Sperm Tail Axoneme Research Articles

Deficiency of the Tbc1d21 gene causes male infertility with morphological abnormalities of the sperm mitochondria and flagellum in mice.

Approximately 2–15% of couples experience infertility, and around half of these cases are attributed to male infertility. We previously identified TBC1D21 as a sterility-related RabGAP gene derived from infertile men. However, the in vivo function of TBC1D21 in male fertility remains unclear. Here, we show that loss of Tbc1d21 in mice resulted in male infertility, characterized by defects in sperm tail structure and diminished sperm motility. The mitochondria of the sperm-tail had an abnormal irregular arrangement, abnormal diameter, and structural defects. Moreover, the axoneme structure of sperm tails was severely disturbed. Several TBC1D21 interactors were selected via proteomic analysis and functional grouping. Two of the candidate interactors, a subunit protein of translocase in the outer membrane of mitochondria (TOMM20) and an inner arm component of the sperm tail axoneme (Dynein Heavy chain 7, DNAH7), confirmed in vivo physical co-localization with TBC1D21. In addition, TOMM20 and DNAH7 detached and dispersed outside the axoneme in Tbc1d21-deficient sperm, instead of aligning with the axoneme. From a clinical perspective, the transcript levels of TBC1D21 in sperm from teratozoospermia cases were significantly reduced when compared with those in normozoospermia. We concluded that TBC1D21 is critical for mitochondrial and axoneme development of mammalian sperm.

Formation and function of the manchette and flagellum during spermatogenesis.

The last phase of spermatogenesis involves spermatid elongation (spermiogenesis), where the nucleus is remodeled by chromatin condensation, the excess cytoplasm is removed and the acrosome and sperm tail are formed. Protein transport during spermatid elongation is required for correct formation of the sperm tail and acrosome and shaping of the head. Two microtubular-based protein delivery platforms transport proteins to the developing head and tail: the manchette and the sperm tail axoneme. The manchette is a transient skirt-like structure surrounding the elongating spermatid head and is only present during spermatid elongation. In this review, we consider current understanding of the assembly, disassembly and function of the manchette and the roles of these processes in spermatid head shaping and sperm tail formation. Recent studies have shown that at least some of the structural proteins of the sperm tail are transported through the intra-manchette transport to the basal body at the base of the developing sperm tail and through the intra-flagellar transport to the construction site in the flagellum. This review focuses on the microtubule-based mechanisms involved and the consequences of their disruption in spermatid elongation.

Tubulin evolution in insects: gene duplication and subfunctionalization provide specialized isoforms in a functionally constrained gene family

BackgroundThe completion of 19 insect genome sequencing projects spanning six insect orders provides the opportunity to investigate the evolution of important gene families, here tubulins. Tubulins are a family of eukaryotic structural genes that form microtubules, fundamental components of the cytoskeleton that mediate cell division, shape, motility, and intracellular trafficking. Previous in vivo studies in Drosophila find a stringent relationship between tubulin structure and function; small, biochemically similar changes in the major alpha 1 or testis-specific beta 2 tubulin protein render each unable to generate a motile spermtail axoneme. This has evolutionary implications, not a single non-synonymous substitution is found in beta 2 among 17 species of Drosophila and Hirtodrosophila flies spanning 60 Myr of evolution. This raises an important question, How do tubulins evolve while maintaining their function? To answer, we use molecular evolutionary analyses to characterize the evolution of insect tubulins.ResultsSixty-six alpha tubulins and eighty-six beta tubulin gene copies were retrieved and subjected to molecular evolutionary analyses. Four ancient clades of alpha and beta tubulins are found in insects, a major isoform clade (alpha 1, beta 1) and three minor, tissue-specific clades (alpha 2-4, beta 2-4). Based on a Homarus americanus (lobster) outgroup, these were generated through gene duplication events on major beta and alpha tubulin ancestors, followed by subfunctionalization in expression domain. Strong purifying selection acts on all tubulins, yet maximum pairwise amino acid distances between tubulin paralogs are large (0.464 substitutions/site beta tubulins, 0.707 alpha tubulins). Conversely orthologs, with the exception of reproductive tissue isoforms, show little sequence variation except in the last 15 carboxy terminus tail (CTT) residues, which serve as sites for post-translational modifications (PTMs) and interactions with microtubule-associated proteins. CTT residues overwhelming comprise the co-evolving residues between Drosophila alpha 2 and beta 3 tubulin proteins, indicating CTT specializations can be mediated at the level of the tubulin dimer. Gene duplications post-dating separation of the insect orders are unevenly distributed, most often appearing in major alpha 1 and minor beta 2 clades. More than 40 introns are found in tubulins. Their distribution among tubulins reveals that insertion and deletion events are common, surprising given their potential for disrupting tubulin coding sequence. Compensatory evolution is found in Drosophila beta 2 tubulin cis-regulation, and reveals selective pressures acting to maintain testis expression without the use of previously identified testis cis-regulatory elements.ConclusionTubulins have stringent structure/function relationships, indicated by strong purifying selection, the loss of many gene duplication products, alpha-beta co-evolution in the tubulin dimer, and compensatory evolution in beta 2 tubulin cis-regulation. They evolve through gene duplication, subfunctionalization in expression domain and divergence of duplication products, largely in CTT residues that mediate interactions with other proteins. This has resulted in the tissue-specific minor insect isoforms, and in particular the highly diverse α3, α4, and β2 reproductive tissue-specific tubulin isoforms, illustrating that even a highly conserved protein family can participate in the adaptive process and respond to sexual selection.

