Characterization Of Epididymal Luminal Proteins And GPI-anchored Proteins On Spermatozoa

Objective:To characterize the epididymal luminal proteins involved in sperm maturation,storage and protection and GPI-anchored proteins on spermatozoa.Methods:1, Establishing Rat model by treating with acetazolamide, the variety of composition in epididymal luminal proteins was analyzed by electrophoretic technique; 2, Separation and identification of the proteins in epididymal luminal fluid from the caput and cauda segments of normal mature rat's epididymidis by two dimensional electrophoresis and mass spectrum, selected proteins were further studied for expression and distribution in Rat's epididymidis with RT-PCR, Western-blot and immunohisto-chemistry; 3, Purification of aerolysin and preparation of anti-aerolysin polyclone antibody. Aerolysin, a bacteriotoxin which can recognize GPI-anchored proteins (GPI-APs) specially, was introduced as a bioprobe to indentify the GPI-APs on spermatozoa by Sandwich Western-blot.Results:1, acetazolamide treated Rat model was successfully established. After treatment with acetazolamide, sperm motility was declined and abnormal spermatozoa increased significantly, however, the composition of epididymal luminal proteins was not changed; 2,12 proteins were identified from 22 differential protein spots separated from the epididymal luminal fluid of caput and cauda epididymidis.11 of them were reported in epididymidis of different species of mammals except the Peroxiredoxin6 (Prdx6). Prdx6 was a novel epididymal protein presented in the luminal fluid of caput and cauda epididymidis. Prdx6 was further studied with RT-PCR, Western-blot and immunohisto-chemistry. It expressed in the epididymal epithelial cell and on spermatozoa, and expression and the expression quantity in caput and cauda epididymis was different; 3, High-purity aerolysin and high specific anti-aerolysin polyclone antibody were prepared by our Lab. Sandwich western-blot showed that there are 12 protein bands recognized by aerolysin which were located among 15-130kD.Conclusion:1, the pathologic changes of epididymal sperm presented after treated with acetazolamide, however, the composition of epididymal luminal proteins was not changed. The pathologic changes of epididymal sperm was not relevant to the composition change of epididymal luminal proteins; 2, Prdx6 protein may play a role in sperm maturation, storage and protection in epididymidis. The concrete mechanism of Prdx6 is worth further investigating; 3, aerolysin could recognize GPI-APs on spermatozoa effectively and there are several GPI-APs on the surface of spermatozoa. Further identification of GPI-APs on spermatozoa is worth investigating.