Pituitary adenylate cyclase activating polypeptide-mediated intracrine signaling in the testicular germ cells

Pituitary adenylate cyclase activating polypeptide (PACAP) is found not only in the brain, but is also abundantly expressed in the testicular germ cells. However, the physiological role of testicular PACAP remains unknown. Autoradiographic studies showed a considerable number of PACAP-specific binding sites in the seminiferous tubules. Immunohistochemistry demonstrated PAC(1)-receptor (R)-like immunoreactivity (h) in the cytoplasm of round spermatids, aggregated in the acrosome and coexpressed with PACAP-li. Spermatid-enriched fractions were examined for the subcellular localization of PACAP binding sites and PAC(1)-R-li. The highest levels of PACAP binding sites and PAC(1)-R-li were found in the cytosolic, followed by the nuclear, and the lowest levels in the membrane fraction. The testicular cytosolic PAC(1)-R-like protein showed a specific competitive inhibition in the radioreceptor assay for PACAP38 and 27, with a K-i of 0.069 nM and 0.179 nM, respectively. The addition of PACAP to the cytosol of spermatids only slightly activated adenylate cyclase, while it markedly stimulated the expression and activation of ERK-type mitogen-activated protein kinase (MAPK). In the PAC(1)-R-like protein-depleted cytosol, a PAC(1)-R-specific agonist, maxadilan, did not activate MAPK, but PACAP and VIP still did. Because VPAC(2)-R, which binds both PACAP and VIP, is expressed in the testis, the findings suggest that cytosolic VPAC(2)-R-like proteins are also present and coupled to MAPK. The MAPK activation does not seem to require a heterotrimeric G-protein. Because PACAP and its receptors are coexpressed in the cytoplasm of spermatids, endogenous PACAP may directly interact with the cytosolic PAC(1)-R-like protein without the ligand being released into the extracellular space. This possibility is supported by the observation that cytosolic endogenous PACAP in spermatids was co-immunoprecipitated with the cytosolic PAC(1)-R. This mechanism may be called intracrine, and its physiological significance is discussed.