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

Activation of the sperm-specific Ca ²⁺ channel CatSper by progesterone evokes rapid changes in intracellular Ca ²⁺ in human sperm that are required for fertilization. However, the mechanisms regulating the progesterone-induced Ca ²⁺ 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 Ca ²⁺ . We show that progesterone evokes a rapid pulse-like depolarization and repolarization. The depolarization is caused by Ca ²⁺ 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 Ca ²⁺ -dependent mechanisms limit the CatSper-mediated Ca ²⁺ 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 /Ca ²⁺ -multiplexing techniques will reveal additional molecular mechanisms underlying CatSper-mediated Ca ²⁺ signaling in human sperm both in health and disease.