Characteristics of Spermatozoa from Five Gall-Midge Species (Diptera, Cecidomyiidae)

The gall-midge family Cecidomyiidae is one of the largest within the animal kingdom. Its characteristics include, among others, a chromosome elimination during spermiogenesis and a bewildering diversity in sperm ultrastructure. The sperm tail, in particular, is unusual in that it deviates from the conventional 9+2 pattern of microtubules. We describe here the spermatozoa of five gall-midge species, all belonging to the same supertribe Cecidomyiidi. In Bremia more than 100 microtubular doublets form a tight spiral around a central mitochondrion. In an undetermined binomen many doublets form a spiral around the mitochondria in the distal end of the sperm tail, but a single row along a peripheral cistern in the main part of the tail. In Coquillettomyia caricis a peripheral row of doublets resides in the proximal part and tightly packed doublets distally. In Arthrocnodax sp. the many doublets also form a peripheral single or double row around a central mitochondrion. In Massalongia bachmaieri there are more than 500 doublets which form several rows around the nucleus and tightly packed doublets distally. The nuclear membrane forms bundles of extensive outpocketings. There is no acrosome. The nucleus consists of both condensed and dispersed material. Only outer dynein arms are present in all this species and the spermatozoa are motile. Cecidomyiidi species that have been examined for sperm structure fall into two groups: those where the mitochondria are confined to the centre of the tail and those in which the mitochondria lie in the head region and have a peripheral location. Most species belong to the first group, while Massalongia belongs to the second one.     

Substructure of solitary cilia in mouse kidney

Summary Recent scanning electron microscopic studies confirm the presence of solitary cilia on most epithelial cells along the mammalian nephron and collecting ducts.By transmission electron microscopy we have found that the axonemata of such cilia consist of a maximal number of 9 doublet and no singlet filaments. 10% of the cross-sectioned cilia contain 9 doublets arranged in a peripheral ring (9+0 pattern). 30 % of the cross-sections contain 8 or 7 doublets in peripheral ring and 1 or 2 doublets in the central region (8+1 and 7+2 patterns). Serial sections and goniometer tilt reveal the central doublets to originate as dislodged peripheral doublets. 60% of the sectioned cilia contain filament numbers between 8 and 4. In patterns of 5 and 4 filaments single microtubules predominate.The functional significance of these atypical cilia is discussed.We are indebted to Prof. B. Afzelius and Prof. Th. Brun for valuable information and discussions during this work. The technical assistance of Miss K. Weltzin, Mr. E. Erichsen, Mr. R. Jensen and Mr. J. Røli is greatly appreciated     

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.     

Comparative immunogold analysis of tubulin isoforms in the mouse sperm flagellum: Unique distribution of glutamylated tubulin

The distribution of different tubulin isoforms in the mouse sperm flagellum was studied using four site-directed antibodies to tubulin: DM1A and DM1B general anti α and β-tubulin, 6-11B-1 anti-acetylated α-tubulin, and GT335 anti-glutamylated α and β-tubulin. Quantitative immunogold analyses were performed on five regions of the flagellum: the middle piece, three successive regions of the principal piece, and the terminal piece. A uniform labeling was observed with DM1A and DM1B along the entire flagellum both for peripheral doublets and the central pair. Similar results were obtained with 6-11B-1 directed to acetylated α-tubulin, an N-terminal-modified tubulin isoform. In contrast, the labeling for glutamylated α and β-tubulin, C-terminal modified isoforms, was not uniform. The highest intensity was found in the middle piece and the terminal piece. The labeling which decreased significantly both for peripheral doublets and central pair along the principal piece was considered as a loss of glutamylated tubulin accessibility. From the middle piece to the end of the principal piece, this labeling was predominant in doublets 1-5-6, corresponding to the plane of the flagellar wave. However, the labeling for doublets 2-3-4-7-8-9 was heterogeneous, showing an increasing asymmetry. These results suggest that in the mammalian sperm cell model, the glutamylated tubulin might be involved in a functional heterogeneity among peripheral doublets of the flagellum. © 1996 Wiley-Liss, Inc.     

