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Synaptonemal complex analysis in spermatocytes and oocytes of rainbow trout, Oncorhynchus mykiss (Pisces, Salmonidae): the process of autosome and sex chromosome synapsis

The surface-spreading synaptonemal complex (SC) technique was employed to analyze spermatocytes and oocytes of rainbow trout in order to visualize the process of autosome and sex chromosome synapsis in this species. The structure of lateral elements
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  NOTES Synaptonemal complex analysis in spermatocytesand oocytes of turbot,  Scophthalmus maximus (Pisces, Scophthalmidae) N. Cuñado, J. Terrones, L. Sánchez, P. Martínez, and J.L. Santos Abstract : A surface-spreading synaptonemal complex (SC) technique was used to analyze spermatocytes and oocytesof turbot ( Scophthalmus maximus ) to visualize the process of chromosome synapsis. The total SC length was 205 ±12  µ m in males and 172 ± 29  µ m in the only female analyzed. A representative SC karyotype of turbot was obtained.Each SC showed lateral elements of equal length. No bivalent exhibiting atypical synaptic behaviour that could be as-sociated with heteromorphic sex chromosomes was observed, either in males or in the female. The DNA content of turbot was evaluated in eight individuals of both sexes by flow cytometry analysis. The 2C mean DNA content of tur-bot (1.308 ± 0.009 pg/cell) was among the lowest observed within fishes. No statistical differences in DNA contentwere revealed between the sexes [Wilcoxon/Mann–Whitney test;  P ( W   x  = 0.243)]. The SC/DNA content ratio observedin turbot was the highest reported to date in bony fishes (Osteichthyes). Key words :  Scophthalmus maximus , fish, DNA content, meiosis, synaptonemal complex. Résumé  : Une technique d’étalement en surface des complexes synaptonémiques (CS) a été employée pour étudier lesspermatocytes et les oocytes du turbot ( Scophthalmus maximus ) afin d’y visualiser le processus de synapse chromoso-mique. La longueur des CS totalisait 205 ± 12  µ m chez les mâles et 172 ± 29  µ m chez la seule femelle examinée. Unkaryotype CS représentatif du turbot a été obtenu. Chaque CS montrait des éléments latéraux d’égale longueur. Aucunbivalent montrant un comportement synaptique atypique pouvant être dû à des chromosomes sexuels hétéromorphes n’aété observé chez les mâles et la femelle. Le contenu en ADN du turbot a été évalué chez huit individus des deux sexespar analyse de cytométrie en flux. La valeur 2C moyenne pour le turbot (1,308 ± 0,009 pg/cellule) figure parmi lesplus faibles observées chez les poissons. Aucune différence statistique n’a été révélée entre les sexes [test deWilcoxon/Mann–Whitney;  P ( W   x  = 0,243)]. Le ratio CS / contenu en ADN observé chez le turbot est le plus élevéparmi toutes les valeurs rapportées à ce jour parmi les poissons osseux (Osteichthyes).  Mots clés  :  Scophthalmus maximus , poisson, contenu en ADN, méiose, complexe synaptonémique.[Traduit par la Rédaction]  Notes 1147 Introduction Unlike studies on higher vertebrates, cytogenetic studieson fishes have traditionally been hampered by the smallchromosome size of this group and the difficulty of obtain-ing serial bands (Medrano et al. 1988). These problems areeven greater in flatfishes because their cellular DNA contentis among the lowest within fishes, only 20% compared withthat of mammals (Ventakesh et al. 2000). Nevertheless, theirchromosome number lies around the modal number ob-served within Teleostei (2 n  = 48; Klinkhardt et al. 1995).Thus, only about 40 of the 570 species of Pleuronectiformes(Nelson 1994) have been karyotyped to date and C banding,Ag staining, and FISH (fluorescence in situ hybridization)have been applied only in a few cases (Bouza et al. 1994;Pardo et al. 2001).Adoption