Bizarre flagellum of thrips spermatozoa (Thysanoptera,Insecta)

The flagellum of the thysanopteran spermatozoon has been examined by electron microscopy and computer-aided image analysis. The flagellum consists of 27 microtubular elements that probably are formed as outgrowths from three separate basal bodies. Nine of the elements are normal microtubular doublets that carry dynein arms and nine are doublets without dynein arms. The remaining nine elements are microtubular singlets that apparently bear dynein arms and have the same appearance as A-subtubules of microtubular doublets. The 27 elements are arranged in a fixed pattern that consists of nine groups, each of which begins with a microtubular singlet and ends with an arm-less microtubular doublet. Computer-aided image analysis has shown that the A-subtubules of the doublets and the microtubular singlets have lumens with very similar patterns. The sperm tail is known to have some motility; it generates fast waves running along its length. The amalgamated axonemes hence act as a functional flagellum. The thysanopteran sperm tail is the only type of flagellum known to us that consists of microtubules in a highly asymmetric array.     

Direction of force generated by the inner row of dynein arms on flagellar microtubules

Our goal was to determine the direction of force generation of the inner dynein arms in flagellar axonemes. We developed an efficient means of extracting the outer row of dynein arms in demembranated sperm tail axonemes, leaving the inner row of dynein arms structurally and functionally intact. Sperm tail axonemes depleted of outer arms beat at half the beat frequency of sperm tails with intact arms over a wide range of ATP concentrations. The isolated, outer arm-depleted axonemes were induced to undergo microtubule sliding in the presence of ATP and trypsin. Electron microscopic analysis of the relative direction of microtubule sliding (see Sale, W. S. and P. Satir, 1977, Proc. Natl. Acad. Sci. USA, 74:2045-2049) revealed that the doublet microtubule with the row of inner dynein arms, doublet N, always moved by sliding toward the proximal end of the axoneme relative to doublet N + 1. Therefore, the inner arms generate force such that doublet N pushes doublet N + 1 tipward. This is the same direction of microtubule sliding induced by ATP and trypsin in axonemes having both inner and outer dynein arms. The implications of this result for the mechanism of ciliary bending and utility in functional definition of cytoplasmic dyneins are discussed.     

A biochemical study of flagellar dynein from starfish spermatozoa: protein components of the arm structure.

Half of the adenosine triphosphatase (dynein) activity of starfish sperm tail axonemes was extracted with 0.6 m KCl-10 mm Tris · HCl (pH 7.8)-0.1 mm EDTA-0.5 mm dithiothreitol (KCl-EDTA), while with 1 mm Tris · HCl (pH 7.8)-0.1 mm EDTA-0.5 mm dithiothreitol (Tris-EDTA) around 90% of the activity was extracted. The main adenosine triphosphatase (ATPase) in the KCl-EDTA extract had a sedimentation coefficient of 20S and that in the Tris-EDTA extract had a sedimentation coefficient of 12S. The effects of divalent cations, pH, and an SH-blocking reagent and the K_m for ATP were different for the activities of the two forms of dynein ATPase. These two forms of dynein can interconvert to some extent when the ionic strength of the medium is changed. In a medium suitable for recombination of dynein to outer doublet microtubules (recombination buffer, 20 mm Tris-HCl (pH 7.6)-2 mm MgCl₂-0.5 mm dithiothreitol), the 20S ATPase converted to a 24S ATPase. Recombination of the ATPase activity from the KCl-EDTA extract was almost complete while that from the Tris-EDTA extract was around 50%. Outer arms disappeared preferentially by the treatment with KCl-EDTA, and the extracted arms could be reconstituted in the recombination buffer. In the case of the Tris-EDTA extraction, both the outer and inner arms disappeared and the reconstitution of the arms could not be confirmed. From the above results it can be considered that the 20 or 24S dynein represented the arm structure. The 20 or 24S ATPase fraction contained two large polypeptide chains as main components having electrophoretic mobilities in the presence of sodium dodecylsulfate similar to those of Tetrahymena ciliary dyneins and of sea urchin sperm flagellar dyneins. The relationship between these chains and dynein subunits is discussed.     

