Association of spectrin with manchette microtubules in mammalian spermatids

Summary— In hamster and mouse spermatozoa a spectrin immunogold labeling was found under the plasma membrane in the principal piece of the flagellum. During spermatid differentiation, the spectrin labeling was associated with the manchette, a transient microtubular network involved in nuclear shaping and organelle translocation.     

The manchette in Stage 14 rat spermatids: A possible structural relationship with the redundant nuclear envelope

Summary Morphologic studies were performed on the summer cell of Stilling in the interrenal gland of the American bullfrog. Ultrastructural studies showed the cell to be packed with membrane-bound granules similar to those of the juxtaglomerular apparatus granules in the mammalian kidney. These granules reacted positively with several stains for granules of cells in the juxtaglomerular apparatus. The Stilling cells are intimately associated with the cortical cells and contain rough endoplasmic reticulum and granules in varying degrees of development associated with the Golgi apparatus. It is concluded that the Stilling cell may secrete a renin-like substance that may function in an aldosterone-stimulating reninangiotensin system.Supported in part by N. I. H. Grant RR 06138 Health Sciences Advancement Award.     

The manchette in Stage 14 rat spermatids: A possible structural relationship with the redundant nuclear envelope

Summary The manchette or caudal tube has been examined in Stage 14 rat spermatids. The microtubules of the caudal tube have been found to be partially sheathed by smooth endoplasmic reticulum which appears to be continuous with the outer nuclear membrane of the redundant nuclear envelope. The microtubules in caudal regions of the manchette have been noted to be interconnected by links of unusual size and morphology. It is suggested that the caudal tube consists at this stage of development of two structures, membrane and microtubules and that the links between the microtubules appear to play a role in the structural order noted in the position of the tubules of the manchette. The possible significance of these links in relation to motility is discussed.Supported by a grant to E. A. MacKinnon by the Medical Research Council of Canada.     

Abnormal manchette development in spermatids of azh/azh mutant mice

A study of manchette development during spermiogenesis in azh/azh mutant mice was carried out by thin-section transmission electron microscopy with the goal of determining which of the initial steps in spermatid development are aberrant. In the homozygous mutant, spermatogenesis was quantitatively normal; but 100% of the sperm nuclei produced had abnormal shapes. The first defect, observed in steps 8-9, was the abnormal positioning of many manchette microtubules. These microtubules were directed towards regions of the plasma membrane not normally associated with manchette formation, in addition to being located at the caudal rim of the acrosome in the normal region of manchette formation. At steps 10-12, sheets of manchette microtubules were often in ectopic positions along the plasma membrane, rather than in association with the nuclear membrane as well. The fine structural appearance of the manchette was generally normal; the defect appeared to be in its positioning within the cell. In many step 8-10 spermatids nuclear invaginations and evaginations were observed, always associated with irregularities in the position of some of the manchette microtubules; these illustrate the capacity of manchette microtubules to deform nuclear shape. The nuclear irregularities remained throughout spermiogenesis. These observations are consistent with the hypothesis that the manchette is involved in at least some aspects of sperm nuclear shaping and that the improper positioning of manchette formation is a likely candidate for the primary abnormality resulting from a defective allele at the azh locus.     

A membrane protein,TMCO5A,has a close relationship with manchette microtubules in rat spermatids during spermiogenesis

We isolated the transmembrane and coiled‐coil domains 5A (Tmco5A) gene using polymerase chain reaction‐based subtraction technique and showed that Tmco5A was predominantly expressed in rat testes starting at 4 weeks of postnatal development. When expressed in COS7 cells, TMCO5A was found to be distributed in the endoplasmic reticulum‐nuclear membrane (ER‐NM) of cells as a membrane‐associated protein, while TMCO5AΔC lacking the transmembrane region (TM) mislocalized and diffused throughout the cytoplasm. The result suggested that TM is responsible for the retention of TMCO5A at the ER‐NM. Immunocytochemical and immunoblotting analyses indicated that TMCO5A was localized along the posterior part of the nuclei in both round and elongated rat spermatids but disappeared from epididymal spermatozoa. Double immunolabeling of isolated spermatids with the anti‐TMCO5A and the anti‐β tubulin antibodies showed that TMCO5A was always found to be closely associated with developing manchette microtubules but did not completely colocalize with them. On the other hand, we found that almost all TMCO5A colocalized with SUN4, a linker of nucleoskeleton and cytoskeleton complex protein present at the posterior part of spermatid nuclei. These data suggested that TMCO5A is located closer to the nuclei than the manchette microtubules. It is likely that TMCO5A, in association with manchette microtubules, is involved in the process of spermiogenesis.     

