ADENOSINE-ANALOGS WITH SPECIFICITY FOR A2 RECEPTORS BIND TO MOUSE SPERMATOZOA AND STIMULATE ADENYLATE-CYCLASE ACTIVITY IN UNCAPACITATED SUSPENSIONS

Adenosine and its analogues, known to stimulate adenylate cyclase acti vity in somatic cells via A2 receptors, can accelerate capacitation in mouse spermatozoa and thereby enhance fertilizing ability in vitro. I ndirect evidence has suggested that adenosine can modulate mouse sperm adenylate cyclase, implicating this enzyme and cAMP in the observed f unctional responses. In the present study we provide evidence that [H- 3]5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analogue with sp ecificity for stimulatory A2 adenosine receptors, can bind to mouse sp ermatozoa. This binding can be displaced by both unlabelled NECA and 2 -chloroadenosine, another A2 receptor agonist, but not by cyclopentyla denosine, an inhibitory A1 receptor agonist, suggesting that the NECA binding is specific for A2 receptors. The presence of S-(p-nitrobenzyl )-6-thioinosine, an adenosine transport inhibitor, did not affect bind ing, indicating an external site for interaction with sperm cells. Sat urable specific binding of [H-3]NECA to mouse spermatozoa incubated at 37-degrees-C was observed, with a B(max) of 5.17 pmol mg-1 protein an d a K(d) value of 930 nmol l-1. Binding data were consistent with the presence of a single major class of receptor. In addition to demonstra ble binding of [H-3]NECA, both NECA and 2-chloroadenosine significantl y stimulated adenylate cyclase activity in a concentration-dependent m anner, with NECA being effective at a lower concentration. Furthermore , the hydrolysis-resistant GTP analogue Gpp(NH)p, alone and in the pre sence of either NECA or 2-chloroadenosine, also significantly stimulat ed enzyme activity. In somatic cells, expression of responses to adeno sine usually requires GTP and G proteins. These results are consistent , therefore, with the hypothesis that adenosine-induced enhancement of sperm functional ability is due to stimulation of adenylate cyclase, via A2 adenosine receptors, and hence increasing availability of intra cellular cAMP. They also suggest that G proteins play a role in the re ceptor-mediated response.