Comparative ultrastructural studies of the epidermis, sperm and nerve fibres of Cleistogamia longicirrus and Seritia stichopi (RhabdocoeIa, UmagiIIidae)

The epidermis of Cleistogamia longicirrus consists of columnar, interdigitated cells with apical mitochondria, short microvilli and cilia which have a single horizontal rootlet anchored in the adjacent cytoplasm by transverse bars and a thin side branch. Cells are held together by desmosomes and intermediary junctions. Epidermal cilia have a terminal, electron dense rod between the central microtubules and doublet 1 and a terminal plate; doublets 1 and 6–9 lose one of their microtubules and gradually all doublets lose one microtubule and peripheral doublets disappear. Spermatozoa have a single closed peripheral row of microtubules, numerous electron dense granules and mitochondria and axonemes are of the 9 +"1" type and free for most of their length. Nerve fibres have microtubules and some nerve fibres are surrounded by lamellae and have invaginations of the fibre wall. The epidermis of Seritia stichopi resembles that of Cleistogamia , but cilia have a single horizontal rootlet anchored by transverse bars in the cytoplasm, without a side branch. Spermatozoa also are similar to those of Cleistogamia . Certain ultrastructural similarities between some Umagillidae and Neodermata are apparently due to convergent evolution and do not allow the conclusion that Umagillidae and Neodermata are particularly closely related.     

Ultrastructure of the flagellar apparatus inPyramimonas octopus (Prasinophyceae)

Summary While green algae usually lack one of the outer dynein arms in the axoneme, flagella of the octoflagellated prasinophytePyramimonas octopus possess dynein arms on all peripheral doublets. The outer dynein arm on doublet no. 1 is modified, and additional structures are associated with doublets no. 2 and 6. The flagellar scales are asymmetrically arranged. Thus the two rows of thick flagellar hairscales are displaced towards doublet no. 6,i.e., in the direction of the effective stroke of each flagellum. The underlayer of small scales includes two nearly opposite double rows scales, arranged in the longitudinal direction of the flagellum. The hairscales emerge from these rows. The double rows are separated on one side by 9, on the other by 11 rows of helically arranged scales. The central pair of microtubules twists, but the axoneme itself (represented by the 9 peripheral doublets), does not seem to rotate. The flagella are arranged in two groups, showing modified 180° rotational symmetry. The effective strokes of the two central flagella are exactly opposite, while the other flagella beat in six intermediate directions.     

Ultrastructure of spermatozoa of the lung fluke, Paragonimus ohirai (Trematoda: Troglotrematidae), in the seminal receptacle

The seminal receptacle of Paragonimus ohirai contains not only mature spermatozoa, but also atypical and degenerate ones, suggesting that abnormal spermatozoa are retained in this organ. The spermatozoon is of a parallel biflagellar type with cortical microtubules, consisting of the anterior region, first mitochondrial region, intermediate (amitochondrial) region, second mitochondrial region, posterior nuclear region (PNR) and tail region (TR). The first third of the spermatozoon exhibits typical undulatory movement, while the middle part shows vibratory movement. At the area between head and midsections (H-M area) the peripheral doublets of axonemes are interrupted, and the external ornamentation is distributed widely around this portion. Throughout the immotile PNR and TR, the axonemes lack the dynein arms of their peripheral doublets. H-M, PNR, and TR ultrastructural characteristics are specific in P. ohirai spermatozoon and seem to be closely related to its pattern of movement.     

Structural biology of cytoplasmic and axonemal dyneins

Dyneins are microtubule-based, ATP-driven motor proteins with six tandemly linked AAA+ domains, a long N-terminal tail and a coiled-coil stalk. Cytoplasmic dyneins function as individual homodimers and are responsible for minus-end-oriented transport along microtubules. Axonemal dyneins of flagella/cilia are anchored in arrays to peripheral microtubule doublets by their N-terminal tails, and generate sliding motions of adjacent microtubule doublets toward the plus end. The coiled-coil stalk is responsible for communication between the AAA+ domains and the microtubule binding domain. A number of isoforms of axonemal dyneins are integrated to generate bending motion. In this article I will review recent structural studies and address the question as to how dyneins generate force and cause bending in flagella/cilia.     

