DEVELOPMENT OF PRESERVATION SYSTEM FOR WILD MOUSE GENES BY FREEZING OF EMBRYOS AND GAMETES.

本研究从基因资源保存的角度出发，旨在通过冷冻胚胎和配子建立野生小鼠基因或基因组的储存系统，并通过一系列实验建立了以卵母细胞为雌性配子的卵巢的成功冷冻条件。组织学检查：采用-7℃播种后，以0.1 ~ 30℃缓慢冷却至-7℃，快速冷却至液氮温度，在冷冻过程的不同时间点对卵巢冷冻损伤进行组织学检查。至-30℃时，卵巢的损伤基本不明显，有大量形态正常的卵母细胞。另一方面，在卵巢浸入液氮后，几乎所有卵母细胞都受到损伤。冷冻卵巢移植：对照移植至-30ºC冷却即可获得新生儿，液氮移植未获得新生儿。这一结果与冷冻卵巢的组织学观察结果有较好的相关性。冷却速率检查：冷冻卵巢移植在冷却速率为0.1、0.2、0.5 ~ -30ºC的条件下获得新生儿。从冷冻卵巢移植到第一批新生儿出生的最短间隔为43,40,141天（对照组41天）。以上结果提示，胚胎冷冻时通常使用的0.5ºC/min的冷冻速率不适合用于卵巢冷冻。冷冻程序的建立：采用0.2ºC/min缓慢冷却至-40ºC，随后快速冷却至-180ºC的程序，成功冷冻卵巢。由于平均产仔数为2.0，且并非所有冷冻卵巢都能获得新生儿，因此卵巢冷冻保存的实际应用似乎需要进一步改善冷冻条件。

This study aimed at establishing a storage system for wild mouse genes or genomes by freezing of embryos and gametes from a view point of the preservation of gene resources, and a succesful freezing condition of an ovary, which included oocytes as female gametes, was developed with a series of experiments.1. Histological examination : In various points of a frozen course, which was made of slow cooling of 0.1ﾟC/min to -30ﾟC after seeding at -7ﾟC and rapid cooling to liquid nitrogen temperature, freezing injuries of the ovary were examined histologically. Till -30ﾟC damage of the ovary was little and a lot of oocytes with normal morphology were observed. On the other hand in the ovary immersed into liquid nitrogen after that almost all oocytes were injured.2. Transplantation of frozen ovary : New borns were obtained from the control transplantation and till the -30ﾟC of cooling, but not from that of liquid nitrogen. This result showed a good correlation to that of the histological observation of the frozen ovary.3. Examination of cooling rate : New borns were obtained from a transplantation of frozen ovary at conditions of cooling rates of 0.1, 0.2, 0.5ﾟC/min to -30ﾟC.The shortest interval between the transfer and the birth of the first new borns derived from the frozen ovary was 43,40,141 days each(control : 41 days). This result suggested that the rate of 0.5ﾟC/min, which was usually used for embryo freezing, was not appropriate for the ovary freezing.4. Establishment of freezing program : Successful freezing of the ovary was performed by using a program which consisted of slow cooling at 0.2ﾟC/min to -40ﾟC and following rapid cooling to -180ﾟC.As an average litter size was 2.0 and new borns were not always obtained from all frozen ovary, more improvements of freezing conditions seemed to be indispensable for the practical use of the cryo-preservation of the ovary.