- Email: [email protected]
Detection of the X chromosomes in a Klinefelter boar using a whole human X chromosome painting probe
Animal Reproduction Science 52 Ž1998. 317–323
Detection of the X chromosomes in a Klinefelter boar using a whole human X chromosome painting probe Auli Makinen ¨
a,)
, Magnus Andersson b, Sanna Nikunen
c
a
b
Department of Animal Science, P.O. Box 28, SF-00014 UniÕersity of Helsinki, Helsinki, Finland Faculty of Veterinary Medicine, Department of Clinical and Veterinary Sciences, UniÕersity of Helsinki, SF-04920 Saari, Finland c Pouttu OY, SF-69 101 Kannus, Finland Accepted 17 July 1998
Abstract In situ hybridization with an X chromosome specific painting probe can be used as a tool for studying the numerical and structural rearrangements of X chromosomes. The commercially available porcine specific X chromosome painting probe is still unable to reliably separate autosomes. However, due to across-species X chromosome homology, the human specific X chromosome painting probe can be used in the identification of X chromosomes in pig metaphases. The commercially available human X chromosome specific painting probe was hybridized to metaphase spreads in a Klinefelter boar with a 2 n s 39,XXY karyotype to characterize the X chromosomes. Klinefelter syndrome with its effects on the male reproductive trait such as testicular hypoplasia, is under the genetic control of some sex-linked genes in the extra X chromosome which have escaped the X inactivation process. Chromosome analysis by chromosome painting using fluorescence in situ hybridization may in future be more widely used in veterinary medicine and the selection of breeding animals. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Pig genetics; Klinefelter syndrome; Painting probe; FISH
)
Corresponding author. Tel.: q358 9 708 5292; fax: q358 9 708 5379; e-mail: [email protected]
0378-4320r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 8 . 0 0 1 3 6 - 5
318
A. Makinen et al.r Animal Reproduction Science 52 (1998) 317–323 ¨
1. Introduction A complete chromosome painting probe is a pool of many probes, each with a sequence complementary to a different region along the length of a similar chromosome. When this pool of probes is used, fluorescence in situ hybridization ŽFISH. relies on the hybridization of chromosome specific DNA probes and chromosome specific painting probes with the chromosome of interest ŽLangford et al., 1993; Yerle et al., 1993.. The corresponding chromosome of interest fluoresces and allows the direct visualization of gross chromosome rearrangements and highlights any aneuploids ŽKonfortova et al., 1995; Breen et al., 1997.. Across-species whole chromosome or chromosome segment homologies are based on conserved DNA sequences between the species under investigation and demonstrate similar banding patterns and gene mapping ŽRettenberger et al., 1995; Goureau et al., 1996.. That fact allows the identification of pig X chromosomes using fluorescence in situ hybridization ŽFISH. with human X chromosome specific paints ŽFronicke et al., 1996.; ¨ chromosome banding and gene homology have been observed between human and pig X chromosomes ŽFrancke, 1994.. Here, we have used a commercially available human chromosome specific X chromosome library as a probe for porcine metaphase chromosomes to study a numerical X chromosome anomaly in domestic male pig Ž Sus scrofa domestica. metaphases.
2. Materials and methods 2.1. Chromosome preparation and fertility studies One Yorkshire breed boar was brought to our attention because it had not produced offspring after mating with 25 sows. It was born in a litter of 11 piglets and selected for breeding purposes whereas the other piglets were reared for meat production. The boar was 15 months old at the time of investigation, its male parent was alive, but its female parent had already been slaughtered. Blood lymphocyte cultures were initiated from the boar and its male parent. Standard methods were applied for cell culturing and preparation of metaphase spreads. The chromosomes were arranged into karyotypes according to the pig standard karyotype ŽCommittee for the Standardized Karyotype of the Domestic Pig, 1988.. An earlier investigation of the GTG-banding ŽSeabright, 1971. of metaphases from cultured lymphocytes had indicated that the karyotype of the boar with Klinefelter syndrome was a 2 n s 39,XXY and that of its male parent was a 2 n s 38,XY karyotype ŽMakinen et ¨ al., 1997.. The fertility records were collected from the male parent but from the female parent they were no longer available for further investigation. Samples from testicular tissue for histological study were collected at the time of boars slaughter. Pieces of testicular tissue were fixed in Bouin-fixative, embedded in paraffin, sectioned and stained with Harris’ haematoxylin-eosin.
