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Development of cryopreservation method of the farm animal ovary

家畜卵巢超低温保存方法的研制

基本信息

批准号：
02660274
负责人：
MIYAMOTO Hajime
金额：
$ 1.54万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

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.

将7日龄大鼠卵巢在2 M甘油中缓慢冷冻或使用改良的VS 1玻璃化法冷却，通过逐步方法或在0.5M蔗糖-PBS中加温并稀释。在每个冷冻保存过程中，对它们进行组织学评价。通过器官培养证实玻璃化冷冻卵巢的活力。玻璃化冷冻和蔗糖稀释的卵巢的活力也进行了测定，通过正交移植到绝育的成年大鼠。将山羊卵巢组织块（0.7-1 mm立方体）在2 M甘油或1.5 M DMSO中缓慢冷却，或通过玻璃化方法快速冷却，温热，稀释并进行组织学和细胞学评价。在大鼠卵巢冷冻保存中，玻璃化冷冻卵巢组织学保存效果较好，冷冻保护剂的渗透和化学作用导致细胞轻度皱缩和固缩，渗透作用和结冰导致细胞轻度破裂。缓慢冷却的卵巢中，颗粒细胞膨胀和破裂，卵母细胞严重破坏和萎缩。培养4d后，在玻璃化冷冻的卵巢中观察到具有2层或更多层颗粒细胞的生长卵泡（24.7*28.8个卵泡/卵巢）。12/17只（70.6%）移植大鼠的卵巢内分泌活动和/或单侧或双侧卵巢生长，但大多数表现为形态和功能异常。他们中的一部分交配，大多数成为假孕，但没有产生后代。结果表明，玻璃化法冷冻保存的大鼠卵巢保持了部分活力。在山羊卵巢组织的冷冻保存件，组织学观察到的损害是类似的大鼠卵巢。在1.5 M DMSO中缓慢冷却的碎片显示出更好的保存。在2 M甘油中缓慢冷却或玻璃化并通过逐步方法稀释的碎片严重损坏。在玻璃化冷冻和蔗糖稀释切片中，部分卵泡的细胞学结构保存良好，但大多数显示轻度损伤。