Calmodulin immunoelectron microscopy: redistribution during ram spermatogenesis and epididymal maturation. II.

Affinity-purified monospecific antibodies and indirect immunogold and immunoferritin labeling on ultra-thin sections of low-temperature Lowicryl K4M-embedded samples were used to study the redistribution of calmodulin in ram spermatids and epididymal spermatozoa at the electron microscopic level. Calmodulin appeared as an integral component of well-defined structures or organelles of these cells. In young spermatids, calmodulin was localized in the nucleus, cytoplasm, and developing acrosome. During spermatogenesis and epididymal maturation, calmodulin left the acrosome to reach the perinuclear substance and finally became concentrated in the post-acrosomal area of the head, although some calmodulin remained associated with the tip of the acrosome. Such a redistribution is consistent with the preferential location of Ca2+ in the post-acrosomal cytoplasm of ejaculated spermatozoa. Calmodulin was also observed in the flagellum associated with the plasma membrane and with the motility apparatus, between coarse fibers and axonemal microtubules. These changes in calmodulin distribution may account for the Ca2+-dependent regulation of spermatogenesis and sperm maturation. Calmodulin therefore appears to be a pleiotropic regulator of male gamete development and functions.

Immunoelectron microscopic localization of calmodulin and phospholipase A2 in spermatozoa. I.

Using the Lowicryl K4M embedding technique, together with indirect immunoferritin or immunogold labeling on ultra-thin sections, tubulin, calmodulin and phospholipase A2 were distinctly localized in ejaculated bull spermatozoa. Calmodulin was concentrated on the plasma membrane, nucleus, post-acrosomal substance, and, in lesser amounts, between coarse fibers and axonemal microtubules of the flagellum. Phospholipase A2 was distributed evenly along the plasma membrane, nucleus, acrosome, post-acrosomal substance, and in the flagellum, on mitochondria, fibrous sheath, coarse fibers, between coarse fibers and axonemal microtubules. Antibodies to tubulin labeled only axonemal microtubules, including the central pair of microtubules. Patterns of tubulin labeling were identical when ferritin granule- or gold particle-conjugated antibodies were tested. In agreement with our previous biochemical studies demonstrating calmodulin binding to phospholipase A2, concomitant with enhancement of phospholipase A2 activity (Arch Biochem Biophys 241:413, 1985), the overlapping distribution of calmodulin and phospholipase A2 in several parts of the sperm suggests that these proteins may play a concerted role in male gamete function in preparation for or during fertilization. The distinct distribution of tubulin along flagellum microtubules indicates their special function in sperm mobility.

Platelet cytoskeleton alpha-actinin in normal and thrombasthenic platelets: distribution and immunologic characterization.

The subcellular localization of alpha-actinin (Mr 100,000) in human skeletal muscle is restricted to the Z line, in which it is believed to anchor actin filaments. Recently, this protein was identified in normal and thrombasthenic human platelets by its antigenic cross-reaction with antibodies to chicken gizzard alpha-actinin. In our study, the biochemical interaction between purified platelet alpha-actinin and striated muscle F-actin was examined by electron microscopy of negatively stained preparations. Like its muscle counterpart, platelet alpha-actinin promotes the cross-linking and bundling of actin filaments. Antibodies prepared to human platelet alpha-actinin cross-reacted with chicken gizzard alpha-actinin as shown by immunoelectrophoresis and the western blotting technique. Immunoblots prepared with normal and thrombasthenic platelets with antibodies to human platelet alpha-actinin revealed that this protein is susceptible to proteolysis. Extracts of freshly drawn platelets showed a protein band of 100 K. When the platelet extracts were incubated at 37 degrees C for various times, the immunoblots showed protein bands of 100 and 80 K. The proportion of the 80 K protein band increased with incubation time. This proteolysis can be prevented by chelating agents such as EDTA or the protease inhibitor leupeptin. Indirect immunofluorescent studies of human skin fibroblasts with antibodies to chicken gizzard actin and human skeletal muscle, chicken gizzard, and platelet alpha-actinin revealed the staining pattern characteristic of each protein. The distribution of alpha-actinin in normal and thrombasthenic platelets was assessed by ferritin-labeled immunoelectron microscopy. Ferritin particles were found in the cytoplasm immediately below the membrane and in some granules. There was no labeling associated with the mitochondria.