BACKGROUND Although in vitro culture system has been optimized in the past few decades, the problem of few or no high quality embryos has been still not completely solved. Accordingly, fully understanding the regulatory mechanism of pre-implantation embryonic development would be beneficial to further optimize the in vitro embryo culture system. Recent studies have found the expression of c-kit in mouse embryo and its promotion effects on mouse embryonic development. However, it is unclear the expression, the role and the related molecular regulatory mechanism of c-kit in human pre-implantation embryo development. Therefore, the present study is to determine whether c-kit is expressed in human pre-implantation embryos, and to investigate the possible regulatory mechanism of c-kit signaling in the process of embryonic development. METHODS The present study includes human immature oocytes and three pronucleus (3PN) embryos collected from 768 women (28-32 ages) undergoing IVF, and normal 2PN embryos collected from ICR mice. Samples were distributed randomly into three different experimental groups: SCF group: G-1™ (medium for culture of embryos from the pro-nucleate stage to day 3) or G-2™ (medium for culture of embryos from day3 to blastocyst stage) + HSA (Human serum album) solution + rhSCF; SCF + imanitib (c-kit inhibitor) group: G-1™ or G-2™ + HSA solution + rhSCF + imanitib; SCF + U0126 (MEK/ERK inhibitor) group: G-1™ or G-2™ + HSA solution + rhSCF + U0126; Control group: G-1™ or G-2™ + HSA solution + PBS; The rate of good quality embryos at day 3, blastulation at day 6 and good quality blastulation at day 6 were analysis. RT-PCR, western blot and immunofluorescence staining were applied to detect the target genes and proteins in samples collected from human or mice, respectively. RESULTS c-kit was expressed ubiquitously in all human immature oocytes, 3PN embryos and 3PN blastocysts. In the experiment of human 3PN embryos, compared with other groups, SCF group showed obviously higher rate of good quality at day 3, better rate of blastocyst formation at day 6 and higher rate of good quality blastocyst formation at day 6. Furthermore, we observed a higher ETV5 expression in SCF group than that in other groups. Similar results were also found in animal experiment. Interestingly, we also found a higher phosphorylation level of MEK/ERK signal molecule in mice embryos from SCF group than those from other groups. Moreover, inhibition of MEK/ERK signaling would remarkably impeded the mice embryonic development, which might be due to the reduced ETV5 expression. CONCLUSIONS The present study firstly revealed that c-kit signaling might promote the human pre-implantation embryonic development and blastocyst formation by up-regulating the expression of ETV5 via MEK/ERK pathway. Our findings provide a new idea for optimizing the in vitro embryo culture condition during ART program, which is beneficial to obtain high quality embryos for infertile patients.

中文翻译：

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C-kit信号传导促进人类植入前3PN胚胎发育和胚泡形成。

背景技术尽管在过去的几十年中已经优化了体外培养系统，但是仍然没有完全解决高质量胚胎很少或没有的问题。因此，充分了解植入前胚胎发育的调控机制将有利于进一步优化体外胚胎培养系统。最近的研究发现c-kit在小鼠胚胎中的表达及其对小鼠胚胎发育的促进作用。然而，目前尚不清楚c-kit在人类植入前胚胎发育中的表达，作用及其相关的分子调控机制。因此，本研究旨在确定c-kit是否在人类植入前的胚胎中表达，并研究c-kit信号在胚胎发育过程中的可能调控机制。方法本研究包括从768名接受IVF的妇女（28-32岁）中收集的人类未成熟卵母细胞和3个原核（3PN）胚胎，以及从ICR小鼠中收集的正常2PN胚胎。将样品随机分配到三个不同的实验组中：SCF组：G-1™（从前核阶段到第3天用于培养胚胎的培养基）或G-2™（从第3天到胚泡阶段从胚胎培养的培养基） + HSA（人类血清相册）溶液+ rhSCF；SCF +伊马替尼（c-kit抑制剂）组：G-1™或G-2™+ HSA溶液+ rhSCF +伊马替尼；SCF + U0126（MEK / ERK抑制剂）组：G-1™或G-2™+ HSA溶液+ rhSCF + U0126；对照组：G-1™或G-2™+ HSA溶液+ PBS；分析第3天，第6天的成胚率和第6天的高质量成胚率。逆转录PCR 免疫印迹和免疫荧光染色分别用于检测从人或小鼠收集的样品中的靶基因和蛋白质。结果c-kit在所有人类未成熟卵母细胞，3PN胚胎和3PN胚泡中普遍表达。在人类3PN胚胎的实验中，与其他组相比，SCF组在第3天显示出较高的优质胚泡率，在第6天显示出较高的胚泡形成率，在第6天显示出较高质量的胚泡形成率。 SCF组的ETV5表达高于其他组。在动物实验中也发现了类似的结果。有趣的是，我们还发现SCF组小鼠胚胎中MEK / ERK信号分子的磷酸化水平高于其他组。而且，抑制MEK / ERK信号传导会显着阻碍小鼠胚胎发育，这可能是由于ETV5表达降低所致。结论本研究首先揭示了c-kit信号通路可能通过上调EK5通过MEK / ERK途径的表达来促进人类植入前胚胎的发育和胚泡的形成。我们的发现为优化ART程序中的体外胚胎培养条件提供了新思路，这对于获得不育患者的高质量胚胎是有益的。