Factors affecting intracellular calcium influx in response to calcium ionophore A23187 in equine sperm

Abstract

Background: Exposure to the calcium ionophore A23187 may present a “universal” sperm treatment for IVF, as it bypasses capacitation pathways. However, success in utilizing A23187 is variable, especially in equine spermatozoa. Notably, albumin is used during A23187 treatment but paradoxically is thought to suppress A23187 action. Essentially no critical data are available on the effects of A23187 and albumin concentrations, ratios, or addition protocols on changes in intracellular calcium ([Ca]i) in any cell type. Objective: To determine factors that affect the action of A23187 on [Ca]i in equine and murine spermatozoa. Methods: Spermatozoa were loaded with Fluo- 4 and changes in fluorescence after A23187 treatment were measured under various conditions using a microplate reader. Results: Concentrations of bovine serum albumin (BSA) and A23187, type of BSA, makeup of A23187 stock solutions (i.e., 1° stock (DMSO) or 2° stock made with medium, water or DMSO), order of addition of spermatozoa and A23187, incubation of media before sperm addition, species of spermatozoa, and time of addition of BSA all affected [Ca]i in response to A23187 treatment. In equine spermatozoa already exposed to 10 μM A23187, addition of BSA to 33 mg/ml to “quench” the A23187 did not affect [Ca]i. When this concentration of BSA was added to spermatozoa exposed to 1 μM A23187, [Ca]i in murine spermatozoa returned to baseline, however, equine spermatozoa continued to exhibit increased [Ca]i. Addition of BSA to 33 mg/ml to media containing 1 μM A23187, prior to addition of spermatozoa, completely inhibited change in [Ca]i i n b oth m urine a nd e quine spermatozoa. Conclusion: These results represent some of the first critical data on the effects of albumin and other procedural factors on A23187- induced changes in [Ca]i in any cell type. Our findings help to explain the variability in reported response of spermatozoa to A23187 among species and among laboratories.