Secreted Epididymal Glycoprotein: MATERIALS AND METHODS(4)
The suspension was gently agitated for 5 min, and the released spermatozoa + fluid was decanted away from tissue fragments and centrifuged at 1000 X g for 5 min. The resulting supernatant was clarified by recentrifugation at 10 000 X g for 5 min and the sperm-free caput or cauda plasma stored frozen at —20°C. The first sperm pellet was then washed once more in eight volumes of PBS/Pefabloc, and proteins were extracted into five volumes of 1% (v/v) NP40 in PBS/Pefabloc at 4°C for 45 min. Detergent extracts of spermatozoa were clarified by centrifugation at 14 000 X g for 10 min and stored frozen at —20°C.
Alternatively, luminal fluid was collected by micropuncture techniques. Although laborious, this method is superior to slicing the tissue, as there is less contamination from blood and lymph. All manipulations were carried out at room temperature using a 10X dissecting microscope. A small area of the surface of the epididymal duct was exposed by careful dissection with fine forceps, washed with PBS, and gently blotted with tissue paper. Glass micropuncture pipettes (25-50-^m tip diameter) filled with PBS/1 mM Pefabloc were inserted into the duct lumen at several superficial sites and the contents drawn out by mild vacuum aspiration. Spermatozoa and their surrounding plasma were then separated and extracted with detergent as described previously. comments