The epididymis regulates sperm maturation through multidimensional mechanisms [1]. During the 10–15 days transit process, key changes such as sperm plasma membrane reorganization, nuclear disulfide bond-mediated DNA compaction, and activation of motility are all dependent on the dynamic microenvironment within the epididymal lumen [2]. The epididymal epithelium not only concentrates sperm by absorbing fluid, but also secretes antioxidant enzymes to neutralize reactive oxygen species, and maintains the quiescent state of sperm tails by regulating pH and secreting inhibitory factors [3]. Recent studies have revealed that epididymosomes, extracellular vesicles secreted by the epididymal epithelium, may serve as the core carriers for delivering maturation factors. For instance, proteins like cysteine-rich secretory protein 1 (CRISP1) and brain serine protease (BSP) family proteins [[4], [5], [6]], along with non-coding RNAs carried by epididymosomes [7], have been shown to transfer to the sperm surface via membrane fusion, directly regulating their motility and fertilization potential. This intercellular communication mechanism explains why immature sperm still leads to increased embryonic lethality even when fertilized through intracytoplasmic sperm injection (ICSI) due to the lack of key molecules accumulated during epididymal transit [8]. The “cargo delivery” model mediated by epididymosomes is emerging as a regulatory network of sperm maturation.

Epididymosomes, as specialized extracellular vesicles, play a pivotal role in orchestrating sperm maturation by delivering essential macromolecules to spermatozoa during their epididymal transit [9,10]. These vesicles are enriched with proteins, lipids, and non-coding RNAs (sncRNAs), which are selectively sorted and transferred to sperm, facilitating critical processes such as membrane remodeling, motility acquisition, and fertilization competence [11]. Proteomic analyses across species have identified epididymosome-derived proteins critical for sperm function, such as ZP-binding protein [12], redox regulator [13], and ADAM family proteases [12], which are spatially regulated along the epididymal segments and androgen-dependent. Equally significant is the sncRNA payload, particularly miRNA [14] and tsRNA [15] species, which exhibit segment-specific enrichment patterns. These sncRNAs not only modulate sperm maturation but also contribute to intergenerational epigenetic inheritance, as evidenced by rescued embryonic developmental defects upon microinjection of cauda-derived sncRNAs into immature sperm-generated embryos [8]. Despite these advances, significant knowledge gaps remain regarding the spatial heterogeneity of epididymal cargoes and the regulatory mechanisms underlying selective cargo sorting, such as S-acylation-mediated protein trafficking in exosome cargo regulation [16].

Emerging evidence highlights the critical role of protein S-acylation, a dynamic post-translational lipid modification [17]. S-acylation, catalyzed by zinc finger DHHC-type palmitoyltransferases (ZDHHCs), modulates protein localization, stability, and function by facilitating membrane association and vesicular trafficking [18]. Our study demonstrates that S-acylation of C4b-binding protein alpha-chain (C4BPA) at Cys15 in the murine epididymis is essential for its enrichment in epididymosomes, which protects sperm from complement C4-mediated attacks and sustains motility [19]. In the context of male reproduction, S-acylation has been implicated in maintaining sperm function and fertility. For instance, ZDHHC19, a testis-enriched palmitoyltransferase, is essential for sperm morphology and motility, as its deficiency leads to male infertility characterized by abnormal sperm structure and impaired motility [20,21]. Furthermore, S-acylation of proteins such as beta-galactosidase-like protein (GLB1L4) in the epididymis has been shown to facilitate their transfer to sperm via epididymosomes, underscoring the importance of this modification in sperm functional maturation [22]. Dysregulation of S-acylation, as observed in palmitic acid-induced models, disrupts the blood-testis barrier and impairs spermatogenesis [23]. Our in vitro study shows that Sertoli cell-derived exosomes loaded with S-acylated guanine nucleotide binding protein, alpha 13 (GNA13) suppress autophagy in spermatogonia [24]. Despite these insights, the spatial dynamics of S-acylation within the epididymis and its role in epididymosome cargo sorting remain poorly understood. Our study employs S-acylation-proteomics to decode the S-acylation-mediated pathways in porcine epididymis and epididymosomes, aiming to elucidate how this modification orchestrates exosome-mediated sperm maturation.