Cyclic AMP efflux, via MRPs and A1 adenosine receptors, is critical for bovine sperm capacitation

OSYCKA-SALUT CLAUDIA; DIEZ FEDERICO; BURDET, JULIANA; GERVASI, MARIA GRACIA; FRANCHI ANA MARIA; BIANCIOTTI LILIANA G; DAVIO CARLOS; PEREZ MARTINEZ SILVINA

MOLECULAR HUMAN REPRODUCTION.

OXFORD UNIV PRESS

Lugar: Oxford; Año: 2014 p. 120 - 130

1360-9947

abstract: Sperm capacitation has been largely associated with an increase in cAMP, although its relevance in the underlying mechanisms ofthis 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 situations and studies from our laboratory stronglysupport this assumption. In the present work we sought to establish the role of cAMP efflux in the regulation of sperm capacitation. Sperm capacitationwasperformed in vitro by exposing bovine spermatozoa to bicarbonate 40 and 70 mM;cAMP; probenecid (a MRPs general inhibitor) andan adenosine type 1 receptor (A1 adenosine receptor) selective antagonist (DPCPX). Capacitation was assessed by chlortetracycline assay andlysophosphatidylcholine-induced acrosome reaction assessed by PSA-FITC staining. Intracellular and extracellularcAMP was measured by radiobindingthe regulatory subunit of PKA under the same experimental conditions. MRP4 was detected by western blot and immunohistochemistryassays. Results showedthat the inhibition of soluble adenylyl cyclase significantly inhibited bicarbonate-induced sperm capacitation. Furthermore,in the presence of 40 and 70 mMbicarbonate bovine spermatozoa synthesized and extrudedcAMP. Interestingly, in the absence of IBMX (a PDEsinhibitor) cAMP efflux still operated in sperm cells, suggesting that cAMP extrusion would be a physiological process in the spermatozoa complementaryto the action of PDE. Blockade of MRPs by probenecid abolished the efflux of the cyclic nucleotide resulting not only in the accumulationof intracellular cAMP but also in the inhibition of bicarbonate-induced sperm capacitation. The effect of probenecid was abolished byexposing sperm cells to cAMP. The high-affinity efflux pump forcAMP, MRP4 was expressed in bovine spermatozoa and localized to the midpieceof the tail as previously reported for soluble adenylyl cyclase and A1 adenosine receptor. Additionally, blockade of A1 adenosine receptor abolishednot only bicarbonate-induced sperm capacitation but also that stimulated by cAMP. Present findings strongly support that cAMP efflux,presumably through MRP4, and the activation of A1 adenosine receptor regulate some events associated with bicarbonate-induced sperm capacitation,and further suggest a paracrine and/or autocrine role for cAMP.