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.     

Linkage of manchette microtubules to the nuclear envelope and observations of the role of the manchette in nuclear shaping during spermiogenesis in rodents.

Structural features of the mouse and rat manchette and the role of the manchette in shaping the spermatid nucleus were investigated. Rod-like elements about 10 nm in diameter and 40-70 nm in length were seen linking the innermost microtubules of the manchette and the outer leaflet of the nuclear envelope in step 8 through step 11 rat and mouse spermatids that either had been routinely fixed for electron microscopy or had been isolated and detergent extracted. Rod-like linkers were also seen joining the nuclear ring to the plasma membrane and nuclear envelope. These linkers may ensure that under normal conditions the manchette remains in a defined position relative to these membranous components. A variety of compounds (taxol, cytoxan, and 5-fluorouracil) were found to perturb the manchette and to affect nuclear shaping. In addition, sys and azh mutant mice were used to determine the consequences of defective manchette formation. These genetic conditions and chemical treatments either produced manchettes that were not in their normal position (azh, sys, and taxol) and/or caused the manchette to appear abnormal (azh, sys, cytoxan, 5-fluorouracil, and taxol), and all resulted in a deformation of the step 9-11 spermatid nucleus. In all instances where the manchette was present, either in normal or ectopic locations, the sectioned nuclear envelope was parallel to the long axis of the microtubules of the manchette. In general, areas of the nuclear envelope where the manchette was not present, or where it was expected to be present but was not, were rounded (normal animals, sys, cytoxan). In addition, there are indications using certain compounds (cytoxan and 5-fluorouracil) as well as in the azh and sys mouse that the manchette may exert pressure to deform the nucleus. It is suggested that the rod-like linkages of the manchette ensure that the nuclear envelope remains at a constant distance from the manchette microtubules and that this is a major factor acting to impart nuclear shape changes on a region of the head caudal to the acrosome during the early elongation phase of spermiogenesis. The manchette microtubules, which are also known to be linked together, may act as a scaffold to deform this part of the nucleus from its spherical shape, perhaps in concert with forces initiated by other structural elements. Evidence from sys animals indicates that structural elements, such as the acrosomal complex over the anterior head (acrosome-actin-nuclear envelope), may affect nuclear shaping over the acrosome-covered portion of the spermatid head.(ABSTRACT TRUNCATED AT 400 WORDS)     

The early development of the tail and the transformation of the shape of the nucleus of the spermatid of the domestic fowl,Gallus gallus

Summary The differentiation of the spermatid, especially in reference to the formation of the flagellum, and transformation of the shape of the nucleus was investigated in the domestic fowl.In the early stage of the spermatid, a prominent Golgi apparatus appears around the centrioles. The Golgi vesicles then surround the axial-filament complex which develops from the distal centriole. These vesicles fuse to form continuous membrane at the earliest stage of flagellar formation, and in the succeeding stage Golgi lamellae are attached to the plasma membrane of the developing flagellum. From these observations, it is assumed that Golgi apparatus may be a source of the membrane system of the flagellum.The microtubules distributed around the nucleus form the circular manchette. The anterior region of the nucleus with the manchette is cylindrical in shape and the posterior region without it remains irregular in shape. When the circular manchette has been completed, the whole nucleus acquires a slender cylindrical shape. The circular manchette then changes into the longitudinal manchette. The nuclei of spermatids without a longitudinal manchette are abnormal in shape. In view of these observations it is assumed that the nuclear shaping of the spermatid may be accomplished by circular manchette and the maintenance of shape of the elongated nucleus by longitudinal manchette.The authors wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices     

Long link induction between the microtubules of the manchette in intermediate stages of spermiogenesis

Summary Links unusual for their length and variable morphology have been described between manchette microtubules in late stages of rat spermiogenesis. In earlier stages of rat spermiogenesis obvious links longer than 160 Å are rare, the majority being 80 or 120 Å in length. The most easily discernible geometric pattern in cross-sections of assemblies of manchette microtubules in intermediate stages of rat spermiogenesis is that of linear arrays sometimes resulting in long and irregularly folded chains of closely linked microtubules. Colcemid disrupts these arrays and is responsible for the formation of more complex geometric patterns. Six hours after drug administration the manchette is dramatically reduced in length. Sheet-like links of variable dimensions and >160 Å in length interconnect not only microtubules but C-type microtubules as well as other links. These links are similar in morphology to those found in later stages of rat spermiogenesis. It is suggested that the formation of these links may perhaps be dependent upon aspects of microtubule disassembly.Supported by a grant to E. A. MacKinnon by the Medical Research Council of Canada.     