of microspreading techniques, srcinally devel-oped for the study of synaptonemal complexes (SCs) inspermatocytes (Counce and Meyer 1973), has allowed theacquisition of high resolution data on the ultrastructure of meiotic chromosome synapsis in several groups of plantsand animals (e.g., von Wettstein et al. 1984; Gillies 1989).In addition, the visualization of whole SCs in fishes has re-sulted in a more accurate chromosome structure, becausesurface-spread pachytene chromosomes are several times Genome  44 : 1143–1147 (2001) © 2001 NRC Canada 1143 DOI: 10.1139/gen-44-6-1143 Received April 10, 2001. Accepted August 2, 2001. Publishedon the NRC Research Press Web site at http://genome.nrc.caon November 7, 2001.Corresponding Editor: W. Traut. N. Cuñado and J.L. Santos.  Departamento de Genética,Facultad de Biología, Universidad Complutense, 28040-Madrid, Spain. J. Terrones, L. Sánchez, and P. Martínez. 1 Departamentode Biología Fundamental, Area de Genética, Universidad deSantiago de Compostela, 27002-Lugo, Spain. 1 Corresponding author (e-mail:  longer than mitotic metaphase chromosomes (Oliveira et al.1995; van Eenennaam et al. 1998; Cuñado et al. 2000).The turbot (Pleuronectiformes; Scophthalmidae;  Scoph-thalmus maximus ) is a marine flatfish of great value foraquaculture. The greater growth of females has promoted theinterest in obtaining all-female populations for culture andhence the interest in this species’ sex determination mecha-nism (Imsland et al. 1997). Previous karyotypic studies onthis species analyzing mitotic chromosomes with differentbanding techniques showed a 2 n  = 44 karyotype without anyapparent sex-related chromosome heteromorphism (Bouza etal. 1994; Pardo et al. 2001). Here, we used a surface-spread © 2001 NRC Canada 1144 Genome Vol. 44, 2001 Fig. 1.  Electron micrographs of two complete pachytene nuclei from female ( a ) and male ( b ) turbot,  Scophthalmus maximus , and thecorresponding female meiotic SC karyotype with 22 SC elements ( c ). Nu, nucleolus. Scale bar = 2.5  µ m.  SC technique in spermatocytes and oocytes of the turbot withthe following objectives: ( i ) to obtain a more accuratepachytene karyotype that could provide evidence of sexualchromosomes; ( ii ) to analyze the process of synapsis; and( iii ) to compare the SC/DNA in this species with that of otherbony fishes. Materials and methods Synaptonemal complex analysis Four male and one female turbot ( Scophthalmus maximus ) wereobtained from the Instituto Oceanográfico, Vigo, Spain. SC spread-ing was carried out according to the method described previouslyby Cuñado et al. (2000). The electron microscope used was a Jeol1200. The absolute lengths of synaptonemal complexes were mea-sured from enlarged photographic prints using an Image Tool 2.00program (Wilcox et al. 1995–1996). DNA content measurement Flow cytometry following standard procedures was used to de-termine DNA content in turbot nuclei. Briefly, blood cells werewashed in cold Hank’s balanced salt solution (HBSS) (0–4°C), andfixed in methanol at –80°C for 15 min. Cells were resuspended inphosphate buffer saline (PBS) before staining. Cell suspension(200  µ L) was mixed with 400  µ L of staining solution (1 mgpropidium iodide/mL , 10 µ g RNAse/mL dissolved in PBS) and in-cubated for 1.5 h. Chicken blood was used as the internal standardfor estimating turbot DNA content (Tiersch et al. 1989). Prior tostaining, cell suspensions were mixed in a proportion of 1 to 3turbot–chicken to obtain the appropriate height of peaks in thecytometer. For each sample more than 50 000 nuclei were exam-ined by excitation with an argon ion laser set at a wavelength of 488 nm in a Coulter EPICS-XL™ flow cytometer. The DNA con-tent was expressed as turbot to chicken ratios, and absolute DNAvalues (2C) were obtained by multiplying these