Paternal exposure to a range of environmental and lifestyle factors elicits distinct changes to the sperm sncRNA profile; modifications that have significant post-fertilization consequences. Despite this knowledge, there remains limited mechanistic understanding of how paternal exposures effect the sperm sncRNA landscape. Here, we report the acute sensitivity of the sperm sncRNA profile to the potent reproductive toxicant, acrylamide. Further, we traced the differential accumulation of acrylamide responsive sncRNAs to coincide with sperm transit of the proximal (caput) segment of the epididymis, wherein acrylamide exposure altered the expression of several transcription factors implicated in the expression of acrylamide-sensitive sncRNAs. We also identified extracellular vesicles secreted from the caput epithelium in relaying altered sncRNA profiles to maturing spermatozoa, the implications of which manifest in the form of dysregulated gene expression during early embryonic development. These data provide a causative mechanistic link to account for how environmental insults can alter the sperm epigenome and compromise the transcriptomic profile of early embryos.

Epididymal epithelial cell preparations from control and acrylamide treated animals were pooled from five to six animals to generate a single biological replicate, with three such replicates being generated for analysis. Samples were prepared as previously described72, 73, 74. In brief, thawed cell suspensions containing lysis buffer, 0.1MNa2CO3 containing protease (Sigma) and phosphatase inhibitors (Roche, Complete EDTA free), were probe tip sonicated at 4°C for 3  10 sec cycles (100% output power) prior to incubation at 4°C for 1 h. A bicinchoninic acid assay (Thermo Fisher Scientific) was conducted to determine the protein concentration of each sample. Protein solutions were diluted in urea (6 M urea, 2 M thiourea), reduced using 10 mM dithiothreitol (DTT) (30 min, room temperature) and alkylated using 20 mM iodoacetamide (30 min, room temperature, in the dark). Proteins were digested with Lys-C/Trypsin using 1:30 ratio of protease to total protein concentration, for 3 h at room temperature. The urea concentration was then reduced to below 1 M by addition of 50 mM tetraethylammonium bromide (TEAB; pH 7.8) and incubated at 37°C overnight. Lipids were precipitated using formic acid (2% v/v final concentration), and peptide populations were purified using desalting columns (Oasis PRIME HLB; Waters, Rydalmere, NSW, Australia). Quantification of peptides was performed using fluorescent quantification (Qubit).Validation of quantitative differences in protein abundance in epididymal epithelial cells detected by nLC-MS/MS was performed using the targeted mass spectrometry strategy, parallel reaction monitoring (PRM). Proteins selected for analysis included those highly abundant in our sample or displaying a significant difference in abundance following acrylamide exposure and implicated in the identified pathways of interest. Peptides were purified as described above and subjected to reversed phase chromatography using Dionex Ultimate 3000RSLC. EasySpray 25cm columns were used in combination with a nano-ESI source. PRM was performed using Exploris 480 mass spectrometer (Thermo Scientific, Bremen, Germany). Methods optimised for collision energy, charge state, and retention times for peptides corresponding to proteins of interest, identified in the discovery TMT dataset. A full MS scan was performed with a resolution of 60,000 and targeted MS2 spectra were acquired using a PRM approach at a resolution of 15,000 employing a normalised AGC target of 100% and a maximum injection time of 120 ms. Raw data was analyzed using Skyline (MacCoss Lab, University of Washington) (REF), where the top six fragment ion intensities for individual peptide sequences were normalized to each sample’s respective total ion count. Quantification was performed by comparing the normalised values between biological triplicates of control and treated cells using a Student’s t-test.

Male mice were administered acrylamide (25 mg/kg bw/day) or vehicle alone (control, phosphate buffered saline; PBS) via intraperitoneal injection once per day for five consecutive days. Mice were then euthanized at one of three different time points following the final injection. In preparation for isolation of epididymal epithelial cells, mice were culled 2-3h following the final injection (AA). Targeted proteomics was carried out on epididymal epithelial cells.