Localization and characterization of adenosine receptor expression in rat testis

Adenosine has long been suspected to play an important role in regulating male reproduction. To determine the sites of adenosine action in testis, the distribution of a family of recently cloned adenosine receptors was examined in rats. Northern blot analysis and in situ hybridization studies revealed high levels of testicular A3 adenosine receptor messenger RNA and lower levels of A1 adenosine receptor messenger RNA. Neither A2a nor A2b adenosine receptor gene expression could be detected. In situ hybridization and comparative polymerase chain reaction studies showed high level A3 receptor gene expression in germ cells (spermatocytes and spermatids), whereas high levels of A1 receptor gene expression was seen in Sertoli cells. To test for the presence of functional A1 and A3 receptors, the regulation of adenylyl cyclase by adenosine analogs was examined in seminiferous tubules and spermatozoa. Treatment of seminiferous tubules with forskolin resulted in a 5-fold increase in cAMP levels. The A1/A3 receptor agonist iodo-N6-aminobenzyladenosine inhibited forskolin-stimulated cAMP levels in the presence of the A1 8-cyclopentyl-1,3-dipropylxanthine antagonist 8-cyclopentyl-1,3-dipropylxanthine, showing that functional A3 receptors were present. The A1-selective agonist N6-cyclopentyladenosine also inhibited forskolin-stimulated cAMP levels, showing that functional A1 receptors were present. Treatment of spermatozoa with forskolin resulted in a 2-fold increase in cAMP levels. However, neither iodo-N6-aminobenzyladenosine nor N6-cyclopentyladenosine altered basal or forskolin-stimulated cAMP levels in sperm. These data show that functional inhibitory adenosine receptors are widely distributed in testis, with different patterns of expression.