Anandamide regulates the expression of GnRH1, GnRH2, and GnRH-Rs in frog testis

Gonadotropin-releasing hormone (either GnRH1 or GnRH2) exerts a local activity in vertebrate testis, including human testis. Relationships between endocannabinoid (eCB) and GnRH systems in gonads have never been elucidated in any species so far. To reveal a cross-talk between eCBs and GnRH at testicular level, we characterized the expression of GnRH (GnRH1 and GnRH2) as well as GnRH receptor (GnRH-R1, -R2, and -R3) mRNA in the testis of the anuran amphibian Rana esculenta during the annual sexual cycle; furthermore, the corresponding transcripts were localized inside the testis by in situ hybridization. The possible endogenous production of the eCB, anandamide (AEA), was investigated in testis by analyzing the expression of its biosynthetic enzyme, Nape-pld. Incubations of testis pieces with AEA were carried out in the postreproductive period (June) and in February, when a new spermatogenetic wave takes place. In June, AEA treatment significantly decreased GnRH1 and GnRH-R2 mRNA, stimulated the transcription of GnRH2 and GnRH-R1, and did not affect GnRH-R3 expression. In February, AEA treatment upregulated GnRH2 and GnRH-R3 mRNA, downregulated GnRH-R2, and did not affect GnRH1 and GnRH-R1 expression. These effects were mediated by type 1 cannabinoid receptor (CB1) since they were fully counteracted by SR141716A (Rimonabant), a selective CB1 antagonist. In conclusion, eCB system modulates GnRH activity in frog testis during the annual sexual cycle in a stage-dependent fashion.