MULTIDRUG RESISTANCE PROTEIN 4 (MRP4/ABCC4) IS PRESENT IN MAMMALIAN SPERM AND IS INVOLVED IN THE ACQUISITION OF FERTILIZING ABILITY

PEREZ MARTINEZ, SILVINA; DI SIERVI, NICOLÁS; PUGA MOLINA, LIS DEL CARMEN; BUFFONE, MARIANO; CASTELLANO, LUCIANA; ALONSO, CARLOS AGUSTÍN ISIDRO; DAVIO, CARLOS; LOTTERO LECONTE, RAQUEL; OSYCKA SALUT, CLAUDIA ELENA

Congreso; Gordon Research Conference; 2017

Sperm capacitation has been largely associated with an increase in cAMP, although its relevance in the underlying mechanisms of this maturation process remains elusive. Increasing evidence shows that the extrusion of cAMP through multidrug resistance associated protein 4 (MRP4) regulates cell homeostasis not only in physiological but also in pathophysiological processes. Previously we studied the role of cAMP efflux in the regulation of bicarbonate-induced sperm capacitation in bovines. Our results showed that the inhibition of soluble adenylyl cyclase (sAC) significantly inhibited bicarbonate-induced sperm capacitation. Furthermore, in the presence of 40 mM bicarbonate and in the absence of IBMX (a PDEs inhibitor) bovine spermatozoa synthesized and extruded cAMP suggesting that cAMP extrusion would be a physiological process in the spermatozoa complementary to the action of PDE. Blockade of MRPs by probenecid abolished the efflux of the cyclic nucleotide resulting not only in the accumulation of intracellular cAMP but also in the inhibition of bicarbonate-induced sperm capacitation. The effect of probenecid was abolished by exposing sperm cells to cAMP. Additionally, blockade of A1 adenosine receptor abolished not only bicarbonate-induced sperm capacitation but also that stimulated by cAMP. Those findings strongly support that cAMP efflux, through MRP4, and the activation of A1 adenosine receptor regulate some events associated with bicarbonate-induced sperm capacitation in bovines. In the present work we aim to deepen into the action pathway of cAMP and its metabolites (5´AMP; ADO). For that, we performed in vitro capacitation in the presence of the cAMP or its metabolites and PLC or PKA inhibitors. The sperm capacitation was evaluated by CTC and LPC-induced acrosome reaction assays. Our results showed that 10 nM of cAMP or 5?AMP induce sperm capacitation, but it was necessary 10 uM ADO to accomplish the same effect. Furthermore, when spermatozoa were incubated with non-capacitating concentrations (NCC) of cAMP plus ADO, they underwent capacitation. The effect of nucleotides was reversed by A1 antagonists.It is known that PKA activation is an event required to trigger an ulterior tyrosine phosphorylation (pTyr) increment during sperm capacitation. We evaluated the kinetic of the PKA activation during bicarbonate-induced capacitation, assessed by Ser-Thr phosphorylation. Results showed an early activation of PKA at 1? with a further decrease at basal levels and finally a second peak of activation between 45? and 90?. This second peak of activation was abolished when sperm was co-incubated with MK571 (MRP4 specific inhibitor). Parallelly, pTyr increased with time, but the augment was stronger in the presence of bicarbonate at 90?. This last augment was also abolished in presence of MK571, indicating that both PKA substrates and Tyr lately phosphorylation is dependent of cAMP extrusion. To support these results we incubated sperm with exogen cAMP, (as a manner to resemble the extruded nucleotide), and evaluated the behaviour of PKA. Results showed that cAMP increased PKA substrate phosphorylation, and the use of specific PKA-inhibitors not only decreased the Ser/Thr phosphorylation as expected, but also inhibited capacitation, suggesting that this kinase could be activated only in the presence of cAMP. We also investigated if the cAMP-activation of PKA was dependent of sAC activity. Results showed that the activation of PKA relies on sAC activity, suggesting whether there?s an intracellular basal tone of cAMP that should stay constant, or the external cAMP has a positive feedback with the intracellular cAMP mediated directly or indirectly by sAC. Finally, we evaluated the kinetic of PKA activation when sperm is incubated with cAMP. Results indicated that there?s an early activation of PKA with a peak between 5? and 15?, and then its activity decays but keeps a phosphorylation level above the unstimulated control. This increase was also inhibited when spermatozoa was pre incubated with KH7, supporting the fact that sAC activity is essential for Ser/thr phosphorylation happen.In conclusion, the previously published data and these new results showed that cAMP extrusion by MRP4 is a critical event associated to capacitation, and that purinergic signalling regulates sperm physiology throughout the activation of signalling pathways that involves PKA activation and pTyr. In the present work we deepen into the metabolism of the extracellular nucleotides, showing that purinergic signalling might not be as linear as it was thought and that A1r is the receptor that triggers capacitation response in sperm. Bicarbonate and cAMP-induced capacitation works through A1r, and both are linked to PKA, directly or indirectly by sAC. Finally, Bicarbonate and cAMP-induced capacitation showed to be different pattern in PKA activation which is expectable since cAMP-induced capacitation resembles the event that allows the late phosphorylation augment of bicarbonate-induced capacitacion to occur. There?s still much to know about the signalling pathways activated by the cAMP, but all this information supports the idea of the cAMP as a novel and important autocrine/paracrine signal involved in capacitation.