A. Makinen et al.r Animal Reproduction Science 52 (1998) 317–323 ¨
319
2.2. The detection of X chromosomes after fluorescence in situ hybridization (FISH) technique using human X chromosome painting probe The human X chromosome specific DNA library used for situ hybridization in this study was the commercially available Oncor, Coatasome w digoxigenin labelled total X chromosome specific probe. In situ hybridization and detection of hybridized DNA was carried out using the procedure described in the manufacturers protocol with one exception, the probe was allowed to hybridize to the chromosomal material for 5 days. The human X chromosome specific DNA library painting probe indicated that there were two X chromosomes in all the metaphase spreads of the Klinefelter boar after FISH ŽFig. 1.. Moreover, only one painting signal was present on the X chromosome in all the metaphase spreads in the boar’s male parent with a 2 n s 38,XY karyotype. The painted X chromosomes were yellow and the other chromosomes were reddish. Although crosshybridization was particularly high with this probe, specific signals on the X chromosomes were clearly observed. The weak signals along all the chromosomes was probably due to nonspecific hybridization of sequences in the probe mixture. These weak signals were distinguishable from the painting signals, and the specific signals were always stronger than the background. While the painting probe of human X chromosome strongly painted the entire porcine X chromosome, a clear hybridization signal was not visible on the pseudoautosomal
Fig. 1. The FISH with a whole human X chromosome-specific painting probe on a metaphase spread from the 2 ns 39,XXY Klinefelter boar. Both X chromosomes are painted Žindicated by arrows.. There is no visible hybridization at the pseudoautosomal region of the Y chromosome Žshown by an arrowhead..
320
A. Makinen et al.r Animal Reproduction Science 52 (1998) 317–323 ¨
region of the pig Y chromosome and the centromeres in the pig X chromosomes lack cross-hybridization as well. The use of human X chromosome painting probe was tested on the boar’s male parent which had a normal pig karyotype. The only chromosome to be highlighted or painted was one X chromosome and here too no visible hybridization were detectable in the pseudoautosomal region of the Y chromosome in the metaphases of this 2 n s 38,XY male pig. So, the use of commercial probes of human X chromosome painting appears efficient enough to investigate the X chromosomes of pig karyotypes. 2.3. Reproduction and phenotype of the Klinefelter boar The boar displayed a vigorous libido but its sperm quality was not studied. Histologically, the testes were predominately composed of normal looking Leydig cells and seminiferous tubules which contained no germinal cells, consisting of irregular shaped masses of collagenous connective tissue ŽFig. 2.. Moreover, light microscopic examinations revealed no meiotic chromosomes. The most prominent difference in this boar’s phenotype was the size of its testes, they were approximately one-third of the usual size for its breed and age. In addition it was observed that its facial appearance was altered, giving the impression that it had a frontal
Fig. 2. A histological photograph from the testes of the XXY boar. The seminiferous tubules lack germ cells Žindicated by arrow., only a few sertoli cells surrounded the epithelium. The Leydig cells appeared to be normal Žshown by an arrowhead..
A. Makinen et al.r Animal Reproduction Science 52 (1998) 317–323 ¨
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bone slightly more convex than usual for Yorkshire boars. However, this phenotypical difference was only subjective. The fertility records of the male parent of this boar were normal for its breed. However, the fertility results from its female parent could not be obtained from the farm records. So, we presume that the Klinefelter syndrome was the result of nondisjunction during meiosis in one of the parents. Thus, the additional X chromosome in the studied offspring was of de novo origin.
