Ultrastructural Study of Spermatogenesis and Mature Spermatozoa of Paravortex cardii (Platyhelminthes, Dalyellioida)

The spermatogenesis and mature spermatozoon of Paravortex cardii were studied by transmission electron microscopy. Meiotic divisions occur without cytokinesis and the spermatid nuclei appear embedded in a common cytoplasmic mass. The mature spermatozoon is filiform, very regular in contour and circular in cross-section. A tubular lining of microtubules lying close to the plasma membrane is found along the spermatozoon. Rows of spherical glycogen particles with helical arrangement lay internal to the cortical microtubules. The spermatozoon of P. cardii may be divided into two regions, nuclear and cytoplasmic regions. The nuclear region contains an elongated nucleus with a densely packed nuclear material. The mitochondria are distributed throughout the cytoplasmic region; they pack tightly together and often fuse to form one large one. This spermatozoon lacks both acrosome and the so-called dense bodies. A ciliary or centriolar apparatus was not observed. Accordingly, the spermatozoon of P. cardii is considered to be aflagellate in type. Spermatozoa are compared among flatworms, and some considerations on the significance of their ultrastructure for phylogeny in the Platyhelminthes are tentatively given.     

Spermiogenesis in a Scutariellid (Platyhelminthes)

Spermiogenesis and spermatozoa were studied by transmission and scanning electron microscopy in Troglocaridicolasp., a scutariellid epizoic on a cavernicolous freshwater shrimp. Spermiogenesis involves elongation of the spermatid in which the nucleus elongates, but remains close to the common cytoplasmic mass. Flagella first grow in opposite direction and at a right angle to the cytoplasmic shaft, and centrioles show associate structures. Later, the two centrioles rotate and the flagella emerge parallel, but still perpendicular to the shaft. An apical process elongates at the extremity of the spermatid shaft. The spermatozoon shows active flagellar beating and undulations of the sperm body. The spermatozoon comprises an anterior ‘corkscrew’ region, the flagellar insertion region, a cytoplasmic region and a posterior nuclear region. The corkscrew contains an electron dense structure, not membrane-bound, originating from the apical process of the spermatid. The flagella show the 9+‘1’ pattern, usual in Platyhelminthes. The cytoplasmic and nuclear regions show a cortical row of about 50 twisted longitudinal microtubules surrounding a row of electron dense, and not membrane-bound, 25-nm granules. These granules are original structures and seem to be known only in a few Platyhelminthes species in which a non-flagellar movement of the spermatozoon occurs. Thus, it is hypothesised that the 25-nm granules play a role in cellular motility. Sperm ultrastructure in Troglocaridicolashows major differences to that in the temnocephalids. It is therefore concluded that the phylogenetic position of the scutariellids within the Temnocephalidea should be reinvestigated.     

Microtubular system during spermiogenesis and in the spermatozoon of Convoluta saliens (platyhelminthes,acoela): Tubulin immunocytochemistry and electron microscopy

Spermiogenesis and the spermatozoon were studied in Convoluta saliens, an acoel platyhelminth, by transmission electron microscopy, labelling of nuclei and immunocytochemistry of tubulin with various antibodies. Spermiogenesis involves formation of a long spermatid shaft containing two axonemes. It is established that the nucleus, after a stage of elongation, does not migrate up to the distal extremity of the spermatid, and that the centriolar derivatives are located at the distal extremity of the shaft. This contrasts with the parasitic Platyhelminthes. The mature spermatozoon, 180 μm in length, comprises a nuclear region, 50 μm in length, and a cytoplasmic region, with a short region of overlap. The cytoplasmic region contains two lateral axonemes with a 9 + 2 pattern of microtubules, granules of two different sizes, and two rows of longitudinal microtubules in the center. Each row consists of 5–6 singlet microtubules, with links between them. Whereas the two axonemes are labelled by antibodies against alpha, acetylated‐alpha, and beta tubulin, the microtubule rows are labelled only by the anti‐beta‐tubulin antibody. This suggests that acetylation does not occur in this part of the cytoskeleton, and that the epitope recognized by the anti‐alpha‐tubulin antibody (DM1A) is different in these units. Mol. Reprod. Dev. 52:74–85, 1999. © 1999 Wiley‐Liss, Inc.     