The reactivation of demembranated human spermatozoa lacking outer dynein arms is independent of pH

The effect of pH, Mg-ATP, and free calcium on activity of the inner dynein arm was investigated using demembranated human spermatozoa lacking the outer dynein arms (LODA). The results were compared with those obtained for demembranated-reactivated normal spermatozoa to evaluate the functional properties of the inner and outer dynein arms in axonemal motility. The reactivation of Triton X-100–demembranated LODA spermatozoa was analysed at various pHs and concentrations of Mg-ATP and calcium using video recordings. The percentage of reactivated LODA spermatozoa as a function of Mg-ATP concentration was not dependent on pH, whereas reactivation of normal human spermatozoa is pH dependent. This suggests that there may be a pH-dependent regulatory mechanism associated with the outer dynein arms. A delay in the principal bend propagation of normal and LODA reactivated cells was found at pH 7.1. This disappeared at pH 7.8 in normal but not in LODA populations. This suggests a role for outer dynein arms in the initiation of the propagation of flagellar bends at alkaline pH. The level of LODA and normal sperm reactivation both depended on the calcium concentration in the medium. At lower free calcium concentrations, the reactivation level and beat frequency of reactivated cells were higher. Our results suggest a functional difference between outer and inner dynein arms of human spermatozoa based on a differential pH sensitivity. Moreover, calcium seems to exert its regulatory action elsewhere than on the outer dynein arms. Mol. Reprod. Dev. 49:416–425, 1998. © 1998 Wiley-Liss, Inc.     

The effect of antidynein 1 serum on the movement of reactivated sea urchin sperm

Rabbit antiserum prepared against an ATPase-containing tryptic fragment of dynein by Ogawa and Mohri (J. Biol. Chem. 250: 6476-6483) specifically inhibited the ATPase activity of dynein 1 and not that of dynein 2. Varying amounts of this antidynein 1 serum were added to demembranated sperm while they were swimming in reactivating solution containing 1 mM ATP. The sperm continued to form regularly propagated flagellar bending waves, but the beat frequency decreased gradually with time, the greater part of the change occurring in the first 15 min. The beat frequency after 1 h was a function of the amount of antiserum used, and could be as low as 1 Hz. The waveforms of the treated sperm resembled those of normal reactivated sperm except that the bend angles of both the principal and reverse bends were larger in the proximal portion of flagellum. The ATPase activity and corresponding beat frequency of sperm which had been pretreated with varying amounts of antidynein 1 serum for 15 min at 0 degrees C and then diluted were both decreased as a function of the amount of antiserum added, the ATPase activity of homogenized, nonmotile sperm also decreased upon pretreatment with antiserum, but the percentage decrease was less than for motile sperm. For moderate to low concentrations of antiserum, the rates of reaction with motile and with rigor sperm were almost identical. The overall results suggest that antidynein 1 inhibits the functioning of the dynein arms, probably by blocking the ATPase sites of the dynein 1.     

Functional recombination of dynein 1 with demembranated sea urchin sperm partially extracted with KC1.

Demembranated sea urchin sperm were extracted with 0.5 M KCl as described earlier and reactivated in a solution containing 1 mM ATP. Their flagellar beat frequency was approximately 13 Hz, while that of standard reactivated sperm which had not been extracted with KCl was approximately 31 Hz at 23°C. Addition of soluble dynein 1 caused a gradual increase in the flagellar beat frequency to approximately 25 Hz after 10 min at room temperature. This restoration of frequency occurred in the absence or presence of ATP. Examination by electron microscopy showed that, whereas KCl-extracted sperm were lacking the majority of the outer arms on the doublet tubules, they had regained most of their outer arms following incubation with soluble dynein 1.     

