Endocannabinoids and stemness: a new role for CB1 receptor in the control of Spermatogonial Stem Cell homeostasis

Abstract Background Spermatogonia (SPGs) are a heterogeneous population of germ cells including undifferentiated (und-) SPGs - specifically spermatogonial stem cells (SSCs) and early progenitors-and differentiated (diff-) SPGs. The SSC niche ensures the life-long production of spermatozoa. Critical SSC niche components are the neurotrophic factor GDNF and the Fibroblast Growth Factor 2 (FGF2), both involved in SSC homeostasis. The cannabinoid receptor CB1 modulates stem cell biology in several tissues but its implications in testis have not yet been investigated. Here, using wild type (WT) and CB1 knock out (CB1 -/- ) mice we characterize the involvement of CB1 activity in SSC homeostasis. Methods Adult WT and CB1 -/- testes were analyzed by western blot and RT-qPCR to assess changes in the expression of ECS components and SPG markers. Morphological and immunohistochemical analyses, including PAS staging and whole-mount tubule imaging, were performed to assess germ cell progression and SSC subset. Adult CB1 +/- testes and prepubertal organotypic cultures were ex vivo treated with selective CB1/CB2 agonists/antagonists or 2-Arachidonoylglycerol (2-AG) hydrolase MAGL inhibitor to evaluate SSC responsiveness and GDNF/FGF2 modulation. Results We demonstrate that Cb1 -deletion negatively affected GDNF/FGF2 production and the expression of pluripotency-associated genes ( Oct4 ; Nanog ; Nanos2 ; Etv5 ), with downstream effects on SSC pool expressing the GDNF family receptor alpha-1 (GFRα1). Concurrently, the expression of commitment-associated genes ( Eomes ; Lin28a ; Sohlh1 ) and markers of early-progenitors commitment (SOX3; NGN3; RARƔ), significantly increased in CB1 -/- testis, unveiling a more proneness of CB1 -/- SSCs to undergo the commitment phase. An inverse relationship between GDNF and MAGL protein levels occurred in the CB1 -/- testis, suggesting that CB1 regulated the 2A-G as its own ligand and, in turn, testicular GDNF levels. Ex vivo pharmacological stimulation of testes with a selective CB1 agonist/antagonist or a MAGL inhibitor confirmed that 2-AG is the endogenous CB1 ligand that modulates GDNF as well as FGF2 levels. Ex vivo testes organotypic culture treated with a CB1 agonist or antagonist also confirmed the direct involvement of CB1 in the self-renewal activity of SSCs. Conclusions Our findings reveal a novel role for CB1 in the molecular pathways that regulate SSC homeostasis and highlight the involvement of the CB1/MAGL molecular axis in regulating GDNF and FGF2 expression.