Fine structure of the spermatozoon of Perinereis macropus (Claparède, 1870) (polychaeta,Nereidae)

The mature spermatozoa of Perinereis macropus were investigated by transmission electron microscopy. The spermatozoon is composed with a large anterior part (head), a short middle piece and a long flagellum. The head contains a large acrosomal complex with a convex acrosomal vesicle, a subacrosomal space, a fibrillar crown and an acrosomal rod which penetrates into the nucleus invagination. The later is U shaped (in longitudinal section). The short middle piece contains about nine to eleven mitochondria and a centriole associated with the flagellum. This centriole, slightly eccentric to the sperm axis, anchors to the plasma membrane by nine satellite rays of the pericentriolar complex. The axoneme has a “9 + 2″ arrangement of microtubules. In cross section, the flagellar membrane extends in two lateral protuberances, aligned with the axis formed by the two central microtubules of the axoneme. The spermatozoon of P. macropus conforms to the primitive type with an acrosomal extension. Nevertheless, the acrosome complex ultrastructure shows noticeable modifications from the basic form. This finding agrees with the previously observed reproductive pattern (broadcast spawning – free-swimming larvae), and may be helpful to classify the sperm type of P. macropus.     

Ultrastructural analysis of spermiogenesis in the Eastern Fence Lizard,Sceloporus undulatus (Squamata: Phrynosomatidae)

Studies on reptilian sperm morphology have shown that variation exists at various taxonomic levels but studies on the ontogeny of variation are rare. Sperm development follows a generalized bauplan that includes acrosome development, nuclear condensation and elongation, and flagellar development. However, minute differences can be observed such as the presence/absence of manchette microtubules, structural organization during nuclear condensation, and presence/absence of a nuclear lacuna. The purpose of this investigation was to examine sperm development within the Sceloporus genus. The process begins with the development of an acrosomal complex from Golgi vesicles followed by nuclear condensation and elongation, which results in the presence of a nuclear lacuna. As the acrosomal complex differentiates, flagellar development commences with elongation of the distal centriole. Spermatid development culminates in a mature spermatid with a highly differentiated acrosomal complex, a condensed nucleus with a nuclear lacuna, and a differentiated flagellum. Although the overall developmental pattern is consistent with other squamate species, minute differences are observed, even within the same genus. For example there is variation in the presence/absence of an endoplasmic reticulum complex during acrosome development, presence/absence of a nuclear lacuna, and presence/absence of manchette microtubules within the three species of Sceloporus studied to date. Future studies concerning sperm morphology in closely related species will aid in our understanding of variation in sperm development and may prove to be useful in testing phylogenetic and evolutionary hypotheses.     

Ultrastructural changes and programmed cell death of trophocytes in the gonad of Isohypsibius granulifer granulifer Thulin, 1928 (Tardigrada,Eutardigrada, Isohypsibiidae)

The studies on the fates of the trophocytes, the apoptosis and autophagy in the gonad of Isohypsibius granulifer granulifer have been described using transmission electron microscope, light and fluorescent microscopes. The results presented here are the first that are connected with the cell death of nurse cells in the gonad of tardigrades. However, here we complete the results presented by Węglarska (1987).The reproductive system of I. g. granulifer contains a single sack-like hermaphroditic gonad and a single gonoduct. The gonad is composed of three parts: a germarium filled with proliferating germ cells (oogonia); a vitellarium that has clusters of female germ cells (the region of oocytes development); and a male part filled with male germ cells in which the sperm cells develop. The trophocytes (nurse cells) show distinct alterations during all of the stages of oogenesis: previtello-, vitello- and choriogenesis. During previtellogenesis the female germ cells situated in the vitellarium are connected by cytoplasmic bridges, and form clusters of cells. No ultrastructural differences appear among the germ cells in a cluster during this stage of oogenesis. In early vitellogenesis, the cells in each cluster start to grow and numerous organelles gradually accumulate in their cytoplasm. However, at the beginning of the middle of vitellogenesis, one cell in each cluster starts to grow in order to differentiate into oocyte, while the remaining cells are trophocytes. Eventually, the cytoplasmic bridges between the oocyte and trophocytes disappear. Autophagosomes also appear in the cytoplasm of nurse cells together with many degenerating organelles. The cytoplasm starts to shrink, which causes the degeneration of the cytoplasmic bridges between trophocytes. Apoptosis begins when the cytoplasm of these cells is full of autophagosomes/autolysosomes and causes their death.     

