DOI: 10.5061/dryad.8pk0p2nkh
title: Data from: Post-ejaculation thermal stress causes changes to the RNA profile of sperm in an external fertiliser

description: 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.
description: This dataset contains data collected from the sperm of
 mussels, <em>Mytilus galloprovincialis</em>, 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
 (<em>hsp70, hsp90, gapdh </em>and <em>actin</em>)
 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.
description: 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<sup>-1</sup>; 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 (<em>hsp70, hsp90,
 gapdh </em>and <em>actin</em>).