Revealing Fertilization-Competent Sperm Subpopulations

1. Department of Biology, East Carolina University, Greenville, NC 

2. Department of Public Health, East Carolina University, Greenville, NC 

3. Flow Cytometry Core, Brody School of Medicine, East Carolina University, Greenville, NC 

4. Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, NC 


Abstract Text: Successful fertilization requires sperm to undergo capacitation, a post-ejaculatory maturation process regulated by both time-dependent and chemical cues. A central event in this process is the acrosome reaction (AR), which enables sperm to bind and penetrate the oocyte. Although the signaling pathways driving capacitation have been extensively studied, the inherent heterogeneity in individual sperm responses makes it difficult to predict which cells are fertilization competent. To better address this challenge, we propose incorporating population-level modeling that can be adapted to describe capacitation. By examining male infertility at the population level rather than focusing on individual cells, we can make more accurate predictions about fertility competence. To test our modified modeling approach, we conducted two spectral flow cytometry experiments—one capturing the time-dependent effects of capacitation and the other focusing on chemical cues. Using detailed measurements of intracellular calcium, viability, and acrosomal status at the single-cell level, we can directly validate our model. Our data reveal that in the CD1 mouse strain, capacitation unfolds in distinct waves, a phenomenon only discernible at the population level. At any given time, a subset of sperm gains fertilization competence, then quickly dies, only to be replaced by a new cohort steadily undergoing capacitation. This cyclical pattern continues until the entire sample eventually follows this pattern or dies before ever reaching the AR. Looking ahead, we will refine this population-level model of capacitation to boost fertilization rates and enhance zygotic development in standard IVF protocols, potentially improving outcomes beyond those achieved with current clinical practices.