Intragonadal somatic cells (ISCs) in the male oyster Crassostrea gigas: morphology and contribution in germinal epithelium structure

The ultrastructure of somatic cells present in gonadal tubules in male oyster Crassostrea gigas was investigated. These cells, named Intragonadal Somatic Cells (ISCs) have a great role in the organization of the germinal epithelium in the gonad. Immunological detection of α-tubulin tyrosine illustrates their association in columns from the basis to the lumen of the tubule, stabilized by numerous adhesive junctions. This somatic intragonadal organization delimited some different groups of germ cells along the tubule walls. In early stages of gonad development, numerous phagolysosomes were observed in the cytoplasm of ISCs indicating that these cells have in this species an essential role in the removal of waste sperm in the tubules. Variations of lipids droplets content in the cytoplasm of ISCs were also noticed along the spermatogenesis course. ISCs also present some mitochondria with tubullo-lamellar cristae.     

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.     

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.     

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.     

High-pressure freezing and freeze-substitution fixation reveal the ultrastructure of immature and mature spermatozoa of the plant-parasitic nematode Trichodorus similis (Nematoda; Triplonchida; Trichodoridae)

The spermatozoa from testis and spermatheca of the plant-parasitic nematode Trichodorus similis Seinhorst, 1963 (Nematoda; Triplonchida; Trichodoridae) were studied with transmission electron microscopy (TEM), being the first study on spermatogenesis of a representative of the order Triplonchida and important to unravel nematode sperm evolution. Comprehensive results could only be obtained using high-pressure freezing (HPF) and freeze-substitution instead of chemical fixation, demonstrating the importance of cryo-fixation for nematode ultrastructural research. The spermatozoa from the testis (immature spermatozoa) are unpolarized cells covered by numerous filopodia. They contain a centrally-located nucleus without a nuclear envelope, surrounded by mitochondria. Specific fibrous bodies (FB) as long parallel bundles of filaments occupy the peripheral cytoplasm. No structures resembling membranous organelles (MO), as found in the sperm of many other nematodes, were observed in immature spermatozoa of T. similis. The spermatozoa from the uterus (mature or activated spermatozoa) are bipolar cells with an anterior pseudopod and posterior main cell body (MCB), which include a nucleus, mitochondria and MO appearing as large vesicles with finger-like invaginations of the outer cell membrane, or as large vesicles connected to the inner cell membrane. The peripheral MO open to the exterior via pores. In the mature sperm, neither FBs nor filopodia were observed. An important feature of T. similis spermatozoa is the late formation of MO; they first appear in mature spermatozoa. This pattern of MO formation is known for several other orders of the nematode class Enoplea: Enoplida, Mermithida, Dioctophymatida, Trichinellida but has never been observed in the class Chromadorea.     

Cytodifferentiation during the spermatogenesis of the hermaphrodite caridea Exhippolysmata oplophoroides

Among the decapods, the caridean Exhippolysmata oplophoroides has been described as a simultaneous protandric hermaphrodite, seeing that it presents a male initial stage followed by a hermaphrodite one in which it can function as male and as female. This work had the aims of characterizing the microscopical morphology of the male portion of the ovotestes gonads from E. oplophoroides, at the different development stages, identifying each cell from the germ lines during spermatogenesis, as well as describing the ultramorphology of spermatozoans in the terminal region of the vasa deferentia. Shrimps were collected in Ubatuba, north coast of São Paulo, and their male gonads and the ampoule were removed, fixed and processed according to histological routine and for scanning electron microscopy. The testicular portion is divided in lobes, inside which cells at the same stage of the spermatogenic cycle are observed, with prevalence of spermatogonia and spermatocytes at stages I, II and V of gonad development, whereas spermatids and spermatozoans are found at stages III and IV, respectively. Ultramorphology of the terminal portion of the vasa deferentia exhibits mature aflagellated spike-shaped spermatozoans, encased in secretion and between membrane foldings that will constitute the spermatophores. Despite presenting reproductive characteristics common to other decapods, E. oplophoroides shows spermatozoans as well as spermatophore with typical morphology, which is important for its identification and taxonomy. Further, this species showed polysaccharide secretions where the spermatozoa are immerse as far as the testicular portion, which could have a role in their transport and nutrition as well as spermatophore constitution and/or fixation; differently, other caridean species begin spermatophore formation during the passage of the gametes through the vasa deferentia.     

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.     

Ultrastructural description of spermiogenesis within the Mediterranean Gecko, Hemidactylus turcicus (Squamata: Gekkonidae)

We studied spermiogenesis in the Mediterranean Gecko, Hemidactylus turcicus, at the electron microscope level and compared to what is known within other Lepidosaurs. In H. turcicus germ cells are connected via cytoplasmic bridges where organelle and cytoplasm sharing is observed. The acrosome develops from merging transport vesicles that arise from the Golgi and subsequently partition into an acrosomal cap containing an acrosomal cortex, acrosomal medulla, perforatorium, and subacrosomal cone. Condensation of DNA occurs in a spiral fashion and elongation is aided by microtubules of the manchette. A nuclear rostrum extends into the subacrosomal cone and is capped by an epinuclear lucent zone. Mitochondria and rough endoplasmic reticulum migrate to the posterior portion of the developing germ cell during the cytoplasmic shift and the flagellum elongates. Mitochondria surround the midpiece as the anlage of the annulus forms. The fibrous sheath begins at mitochondrial tier 3 and continues into the principal piece. Peripheral fibers associated with microtubule doublets 3 and 8 are grossly enlarged. During the final stages of germ cell development spermatids are wrapped with a series of Sertoli cell processes, which exhibit ectoplasmic specializations and differing cytoplasmic consistencies. The results observed here corroborate previous studies, which show the conservative nature of sperm morphology. However, ultrastructural character combinations specific to sperm and spermiogenesis seem to differ among taxa. Further studies into sperm morphology are needed in order to judge the relevance of the ontogenic changes recorded here and to determine their role in future studies on amniote evolution.     

Density functional calculation of the ionization potentials of small metallic particles

A Density Functional Pseudopotential method is used to compute the ionization potential IP of Sodium, Aluminium and Lead microparticles as a function of particle size. The model particles are formed by a central atom surrounded by successive coordination shells of first neighbours, second neighbours, etc. Maxima in IP are obtained for particles with filled coordination shells. The magnitude of IP correlates with the magnitude of the electron density in the region where the electron is extracted.     

Studies on the toxicity of dimethyl sulfoxide, ethylene glycol, methanol and glycerol to loach (Misgurnus fossilis) sperm and the effect on subsequent embryo development

The process of sperm cryopreservation consists of several steps: equilibration of sperm in cryoprotectant medium, freezing of sperm to subzero temperatures, low temperature storage and thawing of the sperm suspension. It has been shown that cryopreservation can cause some damage to the genetic material of cells although the mechanism and significance of these changes are still unknown. The aim of this work was to study the effect of cryoprotectant equilibration process on genetic damage of Loach (Misgurnus fossilis) sperm, using embryo survival as an indicator. Decrease in embryo survival after the 20th stage is generally believed to result from the failure in the genome function of embryos. In the first set of the experiments, Loach sperm were equilibrated in cryoprotectants Me2SO, ethylene glycol, methanol and glycerol (0.6, 1.2, 2.5 M) for 60 min at 10 degree C. The effect of cryoprotectant equilibration on sperm was evaluated based on the survival of embryos derived from cryoprotectant treated sperm. Embryo survival was evaluated at the following stages: 7th, 14th, 17th, 20th, 23rd, 26th, 31st, 34th, 35th, 36th and 37th. Cryoprotectants at concentrations greater than 1.2 M had significant effect on the survival of the embryos after the 20th stage. The effect of glycerol was the most significant with 64.8 +/- 2.4% of embryos survival compared to 77.0 +/- 2.4% for control. Me2SO treatment also effects embryo survival significantly. Possible mechanisms of the genetic instability of cryoprotectants are discussed.     

Ultrastructural immunogold localization of major sperm protein (MSP) in spermatogenic cells of the nematode Acrobeles complexus (Nematoda,Rhabditida)

The nematode spermatozoa represent a highly modified (aberrant) type of male gametes that lack a flagellum but for which the process of spermatogenesis culminates in the production of a crawling spermatozoon on the basis of the cytoskeletal component known as “major sperm protein”, or MSP. MSP is also known as an important hormone triggering oocyte maturation and ovulation in the model nematode Caenorhabditis elegans, where this protein was first identified. However, direct evidence of MSP localization and of its fate in nematode spermatogenic cells is rare. In this study, the spermatogenesis and sperm structure in the rhabditid nematode Acrobeles complexus (Rhabditida: Tylenchina: Cephalobomorpha: Cephaloboidea: Cephalobidae) has been examined with electron microscopy. Morphological observations were followed by high-pressure freezing and freeze-substitution fixation which allows post-embedding immunogold localization of MSP in all stages of sperm development using antibodies raised for MSP of C. elegans. In spermatocytes, synthetic activity results in the development of specific cellular components, fibrous bodies (FB) and membranous organelles (MO), which appear as FB-MO complexes where the filamentous matter of FB has been MSP-labeled. The spermatids subdivide into a residual body with superfluous cytoplasm, and a main cell body which contains nucleus, mitochondria and FB-MO complexes. These complexes dissociate into individual components, MO and FB, with the MSP being localized in FB. Immature spermatozoa from testes are opaque cells where a centrally located nucleus is surrounded by mitochondria, MO and FB clustered together, the MSP still being localized only in FB. Cytoplasm of mature spermatozoa from spermatheca is segregated into external pseudopods lacking organelles and a central cluster of mitochondria with intact MO surrounding the central nucleus. The FB ultimately disappear, and the MSP labeling becomes concentrated in the filamentous content of pseudopods and cytoplasm of the main cell body. Although the spermatogenesis and sperm structure of A. complexus is similar to that of many other rhabditid nematodes, their intact MO makes the morphology of the mature spermatozoa distinct from the “rhabditid pattern” and may be considered as a synapomorphy. The MSP localization in spermatogenic cells of A. complexus also follows the “rhabditid pattern” described in C. elegans and Ascaris spp. Our results and techniques of MSP labeling of A. complexus spermatogeneous cells reveal new possibilities to elucidate different research questions on MSP localization in nematodes related to C. elegans. Furthermore, the laboratory-cultured A. complexus strains can be used as a new and fascinating model to study MO and MSP functions in nematode reproduction.     

