Extracellular cAMP and MRP4 activity influence in vitro capacitation and fertilizing ability of pig spermatozoa

Abstract

cAMP has been reported to be an essential driver of sperm capacitation. In bovine sperm cAMP efflux through multidrug resistance-associated protein 4 (MRP4) has been suggested to maintain intracellular cAMP homeostasis and generate extracellular signaling able to regulate capacitation. The aim of this work was to determine whether extracellular cAMP may influence in vitro pig sperm capacitation and acquisition of fertilizing ability and to evaluate the role of MRP4. In vitro sperm capacitation and gamete coincubation were performed in Brackett and Oliphant's medium (BO) in presence of caffeine (Ctr+) or in BO without caffeine (Ctr-) supplemented with 0, 8, 9, 10 mM cAMP. Despite the percentage of capacitated sperm, assayed by immunolocalization of tyrosine-phosphorylated proteins, was significantly lower in Ctr- compared to Ctr+, it increased supplementing 10 mM cAMP to Ctr- reaching values similar to Ctr+. The absence of caffeine during gamete coincubation reduced the fertilization rate compared to Ctr+, while 10 mM cAMP supplementation to Ctr- increased the fertilization rate reaching values similar to Ctr + .The presence of MRP4 in pig spermatozoa was detected for the first time by western blot and immunohistochemistry assays. To evaluate MRP4 role on pig sperm capacitation, in vitro capacitation and gamete coincubation were performed in Ctr + in presence of MK571, a MRP4 selective inhibitor. MK571 reduced the percentage of capacitated cells and the fertilization rate, while cAMP addition fully reversed MRP4 blockade consequences.Present findings suggest that, under our in vitro conditions, extracellular cAMP and MRP4 activity influence pig sperm capacitating events.