Ultrastructure comparée de la spermiogenèse et du spermatozoïde de trois espèces de Paravortex (Rhabdocoela, "Dalyellioida", Graffillidae), Turbellariés parasites intestinaux de Mollusques

Three species of the genus Paravortex were studied. Spermiogenesis involves progressive lengthening of the spermatid. No centriole nor axoneme were found. Thc mature spermatozoon is threadlike, 30μm long and aflagellate. It contains a nucleus, two longitudinal parallel mitochondria, numerous dense bodies limited by a membrane, a row of cortical microtubules and granules. The spatial arrangement of the microtubules wah deduced from a statistical analysis of their number in transverse section. The microtubules are continuous along the length of the spermatozoon and are arranged along a long cone. The granules, not limited by a membrane, are about 25 nm in diameter. They are arranged in a semicircle and represent a new structure for sperm of Platyhclminthes. A statistical comparison demonstrated that two species may be distinguished by the number of their cortical sperm microtubules. This is the tirst ultrastructural study of an aflagellate spermatozoon in the Rhabdocoela. A rickettsia was found in P. cardii .     

The Homology of Cortical Microtubules in Platyhelminth Spermatozoa: a Comparative Immunocytochemical Study of Acetylated Tubulin

Spermatozoa of certain acoels, a group of primitive Platyhelminthes, and spermatozoa of the most derived Platyhelminthes, the Cercomeridea (parasitic Platyhelminthes), show a general morphological resemblance in that they are long filiform cells with two incorporated axonemes and longitudinal cortical microtubules. A possible way to test the homology of these cortical microtubules in the different groups is to analyze the presence/absence of post-translational modifications of tubulin. An indirect immunofluorescence study showed that the doublet microtubules of the sperm axonemes are labelled by an anti acetylated-alpha-tubulin antibody in all groups, irrespective of the axoneme pattern (9 + 0, 9 + 2 and non-trepaxonematan 9 + “1” in various acoels, and trepaxonematan 9 + “1” of the temnocephalid Troglocaridicola sp., the digenean Echinostoma caproni and the monopisthocotylean monogenean Pseudodactylogyrus sp.). Significant differences are found in the sperm cortical microtubules: they are acetylated in the acoel Actinoposthia beklemischevi, but not in the digenean E. caproni and the temnocephalid Troglocaridicola sp. These results suggest that the sperm cortical microtubules of the acoels are not homologous with the morphologically similar elements found in the higher Platyhelminthes.     

Implications phylogénétiques de I'ultrastructure de la spermatogenèse, du spermatozoïde et de I'ovogenèse du TurbellariéUrastoma cyprinae ("Prolecithophora", Urastomidae)

Spermiogenesis in Urastoma cyprinae (Graff. 1882) involvcs a progressive lengthening of the spermatid. Free flagella are only transitory. The mature spermatozoon is fusibrm. 45μm in length and 2.5 μm in width. It contains two incorporated axonermes of the flatworm 9+"1" pattern, two elongated mitochondria. an elongated nucleus and a row or cortical longitudinal microtubules. Observations on oogenesis concerncd only the immuturc ovary. lmmature oocytes contain few dense granules and accssory cells were not ohserved. Phylogenetic implications of a biflagellate spermatozoon in a Prolecithophora are important. The presence of two 9 +"1" axonemes confirms that Urastoma (and the Prolecithophora) belongs to the taxon Trepaxonemata Ehlers. Previous electron microscope studies on spermitozoa of Prolecithophora (four genera) only dealt with aflagellate spermatozoa. On this basis, Ehlers (1985) proposed two autapomorphics for the taxon Prolecithophora: aflagellate spermatozoon and spermatozoal mitochondrial derivatives with abundant membranes. The present observations on Urastoma contradict these two autapomorphics. The taxon Prolecithophora cannot be defined by autapomorphies of the spermatozoon.     

Ultrastructural study of the spermatozoon of Heterolebes maculosus (Digenea,Opistholebetidae), a parasite of the porcupinefish Diodon hystrix (Pisces,Teleostei)

This paper describes the ultrastructure of the mature spermatozoon of Heterolebes maculosus. It is the first study of this kind concerning the Opistholebetidae (Platyhelminthes, Digenea). The ultrastructural elements observed in the spermatozoon are: two axonemes with 9+“1” pattern of Trepaxonemata and their attachment zones, two mitochondria, a nucleus, cortical microtubules, external ornamentation of the plasma membrane and spine-like bodies. The number and the disposition of cortical microtubules, the organisation of 11 cortical microtubules disposed in semi-circle around the first mitochondrion in the external ornamentation region and the organisation of the posterior part of the spermatozoon are discussed. Three principal types of posterior part of digenean spermatozoa are proposed. The similarity between the spermatozoon of the Opistholebetidae H. maculosus and Opecoelidae enables us to confirm that these two families are closely related.     