Delta-tubulin is a component of intercellular bridges and both the early and mature perinuclear rings during spermatogenesis

Mammalian spermatogenesis involves drastic morphological changes leading to the development of the mature sperm. Sperm development includes formation of the acrosome and flagellum, translocation of nucleus-acrosome to the cell surface, and condensation and elongation of the nucleus. In addition, spermatogenic cell progenies differentiate as cohorts of units interconnected by intercellular bridges. Little is known about the structural components involved in the establishment of conjoined spermatogenic cells and the mechanism of nuclear shaping of the male gamete. We identified two isoforms of delta-tubulin and found that the long isoform is predominantly expressed in testis, while the short isoform is expressed in all tissues examined. We also found that delta-tubulin forms intercellular bridges conjoining sister spermatogenic cells. In addition, delta-tubulin is a component of the perinuclear ring of the manchette, which acts on translocation and elongation of the nucleus. Furthermore, small rings clearly distinct from the intercellular bridges, which might mature to perinuclear ring of the manchette in later stages of spermatogenesis, were detected on the cell surface of round spermatids. These results suggest that delta-tubulin is a component of two types of ring, the intercellular bridges and the perinuclear rings, which may be involved in morphological changes of spermatid to mature sperm.     

Nuclear and manchette development in spermatids of normal and azh/azh mutant mice

Germinal cells or nuclei with attached cytoskeletal elements were prepared from the testes and epididymides of normal mice and mice homozygous for the recessive azh mutation, which results in abnormal sperm heads. To make observations, we utilized phase-contrast microscopy, immunofluorescence microscopy with antitubulin antibodies, and a direct-view stereo electron microscope system developed by A. Cole. Sperm nuclei, tails, manchettes, and other cytoskeletal structures were studied at various stages of development. The tail architectures were similar in the normal and mutant forms, but the shape of the heads at the attachment regions were markedly different. Normal sperm nuclei were very flat, whereas the posterior regions of mutant nuclei were tapered cylinders. The manchette, an organized microtubular structure that girdles the posterior region of the spermatid nucleus, differed in size and configuration between normal and mutant forms. In normal midstage spermatids, the manchette microtubules extended outward at a 45 degree angle from the long axis of the flattened head, whereas in mutant spermatids, the microtubules formed tapered cylinders around the long axis of the caudal part of the nucleus. Radical differences in head shapes between normal and mutant sperm could be related, in part, to the manner in which manchettes formed and matured on the spermatids.     

The cancer/testis antigen CAGE-1 is a component of the acrosome of spermatids and spermatozoa

Cancer/testis antigens (CTAs) are characterized by their restricted expression pattern. In normal individuals their expression is largely restricted to the testis. In the case of cancer patients, CTA expression has also been frequently observed in the tumoral cells. CTAs are considered to be promising targets for immunotherapy. However, almost nothing is known about the properties defined by the vast majority of CTAs. Here, we have investigated the expression pattern and localization of the CTA CAGE-1 during mouse spermatogenesis. We show that protein CAGE-1 is 849 amino acids long. Analysis of the first spermatogenic wave of pubertal mice by RT-PCR and immunoblotting showed that CAGE-1 is predominantly expressed during postmeiotic stages. CAGE-1 localizes to the acrosomal matrix and acrosomal granule, as demonstrated by immunocytochemistry at the light and electron microscopic level. Taken together, our results allowed to define protein CAGE-1 as a novel component of the acrosome of mammalian spermatids and spermatozoa.     

Apoptosis is physiologically restricted to a specialized cytoplasmic compartment in rat spermatids

Cytoplasmic caudal tags of maturing spermatids condense and are detached from the spermatidal cells just before the spermatids are released as spermatozoa. The detached cytoplasmic masses are termed "residual bodies." Features of residual bodies seem to be compatible with those of apoptosis and, just as occurs with apoptotic bodies, residual bodies are phagocytosed by Sertoli cells. Since in vitro studies have demonstrated that nucleus and cytoplasm apoptosis events can be independent phenomena, we reasoned that apoptosis pathways might be restricted to the caudal tag of the maturing spermatids in order to originate residual bodies. Consistent with this idea, here we showed that annexin V specifically bound the membranes of isolated residual bodies and that expression levels of caspase-1, c-jun, p53, and p21 were specifically increased in these cytoplasmic compartments. Electron microscopy of cytoplasmic lobes and residual bodies confirmed that their ultrastructural features were those of apoptosis. These data indicate that the mechanism responsible for the formation of residual bodies is similar to that for apoptotic bodies; and the study presents evidence, for the first time, that apoptotic signaling molecules can be restricted to a cytoplasmic compartment and proceed in the presence of a healthy nucleus.     

Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: a novel class of cytoskeletal elements

The nuclei of bovine spermatids and spermatozoa are surrounded by dense cytoplasmic webs sandwiched between the nuclear envelope and the acrosome and plasma membrane, respectively, filling most of the cytoplasmic space of the sperm head. This web contains a complex structure, the perinuclear theca, which is characterized by resistance to extractions in nondenaturing detergents and high salt buffers, and can be divided into two major subcomponents, the subacrosomal layer and the postacrosomal calyx. Using calyces isolated from bull and rat spermatozoa we have identified two kinds of basic proteins as major constituents of the thecal structure and have localized them by specific antibodies at the light and electron microscopic level. These are an Mr 60,000 protein, termed calicin, localized almost exclusively to the calyx, and a group of multiple-band polypeptides (MBP; Mr 56,000-74,000), which occur in both the calyx and the subacrosomal layer. The polypeptides of the MBP group are immunologically related to each other, but unrelated, by antibody reactions and peptide maps, to calicin. We show that these basic cytoskeletal proteins are first detectable in the round spermatid stage. As we have not detected any intermediate filament proteins and proteins related to nuclear lamins of somatic cells in sperm heads, we conclude that the perinuclear theca and its constituents, calicin and MBP proteins, are the predominant cytoskeletal elements of the sperm head. Immunologically cross-reacting polypeptides with similar properties have been identified in the heads of rat and human spermatozoa. We speculate that these insoluble basic proteins contribute, during spermiogenesis, to the formation of the perinuclear theca as an architectural element involved in the shape changes and the intimate association of the nucleus with the acrosome and the plasma membrane.     

Calreticulin is present in the acrosome of spermatids of rat testis.

We have already succeeded in purifying a calcium-binding protein (CalBP) from rat spermatogenic cells [Nakamura et al., Biochem. Biophys. Res. Commun., 176 (1991) 1358]. In this study, the location of this protein within rat testis was examined, using a rabbit antisera for this protein. The antigen was localized on the developing acrosomes during spermiogenesis. The NH2-terminal amino acid sequence obtained for rat CalBP was identical to that of calreticulin obtained for the skeletal muscle of mice and closely resembled that for rabbit calreticulin. On the immunoblot analysis, the purified rat CalBP reacted with an antibody raised against rabbit skeletal muscle calreticulin. The results indicate that calreticulin is present in the acrosome of spermatids of rat testes.     

Formation of the perinuclear theca in spermatozoa of diverse mammalian species: relationship of the manchette and multiple band polypeptides

The perinuclear theca is a novel cytoskeletal consisting of a densely layered lamina that surrounds the nucleus of mammalian sperm. Using antibodies specific for the multiple band polypeptides present in the perinuclear theca of bull sperm, we show that a heterogeneous group of immunological related proteins are present in the sperm heads of other mammals with greatly different morphologies, including guinea pig, hamster, rat, and mouse. In none of the species were identical groups of immunoreactive polypeptides found, although immunoreactive proteins of molecular weights 65,000 to 80,000 were present in the sperm heads of all species examined. Immunoreactive proteins less than Mr 55,000 were prominent in rat sperm heads and mouse sperm: guinea pig, hamster, and rat sperm heads and mouse sperm had one band in common at approximately Mr 50,000. Different immunoreactive proteins were present in isolated sperm tails. The perinuclear theca first appeared in the subacrosomal space of round to elongating spermatids. Later, with the caudal movement of the manchette, the postacrosomal segment of the perinuclear theca was deposited in a cephalad to caudal direction along the sperm nucleus. Concomitantly, the cytoplasmic space between the nuclear envelope and the plasma membrane narrowed such that only the theca occupied this portion of the sperm head. Immunoreactivity accompanied the ultrastructural appearance of the subacrosomal layer and the postacrosomal segment. The periods of spermiogenesis, in which sub- and post-acrosomal components of the perinuclear theca are formed and the morphogenesis of sperm organelles with which these elements are associated, suggest that components of this cytoskeletal structure function to join the acrosome and the postacrosomal plasma membrane to the nucleus.     