Morphology of the male reproductive system and spermiogenesis in Hypanthidium foveolatum (Alfken, 1930) (Hymenoptera: Apidae: Megachilinae)

The morphological aspects of male reproductive tract, spermiogenesis and spermatozoa are typical for each species and reflect its evolution, establishing a unique source of characters, which has been used to help solve phylogenetic problems. In Hypanthidium foveolatum the reproductive tract is composed of the testes comprising 28 testicular tubules, deferent ducts, seminal vesicles, accessory glands and an ejaculatory duct. The differentiation of spermatids occurs within cysts of up to 128 germ line cells each one. During the early spermatid phase, the nucleus resembles that of somatic cells. There follows a gradual chromatin condensation with an increase in nuclear electron density. In the spermatozoon, the nucleus contains heterogeneous chromatin with a loose appearance. The acrosome, shaped with the active participation of the Golgi complex, shows an electron-dense perforatorium involved by four electron-lucent acrosomal vesicle projections. The sperm tail presents an axoneme with a 9 + 9 + 2 microtubule pattern and two mitochondrial derivatives, which appear with different sizes. A dense crystalloid is formed initially in the mitochondrial matrix of the large derivative. The mitochondrial derivatives’ differentiation occurs concomitantly with an axoneme outgrowth. The centriolar adjunct is observed near the axoneme, anterior to the smaller mithocondrial derivative and exhibits an approximately triangular shape in cross-sections. Microtubules were observed around the head region and flagellar components during spermiogenesis.     

Ultrastructure of the spermatozoa of Cicadella viridis (Linnaeus) and its bearing on the phylogeny of Auchenorrhyncha

The ultrastructure of mature spermatozoa of the leafhopper Cicadella viridis (Linnaeus) was investigated using light and transmission electron microscopy. The spermatozoon is composed of a head containing an acrosome and an elongated nucleus, and a long tail, which consists of a flagellum. The acrosome is conical and invaginated to form a subacrosomal space, and the acrosomal contents are filled with electron-dense tubular substructures. The nucleus is linear and filled with homogeneously condensed chromatin. The centriolar adjunct is parallel to the nucleus and connects the nucleus with the mid-piece/flagellum. The flagellum is formed by a 9+9+2 axoneme, two mitochondrial derivatives and two accessory bodies. The mitochondrial derivatives with an orderly array of peripheral cristae are symmetrical. The accessory bodies are small and slightly elliptical. The end of the axoneme shows progressive loss of microtubules. Comparison of sperm ultrastructure of C. viridis with those of other Auchenorrhyncha families supports the major relationships within Cicadomorpha as (Membracoidea (Cicadoidea, Cercopoidea)).     

Ultrastructural study of spermiogenesis and the spermatozoon of Cavisoma magnum (Southwell, 1927) (Acanthocephala, Palaeacanthocephala, Cavisomidae), from Siganus lineatus (Pisces, Teleostei, Siganidae) (Valenciennes, 1835) in New Caledonia

This paper presents an ultrastructural study of Cavisoma magnum (Acanthocephala, Cavisomatidae) with a Transmission Electron Microscopy tool. This parasite of the fish Siganus lineatus is here reported for the first time from off New Caledonia, South Pacific. It is the first study describing the ultrastructure, spermiogenesis and spermatozoon of a species of the family Cavisomatidae. The young spermatid of C. magnum possesses a centriole constituted of doublets without a central element. After the elaboration of a flagellum of 9+2 pattern, the centriole migrates in a nuclear groove. Then the flagellum migration occurs and gives rise to a spermatozoon. The distribution and the size of the protein granules are reported and the posterior extremity appears like a chromatin lamina wave. Comparative ultrastructural data are presented on sperm and spermiogenesis of the Acanthocephala and Rotifers examined to date and the phylogenetic implications are discussed.     

