Daisuke Miyashiro

Glycolysis plays an important role in energy transfer from the base to the distal end of the flagellum in mouse sperm

Many studies have been conducted to elucidate the relationship between energy metabolic pathways (glycolysis and respiration) and flagellar motility in mammalian sperm, but the contribution of glycolysis to sperm motility has not yet been fully elucidated. In the present study, we performed detailed analysis of mouse sperm flagellar motility for further understanding of the contribution of glycolysis to mammalian sperm motility. Mouse sperm maintained vigorous motility in the presence of substrates either for glycolysis or for respiration. By contrast, inhibition of glycolysis by alpha-chlorohydrine caused a significant decrease in the bend angle of the flagellar bending wave, sliding velocity of outer doublet microtubules and ATP content even in the presence of respiratory substrates (pyruvate or β-hydroxybutyrate). The decrease of flagellar bend angle and sliding velocity are prominent in the distal part of the flagellum, indicating that glycolysis inhibition caused the decrease in ATP concentration threrein. These results suggest that glycolysis potentially acts as a spatial ATP buffering system, transferring energy (ATP) synthesized by respiration at the mitochondria located in the basal part of the flagellum to the distal part. In order to validate that glycolytic enzymes can transfer high energy phosphoryls, we calculated intraflagellar concentration profiles of adenine nucleotides along the flagellum by computer simulation analysis. The result demonstrated the involvement of glycolysis for maintaining the ATP concentration at the tip of the flagellum. It is likely that glycolysis plays a key role in energy homeostasis in mouse sperm not only through ATP production but also through energy transfer.

Quick Shear-Flow Alignment of Biological Filaments for X-ray Fiber Diffraction Facilitated by Methylcellulose

X-ray fiber diffraction is one of the most useful methods for examining the structural details of live biological filaments under physiological conditions. To investigate biologically active or labile materials, it is crucial to finish fiber alignment within seconds before diffraction analysis. However, the conventional methods, e.g., magnetic field alignment and low-speed centrifugations, are time-consuming and not very useful for such purposes. Here, we introduce a new alignment method using a rheometer with two parallel disks, which was applied to observe fiber diffractions of axonemes, tobacco mosaic tobamovirus, and microtubules. We found that fibers were aligned within 5 s by giving high shear flow (1000-5000 s(-1)) to the medium and that methylcellulose contained in the medium (approximately 1%) was essential to the accomplishment of uniform orientation with a small angular deviation (<5 degrees). The new alignment method enabled us to execute structure analyses of axonemes by small-angle x-ray diffraction. Since this method was also useful for the quick alignment of purified microtubules, as well as tobacco mosaic tobamovirus, we expect that we can apply it to the structural analysis of many other biological filaments.

X-ray diffraction recording from single axonemes of eukaryotic flagella.

We report the first X-ray diffraction patterns recorded from single axonemes of eukaryotic flagella with a diameter of only <0.2 μm, by using the technique of cryomicrodiffraction. A spermatozoon isolated from the testis of a fruit fly, Drosophila melanogaster, either intact or demembranated, was mounted straight in a glass capillary, quickly frozen and its 800-μm segment was irradiated end-on with intense synchrotron radiation X-ray microbeams (diameter, ~2 μm) at 74 K. Well-defined diffraction patterns were recorded, consisting of a large number of isolated reflection spots, extending up to 1/5 nm(-1). These reflections showed a tendency to peak every 20°, i.e., the patterns had features of an 18-fold rotational symmetry as expected from the 9-fold rotational symmetry of axonemal structure. This means that the axonemes remain untwisted, even after the manual mounting procedure. The diffraction patterns were compared with the results of model calculations based on a published electron micrograph of the Drosophila axoneme. The comparison provided information about the native state of axoneme, including estimates of axonemal diameter, interdoublet spacing, and masses of axonemal components relative to those of microtubules (e.g., radial spokes, dynein arms, and proteins associated with accessory singlet microtubules). When combined with the genetic resource of Drosophila, the technique presented here will serve as a powerful tool for studying the structure-function relationship of eukaryotic flagella in general.

Chemotactic response with a constant delay-time mechanism in Ciona spermatozoa revealed by a high time resolution analysis of flagellar motility

ABSTRACT During their chemotactic swimming toward eggs, sperm cells detect their species-specific chemoattractant and sense concentration gradients by unknown mechanisms. After sensing the attractant, sperm cells commonly demonstrate a series of responses involving different swimming patterns by changing flagellar beats, gradually approaching a swimming path toward the eggs, which is the source of chemoattractants. Shiba et al. observed a rapid increase in intracellular Ca2+ concentrations in Ciona spermatozoa after sensing chemoattractants; however, the biochemical processes occurring inside the sperm cells are unclear. In the present study, we focused on the timing and sensing mechanism of chemical signal detection in Ciona. One of the most crucial problems to be solved is defining the initial epoch of chemotactic responses. We adopted a high rate of video recording (600 Hz) for detailed analysis of sperm motion and a novel method for detecting subtle signs of beat forms and moving paths of sperm heads. From these analyses, we estimated a virtual sensing point of the attractant before initiation of motility responses and found that the time delay from sensing to motility responses was almost constant. To evaluate the efficiency of this constant delay model, we performed computer simulation of chemotactic behaviors of Ciona spermatozoa.