Treatment with AICAR inhibits blastocyst development, trophectoderm differentiation and tight junction formation and function in mice
Treatment with AICAR inhibits blastocyst development, trophectoderm differentiation and tight junction formation and function in mice
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DOI：
10.1093/molehr/gax050
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发表时间：
2017-11-01
影响因子：
4
通讯作者：
Watson, Andrew J.
中科院分区：
文献类型：
--
作者：
Calder, Michele D.;Edwards, Nicole A.;Watson, Andrew J.
研究问题：腺苷一磷酸激活蛋白激酶（AMPK）激活对囊胚形成、基因表达、紧密连接形成和功能有何影响？总结回答：AMPK的活性必须严格控制才能使胚胎在植入前正常发育和胚泡形成。已知：AMPK亚型在卵母细胞、卵丘细胞和植入前胚胎中均可检测到。培养的胚胎受到许多压力，可以激活AMPK.Study设计，大小，持续时间：进行了两个主要的实验，以确定AICAR处理胚胎发育和囊胚腔的维护的效果。从超数排卵的小鼠中回收胚胎。首先，用浓度系列（0-2000 μ M）的AICAR处理2-细胞胚胎48小时，直到胚泡正常形成。在第二个实验中，用1000 μ M AICAR处理扩张的小鼠囊胚9小时。测量的结果包括发育到囊胚阶段、细胞数量、囊胚体积、AMPK磷酸化、Cdx 2和囊胚形成基因家族表达（使用定量RT-PCR、免疫印迹、免疫荧光测量的mRNA和蛋白质）、紧密连接功能（FITC葡聚糖染料摄取测定）和胚泡ATP水平。AICAR治疗的可逆性进行了评估，使用化合物C（CC），一个众所周知的AMPK抑制剂，单独或与AICAR.Main结果和机会的作用：从2-细胞阶段向前延长治疗AICAR减少囊胚形成，减少总细胞数，胚胎直径，导致滋养外胚层细胞接触和细胞膜蛋白occludens-1染色的损失，并增加核凝聚。单独用CC处理仅在高于正常使用的浓度下抑制胚泡发育。AICAR处理的胚胎显示囊胚形成基因的mRNA和蛋白水平改变。用AICAR处理囊胚9 h可诱导囊胚塌陷，改变囊胚形成基因表达，增加紧密连接通透性，降低CDX 2。处理的囊胚表现出三种表型：那些不受治疗，其中治疗是可逆的，和那些在其中的影响是不可逆的。大规模的数据：不适用。限制，理由回避：我们的研究AICAR治疗的影响，对早期发育。虽然AICAR确实增加AMPK活性，并且这在我们的研究中得到了证明，但AICAR不是AMPK活性的天然调节剂，并且一些结果可能是由脱靶的非AMPK AICAR调节事件引起的。为了支持我们的研究结果，囊胚发育结果证实了与其他两个众所周知的小分子激活剂AMPK，二甲双胍和phenylaldehyde。更广泛的影响的发现：二甲双胍，AMPK激活剂，被广泛用于治疗II型糖尿病和多囊卵巢疾病（PCOS）。我们的研究结果表明，早期胚胎AMPK水平必须严格调节，以确保正常的植入前发育。因此，在植入前和早期胚胎移植后阶段的生育治疗周期中应仔细考虑使用二甲双胍。研究资金和竞争兴趣：加拿大卫生研究院（CIHR）运营资金。没有利益冲突。
STUDY QUESTION: What is the impact of adenosine monophosphate-activated protein kinase (AMPK) activation on blastocyst formation, gene expression, and tight junction formation and function?SUMMARY ANSWER: AMPK activity must be tightly controlled for normal preimplantation development and blastocyst formation to occur.WHAT IS KNOWN ALREADY: AMPK isoforms are detectable in oocytes, cumulus cells and preimplantation embryos. Cultured embryos are subject to many stresses that can activate AMPK.STUDY DESIGN, SIZE, DURATION: Two primary experiments were carried out to determine the effect of AICAR treatment on embryo development and maintenance of the blastocoel cavity. Embryos were recovered from superovulated mice. First, 2-cell embryos were treated with a concentration series (0-2000 mu M) of AICAR for 48 h until blastocyst formation would normally occur. In the second experiment, expanded mouse blastocysts were treated for 9 h with 1000 mu M AICAR.PARTICIPANTS/MATERIALS, SETTING, METHODS: Outcomes measured included development to the blastocyst stage, cell number, blastocyst volume, AMPK phosphorylation, Cdx2 and blastocyst formation gene family expression (mRNAs and protein measured using quantitative RT-PCR, immunoblotting, immunofluorescence), tight junction function (FITC dextran dye uptake assay), and blastocyst ATP levels. The reversibility of AICAR treatment was assessed using Compound C (CC), a well-known inhibitor of AMPK, alone or in combination with AICAR.MAIN RESULTS AND THE ROLE OF CHANCE: Prolonged treatment with AICAR from the 2-cell stage onward decreases blastocyst formation, reduces total cell number, embryo diameter, leads to loss of trophectoderm cell contacts and membrane zona occludens-1 staining, and increased nuclear condensation. Treatment with CC alone inhibited blastocyst development only at concentrations that are higher than normally used. AICAR treated embryos displayed altered mRNA and protein levels of blastocyst formation genes. Treatment of blastocysts with AICAR for 9 h induced blastocyst collapse, altered blastocyst formation gene expression, increased tight junction permeability and decreased CDX2. Treated blastocysts displayed three phenotypes: those that were unaffected by treatment, those in which treatment was reversible, and those in which effects were irreversible.LARGE SCALE DATA: Not applicable.LIMITATIONS, REASONS FOR CAUTION: Our study investigates the effects of AICAR treatment on early development. While AICAR does increase AMPK activity and this is demonstrated in our study, AICAR is not a natural regulator of AMPK activity and some outcomes may result from off target non-AMPK AICAR regulated events. To support our results, blastocyst developmental outcomes were confirmed with two other well-known small molecule activators of AMPK, metformin and phenformin.WIDER IMPLICATIONS OF THE FINDINGS: Metformin, an AMPK activator, is widely used to treat type II diabetes and polycystic ovarian disorder (PCOS). Our results indicate that early embryonic AMPK levels must be tightly regulated to ensure normal preimplantation development. Thus, use of metformin should be carefully considered during preimplantation and early post-embryo transfer phases of fertility treatment cycles.STUDY FUNDING AND COMPETING INTEREST(S): Canadian Institutes of Health Research (CIHR) operating funds. There are no competing interests.