Aberrant distribution of the peri-axonemal structures in the human spermatozoon: possible role of the axoneme in the spatial organization of the flagellar components

A quantitative ultrastructural study combined with stereology was performed on semen samples from four men selected for apparently isolated anomalies of the peri-axonemal structures. Comparison of the results with those of a control group revealed a decrease: in the mean length of the principal piece; in the mean length of the 9 dense fibres and in the difference in length between the 9 dense fibres, all the fibres being approximately as long as the medium length fibres of the normal spermatozoon. In addition, longitudinal columns were single and/or in abnormal position. However, the extent of the dense fibres (along 60% of the principal piece) and their proportion within the flagellum (35.1% of the principal piece per fibre on average) were normal. Analysis of the results suggests that: A-tubules of the axonemal doublets are involved in the spatial arrangement of the peri-axonemal structures; axonemal microtubule-associated proteins (MAPs) may be responsible for this structural function; and three different types of longitudinal doublet differentiations may exist along the flagellar axoneme.     

Male infertility,impaired sperm motility,and hydrocephalus in mice deficient in sperm-associated antigen 6

Gene targeting was used to create mice lacking sperm-associated antigen 6 (Spag6), the murine orthologue of Chlamydomonas PF16, an axonemal protein containing eight armadillo repeats predicted to be important for flagellar motility and stability of the axoneme central apparatus. Within 8 weeks of birth, approximately 50% of Spag6-deficient animals died with hydrocephalus. Spag6-deficient males surviving to maturity were infertile. Their sperm had marked motility defects and was morphologically abnormal with frequent loss of the sperm head and disorganization of flagellar structures, including loss of the central pair of microtubules and disorganization of the outer dense fibers and fibrous sheath. We conclude that Spag6 is essential for sperm flagellar motility and that it is important for the maintenance of the structural integrity of mature sperm. The occurrence of hydrocephalus in the mutant mice also implicates Spag6 in the motility of ependymal cilia.     

THE FLAGELLAR APPARATUS OF THE ZOOSPORE OF UROSPORA PENICILLIFORMIS(CHLOROPHYTA)1

The flagellar apparatus of Urospora penicilliformis (Roth) Aresch. is unique, or at least very unusual among green algae. The flagellar axonemes are rigid, and contain wing-like projections. There are no central microtubules in the most proximal part of the axoneme. The transition region contains a series of electron dense transverse lamellae rather than a single septum, and lacks a stellate pattern. There is no cartwheel pattern in the proximal part of the basal bodies. The latter are associated with four different types of fibrous elements: ascending striated fibers that attach to an electron dense plate in the papillar center, lateral striated fibers that parallel microtubular roots, fibrous elements that link adjacent basal bodies, and finally two massive striated fibers that descend into the cell, passing closely along the nucleus (system II fibers, or rhizoplasts). Each of the four microtubular flagellar roots is sandwiched between two system I striated structures. The roots are probably equal; they contain proximally four, and distally up to eight microtubules. Based on the zoospore flagellar apparatus, it is concluded that the multinucleate U. penicilliformis is related to the Ulvaphyceae. Finally, a possible explanation in functional terms is given for the peculiar external morphology and behavior of the zoospore.     

Ultrastructure of Draparnaldia glomerata (Chaetophorales, Chlorophyceae). 1. The flagellar apparatus of the zoospore

The fine structure of the quadriflagellate zoospores of Draparnaldia glomerata (Vauch.) Agardh is described with emphasis on the flagellar root system and compared with the flagellar apparatus of related green algae. It is demonstrated that the flagellar root system in Draparnaldia is similar to that of the zoospore of Uronema belkae. Common features include presence of a cruciate root system (formula 2–5–2–5), prominent striated distal fibre connecting opposite basal bodies, a system I striated root component associated with the 2–stranded root, association of electron dense material with the 5–stranded root, mode of arrangement of the basal bodies in the absolute configuration model, and presence of four striated peripheral fibres interconnecting adjacent basal bodies. Differences exist in the shape of the striated peripheral fibres, the origin of the 2– and 5– stranded roots in the proximal part of the flagellar apparatus, and the architecture and striation pattern of the proximal part of the system I fibre that detaches from the 2–stranded root between adjacent basal bodies. Both the 2– and 5–stranded roots originate near the basal bodies and descend deeply into the zoospore. One of the 5–stranded roots passes near the eyespot of the chloroplast. The implications of these findings for the taxonomic position of the genus Draparnaldia are discussed. In addition, an evaluation is given of the present status of the order Chaetophorales. Suggestions are given to standardize some aspects of the current terminology of the cruciate flagellar root system in green algae.     