3. Discussion Chromosome painting in fluorescence in situ hybridization is a technique increasingly employed in both clinical and molecular genetics investigations of domesticated animals. Unfortunately, at present, a commercially painting probe for the pig X chromosome is still being developed and so a commercially available human specific painting probe was used in this across-species hybridization. The painting of pig metaphases with a whole X chromosome specific DNA library covering the entire human X chromosome has established correspondences between human and pig chromosomes. This is in spite of the long phylogenetic distance of at least 93 million years of divergent evolution between humans and pigs ŽJanke et al., 1994.. The labelling of pig X chromosomes with a human whole X chromosome painting probe was clearly visualized and permitted the precise delineation of the hybridized pig X chromosomes except for the centromeres, which did not exhibit cross-hybridization, indicating the presence of divergent centromeric satellite DNAs. This result is in accordance with earlier observations by Fronicke et al. Ž1996.. ¨ Probably, due to the heterologous chromosome painting, the pseudoautosomal region between the pig X and Y chromosomes was insufficient to detect, in spite of the region between the X and Y chromosomes sharing DNA sequence homologies ŽSigner et al., 1994.. In connection with this heterologous hybridization using human Y chromosome as a painting probe on pig metaphase spreads gave no signals on the Y chromosome in studies by Goureau et al. Ž1996. and Fronicke et al. Ž1996.. This is thought to be due to ¨ the species-specific repeated sequences on the Y chromosomes, which are insufficient for heterologous chromosome painting. The most important factor in producing a strong intensity of painting was the duration of hybridization as has been earlier pointed out by Rettenberger et al. Ž1995.. In spite of this, the use of commercial painting probes for human X chromosome appears efficient enough to investigate the X chromosomes on the metaphases of pigs. The comparison of genetic data and the exchange of map information will also highlight the importance of studying various reproductive processes that are under the control of genes responsible for reproductive efficiency in farm animals ŽRothschild, 1996.. The presence of an additional X chromosome must have some genetic effect on gonadal development and subsequent function, as the extremely small testicle size of the boar was most likely due to a partial lack of development of the seminiferous tubules
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A. Makinen et al.r Animal Reproduction Science 52 (1998) 317–323 ¨
and a total lack of development of the germinal epithelium within the seminiferous tubules. Even though the libido and service behaviour of the animal may have been normal, however, ejaculates would have been azoospermic. So, genetics play an integral role in the control of the reproductive traits in males which possess two X chromosomes and one Y chromosome. Moreover, the identification of a mosaic karyotype, in which only a small proportion of cells show some variation from the majority karyotype is important, as it may provide diagnostic information about clinical and reproduction defects in Klinefelter syndrome. The FISH technique allows us to more easily identify of Klinefelter individuals with a mosaic structure than conventional chromosome analysis using banding techniques on metaphases. Chromosome painting using FISH may in future result in more attention being paid to structural and numerical chromosomal rearrangements in investigations of the reproduction disorders of farm animals.
Acknowledgements The authors are grateful to the Finnish Animal Breeding Association for providing the fertility results for the male parent of the studied boar.
