Pituitary Adenylate Cyclase Activating Polypeptide Stimulates Rat Leydig Cell Steroidogenesis Through a Novel Transduction Pathway

Abstract

The aim of the present study was to evaluate the effects of pituitary adenylate cyclase activating polypeptide (PACAP) on testosterone production in isolated adult rat Leydig cells and its possible mechanisms of action. PACAP-38 stimulated testosterone secretion in a dose-dependent manner with a minimal and a maximal efficacious dose of 1.0 nM and 100 nM, respectively. PACAP-27 was without effect on testosterone secretion at any dose tested. Similarly, vasoactive intestinal peptide did not stimulate steroidogenesis nor interfere with PACAP-38 activity, as well as preincubation of Leydig cells with the vasoactive intestinal peptide-antagonist [Lys(1), Pro(2,5), Arg(3,4), Tyr(6)]-vasoactive intestinal peptide. Removal of extracellular Ca2+ did not inhibit the stimulatory effects of PACAP-38 on Leydig cell testosterone production. Neither PACAP-38 nor PACAP-27 modified intracellular free Ca2+ and cAMP levels at any dose tested thus excluding a role for Ca2+ and cAMP in the stimulatory effects of PACAP. PACAP-38 was able to induce a plasma membrane depolarization that was dependent on an influx of Na+ from the extracellular medium as confirmed by the monitoring of intracellular Na+ with the Na+-sensitive fluorescent dye sodium benzofuran isophtalate. When Na+ was removed from the extracellular medium, PACAP-38 did not stimulate testosterone production, demonstrating that Na+ influx through the plasma membrane is strictly related to the stimulatory effects of this peptide. In addition, preincubation of Leydig cells in the presence of pertussis-toxin (500 ng/ml for 5 h) significantly reduced PACAP-38-stimulated effects both on plasma membrane depolarization and testosterone secretion. These results demonstrate that PACAP-38 stimulates testosterone secretion in isolated adult rat Leydig cells through the interaction with a novel PACAP receptor subtype coupled to a pertussis toxin sensitive G protein whose activation induces a Na+-dependent depolarization of the plasma membrane and testosterone production.

Delayed testicular aging in pituitary adenylate cyclase-activating peptide (PACAP) null mice

“…Our findings that PACAP is required for testosterone biosynthesis in vivo are consistent with previous in vitro studies in which PACAP was shown to stimulate testosterone secretion by embryonic and adult Leydig cells (20)(21)(22)29). Nevertheless, the low testosterone concentration in PACAP Ϫ/Ϫ mice does not affect the fertility of these animals that routinely breed in our facility.…”

“…A number of studies suggest that PACAP could exert a direct action on Leydig cells (20)(21)(22)29), supporting the argument for a peripheral dysfunction at the testis level. Therefore, we expected higher levels of LH in PACAP Ϫ/Ϫ mice compared with wild-type animals.…”

“…It has been suggested that steroidogenesis itself and, more specifically, the microsomal P450c17 protein (5, 6) participates in the age-induced testis degradation by generating ROS, which have been shown to negatively impact on steroidogenesis in vitro (28) and have been proposed as responsible for the age-related senescence of the Leydig cells in vivo (14). The neuropeptide PACAP has been implicated in regulation of testosterone level in cultured Leydig cells (20)(21)(22)29). It has also been shown to potentially enhance the release of LH in vivo (18).…”

“…PACAP and its mRNA, detected by immunohistochemistry and in situ hybridization in testes, is expressed in the germ cell lineage, including spermatogonia, primary spermatocytes, and round spermatids, but is not expressed in Sertoli or Leydig cells (19), suggesting that germ cell PACAP may act in a paracrine fashion on PAC1 receptor-expressing Leydig cells. Indeed, in cultured Leydig cells from both adult and fetal rat, PACAP induces stimulation of cAMP and testosterone secretion (20)(21)(22), showing that, in vitro, it exerts a direct effect on maturation and function of these cells. Furthermore, PACAP stimulates primordial germ cell proliferation in vitro (23) and induces the secretion of lactate and inhibin by Sertoli cells (24).…”

Novel Alternatively Spliced Exon in the Extracellular Ligand-binding Domain of the Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) Type 1 Receptor (PAC1R) Selectively Increases Ligand Affinity and Alters Signal Transduction Coupling during Spermatogenesis

“…High levels of PACAP are produced by the germ cells of the rat testis (2)(3)(4) and are translated from a testis-specific mRNA (5) transcribed from the PACAP gene using an alternative promoter active in round spermatids (6,7). PACAP regulates gene expression in germ cells (8), increases cAMP in Sertoli cells (9), and stimulates steroidogenesis in Leydig cells (10). Part of the diversity in PACAP actions is due to the existence of multiple receptors.…”