Zhang Chao, Shi Ya-Ran, Liu Xiao-Ran, Cao Yong-Chun, Tian Jin-Ling, Jia Zi-Ye, Zhen Di, Liu Feng-Hua, Gao Jian-Ming
Animal Science and Technology College, Beijing University of Agriculture, Beijing, P.R. China.
Animal Science and Technology College, Beijing University of Agriculture, Beijing, P.R. China.
Theriogenology. 2014 Aug;82(3):461-8. doi: 10.1016/j.theriogenology.2014.05.006. Epub 2014 May 20.
We constructed a model of apoptosis in mouse preimplantation embryos and investigated the effect of the flavonol icariin on embryonic development in vitro in embryos with reduced microRNA-21 (miR-21). The model was generated by microinjecting an miR-21 inhibitor into the cytoplasm of mouse pronuclear embryos, which were cultured in vitro using modified CZB (mCZB) basal medium (model group), or using mCZB medium with 0.6 μg/mL icariin as an experimental group (model-Ica). These were compared with embryos collected in vivo (vivo group) or not microinjected (control group). Developmental rates in vitro of two- and four-cell embryos and blastocysts were observed, and Hoechst 33342 and terminal deoxynucleotidyl transferase dUTP nick end labeling staining were used to count blastocyst cell numbers and apoptotic cell numbers and percentages. The transcriptional levels of miR-21, the apoptotic genes caspase 3 and phosphatase and tensin homolog deleted on chromosome ten (PTEN), and the antiapoptotic gene Bcl-2 were detected by quantitative polymerase chain reaction (qPCR). Western immunoblotting was used to detect the protein levels of caspase 3, PTEN, and Bcl-2. Compared with the model group, icariin treatment significantly improved blastocyst development in vitro (58.43 ± 7.53% vs. 37.85 ± 6.47%; P < 0.01), whereas it was not significantly different to the control group (60.34 ± 9.86%). Icariin treatment significantly increased the blastocyst cell numbers (47.02 ± 4.93 vs. 37.70 ± 5.80; P < 0.01), and reduced the rates of apoptosis (5.51 ± 2.35% vs. 10.11 ± 4.21%; P < 0.01), whereas the blastocyst cell numbers and apoptotic rates revealed no significant differences between the vivo (46.06 ± 6.50, 5.95 ± 2.56%) and control groups (45.77 ± 4.09, 6.18 ± 2.41%). Icariin treatment significantly improved miR-21 expression in all embryo stages, reduced the transcriptional levels of caspase 3 and PTEN, and increased the levels of Bcl-2. The protein expression levels of caspase 3 and PTEN were decreased in blastocysts and the level of Bcl-2 was increased (P < 0.01). These had no significant differences with the vivo and control groups, and the protein levels revealed no significant differences between two- and four-cell embryos. Thus, miR-21 was necessary for preimplantation embryonic development, and embryo quality was closely associated with the apoptosis-related protein expression levels regulated by miR-21. Icariin upregulated miR-21 expression and reduced apoptosis in embryos with reduced miR-21. It also improved embryonic developmental quality in vitro, indicating an important regulatory role for miR-21 in blastocyst development in vitro.
我们构建了小鼠植入前胚胎凋亡模型，并研究了黄酮醇淫羊藿苷对微小RNA-21（miR-21）表达降低的胚胎体外发育的影响。该模型通过将miR-21抑制剂显微注射到小鼠原核胚胎的细胞质中构建而成，这些胚胎使用改良的CZB（mCZB）基础培养基进行体外培养（模型组），或使用添加0.6μg/mL淫羊藿苷的mCZB培养基作为实验组（模型-Ica组）。将这些胚胎与体内采集的胚胎（体内组）或未进行显微注射的胚胎（对照组）进行比较。观察二细胞、四细胞胚胎和囊胚的体外发育率，并用Hoechst 33342和末端脱氧核苷酸转移酶dUTP缺口末端标记染色法对囊胚细胞数、凋亡细胞数及凋亡细胞百分比进行计数。通过定量聚合酶链反应（qPCR）检测miR-21、凋亡基因半胱天冬酶3（caspase 3）和第10号染色体上缺失的磷酸酶及张力蛋白同源物（PTEN）以及抗凋亡基因Bcl-2的转录水平。采用蛋白质免疫印迹法检测caspase 3、PTEN和Bcl-2的蛋白水平。与模型组相比，淫羊藿苷处理显著提高了胚胎体外囊胚发育率（58.43±7.53%对37.85±6.47%；P<0.01），而与对照组相比差异不显著（60.34±9.86%）。淫羊藿苷处理显著增加了囊胚细胞数（47.02±4.93对37.70±5.80；P<0.01），并降低了凋亡率（5.51±2.35%对10.11±4.21%；P<0.01），而体内组（46.06±6.50，5.95±2.56%）和对照组（45.77±4.09，6.18±2.41%）的囊胚细胞数和凋亡率差异不显著。淫羊藿苷处理显著提高了各胚胎阶段的miR-21表达，降低了caspase 3和PTEN的转录水平，并增加了Bcl-2的水平。囊胚中caspase 3和PTEN的蛋白表达水平降低，Bcl-2水平升高（P<0.01）。这些与体内组和对照组无显著差异，且二细胞和四细胞胚胎的蛋白水平差异也不显著。因此，miR-21对植入前胚胎发育是必需的，胚胎质量与miR-21调控的凋亡相关蛋白表达水平密切相关；淫羊藿苷上调miR-21表达并减少miR-21降低的胚胎中的凋亡，还改善了胚胎体外发育质量，表明miR-21在体外囊胚发育中具有重要调控作用。
