Epididymis-specific RNase A family genes regulate fertility and small RNA processing

Sperm small RNAs are implicated in intergenerational transmission of paternal environmental effects. Small RNAs generated by cleavage of tRNAs, known as tRNA fragments (tRFs) or tRNA-derived RNAs (tDRs or tsRNAs), are an abundant class of RNAs in mature sperm, and can be modulated by environmental conditions. The biogenesis of tRFs in the male reproductive tract remains poorly understood. Angiogenin, a member of the Ribonuclease A superfamily (RNase A), cleaves tRNAs to generate tRFs in response to cellular stress. Four paralogs of Angiogenin, namely Rnase9, Rnase10, Rnase11, and Rnase12, are specifically expressed in the epididymis a long, convoluted tubule where sperm mature and acquire fertility and motility. The biological functions of these genes remain largely unknown. Here, by generating mice deleted for all four genes (Rnase9-12-/-, termed KO for Knock Out), we report that these genes regulate fertility and small RNA levels. KO mice showed complete male sterility. KO sperm fertilized oocytes in vitro but failed to efficiently fertilize oocytes in vivo due to an inability of sperm to pass through the utero-tubular junction. Intriguingly, there were decreased levels of fragments of tRNAs (tRFs) and rRNAs (rRNA-derived small RNAs or rsRNAs) in the KO epididymis and epididymal luminal fluid. RNases 9-12 did not show ribonucleolytic activity in-vitro, suggesting that the changes in tRF and rsRNA levels are independent of enzymatic activity of these proteins. Importantly, KO sperm showed a dramatic decrease in the levels of tRFs, demonstrating a role of epididymis-specific Rnase9-12 genes in regulating sperm small RNA composition. Together, our results reveal an unexpected role of four epididymis-specific non-canonical RNase A family genes in regulating fertility and small RNA processing. Small RNA sequencing of various male reproductive tract tissues including sperm, epididymis, and epididymal fluid isolated from wild type mice or mice deleted for four epididymis-specific genes Rnase9, Rnase10, Rnase11 and Rnase12 (Rnase9-12 KO)精子小RNA与父代环境影响的跨代传递密切相关。由转运RNA（transfer RNA, tRNA）剪切产生的小RNA，即tRNA片段（tRNA fragments, tRFs）或称tRNA来源RNA（tRNA-derived RNAs, tDRs或tsRNAs），是成熟精子中一类含量丰富的RNA，其表达水平可受环境条件调控。男性生殖道内tRFs的生物发生机制目前仍不甚明确。血管生成素（Angiogenin）属于核糖核酸酶A超家族（Ribonuclease A superfamily, RNase A），可在细胞应激状态下剪切tRNA以产生tRFs。血管生成素的四个旁系同源基因——Rnase9、Rnase10、Rnase11及Rnase12——特异性表达于附睾（epididymis）——这是一段细长迂曲的管道，精子在此处成熟并获得受精能力与运动能力。这些基因的生物学功能目前仍知之甚少。本研究通过构建四基因敲除（Knock Out, KO）小鼠（Rnase9-12-/-），证实这些基因可调控生育能力与小RNA水平。该敲除小鼠表现为完全雄性不育。敲除小鼠的精子可在体外完成卵母细胞受精，但在体内无法有效使卵母细胞受精，原因是精子无法通过子宫输卵管连接部（utero-tubular junction）。有趣的是，敲除小鼠的附睾及附睾管腔液中，tRFs与核糖体RNA（ribosomal RNA, rRNA）来源小RNA（rRNA-derived small RNAs, rsRNAs）的水平均出现下降。体外实验显示，Rnase9-12并无核糖核酸酶活性，这表明tRF与rsRNA水平的变化并不依赖于这些蛋白的酶活性。尤为重要的是，敲除小鼠的精子中tRFs水平显著降低，这证实了附睾特异性表达的Rnase9-12基因可调控精子的小RNA组成。综上，本研究结果揭示了四个附睾特异性非经典RNase A家族基因在调控生育能力与小RNA加工过程中的意外功能。本研究对野生型（wild type）小鼠及四附睾特异性基因（Rnase9、Rnase10、Rnase11、Rnase12）敲除（Rnase9-12 KO）小鼠的多种男性生殖道组织（包括精子、附睾及附睾液）进行了小RNA测序。