The cAMP efflux, through multidrug resistance protein 4 (MRP4/ABCC4), is involved in the acquisition of fertilizing ability in mammalian spermatozoa

ALONSO CAI; LOTTERO R; MORRELL J; DAVIO C; OSYCKA-SALUT CE; LUQUE GM; DI SIERVI N; VERNAZ ZJ; JOHANNISSON A; BUFFONE MG; PEREZ-MARTINEZ S

Congreso; Reunión Conjunta de la Sociedad Argentina de Investigación Clínica (SAIC), la Sociedad Argentina de Inmunología (SAI) y la Sociedad Argentina de Fisiología (SAFIS); 2018

In order to fertilize an oocyte, mammalian spermatozoa must undergo a number of physiological modifications in the female reproductive tract, collectively known as capacitation. Activation of PKA is essential for capacitation, and therefore cAMP levels are tightly regulated during this process. Although cAMP levels are mainly determined by its synthesis and degradation, we previously demonstrated that cAMP extrusion through multidrug resistance protein 4 (MRP4) is also involved in this modulation in bovine sperm capacitation. Moreover, supplementation of incubation media with non-permeable-cAMP triggers capacitation, suggesting that cAMP efflux is necessary to regulate intracellular nucleotide levels and to provide the extracellular space with molecules that promote purinergic signalling.Our aim was to deepen the role of cAMP/MRP4 efflux system in mammalian sperm capacitation. For this, we propose 1) to elucidate molecular pathways involved in extracellular cAMP-induced in bovine sperm capacitation; 2) to evaluate the possible role of cAMP/MRP4 efflux in mouse sperm capacitation.Bovine spermatozoa were incubated in non-capacitating media with non-permeable-cAMP (ecAMP; 10nM) and enzymes inhibitors of purinergic signaling pathways, and changes associated with capacitation were assessed. Our results indicated that ecAMP exerts a robust response in bovine sperm capacitation activating PLC, PKC and ERK-1/2. Additionally, ecAMP elicited a fast rise in sperm Ca2+ levels that activated sAC and increased pPKA and pY levels, indicating that cAMP exerts a broad range of responses in this species.On the other hand, mouse spermatozoa were incubated in capacitating conditions with MK571 (MRP4 inhibitor) and changes associated with capacitation were assessed. MRP4 inhibition increased intracellular cAMP and pPKA levels. However, MK571 inhibited sperm motility, hyperactivation, induced-acrosomal reaction and in vitro fertilization.Our results suggest that cAMP/MRP4 system has a critical role in the regulation of cAMP-activated signaling pathways in both species and emerges as an important player in sperm physiology.