c-Myc expression in human anagen hair follicles.

The hair follicle represents a very attractive organ system for studying the precise balance between cell proliferation, growth, differentiation, and death of cells, because it periodically and regularly regenerates, retaining its morphogenetic signals throughout its life. One of the most intriguing oncogenes which is able to induce both cell growth and apoptosis, depending upon the environmental conditions, is c-myc. The aim of the present study was to investigate its presence and localization in human hair follicles by immunohistochemistry and immunofluorescence. Our observations demonstrated the consistent presence of two clusters of c-Myc-expressing cells in anagen follicles, located in two annular regions of the inner root sheath, at the border between cells characterized by putative trichohyalin granules and cells which are keratinized. The lower group belongs to Henle's layer, while the upper group belongs to Huxley's layer. c-Myc oncoprotein seems to favour apoptosis/differentiation and may be a marker for terminal differentiation of trichocytes, at least in the inner root sheath. Our findings agree with the interpretation that the complex morphology of the hair follicle reflects its complex function; the extrusion of a highly organized multicellular structure, the hair shaft, driven by another highly organized multicellular structure, the inner root sheath.

Structural and biochemical analysis of the parasite Gordius villoti (Nematomorpha, Gordiacea) cuticle.

The cuticle of the nematomorpha Gordius villoti is a proteinaceous extracellular structure that covers the body during the endoparasitic life in the hemocoelic cavity of insect hosts, and of the free-living adult animals. The ultrastructure of the cuticle has a complex spatial organization with several parallel layers of large diameter fibers, interposed thinner fibrous elements and honeycomb-shaped matrix surrounding the fibers. When adult isolated cuticles were partially solubilized by several compounds, the structure revealed a strong insolubility and the main fibers were always observable. HPLC and spectrophotometric assays carried out to investigate the presence of tyrosine cross-linking, indicated such a mechanism as a key-element in the hardening process of the cuticle. Such data strongly suggest that the Gordius cuticle contains dityrosine compounds, whose formation is probably mediated by endogenous peroxidase activity.

Actin polymerization in boar spermatozoa: fertilization is reduced with use of cytochalasin D.

The aggregational state of actin in boar spermatozoa after capacitation and the acrosome reaction has been examined by several methods. In vitro fertilization (IVF) experiments were conducted in the presence and absence of cytochalasin D (CD) to evaluate the role of actin polymerization in the events of fertilization. The fertilizing capacity was very high in controls, but, when CD (an inhibitor of the polymerization of actin) was added to the capacitation medium, there was a marked decrease in the fertilizing capacity of the boar spermatozoa. There was a further decrease when CD was present during both capacitation and fertilization processes. In addition to the IVF tests, biochemical and immunoelectron microscopic methods were used to analyze the state of aggregation of actin in boar spermatozoa after capacitation, and the acrosome reaction. By immunoelectron microscopy with a phalloidin probe, there were no gold particles, indicating the presence of F-actin on boar sperm heads capacitated and acrosome-reacted in media containing CD. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis there were differences in NP-40 solubility, reflecting actin polymerization, between CD-treated and untreated sperm. These results suggest that actin polymerizes during capacitation and the acrosome reaction and that this polymerization is essential to the fertilization process.

F-actin in acrosome-reacted boar spermatozoa.

Biochemical and immunoelectron microscopic methods have been used to analyze the distribution of actin in boar spermatozoa and its state of aggregation before and after acrosome reaction. F-actin was detected on sperm head and tail by electron microscopy using an improved phalloidin probe: incubation with a fluorescein-phalloidin complex and an anti-fluorescein antibody, followed by labeling with protein A-gold complex. Gold particles, indicating the presence of F-actin, were localized on the sperm surface of the acrosome-reacted spermatozoa. Specific labeling was localized (1) between the outer acrosomal membrane and the plasma membrane in the equatorial region, (2) between the outer surface of the fibrous sheath and the plasma membrane in the postacrosomal region, (3) around the connecting piece and the neck region, and (4) on the external surface of the fibrous sheath in the principal piece of the tail. Furthermore, after NP-40 extraction, the SDS-PAGE revealed a difference in solubility between reacted and unreacted boar spermatozoa, reflecting actin polymerization. We conclude that most actin in the acrosome reacted boar spermatozoa is polymeric.

Electron microscopic localization of F-actin in acrosome reacted boar spermatozoa by means of a phalloidin-FITC complex.

An immunocytochemical study at the ultrastructural level has been performed in boar spermatozoa in order to clarify the aggregation state of actin before and after the acrosome reaction. A new phalloidin probe has been used to detect F-actin: a phalloidin derivative conjugated with FITC, followed by incubation with an anti-FITC antibody. The protein A-gold technique was then applied for the localization of the antigenic sites. Gold particles were localized on the sperm surface only after the acrosome reaction which was induced by the ionophore A23187.

Identification and characterization of the major components of the Oncorhynchus mykiss egg chorion.

The extracellular coat surrounding the fish egg, commonly called the chorion, is a primary envelope that confers biochemical and morphological identity typical of the species. Purified chorions can be easily isolated from either oocytes or ovulated eggs. The aim of this work was to analyze the macromolecular composition of the various chorion components in Oncorhynchus mykiss (Salmonids). SDS-PAGE analysis of purified chorion showed a reproducible pattern of four major components (129, 62, 54, and 47 kD), representing about 80% of total chorion proteins. The 129 and 47 kD polypeptides were periodic-acid Schiff (PAS) and concanavalin A positive. After chemical and enzymatic deglycosylation treatments only the 129 and 47 kD components proved to be glycosylated and to belong to the "asparagine-linked" glycoprotein family. Furthermore, peptide mapping performed on isolated polypeptides showed comigrating fragments on SDS-PAGE. These results suggest that the four main chorion polypeptides might share common structural features.