Bang Soyoung, Shin Hyejin, Song Haengseok, Suh Chang Suk, Lim Hyunjung Jade
Department of Biomedical Science and TechnologyInstitute of Biomedical Science and Technology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, KoreaDepartment of Biomedical ScienceCollege of Life Science, CHA University, Seoul 135-913, KoreaDepartment of Obstetrics and GynecologySeoul National University Bundang Hospital, Seongnam, Gyeonggi-do 463-707, Korea.
Department of Biomedical Science and TechnologyInstitute of Biomedical Science and Technology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, KoreaDepartment of Biomedical ScienceCollege of Life Science, CHA University, Seoul 135-913, KoreaDepartment of Obstetrics and GynecologySeoul National University Bundang Hospital, Seongnam, Gyeonggi-do 463-707, Korea
Reproduction. 2014 Jul;148(1):11-9. doi: 10.1530/REP-14-0036. Epub 2014 Apr 23.
Vitrification involves the use of cryoprotectants (CPAs) and liquid nitrogen (LN2), which may cause osmotic damage and cryoinjury to oocytes. Autophagy is widely recognized as a survival or response mechanism elicited by various environmental and cellular stressors. However, the induction of autophagy in vitrified-warmed oocytes has not been examined. In this work, we investigated whether the vitrification-warming process induces autophagy in mouse oocytes. Metaphase II (MII) oocytes that were vitrified and stored in LN2 for at least 2 weeks were used in the study. In RT-PCR analyses, we observed that several Atg genes such as Atg5, Atg7, Atg12, LC3a (Map1lc3a), LC3b (Map1lc3b), and Beclin1 were expressed in MII mouse oocytes. Slight reduction in mRNA levels of Atg7 and Atg12 in vitrified-warmed oocytes was noted, and expression of these genes was not significantly influenced. Confocal live imaging analysis using oocytes from GFP-LC3 transgenic mice revealed that vitrified-warmed oocytes had a significantly higher number of GFP-LC3 puncta in comparison to fresh oocytes. The expression of BECLIN1 protein was also increased in vitrified-warmed oocytes. Treatment with 3-methyladenine, an inhibitor of autophagy, did not significantly affect the rates of oocyte survival, IVF, and embryonic development after warming and IVF. The results suggest that the observed autophagic activation in vitrified-warmed oocytes is a natural adaptive response to cold stress. Collectively, we show for the first time that vitrified-warmed mouse oocytes exhibit autophagic activation during warming and that this response is not induced by CPA-containing solutions. The induction of autophagy by cold temperature is first reported herein.
玻璃化冷冻涉及使用冷冻保护剂（CPA）和液氮（LN₂），这可能会对卵母细胞造成渗透损伤和冷冻损伤。自噬被广泛认为是由各种环境和细胞应激源引发的一种生存或反应机制。然而，尚未研究玻璃化冷冻-解冻后的卵母细胞中自噬的诱导情况。在这项研究中，我们调查了玻璃化冷冻-解冻过程是否会在小鼠卵母细胞中诱导自噬。研究使用了在LN₂中玻璃化冷冻并储存至少2周的中期II（MII）卵母细胞。在逆转录聚合酶链反应（RT-PCR）分析中，我们观察到一些自噬相关基因，如Atg5、Atg7、Atg12、微管相关蛋白轻链3α（Map1lc3a）、微管相关蛋白轻链3β（Map1lc3b）和Beclin1，在MII小鼠卵母细胞中表达。注意到玻璃化冷冻-解冻后的卵母细胞中Atg7和Atg12的mRNA水平略有降低，但这些基因的表达没有受到显著影响。使用来自绿色荧光蛋白-微管相关蛋白轻链3（GFP-LC3）转基因小鼠的卵母细胞进行共聚焦实时成像分析显示，与新鲜卵母细胞相比，玻璃化冷冻-解冻后的卵母细胞中GFP-LC3斑点的数量显著更多。玻璃化冷冻-解冻后的卵母细胞中Beclin1蛋白的表达也增加了。用自噬抑制剂3-甲基腺嘌呤处理，对解冻后体外受精（IVF）及胚胎发育过程中的卵母细胞存活率、IVF率没有显著影响。结果表明，在玻璃化冷冻-解冻后的卵母细胞中观察到的自噬激活是对冷应激的一种自然适应性反应。总体而言，我们首次表明玻璃化冷冻-解冻后的小鼠卵母细胞在解冻过程中表现出自噬激活，并且这种反应不是由含CPA的溶液诱导的。本文首次报道了低温诱导自噬的现象。
