Defective PDZK1 palmitoylation impairs epididymosome-mediated cholesterol transport and contributes to asthenozoospermia

Asthenozoospermia, a leading cause of male infertility characterized by impaired sperm motility, involves dysregulated cholesterol efflux during epididymal maturation, yet the underlying mechanisms remain elusive. Here, we demonstrate that PDZK1 protein level and its palmitoylation are significantly reduced in asthenozoospermic sperm. Using clinical samples, a 2-bromopalmitate-treated mouse model, and in vitro approaches, we found that palmitoylation inhibition recapitulates the asthenozoospermic phenotype, specifically depleting PDZK1 from sperm and epididymosomes. Moreover, defective PDZK1 palmitoylation led to alterations in cholesterol accumulation, leading to sperm membrane cholesterol accumulation. Mechanistically, we identified cysteine 10 (C10) as a critical palmitoylation site mediated by ZDHHC5, essential for PDZK1 membrane localization, epididymosomal incorporation, and SR-BI interaction. Importantly, palmitic acid supplementation within an optimal dose range or administration of palmitoylated PDZK1-enriched epididymosomes improved sperm motility, further implicating PDZK1 palmitoylation in the regulation of sperm function. Our study reveals a novel pathway wherein ZDHHC5-mediated PDZK1 palmitoylation at C10 ensures its epididymosomal delivery to maintain cholesterol homeostasis and sperm motility, highlighting PDZK1 palmitoylation as a potential mechanistic target for future therapeutic development in asthenozoospermia.