Linggui formula improves asthenozoospermia by modulating seminal plasma exosomes Wnt signaling via the LRP6/GSK3/Septin4 pathway

Ethnopharmacological relevance: Kidney-tonifying herbal formulas have a long history of use in China and Other Asian countries for enhancing sperm motility. However, the therapeutic mechanisms involving seminal plasma exosomes (sEVs) remain unexplored.

Aim of the study: This study employs the kidney-tonifying Chinese herbal medicine Linggui formula (LG) as a representative to investigate its mechanism in treating asthenozoospermia (AS) through the modulation of sEVs.

Materials and methods: In vitro, sEVs were isolated from AS patients, AS patients post-LG treatment, and healthy subjects, followed by analysis of differential Wnt protein expression. Spermatozoa from AS patients were cultured with these sEVs to assess motility and uptake, and sperm p-LRP6, p-GSK3, and Septin4 expression after sEVs treatment was evaluated. In vivo, the Wnt signaling effects of sEVs from different rat epididymal segments, testis, and prostate were compared to identify the therapeutic target of LG for AS.

Results: sEVs were successfully isolated and characterized, revealing that sperm retain the capacity to receive signals in vitro, allowing sEVs-mediated influence on their activity. LG upregulated sEVs-mediated Wnt signaling, activating the phosphorylation of sperm LRP6, reducing GSK3 activity, and promoting Septin4 polymerization, thereby enhancing sperm motility. In vivo studies demonstrated the presence of multiple Wnt ligands with distinct expression patterns across different locations within the reproductive system.

Conclusion: LG may enhance sperm motility by modulating Wnt proteins in the epididymis, testis, and prostate, thereby upregulating sEVs-mediated Wnt signaling via the LRP6/GSK3/Septin4 pathway.

LRP6/GSK3/Septin4 pathway; Linggui formula; Male infertility; Seminal plasma exosomes; Wnt; asthenozoospermia.