ISOLATION OF FLAGELLA AND THEIR MEMBRANES FROM SEA-URCHIN SPERMATOZOA

To facilitate investigation of the ionic mechanisms involved in sperm motility initiation, procedures are described in this chapter to isolate sea urchin sperm flagella and their membranes that are suitable for measurements of ion transport properties. The initiation of sea urchin sperm motility is a Na+-dependent process. On dilution of semen into seawater, motility is activated and is accompanied by acid extrusion. Both of these processes do not occur in the absence of external Na+. Artificial elevation of the internal pH of the sperm with nigericin and/or NH4Cl can overcome the requirement of external Na+. Conversely, lowering the pH of regular seawater reduces the internal pH of the sperm and inhibits motility, which can be reversed by monesin, a Na+/H+ exchange ionophore. These results are consistent with the trigger of motility being elevation of internal pH of the sperm mediated by a Na+/H+ exchanger. These preparations are single-compartment systems, thus allowing unambiguous analysis and interpretation of ion transport measurements. An added advantage of the flagellar membrane preparation is that the ionic conditions in both the internal and external media can be controlled, therefore allowing the determination of which ion(s) is required for the operation of the flagellar Na+/H+ exchanger. Under experimental procedures, the chapter discusses sperm collection, the media, and isolation of sperm flagella and flagellar membranes. The composition of this medium was designed with K+ being the only permeant ion. This simplifies the calculation of membrane potential across the flagellar vesicles and the interpretation of the ion transport measurements. Pyranine is an impermeant pH indicator used as a marker for the intravesicular space and a probe for measuring internal pH of the flagellar vesicles. Sea urchin sperm flagella contain no internal membranous organelles. A simple lysis procedure can be used to release the plasma membrane from the axonemes. © 1995, Academic Press Inc.