Mouse Seminal Vesicle Preparations C4PR_LIV
Ejaculates contain a diverse mixture of sperm and seminal fluid proteins, the combination of which is crucial to male reproductive success under competitive conditions. Males should therefore tailor the production of different ejaculate components according to their social environment, with particular sensitivity to cues of sperm competition risk (i.e. how likely it is that females will mate promiscuously). Here we test this hypothesis using an established vertebrate model system, the house mouse (Mus musculus domesticus), combining experimental data with a quantitative proteomics analysis of seminal fluid composition. Our study tests for the first time how both sperm and seminal fluid components of the ejaculate are tailored to the social environment.
Sample Processing Protocol
In solution tryptic digestions of 50microG quantities of seminal vesicle/fluid preparations. 1 µL ofeach final tryptic digestion dilution was resolved by reversed-phase (C18) UPLC (Waters nanoAcquity) over a 30 min linear organic gradient of 3–40% buffer B (0.1% formic acid in acetonitrile), prior to tandem MS using a LTQ-Orbitrap Velos. High resolution, accurate mass data was acquired in a data-dependent manner, with the top 20 most intense peptides in each MS scan selected for fragmentation. The raw data were processed using Progenesis QI (v2, Nonlinear Dynamics) to determine protein abundances. The data from all raw files were automatically aligned according to retention time to produce an aggregate spectrum, from which charge states +1 and > +4 were excluded.
Data Processing Protocol
The aggregated spectrum was converted into a peak list (.mgf format) enabling protein identifications across all samples by searching the data against a reviewed UniProt Mouse database, using the search engine Mascot (v2.5.1). The .xml file generated by Mascot was imported back into Progenesis QI for feature assignment, normalisation and relative quantification. Downstream data analysis was performed using R and JMP v10.
Dean Hammond, University of Liverpool
Paula Stockley, Mammalian Behaviour and Evolution Group, Institute of Integrative Biology, University of Liverpool, Leahurst Campus, Chester High Road, Neston CH64 7TE, UK ( lab head )
Ramm SA, Edward DA, Claydon AJ, Hammond DE, Brownridge P, Hurst JL, Beynon RJ, Stockley P. Sperm competition risk drives plasticity in seminal fluid composition. BMC Biol. 2015 Oct 27;13:87 PubMed: 26507392