Role of ROCK signaling in formation of the trophectoderm of the bovine preimplantation embryo
Role of ROCK signaling in formation of the trophectoderm of the bovine preimplantation embryo
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DOI：
10.1002/mrd.22976
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发表时间：
2018-05-01
影响因子：
2.5
通讯作者：
Hansen, Peter J.
中科院分区：
文献类型：
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作者：
Negron-Perez, Veronica M.;Rodrigues, Luana T.;Hansen, Peter J.
Rho 相关卷曲螺旋蛋白激酶（ROCK1 和 ROCK2）通过与 RHO GTP 酶结合而被激活，并磷酸化包括放线肌球蛋白在内的多种下游靶标。在小鼠胚胎中，ROCK 信号传导通过极化胚胎的外部细胞使 Hippo 信号传导失活来促进滋养外胚层 (TE) 的形成并抑制内细胞团 (ICM) 的形成 (Kono et al., 2014; Mihajlović and Bruce, 2016)。结果是 YAP1 的核转位和随后的 Cdx2 转录。在这里，我们评估了 ROCK 在牛胚胎囊胚分化中的作用。据推测，对于小鼠来说，抑制 ROCK 将减少 TE 形成并增加 ICM 细胞数量。该假设通过使用选择性抑制 ROCK 的嘧啶衍生物 Y-27632 从桑葚胚发育阶段开始抑制 ROCK 进行了测试（Uehata 等，1997）。与假设的结果相反，ROCK 的抑制增加了 TE 细胞的数量。因此，ROCK 信号传导以与小鼠不同的方式调节牛胚胎的分化。用于本研究的胚胎是按照先前描述的程序在体外产生的（Ortega et al., 2017）。每个重复均来自使用 200-300 个卵丘卵母细胞复合体和来自三头公牛的精子池的单次体外受精程序。每次重复使用不同的公牛池，总共进行 8 次重复。推定受精卵以 25-30 个为一组，在 45 µl 合成输卵管液 - 牛胚胎 2 (SOF-BE2) 的油覆盖微滴中，在湿度为 5%(v/v) O2、5%(v/v) CO2 和平衡氮的 38.5 C 条件下进行培养。在授精后第 5 天，用 5 µl 处理胚胎。 Y-27632 (Selleckchem, Houston, TX, USA) 或载体 [SOF-BE2 含有 1%(v/v) DMSO] 以产生终浓度 10 µM 或等量的载体 [SOF-BE2 含有
Rho-associated coiled-coil containing protein kinases (ROCK1 and ROCK2) are activated by binding to RHO GTPases and phosphorylate a variety of downstream targets including actinomyosin. In the mouse embryo, ROCK signaling acts to promote formation of trophectoderm (TE) and inhibit formation of the inner cell mass (ICM) by polarizing outer cells of the embryo to inactivate Hippo signaling (Kono et al., 2014; Mihajlović and Bruce, 2016). The result is nuclear translocation of YAP1 and subsequent transcription of Cdx2. Here we evaluated the involvement of ROCK in differentiation of the blastocyst of the bovine embryo. It was hypothesized that, as for the mouse, inhibition of ROCK would decrease TE formation and increase number of ICM cells. This hypothesis was tested by inhibition of ROCK beginning at the morula stage of development using the pyrimidine derivative Y-27632 that selectively inhibits ROCK (Uehata et al., 1997). In contrast to the hypothesized results, inhibition of ROCK increased number of TE cells. Thus, ROCK signaling regulates differentiation of the bovine embryo in a different manner from that of the mouse. Embryos used for this study were produced in vitro following the procedures previously described (Ortega et al., 2017). Each replicate was from a single in vitro fertilization procedure using 200-300 cumulus-oocyte-complexes and a pool of sperm from three bulls. A different pool of bulls was used for each replicate and 8 replicates were performed in total. Presumptive zygotes were cultured in groups of 25-30 in 45 µl oil-covered microdrops of synthetic oviductal fluid–bovine embryo 2 (SOF-BE2) in a humidified chamber at 5%(v/v) O2, 5%(v/v) CO2, and the balance nitrogen at 38.5 C. Embryos were treated at Day 5 after insemination with 5 µl Y-27632 (Selleckchem, Houston, TX, USA) or vehicle [SOF-BE2 containing 1%(v/v) DMSO] to produce a final concentration of 10 µM or equivalent amount of vehicle [SOF-BE2 containing