References Breen, M., Langford, C.F., Carter, N.P., Fischer, P.E., Marti, E., Gerstenberg, C., Allen, W.R., Lear, T.L., Binns, M.M., 1997. Detection of equine X chromosome abnormalities in Equids using a horse X whole chromosome paint probe ŽWCPP.. The Veterinary Journal 153, 235–238. Committee for the Standardized Karyotype of the Domestic Pig, 1988. Standard karyotype of the domestic pig. Hereditas 109, 151–157. Francke, U., 1994. Digitized and differentially shaded human chromosome ideograms for genomic applications. Cytogenet. Cell. Genet. 65, 206–219. Fronicke, L., Chowdhary, B.P., Scherthan, H., Gustavsson, I., 1996. A comparative map of the porcine and ¨ human genomes demonstrates ZOO-FISH and gene mapping-based chromosomal homologies. Mammalian Genome 7, 285–290. Goureau, A., Yerle, M., Schmitz, A., Riquet, J., Milan, D., Pinton, P., Frelat, G., Gellin, J., 1996. Human and porcine correspondence of chromosome segments using bidirectional chromosome painting. Genomics 36, 252–262. Janke, A., Feldmaier-Fuchs, G., Thomas, W.K., von Haeseler, A., Paabo, ¨¨ S., 1994. The marsupial mitochondrial genome and the evolution of placental mammals. Genetics 137, 243–256. Konfortova, G.D., Miller, N.G.A., Tucker, E.M., 1995. A new reciprocal translocation Ž7qq;15qy. in the domestic pig. Cytogenet. Cell. Genet. 71, 285–288. Langford, C.F., Telenius, H., Miller, N.G.A., Thomsen, P.D., Tucker, E.M., 1993. Preparation of chromosome-specific paints and complete assignment of chromosomes in the pig flow karyotype. Animal Genetics 24, 261–267. Makinen, A., Andersson, M., Nikunen, S., 1997. Testicular hypoplasia in a boar with 39,XXY karyotype, ¨ 1997. Proceedings of International Conference on Animal Biotechnology, pp. 87–90. Rettenberger, G., Klett, C., Zechner, U., Kunz, J., Vogel, W., Hameister, H., 1995. Visualization of the conservation of synteny between humans and pigs by heterologous chromosomal painting. Genomics 26, 372–378.
A. Makinen et al.r Animal Reproduction Science 52 (1998) 317–323 ¨
323
Rothschild, M.F., 1996. Genetics and reproduction in the pig. Animal Reproduction Science 42, 143–151. Seabright, M., 1971. A rapid banding technique for human chromosomes. Lancet 2, 971–972. Signer, E.N., Gu, F., Gustavsson, I., Andersson, L., Jeffreys, A.J., 1994. A pseudoautosomal minisatellite in the pig. Mamm. Genome 5, 48–51. Yerle, M., Schmitz, A., Milan, D., Chaput, B., Monteagudo, L., Vaiman, M., Frelat, G., Gellin, J., 1993. Accurate characterization of porcine bivariate flow karyotype by PCR and fluorescence in situ hybridization. Genomics 16, 97–103.
Detection of the X chromosomes in a Klinefelter boar using a whole human X chromosome painting probe Auli Makinen ¨
a,)
, Magnus Andersson b, Sanna Nikunen
c
a
b
Department of Animal Science, P.O. Box 28, SF-00014 UniÕersity of Helsinki, Helsinki, Finland Faculty of Veterinary Medicine, Department of Clinical and Veterinary Sciences, UniÕersity of Helsinki, SF-04920 Saari, Finland c Pouttu OY, SF-69 101 Kannus, Finland Accepted 17 July 1998
Abstract In situ hybridization with an X chromosome specific painting probe can be used as a tool for studying the numerical and structural rearrangements of X chromosomes. The commercially available porcine specific X chromosome painting probe is still unable to reliably separate autosomes. However, due to across-species X chromosome homology, the human specific X chromosome painting probe can be used in the identification of X chromosomes in pig metaphases. The commercially available human X chromosome specific painting probe was hybridized to metaphase spreads in a Klinefelter boar with a 2 n s 39,XXY karyotype to characterize the X chromosomes. Klinefelter syndrome with its effects on the male reproductive trait such as testicular hypoplasia, is under the genetic control of some sex-linked genes in the extra X chromosome which have escaped the X inactivation process. Chromosome analysis by chromosome painting using fluorescence in situ hybridization may in future be more widely used in veterinary medicine and the selection of breeding animals. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Pig genetics; Klinefelter syndrome; Painting probe; FISH
)
Corresponding author. Tel.: q358 9 708 5292; fax: q358 9 708 5379; e-mail: [email protected]
0378-4320r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 8 . 0 0 1 3 6 - 5
318
A. Makinen et al.r Animal Reproduction Science 52 (1998) 317–323 ¨
1. Introduction A complete chromosome painting probe is a pool of many probes, each with a sequence complementary to a different region along the length of a similar chromosome. When this pool of probes is used, fluorescence in situ hybridization ŽFISH. relies on the hybridization of chromosome specific DNA probes and chromosome specific painting probes with the chromosome of interest ŽLangford et al., 1993; Yerle et al., 1993.. The corresponding chromosome of interest fluoresces and allows the direct visualization of gross chromosome rearrangements and highlights any aneuploids ŽKonfortova et al., 1995; Breen et al., 1997.. Across-species whole chromosome or chromosome segment homologies are based on conserved DNA sequences between the species under investigation and demonstrate similar banding patterns and gene mapping ŽRettenberger et al., 1995; Goureau et al., 1996.. That fact allows the identification of pig X chromosomes using fluorescence in situ hybridization ŽFISH. with human X chromosome specific paints ŽFronicke et al., 1996.; ¨ chromosome banding and gene homology have been observed between human and pig X chromosomes ŽFrancke, 1994.. Here, we have used a commercially available human chromosome specific X chromosome library as a probe for porcine metaphase chromosomes to study a numerical X chromosome anomaly in domestic male pig Ž Sus scrofa domestica. metaphases.