Ultrastructural morphometry of the human sperm flagellum with a stereological analysis of the lengths of the dense fibres

The dimensions of the various regions of the flagellum and the length of each of the dense fibres has been determined by transmission electron microscopy of a large number of spermatozoa from ten men. The overall mean length of the flagellum was 60.5 micron, and its diameter diminished from 0.88 micron in the midpiece to 0.17 micron at the terminal filament. The midpiece and terminal filament as measured in longitudinal sections had variable lengths among spermatozoa (3.4 +/- 0.5 (S.D.) micron and 3.1 +/- 1.0 micron respectively). Stereological analysis was used to estimate the length of the principal piece (53 micron) and the dense fibres. These latter fibres were of unequal length and extended along 60% of the length of the principal piece. They fell into 3 groups with respect to their lengths: (i) fibres 3 and 8 were short (6 micron); (ii) fibres 4, 2 and 7 were of medium length (17, 18 and 21 micron respectively); and (iii) the longest fibres were 5, 6, 9 (31, 32 and 31 micron respectively) and fibre 1 which was a little longer (35 micron). Although there was variation in the length of the various fibres among spermatozoa, the order of their termination was relatively constant. The relationship between these quantitative data regarding the structural characteristics of the dense fibres and the shape of the flagellar wave is discussed.     

Ultrastructural morphometry of the human sperm flagellum with a stereological analysis of the lengths of the dense fibres

The dimensions of the various regions of the flagellum and the length of each of the dense fibres has been determined by transmission electron microscopy of a large number of spermatozoa from ten men. The overall mean length of the flagellum was 60.5 micron, and its diameter diminished from 0.88 micron in the midpiece to 0.17 micron at the terminal filament. The midpiece and terminal filament as measured in longitudinal sections had variable lengths among spermatozoa (3.4 +/- 0.5 (S.D.) micron and 3.1 +/- 1.0 micron respectively). Stereological analysis was used to estimate the length of the principal piece (53 micron) and the dense fibres. These latter fibres were of unequal length and extended along 60% of the length of the principal piece. They fell into 3 groups with respect to their lengths: (i) fibres 3 and 8 were short (6 micron); (ii) fibres 4, 2 and 7 were of medium length (17, 18 and 21 micron respectively); and (iii) the longest fibres were 5, 6, 9 (31, 32 and 31 micron respectively) and fibre 1 which was a little longer (35 micron). Although there was variation in the length of the various fibres among spermatozoa, the order of their termination was relatively constant. The relationship between these quantitative data regarding the structural characteristics of the dense fibres and the shape of the flagellar wave is discussed.     

The flagellar apparatus of the scaly green flagellatePyramimonas obovata: Absolute configuration

Summary The flagellar apparatus of the quadriflagellate scaly green algaPyramimonas obovata has been studied in detail and the absolute configuration of the flagellar apparatus has been determined. The flagellar root system is cruciate (4-2-4-2-system). 18 major basal body associated fibrous structures connect the four basal bodies with each other. Each basal body is linked to an adjacent basal body by a unique set of connecting fibres, i.e., the flagellar apparatus does not exhibit 180° rotational symmetry. The flagellar apparatus ofPyramimonas obovata is compared with that of quadriflagellate motile cells of theChlorophyceae sensu Stewart andMattox and the phylogenetic relationships are discussed.     

The 3D Structure of the Apical Complex and Association with the Flagellar Apparatus Revealed by Serial TEM Tomography in Psammosa pacifica,a Distant Relative of the Apicomplexa

The apical complex is one of the defining features of apicomplexan parasites, including the malaria parasite Plasmodium, where it mediates host penetration and invasion. The apical complex is also known in a few related lineages, including several non-parasitic heterotrophs, where it mediates feeding behaviour. The origin of the apical complex is unclear, and one reason for this is that in apicomplexans it exists in only part of the life cycle, and never simultaneously with other major cytoskeletal structures like flagella and basal bodies. Here, we used conventional TEM and serial TEM tomography to reconstruct the three dimensional structure of the apical complex in Psammosa pacifica, a predatory relative of apicomplexans and dinoflagellates that retains the archetype apical complex and the flagellar apparatus simultaneously. The P. pacifica apical complex is associated with the gullet and consists of the pseudoconoid, micronemes, and electron dense vesicles. The pseudoconoid is a convex sheet consisting of eight short microtubules, plus a band made up of microtubules that originate from the flagellar apparatus. The flagellar apparatus consists of three microtubular roots. One of the microtubular roots attached to the posterior basal body is connected to bypassing microtubular strands, which are themselves connected to the extension of the pseudoconoid. These complex connections where the apical complex is an extension of the flagellar apparatus, reflect the ancestral state of both, dating back to the common ancestor of apicaomplexans and dinoflagellates.     

