Research The Mechanism Of Semenogelin SGI-52-derived Peptides Inhibit Sperm Movement

Objective:we found human Semenogelin I derived peptides SGI-52have the activity of inhibiting sperm motility in the early studies, on this base,we want to further study whether SGI-52peptide combine sperm, change sperm mitochondrial membrane potential, change sperm membrane potential and change sperm membrane structural or not. To study whether SGI-52peptide can change the concentration of intracellular calcium in spermatogenic cells, and discuss the possible mechanism of Semenogelin SGI-52-derived peptides inhibit sperm movement.Methods:First, we use computer-assisted semen analysis system (CASA) to selecte normal semen specimens in September2012to December2013in line with WHO standard and use discontinuous Percoll density gradient centrifugation to collect viable sperm and divide them into four groups:normal sperm experimental group^normal sperm control group、demembranation sperm in the experimental group、 demembranation sperm in the control group. The control group were added Deionized water, the experimental group were added a final concentration of5mg/ml of SGI-52peptide. Second, The sperm binding site detection:sperm resuspended and rinsed with HTF and adjusted the concentration to1×107/ml, Each experimental group added SGI-52-FITC and each control group added BSA-FITC, all group were incubated at37℃,then,removed the right amount of sperm from the four groups and participate in PBS solution mixed with4%formaldehyde and dyes Hoechst for1min、15min、30min,All samples were rinsed two times with PBS and used70%glycerol resuspend samples into the slide. Last we used OLYMPUS FV-1000laser scanning confocal microscope system and collected fluorescence microscopy images. Third, The sperm membrane structure detection:All experimental group were added SGI-52peptide and incubated37℃for15min,then we joined the final concentration of50μg/ml of the mixing dye SYBR Green I and7-AAD and dark incubate10min, PBS rinsed two times to remove unbound dye, last we detected sperm membrane structure by flow cytometry after adjusting the sperm density of1X106/ml,. Fourth, the sperm membrane potential detection:All sperm suspended in1ml HSS solution containing400nM of DiBAC4(3) and adjusted the sperm density3×106/ml, all experimental group joined SGI-52peptide,37℃after incubation with flow cytometry sperm membrane potential. Fifth, The sperm mitochondrial membrane potential detection: Take6×105/ml sperm resuspended in HTF and added SGI-52peptides for incubating the samples, last we performed by flow cytometry sperm mitochondrial membrane potential using fluorescent dye JC-1monochrome labeling.Six, Seminiferous cells intracellular calcium concentration detection:Get the Testicular specimens from castration surgery under sterile conditions and use mechanical separation methods and discontinuous Percoll gradient centrifugation to collect seminiferous cells, adjust the cells concentration for2×103/ml and plate100μl cell suspension in96well plates overnight at37℃cell culture incubator, after incubation we add30μl a concentration of2mM fluorescent dye Fura-2AM to each well, dark stain1h for incubator at37℃, remove dye and wash with OR2-Ca2+without calcium, then50μl solution containing2mM calcium OR2+Ca2+are added to each well,then we choice well and join final concentration of1mg/ml,3mg/ml,5mg/ml SGI-52peptide dissolved by OR2-Ca2+, then detect the change of concentration of calcium in spermatogenic cells by calcium ions stimulate within the system fluorescence microscopy.Results:1, Confocal show that SGI-52peptide can combine both normal sperm and demembranation sperm of membrane surface binding sites. However, there is a difference degree of the combination. When1min, the fluorescence intensity of normal sperm after SGI-52peptide incubated is dim, and the mainly binding sites are in the sperm tail. Regarding the demembranation sperm we find that SGI-52peptide has been apparent binded the membrane surface of sperm head and the tail. When10min,we find the fluorescence of normal sperm tail is strengthen. Comparison, The SGI-52peptide has penetrated into the acrosome of demembranation sperm. When15min, The SGI-52peptide have been binded the membrane surface of normal sperm head. While the SGI-52peptide has entered into the inside of sperm at the same time.2, SGI-52peptide can increase permeability of the sperm membrane structure. Permeability ratio of normal sperm after a SGI-52peptide treatment are smaller than demembranation sperm after treatment.3, SGI-52peptide can decrease sperm membrane potential, i.e., membrane potential hyperpolarization. Overall of the sperm membrane potential treated by0.1%Triton are higher than normal sperm.4, SGI-52peptide can reduce sperm mitochondrial membrane potential, indicating that SGI-52peptide can inhibt sperm motility. Overall viability of sperm after0.1%Triton deal are lower than normal sperm.5, SGI-52peptide can cause seminiferous cells of intracellular calcium concentration increased.Conclusion:Human Semenogelin derived synthetic peptides SGI-52peptide can binds sperm and lower sperm vitality to inhibit sperm motility by influence and change mitochondrial membrane potential、membrane potential and membrane structure of sperm. it also may play a important role in capacitation.