Membrane potential and feedback dynamics regulate CatSper-mediated progesterone signaling in human sperm

Activation of the sperm-specific Ca²⁺ channel CatSper by progesterone evokes rapid changes in intracellular Ca²⁺ in human sperm that are required for fertilization. However, the mechanisms regulating the progesterone-induced Ca²⁺ signals have remained elusive. Here, we used quantitative kinetic fluorimetry with fast voltage-sensitive fluorescent indicators to investigate how progesterone affects the membrane potential (V_m) of human sperm. Additionally, we employed the FAST^M technique to simultaneously record at millisecond time resolution changes in both V_m and intracellular Ca²⁺. We show that progesterone evokes a rapid pulse-like depolarization and repolarization. The depolarization is caused by Ca²⁺ influx through CatSper, which pulls V_m away from a resting membrane potential (V_rest) of -65 mV set by the sperm-specific K⁺ channel Slo3. We further show that V_m- and Ca²⁺-dependent mechanisms limit the CatSper-mediated Ca²⁺ influx, thereby promoting repolarization and enabling K⁺ efflux through Slo3 channels to restore V_rest. Our findings demonstrate that non-genomic progesterone signaling in human sperm is regulated by negative feedback on CatSper and involves a dynamic interplay between CatSper and Slo3 in controlling V_m. We anticipate that our novel kinetic, quantitative V_m recording and V_m/Ca²⁺-multiplexing techniques will reveal additional molecular mechanisms underlying CatSper-mediated Ca²⁺ signaling in human sperm both in health and disease.