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

Abstract

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 situations and studies from our laboratory strongly support this assumption. In the present work we sought to establish the role of cAMP efflux in the regulation of sperm capacitation. Sperm capacitation was performed in vitro by exposing bovine spermatozoa to bicarbonate 40 and 70 mM; cAMP; probenecid (a MRPs general inhibitor) and an adenosine type 1 receptor (A1 adenosine receptor) selective antagonist (DPCPX). Capacitation was assessed by chlortetracycline assay and lysophosphatidylcholine-induced acrosome reaction assessed by PSA-FITC staining. Intracellular and extracellular cAMP was measured by radiobinding the regulatory subunit of PKA under the same experimental conditions. MRP4 was detected by western blot and immunohistochemistry assays. Results showed that the inhibition of soluble adenylyl cyclase significantly inhibited bicarbonate-induced sperm capacitation. Furthermore, in the presence of 40 and 70 mM bicarbonate bovine spermatozoa synthesized and extruded cAMP. Interestingly, in the absence of IBMX (a PDEs inhibitor) cAMP efflux still operated in sperm cells, 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. The high-affinity efflux pump for cAMP, MRP4 was expressed in bovine spermatozoa and localized to the midpiece of the tail as previously reported for soluble adenylyl cyclase and A1 adenosine receptor. Additionally, blockade of A1 adenosine receptor abolished not 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.

Central role of soluble adenylyl cyclase and cAMP in sperm physiology

“…Interestingly, these initial high cAMP levels are rapidly reduced 43 for human sperm,44 for boar sperm . Decrease of cAMP levels could be attributed either to hydrolysis by phosphodiesterases (see below) or to cAMP extrusion by specific transporters 45 . As expected, the increase in cAMP immediately activates PKA activity 44 .…”

“…Interestingly, the expression of members of the multidrug resistance-associated proteins (MRPs) family has been reported in mammalian sperm 45 . MRPs, also members of the ATP binding cassette (ABC) transporter subfamily C, are known to actively transport cAMP and cGMP and other small non-structurally-related compounds to the extracellular environment One member of this family, MRP4 is localized to the sperm flagellum and to the post acrosomal region of bovine sperm 45 . In addition, this work showed that cAMP is extruded to the extracellular milieu and that MRP inhibition by probenecid abolished this efflux resulting in the accumulation of intracellular cAMP.…”

Functional role of membrane-bound adenylyl cyclases and coupled to them receptors and G-proteins in regulation of fertility of spermatozoa

“…Quite a few works deal with identification of adenosine receptors (AdenR) and elucidation of their role in control of fertility of spermatozoa [50][51][52][53][54]. It is shown by immunocytochemical methods that AdenR of the 1st (Aden 1 R) and 2nd types (Aden 2 R) that by the accordingly inhibiting and excitating way are coupled to AC, are present in the acrosomal area and in flagella, particularly in the main part of flagella enriched in microtubules.…”

“…This agrees with the key role of Aden 1 R in regulation of late capacitation, chemotaxis, and acrosomal reaction, which is necessary for penetration of spermatozoon into the oocyte. In this aspect, of interest seems the point of view expressed by the Argentinian researchers in 2013 about that activation of Aden 1 R at late stages of capacitation is necessary for a decrease of the cAMP level that prior to this was increased due to stimulation of tmAC by hormones and sAC by bicarbonateanions [54]. This decrease is achieved both due to inhibition of the tmAC activity via G i -proteins and owing to the mediated Aden 1 R activation of MRP4 (multidrug resistance associated protein 4), a highly affine transporter for cAMP that at the stage of capacitation colocalizes with Aden 1 R in the anterior part of the spermatozoan head and performs release of cAMP into extracellular space.…”

“…This decrease is achieved both due to inhibition of the tmAC activity via G i -proteins and owing to the mediated Aden 1 R activation of MRP4 (multidrug resistance associated protein 4), a highly affine transporter for cAMP that at the stage of capacitation colocalizes with Aden 1 R in the anterior part of the spermatozoan head and performs release of cAMP into extracellular space. It is shown that both blocking of Aden 1 R with aid of DPCPX, selective Aden 1 R-antagonist of selective Aden 1 R-antagonist, and suppression of activity of MRP4 with probenecide, nonselective inhibitor of cAMP-transporters, leads to the same result-cancellation of capacitation in response to treatment of spermatozoa by millimolar concentrations of NaHCO 3 - [54]. Another mechanism of action of Aden 1 R-agonists on capacitation can involve cAMP-independent cascades including activation of phorbol-sensitive isoforms of protein kinase C and of cascade of mitogen-activated protein kinases, as it is known that Aden 1 R are functionally coupled both to G i -proteins (Gα i2 ) and to G q/11 -proteins [55,58].…”