Pre-spermiogenic initiation of flagellar growth and correlative ultrastructural observations on nuage,nuclear and mitochondrial developmental morphology in the zebrafish Danio rerio

The microstructural and ultrastructural changes of germ cells during spermatogenesis of zebrafish (Danio rerio) were examined using light microscopy (LM) and transmission electron microscopy (TEM). Generally the process of spermatogenesis in zebrafish is similar to that of other teleosts, however, here we describe some peculiar features of zebrafish spermatogenic cells which have a limited report in this species. (1) The basic events of spermiogenesis are asynchronous, location of flagellum finished in initial stage, while chromatin condensation sharply occurred in intermediate stage and elimination of excess cytoplasm mainly taken place in final stages. (2) Surprisingly, the cilia or initial flagellae are created in spermatocytes, approach toward the nucleus of early stage spermatids, and then the centrioles depress into nuclear fossa and change their orientation to each other from right angle to obtuse angle about 125°. (3) During spermatogenesis, the chromatin compaction performs in a distinctive pattern, condensed heterogeneously from granular into chromatin clumps with central electron-lucent areas, round or long, which diminished to small nuclear vacuoles in spermatozoa. This finding demonstrates the origin of nuclear vacuoles in zebrafish spermatozoa for the first time. (4) Nuages are observed in both spermatogonia and spermatocytes. They are connected with the mitochondria and nuclear membrane, and are even located in the perinuclear spaces of spermatogonia nuclei. (5) Mitochondrial morphology and distribution shows diversity in different germ cells. The condensed mitochondria appear in pachytene spermatocytes, and mitochondria including membrane conglomerate exist in both spermatocytes and spermatids. This study was undertaken in order to disclose specific spermatogenic cells features in zebrafish that could be helpful for understanding the correlative function in this model species.     

Comparative sperm ultrastructure of twelve leptodactylid frog species with insights into their phylogenetic relationships

The spermatozoa of representatives of three Neotropical frog subfamilies, Leiuperinae, Leptodactylinae and Paratelmatobiinae, were observed using Transmission Electron Microscopy, with the aim of identifying ultrastructural traits that provide insights into the phylogenetic relationships among these anurans, which are currently unclear. In the leiuperines, spermatozoa of Physalaemus albifrons, P. cicada, P. deimaticus and P. feioi were characterized by an acrosomal vesicle covering the subacrosomal cone that was not observed in the spermatozoa of Physalaemus centralis and P. cuvieri. The tail of the spermatozoa of P. albifrons, P. centralis, P. cicada, P. cuvieri, P. deimaticus, and P. feioi presented a long undulating membrane, whereas Engystomops petersi and E. freibergi, which form a sister clade to Physalaemus, had an axial fiber, which were absent in Physalaemus. Other leiuperine, E. puyango had an abaxonemal bulb-like swelling distally to the paraxonemal rod, which were also absent in Physalaemus. These differences support the revalidation of Engystomops as a true taxon, distinct from Physalaemus. The tail of the spermatozoa of E. petersi and E. freibergi was similar to that of Paratelmatobius poecilogaster (Paratelmatobiinae). The spermatozoa of Leptodactylus natalenis (Leptodactylinae) had undulating membrane and axial fiber, in contrast with Adenomera marmorata, which lacked these structures. Morphological differences between A. marmorata and L. natalensis sperm cells appeared to validate the allocation of A. marmorata into a genus distinct from Leptodactylus. Overall, dissimilarities in the spermatozoa of the leptodactylids provided an important phylogenetic signal for the understanding of their taxonomic relationships.     

Testes of Astyanax altiparanae: The Sertoli cell functions in a semicystic spermatogenesis

The Astyanax altiparanae (lambari) is a South American freshwater fish belonging to the family Characidae. Although some authors have described reproductive aspects of this species, this is the first study about the morphology of the testes throughout the annual reproductive cycle of A. altiparanae. Fish spermatogenesis differs from that in mammals as it occurs in cysts whose borders are defined by cytoplasmic processes of Sertoli cells, thus creating a favorable environment for spermatogenesis. The functions commonly attributed to fish Sertoli cells were investigated using stereological, light and electron microscopy in A. altiparanae. Results showed that when the Sertoli cells of A. altiparanae are in contact with germ cells, they plan a support function that culminates in the production of spermatozoa. After releasing spermatozoa, modified Sertoli cells form the duct epithelium, transform into secretory cells and release a secretion into the duct lumen where spermatids and sperm are located. Thus, the present study revealed important aspects of the testes of A. altiparanae, and propose a sequence of functions played by the Sertoli cells in this species.     