Talin is a post-synaptic component of the rat neuromuscular junction

Talin is a protein, recently discovered in chicken gizzard, which occurs at sites of actin-plasma membrane interaction in several cell types. Vinculin also occurs at many of these sites, possibly in association with talin. In this study, three antisera against talin were used to probe the neuromuscular junction of rat skeletal muscle, which is also a site of vinculin accumulation. By immunofluorescence, all three sera stained the junction strongly in frozen sections of rat diaphragm. The extrajunctional periphery was lightly and irregularly stained in some muscle cells; others seemed not to be stained outside the junction. Staining remained at junctions and increased in extrajunctional regions of muscle denervated 6 weeks before sacrifice. The staining in all cases was abolished by competition with purified talin. One serum tested by immunoblotting recognized one protein at Mr 215 000 (identical with the value for chicken gizzard talin) and traces of a second at Mr 190 000 (corresponding to a known proteolytic fragment of talin). We conclude that rat muscle talin is similar in its general protein structure to chicken gizzard talin, and is a post-synaptic component of the neuromuscular junction.     

Drosophila filamin encoded by the cheerio locus is a component of ovarian ring canals.

BACKGROUND: The ring canals in the ovary of the fruit fly Drosophila provide a versatile system in which to study the assembly and regulation of membrane-associated actin structures. Derived from arrested cleavage furrows, ring canals allow direct communication between cells. The robust inner rim of filamentous actin that attaches to the ring-canal plasma membrane contains cytoskeletal proteins encoded by the hu-li-tao shao (hts) and kelch genes, and is regulated by the Src64 and Tec29 tyrosine kinases. Female sterile cheerio mutants fail to recruit actin to ring canals, disrupting the flow of cytoplasm to oocytes. RESULTS: We have cloned cheerio and found that it encodes a member of the Filamin/ABP-280 family of actin-binding proteins, known to bind transmembrane proteins and crosslink actin filaments into parallel or orthogonal arrays. Antibodies to Drosophila Filamin revealed that Filamin is an abundant ring-canal protein and the first known component of both the outer and inner rims of the ring canal. The cheerio gene also encodes a new Filamin isoform that lacks the actin-binding domain. CONCLUSIONS: Localization of Filamin to nascent ring canals is necessary for the recruitment of actin filaments. We propose that Filamin links filamentous actin to the plasma membrane of the ring canal. Although loss of Filamin in human cells supports a role for Filamin in organizing orthogonal actin arrays at the cell cortex, the cheerio mutant provides the first evidence that Filamin is required in membrane-associated parallel actin bundles, such as those found in ring canals, contractile rings and stress fibers.     

The murine heterochromatin protein M31 is associated with the chromocenter in round spermatids and Is a component of mature spermatozoa

In mature sperm the normal nucleosomal packaging of DNA found in somatic and meiotic cells is transformed into a highly condensed form of chromatin which consists mostly of nucleoprotamines. Although sperm DNA is highly condensed it is nevertheless packaged into a highly defined nuclear architecture which may be organized by the heterochromatic chromocenter. One major component of heterochromatin is the heterochromatin protein 1 which is involved in epigenetic gene silencing. In order to investigate the possible involvement of heterochromatin protein in higher order organization of sperm DNA we studied the localization of the murine homologue of heterochromatin protein 1, M31, during chromatin reorganization in male germ cell differentiation. Each cell type in the testis showed a unique distribution pattern of M31. Colocalization to the heterochromatic regions were found in Sertoli cells, in midstage pachytene spermatocytes, and in round spermatids in which M31 localizes to the centromeric chromocenter. M31 cannot be detected in elongated spermatids or mature spermatozoa immunocytologically, but could be detected in mature spermatozoa by Western blotting. We suggest that M31, a nuclear protein involved in the organization of chromatin architecture, is involved in higher order organization of sperm DNA.     

The major component of the rat sperm fibrous sheath is a phosphoprotein

The fibrous sheath from rat epididymal sperm was isolated by sequential extraction, first with Triton X-100 and dithiothreitol, and then with 6 M urea and dithiothreitol. The latter extraction procedure solubilized most of the sperm components, leaving the head and the fibrous sheath as the only intact structures. This material was purified by sucrose gradient centrifugation. Electron microscopy confirmed the purity of the isolated material and revealed the characteristic structural features of the fibrous sheath. Polyacrylamide gel electrophoresis (in the presence of sodium dodecyl sulfate) of the fibrillar material, showed a complex polypeptide composition. The polypeptides with molecular weights of 80,000, 24,000, and 11,500 accounted for about 65% of the total protein of the fibrous sheath. Peptide map analyses indicated that the components of molecular weights of 80,000 and 24,000 are unrelated to the polypeptides of similar size of the outer dense fibers. On the other hand, it appears that the fibrous sheath and the outer dense fibers share the polypeptide of 11,500 daltons. The component of 80,000 daltons contains on the average about 3 mol of phosphoserine per mol of polypeptide, indicating that the most abundant polypeptide of the fibrous sheath is a phosphoprotein.