From localization to function: comparative analysis of CB1 in sperm across species and its epigenetic role in humans

The endocannabinoid system (ECS) is evolutionarily conserved and regulates key physiological processes, including sperm motility and capacitation. However, the localization and function of cannabinoid receptor 1 (CB1) in human sperm remain debated, with prior widefield microscopy studies producing inconsistent results. Using confocal and Airyscan microscopy, we mapped CB1 distribution in human sperm, revealing a dotted pattern along the tail, presence in some midpieces, and discrete spots in the head. Additionally, a comparative study revealed that CB1 was present in the sperm tail of invertebrates and vertebrates, while it was only detected in the sperm head of roosters (restricted to the acrosomal region) and mammals. Notably in mammalian sperm, a subset of CB1 receptors was detected intracellularly, beneath the plasma and outer acrosomal membranes, extending toward the nuclear region, where it persisted even after the acrosome reaction. These data support additional role beyond sperm motility and capacitation-induced acrosome reaction. Given that CB1 is involved in chromatin remodeling in murine sperm, we investigated whether it plays a similar role in human sperm. Our findings demonstrate that CB1 activation by the specific agonist Arachidonyl-2'-chloroethylamide (ACEA) enhances histone H4 acetylation, restoring levels in asthenoteratozoospermic samples to those of normozoospermic donors. Interestingly, while N-arachidonoylethanolamine (AEA) treatment reduced sperm DNA fragmentation, ACEA had no such effect, evidencing that DNA fragmentation is not CB1-mediated. As established in mammals, the histone-to-protamine transition is a critical phase of chromatin remodeling and our study highlights a conserved role for CB1 in regulating chromatin dynamics during this process.