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대한생식의학회지 제19권 제2호 2010년

투명대 존재/부재 햄스터 난자의 동결보존;1-단계 평형과 2-단계 융해의 효과

서울대학교 의과대학 산부인과학교실;서울대학교 의과대학 산부인과학교실;서울대학교 의과대학 산부인과학교실;서울대학교 의과대학 산부인과학교실;서울대학교 의과대학 산부인과학교실;서울대학교 의과대학 산부인과학교실;서울대학교 의과대학 산부인과학교실;서울대학교 의과대학 산부인과학교실;

정구민;방명걸;김석현;신창재;김정구;문신용;이진용;장윤석;,

Cryopreservation of Zona-intact/-free Hamster Oocytes;Effect of 1-Step Equilibration and 2-Step Thawing

Chung, K.M.;Pang, M.G.;Kim, S.H.;Shin, C.J.;Kim, J.G.;Moon, S.Y.;Lee, J.Y.;Chang, Y.S.;

Department of Obstetrics and Gynecology, College of Medicine, Seoul National University;Department of Obstetrics and Gynecology, College of Medicine, Seoul National University;Department of Obstetrics and Gynecology, College of Medicine, Seoul National University;Department of Obstetrics and Gynecology, College of Medicine, Seoul National University;Department of Obstetrics and Gynecology, College of Medicine, Seoul National University;Department of Obstetrics and Gynecology, College of Medicine, Seoul National University;Department of Obstetrics and Gynecology, College of Medicine, Seoul National University;Department of Obstetrics and Gynecology, College of Medicine, Seoul National University;

The present experiments were focussed to modify a short slow-cooling protocol used for freezing of early stage embryo(Testart et al., 1986) and also to apply the modified method for the cryopreservation of hamster oocytes with Zona or without. The protocol was modified by changing the 4-step equilibration into 1-step and the 1-step thawing into 2-step. The oocytes were added in 1.5M PROH and 0.1M Sucrose, seeded at $-7^{\circ}C$, slow cooled($0.3^{\circ}C$/min) to $-30^{\circ}C$ before plunging to $-196^{\circ}C$. The oocytes were thawed at $23-25^{\circ}C$ air(20sec/150sec) and/or 33-35 water(10sec). The survival of the frozen-thawed oocytes was determined by morphologic criteria and their fertilizing ability was also estimated by Sperm Penetration Assay(SPA) system(Chang et al, 1990) using fertile men semen sample. One-step equilibration showed slightly higher survival rate(83.9% vs. 71.0%) and fertilization rate(83.9% vs. 71.0%) compared with four-step(p>0.05). And two-step thawing(air & water exposing) of oocytes frozen after 1-step equilibration showed significantly higher survival rate(96.3%) than one-step thawing at air(85.2%) or water(65.0%) only(p<0.05). Therefore, by the modified method(l-step equilibration & 2-step thawing), Zona-intact(ZI) and Zona-free(ZF) oocytes were frozen and thawed. ZI-oocytes showed significantly higher survival rate(95.4%, 308/323 vs. 67.6%, 240/355) than ZF-oocytes(P<0.01). But the survival of ZF-oocytes was as high as ZI-oocytes in fourteen of twenty-four replicates. ZI-oocytes was also significantly higher fertilization rate($92.4{\pm}8.9%$ vs. $63.7{\pm}18.5%$) and higher mean number of penetrated sperm($6.2{\pm}4.2$ vs. $3.9{\\pm}3.3$) than ZF-oocytes, but not higher than control(fresh oocytes;$99.3{\pm}2.4%$, $8.4{\pm}4.2$)(P<0.001). Conclusively, this modified method will contribute to freeze effectively the hamster oocytes for simplifing of the logical consideration of performing SPA and also to freeze the human and other animal oocytes.

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