Structural and biochemical features of fractionated spermatid manchettes and sperm axonemes of the azh/azh mutant mouse

The tubulin-containing axoneme and manchette develop consecutively during mammalian spermiogenesis. The nature of their molecular components and developmental sequence are not completely known. The azh/azh (for abnormal sperm headshape) mouse mutant is an ideal model for analyzing tubulin isotypes and microtubule-associated proteins of the manchette and axoneme in light of a potential role of the manchette in the shaping of the sperm head and formation of the tail. We have searched for possible differences in tubulin isotype variants in fractionated manchettes and axonemes of wildtype and azh/azh mutant mice using isotype-specific tubulin antibodies as immunoprobes. Manchettes from wild-type and azh/azh mutant mouse spermatids were fractionated from spermatogenic stage-specific seminiferous tubules and axonemes were isolated from epididymal sperm. We have found that: (1) Fractionated manchettes of azh/azh mutants are longer than in wild-type mice; (2) Manchette and sperm tail axonemes display a remarkable variety of posttranslationally modified tubulins (acetylated, glutamylated, tyrosinated, alpha-3/7 tubulins). Acetylated tubulin was more abundant in manchette than in axonemes; (3) An acidic 62 kDa protein was identified as the main component of the perinuclear ring of the manchette in wild-type and azh/azh mice; (4) Bending and looping of the mid piece of the tail of azh/azh sperm, accompanied by a dislocation of the connecting piece from head attachment sites, were visualized by phase-contrast, immunofluorescence and transmission electron microscopy in about 35% of spermatids/sperm; and (5) A lasso-like tail configuration was predominant in epididymal sperm of azh/azh mutants. We speculate that spermatid and sperm tail abnormalities in the azh/azh mutant could reflect structural and/or assembly deficiencies of peri-axonemal proteins responsible for maintaining a stiffened tail during spermiogenesis and sperm maturation.     

Offspring from normal mouse oocytes injected with sperm heads from the azh/azh mouse display more severe sperm tail abnormalities than the original mutant

Sperm with abnormalities in the position and shape of the head were obtained from the azh/azh mutant and injected into the cytoplasm of mature mouse oocytes to determine whether sperm from the offspring display both head (club shape) and tail (looping, folding, and fusion) abnormalities observed in the mutant donor. Although quantitative differences were observed among the three examined offspring, we found that abnormalities in sperm head shape were less frequent than in the donor mutant, but that tail malformations predominated. In addition, we found that the frequency of tail abnormalities increased during sperm epididymal transit. A typical defect was the multiple folding of the sperm tail and eventual fusion of closely apposed plasma membranes. As a consequence, sperm forward motility and natural fertility were compromised. Results of this study indicate that the azh/azh mutant and offspring generated by intracytoplasmic sperm injection provide a valuable model for determining the role of the manchette and keratin-containing outer dense fibers and fibrous sheath during spermiogenesis. Furthermore, our findings stress the risk of enhancing a phenotypic abnormality caused by mutant male genotypes introduced through bypassing the biologic mechanisms of natural sperm selection during fertilization.     

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.     

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)     

Mapping of the azh locus to mouse chromosome 4.

The azh (abnormal spermatozoon headshape) mutation in the mouse, which results in abnormal sperm head formation, was demonstrated to display an autosomal recessive pattern of inheritance. The azh locus was mapped by crossing mice with the mutation on a relatively pure C57BL/6J(B6) background with C3H/HeKam and backcrossing the F1 mice to B6-azh/azh mice. Up to 60 backcross progeny were typed for azh, by microscopic examination of sperm heads, and for other markers. Eleven loci on chromosomes other than 4 showed no significant linkage with azh. Glucose 6-phosphate dehydrogenase-1 (Gpd-1), located on the distal part of chromosome 4, showed 26% recombination frequency with azh, indicating significant linkage (P less than .001). Linkage with an anonymous DNA probe for the D4Rp1 locus in the central region of chromosome 4 was then analyzed, and only a 5% recombination frequency was observed. The map location indicates that azh is distinct from other known mutations that also result in abnormal sperm heads.     

Intracytoplasmic sperm injection effects in infertile azh mutant mice

Several reports in the literature describe men with infertility resulting from abnormal sperm head shape or decapitation defects of their spermatozoa. These defects are similar to those shown for the spermatozoa from azh (abnormal spermatozoon head shape) mice. The present study examines the efficiency and effects of intracytoplasmic sperm injection (ICSI) in successive generations of azh mice generated with this method. Three successive generations of azh mice were produced with ICSI. In all three ICSI series, more than 80% of 2-cell embryos were obtained, and more than 35% of embryos transferred gave rise to normal live offspring. In addition, ICSI was used to cross homozygous azh/azh males with homozygous azh/azh females, and live offspring were obtained. The ICSI-derived males were tested for their fecundity and abnormalities of sperm morphology. Spermatozoa from ICSI-derived azh/+ males did not show any impairment of fecundity in in vitro fertilization. These spermatozoa successfully fertilized oocytes from both C57BL/6 and B6D2F1 females, with fertilization rates ranging from 70%- 92%. The proportion of morphologically normal spermatozoa was similar in azh/+ males from three successive generations of ICSI (57.8%, 54.8%, and 49.0%, respectively), and no differences were noted when comparing ICSI-derived males with males derived by mating (57.6%) and with wild-type controls (61.6%). Detailed analysis differentiating between specific types of anomalies of sperm morphology did not reveal significant differences among the examined groups. The results of the present study demonstrate that ICSI does not enhance the azh mutation phenotype in the offspring and brings no risks when applied continuously. Moreover, serial (successive generations) ICSI is highly efficient in maintaining valuable mice with fertility problems.     