Swimming behavior and ultrastructure of sperm of<Emphasis Type="Italic"> Lygodium japonicum</Emphasis> (Pteridophyta)

Swimming behavior of the sperm of Lygodium japonicum (Pteridophyta) and the associated ultrastructure of the flagellar apparatus were studied by video microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The sperm has approximately 70 flagella that emerge from a sinistrally-coiled flagellar apparatus, and swims forward by ciliary beat of these flagella. Backward swimming was not observed even after sperm collided with obstacles. Video microscopy showed that the flagella of the swimming sperm are oriented laterally and oblique-anteriorly. TEM and SEM observations revealed that the basal bodies of these flagella are arranged in at least two rows and oriented in the same directions as observed by video microscopy. These basal bodies (flagella) are categorized into two types according to their orientation: group I (laterally directed) and group II (oblique-anteriorly directed). The directionality of the basal bodies appears to be fixed by electron-dense material around their base. The outer dynein arms of the flagellar axoneme are entirely absent. These morphological characteristics of basal bodies (flagella) may relate to the lack of backward swimming behavior of the sperm. Based on these results, the evolution of swimming behavior in the archegoniates is discussed in connection with lack of backward swimming in a distantly related green alga, Mesostigma viride, and the Streptophyta.     

Targeted gene knockout of inner arm 1 in Tetrahymena thermophila

Cilia and flagella contain at least eight different types of dynein arms. It is not entirely clear how the different types of arms are organized along the axoneme. In addition, the role each different type of dynein plays in ciliary or flagellar motility is not known. To initiate studies of dynein organization and function in cilia, we have introduced a mutation into one dynein heavy chain gene (DYH6) in Tetrahymena themophila by targeted gene knockout. We have generated mutant cells that lack wild-type copies of the DYH6 gene. We have shown that the DYH6 gene encodes one heavy chain (HC2) of Tetrahymena 18S dynein and that 18S dynein occupies the I1 position in the ciliary axoneme. We have also shown that Tetrahymena I1 is required for normal motility, normal feeding and normal doubling rate.     

Molecular structure of dynein and motility of a giant sperm axoneme provided with only the outer dynein arm.

The peculiar sperm axoneme of the dipteran Asphondylia ruebsaameni is characterized by an extraordinarily high number of microtubule doublets (up to 2,500) arranged in double parallel spirals. Doublets of the inner row of each spiral are tilted, so that their outer arms point towards the B-tubule of the next doublet in the outer row. Doublets are provided with only the outer arm, and no structure related to the central pair/radial spoke complex is present. When analyzed by quick-freeze, deep-etch electron microscopy, the structure of the dynein arms was shown to share the same organization described in other organisms; however, it appears to be somewhat more complex than that previously found in a related dipteran species, Monarthropalpus flavus, since the foot region of the arms displays a globular extra-domain that is intercalated between adjacent arms. Treatment of demembranated sperm with ATP and vanadate induced conformational changes in the dynein arms. SDS-page suggested the presence of a single dynein high molecular weight band or, in the gels with the best electrophoretic resolution, of two very closely spaced bands. This polypeptide positively reacted with a polyclonal antibody raised against a specific amino acid sequence located in the phosphate-binding loop of the dynein catalytic site. Dynein heavy chain-related DNA sequences corresponding to the catalytic phosphate-binding region were amplified by RT-PCR. Two distinct fragments (Asph-ax1 and Asph-ax2) encoding axonemal dynein sequences were identified. Southern blot analysis performed on genomic DNA using these sequences as a probe showed that they are part of different genes. An intron was identified in the Asph-ax1 fragment at a position corresponding to the site of a nucleotide deletion in the putative pseudogene of Monarthropalpus. Asphondylia spermatozoa exhibited in vivo a whirling movement both in the deferent duct and in the spermatheca, but they were unable to undergo processive movement in vitro. They propagated a three-dimensional wave only when constrained in a bent configuration by some mechanical means. The phylogenetic relationships between the two dipteran species, Monarthopalpus and Asphondylia, based on these biochemical and molecular data are also discussed.     

