精子源性miR-34c介导的ceRNA调控网络影响植入前胚胎发育的作用机制研究

The impact of paternal factors on cleavage stage and blastocyst development was detected by time-lapse imaging study. Moreover, embryos from severe male factor infertilities presents high incidence of meiotic errors, but the underlying mechanism is not clear. Our previous study found that spermatozoa miR-34c levels were expressed at lower levels in idiopathic male infertility patients and patients with lower miR-34c levels were more likely to exhibit lower rates of good-quality embryos, pregnancy and live birth after intracytoplasmic sperm injection. Mouse embryo mRNA levels of Cdk6 was down-regulated after injecting miR-34c inhibitor into male pronuclei of mouse zygotes. Boinformatics analysis revealed that CDK6、E2F3、cyclin D were the target genes of miR-34c, and CDK6/E2F3 were potential ceRNAs. In this study, we will use RNA interference, HITS-CLIP, laser confocal scanning, Co-Immunoprecipitation, etc to clarify the role of miR-34c mediated CDK6/E2F3 ceRNA network in embryo development. Traditionally, it is believed that maternal factors contributed by the egg cytoplasm control early stage embryo development， our study underscores the importance of paternal RNA on preimplantation embryo development and will be beneficial for treatment of severe male factor infertilities.

精子质量会影响胚胎发育，由重度少弱畸精症患者精子形成的胚胎具有较高的非整倍体比例。迄今精子因素导致这些胚胎发育异常的相关机制尚不明确。我们的前期研究发现：男性不育患者精子miR-34c水平显著降低，而精子miR-34c的低表达与优胎率,妊娠率和活产率降低显著相关。小鼠雄原核注射miR-34c的抑制剂后Cdk6等基因在胚胎中的表达降低。生物信息学分析提示：CDK6、E2F3、cyclin D等是miR-34c的靶基因，CDK6/E2F3互为ceRNA。我们拟以小鼠为模型，采用RNA干扰，紫外交联免疫共沉淀高通量测序，免疫共沉淀等技术，阐明miR-34c介导CDK6/E2F3 ceRNA 影响早期胚胎发育的机制。本研究将提供精子源性RNA影响胚胎发育的证据，为提高重度少弱精患者的辅助生殖治疗效果提供理论依据。

精子质量会影响胚胎发育，由重度少弱畸精症患者精子形成的胚胎具有较高的非整倍体比例。迄今精子因素导致这些胚胎发育异常的相关机制尚不明确。我们的前期研究发现：男性不育患者精子miR-34c水平显著降低，而精子miR-34c的低表达与优胎率,妊娠率和活产率降低显著相关。本研究发现：miR-34c在精子和合子中有表达，卵子中的miR-34c的水平极低，据此我们推测合子中的miR-34c来源于精子。 我们还发现：胚胎中E2F3和CDK6的表达从2细胞阶段开始逐渐提高，囊胚的E2F3和CDK6的表达水平显著高于2细胞和4细胞胚胎。而小鼠合子在注射miR-34c抑制剂后， E2F3从2细胞期开始迅速下降，CDK6从4细胞期开始迅速下降，囊胚期CDK6和E2F3降至最低。CDK6和E2F3的表达呈同向性改变。CDK6和E2F3均为细胞周期正向调控因子，可通过调控细胞G1/S期的转变影响植入前胚胎发育。本研究为精子源性RNA影响胚胎发育提供了证据，为提高重度少弱精患者的辅助生殖治疗效果提供了线索。