2. Materials and methods 2.1. Chromosome preparation and fertility studies One Yorkshire breed boar was brought to our attention because it had not produced offspring after mating with 25 sows. It was born in a litter of 11 piglets and selected for breeding purposes whereas the other piglets were reared for meat production. The boar was 15 months old at the time of investigation, its male parent was alive, but its female parent had already been slaughtered. Blood lymphocyte cultures were initiated from the boar and its male parent. Standard methods were applied for cell culturing and preparation of metaphase spreads. The chromosomes were arranged into karyotypes according to the pig standard karyotype ŽCommittee for the Standardized Karyotype of the Domestic Pig, 1988.. An earlier investigation of the GTG-banding ŽSeabright, 1971. of metaphases from cultured lymphocytes had indicated that the karyotype of the boar with Klinefelter syndrome was a 2 n s 39,XXY and that of its male parent was a 2 n s 38,XY karyotype ŽMakinen et ¨ al., 1997.. The fertility records were collected from the male parent but from the female parent they were no longer available for further investigation. Samples from testicular tissue for histological study were collected at the time of boars slaughter. Pieces of testicular tissue were fixed in Bouin-fixative, embedded in paraffin, sectioned and stained with Harris’ haematoxylin-eosin.
A. Makinen et al.r Animal Reproduction Science 52 (1998) 317–323 ¨
319
2.2. The detection of X chromosomes after fluorescence in situ hybridization (FISH) technique using human X chromosome painting probe The human X chromosome specific DNA library used for situ hybridization in this study was the commercially available Oncor, Coatasome w digoxigenin labelled total X chromosome specific probe. In situ hybridization and detection of hybridized DNA was carried out using the procedure described in the manufacturers protocol with one exception, the probe was allowed to hybridize to the chromosomal material for 5 days. The human X chromosome specific DNA library painting probe indicated that there were two X chromosomes in all the metaphase spreads of the Klinefelter boar after FISH ŽFig. 1.. Moreover, only one painting signal was present on the X chromosome in all the metaphase spreads in the boar’s male parent with a 2 n s 38,XY karyotype. The painted X chromosomes were yellow and the other chromosomes were reddish. Although crosshybridization was particularly high with this probe, specific signals on the X chromosomes were clearly observed. The weak signals along all the chromosomes was probably due to nonspecific hybridization of sequences in the probe mixture. These weak signals were distinguishable from the painting signals, and the specific signals were always stronger than the background. While the painting probe of human X chromosome strongly painted the entire porcine X chromosome, a clear hybridization signal was not visible on the pseudoautosomal
Fig. 1. The FISH with a whole human X chromosome-specific painting probe on a metaphase spread from the 2 ns 39,XXY Klinefelter boar. Both X chromosomes are painted Žindicated by arrows.. There is no visible hybridization at the pseudoautosomal region of the Y chromosome Žshown by an arrowhead..