Sperm structure of Trichoptera. I. Integripalpia : Limnephiloidea

Spermal ultrastructure in 16 caddisflies (Trichoptera) belonging to the suborder Integripalpia, superfamily Limnephiloidea, was examined in a search for apomorphic and plesiomorphic features. In all species examined, the sperm tail axoneme was of the 9 + 9 + 2 + type, the axonemal doublets lacked outer dynein arms but had inner ones, and the cell membrane was scalloped with a prominent glycocalyx. The number of protofilaments in the accessory tubules depended on the phylogenetic position: 18 in the family Leptoceridae, 19 in Limnephilidae, Goeridae and Odontoceridae (with a reduction in the number distally), and 20 in Sericostomatidae. Spermatozoa in Leptoccridae are further characterized by the 2 central microtubules being flattened and eccentric and not being surrounded by a central sheath. Spermatozoa of Sericostomatidae have an accessory body and a helicoidal array of the sperm tail.     

Strikingly low ATPase activities in flagellar axonemes of a Chlamydomonas mutant missing outer dynein arms

The ATPase activities in Chlamydomonas axonemes were compared between wild type and a mutant (oda) that lacks entire outer dynein arms, at various ionic strengths and pH values, and in the presence of different concentrations of high-molecular-mass dextran. Over a 0-0.2 M KCl concentration range, the ATPase activity of oda axonemes was found to be 5-12 times lower than that of the wild-type axonemes. The low activity in oda is surprising since outer arm-depleted axonemes of sea urchin sperm have been reported to retain about 50% of the normal activity. In both wild type and oda, the ATPase activity of dynein was higher when contained within the axoneme than when released from it with 0.6 M KCl. The ATPase activation within the wild-type axoneme was inhibited by high ionic strengths or by the presence of dextran. The activation in oda axonemes, on the other hand, was not inhibited by these factors. These significantly different ATPase properties suggest that the inner and outer dynein arms perform somewhat different functions in this organism.     

Widespread absence of outer dynein arms in the spermatozoids of lower plants

Transverse sections of flagellar axonemes from a variety of lower plant spermatozoids were examined by electron microscopy. Motile sperm of four ferns (Marsilea, Pteridium, Lygodium and Aneimia), a horsetail (Equisetum) and a liverwort (Marchantia) were fixed in the presence of tannic acid to visualise the dynein arms. In all cases the inner dynein arms were clearly resolved but the outer arms were absent. Absence of outer arms therefore appears to be a common feature of the archegoniate plants. The implications of these findings to our understanding of the evolution of the land plants and the role of the dynein arms in flagellar beating is discussed.     

Defects in the cytoplasmic assembly of axonemal dynein arms cause morphological abnormalities and dysmotility in sperm cells leading to male infertility

Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males (DNAAF2/ KTU, DNAAF4/ DYX1C1, DNAAF6/ PIH1D3, DNAAF7/ZMYND10, CFAP300/C11orf70 and LRRC6). Besides andrological examinations, we functionally and structurally analyzed sperm flagella of affected individuals by high-speed video- and transmission electron microscopy as well as systematically compared the composition of dynein arms in sperm flagella and respiratory cilia by immunofluorescence microscopy. Furthermore, we analyzed the flagellar length in dynein preassembly mutant sperm. We found that the process of axonemal dynein preassembly is also critical in sperm, by identifying defects of ODAs and IDAs in dysmotile sperm of these individuals. Interestingly, these mutant sperm consistently show a complete loss of ODAs, while some respiratory cilia from the same individual can retain ODAs in the proximal ciliary compartment. This agrees with reports of solely one distinct ODA type in sperm, compared to two different ODA types in proximal and distal respiratory ciliary axonemes. Consistent with observations in model organisms, we also determined a significant reduction of sperm flagellar length in these individuals. These findings are relevant to subsequent studies on the function and composition of sperm flagella in PCD patients and non-syndromic infertile males. Our study contributes to a better understanding of the fertility status in PCD-affected males and should help guide genetic and andrological counselling for affected males and their families.     

