SATO Toshiya Niigata University, Brain Research Institute, Assistant, 脳研究所, 助手 (90359703)
FUJISAWA Nobuyoshi Niigata University, Brain Research Institute, Assistant, 脳研究所, 助手 (50199311)
|Budget Amount *help
¥14,800,000 (Direct Cost: ¥14,800,000)
Fiscal Year 2006: ¥5,600,000 (Direct Cost: ¥5,600,000)
Fiscal Year 2005: ¥9,200,000 (Direct Cost: ¥9,200,000)
To establish an experimental system for practical application by advancing the development and improvement of technologies for mouse ovary cryopreservation as a new line preservation, we examined conditions of donor ovaries, conditions of freezing-thawing, preparation of fertilized eggs derived from frozen ovaries, and conditions of ovary transplant to recipient female mice.
1. To find the conditions of yielding highly productive ovaries for offspring, some cryopreserved ovaries at different ages (days) were transplanted into recipient ovaries and post-transplant productivity was examined. As the result, offspring was produced by frozen ovaries in all groups (0, 10, 21, and 28 days post-partum). In view of the offspring productivity and its experimental procedures, satisfactory productivity was supposed to be generated in the 10-day and 21-day ovaries. Furthermore, favorable productivity was observed in the cryopreserved ovaries of 1 mm or less in size.
2. Histopathological analysis was
performed to investigate whether freezing damage may influence ovary tissues that are frozen and thawed by vitrification. The results showed that very little influence was revealed when soaking time in stock solution was within 30 minutes prior to freezing ; however, it was found that ovary tissues-mainly surrounding areas-began to degenerate when soaking time exceeded 60 minutes.
3. Immature eggs were isolated from cryopreserved ovaries and in vitro maturation and fertilization processed by in vitro incubation was examined. Approximately 50 unfertilized eggs were collected from each newborn ovary at 16, 18, and 22 days of age and almost half of these eggs were successfully matured after incubation. Although immature eggs were comparably obtained from cryopreserved ovaries, the maturity rate of eggs after incubation was low, i.e., remaining at nearly 10%. In addition, in vitro fertilization rate was approximately 40% in fresh ovary-derived eggs; whereas, it was 20% or lower in frozen ovary-derived eggs.
4. For ovary transplant, histocompatibility between "a donor system as the exporter of ovary" and "a recipient system as the importer of ovary" must be taken into consideration. We demonstrated the usefulness of SCID mice whose immune complex functions of T cells and B cells are incomplete as the recipient system. Less