Pathology of the cytoskeleton of the human sperm flagellum: axonemal and peri-axonemal anomalies

A quantitative ultrastructural study was performed on 56 ejaculates showing anomalies of the sperm axonemal complex. The anomalies comprised either the absence of one, or more often several, axonemal structures, or defective elongation of the doublets. Several characteristics relating to the extent and superimposition of the various anomalies could be described and enabled the definition of 6 groups of anomalies. In decreasing order of frequency these were: absence of the doublets and peripheral junctions, absence of the central complex, of the outer dynein arms, of the central junctions, of both dynein arms, and absence of the inner dynein arms and peripheral junctions. Some anomalies caused total immobility, whereas others caused abnormal movement patterns. Abnormalities of the peri-axonemal structures were found in each group. The various light microscopic characteristics of each of the 6 groups represented 6 seminal profiles which should permit their detection during a routine semen analysis. Several specific associations of axonemal and/or peri-axonemal anomalies would suggest some morphogenetic links between them. Relationships between the absence of doublets or the absence of the central complex and disturbances of microtubular polymerization are discussed. Finally, the study has provided new data on the composition of the axoneme.     

STUDIES ON CILIA : II. Examination of the Distal Region of the Ciliary Shaft and the Role of the Filaments in Motility

Termination of peripheral filaments of the axoneme of gill cilia of fresh-water mussels (Elliptio or Anodonta) occurs in characteristic fashion: (a) subfiber b of certain doublets ends leaving a single simplified tubular unit; (b) the wall of the unit becomes thick and may even obliterate the interior; and (c) the filament drops out of the 9 + 2 pattern. The order in which doublets begin simplifying is also characteristic. This may be determined by numbering the filaments, those with the bridge being 5–6, with the direction of numbering determined by the apparent enantiomorphic configuration (I to IV) of the cross-section. Shorter filaments can be identified in simplifying tips with mixed double and single peripheral units. In this material, laterofrontal cirri show a morphological specialization in the region where individual cilia simplify. The cilia studied run frontally from the body of the cirrus and point in the direction of effective stroke. The longest filaments (Nos. 3, 4, 5, 6, 7) appear as the doublets at the bottom of the cross-section, nearest the surface of the cell of origin. Above them, and above the central pair, a dark band (a section of a dense rod) runs through the matrix. The remaining filaments are the single units. Effective-pointing frontal and lateral ciliary tips end in a fashion similar to laterofrontal tips, although no dense band is present. For all effective-pointing tips studied, the order in which the peripheral filaments end appears to be Nos. (9, 1), 8, 2, 7, 6, 3, 4, 5. However, recovery-pointing lateral tips show a different order: Nos. 7, 6, 8, 5, 9, 4, 1 (3, 2), although the longer filaments are still at the bottom of the cross-section. In simple models of ciliary movement involving contraction of the peripheral filaments, filaments at the top of the cross-section should be longer, if any are. Such models are not supported by the evidence here. These results can be interpreted as supporting sliding-filament models of movement where no length change of peripheral filaments occurs.     

Teaching field biology in towns

A three-dimensional, macroscopic model of the ciliary axoneme has been designed and constructed. It allows students to visualize relative displacement of peripheral microtubular doublets at any degree of imposed bending of the cilium. Direct measurement of doublet sliding at desired points is also possible. An example of such measurements is given and compared with corresponding geometrically obtained data.     

Peripheral doublet microtubules and wave generation in eukaryotic flagella

The shape and propagation of waves produced by eukaryotic flagella depend on the three-dimensional arrangement and physical-chemical properties of peripheral substructures. The modeling analysis presented here, which assumes force-moment equilibrium and neglects the viscous resistances of the medium, shows how substructural arrangements characteristic of 9+0, 9+1, and 9+2 axonemes can yield their characteristic wave patterns. When flexural stiffnesses are equal along all axonemal radii, any non-uniform doublet shearing pattern propagated distally at constant rate, with successive pairs $\text{1}\text{9}$ cycle out of phase, should generate helical waves. When stiffnesses differ greatly on different radii, but the stiffness pattern is the same for all cross-sections, any such shearing pattern should yield planar waves resembling sine-generated curves.Propagated axonemal bending results from the active bending moment produced by local shearing of doublet pairs. Uniformly twisting the doublets about the axonemal axis cannot directly alter the magnitude of the active bending moment. If dynein cross-bridges are activated by shear displacement between peripheral doublets, then the resulting distribution of the active bending moment will be appropriate for balancing the elastic moment in a propagated bending wave.     

