Evaluation of vitrification protocol of mouse ovarian tissue by effect of DNA methyltransferase-1 and paternal imprinted growth factor receptor-binding protein 10 on signaling pathways
Evaluation of vitrification protocol of mouse ovarian tissue by effect of DNA methyltransferase-1 and paternal imprinted growth factor receptor-binding protein 10 on signaling pathways
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DNA甲基转移酶1和父本印迹生长因子受体结合蛋白10对信号通路的影响评价小鼠卵巢组织玻璃化冷冻方案
DOI：
10.1016/j.cryobiol.2017.11.008
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发表时间：
2018-02-01
期刊：
影响因子：
2.7
通讯作者：
Wang, Yi-li
中科院分区：
文献类型：
--
作者：
He, Zhong-yi;Wang, Hong-Yan;Wang, Yi-li
冷冻保存的卵巢组织移植被认为是一种很有前途的女性生育保留方法。然而，这种冷冻保存方法易于发生复苏后卵泡增殖和卵母细胞发育停滞，影响后期移植存活。为了评价目前的玻璃化冷冻工作，我们研究了玻璃化冷冻-温热的10日龄幼龄小鼠卵巢中关键途径的变化。我们发现蛋白激酶B（Akt）和丝裂原活化蛋白激酶（Mapk）的磷酸化水平显著降低，在此期间丝氨酸/苏氨酸激酶在协调卵泡和卵母细胞发育和应激反应中起核心作用。Akt和Mapk活性的抑制与印迹胰岛素途径负调控基因之一生长因子受体结合蛋白10（Growth factor receptor binding protein 10，Grb 10）有关，玻璃化冷冻-温热处理的幼龄小鼠卵巢中Grb 10的表达显著高于新鲜组（p < 0.05）。RNAi诱导的Grb 10下调逆转了Akt和Mapk磷酸化的减少。玻璃化-加温组各期卵泡DNA甲基转移酶（Dnmt）1蛋白表达均较新鲜组降低（p < 0.05），GrblO表达的增加部分是由于启动子区的高甲基化所致。玻璃化冷冻组小鼠卵巢Dnmt 1 mRNA和蛋白表达均显著低于新鲜冷冻组（p < 0.05）。Dnmtl过表达显著逆转GrblO上调以及Akt和Mapk磷酸化减少。综上所述，我们的研究结果表明，GrblO表达可能有助于评估玻璃化冷冻的有效性，并被认为是未来进一步改进玻璃化冷冻方案的潜在目标。
Transplantation of cryopreserved ovarian tissue has been considered as a promising way of fertility preservation for women. however, this cryopreservation method is prone to post-resuscitation follicle proliferation and oocyte development stagnation, affecting late transplant survival. To evaluate current vitrification works, we investigated the critical pathway alternations in vitrified-warmed juvenile 10-day-old mouse ovary. We showed a significant decrease of protein kinase B (Akt) and Mitogen-activated protein kinase (Mapk) phosphorylation, during which serine/threonine kinases play central roles in coordinating follicle and oocyte development and stress response. Inhibition of Akt and Mapk activity were associated with one of the imprinted insulin pathway negative regulatory genes, Growth factor receptor-binding protein 10 (Grb10) which remarkably increased in vitrified-warmed juvenile mouse ovary than that of fresh group (p < 0.05). RNAi-induced Grb10 down-regulation reversed the decrease in Akt and Mapk phosphorylation. The increase of GrblO expression was partially caused by the hyper-methylation of the promoter region, associated with the decrease of follicular DNA methyltransferase (Dnmt) 1 protein in different stages of vitrified-warmed group, compared to fresh group (p < 0.05). The mRNA and protein expression of Dnmt1 in ovary of vitrified-warmed juvenile mouse were remarkably lower than those in fresh group (p < 0.05). Dnmtl overexpression dramatically reversed GrblO up regulation and Akt and Mapk phosphorylation reduction. Taken together, our findings suggest that GrblO expression might be helpful in evaluation of effectiveness of vitrification, and considered as a potential target for further vitrification protocols improvement in the future.