Abstract
Mammalian sperm evolutionarily acquired complex mechanisms to regulate their behaviors, which are thought to be crucial in navigating through the female reproductive tract toward fertilization. However, all current knowledge of this process is largely extrapolated from in vitro and ex vivo studies, because in vivo analysis of sperm in their native fertilization environment has not been possible. Here, we report a functional optical coherence tomography approach that allows, for the first time, in vivo three-dimensional tracking of sperm behaviors in the mouse oviduct. Motile sperm are identified with their intrinsic dynamic characteristics. Sperm trajectories are reconstructed in three dimensions with a ∼5 µm spatial resolution, allowing for quantitative analysis of the sperm velocity and location relative to the oviduct. Using this method, we found different behavior patterns, including sperm collection by the oviduct epithelium, spatial dependence of sperm velocity, and sperm grouping and separation as the first in vivo evidence of sperm cooperation in the ampulla, the site of fertilization. This approach opens new avenues to study sperm-oviduct interactions in vivo toward a more complete understanding of fertility and reproductive disorders.