Identification of ejaculated proteins in the house mouse (Mus domesticus) via isotopic labeling

Abstract

BackgroundSeminal fluid plays an important role in successful fertilization, but knowledge of the full suite of proteins transferred from males to females during copulation is incomplete. The list of ejaculated proteins remains particularly scant in one of the best-studied mammalian systems, the house mouse (Mus domesticus), where artificial ejaculation techniques have proven inadequate. Here we investigate an alternative method for identifying ejaculated proteins, by isotopically labeling females with 15N and then mating them to unlabeled, vasectomized males. Proteins were then isolated from mated females and identified using mass spectrometry. In addition to gaining insights into possible functions and fates of ejaculated proteins, our study serves as proof of concept that isotopic labeling is a powerful means to study reproductive proteins.ResultsWe identified 69 male-derived proteins from the female reproductive tract following copulation. More than a third of all spectra detected mapped to just seven genes known to be structurally important in the formation of the copulatory plug, a hard coagulum that forms shortly after mating. Seminal fluid is significantly enriched for proteins that function in protection from oxidative stress and endopeptidase inhibition. Females, on the other hand, produce endopeptidases in response to mating. The 69 ejaculated proteins evolve significantly more rapidly than other proteins that we previously identified directly from dissection of the male reproductive tract.ConclusionOur study attempts to comprehensively identify the proteins transferred from males to females during mating, expanding the application of isotopic labeling to mammalian reproductive genomics. This technique opens the way to the targeted monitoring of the fate of ejaculated proteins as they incubate in the female reproductive tract.

Genetic and phenotypic influences on copulatory plug survival in mice

“…Consistent with a hypothesis of sexual conflict (Stockley, 1997), recently mated females upregulate proteases thought to assist in plug degradation (Kelleher and Pennington, 2009;Dean et al, 2011), whereas male seminal fluid is enriched for protease inhibitors , although proteases and their inhibitors have additional roles in reproduction (Wolfner, 2002;Kawano et al, 2010). Also, plug-forming proteins, proteases and protease inhibitors all tend to evolve rapidly (Dorus et al, 2004;Clark and Swanson, 2005;Kelleher et al, 2007;Lawniczak and Begun, 2007;Ramm et al, 2008;Wong et al, 2008;Dean and Nachman, 2009;Dean et al, 2011) as predicted for genes involved in sexual conflict (Swanson and Vacquier, 2002;Clark et al, 2006). In primates, the evolutionary rate of a key copulatory plug gene, SEMG2, is positively correlated with the inferred intensity of sexual selection (Dorus et al, 2004;Ramm et al, 2009).…”

“…In mice, serine endopeptidases are upregulated by females in response to mating, and serine endopeptidase inhibitors are enriched in male seminal fluid (Dean et al, , 2011. Thrombin is a serine endopeptidase that mimics such proteolytic activity.…”

“…between pairwise genetic distance and pairwise phenotypic difference in plug size or survival (all Mantel's tests, P40.05). Two proteases, Ltf and Klk14, were previously shown to be produced by females in response to mating (Dean et al, 2011) and could be important in plug degradation. The two female strains used here, FVB and C57, are identical at both genes (http://www.…”

“…Coomassie-stained polyacrylamide gels of seminal vesicle fluids revealed four very abundant proteins (Supplementary Figure 3 and Table 2). We did not attempt to identify these four bands using mass spectrometry, but based on previous studies (Lundwall et al, 1997;Lin et al, 2002;Dean et al, 2011;Tseng et al, 2011) and the match to known molecular mass, these four bands most likely consisted of the seminal vesicle-secreted proteins Svs1, Svs2, Svs4 and Svs5 (Supplementary Figure 3). The exact identity of the proteins is not critical as we are only using them as a biomarker of plug composition.…”

“…The full exomes will be published as part of a larger study elsewhere, but we focus here on these five genes as they are either necessary for copulatory plug formation or present in seminal vesicles and plugs at high abundance (Lundwall et al, 1997;Dean et al, , 2011Dean, 2013;Kawano et al, 2014). DNA was sheared using a Bioruptor UCD-200 (Diagenode, Denville, NJ, USA) with 7 rounds of sonication (7 min per round on high, 30 s on and 30 s off) and genomic DNA libraries were constructed using a previously described protocol designed to facilitate multiplexed exome capture (Rohland and Reich, 2012).…”