Comparative ultrastructure of a mucoid strain of Pseudomonas aeruginosa isolated from cystic fibrosis patient and its spontaneous non-mucoid mutant

The ultrastructure of a mucoid strain of Pseudomonas aeruginosa of cystic fibrosis origin and its spontaneous non-mucoid variant was compared by transmission electron microscopy. Negatively-stained preparations of the mucoid strain obtained from plate cultures demonstrated dense, fibrous material projecting from the cell. No such material was observed in thin-sections or in negatively-strained preparations from liquid cultures. Thin-sections of ethanol-precipitated extracellular material from liquid cultures of the mucoid-strain revealed a cottony mesh of thin electron dense fibres. The non-mucoid strain did not produce such material. When prefixed with glutaraldehyde/malachite green mixture, cells of both strains demonstrated electron dense intracellular and extracellular malachite green-stainable structures. The internal complexes were frequently associated with the nucleoid or cell membrane and were replaced by electron transparent areas in cells prefixed with glutaraldehyde alone. Aeruginocins of the R-type were observed in mitomycin C induced cultures of both strains. Bacteriophages with 'claw-shaped' tail-tips were observed in the mucoid strain. Crystalline material was produced by the mucoid strain but only when plated on certain media.     

Structure and significance of cruciate flagellar root systems in green algae: Zoospores ofUlva lactuca (Ulvales,Chlorophyceae)

The ultrastructure of the flagellar apparatus in the quadriflagellate zoospores ofUlva lactuca was examined. The two L-shaped pairs of basal bodies are arranged in mirror image relation. Two apical capping plates connect adjacent basal bodies of different pairs with each other. The flagellar root system is cruciate and exhibits a microtubular part (4-2-4-2 system) and a complex and elaborate fibrillar part. The latter consists of two striated fibres (striation pattern 32 nm) closely associated with the two-stranded roots and four differently patterned fibres (striation pattern 150–160 nm) which are more internally located and run parallel to all four microtubular roots. The presence of four microtubular roots and six striated fibres is at present not known for any other green alga and taxonomic implications are discussed.     

SMP-1, a member of a new family of small myristoylated proteins in kinetoplastid parasites, is targeted to the flagellum membrane in Leishmania

The mechanisms by which proteins are targeted to the membrane of eukaryotic flagella and cilia are largely uncharacterized. We have identified a new family of small myristoylated proteins (SMPs) that are present in Leishmania spp and related trypanosomatid parasites. One of these proteins, termed SMP-1, is targeted to the Leishmania flagellum. SMP-1 is myristoylated and palmitoylated in vivo, and mutation of Gly-2 and Cys-3 residues showed that both fatty acids are required for flagellar localization. SMP-1 is associated with detergent-resistant membranes based on its recovery in the buoyant fraction after Triton X-100 extraction and sucrose density centrifugation and coextraction with the major surface glycolipids in Triton X-114. However, the flagellar localization of SMP-1 was not affected when sterol biosynthesis and the properties of detergent-resistant membranes were perturbed with ketoconazole. Remarkably, treatment of Leishmania with ketoconazole and myriocin (an inhibitor of sphingolipid biosynthesis) also had no affect on SMP-1 localization, despite causing the massive distension of the flagellum membrane and the partial or complete loss of internal axoneme and paraflagellar rod structures, respectively. These data suggest that flagellar membrane targeting of SMP-1 is not dependent on axonemal structures and that alterations in flagellar membrane lipid composition disrupt axoneme extension.     