Microtubule configurations and post-translational alpha-tubulin modifications during mammalian spermatogenesis

The mechanisms underlying cell cycle progression and differentiation are tightly entwined with changes associated in the structure and composition of the cytoskeleton. Mammalian spermatogenesis is a highly intricate process that involves differentiation and polarization of the round spermatid. We found that pachytene spermatocytes and round spermatids have most of the microtubules randomly distributed in a cortical network without any apparent centrosome. The Golgi apparatus faces the acrosomal vesicle and some microtubules contact its surface. In round spermatids, at step 7, there is an increase in short microtubules around and over the nucleus. These microtubules are located between the rims of the acrosome and may be the very first sign in the formation of the manchette. This new microtubular configuration is correlated with the beginning of the migration of the Golgi apparatus from the acrosomal region towards the opposite pole of the cell. Next, the cortical microtubules form a bundle running around the nucleus perpendicular to the main axis of the cell. At later stages, the nuclear microtubules increase in size and a fully formed manchette appears at stage 9. On the other hand, acetylated tubulin is present in a few microtubules in pachytene spermatocytes and in the axial filament (precursor of the sperm tail) in round spermatids. Our results suggest that at step 7, the spermatid undergoes a major microtubular reordering that induces or allows organelle movement and prepares the cell for the formation of the manchette and further nuclear shaping. This new microtubular configuration is associated with an increase in short microtubules over the nucleus that may correspond to the initial step of the manchette formation. The new structure of the cytoskeleton may be associated with major migratory events occurring at this step of differentiation.     

A New Type of Manchette Structure in Spermatids of Culex tigripes(Culicidae)

Abstract In young spermatids of Culex tigripes, microtubules organize a manchette which surrounds the nucleus. When the nucleus elongates, 1–5 expansions appear on the wall of the microtubules. They grow and branch out while the nucleus elongates and chromatin condenses. Expansions are straight or curved in shape. They have the same thickness as the microtubule walls, but they rarely constitute links between microtubules. The manchette disappears naturally at the end of spermiogenesis. The action of colchicine on spermatids leads to the complete disappearance of the microtubules and expansions, and inhibits the lengthening of the nucleus.     

Cytodifferentiation during spermiogenesis in Lumbricus terrestris

The structural changes during spermiogenesis were studied on developing spermatids in seminal vesicles and receptacles of Lumbricus terrestris fixed in glutaraldehyde-osmium tetroxide and embedded in Epon-Araldite. The centriole plays a prominent role in the morphogenesis and organization of the microtubules of the manchette and flagellum. Microtubules arising from the centriole extend anteriorly to encase the developing middle piece, the nucleus, and the acrosome. The manchette not only provides a supporting framework for the cell during elongation, but also may provide the motive force for the elimination of both nucleoplasm and cytoplasm. The manchette participates in segregation and elimination of the nuclear vesicle that contains the nonchromatin nucleoplasm. Compartmentalization and conservation may also be a function of the manchette since those elements which remain within the framework of microtubules are retained, while all the cytoplasm outside the manchette is discarded. At maturation, the endoplasmic reticulum plays a key role in dismantling the manchette and reducing the cytoplasm external to it. During the early stages of middle-piece formation, six ovoid mitochondria aggregate at the posterior pole of the spermatid nucleus. Concurrent with manchette formation, the mitochondria are compressed laterally into elongate wedge-shaped components, and their outer limiting membranes fuse to form an hexagonal framework that surrounds the dense intramitochondrial matrices. Dense glycogen granules are arranged linearly between the peripheral flagellar tubules and the outer membrane of the mature sperm tail.     

