The stathmin-tubulin interaction and the regulation of the microtubule assembly

Stathmin family proteins interact with tubulin and negatively regulate its assembly in microtubules. One stathmin molecule forms a complex with two alphabeta tubulin heterodimers in an interaction that is weakened upon stathmin phosphorylation. The X-ray structure of crystals of the complex reveals a head-to-tail arrangement of the two tubulins which are connected by a long stathmin alpha helix. By holding tubulins in a curved complex that is not incorporated in microtubules, stathmin lowers the pool of "assembly competent" tubulin. An alternate mechanism has been also proposed to account for the stathmin action in vivo; it involves a direct interaction of stathmin with microtubule (+) ends. More experiments are needed to evaluate the relative contribution of this alternative mechanism to the regulation of tubulin assembly by stathmin.     

Purification and characterisation of tubulin from the parasitic nematode, Ascaridia galli

We have developed a method for the purification of tubulin from a parasitic nematode using DEAE-Sephadex column chromatography and temperature-dependent assembly. The resulting microtubules were morphologically similar to those obtained from mammalian brain. The nematode tubulin showed similar properties to mammalian tubulin on one and two dimensional polyacrylamide gels, although certain electrophoretic conditions revealed a slight difference in the α-tubulins from mammals and nematodes. This was confirmed by limited proteolytic peptide mapping. The β subunit of nematode tubulin appeared almost identical to that of mammals. Peptide maps of these tubulins were also compared with those of eukaryotic micro-organisms and these results interpreted in terms of the evolution of the tubulin polypeptides and the sensitivity of helminths to antimicrotubular agents.     

Differential post-translational modifications of microtubules in cells of the seminiferous epithelium of the rat: a light and electron microscope immunocytochemical study

The cells of the seminiferous epithelium of the rat testis are a rich source of microtubules and contain distinct microtubular structures such as the meiotic spindle and manchette. Microtubule diversity can be maintained by differential genetic expression of the multiple alpha- and beta-tubulin polypeptides or by tubulin monomer acetylation and detyrosination, post-translational modifications of alpha-tubulin. In the present analysis, antibodies that specifically recognize acetylated (antiacetylated), tyrosinated (anti-Tyr) and detyrosinated (anti-Glu) alpha-tubulins were employed to examine the distribution of post-translationally modified microtubules in the cells of the seminiferous epithelium. In the light microscope, a distinct pattern of staining for each antibody was detected using immunoperoxidase techniques on paraffin-embedded testicular sections. In the case of the anti-Glu antibody, a dense immunoperoxidase staining was detected in the cytoplasm of steps 4-7 spermatids. Thereafter, staining was noted over the area corresponding to the manchette of steps 8-15 spermatids, but not over their cytoplasm. The tails of spermatids were also reactive with this antibody. The anti-Tyr antibody was observed to be localized over the cytoplasm of Sertoli cells in their basal, supranuclear, and apical regions. A dense immunoperoxidase staining was also noted in the cytoplasm of pachytene spermatocytes, but it was negligible in the cytoplasm of spermatocytes undergoing their meiotic division; in these cells the centrioles and meiotic spindle were reactive. The spermatid's tails were also reactive. The antiacetylated antibody showed reactivity only over the tails of spermatids. With the electron microscope, a similar pattern of labeling was noted using immunogold labeling on Lowicryl K4M embedded testicular sections. The anti-Glu antibody heavily labeled microtubules of the manchette and the axoneme of tails of spermatids as well as microtubules of the proximal and distal centrioles and centriolar adjunct. The anti-Tyr antibody strongly labeled microtubules of Sertoli cells and the meiotic spindle and midbody of dividing spermatocytes. The anti-Tyr antibody also labeled the microtubules of the axoneme, centrioles, and centriolar adjunct of spermatids, but to a lesser degree than the anti-Glu antibodies; the manchette was faintly labeled. Of the three antibodies, the antiacetylated antibody showed the weakest labeling of microtubules of the centrioles, centriolar adjunct, and midbody, whereas those of the manchette and Sertoli cells were unreactive; the axoneme was moderately labeled.(ABSTRACT TRUNCATED AT 400 WORDS)     

Highly degenerated distal centrioles in rhesus and human spermatozoa

In humans and other mammals except rodents, the spermatozoa contribute the proximal centriole during fertilization. The inheritance of the distal centriole is not yet fully clear. In the present work, the distal centrioles of rhesus and human spermatozoa have been studied by transmission electron microscopy. The round and elongating rhesus spermatids possess both proximal and distal centrioles. The distal centriole extends posteriorly as an axoneme while the proximal centriole produces a microtubular adjunct. Ejaculated rhesus and human spermatozoa have intact proximal centrioles, but the distal centrioles have degenerated. The central pair of microtubules of the axoneme extends continuously into the distal centriolar region up to the sperm head. Serial transverse and longitudinal sections of the sperm neck region reveal few scattered microtubule duplexes or triplets in the distal centriolar region. The loss of the centriolar microtubules is more extensive on the ventral side of the neck region, the side where the proximal centriole resides. The distal centriole degenerates caudally from the rostral area. Immunogold electron microscopy with anti-beta-tubulin antibody showed that the distal centriolar regions possess 50% fewer gold particles than the proximal centrioles, indicating a significant loss of centriolar microtubules in the distal centriolar region. The A-tubules of the remaining triplets are filled with a dense material, as observed in the axoneme. Thus, rhesus and human spermatozoa introduce only proximal centrioles intact, whereas the distal centrioles are mostly disorganized in the mature spermatozoa.     

'9 + 0' axoneme in spermatozoa and some nasal cilia of a patient with totally immotile spermatozoa associated with thickened sheath and short midpiece

A patient is described with 100% immotile spermatozoa. The sperm cells lack the central pair of microtubules or the complete axoneme. This defect is associated with a thickened fibrous sheath and a shortened midpiece containing defective mitochondria. Fifteen per cent of the nasal cilia of this patient lack the central pair of microtubules. Eighteen similar cases have been described in the literature suggesting that this association of ultrastructural defects may be considered a syndrome.     

Cytoskeletal organization defects and abortive activation in human oocytes after IVF and ICSI failure

In this study, we analysed the distribution of beta tubulins to detect spindle and cytoplasmic microtubules, alpha acetylated tubulins for sperm microtubules and chromatin configuration in oocytes showing fertilization failure after conventional IVF or intracytoplasmic sperm injection (ICSI). A total of 450 human oocytes that failed to fertilize were studied 20-40 h after IVF or ICSI. In all, 287 oocytes were stained for immunofluorescence and chromosomal spreads were performed by Tarkowski's air-drying method in 163 IVF or ICSI oocytes that did not develop pronuclei after the extrusion of a second polar body. Immunofluorescence analysis showed that the main reason of fertilization failure after IVF was no sperm penetration (55.5%). The remaining oocytes showed different abnormal patterns, e.g. oocyte activation failure (15.1%) and defects in pronuclei apposition (19.2%). On the other hand, fertilization failure after ICSI was mainly associated to incomplete oocyte activation (39.9%), and to a lesser extent with defects in pronuclei apposition (22.6%) and failure of sperm penetration (13.3%). A further 13.3% of the ICSI oocytes arrested their development at the metaphase of the first mitotic division. The chromosomal spreads allowed the analysis of abortive activations, in which no pronuclei formed but a second polar body was extruded. Immunofluorescence and cytogenetic analysis provided a useful tool to improve infertility diagnosis and prognosis in each particular case.     

A quick molecular method for the simultaneous detection in spermatozoa of nuclear, acrosomal and axonemal structure by fluorescent microscopy

Ejaculated spermatozoa from infertile men presenting to our laboratory for semen analysis were processed with a new molecular method which reveals simultaneously, in the same sperm cell, the status of the acrosome, by testing the hyaluronidase content, the texture of the nucleus, by checking the DNA strands breaks, and the structure of the axoneme, revealing the tubulin content. The presence of hyaluronidase and tubulin is essential for the sperm function, and the analysis of the DNA status reveals the eventual apoptotic process. Using this method in normal spermatozoa, the staining of the acrosomal hyaluronidase reveals, by yellow-green fluorescence, the shape of the acrosomal complex and its texture. At the same time, in the same sperm cell, the staining of the axonemal tubulin demonstrates, by a red labeling, the presence of the protein and therefore the consistence of the axonemal structure. Simultaneously, at the head level, the absence of red labeling from nuclear DNA indicates that the apoptotic process is not present. This protocol allows quantification of the frequency of the presence of normal or abnormal spermatozoa, by an easy scoring and calculation of the apoptotic sperm or of the sperm with generic defects at acrosomal or flagellar level. The percentage of normal spermatozoa evaluated by the triple staining method has been compared with the results of the PAP staining and of the ultrastructural analysis, statistically elaborated. Triple staining results more severe than the PAP method, but TEM analysis is the finest technique to detect sperm abnormality because it considers the entire panel of sperm defects.     

Sperm morphology of mud dauber Sceliphron fistularium dahlbom (Hymenoptera: Apoidea: Sphecidae), as an indication of bees relation

The morphology of spermatozoon of Sceliphron fistularium is very similar to that described for bees. In particular, the response to E-PTA stains is similar to that observed in corbiculated Apidae, especially Meliponini bees. Spermatozoa measure 285 microm and are composed of 1) a bilayered acrosome (acrosomal vesicle and perforatorium); 2) a homogeneous and compact nucleus; 3) a 9+9+2 axoneme; 4) a rod-shaped centriolar adjunct; 5) two asymmetrical mitochondrial derivatives with paracrystalline material exclusively in the larger one, and 6) two accessory bodies. Only the accessory microtubules of axoneme and the paracrystalline material are E-PTA positive. Comparison of S. fistularium sperm to data on Hymenoptera corroborates their proximity with bees.     

Characterization of tubulin from Brugia malayi and Brugia pahangi

The tubulins of Brugia malayi and B. pahangi were similar with respect to concentration (mg tubulin per mg soluble protein), electrophoretic and isoelectric mobility, reaction in Western blots with anti-tubulin monoclonal antibodies, and isoform patterns. Tubulin was estimated to account for 2.8% and 2.9% of soluble protein in B. malayi and B. pahangi extracts, respectively. Tubulins from Brugia nematodes have been partially purified by polylysine agarose chromatography and with taxol. Western blots with alpha- and beta-tubulin monoclonal antibodies confirmed the presence of tubulin. The mobility of Brugia tubulins on sodium dodecyl sulfate polyacrylamide gel electrophoresis was very similar to that of N. brasiliensis and rat brain tubulins. The isoelectric range for Brugia alpha- and beta-tubulin isoforms was pH 5.4-4.7. Western blots with anti-tubulin monoclonal antibodies revealed 4-5 isoforms of alpha-tubulin and 4-5 isoforms of beta-tubulin for Brugia nematodes.     

Anti-tubulin antibodies in rabbits before and after immunization with pig tubulin

Sera from rabbits before and after repeated injections of pig tubulin in complete Freund's adjuvant were examined for antibody activity against pig and rabbit tubulins and against a panel of antigens: actin, myosin, DNA, TNP/BSA. Antibody activity against all the antigens of the panel (PAg) increased moderately after the first but not after subsequent injections. Antibody activity against pig and rabbit tubulins strongly increased after the second immunization when the maximum was reached. Isolation of anti-tubulin antibodies from normal or immune sera on tubulin-immunoadsorbent demonstrated the presence of three different antibody populations: (1) polyspecific IgM reacting with the PAg and the tubulins, present in substantial amounts in normal sera and moderately increased in immune sera; (2) small amounts of polyspecific IgG detected only in immune sera; (3) high amounts of specific IgG reacting with pig and rabbit tubulins, present in immune but not normal sera. Western blot analysis of the specific IgG population showed that it contained antibodies reacting with both native pig and rabbit tubulins, as well as antibodies recognizing only the 30,000 proteolytic fragment of pig, but not that of rabbit tubulin. The results indicate that the immunization of rabbits with heterologous tubulin induced specific IgG anti-tubulin antibodies which recognize the self and non-self antigens differently.     

Sperm flagella: comparative and phylogenetic perspectives of protein components

Sperm motility is necessary for the transport of male DNA to eggs in species with both external and internal fertilization. Flagella comprise several proteins for generating and regulating motility. Central cytoskeletal structures called axonemes have been well conserved through evolution. In mammalian sperm flagella, two accessory structures (outer dense fiber and the fibrous sheath) surround the axoneme. The axonemal bend movement is based on the active sliding of axonemal doublet microtubules by the molecular motor dynein, which is divided into outer and inner arm dyneins according to positioning on the doublet microtubule. Outer and inner arm dyneins play different roles in the production and regulation of flagellar motility. Several regulatory mechanisms are known for both dyneins, which are important in motility activation and chemotaxis at fertilization. Although dynein itself has certain properties that contribute to the formation and propagation of flagellar bending, other axonemal structures-specifically, the radial spoke/central pair apparatus-have essential roles in the regulation of flagellar bending. Recent genetic and proteomic studies have explored several new components of axonemes and shed light on the generation and regulation of sperm motility during fertilization.     

Spermatozoon cytoarchitecture of Amphilina foliacea (Platyhelminthes, Amphilinidea)

The mature spermatozoon of Amphilina foliacea Rudolphi, 1819 has been examined using transmission electron microscopy. The male gamete is filiform and tapered at both extremities. Its moderately electron-dense cytoplasm possesses two parallel axonemes of unequal lengths with the 9?+?“1” trepaxonematan pattern, a mitochondrion, a nucleus, parallel cortical microtubules, four electron-dense attachment zones, and electron-dense glycogen granules. A crested body is absent. The anterior extremity of the cell exhibits a single axoneme. The anteriormost cortical microtubules have been observed with the appearance of the second axoneme. The number of cortical microtubules reaches a maximum (up to 25) in the nucleated region III of the spermatozoon. A single mitochondrion extends from the middle of region II to the end of region III of the cell. Both axonemes have become disorganized in a similar way: the axonemal doublets disappear first, followed by the central core. The nucleus is surrounded by a few cortical microtubules in the proximal part of region V. In the distal extremity of the mature spermatozoon, there is only the nucleus. Differences of spermatozoon ultrastructure within Amphilinidea and other Neodermata are discussed.     

Ultrastructural studies on spermatogenesis in a sex-ratio-mutant strain of Drosophila simulans

The aberrant sex-ratio mutation in D. simulans used for this study is a temperature-sensitive autosomal recessive. Homozygous males raised at 16°C produce about 2% in F₁, but those raised at 26°C, have a normal sex ratio. Ultrastructural studies of spermatogenesis have revealed many anomalies in the germ cells of flies raised at 16°C, but the same flies raised at 26°C had few anomalies. The earliest spermatogenic stage with noticeable abnormalities was the primary spermatocyte. In later stages there were pronounced abnormalities in nucleoli, chromatin condensation, nuclear shape, Golgi complex, acrosome, and microtubules. There is asynchronous differentiation of spermatids within a bundle. Some of the abnormalities encountered are disorganization or loss of microtubules of the axoneme, degenerating nebenkern derivatives, and increased numbers of lysosomes, multilamellate bodies, and multivesicular bodies. At the lower temperature, more than half of the sperm within the same bundle were found in different stages of degeneration. Genetic analysis suggests that the sex-ratio gene causes abnormalities and degeneration of most of the Y-bearing sperm. However, counts of abnormal sperm at the ultrastructural level indicate that some X-bearing sperm must also undergo degeneration. These observations show that the sex-ratio gene is of variable penetrance at different temperatures in the primary spermatocyte and of differential penetrance in X- and Y-bearing sperm.     

Taenia hydatigena

Summary The process of cell division during spermatogenesis in Taenia hydatigena followed the general pattern reported by Rybicka (1966) for other cestodes. Sixty-four spermatozoa were formed from each primary spermatogonium after a series of five nuclear divisions. During spermiogenesis, changes in the organisation of sperm tail cytoplasm were evident with four distinct regions along the length of the spermatozoon being distinguishable. The axoneme which was normally single in each sperm tail, had the typical 9 plus 1 structure found in platyhelminths. Nuclear material in the mature spermatozoon was arranged spirally around the axoneme.     

Tubulin acetylation and histone deacetylase 6 activity in the lung under cyclic load

Previous studies from our lab have demonstrated that upon exposure to physiologic levels of cyclic stretch, alveolar epithelial cells demonstrate a significant decrease in the amount of polymerized tubulin (Geiger et al., Gene Therapy 2006;13:725-731). However, not all microtubules are disassembled, although the mechanisms or implications of this were unknown. Using immunofluorescence microscopy, Western blotting, and immunohistochemistry approaches, we have compared the levels of acetylated tubulin in stretched and unstretched A549 cells and in murine lungs. In cultured cells exposed to cyclic stretch (10% change in basement membrane surface area at 0.25 Hz), nearly all of the remaining microtubules were acetylated, as demonstrated using immunofluorescence microscopy. In murine lungs ventilated for 20 minutes at 12 to 20 ml/kg followed by 48 hours of spontaneous breathing or for 3 hours at 16 to 40 ml/kg, levels of acetylated tubulin were increased in the peripheral lung. In both our in vitro and in vivo studies, we have found that mild to moderate levels of cyclic stretch significantly increases tubulin acetylation in a magnitude- and duration-dependent manner. This appears to be due to a decrease in histone deacetylase 6 activity (HDAC6), the major tubulin deacetylase. Since it has been previously shown that acetylated microtubules are positively correlated to a more stable population of microtubules, this result suggests that microtubule stability may be increased by cyclic stretch, and that tubulin acetylation is one way in which cells respond to changes in exogenous mechanical forces.     

Gamma-tubulin during the differentiation of spermatozoa in various mammals and man

The distribution of gamma-tubulin as a marker of microtubule organizing centres (MTOC) was studied during spermiogenesis in rodents and in rabbit, monkey and man. A polyclonal antibody directed against human gamma-tubulin was used both for indirect immunofluorescence (IIF) and post-embedding immunogold procedures. In all species, gamma-tubulin was detected in the proximal and distal centrioles of round spermatids. In elongating spermatids, gamma-tubulin was predominantly found in the pericentriolar material (PCM) of both centrioles and particularly around the adjunct of the proximal centriole. At this level, some labelling was also associated with manchette microtubules, but other parts of the manchette and the nuclear ring were never labelled. We propose a role for distal centriole gamma-tubulin in axoneme nucleation and centriolar adjunct gamma-tubulin in manchette nucleation. The disappearance of gamma-tubulin in mature spermatozoa indicates that sperm aster nucleation should be dependent on oocyte gamma-tubulin. Remnants of gamma-tubulin in some human spermatozoa suggest that paternal gamma-tubulin also could contribute to sperm aster formation.      

In vitro interaction of tubulin with the photoreceptor cGMP phosphodiesterase γ-subunit

The α and β tubulins compose the microtubule cytoskeleton which is involved in many cellular processes such as vesicular transport. The photoreceptor cells in the retina are neurons specialized for phototransduction. Here we report a novel interaction between tubulin and the photoreceptor cGMP phosphodiesterase (PDE6) gamma subunit (PDEγ). The specificity and molecular details of the PDEγ:tubulin interaction were analyzed through the experiments of pull down, microtubule co-sedimentation, and NMR spectroscopy. The tubulin-interacting site was identified to be in the PDEγ C-terminal I67-G85 region, and the interaction interface appeared to be distinct from those with the other PDEγ targets in phototransduction. We also observed that PDEγ interacted with tubulin in a GTP-dependent manner. Our findings offer implications for non-phototransduction role(s) of PDEγ in the photoreceptor neurons.     

Fertilization and early embryology: Intracytoplasmic sperm injection for Rhesus monkey fertilization results in unusual chromatin, cytoskeletal, and membrane events, but eventually leads to pronuclear development and sperm aster assembly

The disassembly and reorganization of sperm-derived structuresare landmarks for the onset of embryonic development. Sincecomplete information on these events is not yet available, weexamined the disassembly of the sperm axoneme, the formationof the sperm aster, and the decondensation and development ofthe male and female pronuclel in inseminated Rhesus monkey oocytesconceived by in-vitro fertilization (IVF) or by intracytoplassnicsperm injection. During IVF, the spermatozoa lose their acrosomesafter contacting the zona pellucida, and the plasma membraneand nuclear envelope disappear after fusion with the oolemma.Subsequently, a sperm aster of microtubules forms around theproximal centriole, which is bound to the sperm connecting piece.This process is then followed by the formation of both pronuclei.The single sperm centriole later duplicates and the bipolarmitotic apparatus is observed. Following sperm injection, thespermatozoa have both an intact plasma membrane and acrosome.Although the microtubules form the sperm aster in a fashionidentical to that seen during IVF, the presence of an intactacrosome appears to be associated with a heterogeneity in thedecondensation of sperm chromatin. While this may indicate anabnormal pattern of chromatin decondensation during the formationof the male pro-nucleus following sperm injection, the malepronucleus eventually fully decondenses, as during 1W. Spermmito chondria are displaced as the sperm centriole is exposed.Annulate lamellae and a previously undescribed organelle whichseems to contain annulate lamellae precursors, as well as maternalmitochondria, are found in association with the developing pronuclearenvelopes. This information increases understanding of fertilizationin primates, and may also be of significance for use in assistedhuman reproduction as well as in the preservation of endangeredmammalian species. In addition, these results demonstrate thesimilarities between fertilization in Rhesus monkeys and humans,providing additional evidence for the use of this non-humanprimate as a model system in which to investigate the cellularand molecular biological basis of human reproduction.     

Sperm membrane protein (hSMP-1) and RanBPM complex in the microtubule-organizing centre

hSMP-1 is a human sperm membrane protein expressed during development. It is a testis-specific component produced during male germ cell differentiation. Proteins that interact with hSMP-1 were identified by the application of the yeast two-hybrid system. One of the components, RanBPM, was found to be associated with hSMP-1 under both in vitro and in vivo conditions. In the human testis, RanBPM is produced in spermatogonia and primary spermatocytes, suggesting expression during the early stages of spermatogenesis; whereas in the rat testis, it is located in round and elongated spermatids, similar to hSMP-1, suggesting expression of both components during spermiogenesis. Images obtained by immunofluorescence and confocal scanning microscopy of CHO-K1 cells co-transfected with pEGFP-C1-hSMP-1 and pDsRed1-Nl-RanBPM revealed that RanBPM and hSMP-1 are distributed in discrete loci throughout the cytoplasm. When superimposed, the stained spots appeared as congruent yellow areas, indicative of co-localization and probable complex formation of these two components. This interaction between hSMP-1 and RanBPM may be involved in the process of male germ cell differentiation. In CHO-Kl cells transfected with pEGFP-Cl-hSMP-1, the exogenously expressed hSMP-1 was found to co-localize with -tubulin. Depolymerization of microtubules can be induced in CHO-Kl cells by cold treatment. In cells transfected with the pEGFP-Cl vector, the dispersed tubulins promptly reassembled upon warming. However, in cells transfected with pEGFP-Cl-hSMP-1, reassembly of the dispersed tubulins was blocked even upon rewarming of the cells. These findings suggest that hSMP-1 interacts with tubulins and thereby may modulate microtubule assembly and/or activity.Abbreviations RanBPM Ran binding protein in the microtubule organizing centre - hSMP-1 Human sperm membrane protein - MBP Maltose binding protein