RNA sequencing of single blastocyst-stage embryos (E3.5) from zygotes which were injected with sperm sRNAs from normal or depressive male mice.

We demonstrated that sperm sRNAs contribute to paternal transmission of depression-like symptoms.To further investigated the mechanism by which sperm sRNAs contribute to depression inheritance. Since the early embryonic period represents a window of plasticity important for adult phenotypes, we injected sperm sRNAs from mice of normal(F0-Ctl) or stress induced depressive(F0-Dep)into zygotes and assessed transcriptional changes when the embryos developed to the E3.5 blastocyst stage (sRNA-Dep-E3.5 vs sRNA-Ctl-E3.5). Overall design: sRNAs isolated from normal (F0-Ctl) or stress induced depressive (F0-Dep) mouse sperm with a concentration of 2 ng/µl were microinjected into zygotes of the C57Bl/6J background. The zygotes were then cultured in M16 medium at 37 °C in 5% CO2. On the second day, the 2-cell embryos were transferred to potassium-supplemented simplex optimized medium. Embryos at the blastocyst stage (E3.5) were collected at approximately 96 h after microinjection.A single blastocyst-stage embryo was suspended in RNA lysis reagents and frozen at -80 °C, ready for RNA-sequencing processing.

我们已证实，精子小RNA（small RNA，sRNA）参与了抑郁样症状的父系传递。为进一步探究精子sRNA介导抑郁遗传的分子机制，鉴于早期胚胎阶段是调控成年表型的关键可塑性窗口，我们将正常（F0-Ctl）或应激诱导抑郁（F0-Dep）小鼠的精子sRNA注射至受精卵中，并在胚胎发育至E3.5囊胚阶段时检测转录组变化（分组为sRNA-Dep-E3.5与sRNA-Ctl-E3.5）。实验设计：将浓度为2 ng/µl的正常（F0-Ctl）或应激诱导抑郁（F0-Dep）小鼠精子sRNA，显微注射至C57Bl/6J背景的受精卵内。随后将受精卵置于37℃、5% CO₂的培养环境中，使用M16培养基进行体外培养。培养至次日，将2细胞胚胎转移至钾补充型简化优化培养基中。于显微注射后约96小时，收集处于囊胚阶段（E3.5）的胚胎。将单个囊胚悬浮于RNA裂解试剂中，于-80℃冷冻保存，以待进行RNA测序（RNA-seq）处理。