8 mM CaCl2 for a similar period. Moreover, the addition of 5 mM EDTA to fresh, motile human sperm induced a similar release of surface attached SAP within minutes. However, less than 10% of the gametes total SAP con tent was released by EDTA treatment, indi cating that the majority of SAP molecules are associated click here with the human sperm in a calcium independent manner. Immunofluorescence microscopy confirmed the presence of SAP on the surface of intact, viable human sperm. SAP was localized to the membrane overlying the neck, midpiece and tail regions of fresh, motile sperm. The patches of SAP staining were confined to the proximal section of the principal piece in the majority of cases. IF staining of permeabi lized fixed sperm revealed intracellular SAP antigen in the neck region of some human sperm.

Antiserum against SAP induced mixed agglutination of swim up harvested human sperm in the standard slide agglutination assay, consistent with the broad distribution of the antigen observed by IF. The mixed agglutination pattern obtained with highly motile cells implies that Inhibitors,Modulators,Libraries SAP is tightly bound to the plasma membrane overlying both the neck and tail regions of human sperm. Discussion Calcium binding Inhibitors,Modulators,Libraries proteins detected by 45Ca binding assay Five of the nine human sperm 45 Ca binding proteins identified in this study contain at least one calcium binding helix loop helix structure. This sug gests that the Inhibitors,Modulators,Libraries EF hand is either resistant to the denatur ing effect of SDS to which the proteins are exposed to during the second dimension electrophoresis or that the domain is readily refolded during the subsequent mild electrotransfer, washing and blocking procedures.

Both tubulin and HSP70 chaperones, which lack an EF hand domain, have previously been shown to bind calcium, confirming the specificity of the 45Ca overlay procedure Inhibitors,Modulators,Libraries employed in the present study. Five of the calcium binding proteins were found to be accessible for radioiodination on the surface of ejacu lated human sperm HYOU1, HSPA5, HSPA2, SAP, and 80K H. The three heat shock protein 70 family mem bers HYOU1, HSPA5 and HSPA2 have previously been demonstrated on the surface of both the male and the female gamete. Indeed, HSP70 antigens have been localized over the entire human sperm sur face by immunofluorescence analysis. Serum amyloid P component in association with spermatozoa SAP has been localized to the human sperm surface by vectorial labelling, immunohistochemistry, and flow cytometry analysis. The presence of SAP in the sperm free seminal fluid from a vasectomized Inhibitors,Modulators,Libraries man sug gested that SAP associates with the sperm membrane after the epididymal contents mix with secretions selleck chemicals llc from the accessory glands.