ratios by the esti-mated DNA content of chicken (2C = 2.44 pg/nucleus; Tiersch etal. 1989). Four replicates were analyzed in each of the eight turbotindividuals (four males and four females), and the mean, standarderror, and coefficient of variation (CV) were obtained for each of them, as well as for both sexes. A Wilcoxon/Mann–Whitneynonparametric test (Siegel and Castellan 1988) was used to check for differences in DNA content between sexes. Results and discussion Electron micrographs of 5 zygotene and 42 pachytene nu-clei were obtained. The entire SC complement was presentin all pachytene nuclei examined, with the zygotene nucleihaving only partial spreads available for analysis. Chromo-somal axes represented by the lateral elements (LEs) of theSCs were well resolved, and centromeric regions appeared tooverlap and span the LEs of synapsed bivalents in most of the nuclei. Attachment plaques were rarely observed.Zygotene nuclei were characterized by incompletesynapsis, generally beginning in one region near the chromo-some ends, although in some bivalents, synaptic initiationfrom both ends was also observed. This pattern has been therule in fish species studied to date (Oliveira et al. 1995; vanEenennaam et al. 1998; Cuñado et al. 2000).All pachytene nuclei showed 22 SCs (Figs. 1 a –1 b ). Themean length of SC complement in males was 205 ± 12  µ m(32 nuclei analyzed), ranging from 170 to 226  µ m; in the fe-male the length was 172 ± 29 µ m (10 nuclei). A representativemeiotic karyotype of turbot consisted of 2 submetacentric, 11subtelocentric, and 9 telocentric pairs (Fig. 1 c ). Nucleolar or-ganizer regions (NORs) were localized in the short arms of asubtelocentric chromosome pair.These results are in broad agreement with the mitotickaryotype reported by Bouza et al. (1994) not only in thediploid number (2 n  = 44), but also in chromosome morphol-ogy. Nevertheless, the decondensed stage of pachytene chro-mosomes and the consistent identification of centromereshave allowed us the precise determination of the number of subtelocentric and telocentric chromosome pairs, 11 and 9,respectively. © 2001 NRC Canada Notes 1145 Fig. 2.  Representative flow histograms of a male (A) and female(B) turbot ( Scophthalmus maximus ). Peak ( a ) refers to turbot andpeak ( b ) refers to chicken ( Gallus domesticus ), the species usedas the internal standard. Nuclear DNA content is proportional tofluorescence intensity, expressed as channel numbers.  SC studies have been useful in detecting atypical synapsisamong the well-differentiated sex chromosomes of mammalsand birds (Solari 1972, 1992) or even in those with a smalldegree of differentiation, such as fishes. In the fish Orechromis niloticus  (Nile tilapia), SC studies have shownan asynaptic segment in the terminal region of the largest bi-valent, suggesting an XX/XY system in this species(Carrasco et al. 1999). However, full synapsis was observedbetween the LEs of this bivalent in 74.3% of the cases, prob-ably because of the mechanism termed axial equalization.This behaviour is also characteristic of the sex chromosomesof insects, birds, and mammals and implies nonhomologoussynapsis throughout the pachytene stage (Weith and Traut1986; Solari 1992). No evidence of atypical synapsis per-haps characterizing a heteromorphic sex bivalent was foundin turbot, and the 22 bivalents showed regular synapsis withthe LEs uniformly arranged between the homologues in allpachytene nuclei analyzed. The existence of axial equaliza-tion can be discarded in the case of turbot because the SCsets chosen for measurement represented a random sampleof pachytene substages. Therefore, the data obtained do notsupport the presence of sexual heteromorphism in turbot.Estimation of 2C DNA content in turbot by flowcytometry showed a good resolution with CVs