) Zytogenetischer Vergleich to proceed on the proto-sex chromosomes. Further der Karyotypen verschiedener Rassen und Arten lebendge more, in a number ofrecent studies, simple repeti bärender Zahnkarpfen der Gattung Xiphophorus. Zool. 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