Three dizygotic, heterosexual twins with chimerisms carrying marker chromosomes are described. Phenotypic and cytogenetic methods were used to identify these animals. The occurrence of germinal cell migration causing gonad chimerism can be detected by the marker chromosome event under conditions described in this repor

Dolf, G.

Fries, R.

Stocker, H.

Stranzlnger, G.

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The Journal of Heredity 72: 360-362. 1981.Some rare cases of chimerismin twin cattle and theirproposed use in determininggerminal cell migrationG. Stranzlnger, G. Dolf, R. Fries, andH. StockerABSTRACT: Three dizygotic, heterosexual twins withchimerlsms carrying marker chromosomes are de-scribed. Phenotypic and cytogenetlc methods were usedto identify these animals. The occurrence of germinalcell migration causing gonad chimerism can be detectedby the marker chromosome event under conditions de-scribed in this report.T H E PHENOMENON of chimerism has beenstudied and discussed frequently in recentyears1. In embryo transfers for twin produc-tion in cattle chimerism is a potential problembecause females with male twins are likely tobecome sterile10. Such females are unsuitablefor breeding and have subnormal weight-gainefficiency compared to males. Freemartinsamong twins are generally identified by rectalpalpation of adult animals8, as well as by theThe authors are professor and graduate students,respectively, at the Swiss Federal Institute ofTechnology (ETH), Institute of Animal Production,CH-8902 Zurich. They are grateful to Mrs. K. Emler,Miss Keeman, Mrs. Jenny, and Mrs. Kirianoff fortheir assistance in the preparation, translation andtyping of the manuscript. These studies have beenfinanced by the AGRO-Fonds ETH and the SwissAssociation for Artificial Insemination. The authorsthank Professor H. Abplanalp for his critical reviewof the manuscript.© 1981, American Genetic Association.normalcow ^ x heterozygotecentric fusion It/29© ©egg cell poll callsperm populationXXchJm. case A sterile•perm populationXX * female chromosome*XY= maleM : marker chromosome 1/29 7 \FIGURE 1 Fertilization of a normal egg cell with sperm carrying the centromere fusion chromosome.Also shown are the potential combinations of various germ cells (see text).size and form of the clitoris. A blood groupanalysis is successful only if a sufficientnumber of factors are tested so as to distin-guish the two erythrocyte populations. Suchstudies become more reliable if additionalbiochemical markers13 or an H-Y antigentest12 are included. A further diagnostic pos-sibility is a cytogenetic examination as de-scribed by Herschler and Fechheimer4'5.Given sufficient cell material one can deter-mine the origin of cells on the basis of sexchromosomes. However, the cell proportionis of little value when considering the degreeof virilization.A high proportion of affected females arefreemartins. The sex differentiation of themale partner seems to be quite normal, butaccording to various reports1 fertility troublesdo occur causing increased rates of cullingamong affected males. Thus, the presence ofXX primordial germ cells in testes9'16 couldlead to disturbances in spermatogenesis andthus alter reproductive fitness as well as sexratio among offspring. However, the time andTable I. Metaphases counted from short-term lymphocyte cultures and one long-term flbroblast culture (muscle cells) of chimeric twin pairsA, B, and C and repeat investigations in bulls B and CDate ofMaterial examinationBlood 24-9-799-10-7922-4-8017-7-8014-8-8025-9-809-10-8020-11-8029-1-8112-2-8126-2-81Fibroblast 15-5-81cellXYM27(20)23(22)Pair A*male femaletype cellXX XYM110(80) 28(24)82(78) 24(24)typeXX90(76)77(76)maletypeXX89(89)85(85)84(84)97(97)95(95)98(98)93(93)97(97)92(92)21(87)PairtypeXXM11(11)15(15)16(16)3(3)5(5)2(2)7(7)3(3)8(8)3(13)BfemalecellXY93(93)typeXXM7(7)malecellXY32(44)32(39)34(34)38(38)51(51)28(28)50(50)57(57)54(54)11(48)PairCtypeXXM41(56)61(61)66(66)62(62)49(49)72(72)50(50)43(43)46(46)12(52)femalecell typeXY XXM23(35) 42(65)* X, Y = sex chromosomes; M = marker