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 in both murine and equine 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.

Antioxidant Nanoparticles Restore Cisplatin-Induced Male Fertility Defects by Promoting MDC1-53bp1-Associated Non-Homologous DNA Repair Mechanism and Sperm Intracellular Calcium Influx

“…To evaluate whether nHNK actually affects extracellular calcium influx in sperm, we performed a kinetic intracellular calcium measurement using Fluo-4 AM as an indicator in live sperm. 37 Baseline values Figure 5 nHNK promoted sperm calcium influx. (A) Genes responsible for calcium regulation were affected.…”

Correction to: Successful in vitro fertilization in the horse: production of blastocysts and birth of foals after prolonged sperm incubation for capacitation

“…Then, 5 μL of an A23187 stock solution (100 μM A23187 in 5.2% DMSO) was added to the HBSS-sperm suspension to a final concentration of 1 μM A23187. One minute after A23187 addition, 25 μL of HBSS containing 100 mg/mL BSA was added to quench the A23187 action (final concentration of BSA 4 mg/mL) [30,31]. The suspension was centrifuged, the supernatant was removed, and the sperm pellet (20-30 μL) was combined with a modified TALP (TALP-R, see Supplemental Materials and Methods), previously equilibrated at 5% CO 2 in air, to achieve a final volume of 60 μL.…”