Mature sperm carry thousands of RNAs, including mRNAs, lncRNAs, tRNAs, rRNAs and sncRNAs, though their functional significance is still a matter of debate. Growing evidence suggests that sperm RNAs, especially sncRNAs, are selectively retained during spermiogenesis or specifically transferred during epididymis maturation, and are thus delivered to the oocyte at fertilization, providing resources for embryo development. However , a deep characterization of the sncRNA content of bull sperm and its expression profile across breeds is currently lacking. To fill this gap, we optimized a guanidinium–Trizol total RNA extraction protocol to prepare high-quality RNA from frozen bull sperm collected from 40 representative bulls from six breeds. Deep sequencing was performed (40 M single 50-bp reads per sample) to establish a comprehensive repertoire of cattle sperm sncRNA. Our study showed that it comprises mostly piRNAs (26%), rRNA fragments (25%), miRNAs (20%) and tRNA fragments (tsRNA, 14%). We identified 5p-halves as the predominant tsRNA subgroup in bull sperm, originating mostly from Gly and Glu isoacceptors. Our study also increased by ~ 50% the sperm repertoire of known miRNAs and identified 2022 predicted miRNAs. About 20% of sperm miRNAs were located within genomic clusters, expanding the list of known polycistronic pri-miRNA clusters and defining several networks of co-expressed miRNAs. Strikingly, our study highlighted the great diversity of isomiRs, resulting mainly from deletions and non-templated additions (A and U) at the 3p end. Substitutions within miRNA sequence accounted for 40% of isomiRs, with G>A, U>C and C>U substitutions being the most frequent variations. In addition, many sncRNAs were found to be differentially expressed across breeds. Our study provides a comprehensive overview of cattle sperm sncRNA, and these findings will pave the way for future work on the role of sncRNAs in embryo development and their relevance as biomarkers of semen fertility.

中文翻译：

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牛精子携带的小型非编码RNA及其在不同品种中的多样性的全面概述

成熟的精子携带成千上万的RNA，包括mRNA，lncRNA，tRNA，rRNA和sncRNA，尽管它们的功能意义尚有争议。越来越多的证据表明，精子RNA，尤其是sncRNA，在精子发生过程中被选择性保留，或在附睾成熟过程中被特异性转移，因此在受精时被传递到卵母细胞，为胚胎发育提供了资源。但是，目前尚缺乏对公牛精子中sncRNA含量及其在不同品种间表达情况的深入表征。为了填补这一空白，我们优化了胍-三唑总RNA提取方案，以从六个品种的40头代表性公牛中收集的冷冻公牛精子中制备高质量RNA。进行了深度测序（每个样品40 M单次50 bp读数），以建立牛精子sncRNA的全面库。我们的研究表明，它主要包含piRNA（26％），rRNA片段（25％），miRNA（20％）和tRNA片段（tsRNA，14％）。我们确定了5p-一半是公牛精子中主要的tsRNA亚组，主要来源于Gly和Glu异构受体。我们的研究还使已知miRNA的精子库增加了约50％，并确定了2022个预测的miRNA。大约20％的精子miRNA位于基因组簇内，扩大了已知的多顺反子pri-miRNA簇的列表，并定义了几种共表达miRNA的网络。令人惊讶的是，我们的研究强调了isoomiR的多样性，这主要是由于3p末端的缺失和非模板化添加（A和U）所致。miRNA序列内的取代占isomiRs的40％，其中G> A，U> C和C> U取代是最常见的变异。此外，发现许多sncRNA在不同品种之间差异表达。我们的研究提供了对牛精子sncRNA的全面概述，这些发现将为sncRNA在胚胎发育中的作用及其作为精液育性的生物标志物的相关性铺平道路。