Ultrastructural study of the epidermis in two free-living Platyhelminthes, Mesostoma viaregginum and M. productum (Rhabdocoela, Typhloplanida)

Abstract. The epidermis of the free-living typhloplanids Mesostoma viaregginum and M. productum (Mesostominae) is described. In both species, the epidermis has polarized cells with nuclei located at the basal part of the cell, whereas mitochondria are in the apical one. The epidermis is entirely covered by microvilli and locomotory cilia anchored in the cytoplasm by vertical and horizontal rootlets. Rootlets exhibit distinct length and periodic structure in the two species. Furthermore, in each species vertical and horizontal rootlets possess different periodic structure. The pattern of termination of microtubules in epidermal cilia is described for the first time in the Typhloplanida; central microtubules shift along one axonemal side, doublets 1 and 6–9 lose their microtubule B, and gradually peripheral doublets become singlets. Finally, an electron-dense material caps the tip of the cilia. This pattern of termination closely resembles that of Temnocephalida, Kalytorhynchia, and Dalyelliida examined so far, but differences exist.     

Spontaneous multiple mutations show both proximal spacing consistent with chronocoordinate events and alterations with p53-deficiency

Analysis of spontaneous multiple mutations in normal and tumor cells may constrain hypotheses about the mechanisms responsible for multiple mutations and provide insight into the mutator phenotype. In a previous study, spontaneous doublets in Big Blue mice were dramatically more frequent than expected by chance and exhibited a mutation pattern similar to that observed for single mutations [Mutat. Res. 452 (2000) 219]. The spacing between mutations in doublets was generally closer than expected by chance and the distribution of mutation spacing fit an exponential, albeit with substantial scatter. We now analyze 2658 additional mutants and confirm that doublets are enhanced dramatically relative to chance expectation. The spacing, frequency and pattern of spontaneous doublets and multiplets (domuplets) are examined as a function of age, tissue type, p53-deficiency and neoplasia in the new and combined data. The new and combined data confirm that the distribution of the spacing between mutations in doublets is non-random with the mutations more closely spaced than expected by chance (P < 0.0005; combined data), consistent with temporally coordinate (chronocoordinate) events. An exponential provides an excellent fit to the distribution (R2 = 0.98) and estimates that half of doublets have mutations separated by 120 nucleotides or less (the "half-life of mutation spacing"). We make several novel observations: (i) singlets and doublets show similar overall increases in frequency with age (ii) doublet frequency may be lower in the male germline, consistent with the generally reduced mutation frequency in the male germline (iii) doublet frequencies are elevated in somatic tissues of p53-deficient mice (Li-Fraumini cancer syndrome model; P = 0.005) and (iv) doublets and singlets in tumors from p53-deficient mice have a different mutation pattern (P = 0.007). The observations are consistent with chronocoordinate occurrence of spontaneous doublets and multiplets due to a transient error-prone condition and do not suggest a major role for the recently discovered Y family of error-prone polymerases. The enhancement of doublets in p53-deficient mice may contribute to cancer risk.     

Describing RNA structure by libraries of clustered nucleotide doublets

The rapidly increasing wealth of structural information on RNA and knowledge of its varying roles in biology have facilitated the study of RNA structure using computational methods. Here, we present a new method to describe RNA structure based on nucleotide doublets, where a doublet is any two nucleotides in a structure. We restrict our search to doublets that are close together in space, but not necessarily in sequence, and obtain doublet libraries of various sizes by clustering a large set of doublets taken from a data set of high-resolution RNA structures. We demonstrate that these libraries are able to both capture structural features present in RNA and fit local RNA structure with a high level of accuracy. Libraries ranging in size from ten to 100 doublets are examined, and a detailed analysis shows that a library with as few as 30 doublets is sufficient to capture the most common structural features, while larger libraries would be more appropriate for accurate modeling. We anticipate many uses for these libraries, from annotation to structure refinement and prediction.     

Spermiogenesis in the Flatworm, Notoplana Japonica with Special Attention to the Organization of an Acrosome and Flagella

Ultrastructural changes during spermiogenesis in the flatworm, Notoplana japonica were studied with special attention to organizing process of an acrosome and flagella. During spermiogenesis, the G olgi complex develops conspicuously but it fails to organize the structure of an acrosomal vesicle. Consequently, no acrosome is formed at the apex of the sperm. As a substitute for an acrosomal structure, the slender process at the tip of the mature sperm is prominently occupied with glycogen granules.
The axoneme of the flagellum is formed from the basal body in the protrusion which is juxtaposed to the nucleus of the early spermatid. Two flagella associated with an electron-dense structure (EDS) extend superficially from the spermatid body in opposite directions. Progressively, they take an acute angle to each other and finally run alongside the sperm body. The axoneme consits of nine peripheral doublets with arms, a central cylinder containing an electron dense core, a less dense intermediate zone and fine spokes between the cylinder and doublets.     

