Using stable isotopes to track sperm competition

Paula Stockley, Catarina Franco, Amy J. Claydon, Amanda Davidson, Dean E. Hammond, Philip J. Brownridge, Jane L. Hurst, and Robert J. Beynon (2020) Revealing mechanisms of mating plug function under sexual selection. PNAS USA

Promiscuous mating by females leads to competition between males for fertilization success. When fertilization is internal, this means that rival males’ sperm must compete within the female reproductive tract to reach the eggs. Males of diverse species deposit a mating plug during copulation, which is hypothesized to assist in the race for fertilization following multiple mating. Here, we tested this by using stable isotope labeling to discriminate the ejaculates of competing male voles in direct competition. This revealed that the mating plug simultaneously inhibits the sperm of rival males while promoting transport of a male’s own sperm, both of which are beneficial in the competition for fertilizations.

Mating plugs are produced by many sexually reproducing animals and are hypothesized to promote male fertilization success under promiscuous mating. However, tests of this hypothesis have been constrained by an inability to discriminate ejaculates of different males in direct competition. Here, we use stable isotope labeling in vivo and proteomics to achieve this in a promiscuous rodent, Myodes glareolus. We show that, although the first male’s plug is usually dislodged, it can be retained throughout the second male’s copulation. Retained plugs did not completely block rival sperm but did significantly limit their numbers. Differences in the number of each male’s sperm progressing through the female reproductive tract were also explained by natural variation in the size of mating plugs and reproductive accessory glands from which major plug proteins originate. Relative sperm numbers in turn predicted the relative fertilization success of rival males. Our application of stable isotopes to label ejaculates resolves a longstanding debate by revealing how rodent mating plugs promote fertilization success under competitive conditions. This approach opens new opportunities to reveal cryptic mechanisms of postcopulatory sexual selection among diverse animal taxa.