Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial  dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.