chromosome; () = percentage360 Journal of Heredity Notesways of migration of XX cells into an XY malegonad is very uncertain. For one thing, im-plantation of the embryo occurs relatively late,approximately 20 days after fertilization, andthe development of placental vessels is evenfurther delayed. According to Jost et al.7, go-nadal differentiation is nearly complete at thattime, thus preventing the entry of female cellsinto the gonad from a twin.The positive identification of chimeric cellsin the male gonad and their subsequent de-velopment into spermatozoa that fertilize andgive rise to offspring can be achieved by coatcolor marker genes11, or more easily bymarker chromosomes such as the 1/29 centricfusion chromosome found quite often in cattlepopulations.Materials and MethodsAn experiment was conducted using mixedsemen from three bulls of the Angus, Sim-mental, and Bavarian Brown breeds andhaving diploid numbers of 60, 59, and 58chromosomes, respectively. The last two bullsare carriers of the 1/29 centric fusion eitherheterozygously or homozygously. As a firststep, twin calves of different sex sired by theheterozygous translocation carrier (59) wereproduced. Second, a cytogenetic investigationwas carried out to find animals with the nec-essary chromosome composition to prove go-nodal chimerism. Only male calves wereused, since the female partners were morelikely to be sterile. Several heterosexual twinpairs were obtained sired by the heterozygoteSimmental bull known to carry the centricfusion 1/29. In all cases, the males appearednormal, while females showed definitesymptoms of freemartinism.Standard techniques for leucocyte cultureand chromosome preparations were used14 tostudy the three twin pairs A, B, and C. Re-peated cultures (pair A, two times; the bullsfrom B and C, nine times) were made to assureaccurate results especially as related to thepossible influence of cell selection in vitro.Finally, muscle biopsies were taken from bullsB and C and fibroblast cultures established toget indications of chimerism from the chro-mosome preparations.Results and DiscussionThe cytogenetic analysis showed that intwin pair A the male resulted from the fertil-ization of a normal egg cell with spermcarrying the centromere fusion chromosome'(designated as M in Figure 1), while the fe-male developed from fertilization with anormal sperm. In the twin pairs B and C thereverse situation prevailed with femalescarrying the marker chromosomes. The di-agram in Figure 1 illustrates the initial situa-tion and the potential combinations of thevarious germ cells. For the sake of simplicity,all autosomes that are not essential in this di-agram have been omitted. The diagram in-cludes gametes expected from male twinswith hypothetically chimeric gonads includingthose of animals B and C that may eventuallylead to conclusive progeny tests. In Table I theresults of the number of cell populations fromthe three twin pairs and the results of repeatanalyses of bulls B and C from blood culturesand one fibroblast study are presented. Allthree cases show consistent results. The whiteblood cells from the bull of pair C varies in themale cells from 28 to 57 percent and in thefemale cells from 41 to 72 percent. The bull inpair B had a very low proportion of femalecells (2-16 percent); therefore, the animalsrepresent, to some extent, both possibilities ofdominating cells. Figure 2 shows the meta-phases of the four different cell types and twokaryograms including the sex chromosomesand marker chromosomes of the chromosomesituation in pair B. In the fibroblast culturesthe ratio of the two cell types are in the rangeof the lymphocytes (bull B, 87/13 percent; bullC, 48/52 percent). Therefore, the chimeric cellmixture is comparable in both tissues. Thebulls from twin pairs B and C can thus be usedin subsequent matings to further investigategonodal chimerism because if their testes alsoare chimeric they should transmit the markerchromosome to some female offsprings de-rived from their twin sister.The important factor in this unusual case ofchimerism is the use of translocation