Novel aspect of perinuclear theca assembly revealed by immunolocalization of non-nuclear somatic histones during bovine spermiogenesis

The perinuclear theca (PT) is an important accessory structure of the sperm head, yet its biogenesis is not well defined. To understand the developmental origins of PT-derived somatic histones during spermiogenesis, we used affinity-purified antibodies against somatic-type histones H3, H2B, H2A, and H4 to probe bovine testicular tissue using three different immunolocalization techniques. While undetectable in elongating spermatid nuclei, immunoperoxidase light microscopy showed all four somatic histones remained associated to the caudal head region of spermatids from steps 11 to 14 of the 14 steps in bovine spermiogenesis. Immunogold electron microscopy confirmed the localization of somatic histones on two nonnuclear structures, namely transient manchette microtubules of step-9 to step-11 spermatids and the developing postacrosomal sheath of step-13 and -14 spermatids. Immunofluorescence demonstrated somatic histone immunoreactivity in the developing postacrosomal sheath, and on anti-beta-tubulin decorated manchette microtubules of step-12 spermatids. Focal antinuclear pore complex labeling on the base of round spermatid nuclei was detected by electron microscopy and immunofluorescence, occurring before the nucleoprotein transition period during spermatid elongation. This indicated that, if nuclear histone export precedes their degradation, this process could only occur in this region, thereby questioning the proposed role of the manchette in nucleocytoplasmic trafficking. Somatic histone immunodetection on the manchette during postacrosomal sheath formation supports a role for the manchette in PT assembly, signifying that some PT components have origins in the distal spermatid cytoplasm. Furthermore, these findings suggest that somatic histones are de novo synthesized in late spermiogenesis for PT assembly.     

Sperm head shaping in ratites: New insights,yet more questions

Head shaping in mammalian sperm is regulated by a number of factors including acrosome formation, nuclear condensation and the action of the microtubular manchette. A role has also been suggested for the attendant Sertoli cells and the perinuclear theca (PT). In comparison, relatively little information is available on this topic in birds and the presence of a PT per se has not been described in this vertebrate order. This study revealed that a similar combination of factors contributed to head shaping in the ostrich, emu and rhea, although the Sertoli cells seem to play a limited role in ratites. A fibro-granular structure analogous to the mammalian PT was identified, consisting of sub- and post-acrosomal components. The latter was characterized by stage-specific finger-like projections that appeared to emanate from the cytoplasmic face of the nuclear envelope. They were particularly obvious beneath the base of the acrosome, and closely aligned, but not connected to, the manchette microtubules. During the final stages of chromatin condensation and elongation of the sperm head the projections abruptly disappeared. They appear to play a role in stabilizing the shape of the sperm head during the caudal translocation of the spermatid cytoplasm.     

Ultrastructural differentiation of spermiogenesis in Scincus scincus (Scincidae,Reptilia)

BackgroundKnowledge of spermiogenesis in reptiles, especially in lizards, is very limited. Lizards found in Arabian deserts have not been considered for detailed studies due to many reasons and the paucity of these animals. Therefore, we designed a study on the differentiation and morphogenesis of spermiogenesis, at an ultrastructural level, in a rare lizard species, Scincus scincus.ResultsThe spermiogenesis process includes the development of an acrosomal vesicle, aggregation of acrosomal granules, formation of subacrosomal nuclear space, and nuclear elongation. A role for manchette microtubules was described in nuclear shaping and organelle movement. During head differentiation, the fine granular chromatin of the early spermatid is gradually replaced by highly condensed contents in a process called chromatin condensation. Furthermore, ultrastructural features of sperm tail differentiation in S. scincus were described in detail. The commencement was with caudal migration toward centrioles, insertion of the proximal centriole in the nuclear fossa, and extension of the distal centrioles to form the microtubular axoneme. Subsequently, tail differentiation consists of the enlargement of neck portion, middle piece, the main and end pieces.ConclusionsThis study aids in the understanding of different aspects of spermiogenesis in the lizard family and unfurls evolutionary links within and outside reptiles.     

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.     

Microtubules in the spermatid of the rabbit

Summary In the cytology of the developing rabbit spermatid two systems of microtubules are conspicuous features. One corresponding to the manchette of light microscopy is in correlation with changes in the nuclear membrane giving rise to the posterior nuclear space. This system disappears as the elongation of the nucleus is accomplished without leaving any remnants. In the late spermatid another system of microtubules is observed just anterior to the fibrous sheath inside the periflagellar invagination giving this part of the flagellum a spindle shaped form. — The possible functions of the microtubules and related cellular events are discussed.This investigation was supported by United States Public Health Service, National Institutes of Health (HD 02493) and the Lalor Foundation.     

