Real-Time Imaging of Calcium Dynamics in Human Sperm After Precise Single-Cell Stimulation

Calcium signaling is a critical regulator of sperm activation and function during the processes of capacitation and fertilization. Here, we describe a combined method for calcium imaging of single, live human sperm in response to stimuli administered with a precisely targeted delivery technique. This protocol is an adaptation of techniques developed for studies of murine sperm [1, 2], and enables real-time monitoring of human sperm calcium dynamics with high spatiotemporal resolution and concurrent detection of acrosome exocytosis (AE), a functional endpoint of sperm capacitation and requirement for physiological fertilization.The described imaging technique provides a valuable tool for exploration of calcium regulation in human sperm, which is essential to answer important questions and knowledge gaps regarding the link between calcium dynamics, AE, and fertilization. The versatility of this technique can be amplified through use of various indicator dyes or integration with pharmacological strategies such as pre-treating sperm with inhibitors or activators targeting specific receptors, channels, or intracellular signaling pathways of interest. Beyond fundamental inquiries into sperm physiology, this method can also be applied to assess the impact of potential contraceptive compounds on calcium signaling, AE, and membrane integrity.