Ultrastructure of the biflagellate spermatozoon of tomopteris helgolandica greef, 1879 (annelida,polychaeta)

The spermatozoon of the polychaete Tomopteris helgolandica is of an aberrant type with two flagella, each measuring about 40μm. The nucleus is roughly conical and weakly bent. At the anterior end it is rounded and covered only by the nuclear and plasma membranes. Membraneous, electron-dense structures are applied laterally to the nucleus. These structures may have a helical arrangement. The middle piece contains about ten mitochondria, two centrioles, and two centriolar satellite complexes. The centriolar regions are connected with the posterior part of the nucleus. The axonemes of the two tail flagella lack the usual central complex with central tubules, radial spokes, or related structures. No arms seem to be present on the A tubules of the doublets. In the middle piece the tail flagella are surrounded by invaginations of the plasma membrane forming flagellar canals. The sperm has a bilateral symmetry whereas the primitive sperm has a radial symmetry. The occurrence of two tail flagella in this spermatozoon has no phylogenetical connection with biflagellate spermatozoa in other animal groups. A series of mutations has resulted in the development of two flagella emerging from the two centrioles, the lack of a central complex in the axoneme, and the lack of a typical acrosome. In the Polychaeta, sperm structure is generally more related to function that to phylogenetics. During swimming the spermatozoon of Tomopteris rotates around its longitudinal axis.     

A biflagellate spermatozoon in the African bonytongue Heterotis niloticus (Teleostei,Osteoglossidae)

In this transmission electron microscopy study, we describe the ultrastructure of the spermatozoon of Heterotis niloticus (Osteoglossiformes), which is distinguished by having two flagella. Our investigation also highlights the great diversity of sperm cell structures observed across osteoglossiform families, such as aflagellate (Gymnarchidae, Mormyridae), monoflagellate (Notopteridae, Pantodontidae) and biflagellate spermatozoa. As biflagellate spermatozoa are rare in vertebrates, we also summarize the orders and families known to possess this ultrastructural character, most of which are fishes.     

The role of structural integrity of the fertilising spermatozoon in early human embryogenesis.

While the fertilising spermatozoon supplies the active centre directing the human zygote's first mitotic division, the relative contributions of the sperm head and tail (as well as the importance of the sperm's general structural integrity) to subsequent developmental processes remain incompletely studied. The sperm nucleus contains paternal chromatin necessary for restoration of a diploid genome, but the functional role of the sperm tail (either attached or dissected) in early human embryonic growth is not known. In this investigation using oocytes donated by in vitro fertilisation patients, human oocytes were injected with isolated sperm heads (n = 73), isolated sperm flagella (n = 11) or both (dissected sperm heads + free sperm tails, n = 26). The formation of bipronucleate zygotes was recorded for each method. Among oocytes surviving injection with isolated sperm heads, 44 of 66 (67%) formed two pronuclei. Of oocytes receiving only sperm tails, 2 of 11 (18%) displayed two pronuclei, but a single polar body was evident in both cases. When dissected spermatozoa parts (head + tail) were jointly injected, 12 of 26 (46%) developed two pronuclei. From embryos resulting from each of these three fertilisation regimes, blastomere biopsies were obtained and subjected to multiprobe fluorescent in situ hybridisation (FISH) analysis to detect mosaicism or aneuploidy arising from these experimental treatments. Only embryos with growth sufficient to permit sampling of at least two blastomeres were evaluated, and FISH analysis was successful in 25 of 29 (86%) embryos tested. Of 12 embryos derived from injection of an isolated sperm head, only one was normal diploid; the remaining 11 were mosaic. Both embryos resulting from injection of an unattached sperm tail were mosaic. Of 11 embryos generated from oocyte injection with sperm head + tail segments, 10 (91%) were mosaic and only one was normal diploid. Results from this study show that injection of isolated sperm segments can permit oocyte activation and bipronuclear formation. However, a high rate of mosaicism in human embryos originating from disrupted sperm or sperm components suggests that more than a 'sum of parts' is needed for later development. The structural integrity of the intact fertilising spermatozoon appears to contribute to normal human early embryogenesis.     

The equation of motion for sperm flagella.

The equation of motion for sperm flagella, in which the elastic bending moment and the active contractile moment are balanced by the moment from the viscous resistance of the surrounding fluid, is solved for a wave solution that superimposes partial solutions. Substitution of the expression for the wave solution into the equation leads to an expression for the active contractile moment. This active moment can be decomposed into two parts. The first part describes an active moment that travels over the flagellum with the mechanical flagellar wave, the second part represents a moment in phase over the entire length of the flagellum, which decreases linearly towards the distal tip. The linear synchronous moment, to which an amount of traveling moment has been added as a perturbation, leads to wave solutions that closely resemble flagellar waves. Properties such as wavelength and wave amplitudes and also the shape of the waves in sea urchin sperm flagella at different frequencies are accurately described by the theory. The change in wave shape in sea urchin sperm flagella at raised viscosity is predicted well by the theory. The different wave properties caused in bull sperm flagella by different boundary conditions at the proximal junction are explained. When only a traveling active moment is present in a flagellum, the wave solutions describe waves of a small wave length in a long flagellum. Some properties of the wave motion of sperm flagella are derived from the theory and verified experimentally.     

FERTILIZATION IN OEDOGONIUM. I. PLASMOGAMY12

Events prior to, during, and immediately following plasmogamy have been investigated in Oedogonium cardiacum using combined techniques of light and electron microscopy. Maturation of the oogonium involves the formation of an oogonial pore and the differentiation of the single egg from the larger oogonial protoplast from which it is formed. The fine structure of the sperm cell at the time of plasmogamy is described as well as the nature of its entrance into the oogonium. Cinematographic films were used to analyze the movements of the spermatozoids prior to plasmogamy and, similarly, 26 complete acts of gametic fusion were recorded and analyzed. Prior to plasmogamy the flagella-bearing anterior extremity of the spermatozoid typically becomes elongate and is thereafter capable of flexible movements and rapid changes in shape which appear more or less independent of the rest of the cell. The sperm cell always makes initial contact with the egg surface by means of this agile, proboscis-like, anterior end. Contact results through a combination of thrusting movements of the entire sperm cell and rapid, lateral sweeping movements of its flagellated anterior extremity against the egg surface. Gametic fusion is initiated with violent, vibrational movements of the sperm cell accompanied by loss of its flagella. Apparent fusion of the gamete membranes unites their protoplasts by a narrow cytoplasmic bridge which gradually increases in size as the sperm cell cytoplasm flows into the egg. An average time of 30.5 sec was required for complete fusion as determined from 25 typical sequences of plasmogamy recorded cinematographically. Fusion occurs even more rapidly when diploid oogonia are substituted for daploid oogonia. The entire sperm cell, with the exception of the flagella, fuses with the egg during plasmogamy. The dissimilar gamete nuclei are clearly distinguished ultrastructurally in the binucleate fusion cell. Concentrations of sperm cell mitochondria and remains of the flagellar apparatus (but no flagella) are readily recognized in the fusion cell. The fate of these and other cytoplasmic constituents of the sperm cell is discussed. Immediately after plasmogamy, and prior to karyogamy, a thin, finely fibrous layer is formed us an investment exterior to the fusion cell. Karyogamy follows shortly after plasmogamy, and both events may take place within 15 min after mixing eggs and spermatozoids.     

The spermatozoon flagella in Diphyllobothrium latum (fish tapeworm)

Summary The spermatozoon flagella of the fish tapeworm (Diphyllobothrium latum) is studied. The flagella consist of two axial filament complexes, which represent the unusual nine-plus-one pattern. The inside of the spermatozoon sheath is lined by single filaments. The mechanism of formation of this type of flagella is discussed. The spermatozoon flagella of the flatworms hitherto described seems to show some deviating features as compared with those in other species.It is a pleasure to acknowledge the skilful technical assistance of Miss Tellervo Huima. This study is supported by Finnish Medical Research Council.     

THE EFFECTS OF COLCHICINE ON SPERMATOGENESIS IN NITELLA

Treatment of Nitella antheridia with colchicine results in various sperm abnormalities, depending upon duration of exposure and subsequent recovery. Early effects of treatment include disappearance of spindle fibers and a cessation of ordered cell wall formation in dividing cells. Sperm released from antheridia treated for 24 hr and allowed to recover for 4–5 days possess branched flagella. After a recovery period of 6–10 days the sperm appear normal; however, following longer recovery periods, the sperm exhibit variations in size and number of flagella. Branched flagella contain a variety of microtubule patterns ranging from branches containing a single microtubule to flagella with an excess of microtubules. Spermatids which differentiate in the presence of colchicine lack flagella and a microtubular sheath. Nuclear contents undergo condensation stages; however, the nucleus as a whole does not undergo the orderly elongation and coiling characteristic of untreated Nitella spermatids. Long-term colchicine treatment followed by a recovery period produces atypical microtubules and microtubular aggregations in the spermatid. The results indicate that colchicine affects not only polymerization of microtubule subunits but also factors responsible for their ordered spatial relationships in the cell. The presence of microtubules is a prerequisite for normal morphological changes during spermiogenesis.     

Spermiogenesis and spermatozoon ultrastructure of the digenean Neoapocreadium chabaudi (Apocreadiidae), a parasite of Balistes capriscus (Pisces,Teleostei)

Spermiogenesis and the ultrastructural organization of the spermatozoon of the digenean Neoapocreadium chabaudi are described by means of transmission electron microscopy.Spermiogenesis follows the usual pattern found in the digeneans. It begins with the formation of a zone of differentiation bordered by cortical microtubules, characterized by the presence of an intercentriolar body composed of seven electron-dense plates situated between two striated rootlets and two centrioles. These centrioles give rise to two free flagella. Later, both flagella undergo a rotation of 90° and fuse with the median cytoplasmic process. Spermiogenesis finishes when the ring of arched membranes constricts. The mature spermatozoon of N. chabaudi is characterized by the presence of 2 axonemes of different lengths presenting the 9 + “1” trepaxonematan pattern, 2 bundles of parallel cortical microtubules, 2 mitochondria, a nucleus, and granules of glycogen. Nevertheless, several characters such as the morphology of sperm extremities and the presence of spinelike bodies allow us to distinguish N. chabaudi from other digenetic trematodes. The present paper provides the first ultrastructural results of a digenean belonging to the family Apocreadiidae that may be useful for the understanding of digenean relationships and phylogenetic studies.     

Ultrastructure of spermiogenesis and the spermatozoon in Castrada cristatispina Papi, 1951 (Platyhelminthes, Rhabdocoela, Typhloplanida)

Spermiogenesis in Castrada cristatispina begins with the formation of a zone of differentiation containing two centrioles with associated striated rootlets and an intercentriolar body between them. The centrioles give rise to two parallel, free flagella of the Trepaxonemata 9 + '1' pattern, growing out in opposite directions. Spermatids undergo a latero-ventral rotation of the flagella and a subsequent disto-proximal rotation of centrioles, and a distal cytoplasmic projection appears. The former rotation involves the compression of a row of microtubules and allows the recognition of a ventral side and a dorsal side. At the end of the differentiation, the centrioles and cortical microtubules lie parallel to the sperm axis. The modifications of the intercentriolar body and the migration of the nucleus and the centrioles toward the distal projection are described. The mature spermatozoon of C. cristatispina is filiform, tapered at both ends and shares several features with the other Rhabdocoela gametes. Nevertheless, the posterior extremity is capped by an electron-dense material. A gradient between mitochondria and dense bodies exists along the sperm axis. This study has enable us a phylogenetic approach of the Rhabdocoela through a comparison of the ultrastructural features of C. cristatispina with the other Rhabdocoela taxa. We propose the disto-proximal rotation of centrioles as a synapomorphy of the Rhabdocoela.     

MNS1 is essential for spermiogenesis and motile ciliary functions in mice

During spermiogenesis, haploid round spermatids undergo dramatic cell differentiation and morphogenesis to give rise to mature spermatozoa for fertilization, including nuclear elongation, chromatin remodeling, acrosome formation, and development of flagella. The molecular mechanisms underlining these fundamental processes remain poorly understood. Here, we report that MNS1, a coiled-coil protein of unknown function, is essential for spermiogenesis. We find that MNS1 is expressed in the germ cells in the testes and localizes to sperm flagella in a detergent-resistant manner, indicating that it is an integral component of flagella. MNS1-deficient males are sterile, as they exhibit a sharp reduction in sperm production and the remnant sperm are immotile with abnormal short tails. In MNS1-deficient sperm flagella, the characteristic arrangement of "9+2" microtubules and outer dense fibers are completely disrupted. In addition, MNS1-deficient mice display situs inversus and hydrocephalus. MNS1-deficient tracheal motile cilia lack some outer dynein arms in the axoneme. Moreover, MNS1 monomers interact with each other and are able to form polymers in cultured somatic cells. These results demonstrate that MNS1 is essential for spermiogenesis, the assembly of sperm flagella, and motile ciliary functions.     

The fine structure of the spermatozoon of Pennaria tiarella (coelenterata)

Spermatozoa of the hydroid Pennaria tiarella were examined with the electron microscope. The anterior region is characterized by the presence of 30–40 membrane-bounded vesicles which lie anterior to the nucleus. These vesicles are apparently derived from the Golgi apparatus. The nucleus is conical in shape with a protrusion at the anterior end. Posteriorly it is indented by four radially arranged mitochondria. Lying within the fossa formed by the mitochondria are proximal and distal (filament forming) centrioles. The distal centriole is characterized by nine centriole satellite projections which emanate from its matrix. The tubules of the distal centriole are continuous with the alpha filaments of the tail. The tails are typical 9 + 2 flagella with 9 peripheral doublet (or alpha) filaments surrounding two central (or beta) filaments.     

Formation of temporary flagellar structures during insect organogenesis

Cilia and flagella are rare in nongerminal tissues of anthropods, and are generally thought to be restricted to sperm and sensory cells in insects (2). Whitten (5) has reported the presence of kinetosomes at the base of mitotrichia in the dipteran fly Sarcophaga bullata, but reports no evidence of the organization of fibrous elements characteristic of cilia and or flagella. During an ultrastructural analysis of morphogenesis of the colleterial gland of the silk moth Hyalophora cecropia, we found the first example of paired flagella associated with an insect secretory cell. These structures are also unusual in that they serve a temporary role in morphogenesis and subsequently disappear at the terminal stages of differentiation.     

Spermiogenesis and the spermatozoon ultrastructure of Robphildollfusium fractum (Digenea: Gyliauchenidae), an intestinal parasite of Sarpa salpa (Pisces: Teleostei)

Spermiogenesis in Robphildollfusium fractum begins with the formation of a differentiation zone containing: two centrioles, each bearing striated rootlets, nucleus, several mitochondria and an intercentriolar body constituted by seven electron-dense layers. The two centrioles originate two free flagella growing orthogonally to the median cytoplasmic process. Later, the free flagella rotate and undergo proximodistal fusion with the median cytoplasmic process. Nuclear and mitochondrial migrations occur before this proximodistal fusion. Finally, the young spermatozoon detaches from the residual cytoplasm after the constriction of the ring of arched membranes. The spermatozoon of R. fractum exhibits two axonemes of different length of the 9 + ‘1’ trepaxonematan pattern, nucleus, two mitochondria, two bundles of parallel cortical microtubules, external ornamentation of the plasma membrane, spine-like bodies and granules of glycogen. Additionally, a shorter axoneme, which does not reach the nuclear region, the presence of an electron-dense material in the anterior spermatozoon extremity and the morphologies of both spermatozoon extremities characterize the mature sperm of R. fractum.     

A scanning electron microscopical study of sperm development and activation in Arenicola marina L. (Annelida: Polychaeta)

Abstract

The lugworm, Arenicola marina L. has an annual cycle of reproduction with epidemic spawning and external fertilisation. The spermatozoa of Arenicola are unusual in that they are held immotile (as plates of several hundred cells known as morulae) in the coelomic fluid until activated just prior to spawning. Activation of Arenicola sperm is brought about by a sperm maturation factor (SMF) from the prostomium and can be carried out in vitro using an assay technique developed by Bentley (1985). Scanning electron microscopy is used here to examine the changes which occur during in vitro activation. This revealed that the bundles of flagella of inactive sperm become disorganised as flagella beating commences but the flagella at this stage are still bound together at their tips. The sperm heads then become separated from the cytophore and finally the distal binding of the flagella is broken to give free-swimming spermatozoa. Coelomocytes present in the coelomic fluid resorb unspawned gametes prior to the initiation of the next gametogenic phase.     

Ultrastructure of spermatogenesis and mature spermatozoa in the flatworm Prosthiostomum siphunculus (Polycladida,Cotylea)

This is the first study investigating spermatogenesis and spermatozoan ultrastructure in the polyclad flatworm Prosthiostomum siphunculus. The testes are numerous and scattered as follicles ventrally between the digestive ramifications. Each follicle contains the different stages of sperm differentiation. Spermatocytes and spermatids derive from a spermatogonium and the spermatids remain connected by intercellular bridges. Chromatoid bodies are present in the cytoplasm of spermatogonia up to spermatids. During early spermiogenesis, a differentiation zone appears in the distal part of spermatids. A ring of microtubules extends along the entire sperm shaft just beneath the cell membrane. An intercentriolar body is present and gives rise to two axonemes, each with a 9 + “1” micro‐tubular pattern. Development of the spermatid leads to cell elongation and formation of a filiform, mature spermatozoon with two free flagella and with cortical microtubules along the sperm shaft. The flagella exit the sperm shaft at different levels, a finding common for acotyleans, but so far unique for cotylean polyclads. The Golgi complex produces numerous electron‐dense bodies of two types and of different sizes. These bodies are located around a perinuclear row of mitochondria. The elongated nucleus extends almost along the entire sperm body. The nucleus is wide in the proximal part and becomes narrow going towards the distal end. Thread‐like chromatin mixed with electron‐dense intranuclear spindle‐shaped bodies are present throughout nucleus. The general sperm ultrastructure, the presence of intranuclear bodies and a second type of cytoplasmic electron‐dense bodies may provide characters useful for phylogenetic analysis.     

The spermatozoon of arthropoda. XXIV. Sperm metamorphosis in the diplopod Polyxenus

Specimens, representative of each of the major taxa of mosquitoes, were fixed in copula and the external genitalia examined by scanning electron microscopy. The periphery of the basin-like everted aedeagus of Aedus aegypti precisely matches that of the everted atrial membrane of the female. These structures are appressed during coitus and sealed by pressure of the paraprocts, aedeagal pouch and proctiger. When everted, the aedeagus of male Culex pipiens reveals a ridged dome that surrounds the genital opening. This dome seals itself laterally into a gutter formed by pad-like extensions of the female's genital lips and is sealed dorsally by pressure of the aedeagal apodeme. The aedeagus of another culicine species, Wyeomyia smithii, bears the gonopore at the apex of a spined tube. This tube is inserted between the female's genital lips and is sealed within the genital atrium. The aedeagus of the toxorhynchitine species Toxorhynchitis brevipalpus is immobile and is inserted deep within the genital atrium of the female where it is sealed by pressure of the atrial walls. Males of each of these mosquitoes deliver a mixture of semen and sperm to the copulatory bursa of the female. After withdrawal of the aedeagus, sperm is transferred to the spermathecae. In contrast, sperm of Anopheles quadrimaculatus are delivered directly to the spermathecal duct. The tube-like aedeagus is positioned by its leaflets during sperm transfer and is driven deep into the atrium, where a mixture of semen and sperm is ejaculated. The significance of mechanical barriers to mating between species is discussed.     

The restructuring of the flagellar base and the flagellar necklace during spermatogenesis of Ephestia kuehniella Z. (Pyralidae,Lepidoptera)

Summary Transmission electron microscopy was used to study the development of the flagellar base and the flagellar necklace during spermatogenesis in a moth (Ephestia kuehniella Z.). Until mid-pachytene, two basal body pairs without flagella occur per cell. The basal bodies, which contain a cartwheel complex, give rise to four flagella in late prophase I. The cartwheel complex appears to be involved in the nucleation of the central pair of axonemal microtubules. In spermatids, there is one basal body; this is attached to a flagellum. At this stage, the nine microtubular triplets of the basal body do not terminate at the same proximal level. The juxtanuclear triplets are shifted distally relative to the triplets distant from the nuclear envelope. Transition fibrils and a flagellar necklace are formed at the onset of axoneme elongation. The flagellar necklace includes Y-shaped elements that connect the flagellar membrane and the axonemal doublets. In spindle-containing spermatocytes, the flagellar necklace is no longer detectable. During spermatid differentiation, the transition fibrils move distally along the axoneme and a prominent middle piece appears. Our observations and those in the literature indicate certain trends in sperm structure. In sperms with a short middle piece, we expect the presence of a flagellar necklace. The distal movement of the transition fibrils or equivalent structures is prevented by the presence of radial linkers between the flagellar membrane and the axonemal doublets. On the other hand, the absence of a flagellar necklace at the initiation of spermiogenesis enables the formation of a long middle piece. Thus, in spermatozoa possessing an extended middle piece, a flagellar necklace may be missing.     

Microtubule polyglutamylation in Drosophila melanogaster brain and testis.

The presence of glutamylated tubulin, a widespread posttranslational modification of alpha- and beta-tubulin, has been investigated in Drosophila melanogaster using the specific monoclonal antibody GT335. We show here that this modification is strongly detected in brain and testis whereas other tissues analyzed did not appear to contain any glutamylated isoforms. Neuronal microtubules are glutamylated on alpha-tubulin only whereas sperm flagella showed a strong modification of both alpha- and beta-tubulin. These results argue for an essential role for glutamylation in differentiation processes that require microtubule stabilization.     

The effects of male social environment on sperm phenotype and genome integrity

Sperm function and quality are primary determinants of male reproductive performance and hence fitness. The presence of rival males has been shown to affect ejaculate and sperm traits in a wide range of taxa. However, male physiological conditions may not only affect sperm phenotypic traits but also their genetic and epigenetic signatures, affecting the fitness of the resulting offspring. We investigated the effects of male‐male competition on sperm quality using TUNEL assays and geometric morphometrics in the zebrafish, Danio rerio. We found that the sperm produced by males exposed to high male–male competition had smaller heads but larger midpiece and flagellum than sperm produced by males under low competition. Head and flagella also appeared less sensitive to the osmotic stress induced by activation with water. In addition, more sperm showed signals of DNA damage in ejaculates of males under high competition. These findings suggest that the presence of a rival male may have positive effects on sperm phenotypic traits but negative effects on sperm DNA integrity. Overall, males facing the presence of rival males may produce faster swimming and more competitive sperm but this may come at a cost for the next generation.