chro-mosome 1/29, as first suggested by Herschlerand Fechheimer4. Their marker chromosomein a set of bovine triplets and our case is the1/29 centric fusion type that was studied ex-tensively by Gustavsson3 and others2.Since twinning in cattle occurs in less than5 percent of births6 and the frequency ofcentric fusions also is low in most breeds,special matings involving bulls known to betranslocation heterozygotes are essential forinvestigations of chimerism, as proposed inthis paper. Numerous biochemical and ge-netical phenomena may be associated withsexual chimerism of cattle twins, but these9 ^ Vrtfi!flO;flnHit fi.Oifi 0Iv^) wfn fffl10pmD ft M 0/>r>OA^vf»ift* A ItwCase ACase B O o « i cmmFIGURE 2 Metaphases (case A and B) and two karyotypes (case A) and additional significant chromosomes (case B) of the chimeric cattle twins.Notes September / (Dctober 1981 361may be masked by embryonic mortality.Therefore, the described cases could serve asmodel examples with which to better under-stand certain events involved in sex differ-entiation or genetic expression. Since chim-erism is commonly found among heterosex-ual, dizygotic twins we believe that chimerismbetween like-sexed twins is equally frequentand should receive further attention in futurestudies, especially with animals from embryotransfers.A single offspring with a centromeric fusionchromosome from the described males B andC would support the theory of germ cellchimerism. Such studies are now in prog-ress.References1. DUNN, H. O., K. MCENTEE, C. E. HALL, R. H.JOHNSON, JR., and W. H. STONE. Cytogeneticand reproductive studies of bulls born co-twinwith freemartins. ]. Reprod. Fert. 57:21-30.1979.2. ELDRIDGE, F. E. High frequency of a Robertso-nian translocation in a herd of British Whitecattle. Vet. Rec. 96:71-73.1975.3. GUSTAVSSON, I. Cytogenetics, distribution andphenotypic effects of a translocation in Swedishcattle. Hereditas 63:68-169.1969.4. HERSCHLER, M. S. and N. S. FECHHEIMER.Centric fusion of chromosomes in a set of bovinetriplets. Cylogenetics 5:307-312.1966.5. and . The role of sex chromosomechimerism in alterning sexual development ofmammals. Cytogenetics 6:204.1967.6. JOHANSSON, I., B. LINDHE, and F. PIRCHNER.Causes of variation in the frequency of monoz-ygous and dizygous twinning in various breedsof cattle. Hereditas 78:201-234.1974.7. |OST, A., B. VIGIER, and J. PREPIN. Freemartinsin cattle: the first step of sexual organogenesis./. Reprod. Fert. 29:249-379.1972.8. KAESTLI, F. Neuere Untersuchungen uberdie Rinderzwicke—eine Literaturubersicht.Schwei'z. Arch. Tierheilk. 121:425-429.1979.9. OHNO, S., J. M. TRUIILLO, C. STENIUS, L. C.CHRISTIAN, and R. L. TEPLITZ. Possible germcell chimeras among newborn dizygotic twincalves (Bos taurus). Cytogenetics 1:258-265.1962.10- . The problem of the bovine freemartin. J.Reprod. Fert. (Suppl.) 7:53-61.1969.11. ROWSON, L. E. A., and R. NEWCOMB. A methodof determining whether germinal cells migratein bovine chimaeras. VIIIth Intern. Congress ofAnimal Reprod. and Artificial Insemination.Cracow, p. 266-268.1976.12. STOCKER, H. and G STRANZINGER. H-Y Anti-gen: Genetische Aspekte und Nachweismeth-oden (ein Literaturuberblick). Zuchthyg. 16:1-10.1981.13. STONE, W. H., D. T. BERMAN, W. J. TYLER, andM. R. IRWIN. Blood types of the progeny of apair of cattle twins showing erythrocyte mosai-cism. /. Hered. 51:136-140.1960.14. STRANZINGER, G. Die Wirkung verschiedenerBelastungseinflusse auf die Mitoseaktivitat derLeukozyten von weiblicheri Kalbern. Z.Tierzuchtg. Zuchtungsbiol. 92:27-34.1975.15. . New approach of demonstrating fertil-izing capacity of bulls with centric fusion chro-mosomes. 4th Eur. Colloq. Cytogenet. Domest.Anim. p. 220-224.1980.16. TEPLITZ, R. L., Y. S. MOON, and P. k. BASRUR.Further studies of chimerism in heterosexualcattle twins. Chromosoma 22:202-209.1967.The Journal of Heredity 72:362-363. 