Chromosome Analyses of Spermatozoa and Embryos Derived from Bulls Carrying the 7/21 Robertsonian Translocation

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1 Chromosome Analyses of Spermatozoa and Embryos Derived from Bulls Carrying the 7/21 Robertsonian Translocation Hirofumi HANADA, Masaya GESHI* and Osamu SUZUKI** National Institute of Animal Industry, Tsukuba Norin Kenkyu Danchi, Ibaraki-ken 305 * Tohoku National Agricultural Experiment Station, Morioka-shi ** National Grassland Research Institute, Nishinasuno-machi Tochigi-ken (Received June 27, 1995) Abstract To investigate the effect of the 7/21 Robertsonian translocation on fertility in Japanese Black Cattle, sperm chromosomes of the carrier bulls were analyzed using an interspecific in vitro fertilization system with zona-free golden hamster oocytes. Further studies were performed on embryos at the 8-cell stage derived from bulls heterozygous for the 7/21 translocation. No chromosomally unbalanced spermatozoa, due to the 7/21 translocation, were observed in a homozygous bull. In bulls heterozygous for the 7/21 translocation, however, unbalanced spermatozoa resulting from adjacent meiotic segregation were detected but only at a rate of 3.4%. A few unbalanced embryos were also observed in chromosome analysis of embryos sired by bulls heterozygous for the 7/21 translocation. These results suggest that the 7/21 translocation in a heterozygous state causes only a slight reduction in fertility. Anim. Sci. Technol. (Jpn.) 66 (11): , 1995 Key words: cattle, 7/21 translocation, sperm chromosome, bovine embryo analysis The 7/21 Robertsonian translocation is an individual chromosome aberration observed only in Japanese Black Cattle4,8). Recently, it has been reported that bulls heterozygous for the 7/21 translocation produced chromosomally unbalanced spermatozoa12) and resulted in abnormal embryos6,7). This translocation apparently causes a reduction in reproductive efficiency due to the death of unbalanced embryos during gestation. The degree depends on the incidence of chromosomally abnormal embryos resulting from the fertilization of unbalanced gametes and the time of their loss. However, there are few data about the incidence of unbalanced gametes and embryos due to the technical difficulties in preparing chromosome slides from spermatozoa and the limited number of embryos available for chromosome analysis. To investigate the effect of the 7/21 translocation on fertility in detail, we carried out a chromosome analysis of spermatozoa of bulls carrying the 7/21 Robertsonian translocation, and embryos produced by in vitro fertilization Materials and Methods of the semen. Chromosome analysis of spermatozoa: Frozen semen samples from five Japanese Black bulls were used in this experiment. Of these bulls, two were heterozygotes with 59,

2 HANADA, GESHI and SUZUKI XY, t (7q 21q) and one was a homozygote with 58, XY, t (7q 21q) t (7q 21q). The remaining two bulls had a normal karyotype of 60, XY. Chromosome analysis of embryos: Results The results of chromosome analysis of spermatozoa are shown in Table 1. In normal bulls, 41 out of a total of 42 spermatozoa karyotyped had normal haploid chromosome complements, 30, Y and 30, X. The remaining one was sperm with 29, Y. Except for one with 28, X, t, all of the analyzable spermatozoa had balanced chromosome complements in the 7/21 Table 1. Segregation of sperm chromosomes in the normal and the 7/21 translocation carrier bulls

3 Analyses of Bull Spermatozoa and Embryos translocation homozygous bull. In bulls heterozygous for the 7/21 translocation, chromosome analysis was successfully carried out on a total of 118 spermatozoa, of which 59 had normal chromosome complements and 53 were balanced spermatozoa. Four out of the remaining six spermatozoa were hyperhaploids with 30, Y, t and 30, X, t (Fig.1) chromosome complements, and two were unbalanced spermatozoa with 29, X and 31, X. The results of chromosome analysis of embryos are shown in Table 2. In the group sired by normal bulls, all of the analyzable embryos showed normal chromosome complements, 60, XY and 60, XX. While, two (5.1%) out of 39 embryos sired by the 7/21 translocation heterozygous bulls were monosomy (Fig. 2a) and trisomy (Fig.2b). The incidence corresponds to that of unbalanced spermatozoa (5.1%). In normal bulls and the 7/21 translocation homozygous bull, only % of spermatozoa analyzed was chromosomally unbalanced cells resulting from nondisjunction of the other chromosomes unrelated to the 7/21 translocation at the first division. No abnormal embryos were also observed in the group sired by normal bulls. These results suggest that Table 2. Chromosome analysis of embryos sired by the normal and heterozygous bulls for the 7/21 translocation Fig.1. Chromosome plate and karyotype of an unbalanced sperm with n=30, X, t (7q21q). Fig.2. Chromosome metaphase spreads of embryos sired by bulls heterozygous for the 7/21 Robertsonian translocation. Arrow shows the translocation chromosome. a : Monosomic embryo with 2n=59, XX. b : Trisomic embryo with 2n=60, XX, t (7q21q).