P granules phase transition induced by cytoplasmic streaming in Caenorhabditis elegans embryo

P granules are germ granules contained in Caenorhabditis elegans germ cells.The first germ cell is specified by the one-cell embryo in which P granules localize to the posterior.Previous studies suggested that the mechanism of the localization phenomena is induced by liquid-liquid phase transition(LLPT),in which the polarity proteins control the saturation point of P granules.In the present study,we propose that the P granules phase transition can be triggered by the cytoplasmic streaming.A two-phase flow model is employed to simulate the localization of P granules,i.e.,the cytoplasm is considered as a liquid phase,and the droplet-like P granules are another liquid phase.With the presence of the cytoplasmic streaming,P granules,initially distributing uniformly in the entire one-cell embryo,eventually condense/dissolve in the cytoplasm phase,regulated by difference between the saturation pressure and the hydrodynamic pressure.The numerical results reveal that the cytoplasmic streaming has significant effects on the localization of P granules,as well as the embryo division.     

Topological alterations in human spermatozoa associated with the polyelectrolytic effect of RISUG

A new method of male contraception has been developed which results in long-term infertility and has the potential advantage of being reversible. The contraceptive, given the name RISUG (an acronym for Reversible Inhibition of Sperm Under Guidance) is a polyelectrolytic compound and when injected into the lumen of the vas deferens, induces a surface charge imbalance on the sperm membrane system leading to its destabilization. In the present study, morphological and topological alterations in human spermatozoa induced by RISUG have been investigated using atomic force microscopy (AFM). Complete disintegration of the plasma membrane with subsequent rupture and dispersion of the acrosomal contents is observed on treatment with RISUG in vitro. Considerable damage to the midpiece region with significant clustering of the mitochondria and its fusion with the head region is also observed. These observations are in agreement with the significant increase in the volume of RISUG-treated sperm-head region. Topological alterations in the flagellar and midpiece region of RISUG-treated spermatozoa have also been studied.     

Ultrastructure of germ cells,Sertoli cells and mitochondria during spermatogenesis in mature testis of the Chinese Taihang black goats (Capra hircus)

The objective of this study was to describe the ultrastructure of germ cells, Sertoli cells and mitochondria in mature testis of the Chinese Taihang black goat. The characteristics of germ cell nucleus and mitochondria changing during spermatogenesis were investigated by transmission electron microscopy (TEM). The results showed that the spermatogonium was elliptical, and its nucleus was about 4–5 μm. The round mitochondria can be observed throughout the cytoplasm around the nucleus. Small patches of heterochromatin were distributed throughout the nucleus. Spermatocyte was oval-shaped with a nucleus of about 4–4.5 μm in diameter. The heterochromatin began to attach to the inner surface of the nuclear membrane. Spermatid was about 4 μm and oval in shape. Its nucleus was oval or round and approximately 2–3 μm in diameter. The borderline between nucleus membrane and karyoplasm was distinct. During spermiogenesis, spermatid nucleus was condensed and elongated, and chromatin reached the highest condensation in the mature spermatozoon. The mid-piece was surrounded by mitochondria at the neck region. The sperm tail showed the typical “9 + 2″ structure, contained axoneme and central singlet microtubules. The nuclei of the Sertoli cells were irregular shaped and showed indentations in the membrane. In the mature testes of goat bucks, abundant mitochondria were around the germ cells and Sertoli cells. The scattered mitochondria were aggregated around the base of the flagellum (axoneme) during the spermatid differentiation stage. In conclusion, the present study showed that the spermatogenic process of Taihang black goat followed the pattern of mammals with some specific.     

The sperm ultrastructure and spermiogenesis of Tribolium castaneum (Coleoptera: Tenebrionidae) with evidence of cyst degeneration

Previous studies on the spermatogenesis of tenebrionid beetles showed the unusual formation of two antiparallel sperm bundles per cyst. In this work we reported this feature also in Tribolium castaneum using light and transmission electron microscopy. The sperm structure of T. castaneum, similar to other tenebrionids, consists of a three-layered acrosome, an elongated nucleus and a flagellum with a 9 + 9 + 2 axoneme, two accessory bodies and two asymmetric mitochondrial derivatives. The presence of two antiparallel sperm bundles per cyst also in Meloidae and Rhipiphoridae suggests that it is a strong trait synapomorphic for Tenebrionoidea. The huge degeneration of whole sperm cells in several cysts of testes during spermiogenesis is also described.     

