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

Abstract

Background: Seminal 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.

Results: We 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.

Conclusion: Our 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.

Figure 1

A cumulative distribution showing new genes discovered across technical replicates. The sixth and seventh technical replicate added a combined total of four new genes (out of 69 total), suggesting we have approached an asymptote of new gene discovery.

Figure 2

Genes discovered in the first replicate had higher NSAF (arcsin square root transformed) than genes discovered in later replicates. This pattern was seen in all four biological samples; we present one of the copulatory plug samples here. This result suggests that we have identified all reasonably abundant (and detectable) proteins under the experimental conditions employed.

Figure 3

Genes identified in the present study, including 15 unique to ejaculates (ejac.) and 54 that overlapped with our previous study of the male reproductive tract (RT), evolve significantly more rapidly than the 429 non-overlapping proteins identified in our previous study [49].