Development of the sperm head in the caddis fly Potamophylax rotundipennis (Insecta,Trichoptera)

The restructuring of the sperm head has been examined in a caddis fly, Potamophylax rotundipennis (Limnephilidae), using light and electron microscopy. The roughly spherical nuclei of young spermatids are transformed into needle-shaped elements in advanced spermatids. During this process, the nuclei transiently become sickle-shaped. Prominent structural changes occur within the nucleus during spermiogenesis. The chromatin of spherical and slightly elongated nuclei has an amorphous appearance, then coarse granules become apparent, chromatin threads are visible in fully elongated nuclei and finally lamellar elements appear. During the changes in chromatin texture, a dense layer, the chromatin rim, develops transiently. This feature of the chromatin surface is interpreted as the structural expression of exchanges between nucleus and cytoplasm. A microtubular manchette is formed at the cytoplasmic face of the nuclear envelope. Whereas the manchette covers the full perimeter of the nucleus in early stages of elongation, gaps in the palisade of microtubules appear before the nuclear diameter decreases and needle-shaped nuclei develop. It is possible that the intermittent deployment of manchette microtubules is involved in reducing the nuclear diameter towards the end of nuclear elongation. The delayed detachment of the chromatin from the posterior pole of the nucleus, observed at the onset of nuclear clongation, points to local modifications of the nuclear envelope responsible for the connection of the centriole adjunct and the flagellum with the posterior pole of the nucleus.     

TLRR (lrrc67) interacts with PP1 and is associated with a cytoskeletal complex in the testis

Background information. Spermatozoa are formed via a complex series of cellular transformations, including acrosome and flagellum formation, nuclear condensation and elongation and removal of residual cytoplasm. Nuclear elongation is accompanied by the formation of a unique cytoskeletal structure, the manchette. We have previously identified a leucine‐rich repeat protein that we have named TLRR (testis leucine‐rich repeat), associated with the manchette that contains a PP1 (protein phosphatase‐1)‐binding site. Leucine‐rich repeat proteins often mediate protein–protein interactions; therefore, we hypothesize that TLRR acts as a scaffold to link signalling molecules, including PP1, to the manchette near potential substrate proteins important for spermatogenesis. Results. TLRR and PP1 interact with one another as demonstrated by co‐immunoprecipitation and the yeast two‐hybrid assay. TLRR binds more strongly to PP1γ2 than it does to PP1α. Anti‐phosphoserine antibodies immunoprecipitate TLRR from testis lysate, indicating that TLRR is a phosphoprotein. TLRR is part of a complex in testis that includes cytoskeletal proteins and constituents of the ubiquitin–proteasome pathway. The TLRR complex purified from 3T3 cells contains similar proteins, co‐localizes with microtubules and is enriched at the microtubule‐organizing centre. TLRR is also detected near the centrosome of elongated, but not mid‐stage, spermatids. Conclusion. We demonstrate here that TLRR interacts with PP1, particularly the testis‐specific isoform, PP1γ2. Immunoaffinity purification confirms that TLRR is associated with the spermatid cytoskeleton. In addition, proteins involved in protein stability are part of the TLRR complex. These results support our hypothesis that TLRR links signalling molecules to the spermatid cytoskeleton in order to regulate important substrates involved in spermatid transformation. The translocation of TLRR from the manchette to the centrosome region suggests a possible role for this protein in tail formation. Our finding that TLRR is associated with microtubules in cultured cells suggests that TLRR may play a common role in modulating the cytoskeleton in other cell types besides male germ cells.     

Nuclear morphogenesis during spermiogenesis in the nudibranch molluscHypselodoris tricolor (Gastropoda,opisthobranchia)

An electron microscope study was carried out on Hypselodoris tricolor spermatids to describe the development of the nuclear morphogenesis and investigate the possible cause(s) of the change in the shape of the spermatid nucleus during spermiogenesis. Three different stages may be distinguished in the course of the nuclear morphogenesis on the basis of the morphology and inner organization of the nucleus. Stage 1 spermatid nuclei are spherical or ovoid in shape and the nucleoplasm finely granular in appearance. Stage 2 nuclei exhibit a disc- or cup-shaped morphology, and the chromatin forms short, thin filaments. During stage 3, a progressive nuclear elongation takes place, accompanied by chromatin rearrangement, first into fibers and then into lamellae, both formations helically oriented. A row of microtubules attached to the nuclear envelope completely surrounds the nucleus. Interestingly, the microtubules always lie parallel to the chromatin fibers adjacent to them. Late stage 3 spermatids show the highest degree of chromatin condensation and lack the manchette at the end of spermiogenesis. Our findings indicate the existence of a clear influence exerted on the chromatin by the manchette microtubules, which appear to be involved in determining the specific pattern of chromatin condensation in Hypselodoris tricolor.     

