The structure and function of the Cnidarian (Hydractinia Echinata) sperm centriolar complex



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Cnidarian sperm centrioles associate, in a sequential fashion, with numerous specialized structures (microtubular nucleating satellites, centriolar plaques, centriolar rootlets, and pericentriolar processes) during the various stages of spermiogenesis. Satellites are ovoid amorphous structures present early in spermiogenesis. They are responsible for the nucleation of microtubules that influence the morphologic events of sperm differentiation. Satellites are often attached to distal centrioles by a tapered stalk which is fixed to a centriolar plaque. Rootlets are tapered striated structures present for a short time midway through spermiogenesis. They appear responsible for the spatial arrangement of centriolar pairs during flagellar differentiation. Pericentriolar processes consist of nine primary processes radiating from the centriolar matrix between triplet microtubules. Three secondary processes radiate from each primary process and each has tertiary filamentous extensions. Similarities in size and structure between these filaments and microfilaments were noted. Though distinct from rootlets, the processes appear striated with a minor band periodicity similar to that of centriolar rootlets. The pericentriolar process complex forms a "cradle" around the midpiece of Hydractinia sperm, extending from the distal centriole anteriorly to the nuclear membrane. A technique for the isolation of Cnidarian sperm distal centrioles with intact accessory structures (pericentriolar processes) was developed to facilitate structural and biochemical analysis. Spermatids or sperm of uniform maturity were subjected to swelling in a hypotonic buffer (10 mM NaCl, 10 mM Mg SO[subscript 4], 10 mM Tris HCl, pH 7.2) and disruption by ultrasonication. Centriolar rich fractions were obtained by differential sedimentation over sucrose and isolates of centriolar complexes were prepared for whole mount electron microscopy by staining with 0.2% uranyl acetate to enhance image contrast. Protein extracts of these isolated centrioles were compared with whole sperm extracts on sodium dodecyl sulfate (SDS) gel electrophoresis. Major protein bands which co-migrate with bovine brain tubulin, rabbit skeletal muscle actin and myosin were observed. Densitometric quantitation of these SDS gels indicate that a major portion of the actin co-migrating protein present in whole sperm is located in the centriolar fraction. This fraction contains distal centrioles with attached pericentriolar processes and axonemal fragments. The specific identification and localization of actin in Hydractinia sperm was accomplished by indirect immunofluorescent microscopy. The fluorescent labeling was conducted with an antibody to shrimp tail muscle actin. The antigen for antibody production was purified and denatured by means of SDS gel electrophoresis. This highly purified antigen with increased antigenicity due to SDS denaturation produced high titers of monospecific antibody in rabbits. An ammonium sulfate precipitated gamma globulin fraction of the anti-actin sera has been demonstrated to be highly specific for native actin by immunodiffusion and immunoelectrophoresis. Indirect immunofluorescent labeling of Hydractinia sperm with mono-specific anti-actin demonstrated a fluorescent "cradle" identical with the midpiece position of the pericentriolar processes. The presence of actin and myosin co-migrating proteins in centriolar isolates containing pericentriolar processes and the specific labeling of these processes in the midpiece region of the sperm with anti-actin support the theory that the pericentriolar processes complex may be contractile in nature. Such a contractile unit located in the midpiece of the sperm firmly attached to the flagellum and distal centriole, could alter the midpiece symmetry (sperm head-flagellar angle). These alterations in sperm morphology may facilitate the directional selectivity these sperm demonstrate when presented with a chemo-attractant.