Methods for Collecting and Analyzing Post-Ejaculatory Uterine Fluid and the Uterus in Mice

In mammals, the semen is ejaculated into the female reproductive tract, and the sperm travel to the oviduct to fertilize the egg. A comprehensive understanding of the pre- and post-ejaculatory intrauterine environment is one of the key points for overcoming infertility; however, the dynamics of the intrauterine environment and its physiological role in the uterus, namely in the internal fertilization process, remain unclear. Conventional methods for collecting uterine fluids from the uterus post-ejaculation of mice show challenges regarding the ambiguous ejaculation timing. Here, we established a method for a mating environment with exact ejaculation timing. We also created a simple method for collecting pre- and post-ejaculatory uterine fluid without using forceps. Our methods achieved time-dependent biochemical and histological analyses of uterine fluids to provide fundamental information regarding protein composition and uterine structure changes during pre- and post-ejaculation. This protocol is suitable for analyzing temporal changes in reproductive phenomena, thereby contributing to elucidating the physiological role of the uterus in the process of intrauterine fertilization. Key features • This protocol is used for the simple collection of pre- and post-ejaculatory uterine fluid. • Changes in the pre- and post-ejaculatory intrauterine environment can be examined by controlling the dissection time of females after ejaculation. • An estrous female can be determined without a vaginal smear test in this protocol. • This protocol can be used to analyze the protein composition of post-ejaculatory uterine fluid and is applicable to analyze sperm within the uterus post-ejaculation.