within indi-viduals less than 1.5% in the eight specimens analyzed(Fig. 2). The differences observed between sexes (males,1.305 ± 0.003; females, 1.312 ± 0.019) were not significantas evidenced by the Wilcoxon/Mann–Whitney test [ P ( W   x ) =0.243]. The 2C DNA content obtained in turbot when all in-dividuals were pooled was 1.308 ± 0.009 pg/cell. This valuelies within the rank observed in other species of the orderPleuronectiformes (1.3–2.2; Ventakesh et al. 2000). The ge-nome of turbot appears among the smallest within Verte-brates, with a DNA content close to that observed in thehighly compacted genome of   Fugu rubripes  (Ventakesh etal. 2000). Considering that the 4C DNA content of turbot is2.62 pg/cell, the mean SC/4C DNA ratio ( µ m/pg) is 71.95.As far as we know, this value is higher than those found inplants (Anderson et al. 1985) and vertebrates (Peterson et al.1994), with the only exception of   Xenopus laevis  females(Loidl and Schweizer 1992), and indicates that thepachytene chromatin compaction in this species is low. InTable 1, the relationship between SC length and genome size( µ m/pg) of turbot is compared with the values obtained inbony fishes (Osteichthyes). From these data, it is clear thatturbot exhibits the lowest SC chromatin compaction withinOsteichthyes and that this group of fish show a very widerange in SC/DNA, higher than that shown by birds andmammals (Peterson et al. 1994). Anderson et al. (1985)found an excellent correlation between SC length and ge-nome size in Angiosperms, but this is far from being ob-served in fishes. A possible explanation for this observationcould be the low number of fish species analyzed within thishighly variable group of vertebrates. Also, Peterson et al.(1994) suggested that the SC/DNA might be positively cor-related to recombination frequency. On these grounds, ahigh chiasma frequency would be expected in male andfemale turbot. Further investigations are necessary to ascer-tain the biological significance of these findings.  Acknowledgements N. Cuñado and J. Terrones contributed equally to thiswork and are both considered first authors. We are indebtedto J. Barrios for valuable technical assistance. We also thank R.M. Cal (Instituto Español de Oceanografía, Vigo) for sup-plying biological material for SC and DNA content analyses.S. Vidal provided valuable support for flow cytometry analy-sis. This work was supported by grant PB98-0107, awardedby the Dirección General de Enseñanza Superior (DGES,Spain), and by the Spanish Goverment FEDER grant1FD1997-2404. References Anderson, L.K., Stack, S.M., Fox, M.H., and Chuanshan, Z. 1985.The relationship between genome size and synaptonemal com-plex length in higher plants. Exp. Cell Res.  156 : 367–378.Bouza, C., Sánchez, L., and Martínez, P. 1994. Karyotype charac-terization of turbot ( Scophthalmus maximus ) with conventional,fluorochrome and restriction endonuclease-banding techniques.Mar. Biol.  120 : 609–613.Carrasco, L.A.P., Penman, D.J., and Bromage, N. 1999. Evidencefor the presence of sex chromosomes in the Nile tilapia( Oreochromis niloticus ) from synaptonemal complex analysis of XX, XY and YY genotypes. Aquaculture,  173 : 207–218.Counce, S.J., and Meyer, G.F. 1973. Differentiation of thesynaptonemal complex and the kinetochore in  Locusta  sper- © 2001 NRC Canada 1146 Genome Vol. 44, 2001 Species Sex a Mean SC length SD ( µ m) 4C (pg) SC/DNA ( µ m/pg) Reference Pomoxis annularis  M 127±13 4.16 30.5 Peterson et al. 1994  Lepomis macrochirus  M 130±70 4.04 32.2 Peterson et al. 1994  Micropterus salmoides  M 143±18 4.08 35.0 Peterson et al. 1994  Dorosoma cepedianum  M 78±11 3.96 19.7 Peterson et al. 1994 Oncorhynchus mykiss  M 144±16 11.00 13.1 Peterson et al. 1994M 259±49 9.32 27.8 Oliveira et al. 1995F 223±34 9.32 23.9 Oliveira et al. 1995  Acipenser transmontanus  M 482±56 18.92 25.5 van Eenennaam et al. 1998  Danio rerio  M 280 6.44 43.5 Loidl 2000 Scophthalmus maximus  M 205±12 2.61 77.65 This paperF 172±29 2.62 65.15 This paper a M, male; F, female. Table 1.  