Identification of the human ortholog of the t-complex-encoded protein TCTE3 and evaluation as a candidate gene for primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a heterogeneous autosomal recessive disease that is caused by impaired ciliary and flagellar functions. About 50% of PCD patients show situs inversus, denoted as Kartagener syndrome. In most cases, axonemal defects in cilia and sperm tails can be demonstrated by electron microscopy, i.e. PCD patients often lack inner and/or outer dynein arms in their sperm tails and cilia, supporting the hypothesis that mutations in dynein genes may cause PCD. In order to identify novel PCD genes we have isolated the human ortholog of the murine TCTE3 gene. The human TCTE3 gene encodes a dynein light chain and shares high similarity to dynein light chains of other species. The TCTE3 gene is expressed in tissues containing cilia or flagella, it is composed of four exons and located on chromosome 6q25-->q27. To elucidate the role of TCTE3 as a candidate gene for PCD a mutational analysis of thirty-six PCD patients was performed. We detected five polymorphisms in the coding sequence and in the 5' UTR of the TCTE3 gene. In one patient a heterozygous nucleotide exchange was identified resulting in an arginine to isoleucine substitution at the amino acid level. However, this exchange was also detected in one control DNA. Our results indicate that mutations in the TCTE3 gene are not a main cause of primary ciliary dyskinesia.     

Pih1d3 is required for cytoplasmic preassembly of axonemal dynein in mouse sperm

Axonemal dynein complexes are preassembled in the cytoplasm before their transport to cilia, but the mechanism of this process remains unclear. We now show that mice lacking Pih1d3, a PIH1 domain–containing protein, develop normally but manifest male sterility. Pih1d3^−/− sperm were immotile and fragile, with the axoneme of the flagellum lacking outer dynein arms (ODAs) and inner dynein arms (IDAs) and showing a disturbed 9+2 microtubule organization. Pih1d3 was expressed specifically in spermatogenic cells, with the mRNA being most abundant in pachytene spermatocytes. Pih1d3 localized to the cytoplasm of spermatogenic cells but was not detected in spermatids or mature sperm. The levels of ODA and IDA proteins were reduced in the mutant testis and sperm, and Pih1d3 was found to interact with an intermediate chain of ODA as well as with Hsp70 and Hsp90. Our results suggest that Pih1d3 contributes to cytoplasmic preassembly of dynein complexes in spermatogenic cells by stabilizing and promoting complex formation by ODA and IDA proteins.     

Arrangement of inner dynein arms in wild-type and mutant flagella of Chlamydomonas

We have used computer averaging of electron micrographs from longitudinal and cross-sections of wild-type and mutant axonemes to determine the arrangement of the inner dynein arms in Chlamydomonas reinhardtii. Based on biochemical and morphological data, the inner arms have previously been described as consisting of three distinct subspecies, I1, I2, and I3. Our longitudinal averages revealed 10 distinguishable lobes of density per 96-nm repeating unit in the inner row of dynein arms. These lobes occurred predominantly but not exclusively in two parallel rows. We have analyzed mutant strains that are missing I1 and I2 subspecies. Cross-sectional averages of pf9 axonemes, which are missing the I1 subspecies, showed a loss of density in both the inner and outer portions of the inner arm. Averages from longitudinal images showed that three distinct lobes were missing from a single region; two of the lobes were near the outer arms but one was more inward. Serial 24-nm cross-sections of pf9 axonemes showed a complete gap at the proximal end of the repeating unit, confirming that the I1 subunit spans both inner and outer portions of the inner arm region. Examination of pf23 axonemes, which are missing both I1 and I2 subspecies, showed an additional loss almost exclusively in the inner portion of the inner arm. In longitudinal view, this additional loss occurred in three separate locations and consisted of three inwardly placed lobes, one adjacent to each of the two radial spokes and the third at the distal end of the repeating unit. These same lobes were absent ida4 axonemes, which lack only the I2 subspecies. The I2 subspecies thus does not consist of a single dynein arm subunit in the middle of the repeating unit. The radial spoke suppressor mutation, pf2, is missing four polypeptides of previously unknown location. Averages of these axonemes were missing a portion of the structures remaining in pf23 axonemes. This result suggests that polypeptides of the radial spoke control system are close to the inner dynein arms.     