Ultrastructure of distal flagellar swellings in spermatocytes and spermatids of Ephestia kuehniella Z. (Pyralidae,Lepidoptera)

Summary Development of flagella was investigated by transmission electron microscopy in spermatocytes and spermatids of the Mediterranean mealmoth, Ephestia kuehniella Z. Growing flagella displayed voluminous distal swellings. In short flagella the apical portion of the swellings contained an amorphous, dense accumulation. In more developed flagella a less dense proximal extension of the apical accumulation was formed, which in turn was in contact with the elongating flagellar microtubules. The material of the flagellar tip is interpreted as being a precursor of the axoneme containing mainly tubulin. The material may be converted into the axoneme.     

New insights into the assembly of the periaxonemal structures in mammalian spermatozoa

Disruption of Ube2b in the mouse has revealed that the regular and symmetric organization of the fibrous sheath of the sperm flagella is dependent on expression of the ubiquitin-conjugating enzyme UBE2B. These data could cast light on how a component of the ubiquitin-proteasome pathway participates in the assembly of flagellar periaxonemal structures. Data in the literature support the notion of involvement of ubiquitin-proteasome pathways in the assembly of cytoskeletal components in somatic cells. This review attempts to integrate recent knowledge regarding flagellar components that could be related to proteasome components and, therefore, could be targets of UBE2B in the spermatid. An attempt is made to characterize the human flagellar anomalies of infertile patients, which are the closest to those of Ube2b-deficient mice. These new insights regarding the assembly of mammalian sperm flagella provide a basis for studying the ontogenesis of flagellar accessory structures and suggest leads for medical and genetic investigations.     

Ultrastructure of free nerve endings in respiratory and squamous epithelium on the rat nasal septum

The distribution of nerve fibres in the mucosa of the nasal septum of the rat was investigated by means of transmission electron microscopy on transverse and tangential ultrathin sections. Near the basement membrane of respiratory and squamous epithelium, a rather dense network of unmyelinated nerve fibres occurs. Some fibres in the respiratory epithelium ascend between the epithelial cells to reach up to the tight junctions. These fibres appeared in transverse sections to end as hooks or boutons, sometimes with branches. These shapes resemble the free nerve endings that are considered to act as nociceptors. The small intraepithelial fibres, with diameters of about 0.5–1 m, contain both dense granules and clear vesicles comparable to synaptic vesicles. Substance P was found in dense granules in basal fibres; vasoactive intestinal peptide was absent throughout the epithelium. Acetylcholinesterase activity was observed closely associated with the basal fibres; the apical fibres showed little if any activity. Membrane specializations pointing to an efferent function as well as structures usually associated with mechanoreceptive functions were lacking in both respiratory and squamous epithelium.Part of this work was presented at the Annual Conference of the Netherlands Society for Electron Microscopy, 28–29 Nov 1985, Wageningen, The Netherlands. See Spit BJ and Hendriksen EGJ (1986) Ultramicroscopy 19:102–103     

Morphological changes in the testis and epididymis of rats treated with cyclophosphamide: a quantitative approach

Cyclophosphamide is a widely used anticancer and immunosuppressive drug that affects fertility in men. In a previous study, we found that chronic, daily treatment of male rats with low doses of cyclophosphamide had no apparent effect on the pituitary-gonadal axis, whereas it had time- and dose-dependent effects on male reproductive organ weights, the hematologic system, and on pregnancy outcome. To determine whether cyclophosphamide induces morphological changes within the male reproductive system, a detailed qualitative and quantitative evaluation of changes in the histology of the testis and epididymis was undertaken. Adult male Sprague-Dawley rats were gavage-fed for 1, 3, 6, and 9 wk with saline (control), 5.1 (low dose) or 6.8 (high dose) mg/kg/day of cyclophosphamide; the testes and epididymides were prepared for light and electron microscopy. At the light microscopic level, the orderly process of spermatogenesis in the seminiferous tubules was not affected at any time point with either dose of the drug. A number of time-dependent drug-induced changes in the histology of the epididymis, however, were apparent: 1) an increase in the relative number and a change in the distribution of halo cells in the caput epididymidis, 2) an increase in the number and size of clear cells in the caput and/or cauda epididymidis, and 3) an increase in the size of clear cells in both the caput and cauda epididymides; these changes were time dependent. At the electron microscopic level, there was a dose-dependent, two- to threefold increase in the number of spermatozoa with abnormal flagellar midpieces in the lumen of both the caput and cauda epididymides. Although the 9 plus 2 axonemal complex and the 9 outer dense fibers were present and appeared normal, the close approximation of these two structures was lost in these abnormal spermatozoa. Such abnormal flagellar midpieces were also found in the testes of control and treated rats. Electron microscopic examination of the testis revealed that both Sertoli and Leydig cells were normal in appearance. The type and timing of the effects of cyclophosphamide on the histology of the testis and epididymis suggest that the drug could be affecting germ cells by 1) inducing changes in the developing spermatozoa in the testis, some of which are seen microscopically in the epididymal lumen, and/or 2) affecting epididymal morphology and function.     