Axoneme‐dependent tubulin modifications in singlet microtubules of the Drosophila sperm tail

Drosophila melanogaster sperm tubulins are posttranslationally glutamylated and glycylated. We show here that axonemes are the substrate for these tubulin C-terminal modifications. Axoneme architecture is required, but full length, motile axonemes are not necessary. Tubulin glutamylation occurs during or shortly after assembly into the axoneme; only glutamylated tubulins are glycylated. Tubulins in other testis microtubules are not modified. Only a small subset of total Drosophila sperm axoneme tubulins have these modifications. Biochemical fractionation of Drosophila sperm showed that central pair and accessory microtubules have the majority of poly-modified tubulins, whereas doublet microtubules have only small amounts of mono- and oligo-modified tubulins. Glutamylation patterns for different beta-tubulins experimentally assembled into axonemes were consistent with utilization of modification sites corresponding to those identified in other organisms, but surrounding sequence context was also important. We compared tubulin modifications in the 9 + 9 + 2 insect sperm tail axonemes of Drosophila with the canonical 9 + 2 axonemes of sperm of the sea urchin Lytichinus pictus and the 9 + 0 motile sperm axonemes of the eel Anguilla japonica. In contrast to Drosophila sperm, L. pictus sperm have equivalent levels of modified tubulins in both doublet and central pair microtubule fractions, whereas the doublets of A. japonica sperm exhibit little glutamylation but extensive glycylation. Tubulin C-terminal modifications are a prevalent feature of motile axonemes, but there is no conserved pattern for placement or amount of these

Effect of Fenbendazole on Turkey Semen Quality

Fenbendazole (FBZ) is an anthelmintic recently approved to treat and control nematode infections in growing turkeys. When administered to growing turkeys there are no detrimental side effects. However, when we used FBZ to treat nematodes in mature breeder toms, we observed a decrease in semen quality and a subsequent precipitous decline in fertility to less than 20% within 6 wk of administration. An experiment was designed to determine the impact of FBZ administration on aspects of spermatogenesis and semen quality. We discovered that although sperm viability and concentration was not significantly affected by FBZ, this drug significantly reduced sperm mobility. However, normal mobility resumed within 6 wk after FBZ administration. Fenbendazole binds to tubulin and interferes with microtubule assembly. Based on testes histology and the immunocytochemical localization of tubulin in spermatids and mature sperm, FBZ had no affect on any aspect of spermatogenesis. We suggest that FBZ may be affecting sperm mobility by some molecular alteration of the sperm tail axoneme or midpiece, both of which are tubulin-containing structures. If a breeding flock of toms become infected with nematode parasites, a flock manager has the following options: do not treat the toms, find an alternative to FBZ, treat the toms with FBZ and increase the sperm number and frequency of artificial inseminations, or treat the toms with FBZ and secure semen from an uninfected tom flock.

Functional constraint underlies 60 million year stasis of Dipteran testis‐specific β‐tubulin

How do proteins evolve while maintaining their function? Previous studies find a highly stringent structure/function relationship between the Drosophila melanogaster testis-specific tubulin beta2 and the spermtail axoneme, such that small changes in the beta2 protein render it unable to generate a motile axoneme. This raises the question, how does beta2 evolve while maintaining its function? To address this question we cloned full- and partial-length beta2 sequences from 17 species of Drosophila and Hirtodrosophila flies spanning 60 Myr of evolution. Not a single amino acid difference is coded among them-beta2 maintains its function by not evolving. We also performed gene genealogical analyses to determine ortholog/paralog relationships among insect tubulins. We find that the Lepidopteran and Dipteran testis-specific beta-tubulins are likely orthologs, and surprisingly, despite functioning in the same structure, the Lepidopteran orthologs are evolving rapidly. We argue that differences in tubulin isoform use in the testes cause the Dipteran axoneme to be less evolvable than the Lepidopteran axoneme, which has facilitated the evolution of a unique amino acid synergism in Drosophila and Hirtodrosophilabeta2 that is resistant to change, contributing to its evolutionary stasis.

Rat Spag5 associates in somatic cells with endoplasmic reticulum and microtubules but in spermatozoa with outer dense fibers

The leucine zipper motif has been identified as an important and specific interaction motif used by various sperm tail proteins that localize to the outer dense fibers. We had found that rat Odf1, a major integral ODF protein, utilizes its leucine zipper to associate with Odf2, another major ODF protein, Spag4 which localizes to the interface between ODF and axonemal microtubule doublets, and Spag5. The rat Spag5 sequence indicated a close relationship with human Astrin, a microtubule-binding spindle protein suggesting that Spag5, like Spag4, may associate with the sperm tail axoneme. RT PCR assays indicated expression of Spag5 in various tissues and in somatic cells Spag5 localizes to endoplasmic reticulum and microtubules, as expected for an Astrin orthologue. MT binding was confirmed both in vivo and in in vitro MT-binding assays: somatic cells contain a 58 kDa MT-associated Spag5 protein. Western blotting assays of rat somatic cells and male germ cells at different stages of development using anti-Spag5 antibodies demonstrated that the protein expression pattern changes during spermatogenesis and that sperm tails contain a 58 kDa Spag5 protein. Use of affinity-purified anti-Spag5 antibodies in immuno electron microscopy shows that in rat elongated spermatids and epididymal sperm the Spag5 protein associates with ODF, but not with the axonemal MTs. This observation is in contrast to that for the other Odf1-binding, MT-binding protein Spag4, which is present between ODF and axoneme. Our data demonstrate that Spag5 has different localization in somatic versus male germ cells suggesting the possibility of different function.

The proximal region of the β-tubulin C-terminal tail is sufficient for axoneme assembly

We have used Drosophila testis-specific beta2-tubulin to determine sequence requirements for different microtubules. The beta2-tubulin C-terminal tail has unique sperm-specific functions [Dev Biol 158:267-286 (2003)] and is also important for forming stable heterodimers with alpha-tubulin, a general function common to all microtubules [Mol Biol Cell 12(7):2185-2194 (2001)]. beta-tubulins utilized in motile 9 + 2 axonemes contain a C-terminal sequence "axoneme motif" [Science 275 (1997) 70-73]. C-terminal truncated beta2-tubulin cannot form the sperm tail axoneme. Here we show that a partially truncated beta2-tubulin (beta2Delta7) containing only the proximal portion of the C-terminal tail, including the axoneme motif, can support production of functional motile sperm. We conclude that these proximal eight amino acids specify the binding site for protein(s) essential to support assembly of the motile axoneme. Males that express beta2Delta7, although they are fertile, produce fewer sperm than wild type males. Beta2Delta7 causes a slightly increased error rate in spermatogenesis attributable to loss of stabilizing properties intrinsic to the full-length C-terminal tail. Therefore, beta2Delta7 males would be at a selective disadvantage and it is likely that the full-length C-terminus would be essential in the wild and in evolution.

The Drosophila fragile X-related gene regulates axoneme differentiation during spermatogenesis

Macroorchidism (i.e., enlarged testicles) and mental retardation are the two hallmark symptoms of Fragile X syndrome (FraX). The disease is caused by loss of fragile X mental retardation protein (FMRP), an RNA-binding translational regulator. We previously established a FraX model in Drosophila, showing that the fly FMRP homologue, dFXR, acts as a negative translational regulator of microtubule-associated Futsch to control stability of the microtubule cytoskeleton during nervous system development. Here, we investigate dFXR function in the testes. Male dfxr null mutants have the enlarged testes characteristic of the disease and are nearly sterile (>90% reduced male fecundity). dFXR protein is highly enriched in Drosophila testes, particularly in spermatogenic cells during the early stages of spermatogenesis. Cytological analyses reveal that spermatogenesis is arrested specifically in late-stage spermatid differentiation following individualization. Ultrastructurally, dfxr mutants lose specifically the central pair microtubules in the sperm tail axoneme. The frequency of central pair microtubule loss becomes progressively greater as spermatogenesis progresses, suggesting that dFXR regulates microtubule stability. Proteomic analyses reveal that chaperones Hsp60B-, Hsp68-, Hsp90-related protein TRAP1, and other proteins have altered expression in dfxr mutant testes. Taken together with our previous nervous system results, these data suggest a common model in which dFXR regulates microtubule stability in both synaptogenesis in the nervous system and spermatogenesis in the testes. The characterization of dfxr function in the testes paves the way to genetic screens for modifiers of dfxr-induced male sterility, as a means to efficiently dissect FMRP-mediated mechanisms.

Mutations in a novel locus on mouse chromosome 11 resulting in male infertility associated with defects in microtubule assembly and sperm tail function.

Traditional gene knock-out approaches using homologous recombination in embryonic stem cells are routinely used to provide functional information about genes involved in reproduction. In the present study, we examined a novel approach using N-ethyl-N-nitrosourea (ENU) together with a balancer chromosome mating strategy to identify new loci with functional roles in male fertility. Our genetic strategy is a forward-genetic approach; thus, our phenotypic investigation begins with the discovery of an abnormal phenotype without previous knowledge of the mutant locus. We isolated eight recessive mutations on chromosome 11 that resulted in male or female infertility from a screen of 184 founder pedigrees from ENU-treated males. After testing the six male infertile and two female infertile mutations for their ability to complement, we found that three independent recessive male infertile mutations failed to complement each other. The male infertility was associated with reduced epididymal sperm count, a block in late-spermatid differentiation, and increased apoptosis. Furthermore, the three male infertile mutants had severe defects in epididymal sperm morphology associated with incorrect microtubule assembly. Electron microscopy revealed unique defects in sperm head and tail morphology for each of the three alleles. One allele had an abnormal manchette assembly of the sperm head. The other two alleles had different abnormalities in the 9+2 patterning of the microtubules in the sperm tail axoneme, with one containing only five of the microtubule doublets and the other containing an extra doublet. The isolation of this allelic series identifies a new locus on mouse chromosome 11 that is required for spermiogenesis and male fertility.

The best of all worlds or the best possible world? Developmental constraint in the evolution of β‐tubulin and the sperm tail axoneme

Through evolutionary history, some features of the phenotype show little variation. Stabilizing selection could produce this result, but the possibility also exists that a feature is conserved because it is developmentally constrained--only one or a few developmental mechanisms can produce that feature. We present experimental data documenting developmental constraint in the assembly of the motile sperm tail axoneme. The 9+2 microtubule architecture of the eukaryotic axoneme has been deeply conserved. We argue that the quality of motility supported by axonemes with this morphology explains their long conservation, rather than a developmental necessity for the 9+2 architecture. However, our functional tests in Drosophila spermatogenesis reveal considerable constraint in the coevolution of testis-specific beta-tubulin and the sperm tail axoneme. The evolution of testis beta-tubulins used in insect sperm tail axonemes is highly punctuated, indicating some pressure acting on their evolution. We provide a mechanistic explanation for their punctuated evolution by testing structure-function relationships between testis beta-tubulin and the motile axoneme in D. melanogaster. We discovered that a highly conserved sequence feature of beta-tubulins used in motile axonemes is needed to specify central pair formation. Second, our data suggest that cooperativity in the function of internal beta-tubulin amino acids is needed to support the long axonemes characteristic of Drosophila sperm tails. Thus, central pair formation constrains the evolution of the axoneme motif, and intramolecular cooperativity makes the evolution of the internal residues path dependent, which slows their evolution. Our results explain why a highly specialized beta-tubulin is needed to construct the Drosophila sperm tail axoneme. We conclude that these constraints have fixed testis-specific beta-tubulin identity in Drosophila.

Axoneme-specific β-tubulin specialization: a conserved C-terminal motif specifies the central pair

Axonemes are ancient organelles that mediate motility of cilia and flagella in animals, plants, and protists. The long evolutionary conservation of axoneme architecture, a cylinder of nine doublet microtubules surrounding a central pair of singlet microtubules, suggests all motile axonemes may share common assembly mechanisms. Consistent with this, α- and β-tubulins utilized in motile axonemes fall among the most conserved tubulin sequences [1, 2], and the β-tubulins contain a sequence motif at the same position in the carboxyl terminus [3]. Axoneme doublet microtubules are initiated from the corresponding triplet microtubules of the basal body [4], but the large macromolecular “central apparatus” that includes the central pair microtubules and associated structures [5] is a specialization unique to motile axonemes. In Drosophila spermatogenesis, basal bodies and axonemes utilize the same α-tubulin but different β-tubulins [6–13]. β1 is utilized for the centriole/basal body, and β2 is utilized for the motile sperm tail axoneme. β2 contains the motile axoneme-specific sequence motif, but β1 does not [3]. Here, we show that the “axoneme motif” specifies the central pair. β1 can provide partial function for axoneme assembly but cannot make the central microtubules [14]. Introducing the axoneme motif into the β1 carboxyl terminus, a two amino acid change, conferred upon β1 the ability to assemble 9 + 2 axonemes. This finding explains the conservation of the axoneme-specific sequence motif through 1.5 billion years of evolution.

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.

The Y chromosomal fertility factor Threads in Drosophila hydei harbors a functional gene encoding an axonemal dynein beta heavy chain protein.

To understand the contradiction between megabase-sized lampbrush loops and putative protein encoding genes both associated with the loci of Y chromosomal fertility genes of Drosophila on the molecular level, we used PCR-mediated cloning to identify and isolate the cDNA sequence of the Y chromosomal Drosophila hydei gene DhDhc7(Y). Alignment of the sequences of the putative protein DhDhc7(Y) and the outer arm dynein beta heavy chain protein DYH2 of Tripneustes gratilla shows homology over the entire length of the protein chains. Therefore the proteins can be assumed to fulfill orthologous functions within the sperm tail axonemes of both species. Functional dynein beta heavy chain molecules, however, are necessary for the assembly and attachment of outer dynein arms within the sperm tail axoneme. Localization of DhDhc7(Y) to the fertility factor Threads, comprising at least 5.1 Mb of transcriptionally active repetitive DNA, results from an infertile Threads- mutant where large clusters of Threads specifically transcribed satellites and parts of DhDhc7(Y) encoding sequences are missing simultaneously. Consequently, the complete lack of the outer dynein arms in Threads- males most probably causes sperm immotility and hence infertility of the fly. Moreover, preliminary sequence analysis and several other features support the hypothesis that DhDhc7(Y) on the lampbrush loops Threads in D. hydei and Dhc-Yh3 on the lampbrush loops kl-5 in Drosophila melanogaster on the heterochromatic Y chromosome of both species might indeed code for orthologous dynein beta heavy chain proteins.

Structurally similar Drosophila alpha-tubulins are functionally distinct in vivo.

We used transgenic analysis in Drosophila to compare the ability of two structurally similar alpha-tubulin isoforms to support microtubule assembly in vivo. Our data revealed that even closely related alpha-tubulin isoforms have different functional capacities. Thus, in multicellular organisms, even small changes in tubulin structure may have important consequences for regulation of the microtubule cytoskeleton. In spermatogenesis, all microtubule functions in the postmitotic male germ cells are carried out by a single tubulin heterodimer composed of the major Drosophila alpha-84B tubulin isoform and the testis-specific beta 2-tubulin isoform. We tested the ability of the developmentally regulated alpha 85E-tubulin isoform to replace alpha 84B in spermatogenesis. Even though it is 98% similar in sequence, alpha 85E is not functionally equivalent to alpha 84B. alpha 85E can support some functional microtubules in the male germ cells, but alpha 85E causes dominant male sterility if it makes up more than one-half of the total alpha-tubulin pool in the spermatids. alpha 85E does not disrupt meiotic spindle or cytoplasmic microtubules but causes defects in morphogenesis of the two classes of singlet microtubules in the sperm tail axoneme, the central pair and the accessory microtubules. Axonemal defects caused by alpha 85E are precisely reciprocal to dominant defects in doublet microtubules we observed in a previous study of ectopic germ-line expression of the developmentally regulated beta 3-tubulin isoform. These data demonstrate that the doublet and singlet axoneme microtubules have different requirements for alpha- and beta-tubulin structure. In their normal sites of expression, alpha 85E and beta 3 are coexpressed during differentiation of several somatic cell types, suggesting that alpha 85E and beta 3 might form a specialized heterodimer. Our tests of different alpha-beta pairs in spermatogenesis did not support this model. We conclude that if alpha 85E and beta 3 have specialized properties required for their normal functions, they act independently to modulate the properties of microtubules into which they are incorporated.