Total SC length, 4C DNA content and SC/DNA for different species of Osteichthyes.  © 2001 NRC Canada Notes 1147 matocytes studied by whole mount electron microscopy.Chromosoma (Berlin),  44 : 231–253.Cuñado, N., Garrido-Ramos, M.A., de la Herrán, R., Ruíz-Rejón,C., Ruíz-Rejón, M., and Santos, J.L. 2000. Organization of re-petitive DNA sequences at pachytene chromosomes of giltheadseabream  Sparus aurata  (Pisces, Perciformes). ChromosomeRes.  8 : 67–72.Gillies, C.B. 1989. Chromosome pairing and fertility in polyploids.  In  Fertility and chromosome pairing: recent studies in plants andanimals.  Edited by  C.B. Gillies. CRC Press, Boca Raton, Fla.pp. 137–176.Imsland, A.K., Folkvord, A., Grung, G.L., Stefansson, S.O., andTaranger, G.L. 1997. Sexual dimorphism in growth and matura-tion of turbot,  Scophthalmus maximus  (Rafinesque, 1810).Aquac. Res.  28 : 101–114.Klinkhardt, M., Tersche, M., and Greven, H.J. 1995. Databaseof fish chromosomes. Westasp Wissenschaften, Magdeburg,Germany.Loidl, J., and Schweizer, D. 1992. Synaptonemal complex of   Xenopus laevis . J. Hered.  83 : 307–309.Medrano, L., Bernardi, G., Couturier, J., Dutrillaux, B., andBernardi, G. 1988. Chromosome banding and genomecompartimentalization in fishes. Chromosoma (Berlin),  96 :178–183.Nelson, J.S. 1994. Fishes of the world. John Wiley & Sons, Inc.,New York.Oliveira, C., Foresti, F., Rigolino, M.C., and Tabata, Y.A. 1995.Synaptonemal complex analysis in spermatocytes and oocytes of rainbow trout,  Oncorhynchus mykiss  (Pisces, Salmonidae): theprocess of autosome and sex chromosome synapsis. Chromo-some Res.  3 : 182–190.Pardo, B.G., Bouza, C., Castro, J., Martínez, P., and Sánchez, L.2001. Localization of ribosomal genes in Pleuronectiformes us-ing Ag- and CMA 3  banding and  in situ  hibridization. Heredity, 86 : 1–6.Peterson, D.G., Stack, S.M., Healy, J.L., Donohoe, B.S., and Ander-son, L.K. 1994. The relationship between synaptonemal complexlength and genome size in four vertebrate classes (Osteicthyes,Reptilia, Aves, Mammalia). Chromosome Res.  2 : 153–162.Siegel, S., and Castellan, N.J. 1988. Non-parametric statistics forthe behavioral sciences. McGraw-Hill, New York.Solari, A.J. 1972. Ultrastructure and composition of synaptonemalcomplex in spread and negatively stained spermatocytes of golden hamster and the albino rat. Chromosoma (Berlin),  39 :237–263.Solari, A.J. 1992. Equalization of Z and W axes in chicken andquail oocytes. Cytogenet. Cell Genet.  59 : 52–56.Tiersch, T.R., Chandler, R.W., Wachtel, S.S., and Sherman, E.1989. Reference standards for flow cytometry and application incomparative studies of nuclear DNA content. Cytometry,  10 :706–710.van Eenennaam, A.L., Murray, J.D., and Mediano, J.F. 1998.Synaptonemal complex analysis in spermatocytes of white stur-geon,  Acipenser transmontanus  Richardson (Pisces,Acipenseridae), a fish with a very high chromosome number.Genome,  41 : 51–61.Ventakesh, B., Gilligan, P., and Brenner, S. 2000. Fugu: a compactvertebrate reference genome. FEBS Lett.  476 : 3–7.von Wettstein, D., Rasmussen, S.W., and Holm, P.B. 1984. Thesynaptonemal complex in genetic segregation. Annu. Rev.Genet.  18 : 331–413.Weith, A., and Traut, W. 1986. Synaptic adjustment, non-homo-logous pairing, and non-pairing of homologous segments in sexchromosome mutants of   Ephestia kuehniella  (Insecta,Lepidoptera). Chromosoma (Berlin),  94 : 125–131.Wilcox, D., Dove, B., McDavid, D., and Green, D., 1995–1996.Image tool for Windows, Version 2.00. University of TexasHealth Science Centre, San Antonio, Tex.
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