Hirohito Yamakawa

Introduction of α(1,2)‐fucosyltransferase and its effect on a‐Gal epitopes in transgenic pig

Abstract: Hyperacute rejection (from pig to human) is thought to result from activation of complement initiated by the binding of host natural antibodies to α‐galactosyl (α‐Gal) epitopes of donor endothelial cells. However, α‐Gal epitope shares a common precursor with H antigen in humans. This means that H antigens as well as α‐Gal epitopes are synthesized in a competitive manner by different enzymes. We thought that it would be possible to convert α‐Gal epitopes into H antigens by introducing cDNA of α(1,2)‐fucosyltransferase (α1–2FT) into porcine cells, and so, pig embryos were microinjected with αl‐2FT cDNA. Transgenic pigs that carried α1–2FT were thus established. Cytotoxicity of fibrocytes derived from skin of transgenic pig was measured by 51Cr release assay, which showed that H antigen‐expressing cells were significantly resistant to a challenge with human sera. These experiments indicate that our method provides a new strategy which contributes to a successful discordant xenotransplantation.

The survival of whole and bisected rabbit morulae after cryopreservation by the vitrification method.

The survival of whole and bisected rabbit morulae cryopreserved by the vitrification method was investigated. The embryos were loaded in a column of vitrification solution (VS, a mixture of 25% glycerol and 25% 1, 2-propanediol in PBS+16% calf serum), which was located between two columns of 1 M sucrose solution in a plastic straw. The embryos were frozen by being plunged into liquid nitrogen and thawed in a water bath at 20 degrees C. Two methods of loading embryos into straws were used: the single and double column vitrification solution methods. The embryonic survival rates between these two methods were compared. Seventy-one (86.6%) out of 82 morulae vitrified in double column straws developed into the blastocyst stage in vitro. Eleven (18.3%) live fetuses were obtained after the transfer of 60 frozen-thawed morulae to four recipients. By contrast, the survival rate (36.5%, 27 74 ) of embryos vitrified in the single column straws was significantly lower (P<0.05). The vitrification solution of the single column straws became opaque, indicating ice-crystal formation, upon thawing in 5 of 11 straws, which was assumed to have damaged the embryos. More than 80% (29 36 ) of the bisected morulae frozen and thawed in the double column straws developed to the blastocyst stage in vitro when cryoprotectant was diluted stepwise with 1 M and 0.25 M sucrose solution. When the cryoprotectant was removed by one-step dilution with 1 M sucrose solution, swelling in blastomeres was observed and the development rate of the recovered embryos decreased (45.8%, 11 24 ). These results indicate that the vitrification method employed in our experiment is not only efficient for the cryopreservation of rabbit morulae, but it can also be used for the preservation of bisected rabbit morulae, which had not been successful using previous methods.

Are fluorescent bodies of Y-spermatozoa detectable in common with mammalan species?*

An attempt was made to detect the fluorescent bodies (F-body), using Quinacrine mustard (Q-M) staining in the spermatozoa from eight mammalian species (human, bull, boar, dog, rabbit, rat, mouse, and mastomys) as well as in the cock (used as negative control). Sperm suspension, prepared after rinsing by repeated centrifugation with phosphate buffered saline (PBS), was either stained with Q-M for 24 h or treated with protease and then stained with Q-M for 60 min. The final concentration of Q-M in the mixed staining sperm suspension was 0.025 mg/ml. The examination using a reflecting fluorescent microscope revealed that the F-body found in human sperm was also present in the sperm of all the mammals but not in the cock after 24 h of staining. The enzyme-treated specimens showed higher incidences of F-bodies than specimens stained for 24 h without enzymatic digestion. These findings strongly suggest that the F-body is commonly present in the spermatozoa of many mammalian species.