Data from: Post-ejaculation thermal stress causes changes to the RNA profile of sperm in an external fertiliser

Sperm cells experience considerable post-ejaculation environmental variation. However, little is known about whether this affects their molecular composition, likely due to the assumption that sperm are transcriptionally quiescent. Nevertheless, recent evidence shows sperm have distinct RNA profiles that affect fertilisation and embryo viability. Moreover, RNAs are expected to be highly sensitive to extracellular changes. One such group of RNAs are heat shock protein (hsp) transcripts, which function in stress responses and are enriched in sperm. Here, we exploit the tractability of the mussel Mytilus galloprovincialis to exposed paired samples of ejaculated sperm to ambient (19 °C) and increased (25 °C) temperatures, then measure (a) sperm motility phenotypes, and (b) mRNA levels of two target genes (hsp70 and hsp90) and several putative reference genes. We find no phenotypic changes in motility, but reduced mRNA levels for hsp90 and the putative reference gene gapdh at 25 °C. This could reflect either decay of specific RNAs, or changes in translation and degradation rates of transcripts to maintain sperm function under stress. These findings represent the first evidence for changes in sperm RNA profiles due to post-ejaculation environments, and suggest that sperm may be more vulnerable to stress from rising temperatures than currently thought.,This dataset contains data collected from the sperm of mussels, Mytilus galloprovincialis, during a controlled, split-ejaculate experiment. Sperm from each male were split into two aliquots; one was kept at ambient temperature (18C) and the other was treated to a high temperature (25C) for 10 minutes. For a subset of males, we measured motility traits of sperm in each treatment using computer-assisted sperm analysis (CASA). For another subset of males, we measured the abundance of RNA transcripts in sperm from four genes (hsp70, hsp90, gapdh and actin) using reverse-transription quantitative polymerase chain reaction (qPCR). Note that for logistical reasons, some males had both data types collected (CASA and qPCR), while other males had one or the other. The cycle threshold outputs from qPCR reactions were converted into molecule counts for analysis using the R package 'MCMC.qpcr' (https://cran.r-project.org/web/packages/MCMC.qpcr/index.html), based on amplification efficiencies for each gene calculated from standard curves.,The dataset contains three spreadsheets. The sheet titled "CASA" contains sperm motility data, with rows for sperm samples and columns for block (experimental day of collection), unique male ID, temperature treatment (A=ambient, H=high), sample ID, and the following CASA measurements: amplitude of lateral head displacement (ALH), beat cross frequency (BCF), curvilinear velocity (VCL), average path velocity (VAP), straight-line velocity (VSL), linearity (LIN), straightness (STR), number of motile sperm, number of immotile sperm, and total number of sperm cells tracked. The sheet titled ""qPCR_standard_curves" contains results from qPCR standard cDNA curves of five-fold serial dilutions (10, 2, 0.4, 0.08, and 0.016 ng µL-1; each dilution was prepared in triplicate) for each gene, prepared using a non-experimental mussel sperm sample at ambient temperature. Columns denote cDNA concentration (dna), cycle threshold output (cq) and gene ID (gene). The sheet titled "qPCR_gene_assays" contains the results from qPCR assays for each gene on experimental sperm samples. Each sperm sample (i.e. each male by treatment combination) had reactions prepared in triplicate for each gene. Columns denote sample ID, block (experimental day of collection), unique male ID, temperature treatment (A=ambient, H=high), and the cycle threshold output for each gene (hsp70, hsp90, gapdh and actin).,精子细胞在射出后会经历显著的环境变异，但目前对该变异是否会影响其分子组成仍知之甚少，这大概率源于此前认为精子处于转录静默状态的认知。不过，近期研究表明精子拥有独特的RNA谱，该谱会影响受精过程与胚胎存活率。此外，RNA分子对胞外环境变化高度敏感，热休克蛋白（heat shock protein, hsp）转录本便是其中一类，这类RNA参与应激反应且在精子中富集。本研究利用贻贝（Mytilus galloprovincialis）的可操作性，将成对的射出精子样本分别置于环境温度（19 °C）与升高温度（25 °C）环境中处理，随后开展两项检测：(a) 精子运动表型；(b) 两个靶基因（hsp70与hsp90）及若干候选内参基因的mRNA水平。研究结果显示，精子运动能力未出现显著变化，但在25 °C处理组中，hsp90与候选内参基因gapdh的mRNA水平有所降低。该现象可能反映了特定RNA的降解，或是为维持应激状态下精子功能而发生的转录本翻译与降解速率改变。本研究首次证实了射出后环境变化会改变精子的RNA谱，同时表明精子对升温带来的应激压力可能比当前认知中更为脆弱。 本数据集源自贻贝（Mytilus galloprovincialis）精子的控制性分样实验。将每只雄性个体的精子分为两份等分试样：一份置于环境温度（18 °C）中保存，另一份置于高温（25 °C）环境处理10分钟。针对部分雄性个体，采用计算机辅助精子分析系统（computer-assisted sperm analysis, CASA）检测各处理组的精子运动性状；针对另一部分雄性个体，采用反转录定量聚合酶链式反应（reverse-transcription quantitative polymerase chain reaction, qPCR）检测精子中4个基因（hsp70、hsp90、gapdh与肌动蛋白（actin））的RNA转录本丰度。需注意，受后勤条件限制，部分雄性个体同时获取了CASA与qPCR两类数据，其余个体仅获取其中一类数据。qPCR反应的循环阈值（cycle threshold, Ct）输出结果通过R包‘MCMC.qpcr’（https://cran.r-project.org/web/packages/MCMC.qpcr/index.html）转换为分子计数用于分析，转换依据为通过标准曲线计算得到的每个基因的扩增效率。 本数据集包含三张电子表格： 1. 名为"CASA"的工作表：存储精子运动数据，行代表精子样本，列包含实验批次（样本收集的实验日期）、唯一雄性个体ID、温度处理组（A=环境温度，H=高温）、样本ID，以及以下CASA检测指标：精子头部侧摆幅度（amplitude of lateral head displacement, ALH）、鞭打交叉频率（beat cross frequency, BCF）、曲线速度（curvilinear velocity, VCL）、平均路径速度（average path velocity, VAP）、直线速度（straight-line velocity, VSL）、线性度（linearity, LIN）、直线性（straightness, STR）、活动精子数量、非活动精子数量以及追踪到的总精子细胞数。 2. 名为"qPCR_standard_curves"的工作表：存储各基因的qPCR标准cDNA曲线结果，采用非实验用环境温度下的贻贝精子样本制备，进行5倍系列稀释（浓度依次为10、2、0.4、0.08与0.016 ng µL⁻¹，每个稀释度设置三次重复）。列信息包括cDNA浓度（dna）、循环阈值（cq）与基因ID（gene）。 3. 名为"qPCR_gene_assays"的工作表：存储实验精子样本的各基因qPCR检测结果。每个精子样本（即每个雄性个体与处理组的组合）为每个基因设置三次重复反应。列信息包括样本ID、实验批次（样本收集的实验日期）、唯一雄性个体ID、温度处理组（A=环境温度，H=高温），以及每个基因的循环阈值输出结果（hsp70、hsp90、gapdh与actin）。