320
A. Makinen et al.r Animal Reproduction Science 52 (1998) 317–323 ¨
region of the pig Y chromosome and the centromeres in the pig X chromosomes lack cross-hybridization as well. The use of human X chromosome painting probe was tested on the boar’s male parent which had a normal pig karyotype. The only chromosome to be highlighted or painted was one X chromosome and here too no visible hybridization were detectable in the pseudoautosomal region of the Y chromosome in the metaphases of this 2 n s 38,XY male pig. So, the use of commercial probes of human X chromosome painting appears efficient enough to investigate the X chromosomes of pig karyotypes. 2.3. Reproduction and phenotype of the Klinefelter boar The boar displayed a vigorous libido but its sperm quality was not studied. Histologically, the testes were predominately composed of normal looking Leydig cells and seminiferous tubules which contained no germinal cells, consisting of irregular shaped masses of collagenous connective tissue ŽFig. 2.. Moreover, light microscopic examinations revealed no meiotic chromosomes. The most prominent difference in this boar’s phenotype was the size of its testes, they were approximately one-third of the usual size for its breed and age. In addition it was observed that its facial appearance was altered, giving the impression that it had a frontal
Fig. 2. A histological photograph from the testes of the XXY boar. The seminiferous tubules lack germ cells Žindicated by arrow., only a few sertoli cells surrounded the epithelium. The Leydig cells appeared to be normal Žshown by an arrowhead..
A. Makinen et al.r Animal Reproduction Science 52 (1998) 317–323 ¨
321
bone slightly more convex than usual for Yorkshire boars. However, this phenotypical difference was only subjective. The fertility records of the male parent of this boar were normal for its breed. However, the fertility results from its female parent could not be obtained from the farm records. So, we presume that the Klinefelter syndrome was the result of nondisjunction during meiosis in one of the parents. Thus, the additional X chromosome in the studied offspring was of de novo origin.
3. Discussion Chromosome painting in fluorescence in situ hybridization is a technique increasingly employed in both clinical and molecular genetics investigations of domesticated animals. Unfortunately, at present, a commercially painting probe for the pig X chromosome is still being developed and so a commercially available human specific painting probe was used in this across-species hybridization. The painting of pig metaphases with a whole X chromosome specific DNA library covering the entire human X chromosome has established correspondences between human and pig chromosomes. This is in spite of the long phylogenetic distance of at least 93 million years of divergent evolution between humans and pigs ŽJanke et al., 1994.. The labelling of pig X chromosomes with a human whole X chromosome painting probe was clearly visualized and permitted the precise delineation of the hybridized pig X chromosomes except for the centromeres, which did not exhibit cross-hybridization, indicating the presence of divergent centromeric satellite DNAs. This result is in accordance with earlier observations by Fronicke et al. Ž1996.. ¨ Probably, due to the heterologous chromosome painting, the pseudoautosomal region between the pig X and Y chromosomes was insufficient to detect, in spite of the region between the X and Y chromosomes sharing DNA sequence homologies ŽSigner et al., 1994.. In connection with this heterologous hybridization using human Y chromosome as a painting probe on pig metaphase spreads gave no signals on the Y chromosome in studies by Goureau et al. Ž1996. and Fronicke et al. Ž1996.. This is thought to be due to ¨ the species-specific repeated sequences on the Y chromosomes, which are insufficient for heterologous chromosome painting. The most important factor in producing a strong intensity of painting was the duration of hybridization as has been earlier pointed out by Rettenberger et al. Ž1995.. In spite of this, the use of commercial painting probes for human X chromosome appears efficient enough to investigate the X chromosomes on the metaphases of pigs. The comparison of genetic data and the exchange of map information will also highlight the importance of studying various reproductive processes that are under the control of genes responsible for reproductive efficiency in farm animals ŽRothschild, 1996.. The presence of an additional X chromosome must have some genetic effect on gonadal development and subsequent function, as the extremely small testicle size of the boar was most likely due to a partial lack of development of the seminiferous tubules
322
A. Makinen et al.r Animal Reproduction Science 52 (1998) 317–323 ¨
and a total lack of development of the germinal epithelium within the seminiferous tubules. Even though the libido and service behaviour of the animal may have been normal, however, ejaculates would have been azoospermic. So, genetics play an integral role in the control of the reproductive traits in males which possess two X chromosomes and one Y chromosome. Moreover, the identification of a mosaic karyotype, in which only a small proportion of cells show some variation from the majority karyotype is important, as it may provide diagnostic information about clinical and reproduction defects in Klinefelter syndrome. The FISH technique allows us to more easily identify of Klinefelter individuals with a mosaic structure than conventional chromosome analysis using banding techniques on metaphases. Chromosome painting using FISH may in future result in more attention being paid to structural and numerical chromosomal rearrangements in investigations of the reproduction disorders of farm animals.
