Andrology · 2026
A Scalable Flow Cytometry Assay to Evaluate CatSper-mediated Ca2+ Influx Triggered by High-K+/High-pH Stimulation in Mouse Sperm.
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Gomez-Olivieri Lucila R, Schiavi-Ehrenhaus Liza J, Jabloñski Martina, Buffone Mariano G, Luque Guillermina M
Аннотация
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.
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.