Properties of an antiserum against native dynein 1 from sea urchin sperm flagella

Effects of an antiserum against native dynein 1 from sperm flagella of the sea urchin Strongylocentrotus purpuratus were compared with effects of an antiserum previously obtained against an ATPase-active tryptic fragment (fragment 1A) of dynein 1 from sperm flagella of the sea urchin, Anthocidaris crassispina. Both antisera precipitate dynein 1 and do not precipitate dynein 2. Only the fragment 1A antiserum precipitates fragment 1A and produces a measurable inhibition of dynein 1 ATPase activity. Both antisera inhibit the movement and the movement-coupled ATP dephosphorylation of reactivated spermatozoa. The inhibition of movement by the antiserum against dynein 1 is much less than by the antiserum against fragment 1A, suggesting that a specific interference with the active ATPase site may be required for effective inhibition of movement. Both antisera reduce the bend angle as well as the beat frequency of reactivated S. purpuratus spermatozoa, suggesting that the bend angle may depend on the activity of the dynein arms which generate active sliding.     

Mutations at twelve independent loci result in absence of outer dynein arms in Chylamydomonas reinhardtii

35 strains of Chlamydomonas mutant missing the entire outer dynein arm were isolated by screening slow-swimming phenotypes. They comprised 10 independent genetic loci (odal-10) including those of previously isolated mutants oda38 and pf28. The 10 loci were distinct from pf13 and pf22, loci for nonmotile mutants missing the outer arm. These results indicate that at least 12 genes are responsible for the assembly of the outer dynein arms. There were no mutants lacking partial structures of the outer arm, suggesting that lack of a single component results in failure of assembly of entire outer arms. Temporary dikaryons derived from mating of two different oda strains often, but not always, recovered the wild-type motility within 2 h of mating. Hence, outer arms can be transported and attached to the outer doublets independently of flagellar growth.     

Substructure of the axoneme of pterygote insect spermatozoa: Phylogenetic considerations

Spermatozoa from a great number of insect species were fixed in a tannic acid-containing fixative and the ultrastructure of the flagellar axoneme was examined in a search for apomorphies. Most of the examined species, representing a majority of insect orders. have accessory tubules outside the axoneme (hence a 9 + 9 + 2 pattern), and these consist of 16 protofilaments. Some important apomorphies concern the number of protofilaments in the accessory tubules: 13 (plus 7 inner elements) in Ephemeroptera, 13 in the (elliptic) tubules of Psocoptera + Anoplura + Mallophaga (thus a synapomorphy), 13 in Tipulidae + Brachycera, 15 in the dipteran families Dixidae + Chironomidae (with a 9 + 9 + 2 axoneme) and Culicidae + Bibionidae (with a 9 + 9 + “1” axoneme), 17 in Phasmatodea, and 17–20 in Trichoptera. Other apomorphies concern the appearance of the so-called intertubular material outside the microtubular doublets, the appearance of the interior of the various microtubules, and the loss, in some taxa, of outer or inner dynein arms of both dynein arms. In some cases, the flagellum is completely abnormal; the sperm tail of Thysanoptera, for example, consists of 27 elements of 3 different kinds. The different taxa within orders Diptera and Trichoptera have sperm tail axonemes of different appearances, where those from other orders have a rather uniform appearance. The conclusions that can be drawn from this spermatological study, generally agree with data from classical studies, except with some variations, in some cases.