Involvement of MRP4 in the regulation of cAMP-dependent signaling pathway associated with sperm capacitation in humans

SOFÍA MARIA RIO; CAMILA ARROYO SALVO; BOGETTI MARIA EUGENIA; SILVINA PEREZ MARTINEZ

Conferencia; Fertilization and Activation of Development Gordon Research Conference; 2023

Involvement of MRP4 in the regulation of cAMP-dependent signaling pathway associated with sperm capacitation in humans.Río S, Arroyo-Salvo C, Alonso C A I, Bogetti M E, Morales M F, Arenas G, Dotto C, ReyValzacchi G, Marín-Briggiler C I ,Buffone M Yanef Af, Davio C, Pérez-Martínez S.Cyclic AMP (cAMP) has been informed to be essential for events associated with sperm capacitation, including regulation of motility and changes in motility pattern known as hyperactivation. There is also evidence that many of cAMP effects on sperm are mediated by activation of PKA and its downstream dependent signaling pathways. Previously, we demonstrated that during capacitation, both mouse and bovine spermatozoa extrude cAMP through the multidrug resistance-associated protein 4 (MRP4, also known as ABCC4), which regulates intracellular levels of the nucleotide and provides cAMP to the extracellular space. In addition, we characterized MRP4 in human sperm and determined the exclusion of cAMP to extracellular space. Here, we report the presence of a functional MRP4 in human spermatozoa, since its pharmacological inhibition with MK571 decreased levels of extracellular cAMP. We also evaluated the role of MRP4 in cAMP-dependent phosphorylation pathway during sperm capacitation. In this study, spermatozoa from 32 normozoospermic samples were used. First, we confirmed the presence of MRP4by western blotting (~150 kDa) and its cellular localization by immunofluorescence in the post-acrosomal region and midpiece (~80 % were positive for this pattern). Sperm capacitation correlates with an increase in the levels of tyrosine-phosphorylated proteins (pY) mediated by the cAMP/protein kinase A (PKA) pathway. Therefore, we incubated the sperm under non-capacitating (NC) and capacitating (CAP) conditions (HTF medium; 25 mM bicarbonate and 5 mg/ml BSA) for 0, 5, 30, 60, and 360 min, and evaluated the levels of pY and PKA-phosphorylated residues (pPKA). We observed a significant increase of pPKA levels in spermatozoa incubated in CAP medium compared to NC at 5, 30 and 360 min, and a marked increase of pY levels in spermatozoa incubated in CAP medium compared to NC at 360 min (p