Ultrastructure of the Harmful Unarmored Dinoflagellate Cochlodinium polykrikoides (Dinophyceae) with Reference to the Apical Groove and Flagellar Apparatus

ABSTRACT. The external and internal ultrastructure of the harmful unarmored dinoflagellate Cochlodinium polykrikoides Margalef has been examined with special reference to the apical groove and three‐dimensional structure of the flagellar apparatus. The apical groove is U‐shaped and connected to the anterior sulcal extension on the dorsal side of the epicone. The eyespot is located dorsally and composed of two layers of globules situated within the chloroplast. A narrow invagination of the plasma membrane is associated with the eyespot. The nuclear envelope has normal nuclear pores similar to other eukaryotes but different from the Gymnodinium group with diagnostic nuclear chambers. The longitudinal and transverse basal bodies are separated by approximately 0.5–1.0 μm and interconnected directly by a striated basal body connective and indirectly by microtubular and fibrous structures. Characteristic features of the flagellar apparatus are as follows: (1) a nuclear extension projects to the R1 (longitudinal microtubular root) and is connected to the root by thin fibrous material; (2) fibrillar structures are associated with the longitudinal and transverse flagellar canal; and (3) a striated ventral connective extends toward the posterior end of the cell along the longitudinal flagellar canal. We conclude, based on both morphological and molecular evidence, that Cochlodinium is only distantly related to Gymnodinium.     

Human spermatozoa with large heads and multiple flagella: a quantitative ultrastructural study of 6 cases

Macrocephalic spermatozoa of six men were studied. In all cases, sperm concentration, proportion of live spermatozoa and sperm motility were very low. A range of ultrastructural abnormalities was found, essentially comprising a threefold increase in nuclear volume and acrosomal hyperdevelopment and malformation. There were on average 3.6 flagella for each sperm head found in the semen, some tails were separate from heads. The various defects appeared with great constancy in all of the six cases: this homogeneity indicated the existence of a defined semen profile whose most significant expression was sterility. In four of the cases large incidences of different flagellar abnormalities were also noted; whether these flagellar abnormalities are intrinsic to the above profile is not clear. Although the increase in nuclear volume suggests a disturbance in meiosis, its association with defective nuclear elongation would also indicate the existence of one or more anomalies of spermiogenesis. These results were discussed in relation to abnormalities already reported in other species either spontaneously in cases of mutations, or by experimental inhibition of microtubular structures.     

Craniofacial, caudal, and visceral anomalies associated with mutant sirenomelic mice

Craniofacial anomalies were correlated with mutant murine sirenomelia. Ninety-eight newborn sirens from heterozygous matings were examined and analyzed. In the 96 sirens that had intact craniofacial structures, micrognathia was seen in 39% of the sirens, microstomia in 34%, macroglossia in 26%, and cleft palate in 21%. Even when not cleft, the siren palates were narrower and more highly arched than those of nonsiren littermates. The frequency of abnormal craniofacial development was greater in those sirens that were more severely affected caudally. Even though some earlier studies had indicated a preponderance of males, 46 of the 95 sirens with intact pelvic viscera were females. Fifty-three percent of the sirens were monopodal, 35% were apodal, and 11% were dipodal. A penile-like projection on the genital tubercle occurred on 15 apodal sirens and four monopodal sirens; all but three of these sirens were males. Bladder agenesis was seen in 100% of the sirens, anal atresia in 80%, and bilateral renal agenesis in 43%. No siren was found with bilaterally normal kidneys. The srn gene responsible for sirenomelia might either directly affect the embryo at both the caudal and cranial regions or indirectly affect the embryo by producing lateral mechanical compression at both these sites. The srn gene was earlier characterized as autosomal-recessive; our data confirm this. Sirenomelia was found in only 11% of the newborns from crosses of carrier mice in the colony. Analyses of uterine contents at days 12-14 suggest that the srn gene is fully penetrant, but often lethal, during the fetal period.