Spermiogenesis and sperm ultrastructure of Machilontus sp (Insecta: Archaeognatha) with phylogenetic consideration

The sperm structure of the jumping bristletail Machilontus sp has been described. The species shares several sperm characteristics with other genera of the same order Archaeognatha. During late spermiogenesis the spermatid bends at half of its length with the two limbs closely apposed within the same plasma membrane. The sperm has a helicoidal bi-layered acrosome with a filamentous perforatorium and a long nucleus. The elongated flagellum consists of an axoneme with 9 accessory microtubules external to the 9+2, two rows of conventional mitochondria and two accessory bodies. The accessory bodies are located lateral to the axoneme and are probably responsible for the shifting of the accessory tubules in two opposite groups of 5 and 4 tubules, respectively. These sperm characteristics represent common traits of all Archaeognatha.     

Comparative ultrastructural study of spermatozoa in some oyster species from the Asian-Pacific Coast

Sperm organization in the oysters Crassostrea gigas, Crassostrea nippona, Crassostrea cf. rivularis and Saccostrea cf. mordax inhabiting Asian Pacific coast was studied. The spermatozoa of all studied species had a number of common morphological characters such as a cup-like acrosome with heterogeneous matrix on its top, an axial rod in the subacrosomal space, a barrel-shaped nucleus, four mitochondria in the midpiece, pericentriolar complexes, and a 9+2-organized flagellum. The spermatozoa of C. cf. rivularis differed from the other species by having cytoplasm processes in the midpiece region. Such structures have never been described in the Ostreidae. Additionally, each species could be identified by the shape and size of sperm compartments (acrosome, nucleus, anterior nuclear fossa). The most significant interspecific difference was found in the size of an anterior nuclear fossa. The smallest anterior nuclear fossa was found in C. cf. rivularis (about 0.24 μm in length reaching about 22% of the nuclear length) while the biggest in C. gigas from the Sea of Japan (about 0.53 μm in length reaching about 46% of the nuclear length). The spermatozoa of C. gigas collected from the Sea of Japan and Taiwan Strait differed significantly in almost all the studied parameters. Since sperm morphology has been successfully used for species differentiation, this suggests the existence of two species rather than two populations. The data obtained indicate the species-specific difference in the sperm ultrastructure within the Ostreidae, which may be identified both ultrastructurally and morphometrically.     

Nonlinear optical effects of near-IR femtosecond laser radiation on the morphology and structure of a nerve cell in the field of an optical trap

Changes in the morphology and structure of a neuron cell in the process of manipulating it with an femtosecond laser tweezers operating at a wavelength of 800 nm are examined. The changes in the morphology and structure are caused by the nonlinear optical absorption and multiphoton excitation of cell biomacromolecules by femtosecond pulses of light. The cell nucleus is demonstrated to be destroyed by focused femtosecond laser radiation (FLR). Changes in the state of the cytoplasmic nucleoproteins and the hydrophobicity of the plasmatic membrane under the action of FLR focused inside the cell are observed. By the example of the simultaneous displacement of the neuron with a cw laser and cutting of a neuron with FLR, the operation of holographic optical manipulator and a scalpel based on the use of femtosecond and cw lasers is considered. The possibility of the simultaneous microsurgical operation with several optical foci of FLR is demonstrated.     

Cellular organisation of the mature testes and stages of spermiogenesis in Danio rerio (Cyprinidae; Teleostei)--structural and ultrastructural studies

The male gonads of Danio rerio occupy a position typical of the Teleostei species. The structure of the testes corresponds to the anastomosing tubular type with unrestricted spermatogonia and represents a cystic type of spermatogenesis. The results of this study indicate that four distinct stages of cell differentiation can be identified during spermiogenesis. These stages are characterised by chromatin condensation, the development of flagellum, nuclear rotation, the formation of nuclear fossa and the elimination of excess cytoplasm. A round head and the absence of an acrosome characterise the differentiated sperm. The midpiece is short and large, and C-shaped mitochondria form a ring surrounding the initial region of the flagellum. The axoneme shows a 9 + 2 pattern. In the D. rerio spermatozoa the flagellar axis is at an angle of 110° to the nucleus diameter running through the centriole.     