4 HANADA, GESHI and SUZUKI malsegregation occur very rarely in normal bull and a homozygous bull. We have reported that bulls heterozygous for the 7/21 translocation produced aneuploid secondary spermatocytes with a higher incidence5). The percentage of nondisjunction was 7.9% in the heterozygous state versus 2.3% in the normal bulls. These aneuploid cells from adjacent segregation were thought to be eliminated during spermatogenesis in sheep, because no chromosomally unbalanced blastocysts had been found in chromosome analysis of embryos9). Whereas, bulls heterozygous for the 7/21 translocation produced chromosomally unbalanced spermatozoa as reported by Tateno et al.12). Further study of embryos apparently indicated that these spermatozoa were capable of fertilization and resulted in abnormal embryos. However, we have failed to find any significant difference in both the fertilization rate and proportion of embryos developed to the blastocyst stage between the normal and the 7/21 translocation heterozygous bulls3). This may be due to the fact that unbalanced embryos occurred only rarely and they mostly survived to develop at least to the blastocyst stage. Nullisomic and disomic spermatozoa must be produced in equal frequency in bulls heterozygous for the 7/21 translocation. However, no unbalanced nullisomic spermatozoa were observed in the previous study12). Similarly, presumptive nullisomic sperm was the only one with 29, X in our study, which may have resulted from nondisjunction of the other chromosomes unrelated to the 7/21 translocation. While, four spermatozoa were disomic cells with 30, Y, t and 30, X, t chromosome complements, presumably resulting from adjacent segregation of the trivalent chromosome. Until now, we have analyzed a total of 119 embryos including 80 embryos at the blastocyst stage in the previous studies6,7). Nevertheless, monosomy was the only one at the 8-cell stage. Whereas six out of seven embryos with unbalanced karyotypes were trisomies for either chromosome 7 or 21 resulting from the fertilization of normal ova by aneuploid spermatozoa. Some studies in mice and hamsters suggest that a nullisomic state results in reduced fertilizing capability of sperm and a failure in fertilization1,10,13). Though preferential selection against the nullisomic spermatozoa may take place in the 7/21 translocation, there were not enough to draw a conclusion. In conclusion, the 7/21 translocation in a heterozygous state may be associated with a reduction in reproductive efficiency. However, the unbalanced gametes from adjacent segregation and the subsequent abnormal embryos were very rare. Most spermatozoa in the 7/21 translocation carriers resulted from alternate segregation of a trivalent chromosome. The 7/21 translocation seemed to cause only a slight reduction in fertility. Acknowledgements The authors thank Dr. H. Tateno, Asahikawa Medical College, for suggestions on analysis of bovine sperm chromosomes. References 1) Aranha I, Martin-DeLeon PA. The murine Rb (6.16) translocation: evidence for sperm selection and modulating effect of aging. Hum. Genet., 87: ) Geshi M, Yonai M, Hanada H, Komiyama T, Takahashi M. Effect of serum on the in vitro development of bovine embryos derived from in vitro maturation and fertilization. Anim. Sci. Technol. (Jpn.), 64: ) Geshi M, Sakaguchi M, Yonai M, Nagai T, Suzuki O, Hanada H. Effects of the 7/21 Robertsonian translocation of bull on fertilization rates and early development of bovine embryos in vitro. Theriogenology, 41: ) Hanada H, Muramatsu S, Abe T, Fukushima T. Robertsonian chromosome polymorphism found in a local herd of the Japanese Black cattle. Ann. Genet. Sel. anim., 13: ) Hanada H, Muramatsu S. A study of meiotic

5 Analyses of Bull Spermatozoa and Embryos chromosomes in Japanese Black bulls carrying the 7/21 Robertsonian translocation. Jpn. J. Zootech. Sci., 60: ) Hanada H, Geshi M, Sakaguchi M, Yonai M. Chromosome analyses of embryos sired by bulls heterozygous for the 7/21 Robertsonian translocation. Anim. Sci. Technol. (Jpn.), 64: ) Hanada H, Geshi M, Suzuki O. Additional evidence of formation of unbalanced embryos in cattle with the 7/21 Robertsonian translocation. Theriogenology, 44: ) Masuda H, Takahashi T, Soejima A, Waide Y. Centric fusion of the chromosomes in a Japanese Black bull and his offsprings. Jpn. J. Zootech. Sci., 49: ) Scott ISA, Bruere AN. Distribution of heterozygous translocations and aneuploid spermatocyte frequency in domestic sheep. J. Hered., 78: ) Sonta S, Yamada M, Tsukasaki M. Failure of chromosomally abnormal sperm to participate in fertilization in the Chinese hamster. Cytogenet. Cell Genet:, 57: ) Tateno H, Mikamo K. A chromosome method to distinguish between X- and Y- bearing spermatozoa of the bull in zona-free hamster ova. J. Reprod. Fert., 81: , ) Tateno H, Miyake Y-I, Mori H, Kamiguchi Y, Mikamo K. Sperm chromosome study of two bulls heterozygous for two different Robertsonian translocation. Hereditas, 120: ) Zackowski JL, Martin-DeLeon PA. Segregation products of male mice doubly heterozygous for the Rb (6. 16) and Rb (16. 17) translocation: influence of sperm karyotype on fertilizing competence under varying mating frequencies. Gamete Res., 22: