Adenosine analogues with specificity for A2 receptors bind to mouse spermatozoa and stimulate adenylate cyclase activity in uncapacitated suspensions

Abstract

Adenosine and its analogues, known to stimulate adenylate cyclase activity in somatic cells via A2 receptors, can accelerate capacitation in mouse spermatozoa and thereby enhance fertilizing ability in vitro. Indirect evidence has suggested that adenosine can modulate mouse sperm adenylate cyclase, implicating this enzyme and cAMP in the observed functional responses. In the present study we provide evidence that [3H]5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analogue with specificity for stimulatory A2 adenosine receptors, can bind to mouse spermatozoa. This binding can be displaced by both unlabelled NECA and 2-chloroadenosine, another A2 receptor agonist, but not by cyclopentyladenosine, 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 binding, indicating an external site for interaction with sperm cells. Saturable specific binding of [3H]NECA to mouse spermatozoa incubated at 37 degrees C was observed, with a Bmax of 5.17 pmol mg-1 protein and a Kd value of 930 nmol l-1. Binding data were consistent with the presence of a single major class of receptor. In addition to demonstrable binding of [3H]NECA, both NECA and 2-chloroadenosine significantly stimulated adenylate cyclase activity in a concentration-dependent manner, with NECA being effective at a lower concentration. Furthermore, the hydrolysis-resistant GTP analogue Gpp(NH)p, alone and in the presence of either NECA or 2-chloroadenosine, also significantly stimulated enzyme activity. In somatic cells, expression of responses to adenosine usually requires GTP and G proteins.(ABSTRACT TRUNCATED AT 250 WORDS)