A Scalable Flow Cytometry Assay to Evaluate CatSper-mediated Ca2+ Influx Triggered by High-K+/High-pH Stimulation in Mouse Sperm.

BACKGROUND

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 nonhormonal male contraceptives. However, its structural complexity and the absence of functional heterologous expression systems have limited inhibitor discovery.

OBJECTIVES

To develop a scalable and multiplexed flow cytometry assay to identify compounds capable of blocking CatSper activity in mammalian sperm.

MATERIALS AND

METHODS

The assay 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. Additionally, a compound pooling strategy was implemented 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.

RESULTS

Fluorescent cell barcoding greatly reduced acquisition time and reagent consumption by allowing multiple experimental conditions to be pooled and analyzed simultaneously within a single acquisition. The compound pooling strategy facilitated large-scale screening experiments. Validation confirmed that both genetic deletion and pharmacological blockade of CatSper abolish the K8.6-induced Ca2+ response.

CONCLUSION

These results establish a robust, scalable, and high-throughput screening platform for identifying novel CatSper blockers and potential male contraceptive candidates.