Epididymal acquired sperm microRNAs modify post-fertilization embryonic gene expression

Sperm small RNAs have emerged as important non-genetic contributors to embryogenesis and offspring health. A subset of sperm small RNAs are thought to be acquired during epididymal transit. However, the transfer of RNAs from the somatic epididymis to the sperm has been questioned, and the identity of the specific small RNAs transferred remains unclear. Here, we employ Cre/Lox genetics to generate germline- and epididymal-specific Dgcr8 conditional knockout mice to investigate the dynamics of sperm microRNAs and their function in the early embryo. Interestingly, sperm from germline specific Dgcr8 knockout males restored the levels of 58 of the 98 (59%) miRNAs that were lost in testicular sperm during epididymal transit. Conversely, sperm from epididymal Dgcr8 knockouts displayed a 5-fold reduction in 25 miRNAs. This substantial loss of epididymal miRNAs in sperm was accompanied by transcriptomic changes in the embryo which was rescued by microinjection of epididymal miRNAs. These findings ultimately demonstrate the acquisition of miRNAs by sperm during epididymal transit and their regulation of post-fertilization embryonic gene expression. To examine the impact of ablation of the miRNA pathway on male fertility and the regulation of gene expression in the male reproductive tract we sequenced the small RNA fraction of testicular and cauda sperm from 2 Dgcr8 conditional knockout mice and their respective controls and performed RNA-seq on epididymal tissue from these mice. Additionally, in examining the impact of altered sperm miRNA levels on gene expression in the early embryo we sequenced single embryos (4-cell and morula stage) fertilized by control and mutant male mice.精子小RNA（sperm small RNAs）现已被证实为胚胎发生及子代健康的关键非遗传影响因子。研究表明，部分精子小RNA是在附睾转运过程中获得的。然而，从附睾体细胞向精子转移RNA这一过程受到了广泛质疑，且已确认的特异性转移小RNA种类仍不明确。本研究借助Cre/Lox基因遗传学系统，构建了生殖系特异性与附睾特异性的Dgcr8条件性敲除（conditional knockout）小鼠模型，以探究精子微RNA（microRNAs，miRNAs）的动态变化及其在早期胚胎中的功能。值得注意的是，生殖系特异性Dgcr8敲除雄性小鼠的精子，可恢复睾丸精子在附睾转运过程中丢失的98种微RNA中的58种（占比59%）。与之相反，附睾特异性Dgcr8敲除小鼠的精子中，25种微RNA的表达水平下降了5倍。精子中这类附睾源性微RNA的显著丢失，会引发胚胎的转录组改变，而通过显微注射附睾源性微RNA可逆转这一异常表型。上述研究结果最终证实，精子可在附睾转运过程中获取微RNA，并对受精后的胚胎基因表达进行调控。为探究微RNA通路敲除对雄性生育能力以及雄性生殖道基因表达调控的影响，本研究对2株Dgcr8条件性敲除小鼠及其对应对照组的睾丸精子与附睾尾精子（cauda sperm）的小RNA组分进行了测序，并对这些小鼠的附睾组织开展了RNA测序（RNA-seq）。此外，为探究精子微RNA水平改变对早期胚胎基因表达的影响，本研究对由野生型与突变型雄性小鼠受精得到的单个早期胚胎（4细胞期与桑椹胚期）进行了测序。