Abstract
Spermatozoa of mutated mice whose appearance quite resembles the rhino mouse were frozen and thawed, then submitted to in vitro fertilization and embryo transfer (IVF-ET). The cryopreservation solution was an 18% raffinose and 3% skim milk mixture, and the spermatozoa were stored in liquid nitrogen at -196°C. Two and half months later, the samples were thawed and fertilized with ICR mouse oocytes in vitro. The motility rate after thawing was approximately 20% and the fertilization rate was 14% (13/95), but 92% (12/13) of the fertilized eggs developed into the blastocyst stage. These twelve embryos were transferred into the uterine horns of a pseudopregnant recipient, and 75% (9/12) were born. The heterozygous F1 offspring did not show signs of the rhino-like phenotype, such as wrinkled skin without hair, but among a total of 39 offspring (F2) derived from brother-sister mating of F1 siblings, 11 (5 males and 6 females) pups showed signs of the rhino-like phenotype, e.g. at two- to three-weeks of age hair fell out, and at 6 months their skin became thick and wrinkled. Histopathological observations showed pilary canal cysts in the cortex of the skin, and dermal cysts were observed in the middle and inner area. These results demonstrate that the gene resources of rhino-like mutated mice can be preserved by the cryopreservation of spermatozoa and re-expressed via IVF-ET.