Assembly of spermatid acrosome depends on microtubule organization during mammalian spermiogenesis

The acrosome is a secretory vesicle attached to the nucleus of the sperm. Our hypothesis is that microtubules participate in the membrane traffic between the Golgi apparatus and acrosome during the first steps of spermatid differentiation. In this work, we show that nocodazole-induced microtubule depolarization triggers the formation of vesicles of the acrosomal membrane, without detaching the acrosome from the nuclear envelope. Nocodazole also induced fragmentation of the Golgi apparatus as determined by antibodies against giantin, golgin-97 and GM130, and electron microscopy. Conversely, neither the acrosome nor the Golgi apparatus underwent fragmentation in elongating spermatids (acrosome- and maturation-phase). The microtubule network of round spermatids of azh/azh mice also became disorganized. Disorganization correlated with fragmentation of the acrosome and the Golgi apparatus, as evaluated by domain-specific markers. Elongating spermatids (acrosome and maturation-phase) of azh/azh mice also had alterations in microtubule organization, acrosome, and Golgi apparatus. Finally, the spermatozoa of azh/azh mice displayed aberrant localization of the acrosomal protein sp56 in both the post-acrosomal and flagellum domains. Our results suggest that microtubules participate in the formation and/or maintenance of the structure of the acrosome and the Golgi apparatus and that the organization of the microtubules in round spermatids is key to sorting acrosomal proteins to the proper organelle.     

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.     

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.     

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 single amino acid mutation in the mouse MEIG1 protein disrupts a cargo transport system necessary for sperm formation

Mammalian spermatogenesis is a highly coordinated process that requires cooperation between specific proteins to coordinate diverse biological functions. For example, mouse Parkin coregulated gene (PACRG) recruits meiosis-expressed gene 1 (MEIG1) to the manchette during normal spermiogenesis. Here we mutated Y68 of MEIG1 using the CRISPR/cas9 system and examined the biological and physiological consequences in mice. All homozygous mutant males examined were completely infertile, and sperm count was dramatically reduced. The few developed sperm were immotile and displayed multiple abnormalities. Histological staining showed impaired spermiogenesis in these mutant mice. Immunofluorescent staining further revealed that this mutant MEIG1 was still present in the cell body of spermatocytes, but also that more MEIG1 accumulated in the acrosome region of round spermatids. The mutant MEIG1 and a cargo protein of the MEIG1/PACRG complex, sperm-associated antigen 16L (SPAG16L), were no longer found to be present in the manchette; however, localization of the PACRG component was not changed in the mutants. These findings demonstrate that Y68 of MEIG1 is a key amino acid required for PACRG to recruit MEIG1 to the manchette to transport cargo proteins during sperm flagella formation. Given that MEIG1 and PACRG are conserved in humans, small molecules that block MEIG1/PACRG interaction are likely ideal targets for the development of male contraconception drugs.     

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     

Ultrastructure of spermiogenesis and synthesis of enigmatic cytoplasmic inclusions in the spirally shaped spermatozoon of the polychaete Spio setosa (Annelida: Spionidae)

The sperm of Spio setosa (Polychaeta, Spionidae) are known to be very unusual in form; here, spermiogenesis and the structure of the spermatozoon in this species are described by transmission electron microscopy. While spermiogenesis is similar to that described for many other polychaetes, two notable exceptions to this process include the synthesis of abundant ring‐shaped and tubular, membrane‐bounded cytoplasmic inclusions in the midpiece, and the differentiation of a spirally shaped sperm head. Spermatids develop as free‐floating tetrads in the male's coelom. A microtubular manchette does not develop during chromatin condensation and nuclear elongation, and the spiral acrosome forms as a single Golgi‐derived vesicle that migrates anteriorly to become housed in a deep anterior nuclear fossa. Early in spermiogenesis, numerous Golgi‐derived, membrane‐bounded cytoplasmic inclusions appear in the cytoplasm; these ultimately occupy the sperm midpiece only. The mature spermatozoon in the male has a 15‐μm‐long head consisting of a nucleus coiled like a spring and a spiral acrosome with differentiated substructure, the posterior two thirds of which sits in an anterior nuclear fossa. The midpiece is wider than the rest of the spermatozoon and contains 9–10 spherical mitochondria surrounding the two centrioles, as well as numerous membrane‐bounded conoid and tubular cytoplasmic inclusions. The axoneme has a 9 + 2 arrangement of microtubules. By contrast, stored sperm in the female's seminal receptacles have lost the midpiece inclusions but contain an abundance of glycogen. The function of the midpiece inclusions remains unresolved, and the significance of their absence in stored sperm within the seminal receptacle and the appearance of midpiece glycogen stores remains unclear and requires additional investigation.     

