Calcium influx regulates sperm – IPVL interaction in Chickens (Gallus gallus domesticus)

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[Introduction] Once ejaculated from the male reproductive tract, avian sperm undergo acrosome reaction (AR) by binding to inner peri-vitelline layer (IPVL), which enables them to release proteolytic enzymes generating holes for their penetration into an egg. Although numerous in-vitro studies demonstrated that extracellular Ca²⁺ is essential for AR induction beyond species, functional role of Ca²⁺ in AR is poorly understood in birds. This motivated us to examine the role of Ca²⁺ in IPVL-mediated changes in chicken sperm. [Materials & Methods] Ejaculated sperm were obtained from Rhode Island Red. IPVL was prepared from freshly laid eggs. Sperm were induced with AR by co-incubating with 0.1 µg/µl IPVL under presence or absence of chelators, and subjected to immunodetection of PKA substrate phosphorylation (p-PKA), [Ca²⁺]_i measurement and motility analysis with SMAS. Sperm penetrability was assessed, based on number of holes formed on co-incubated IPVL sheet. [Results] Although p-PKA did not differ, [Ca²⁺]_i level and motility parameters, such as VAP and ALH, changed in response to IPVL treatment. However, the changes in motility were abrogated when EGTA-AM or EDTA were present. Further, sperm penetrability was inhibited by these chelators with a prominent reduction by EDTA. [Conclusion] This study demonstrates that IPVL treatment triggers calcium influx together with changes in motility, resulting in elevated penetration ability in chicken sperm. Furthermore, these results reinforce a primary role of extracellular Ca²⁺ in penetrability, together suggesting calcium-regulated pathway supporting sperm-IPVL interaction.