Acknowledgements The authors are grateful to the Finnish Animal Breeding Association for providing the fertility results for the male parent of the studied boar.
References Breen, M., Langford, C.F., Carter, N.P., Fischer, P.E., Marti, E., Gerstenberg, C., Allen, W.R., Lear, T.L., Binns, M.M., 1997. Detection of equine X chromosome abnormalities in Equids using a horse X whole chromosome paint probe ŽWCPP.. The Veterinary Journal 153, 235–238. Committee for the Standardized Karyotype of the Domestic Pig, 1988. Standard karyotype of the domestic pig. Hereditas 109, 151–157. Francke, U., 1994. Digitized and differentially shaded human chromosome ideograms for genomic applications. Cytogenet. Cell. Genet. 65, 206–219. Fronicke, L., Chowdhary, B.P., Scherthan, H., Gustavsson, I., 1996. A comparative map of the porcine and ¨ human genomes demonstrates ZOO-FISH and gene mapping-based chromosomal homologies. Mammalian Genome 7, 285–290. Goureau, A., Yerle, M., Schmitz, A., Riquet, J., Milan, D., Pinton, P., Frelat, G., Gellin, J., 1996. Human and porcine correspondence of chromosome segments using bidirectional chromosome painting. Genomics 36, 252–262. Janke, A., Feldmaier-Fuchs, G., Thomas, W.K., von Haeseler, A., Paabo, ¨¨ S., 1994. The marsupial mitochondrial genome and the evolution of placental mammals. Genetics 137, 243–256. Konfortova, G.D., Miller, N.G.A., Tucker, E.M., 1995. A new reciprocal translocation Ž7qq;15qy. in the domestic pig. Cytogenet. Cell. Genet. 71, 285–288. Langford, C.F., Telenius, H., Miller, N.G.A., Thomsen, P.D., Tucker, E.M., 1993. Preparation of chromosome-specific paints and complete assignment of chromosomes in the pig flow karyotype. Animal Genetics 24, 261–267. Makinen, A., Andersson, M., Nikunen, S., 1997. Testicular hypoplasia in a boar with 39,XXY karyotype, ¨ 1997. Proceedings of International Conference on Animal Biotechnology, pp. 87–90. Rettenberger, G., Klett, C., Zechner, U., Kunz, J., Vogel, W., Hameister, H., 1995. Visualization of the conservation of synteny between humans and pigs by heterologous chromosomal painting. Genomics 26, 372–378.
A. Makinen et al.r Animal Reproduction Science 52 (1998) 317–323 ¨
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Rothschild, M.F., 1996. Genetics and reproduction in the pig. Animal Reproduction Science 42, 143–151. Seabright, M., 1971. A rapid banding technique for human chromosomes. Lancet 2, 971–972. Signer, E.N., Gu, F., Gustavsson, I., Andersson, L., Jeffreys, A.J., 1994. A pseudoautosomal minisatellite in the pig. Mamm. Genome 5, 48–51. Yerle, M., Schmitz, A., Milan, D., Chaput, B., Monteagudo, L., Vaiman, M., Frelat, G., Gellin, J., 1993. Accurate characterization of porcine bivariate flow karyotype by PCR and fluorescence in situ hybridization. Genomics 16, 97–103.