Ultrastructural studies on the reproductive system of gyrocotylidea and amphilinidea (Cestoda): Spermatogenesis, spermatozoa, testes and vas deferens of Gyrocotyle

The ultrastructure of the vas deferens, testes, spermatogenesis and spermatozoa of Gyrocotyle urna and G. parvispinosa is described. The vas deferens is ciliated and syncytial. Within the testes primary spermatocytes arise from the primary spermatogonia by incomplete mitotic divisions; the primary spermatocytes undergo two meiotic divisions leading to spermatids. In early spermatids microtubules are formed at the cell periphery. Later the spermatozoal cytoplasm (the ‘middle-piece’) grows out and the two spermatozoal flagella with their typical 9 + ‘1’ axonemes are formed. During ciliogenesis the flagella are at an angle of about 60° to the axis of the middle-piece. The flagella are inserted into basal bodies terminating in striated rootlets. Subsequently, the nucleus and isolated mitochondria migrate into the central axis. The angle between the flagella and the axis decreases; the flagella are incorporated to form the spermatozoon. In mature spermatozoa no basal body or rootlet elements were found. The phylogeny of parasitic Platyhelminthes is discussed with respect to the evolution of spermatozoa. The reduction of the acrosinoid granules which are found in spermatozoa of free-living Platyhelminthes and the incorporation of the spermatozoal flagella into the sperm body constitute autapomorphies of the Neodermata (the parasitic Platyhelminthes). Included in the Cestoda because of several common derived characters, Amphilinidea and Gyrocotylidea are the only cestodes with spermatozoa containing mitochondria. Their absence in Cestoidea—all taxa with a six-hooked larva and other characteristics—is an autapomorphy of this group.     

Ultrastructural Observations on the Spermatozoon,Oocyte and Fertilization Process in Gonapodasmius,a Gonochoristic Trematode (Trematoda: Digenea: Didymozoidae)

Observations were performed in the uterus of a female Gonapodasmius sp., a gonochoristic didymozoid Trematode. The oocyte is a round cell 6 μm in diameter, which shows a ‘nucleolus-like cytoplasmic body’ and cortical granules. The spermatozoon is filiform, mobile and about 50 μm long. There is no acrosome. The anterior tip of the spermatozoon contains two centrioles made up of singlets and cortical microtubules with associated glycocalyx. The centrioles are continued as two axonemes of the classical 9 + ‘1’ pattern of flatworms, accompanied by a mitochondrion and a short row of cortical longitudinal microtubules. It is the posterior part of the sperm cell which contains the nucleus. At the outset of fertilization, the anterior part of the spermatozoon coils around the oocyte and penetrates it by lateral fusion. The posterior region of the spermatozoon, with the nucleus, is the last part to enter the oocyte, after passing through a perforation in the forming eggshell. The whole spermatozoon thus penetrates the female cell.     

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.     

STUDIES OF SPERMIOGENESIS IN A NEMATODE, NIPPOSTRONGYLUS BRASILIENSIS

The fine structure of the developing spermatids and the mature sperm of Nippostrongylus brasiliensis was investigated. Immature spermatids are found at one end of the tubelike testis, and the mature sperm at the other. The spermatid has a prominent nucleus, with the chromatin clumped at the margin. It also contains a pair of centrioles, located near the nucleus. The cytoplasm is filled with ribosomal clusters, but it lacks an organized Golgi area or endoplasmic reticulum. Besides the normal mitochondria, the spermatid has specialized mitochondrionlike inclusions with dense matrix, few broad cristae, and a crystalloid structure always facing the nucleus. As spermiogenesis proceeds, the nucleus elongates, comes to lie at one end, and later evaginates to form a separate head structure, leaving the mitochondria and other cytoplasmic organelles in a broad cytoplasmic region. The nuclear material becomes filamentous and spiral, and the centrioles come to lie at one end near the junction of the head and the cytoplasmic portion of the sperm. Microtubules are found in the cytoplasmic region extending from the tubelike nucleus. The specialized mitochondria are about eighteen in number, and are arranged in rows in staggered groups of three around the microtubules in the cytoplasmic region. The mature sperm is aflagellate and lacks an acrosome. No movement of the sperm was ever observed.     

Spermiogenesis and spermatozoon ultrastructure of the cestode Mosgovoyia ctenoides (Cyclophyllidea: Anoplocephalidae), an intestinal parasite of Oryctolagus cuniculus (Lagomorpha: Leporidae)

Ultrastructural characters in spermiogenesis and spermatozoa are considered important tools to elucidate the phylogenetic relationships within the Platyhelminthes. In the Anoplocephalidae, ultrastructural data refer to the spermatozoon of 14 species, whereas data on spermiogenesis refer to only 7 species. The present study focused on the spermiogenesis and spermatozoon of the anoplocephalid cestode Mosgovoyia ctenoides, as revealed by transmission electron microscopy. Type IV spermiogenesis was detected, beginning with the formation of a differentiation zone containing 2 centrioles, with a centriolar adjunct and vestigial striated rootlets. Different forms of the latter character have been described in other anoplocephalids. This study supports spermiogenesis of type IV as the most frequent in the Anoplocephalidae and confirms the presence of a centriolar adjunct in yet another type IV spermiogenesis species. The spermatozoon of M. ctenoides possesses 1 axoneme of the 9+ '1' trepaxonematan type, 2 crestlike bodies, dense plates, and granules of electron-dense cytoplasmic material, nucleus, and twisted cortical microtubules. It was again confirmed that the presence of granular material and the absence of both a periaxonemal sheath and intracytoplasmic walls are constant characters in the spermatozoa of all the Anoplocephalinae.     

Ultrastructural study of spermiogenesis and the spermatozoon of Microcotyle pancerii (Monogenea: Polyopisthocotylea: Microcotylidae), parasite of meagre Argyrosomus regius (Pisces: Teleostei)

The present work deals with the ultrastructure of spermiogenesis and the spermatozoon of Microcotyle pancerii, a gill parasite of meagre Argyrosomus regius collected in Corsican fish farms. Spermiogenesis was rather similar to that observed in other polyopisthocotylean Monogenea. The intercentriolar body was different from that described in digeneans. The nuclear condensation occurred in 2 successive stages. First, during the nuclear migration in the median cytoplasmic process, the nucleus developed a honeycomb-like appearance. Then, after the flagellar fusion, a discontinuous twisting of the chromatin appeared along the nucleus, with this process ending in total nuclear condensation. The structure of the spermatozoon is characterized by 2 axonemes (9 + "1" pattern), a single and continuous field of cortical microtubules, a mitochondrion, and a nucleus. Our findings were compared with various ultrastructural features in order to highlight variability within the group.     

The transformation of male sex cells of Tetranychus urticae K. (Acari,Tetranychidae) during passage from the testis to the oocytes: an electron microscopic study

The fine structure of the spermatozoon of Tetranychus urticae is described during passage from the testis to the site of insemination in the ovary. The male sex cells differentiate from a cytoplasmic mass which is characterised by nuclei bearing tubule-like structures. Infoldings appear in peripheral membrane of the germ cells at the beginning of spermiogenesis, chromatin condenses, and the nuclear membrane is reduced. The spermatozoon is surrounded by a double membrane: the inner one is the sperm membrane and the outer one is of somatic origin. The sperm reach the glanular region of the testis where they are transformed into amoeboid cell and are next collected in the seminal vesicle.

After copulation, the sperm can be observed in the lumen of the receptaculum seminis of the female from which they soon enter the epithelial cells. Still surrounded by a double membrane, the sperm, which are now packed in clusters, develop microtubules immediately beneath the inner membrane and enlarge by decondensation of chromatin and by infiltration of cytoplasmic material. Insemination takes place in the vitellogenic region of the ovary just before the eggs close their pores; the sperm have now reached ten times their original size.     

