Objective: Human semen is the jelly-like substance mainly containing semenogelin 1(Sgl)and fibronectin(Fn)with the characteristics of coagulation and liquefaction in a short time.In our previous study,we have identified that Eppin could interact with Sg.Eppin75-133 C-terminal fragment bind the Sg164-283 fragment containing the only cysteine in human Sg I(Cys-239).Besides that,during semen liquefaction,physiologically prostate specific antigen(PSA)hydrolyzes some region of Sgl which inhibits sperm motility.Therefore,complex interaction among Eppin,Sg and PSA plays a major role in regulating semen liquefaction process.The aim of this study is to investigate the molecular pathways during semen liquefaction.Methods: Molecular cloning was used to recombination in vitro 6-His-Eppin protein with N-and C-terminal fragments.Protein in seminal vesicle fluid was transferred to immobilon-P polyvinylidene difuoride membrane by western blot analysis,followed by incubation with 6-His-Eppin protein,eppin283-423 C-terminal fragment and eppin73-288 N-terminal fragment at 4 ℃ overnight respectively in order to find the protein which can be bind to 6-HisEppin protein.2-D electrophoresis was used to identification of Eppin binding partners.After that,anti-His was used to visualise using enhanced chemiluminescence and mass spectrometry(MS)to identify the sequence of protein.Results: We found that the protein specifically binding to Eppin through Far-western immunoblot analysis demonstrated only the N-terminal of recombinant epididymal protease inhibitor(N-rEppin)and rEppin can binds to reduce seminal plasma protein,while MS identified that Fn can specifically bind to Eppin.Our study was the first evidence that some protein existed in seminal vesicle fluid does bind to Eppin,regulating the semen coagulation and liquefaction.Conclusions: Two molecular pathways occurred in semen liquefaction.Eppin C-and N-terminal fragment interacted with Sg and Fn.Eppin N-terminal has a binding site of fibronectin,which is an important protein for semen coagulation.Eppin regulated the process of semen coagulation and liquefaction through its N-terminal and C-terminal bound to fibronectin and Sg respectively,influencing sperm capacitation.Whats more,the C-and N-terminal fragment of Eppin self-formed double ring type molecular structure respectively and closely bound to the core structure named β-sheet by four disulfide,which make the combination of Wap and Kunitze type inhibitor external surface just in the opposite two terminals of the transection of molecular structure.This structure contributes to interact with other proteins in increasing its roles.During human ejaculation,spermatozoa pass through the ampulla of the vas deferens and then move into the proximal extension of the seminal vesicle and finally enter into the ejaculatory duct.At this juncture spermatozoa are first mixed with copious secretion from the seminal vesicles.Thereafter the spermatozoa and seminal fluid is mixed with prostatic secretions when they enter into the prostatic urethra.It can be imagined that after spermatozoa enter into the ejaculatory ducts their surface Eppin would be saturated by binding with Sg and Fn.This process inhibits human sperm capacitation,making the initial ejaculated spermatozoa be in an immotile state.Purified plasma Fn,added at various concentrations to a preparation of live spermatozoa,was found to inhibit sperm motility in a dose dependent manner.During semen liquefaction,physiologically PSA hydrolyzes Sg and Fn to increase sperm motility.Therefore,seminal liquefaction is a process increasing the capacitation of sperm progressive motility and fertilization.Fibronectin could affect the process of sperm coagulation and liquefaction through specificity combined with Eppin,involving in sperm capacitation and fertilization.Our findings revealed novel molecular pathways of semen liquefaction.