No abstract

Fertilization is a matter of life or death. In animals of sexual reproduction, the appropriate communication between mature and competent male and female gametes determines the generation of a new individual. Ion channels are key elements in the dialogue between sperm, its environment, and the egg. Components from the outer layer of the egg induce ion permeability changes in sperm that regulate sperm motility, chemotaxis, and the acrosome reaction. Sperm are tiny differentiated terminal cells unable to synthesize protein and difficult to study electrophysiologically. Thus understanding how sperm ion channels participate in fertilization requires combining planar bilayer techniques, in vivo measurements of membrane potential, intracellular Ca2+ and intracellular pH using fluorescent probes, patch-clamp recordings, and molecular cloning and heterologous expression. Spermatogenic cells are larger than sperm and synthesize the ion channels that will end up in mature sperm. Correlating the presence and cellular distribution of various ion channels with their functional status at different stages of spermatogenesis is contributing to understand their participation in differentiation and in sperm physiology. The multi-faceted approach being used to unravel sperm ion channel function and regulation is yielding valuable information about the finely orchestrated events that lead to sperm activation, induction of the acrosome reaction, and in the end to the miracle of life.

Two vesicle pools, readily releasable (RRP) and reserve (RP) pools, are present at Drosophila neuromuscular junctions. Using a temperature-sensitive mutant, shibire(ts), we studied pool sizes and vesicle mobilization rates. In shibire(ts), due to lack of endocytosis at nonpermissive temperatures, synaptic currents continuously declined during tetanic stimulation until they ceased as the result of vesicle depletion. By then, approximately 84,000 quanta were released. Vesicles were mobilized from RP at a rate 1/7-1/10 of RRP. Cytochalasin D inhibited mobilization of vesicles from RP, allowing us to estimate the size of RRP as 14%-19% of all vesicles. Vesicle recycling supports synaptic transmission during prolonged tetanic stimulation and the maximum recycling rate was 1000 vesicles/s.