Glycopolymer induction of mouse sperm acrosomal exocytosis shows highly cooperative self-antagonism

Identifying inducers of sperm acrosomal exocytosis (AE) to understand sperm functionality is important for both mechanistic and clinical studies in mammalian fertilization. Epifluorescence microscopy methods, while reproducible, are laborious and incompatible for high throughput screening. Flow cytometry methods are ideal for quantitative measurements on large numbers of samples, yet typically rely on the use of lectins that can interfere with physiologic AE-inducers. Here, we present an optimized triple stain flow cytometric method that is suitable for high-throughput screening of AE activation by glycopolymers. SYTO-17 and propidium iodide (PI) were used to differentiate cells based on their membrane integrity or viability, and membrane impermeable soybean trypsin inhibitor (SBTI) was used to monitor acrosome exocytosis. The SBTI/PI/SYTO-17 combination provides a positive screen for viability and AE of live sperm cells with minimal noise or false positives. A scattering gate enables the use of samples that may be contaminated with non-cellular aggregates, e.g., cryopreservation agents. This assay format enabled detailed analysis of glycopolymer dose response curves. We found that fucose polymer has a narrow effective dose range (EC₅₀ = 1.6 μM; IC₅₀ = 13.5 μM); whereas mannose polymer and β-N-acetylglucosamine polymer have broader effective dose ranges (EC₅₀ = 1.2 μM and 3.4 μM, respectively). These results highlight the importance of testing inducers over a large concentration range in small increments for accurate comparison.