Autophagic activation in vitrified-warmed mouse oocytes
Autophagic activation in vitrified-warmed mouse oocytes
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DOI：
10.1530/rep-14-0036
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发表时间：
2014-07-01
期刊：
影响因子：
3.8
通讯作者：
Lim, Hyunjung Jade
中科院分区：
文献类型：
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作者：
Bang, Soyoung;Shin, Hyejin;Lim, Hyunjung Jade
玻璃化冷冻涉及冷冻保护剂（CPA）和液氮（LN 2）的使用，这可能会导致卵母细胞的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引起的生存或反应机制。然而，在玻璃化冷冻-温热的卵母细胞中诱导自噬还没有被研究。在这项工作中，我们研究了玻璃化-升温过程是否诱导小鼠卵母细胞自噬。在研究中使用玻璃化冷冻并在液氮中储存至少2周的中期II（MII）卵母细胞。在RT-PCR分析中，我们观察到几个Atg基因，如Atg 5，Atg 7，Atg 12，LC 3a（Map 1 lc 3a），LC 3b（Map 1 lc 3b）和Beclin 1在MII小鼠卵母细胞中表达。在玻璃化冷冻-温热的卵母细胞中，Atg 7和Atg 12的mRNA水平略有降低，这些基因的表达没有受到显著影响。使用来自GFP-LC 3转基因小鼠的卵母细胞的共聚焦活体成像分析显示，玻璃化温热的卵母细胞与新鲜卵母细胞相比具有显著更高数量的GFP-LC 3斑点。BECLIN 1蛋白在玻璃化冷冻卵母细胞中的表达也增加。用3-甲基腺嘌呤（一种自噬抑制剂）治疗，对卵母细胞存活率、IVF率以及加温和IVF后的胚胎发育率没有显著影响。结果表明，观察到的自噬激活在玻璃化冷冻温暖的卵母细胞是一种自然的适应性反应，冷应激。总的来说，我们第一次表明，玻璃化温热的小鼠卵母细胞在升温过程中表现出自噬激活，这种反应不是由含有CPA的溶液诱导的。本文首次报道了低温诱导自噬。
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.