The peculiar extra-acrosomal structure of the Collembola (Hexapoda) spermatozoa.

The springtail Collembola are characterized by having rolled spermatozoa, with a long cylindrical extracellular structure adhering to the acrosome. This structure is produced by the secretory activity of the testes epithelial cells at almost the end of spermiogenesis. At the beginning of its formation, it is a thin extension with a helical wall and a dense axial region. Later the cylindrical structure shows an inner organization which is different in the several species examined: species of Entomobryidae contain material with a paracrystalline structure, whilst some of Symphypleona contain ovoid structures. The outer envelope of the extracellular structure consists of two overlapped layers orthogonally arranged, clearly identified by cryo-preparations. Immunoblot analysis and lectin stainings have indicated that the cylindrical structure has a glycoproteic composition. As the structure is no longer visible after the sperm transfer into the female spermatheca, it is suggested that it could contain enzymes able to activate the sperm unwinding process and possibly allowing the reacquisition of sperm motility.

The evolution of sperm axoneme structure and the dynein heavy chain complement in cecidomid insects.

The 9 + 2 axoneme of cilia and flagella is specialized machinery aimed at the production of efficient, finely tuned motility, and it has been evolutionarily conserved from protists to mammals. However, the sperm cells of several insects express unconventional axonemes, which represent unique models for studying the structural-functional relationships underlying axonemal function and evolution. Cecidomids comprise a group of dipterans characterized by an overall tendency to deviate from the standard axonemal pattern. In particular, the subfamily Cecidomyiinae shows a series of progressive modifications of the sperm axoneme. We previously analyzed the unusual sperm axonemes of Asphondylia ruebsaameni (Asphondyliidi) and Monarthropalpus buxi (Cecidomyiidi), which are characterized by the absence of any structure related to the control of motility (that is, the central pair complex, radial spokes and inner dynein arms); however, these sperm are motile, and motility is driven by the outer dynein arms only. This simplification of the motility machinery is accompanied by a parallel reduction in the dynein isoform complement. Here, we complete our survey of the axonemal organization and the parallel evolution of sperm dynein complement in cecidomids with the characterization of both the sperm ultrastructure and the dynein genes in Dryomyia lichtensteini, a representative of Lasiopteridi, the cecidomid taxon with aberrant and immotile sperm cells. On the basis of the whole set of our data, we discuss the potential molecular mechanism(s) underlying the progressive modification of axoneme in cecidomids, leading first to a reduction of dynein genes and eventually to the complete loss of motility.

New insights into the cell biology of insect axonemes.

Insects do not possess ciliated epithelia, and cilia/flagella are present in the sperm tail and--as modified cilia--in mechano- and chemosensory neurons. The core cytoskeletal component of these organelles, the axoneme, is a microtubule-based structure that has been conserved throughout evolution. However, in insects the sperm axoneme exhibits distinctive structural features; moreover, several insect groups are characterized by an unusual sperm axoneme variability. Besides the abundance of morphological data on insect sperm flagella, most of the available molecular information on the insect axoneme comes from genetic studies on Drosophila spermatogenesis, and only recently other insect species have been proposed as useful models. Here, we review the current knowledge on the cell biology of insect axoneme, including contributions from both Drosophila and other model insects.

Sperm ultrastructure and spermiogenesis of Coniopterygidae (Neuroptera, Insecta).

The spermiogenesis and the sperm ultrastructure of several species of Coniopterygidae have been examined. The spermatozoa consist of a three-layered acrosome, an elongated elliptical nucleus, a long flagellum provided with a 9+9+3 axoneme and two mitochondrial derivatives. No accessory bodies were observed. The axoneme exhibits accessory microtubules provided with 13, rather than 16, protofilaments in their tubular wall; the intertubular material is reduced and distributed differently from that observed in other Neuropterida. Sperm axoneme organization supports the isolated position of the family previously proposed on the basis of morphological data.

Molecular structure of dynein and motility of a giant sperm axoneme provided with only the outer dynein arm.

The peculiar sperm axoneme of the dipteran Asphondylia ruebsaameni is characterized by an extraordinarily high number of microtubule doublets (up to 2,500) arranged in double parallel spirals. Doublets of the inner row of each spiral are tilted, so that their outer arms point towards the B-tubule of the next doublet in the outer row. Doublets are provided with only the outer arm, and no structure related to the central pair/radial spoke complex is present. When analyzed by quick-freeze, deep-etch electron microscopy, the structure of the dynein arms was shown to share the same organization described in other organisms; however, it appears to be somewhat more complex than that previously found in a related dipteran species, Monarthropalpus flavus, since the foot region of the arms displays a globular extra-domain that is intercalated between adjacent arms. Treatment of demembranated sperm with ATP and vanadate induced conformational changes in the dynein arms. SDS-page suggested the presence of a single dynein high molecular weight band or, in the gels with the best electrophoretic resolution, of two very closely spaced bands. This polypeptide positively reacted with a polyclonal antibody raised against a specific amino acid sequence located in the phosphate-binding loop of the dynein catalytic site. Dynein heavy chain-related DNA sequences corresponding to the catalytic phosphate-binding region were amplified by RT-PCR. Two distinct fragments (Asph-ax1 and Asph-ax2) encoding axonemal dynein sequences were identified. Southern blot analysis performed on genomic DNA using these sequences as a probe showed that they are part of different genes. An intron was identified in the Asph-ax1 fragment at a position corresponding to the site of a nucleotide deletion in the putative pseudogene of Monarthropalpus. Asphondylia spermatozoa exhibited in vivo a whirling movement both in the deferent duct and in the spermatheca, but they were unable to undergo processive movement in vitro. They propagated a three-dimensional wave only when constrained in a bent configuration by some mechanical means. The phylogenetic relationships between the two dipteran species, Monarthopalpus and Asphondylia, based on these biochemical and molecular data are also discussed.

Accessory tubules and axonemal microtubules of Apis mellifera sperm flagellum differ in their tubulin isoform content.

In the insect sperm flagellum, an extra set of nine additional microtubules, named accessory tubules, is present surrounding the axoneme. Using a sarcosyl/urea extraction, we were able to fractionate the microtubular cytoskeleton of the sperm flagellum of the insect Apis mellifera resulting in the dissociation of the axonemal microtubule protein components and the accessory tubules. This has allowed us to compare the tubulin isoform content of axonemal microtubules and accessory tubules by immunoelectron microscopy and immunoblotting using a panel of monoclonal antibodies directed against different tubulin post-translational modifications (PTMs). All the PTMs occurring in axonemal tubulin are also present in accessory tubules, which indicates the close relativeness of accessory tubules to axonemal rather than to cytoplasmic microtubules. However, our results demonstrate the presence of significant differences in the tubulin isoform content of axonemal microtubules and accessory tubules. First, the tubulin tyrosination extent of accessory tubules is far lower than that of axonemal microtubules, thus confirming at the molecular level their morphogenetic origin as outgrowths from the B-subtubule of each microtubular doublet. Second, although polyglycylation seems to occurr at the same extent in both microtubular systems, alpha-tubulin exhibits a larger amount of monoglycylated sites in axonemal microtubules than in accessory tubules. Third, a greater amount of beta-tubulin molecules is glutamylated in axonemal microtubules than in accessory tubules. Moreover, highly acidic isoforms, likely molecules with longer polyglutamate side chains, are present only in axonemal microtubules. Taken together, our data are indicative of a higher level of tubulin heterogeneity in axonemal microtubules than in accessory tubules. They also show a segregation of post-translationally modified isoforms between accessory tubules and axonemal microtubules and suggest the implication of PTMs in the functional specialization of the two microtubular systems.

Morphogenesis of the giant sperm axoneme in Asphondylia ruebsaameni Kertesz (Diptera, Cecidomyiidae).

The formation of the sperm giant axoneme of the gall-midge fly Asphondylia ruebsaameni is described here. The axoneme consists of a great number of microtubular doublets (up to 2,500) arranged in a double spiral wrapping around an axial cluster of mitochondria. Each microtubular doublet is provided with an outer arm only. In the early spermatid the occurrence of a large system of curved multi-layered filamentous material associated with membranous cisternae has been observed in the perinuclear region. Such a system extends throughout the cytoplasm to contact the plasma membrane. The filamentous material appears to act as a nucleating centre for the assembly of the microtubular doublets, which initially have a submembranous location and later are distributed in the interior of the cell. After their assembly, microtubular doublets are associated pairwise and are arranged in a single microtubular row with a zig-zag configuration. This configuration changes during spermiogenesis as a consequence both of a rotation of the microtubular doublet pairs and a compaction of the axonemal complex due to the elimination of the excess cytoplasm. As a result of this process, a double parallel spiral of microtubular doublets is formed.

Structural and molecular characterization of dynein in a gall-midge insect having motile sperm with only the outer arm.

The dipteran Monarthropalpus flavus possesses a peculiar sperm axoneme, characterized by multiple rows of microtubular doublets linked by the outer dynein arms only, lacking any equivalent of the central pair/radial spoke complex. The structure of these dynein molecules was studied by electron microscopy (EM). Using the quick-freeze, deep-etch method of EM, they were found to be similar to outer dynein arms described previously. Two globular "heads," each subdivided by a cleft, are clearly discernible. "Stalks" extend from proximal head to contact the B-tubule of the adjacent doublet. Unlike the situation in vertebrate sperm, the stalks sometimes branch into two thinner strands that contact the B-tubule at different sites. Treatment of demembranated sperm cells with ATP and vanadate induces conformational changes in the dynein outer arms. These are interpreted as the result of rotation of the dynein head with respect to what is observed in axonemes in rigor condition (after ATP depletion). SDS-PAGE indicates that the high-molecular-weight complement of this molecule comprises a single heavy chain. Specific dynein heavy chain-related DNA sequences corresponding to the catalytic-phosphate binding region were amplified by RT-PCR. Only one axonemal dynein sequence was identified among all amplified fragments. Southern blot analysis performed on genomic DNA using this sequence as a probe identified two hybridizing genes, only one of which is able to encode a functional product. Thus, genetic analysis indicates that this axonemal outer arm dynein is a homodymer of a single heavy chain subunit. In vivo, spermatozoa of this species are stored in a rolled configuration in female spermatheca, where they move rapidly with a wave-like motion. This movement could not be reproduced in vitro, except when spermatozoa were constrained in a bent configuration by some mechanical impediment. We propose that, in the absence of both the central pair/radial spoke complex and the inner arms, a curvature-dependent activation acts to trigger motility in these spermatozoa.

Intermediate filament proteins immunologically related to cytokeratins in the oocyte of the fish Cyprinus carpio.

We have used monoclonal antibodies specific for different sets of human cytokeratins and the anti-IFA (Intermediate Filament Antigen) antibody to investigate the expression of intermediate filament proteins in the mature oocyte of the teleost Cyprinus carpio. Several polypeptides have been identified, showing molecular weights ranging from 43 to 65 kDa. Two-dimensional analysis of the immunoreactive species revealed the presence of at least six major protein spots and a series of minor components, grouped in quite a narrow pI range from 5.52 to 6.28. The general complexity of the carp oocyte cytokeratin-related cytoskeleton appears to be higher than those described for oocytes of other vertebrate species.

Heterogeneous localization of epitopes along axonemes of mammalian cilia.

The specificity of four monoclonal antibodies, raised against mammalian ciliary axonemes, was determined by both immunofluorescence and immunoblot experiments. Three antibodies reacted with epitopes which are differentially located along axonemal length. Among these, antibody 3.12 recognized an epitope common to different dynein heavy chains, reacted only with tracheal cilia and specifically stained the proximal portion of the ciliary axoneme.

Immunological and charge properties of GFAP in lower vertebrates.

1. An antiserum specific for bovine GFAP was employed in a comparative study of this protein in several species of bony fish and in an anuran species. 2. The immunological properties of this protein are conserved in a remarkable way in all the species examined. 3. Analysis of trout and bovine GFAP by two-dimensional gel electrophoresis indicated that the charge properties of this protein have remained quite constant from fish to mammals.

Evolutionary trends of neurofilament proteins in fish.

1. Neurofilament complement was studied in an early chordate (Ciona intestinalis) and six fish species by immunoblot with antisera specific for each of the three mammalian NF subunits. 2. The anti-NF-H and anti-NF-M antisera were characterized as strictly specific for phosphorylated epitopes located in the carboxyterminal domain. 3. The NF-L subunit is absent in primitive chordates and appears first in fish; it can be identified on the basis of its apparent mol. wt, its reactivity with the anti-IFA antibody and with polyclonal antibodies raised to the NF-L subunit of mammals. 4. Primitive chordate neurofilaments are constituted by a single polypeptide of ca 160,000 mol. wt exhibiting only M-type phosphorylation-dependent epitopes. 5. Primitive fish (Acipenser transmontanus, Salmo gairdneri, Scorpaena porcus, Serranus scriba) possess only a single high mol. wt NF subunit reacting with both anti-NF-H and anti-NF-M antiserum while more recent species (Mugil saliens, Perca fluviatilis) possess two high mol. wt NF subunits which are immunologically distinct as to their phosphorylation structures. 6. The existence in some fish species of two high mol. wt NF polypeptides suggests that the process of gene duplication and diversification supposed to have given rise to the two high mol. wt NF subunits of mammals and birds has occurred repeatedly in vertebrate evolution, and may be regarded as a case of convergent evolution.

Evolution of the "titin epitope" in neurofilament proteins.

1. The specificity of a monoclonal antibody raised to human titin was characterized. The antibody reacts with an epitope which is common to titin and the high mol. wt subunits NF-H and NF-M of mammalian neurofilaments. 2. Mapping of the epitope indicated that it is located in the carboxyterminal extension of NF-H and NF-M, and that its reactivity does not depend on the phosphorylation state of the molecule. 3. A comparative study on neurofilament protein of lower vertebrates revealed that this epitope has been conserved during vertebrate evolution.

Expression of phosphorylated and non-phosphorylated neurofilament subunits and cytokeratins in neuroendocrine lung tumors.

In this study antibodies specific for different intermediate-sized proteins (cytokeratins and neurofilaments) have been tested on a series of neuroendocrine (NE) lung tumors in order to evaluate their diagnostic validity. In particular we used a panel of polyclonal anti-neurofilament 200-kilodalton subunits whose reactivity against phospho-dependent epitopes was known. At least one NF subunit was constantly present and in all cases coexpression of cytokeratins and neurofilaments was confirmed. However, in cases of carcinoid tumor (CT) the results were homogeneous, while the cases of small cell lung carcinoma (SCLC) showed a much wider range of immunostaining. Our investigation confirms the hypothesis that the phosphorylation state is a significant determinant of immunohistochemical properties of neurofilaments. This might explain the large number of negative results obtained in previous investigations on NE tumors. The phosphorylation of neurofilaments may also be considered an indication of the degree of differentiation of the tumor.

Expression of intermediate filaments and synaptophysin show neuronal properties and lack of glial characteristics in Y79 retinoblastoma cells.

The expression of intermediate filaments and synaptophysin in Y79 retinoblastoma cells was studied. The cells grew normally in suspension as floating aggregates. Sodium butyrate and dibutyryl cyclic AMP induced adherence and rapid spreading of Y79 cells on laminin-coated substratum and many of the cells presented long cellular extensions. As judged by immunostaining with monoclonal and polyclonal antibodies, most untreated Y79 cells expressed synaptophysin, whereas only vimentin intermediate filaments were detected in a few cells. A more bright fibrillar vimentin-positivity, which responded by coiling to disruption of microtubules, could be seen in the spread cells. Furthermore, especially when exposed to butyrate, a distinct fibrillar positivity could be revealed in some of the spread cells with antibodies recognizing a phosphorylated epitope in neurofilaments. Western blotting results showed the presence of the molecular weight 57,000 vimentin polypeptide and the molecular weight 38,000 synaptophysin polypeptide in both undifferentiated and spread Y79 cells, and a phosphorylated molecular weight 200,000 neurofilament polypeptide in spread cells only. In contrast, both immunostaining and Western blotting results with several antibodies indicated lack of glial fibrillary acidic protein in Y79 cells suggesting hence, a lack of glial differentiation. The present results do not support the hypothesis that retinoblastomas would originate from a common precursor cell of neurons and glia, and show the presence of mainly neuronal features in Y79 retinoblastoma cells.

Phosphorylated epitopes of neurofilaments have been conserved during chordate evolution.

We have characterized some rabbit polyclonal responses as strictly specific for phosphorylated epitopes located in the carboxyterminal (tail) domain of the H or the M subunits of mammalian neurofilaments. These antibodies have been used to confirm the occurrence in lizard neurofilaments of a single heavy subunit cross-reacting with both H and M from mammals. A heavy subunit with similar cross-reactivity has been detected in neurofilaments preparations from fishes, whereas more primitive Chordata possess a HMW polypeptide cross-reacting with only the M subunit. We could also demonstrate in frog spinal cord two distinct heavy subunits cross-reacting with either the M or the H subunit from mammals, a fact which suggests a convergent evolution for phosphorylated epitopes of neurofilaments.