LRGUK-1 Is Required for Basal Body and Manchette Function during Spermatogenesis and Male Fertility

Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.     

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.     

Acroplaxome, an F-actin-keratin-containing plate, anchors the acrosome to the nucleus during shaping of the spermatid head

Nuclear shaping is a critical event during sperm development as demonstrated by the incidence of male infertility associated with abnormal sperm ad shaping. Herein, we demonstrate that mouse and rat spermatids assemble in the subacrosomal space a cytoskeletal scaffold containing F-actin and Sak57, a keratin ortholog. The cytoskeletal plate, designated acroplaxome, anchors the developing acrosome to the nuclear envelope. The acroplaxome consists of a marginal ring containing keratin 5 10-nm-thick filaments and F-actin. The ring is closely associated with the leading edge of the acrosome and to the nuclear envelope during the elongation of the spermatid head. Anchorage of the acroplaxome to the gradually shaping nucleus is not disrupted by hypotonic treatment and brief Triton X-100 extraction. By examining spermiogenesis in the azh mutant mouse, characterized by abnormal spermatid/sperm head shaping, we have determined that a deformity of the spermatid nucleus is restricted to the acroplaxome region. These findings lead to the suggestion that the acroplaxome nucleates an F-actin-keratin-containing assembly with the purpose of stabilizing and anchoring the developing acrosome during spermatid nuclear elongation. The acroplaxome may also provide a mechanical planar scaffold modulating external clutching forces generated by a stack of Sertoli cell F-actin-containing hoops encircling the elongating spermatid nucleus.     

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.     

Spermiogenesis in Polyplacophora,with special reference to acrosome formation (Mollusca)

Summary The present study examines spermiogenesis, and in particular the formation of the acrosome, in ten species of chitons belonging to four families. This study emphasizes the formation of the acrosome but brings to light several other structures that have received little or no mention in previous studies. The process of spermiogenesis is essentially similar in each species, although Chaetopleura exhibits some significant differences. In early spermiogenesis the Golgi body secretes numerous small pro-acrosomal vesicles that gradually migrate into the apical cytoplasm. The chromatin condenses from granules into fibres which become twisted within the nucleus. A small bundle of chromatin fibres projects from the main nuclear mass into the anterior filament; this coincides with the appearance of a developing manchette of microtubules around the nucleus that originates from the two centrioles. Radiating from the distal centriole is the centriolar satellite complex, which is attached to the plasma membrane by the annulus. The distal centriole produces the flagellum posteriorly and it exits eccentrically through a ring of folded membrane that houses the annulus. Extending from the annulus on one side of the flagellum, in all but one species, is a dense fibrous body that has not been previously reported. The proximal centriole lies perpendicular to the end of the distal centriole and is attached to it by fibro-granular material. Pro-acrosomal vesicles migrate anteriorly through the cytoplasm and move into the anterior filament to one side of the expanding nucleus. Eventually these vesicles migrate all the way to the tip of the sperm, where they fuse to form one of two granules in the acrosome. In mature sperm the nucleus is bullet-shaped with a long anterior filament and contains dense chromatin with occasional lacunae. The mitochondria vary in both number and position in the mature sperm of different species. Both centrioles are housed eccentrically in a posterior indentation of the nucleus, where the membranes are modified. The elongate flagellum tapers to a long filamentous end-piece that roughly corresponds to the anterior filament and may be important in sperm locomotion for hydrodynamic reasons. An acrosome is present in all ten species and stained positively for acid phosphatase in three species that were tested.     

Mammalian Fused is essential for sperm head shaping and periaxonemal structure formation during spermatogenesis

During mammalian spermatogenesis, the diploid spermatogonia mature into haploid spermatozoa through a highly controlled process of mitosis, meiosis and post-meiotic morphological remodeling (spermiogenesis). Despite important progress made in this area, the molecular mechanisms underpinning this transformation are poorly understood. Our analysis of the expression and function of the putative serine–threonine kinase Fused (Fu) provides critical insight into key steps in spermatogenesis. In this report, we demonstrate that conditional inactivation of Fu in male germ cells results in infertility due to diminished sperm count, abnormal head shaping, decapitation and motility defects of the sperm. Interestingly, mutant flagellar axonemes are intact but exhibit altered periaxonemal structures that affect motility. These data suggest that Fu plays a central role in shaping the sperm head and controlling the organization of the periaxonemal structures in the flagellum. We show that Fu localizes to multiple tubulin-containing or microtubule-organizing structures, including the manchette and the acrosome–acroplaxome complex that are involved in spermatid head shaping. In addition, Fu interacts with the outer dense fiber protein Odf1, a major component of the periaxonemal structures in the sperm flagellum, and Kif27, which is detected in the manchette. We propose that disrupted Fu function in these structures underlies the head and flagellar defects in Fu-deficient sperm. Since a majority of human male infertility syndromes stem from reduced sperm motility and structural defects, uncovering Fu?s role in spermiogenesis provides new insight into the causes of sterility and the biology of reproduction.