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Proteome analysis of spermathecal fluid and seminal plasma reveals the mechanism of sperm storage in Amphioctopus fangsiao

ABSTRACT: The development of males and females of the cephalopod Amphioctopus fangsiao is asyn-chronous. The male produces sperm after maturity for storage in a spermatophore prior to mating. After mating, the sperm enters the female spermatheca for storage until ovulation occurs, a period that lasts for 8 months. This is a biologically uncommona rare phenomenon, because sperm cells generally fail to maintain their ability to fertilize for a long time after being ejaculated However, the molecular mechanisms about this phenomenon are still not clear. Sperm cells are stored in the male spermatophore and the female spermatheca, each of which provides a suitable environment. To determine the molecular basis of the sperm stor-age mechanisms in A. fangsiao, protein profiles from spermathecal fluid and seminal plasma separately were characterized using mass spectrometry-based proteomics. The antioxidant enzymes superoxide dismutase (SOD), glutathione S-transferase (GST), and Thioredoxin (Trx) and the glycolytic enzymes lactate dehydrogenase (LDH), hexokinase (HK), pyruvate dehydrogenase kinase (PDK), and ATP synthase were significantly enriched in the sperma-thecal fluid. Catalase (CAT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tri-osephosphate isomerase (TIM), and phosphoglycerate kinase (PGK) were significantly en-riched in the seminal plasma. The antimicrobial proteins transforming growth factor beta regulator 1 (TBRG1) and interleukin enhancer binding factor 2 (ILF2) and the extracellular matrix-related proteins transforming growth factor beta induced protein (TGFBIp) and thrombospondin type-1 domain-containing protein 4 (THSD4) were also significantly ex-pressed in the spermathecal fluid. These proteins may be crucial for successful long-term sperm storage. We measured the activities of four antioxidant enzymes based on the proteomic results, with the enzyme activity results showing consistency with the proteomic data. This study enhances our understanding of the sperm storage ability of A. fangsiao

ORGANISM(S): Amphioctopus Fangsiao

SUBMITTER: Yanwei Feng

PROVIDER: PXD071103 | iProX | Thu Nov 20 00:00:00 GMT 2025

REPOSITORIES: iProX

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Project description: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 3,195 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 (LMW-PTP) 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.

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Proteome analysis of spermathecal fluid and seminal plasma reveals the mechanism of sperm storage in Amphioctopus fangsiao

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