Background: Obesity is a multifactorial disorder driving systemic inflammation and male reproductive decline. However, the molecular links between metabolic dysfunction and compromised semen quality remain elusive. Circular RNAs (circRNAs) - covalently closed transcripts generated via backsplicing - are crucial regulators of spermatogenesis and gamete function. Although metabolic stress alters the sperm transcriptome, its impact on the circRNA cargo and trafficking during epididymal maturation remains unexplored. Here, we investigated obesity-induced disruptions in epididymal circRNA sorting via altered sphingosine-1-phosphate (S1P) signaling.
Methods: Male mice were fed either a high-fat diet (HFD) or a standard control diet (n=5 per group). Total sperm circRNA expression was profiled via microarray, with key candidates validated in both testes and epididymal extracellular vesicles (EVs). To evaluate S1P signaling, S1PR1–3 expression was assessed in spermatozoa, followed by functional assays in DC2 cells treated with S1P, with or without the S1PR1–3 antagonist VPC23019. Finally, RNA immunoprecipitation and EV shuttling assays were employed to elucidate circRNA biogenesis and trafficking mechanisms.
Results: Microarray identified 104 differentially expressed circRNAs in HFD spermatozoa (14 upregulated and 90 downregulated; p<0.01). Six downregulated candidates (circInpp5, circMed1, circTtbk2, circPdlim5, circPrkcb, and circEts1) were enriched in HFD caput EVs, indicating impaired vesicle-to-sperm transfer. This was mirrored by S1PR1–3 downregulation, suggesting defective S1P-dependent EV internalization. In DC2 cells, S1P stimulation exposed two regulatory pathways: circInpp5, circTtbk2, and circPdlim5 were induced by S1P and released into EVs, whereas circMed1, circPrkcb, and circEts1 were preferentially sorted into EVs without increased backsplicing. Mechanistically, S1P signaling promoted FUS–QKI interaction, while shuttling assays confirmed S1PR-dependent transfer of epididymal EV-circRNAs to spermatozoa.
Conclusion: These findings identify the S1P pathway as a previously unrecognized regulator of epididymal circRNA biogenesis and EV-mediated RNA transfer to spermatozoa. This disrupted signaling axis mechanistically links metabolic dysfunction to altered sperm circRNA cargo.