1981.Left ostium straight: aninherited anomaly of the femalegenital tract in the rabbitD. D. Crary and R. R. FoxABSTRACT: An inherited anomaly resulting in the leftoviduct extending lateral to the kidney with the ostiumopening upward in an otherwise normal animal is de-scribed. This may be the result of a mechanical problemcaused by the persistence of early embryonic mesen-teries. Inheritance appears to be due to an autosomalrecessive gene with incomplete penetrance. We pro-pose the symbol tos for the gene responsible lor thiscondition.ABNORMALITIES of the female genital tractare relatively rare in wild and domestic ani-mals and most are associated with abnor-malities of the urinary tract. The MullerianThe authors are, respectively, research associate,and senior staff scientist, The Jackson Laboratory,Bar Harbor, Maine 04609. This investigation wassupported in part by NIH research grant RR-00251from the Division of Research Resources to theJackson Laboratory, and in part by institutionalfunds of the Jackson Laboratory. The Jackson Lab-oratory is fully accredited by the American Associ-ation for Accreditation of Laboratory AnimalCare.© 1981, American Genetic Association.ducts are formed from invaginations of thecoelomic epithelium of the urogenital ridges7.Details of their growth are not well known butearly stages of their development suggest aclose relationship between Mullerian andWolffian ducts7. When a portion of the Wolf-fian duct is absent, the Mullerian duct willgrow only as far as the terminal end of theWolffian duct.The developing uterine tubes fail to matchthe elongation of the trunk as a whole and theflaring ostial ends finally lie some 13 segmentsposterior to the level of their origin1. Normallythe ostium is tied to the ovary by the fimbriae7and is pulled around mechanically when theovary migrates posteriorly so that the open endlies over the anterior end of the gonad.In 1955 when an adult rabbit of strain AX/Jwas necropsied, it was shown to be normal inevery respect except that the anterior end ofthe left oviduct projected anteriorly and lat-erally to the kidney with the ostium projectingstraight up instead of curling around the ovary.In 1958 six more cases were observed. Sincethat time many more such animals have beenseen both in strain AX/J and in its sublineAXBU/J. We know of no similar condition inany other animal. In this communication wereport the mode of inheritance and describethe pathology of this new condition in therabbit.Materials and MethodsOur data were obtained from the necropsyrecords of the rabbit colony of the JacksonLaboratory. All rabbits that die or are killed forexperimental purposes are necropsied andchecked for gross abnormalities of all majororgan systems3. The data for this report con-cern two partially inbred strains, AX/J andAXBU/J5.Standard genetic and statistical analyseswere used to determine the mode of inheri-tance.ResultsDescription. Figure 1 shows the relativeposition of the oviducts and ostia in a rabbitwith left ostium straight. The right oviduct andostium are normal with the ostium tubaecurving around its ovary. The left oviduct isstraight and projects craniad lateral to thekidney. The ostium tubae opens as a straightextension of the oviduct. In some specimensa thin mesentery tied the ostium tubae to thespleen, but most lacked such a mesentery. Itmay have existed temporarily in earlier de-velopment. The left ovary is in its normal po-sition, but the left ostium tubae remains closerto its primitive location.Litter size is significantly reduced in af-fected animals (Table I) of both strains.However, we have evidence that at least someof the eggs from the left ovary of affected fe-males can make the long journey to, and bepicked up by, the straight left ostium tubae.Genetics. From September 1969 throughDecember 1977, 1909 strain AX/J femaleswere examined and 106 (5.6 percent) werefound with straight left ostia. Similarly, among1397 strain AXBU/J females, 99 (7.1 percent)had the left ostium straight. In contrast, of12,133 females from all other strains examinedduring the same time period, only 5 (0.0004362 Journal of Heredity Notes

Some rare cases of chimerism in twin cattle and their proposed use in determining germinal cell migration

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