Pin1抑制剂对小鼠植入前胚体外发育的影响

Effects of Pin1 antagonist on mouse preimplantation embryo development in vitro

目的观察Pin1在小鼠植入前胚中的定位分布及其抑制剂胡桃醌(Juglone)对小鼠植入前胚体外发育的影响。方法免疫荧光染色观察不同发育阶段植入前胚中Pin1的分布;收集小鼠1-细胞胚,以KSOM培养液为对照组,以培养液中添加有不同浓度的Juglone为实验组,观察各组1-细胞胚体外发育情况;Real-time PCR检测体外发育2-细胞胚内干细胞转录因子(Sox2、Oct4、c-Myc和Klf4)m RNA表达水平。结果小鼠不同发育阶段植入前胚中都存在Pin1的表达,胞质和胞核内均有分布,且核内荧光着色明显比胞质强;10μmol/L和25μmol/L胡桃醌持续作用93h(注射人绒毛膜促性腺激素后27h^120h)使1-细胞胚阻滞于2-细胞阶段,极少能过渡至4-细胞胚(P<0.01)。25μmol/L胡桃醌短时间作用18h(注射人绒毛膜促性腺激素后27~45h)同样使1-细胞胚发育阻滞于2-细胞胚(P<0.01);25μmol/L胡桃醌组2-细胞胚内Sox2 m RNA表达水平低于KSOM组(P<0.05),而Klf4、cMyc和Oct4 m RNA表达水平无明显变化(P>0.05)。结论小鼠1-细胞胚之后,Pin1主要定位在细胞核内,提示其可能参与细胞转录活动。且Pin1抑制剂(胡桃醌)能使2-细胞胚至4-细胞胚过渡率明显降低,并使干细胞转录因子Sox2 m RNA表达下调,提示Pin1在小鼠植入前胚早期发育阶段具有一定作用,其参与调控合子基因组激活与干细胞转录因子Sox2有关。

Objective To observe the distribution of Pin1 in mouse preimplantation embryos and the effect of Pinl antagonist （Juglone） on the development of mouse preimplantation embryo in vitro. Methods The immunofluorescence technique was used to localize the distribution of Pinl protein in the preimplanration mouse embryo. The 1-cell embryos of Kunming mice were cuhured in KSOM culture medium supplemented various concentrations Pinl inhibitor （Juglone） , respectively. The different development potentials were observed under an inverted microscope. The mRNA expression levels of Sox2, Oct4, Klf4 and c-Myc were detected by Real-time PCR. Results Pinl protein was distributed in both cytoplasm and nucleus of mouse preimplantation embryos. The immunofluorescent intensities in nuclei were higher than those in the complasma. The 1-cell embryos treated with 10μmol/L and 25μmol/L Juglone for 93hours （from post hCG 27hours to 120hours） in vitro hardly developed to 4-cell embryos （P 〈 0.01 ）. The 1-cell embryos treated with 25 μmol/L Juglone for 18hours （from post hCG 27hours to 45hours） in vitro were arrested at 2-cell stage （ P 〈 0. 01 ）. In 25μmol/L Juglone group, the Sox2 mRNA levels were significantly low compared with KSOM group （P 〈 0.05） , while the mRNA levels of Oct4, c-Myc and Klf4 were no significant differences compared with KSOM group （P 〉 0. 05）. Conclusion Pin1 is mainly distributed in the nucleus of mouse preimplantation embryos. The resuhs suggest that Pinl may participate in gene transcription. The development rate of mouse preimplantatiom embryos from 2-cell to 4-cell is markedly decreased and the mRNA expression levels of stem cell factor （Sox2） are down-regulated by Juglone treatment, which indicates Pinl may play an important role in the early development of mouse preimplantation embryos by regulating zygotic genome activation.