Ultrastructural study of spermatogenesis and the spermatozoon in Postorchigenes gymnesicus (Trematoda,Lecithodendriidae)

An ultrastructural study of spermatogenesis, spermiogenesis, and spermatozoa in Postorchigenes gymnesicus is presented. Cytoplasmic projections originating in nurse cells surround the spermatogonia, which are located at the periphery of the testes. Primary spermatocytes attached to a cytophore show synaptonemal complexes and a pair of centrioles. Spermiogenesis begins with the appearance of a cytoskeletal structure formed by an intercentriolar body and two perpendicular centrioles. An axoneme and a striated rootlet emerge from each centriole. The progressive rotation and fusion of both flagella with the median process occurs simultaneously with the migration of nucleus to the distal tip of the forming spermatozoon. The mature spermatozoon consists of three regions: (1) the nuclear region, containing the nucleus, one mitochondrion, two 9+1 axonemes, and cortical microtubules; (2) the intermitochondrial region, containing two axonemes; and (3) the mitochondrial region with another mitochondrion, two axonemes, cortical microtubules, and external ornamentation symmetrically and asymmetrically arranged coincidental with the cortical microtubules. Glycogen particles, absent in testicular cells, are abundant in the spermatozoon. Ultrastructural features of the non-nuclear region of the spermatozoon are specific for P. gymnesicus and are proposed to characterize the spermatozoon of digenean species. J. Morphol. 234:223–232, 1997. © 1997 Wiley-Liss, Inc.     

Acoel spermatozoa: ultrastructure and immunocytochemistry of tubulin

Acoel spermatozoa are filiform and contain two parallel axonemes, which do not show the trepaxonematan 9 + ‘1’ pattern, but instead, another kind of 9 + ‘1’ pattern, or a 9 + 0 or 9 + 2 pattern. Spermatozoa have either cortical singlet microtubules or central microtubules. Identification of these groups of microtubules and recognition of homologies between species is difficult with electron microscopy. In addition to conventional electron microscopy, indirect immunofluorescence of tubulin was performed on three species (Symsagittifera schultzei, Symsagittifera psammophila, and Actinoposthia beklemischevi). This technique facilitated understanding of the general morphology of the filiform spermatozoon and of the arrangement of the microtubular organelles along its length. We have found that different monoclonal antibodies (anti-alpha-, anti-alpha-acetylated- and anti-beta-tubulin) can distinguish distinct subcellular populations of microtubules. The axonemes were labelled by the three antibodies in all species. The cortical microtubules (in Actinoposthia beklemischevi) were labelled by the three antibodies. The central microtubules (in Symsagittifera schultzei and S. psammophila) were labelled with the anti-beta-tubulin antibody and not labelled by the anti-alpha- and anti-alpha-acetylated-tubulin. Similar experiments were performed on other Platyhelminthes and indicated that immunocytochemistry of spermatozoa may provide new characters for phylogenetic studies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Silver staining analysis on spermatozoa of Nucella lapillus (Gastropoda,Prosobranchia)

Summary

Sperm of Nucella lapillus was studied by electron microscopy, including the application of a cytochemical silver method. Using silver impregnation a dense precipitation of Ag granules in spermatocyte II nucleoli was seen over the fibrillar component and a slight one in the granular component. On longitudinal sections of the spermatozoon the results demonstrate that argyrophilic proteins are located in the external limiting zone of the acrosome in the anterior portion of the nucleus between the cytoplasmic and the nuclear membranes, in the posterior end of the nucleus and in the terminal portion of the middle region. These data indicate an affinity for silver in areas of the cytoplasm containing microtubules and in zones of transition.     

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.     

Spermatozoon ultrastructure of Aponurus laguncula (Digenea: Lecithasteridae), a parasite of Aluterus monoceros (Pisces: Teleostei)

The mature spermatozoon of Aponurus laguncula, a parasite of the unicorn leatherjacket Aluterus monoceros, was studied by transmission electron microscopy. The spermatozoon possesses 2 axonemes of the 9 + “1” trepaxonematan pattern, attachment zones, a nucleus, a mitochondrion, external ornamentation of the plasma membrane and cortical microtubules. The major features are the presence of: 1) external ornamentation in the anterior part of the spermatozoon not associated with cortical microtubules; 2) one mitochondrion; and 3) cortical microtubules arranged as a single field in the ventral side. The maximum number of microtubules is in the nuclear region. The extremities of the axonemes are characterized by the disappearance of the central core and the presence of microtubule doublets or singlets. This study is the first undertaken with a member of the Lecithasteridae and exemplifies the sperm ultrastructure for the superfamily Hemiuroidea.