Temporal correlations and coding of information in the visual cortex of the cat

We have observed repeated patterns in evoked spike trains recorded from the primary visual cortex of the cat. These patterns are called "triplets" and "ghost doublets". Triplets are groups of three pulses, that may or may not be adjacent to one other, the mutual intervals of which are replicated in one other group of three spikes with a precision higher than 0.15 ms. Ghost doublets are doublets of pulses whose interval replicates, with the above precision, one of the intervals of the repeated triplets and are also present in the record. In one of the 9 recorded cells, in which pulses were clearly emitted in bursts in phase with the drifting of the sinusoidal grating used as a stimulus, we could show that local temporal correlations in the form of replicating triplets and ghost doublets correspond very precisely to the temporal phase of the grating: the study of the distance between triplets, or between triplets and ghost doublets, gives a remarkably precise value of the time frequency of the grating.     

Positional reorganization in compound janus cells of Tetrahymena thermophila

The janus mutations of Tetrahymena thermophila convert the large-scale organization of the dorsal surface of the cell into a mirror-image of the ventral surface, which is characterized by a second, abnormal, oral apparatus and by contractile vacuole pores to the left of the second oral area rather than the usual right. This conversion could be due either to a local change in the response to an unaltered positional system or to a more global reorganization of the system itself. janus homopolar doublets were used to distinguish between these two alternatives. Homopolar doublets can be made by fusing two similarly oriented cells in side-by-side parabiosis. Non-janus homopolar doublets typically possess two sets of normal oral structures with contractile vacuole pores to the right of each of them. In janus doublets, there are up to four sets of oral structures, with the abnormal oral structures located between the two sets of normal oral structures; contractile vacuole pores are situated to the right of the normal oral areas and to the left of the abnormal oral structures. Non-janus homopolar doublets are known to propagate their compound condition for a number of cell divisions, but also to regulate toward the singlet state through a progressive reduction in number of ciliary rows followed by loss of one of the two sets of major cell surface structures. janus homopolar doublets go through a corresponding regulation. As a consequence, the location of the abnormal oral structures relative to the normal ones is more variable in janus doublets than in janus singlets. Sometimes the abnormal oral structures shift to a position close to their normal counterparts and then the intervening CVP sets disappear. There is evidence for occasional fusion of an abnormal oral area with an adjacent normal oral apparatus, a condition that may be transitional to the singlet state. These observations are inconsistent with the idea of a fixed positional system and strongly suggest a global reorganization of the surface pattern in a manner consistent with predictions of an intercalation model that was first proposed to explain the regulation of non-janus doublets to singlets.     

One of the Nine Doublet Microtubules of Eukaryotic Flagella Exhibits Unique and Partially Conserved Structures

The axonemal core of motile cilia and flagella consists of nine doublet microtubules surrounding two central single microtubules. Attached to the doublets are thousands of dynein motors that produce sliding between neighboring doublets, which in turn causes flagellar bending. Although many structural features of the axoneme have been described, structures that are unique to specific doublets remain largely uncharacterized. These doublet-specific structures introduce asymmetry into the axoneme and are likely important for the spatial control of local microtubule sliding. Here, we used cryo-electron tomography and doublet-specific averaging to determine the 3D structures of individual doublets in the flagella of two evolutionarily distant organisms, the protist Chlamydomonas and the sea urchin Strongylocentrotus. We demonstrate that, in both organisms, one of the nine doublets exhibits unique structural features. Some of these features are highly conserved, such as the inter-doublet link i-SUB5-6, which connects this doublet to its neighbor with a periodicity of 96 nm. We also show that the previously described inter-doublet links attached to this doublet, the o-SUB5-6 in Strongylocentrotus and the proximal 1–2 bridge in Chlamydomonas, are likely not homologous features. The presence of inter-doublet links and reduction of dynein arms indicate that inter-doublet sliding of this unique doublet against its neighbor is limited, providing a rigid plane perpendicular to the flagellar bending plane. These doublet-specific features and the non-sliding nature of these connected doublets suggest a structural basis for the asymmetric distribution of dynein activity and inter-doublet sliding, resulting in quasi-planar waveforms typical of 9+2 cilia and flagella.