|Budget Amount *help
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1992: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 1991: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1990: ¥1,600,000 (Direct Cost: ¥1,600,000)
Seven day-old rat ovaries were cooled by slow freezing in 2 M glycerol or by the vitrification method using modified VS1, warmed and diluted by stepwise method or in 0.5 M sucrose-PBS. In each cryopreservation process, they were evaluated histologically. Viability of vitrified ovaries were confirmed by organ culture. Viability of vitrified and sucrose-diluted ovaries were also assayed by orthotropic transplantation into spayed adult rats. Pieces of goat ovarian tissue (0.7-1 mm cubes) were cooled slowly in 2 M glycerol or 1.5 M DMSO, or cooled rapidly by vitrification method, warmed, diluted and evaluated histologically and cytologically. In cryopreservation of rat ovaries,vitrified ovaries showed better histological preservation, with mild cell shrinkage and pyknosis caused by osmotic and chemical effects of cryoprotectants and mild cell rupture by osmotic effect and ice formation. In ovaries cooled slowly, expansion and rupture of granulosa cells, and severe destruction and shrinkage of oocytes were noted. After 4 day-culture, growing follicles with 2 or more layers of granulosa cells were observed in vitrified ovaries (24.7*28.8 follicles/ovary). Endocrine activity of ovaries and/or growth of unilateral or bilateral ovaries were observed in 12/17 (70.6%) rats with grafts, but most showed abnormality in morphology and function. A part of them were mated and most became pseudopregnant, but none yielded progenies. These results indicate that rat ovaries cryopreserved by vitrification method have kept partial viability. In cryopreserved pieces of goat ovarian tissue, damage observed histologically were similar to those in rat ovaries. Pieces cooled slowly in 1.5 M DMSO showed better preservation. Pieces cooled slowly in 2 M glycerol or vitrified and diluted by stepwise method were severely damaged. In vitrified and sucrose-diluted pieces, cytological structure was well preserved in some of follicles, however, most showed mild damage.