A high-throughput flow cytometry assay to evaluate CatSper-mediated Ca2+ influx triggered by high-K+/high-pH stimulation in mouse sperm

The sperm-specific Ca2+ channel CatSper is essential for male fertility, as it mediates Ca2+ influx required for sperm hyperactivation. Because of its restricted expression in sperm and crucial role in fertilization, CatSper represents a promising target for the development of non-hormonal male contraceptives. However, its structural complexity and the absence of functional heterologous expression systems have limited inhibitor discovery. Here, we developed a high-throughput flow cytometry assay to identify compounds capable of blocking CatSper activity in mammalian sperm. The method relies on the stimulation of CatSper by exposing live mouse sperm to a high-K+/high-pH solution (K8.6), which induces membrane depolarization and intracellular alkalinization, triggering Ca2+ entry through CatSper. Changes in intracellular Ca2+ levels were monitored using the fluorescent indicator Fluo-4. To increase throughput and minimize variability, this assay was combined with fluorescent cell barcoding, which assigns each sperm sample a unique fluorescent signature, enabling the simultaneous acquisition of multiple conditions within a single tube. This integration greatly reduces acquisition time and reagent consumption, thereby facilitating large-scale screening experiments. Moreover, we implemented a compound pooling strategy to further improve assay efficiency, allowing the rapid identification of active compounds within a library. The assay was validated using CatSper1 knockout sperm and known pharmacological inhibitors, confirming that both genetic deletion and pharmacological blockade of CatSper abolish the K8.6-induced Ca2+ response. Together, these results establish a robust, scalable, and high-throughput screening platform for identifying novel CatSper blockers and potential male contraceptive candidates.