Proteomic analysis of spermathecal fluid in Amphioctopus fangsiao: molecular insights into long-term sperm storage

Amphioctopus fangsiao is an economically important cephalopod species in northern China. A notable reproductive adaptation of this species is the prolonged storage of sperm in the female spermatheca for up to 8 months post-mating, until oocyte maturation and release. Spermathecal fluid, as a critical component of the microenvironment directly interacting with stored sperm, is essential for maintaining sperm long-term viability and fertilizing capacity. To explore the regulatory mechanism of the A. fangsiao spermathecal microenvironment during sperm storage, this study employed data-independent acquisition (DIA)-based quantitative proteomic to compare changes in protein expression between spermathecal fluid with stored sperm and without stored sperm. A total number of 3195 proteins were identified in the two groups, and 200 differentially expressed proteins were screened - 94 upregulated, 106 downregulated in spermathecal fluid with stored sperm. Functional enrichment analysis revealed that these DEPs were mainly involved in metabolic processes, antioxidant activity, and immune system processes, with significant enrichment in the glycolysis/gluconeogenesis and TCA cycle pathways. After sperm storage, glycolysis-related enzymes (PGK1, PGM1, ADPGK, ENO1) in the spermathecal fluid were significantly upregulated, while TCA cycle-related enzymes (SUCLG, IDH3) and dephosphorylases (ACP1) were significantly downregulated. This suggests that the energy supply mode in the spermatheca may shift from TCA cycle to glycolysis-dominated anaerobic metabolism to reduce the production of reactive oxygen species. Meanwhile, the dynamic expression of antioxidant proteins (TALDO1, PHGDH, UROD) may synergistically protect sperm from oxidative stress, and the downregulation of immune factors (IL17, PNP) may inhibit local immune responses to prevent stored sperm from being attacked. This study demonstrates that spermathecal fluid may ensure sperm viability through three aspects: altering energy metabolism modes, providing antioxidant defense, and suppressing immune responses. These results contribute to a deeper understanding of the sperm storage mechanism of A. fangsiao and provide a scientific basis for developing sperm storage technology in vitro.