동결 생쥐 난자에서의 칼슘전류 = Crypreservation does not affect Ca^(2+) entry across the membrane of the mouse oocyte

다국어 초록 (Multilingual Abstract)

Cryopreservation is commonly used as an efficient storing means of semen, oocytes, embryos, but has disadvantage in the survival and development of the post-thawed eggs. The high risk after thawing is thought to be caused by inappropriate internal regulation of Ca^2+ and/or formation of intracellular ice crystals. Most cryopreservation techniques utilize slow-freezing method using low concentration of cryoprotectants and very slow cooling rates. However, slow cooling of oocytes results in zona hardning and disruption of the chromosomes. Vitrification is alternative method to solve this problem of the slow-freezing. It has advantage that minimize intracellular ice formation due to the high concentrations of cryoprotectant and rapid cooling.
To confirm if an inappropriate regulation of intracellular Ca^2+ is critical risk factor in post-thawed eggs, we tested alteration in Ca^2+ current (iCa), a decisive factor to Ca^2+ entry, might be changed after thawing by using whole cell voltage clamp technique.
The quality and survival rates of the oocytes derived from both fresh and frozen groups were examined by morphology and FDA-test. Frozen-thawed oocytes were incubated for 4 hr after thawing and then donated to this experiment. We have compared on survival rate in vitro of oocytes frozen with a vitrification and slow-freezing method. The survival rates in vitrified oocytes (VOs) were similar to those of oocytes frozen with slow freezing method. Therefore, we used vitrification as a method for cryopreservation of oocytes in this experiment. There is no significant different among three types of cryoprotectant (EFG, EFT, EFS) in the survival rate of VOs, but survival rate of oocytes vitrified with EFG was higher about 10% than those vitrified with EFS and EFT. The survival rates in VOs vitrified with EFG under the Ca^2+-free condition were showed very low compared to those vitrified with EFG containing Ca^2+.
To record Ca^2+ current in fresh and vitrified oocytes, the membrane potential was held at -80 mV and step depolarizations of 250 ms were applied from -50 mV to 50 mV in 10 mV increments. In the fresh metaphase II oocytes (FOs), current-voltage (I-V) relationship showed that iCa began to activate at -40 mV and reached its maximum at -10 mV. With same voltage pulses, inward currents were elicited in VOs. I-V relationships observed in VOs were similar to those in FOs. Time constants of activation and inactivation of the inward current shown in VOs were not different to those in FOs. This accordance in I-V relations and time constants in FOs with those in VOs indicates that the inward currents in FOs are unaltered by vitrification and thawing.
Therefore, vitrification with EFG does not play as a factor to deteriorate Ca^2+ entry across the membrane of the oocytes.

목차 (Table of Contents)

• 목차
• 약어표(Abbreviation) = iii
• 영문초록(Abstract) = iv
• I. 서론 = 1
• II. 재료 및 방법 = 4
• 1. 실험동물 및 사양관리 = 4
• 2. 과배란유도 및 난자의 채란 = 4
• 3. 실험용액 = 5
• 4. 동결 보존액 및 희석액의 제조 = 5
• 5. 난자의 동결·융해 = 6
• 6. 동결 난자의 생존성 = 9
• 7. Ca^2+ 전류의 기록 = 9
• 8. 통계학적 분석 및 실험성적의 처리 = 10
• III. 결과 = 11
• 1. 생쥐 난자의 동결·융해 후 생존성 = 11
• 2. 동결보호제의 종류에 따른 난자의 생존율 = 11
• 3. 동결에 있어서 Ca^2+의 영향 = 13
• 4. 생쥐난자의 동결·융해 후 Ca^2+ 전류 = 14
• IV. 고찰 = 17
• V. 적요 = 21
• VI. 참고문헌 = 23
• VII. 사사 = 32