Immunocytochemical electron microscopic study and Western blot analysis of troponin in striated muscle of the fruit fly Drosophila melanogaster and in several muscle cell types of the earthworm Eisenia foetida.

BACKGROUND: There is little information about troponin in invertebrate muscles, and no previous references to this protein in annelid muscles have been found. The aim of this paper was to study the presence and distribution of troponin in different muscle cell types from the earthworm Eisenia foetida (the muscular body wall, and the inner and outer muscular layer of the pseudoheart). These results were compared with those obtained in the transversely striated muscle of Drosophila melanogaster and in skeletal and smooth muscles of the mouse. METHODS: Immunocytochemical electron microscopic study and Western blot analysis using anti-TnT antibodies were employed in this study. RESULTS: Troponin immunoreaction was detected in the mouse skeletal muscle, the fly flight muscle, and earthworm obliquely striated muscles (body wall musculature and inner muscular layer of the pseudoheart). Immunolabeling for TnT in all these muscle cells appeared in moderate amounts at any point along the sarcomere length, except for the central zone of the A band (H band). This suggests that troponin molecules were located along the thin filaments. The density of immunogold particles was similar in the three muscles, and thus the amount of troponin in each muscle type was proportional to the number and length of actin filaments in each. Troponin was found in neither the mouse smooth muscle nor the outer muscular layer of the earthworm pseudoheart. The latter muscle showed an ultrastructural pattern that was intermediate between obliquely striated and smooth muscle. The estimated molecular weight for TnT in the earthworm was 55 kDa; this is higher than the weight of this protein in the mouse skeletal muscle (40 kDa) but similar to that of the D. melanogaster muscle (52 kDa). CONCLUSIONS: Troponin is present in both types of striated muscle (transversely striated and obliquely striated) of the earthworm with a distribution that is very similar to that observed in the mammalian striated muscle. As in vertebrates, troponin is absent in the smooth muscle of the earthworm. Discrepancies in the classification of some invertebrate muscles are common in the literature, and the use of distinctive markers, such as troponin, may improve our understanding of the nature and properties of many invertebrate muscles showing an ultrastructural pattern that does not resemble any of the classic muscle types.

Cytoskeleton in Sertoli cells of the mosquito fish (Gambusia affinis holbrooki).

BACKGROUND: There is little information about the distribution of cytoskeletal components in the testes of teleost fish. The aim of this paper was to know the distribution of some major cytoskeletal proteins (tubulin, actin, vimentin, desmin, and cytokeratins) in the Sertoli cells of Gambusia affinis holbrooki and in their efferent duct epithelial cells which are possibly originated from the Sertoli cells. METHODS: Light and electron microscopic immunocytochemical studies and Western blotting analysis were performed in G. affinis testis. RESULTS: Actin immunoreaction was observed in the Sertoli cells at all spermatogenic stages, although the intensity of this reaction varied from one stage to another. Sertoli cells that support spermatogonia or spermatocytes showed a weak immunoreaction which was uniformly distributed throughout the cytoplasm and somewhat more concentrated at the level of the inter-Sertoli specialized junctions. Immunoreaction to actin increased during the first stages of spermiogenesis and was mainly localized beneath the plasma membrane. This immunoreaction was more intense in the basal than in the apical cytoplasm of Sertoli cells. In a more advanced stage of spermiogenesis, actin immunoreaction become stronger in the apical cytoplasm where Sertoli cells displayed cytoplasmic projections around each spermatid. After sperm release, the apical Sertoli cell cytoplasm still showed an intense actin immunoreaction. Intense immunoreaction to actin was also observed in the epithelial cells lining the efferent ducts. Immunoreaction to tubulin was diffuse throughout the Sertoli cell cytoplasm. No immunoreaction to vimentin or desmin was observed in the Sertoli cells during the spermatogenic process. Immunoreaction to both vimentin and desmin was observed in the efferent duct cells. Desmin immunoreaction was also observed in the seminiferous tubule boundary cells, mainly in the sections showing germ cell cysts at the last stages of spermiogenesis and in the peritubular cells that surrounded the efferent duct epithelium. Immunoreaction to cytokeratins was found in the endothelium of testicular blood vessels but not in the Sertoli cells or in the efferent duct epithelium. CONCLUSIONS: Immunoreaction pattern to cytoskeletal proteins in the Sertoli cells of G. affinis differs from that reported in mammalian Sertoli cells. These differences include the distribution of actin filaments and the absence of detectable vimentin immunoreaction in G. affinis Sertoli cells.