Determination of Microtubule Polarityin Vitroby the Use of Video-Enhanced Differential-Interference Contrast Light Microscopy andChlamydomonasFlagellar Axonemal Pieces

Microtubules nucleated by sea urchin sperm-tail axonemes have polar ends that differ both functionally and structurally but cannot be distinguished from one another when viewed by light microscopy. Ambiguity and circularity surround any classification of microtubule polarity by conventional methods.Chlamydomonasflagellar axonemal pieces have distinct morphological differences at their plus- and minus-ends, and microtubules nucleated from these pieces can be distinguished as plus- or minus-ended based on the morphological differences present in theChlamydomonasflagellar axonemal pieces. Plus- and minus-ended microtubules were polymerized in this fashion and analyzed for differences in growth rates, shortening rates, and frequencies of transitions. The results were in good agreement with similar data generated by the more time-consuming and difficult use of kinesin-coated beads (R. J. Kowalski, and R. C. Williams, Jr. (1993)Cell Motil. Cytoskeleton26, 282–290) to determine microtubule polarity. This is a relatively simple and effective method for determining the polarity of microtubulesin vitroby video-enhanced differential-interference contrast light microscopy.

Reactivated movement of decondensed rat sperm models and a description of their ultrastructure

This study aimed at finding optimal conditions to decondense rat sperm nuclear chromatin with minimal damage. This was judged by the ability of the sperm-tail axoneme in the partially decondensed sperm models to be reactivated. Decondensation was assessed by phase contrast microscopy. Partial decondensation was judged to occur when the bright refractive appearance of the sperm nucleus turned black, and full decondensation when the nucleus turned pale and increased in volume. Demembranation was shown to have occurred by electron microscopy. With 0.03% Triton X-100 rat caudal epididymal sperm were partially demembranated to produce sperm models. Demembranation using a 0.1% solution of Triton X-100 was complete, but as with the solution of 0.05% Triton X-100, resulted in poorer reactivation of the partially decondensed sperm models. Reactivated movement of decondensed sperm models was used to assess the effect of the decondensing agents DTT and heparin. We were only able to achieve reactivation of sperm models that had undergone partial decondensation. Optimal reactivation was obtained after rat sperm models had decondensed in the decondensation solution containing 5 mM DTT, 6 mM EDTA, and 27.3 or 34.1 USP/ml heparin. Concentrations of heparin above or below these values resulted in a decrease in the number of sperm models reactivated. Ultrastructurally, sperm partially decondensed with 5 mM DTT, 6 mM EDTA, and 34.1 USP/ml heparin had their plasma membrane further extracted compared with sperm treated with 0.03% Triton X-100 alone. Decondensation was greatest in the peripheral regions of the nucleus with extraction of the acrosome but not of the perforatorium. The decondensed regions had a filamentous appearance. This procedure will allow access to sperm nuclear chromatin for experimental manipulation in rat sperm models.

Three-Dimensional Reconstructions of Accessory Tubules Observed in the Sperm Axonemes of Two Insect Species

The sperm-tail axonemes of several insect species are known to have accessory microtubules with a diameter greater than that of ordinary cytoplasmatic microtubules. This provides an opportunity to investigate the three-dimensional organization of naturally occurring tubules with more than 13 protofilaments. For this purpose electron micrographs of negatively stained axonemes of a dipteran species, Exechia seriata, and a trichopteran, Limnephilus bipunctatus , were taken for computer analysis. The original micrographs contained long stretches of somewhat curved microtubules that, in a first step, were straightened by an appropriate algorithm. The results were used to determine helical models from spectral analysis, to obtain filtered images of the front and back tubule sides, and to compute three-dimensional reconstructions by Fourier methods. The tubule segments used in the present study were about 500 nm long. All accessory microtubules analyzed consisted of a double-stranded helix of clearly distinguishable heterodimers with a pitch of 16 nm and skewing protofilaments. Most segments of the dipteran accessory tubules consisted of 16 protofilaments, although 15 were found in some images portraying the distal region of the flagellum. The accessory tubules of the trichopteran had 19 protofilaments. The repeat periods that describe the skewness of protofilaments (128 nm for 16 and 320 nm for 19 protofilaments) are in any case considerably thicker than an electron microscopical section which encompasses about 50 nm. It is hence valid to count the number of protofilaments in cross-sectioned axonemes.

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.

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.