Chapter 9 Isolation of Fish Sperm Flagella

Abstract

Publisher Summary This chapter describes methods for purifying axonemes and the focus is on preparing intact flagella to analyze their membrane proteins and reactivate them to study axonemal motility mechanisms. Fish spermatozoa offer a good source from which to isolate and purify large amounts of vertebrate flagella and axonemes. Sperm from two teleost species, trout and carp, have been used extensively to study the mechanisms involved in the initiation of sperm motility. During the spawning season, an adult male trout produces a large number of sperm. In carp, sperm production can be induced throughout the year by repetitive injection of pituitary extracts. Trout and carp spermatozoa are quite simple and their flagella contain a classical 9+2 axoneme without periaxonemal structures. The absence of an acrosomal vesicle decreases the risk of contamination of the preparation with proteases. Trout sperm motility can be inhibited by media containing large amounts of potassium or having a low Ph. Carp sperm activation is controlled by a decrease in the external osmotic pressure and is not dependent on the external ions. It is advisable to include different protease inhibitors in extraction buffer (IMEN), as proteasomes are present on trout sperm plasma membrane. For trout, the water should be aerated with bubbling and frequently renewed. Chlorine must be avoided. For injection or sperm collection, fish must be anesthetized. For carp, spermiation can be induced throughout the year by intraabdominal injection of carp pituitary extract. Only a fraction of the sperm obtained from a single trout can be used directly for axoneme isolation. Therefore, for biochemical studies, some of the sperm may be frozen in liquid nitrogen for later use. The chapter discusses isolation of axonemes and flagella, and thereafter the demembranation and ATP reactivation of isolated carp sperm flagella.