Sperm morphology of Trichospilus diatraeae and Palmistichus elaeisis (Hymenoptera: Chalcidoidea: Eulophidae)

In this study, the sperm morphology of the parasitoids Trichospilus diatraeae and Palmistichus elaeisis (Eulophidae) was investigated using light and transmission electron microscopy. In the two species, the sperm are spiral along their entire length and measure about 130 μm and 195 μm in length, respectively. The head region consists of the acrosome and nucleus. The acrosome is composed of an acrosomal vesicle and, in P. elaeisis, a perforatorium. In both species, an extracellular layer in which several filaments are radiated covers the acrosome and the anterior nuclear region. The nuclei are filled with homogeneous and compact chromatin and measure about 50 μm in length in P. elaeisis and 20 μm in T. diatraeae. The flagellum consists of an axoneme with the 9 + 9 + 2 microtubule arrangement spiraled in a long helix, two mitochondrial derivatives coiling around the axoneme and, in P. elaeisis, two accessory bodies. In T. diatraeae were observed transverse striations throughout the central region of the axoneme, whereas the central pair of microtubules was rarely observed. In the final flagellar region in T. diatraeae, different from P. elaeisis, one mitochondrial derivative ends well before the other and both end before the axoneme. The sperm of these two species exhibit features that discriminate one species from each other, as well as characteristics suggest that Eulophidae is closely related to Trichogrammatidae and both of these families are more similar to Eurytomidae than Agaonidae.     

Meiotic nucleolar cycle and chromatoid body formation during the rat (Rattus novergicus) and mouse (Mus musculus) spermiogenesis

The aims of the present study were to follow the nucleolar cycle in spermiogenesis of the laboratory rodents Rattus novergicus and Mus musculus, to verify the relationship between the nucleolar component and chromatoid body (CB) formation and to investigate the function of this cytoplasmic supramolecular structure in spermatogenic haploid cells. Histological sections of adult seminiferous tubules were analyzed cytochemically by light microscopy and ultrastructural procedures by transmission electron microscopy. The results reveal that in early spermatids, the CB was visualized in association with the Golgi cisterns indicating that this structure may participate in the acrosome formation process. In late spermatids, the CB was observed near the axonema, a fact suggesting that this structure may support the formation of the spermatozoon tail. In conclusion, our data showed that there is disintegration of spermatid nucleoli at the beginning of spermatogenesis and a fraction of this nucleolar material migrates to the cytoplasm, where a specific structure is formed, known as the "chromatoid body", which, apparently, participates in some parts of the rodent spermiogenesis process.     

Excimer laser irradiation at 248 nm of wooden archaeological objects and polymeric consolidants used in conservation: a study of cone formation and optimum cleaning parameters

Samples of a prehistoric consolidated vegetable basketry, coming from an important pile building in the North-East of Italy (Fiavé Carrera, Trento), had been irradiated with excimer laser pulses to remove the consolidant layer and restore the artifact. Some problems, related to the nature of the consolidant resin, were found in the preliminary laser cleaning process. The two main obstacles were related to the unknown composition of the polymeric resin and to the irregular surface morphology of the archaeological find. We observed that large numbers of laser pulses, for selected laser parameters, strongly modify the resin surface morphology with formation of a large number of cones. After cone formation, the surface was so irregular that it was almost impossible to complete the laser cleaning procedure. To find a solution to the problem, we here try to understand the mechanisms of cone formation in the present polymeric material. Scanning electron microscopy (SEM) and Fourier transformation infrared spectroscopy (FTIR) have been used to study surface morphology and chemical modification after selected irradiation processes. To understand the influence of laser irradiation on surface modifications and to establish a cleaning procedure for this specific application, investigations have been carried out separately on models of resin consolidants and wood (both dried and buried wood sample). The consolidants were commercial resins, typically used in archaeological conservation (Plexisol and Paraloid), deposited on a silicon substrate. Silicon was used because it is an appropriate substrate in FT-IR analysis. Finally, we chose wood because of its wide use in prehistoric artefact (Viburnum lantana). PACS  81.65.Cf; 82.50.Hp; 87.64.Ee; 87.64.km