Local Inhibition of Flagellar Activity That Regulate Sperm Flagellar Movement.

Abstract

It has been well known that any part of flagellum has the ability to produce bend. However, it is not clearly understood how flagella generate the successive bending wave spontaneously. Some micromanipulation experiments have suggested the requirement of the base of flagella. On the contrary, counterarguments have been made from simulation experiments. In order to elucidate this subject, we developed a new technique for introducing local inhibition at any place of the flagellum. Sperm were demembranated, incubated with PRODAN, a fluorescent dye, and reactivated. Then, UV spot irradiation was applied on the flagellum. The irradiated area of flagellar axoneme rapidly lost the activity to move and exhibited "rigor-like" property. When the local irradiation of UV, as small as 2.5 μm in diameter, was introduced at any place of the flagellum, the non-inhibited area of the flagellum maintained spontaneous beating. However, cAMP was necessary for beating at high ATP concentration when the proximal area of flagellum was inhibited. When the local inhibition was introduced at the mid region of flagellum the bending waves occurred in both the proximal and distal area of flagellum. The results suggested that any part of flagella could produce spontaneous beating. The beat frequency of beating flagella depended on the length of bending area of flagella. The shorter the length was, the higher the beat frequency became. In addition, we found that the spontaneous beating occurred neither with sperm head nor basal body. In that experiment, the proximal part of flagellum was inhibited by PRODAN-UV. Then, the head and basal part of flagellum was dissected out by glass micro needle remaining a short inhibited area. Thus, the microtubules were artificially tied together at the proximal edge of the axoneme. The flagellum exhibited spontaneous beating by itself. Thus, we conclude that flagella require the fixed end for generating spontaneous beating at which microtubules are tightly tied together not to disintegrate. We also observed that the response of flagellar beating to PRODAN-UV treatment exhibited time and spatial differences along flagellar axis. In addition, the time course of the propagating wave rapidly dissolved and got replaced by the standing wave like motion, suggesting that the regulatory mechanism for bend propagation might be more precisely regulated in the sophisticated nano-machine, sperm flagella.