Ultrastructure of spermiogenesis in the Cottonmouth,Agkistrodon piscivorus (Squamata: Viperidae: Crotalinae)

To date multiple studies exist that examine the morphology of spermatozoa. However, there are limited numbers of data detailing the ontogenic characters of spermiogenesis within squamates. Testicular tissues were collected from Cottonmouths (Agkistrodon piscivorus) and tissues from spermiogenically active months were analyzed ultrastructurally to detail the cellular changes that occur during spermiogenesis. The major events of spermiogenesis (acrosome formation, nuclear elongation/DNA condensation, and flagellar development) resemble that of other squamates; however, specific ultrastructural differences can be observed between Cottonmouths and other squamates studied to date. During acrosome formation vesicles from the Golgi apparatus fuse at the apical surface of the nuclear membrane prior to making nuclear contact. At this stage, the acrosome granule can be observed in a centralized location within the vesicle. As elongation commences the acrosome complex becomes highly compartmentalized and migrates laterally along the nucleus. Parallel and circum‐cylindrical microtubules (components of the manchette) are observed with parallel microtubules outnumbering the circum‐cylindrical microtubules. Flagella, displaying the conserved 9 + 2 microtubule arrangement, sit in nuclear fossae that have electron lucent shoulders juxtaposed on either side of the spermatids basal plates. This study aims to provide developmental characters for squamates in the subfamily Crotalinae, family Viperidae, which may be useful for histopathological studies on spermatogenesis in semi‐aquatic species exposed to pesticides. Furthermore, these data in the near future may provide morphological characters for spermiogenesis that can be added to morphological data matrices that may be used in phylogenetic analyses. J. Morphol. 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Characteristics of the sperm tail of the velvet worm, Euperipatoides leuckarti (Onychophora)

The onychophoran sperm tail contains several kinds of microtubulcs; probably more than that of any other animal group. There are thus a peripheral manchette consisting of many tightly spaced microtubules, a ring of nine 'peripheral singlets' and a central axoneme of the classical 9 + 2 type (nine doublets and two central singlets). The protofilament organization of these various microtubules was examined and compared to the structure and mode of formation of the peripheral singlets with that of its analogues in other animal groups. The onychophoran peripheral singlets were found to differ in two respects from those in insects: they are formed from the manchette rather than from the axonemal doublets and their transient connection to the axoneme is to the A-subtubules of the doublet rather than to the B-subtubules. The manchette microtubules as well as the peripheral singlets consist of 13 protoh'laments. The manchette may serve a mechanical function (to strengthen the unusually thick sperm tail) hut the role of the peripheral singlets remains unknown.     

Perinuclear cytoskeleton of acrosome-less spermatids in the blind sterile mutant mouse.

The perinuclear cytoskeleton of mammalian spermatids is thought to play a major role in nucleus-acrosome association and in shape changes of the head during spermiogenesis. To test these hypotheses acrosome-less spermatids in blind-sterile mutant mice were investigated for the development of the subacrosomal layer. Immunogold procedures were used for the detection of actin and calmodulin. In addition to various other abnormalities many acrosome-less round and elongating spermatids developed a subacrosomal layer with an actin and calmodulin distribution similar to that observed in normal spermatids. However, in mutant elongating spermatids the apical part of the nucleus was truncated and/or folded. The expected elongation and shaping of the nucleus only occurred in its caudal part associated with an hypertrophied and somewhat ectopic manchette. These abnormalities and those previously observed in mutant and experimental models indicated that the subacrosomal layer may form independently of the acrosome. It is suggested that the subacrosomal filamentous actin is a transitory scaffolding which might be involved in the assemblage of other proteins of the perinuclear cytoskeleton. However, by itself, this layer is not sufficient to ensure a normal shaping of the nucleus. Acrosome-nucleus interactions mediated by the subacrosomal layer seem necessary to shape the cranial spermatid head. The manchette appears to be involved only in the caudal nuclear shaping.