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.     

Ultrastructural analysis of the nucleolar aspects at spermiogenesis in triatomines (Heteroptera,Triatominae)

In this study the ultrastructural technique was used to analyze seminiferous tubule cells of the triatomine species Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans. The data obtained provided evidence of the phenomenon known as persistence of the nucleolar material in initial spermatids at early differentiation. Our results confirmed the presence of the nucleolus and its products during spermiogenesis up to the formation of the axoneme and during spermatid elongation in all three species studied, similar to the process that takes place during cell division. In early spermatids, the nucleoli had a reticulate appearance and a well defined nucleolonema in P. megistus; showed a clear distinction between the fibrillar and the granular component in T. infestans; and had a compact aspect in R. pallescens. In this study, ultrastructural analyses at spermiogenesis indicated that these nucleolar products may represent RNP complexes that will probably be needed at early spermiogenesis when important changes such as chromatin condensation and acrosome and flagellum formation take place. Therefore, it was concluded from the ultrastructural analysis that the triatomine nucleolus does not totally disappear but remains as corpuscles that gather to form the next nucleolar cycle that in the case of meiosis, will be completed if fertilization occurs and a zygote is formed.     

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.     

Cohesive energy of small metallic particles

The cohesive energy of sodium microparticles is calculated using the density functional formalism. The model particles are formed by a central atom surrounded by successive coordination shells of first neighbours, second neighbours, etc. Maxima in the cohesive energy per atom are obtained for particles with filled coordination shells. The sublimation energy is also studied. Insight is gained on the nature of “magic numbers” observed in the experimental size distribution.     

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)).     

Ultrastructure of spermatogenesis in the white-lined broad-nosed bat, Platyrrhinus lineatus (Chiroptera: Phyllostomidae)

Spermatogenesis, the remarkable process of morphological and biochemical transformation and cell division of diploid stem cells into haploid elongated spermatozoa, is one of the most complex cell differentiations found in animals. This differentiation process has attracted extensive studies, not only because the process involves many radical changes in the cell shape and biochemistry, but also because the phases and steps of differentiation have provided a better basis for analyzing the seminiferous epithelium cycle. Thus, this study aimed to characterize ultrastructurally the spermatogenesis process in the bat Platyrrhinus lineatus in order to provide a basis for determining the stages of spermatogenesis and to facilitate comparisons of the process between bat species and other vertebrates. Based on ultrastructural characteristics three main types of spermatogonia could be accurately identified: A(d), A(p) and B; the differentiation of spermatids was clearly divided into 12 steps (steps 1-3: Golgi phase, steps 4-5: cap phase, steps 6-9: acrosomal phase and steps 10-12: maturation phase). The ultrastructure of spermatozoa, Leydig cells and Sertoli cells was characterized; and some processes including nucleolar disorganization and the formation of synaptonemal complexes, acrosome and chromatoid body were discussed. Based on our results we may conclude that the spermatogenic process of P. lineatus follows the pattern of mammals with some specificity, as the process of formation of the acrosome and the presence of the perfuratorium. By other side, the simpler ultrastructure of its spermatozoon shows a pattern more closely related to the sperm cells of humans and other primates.     

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.     

Focusing of explosion waves: Three-dimensional mathematical modeling

The propagation and focusing explosionlike waves in a cubic volume filled with a gas was considered. The wave is generated by the expansion of a small volume of a gas with enhanced parameters located at different points inside the cube. This formulation of the problem simulates the process initiated by an explosion in a volume filled with a gas and the character of loading on the walls of the volume. 3D calculations of the propagation and cumulation of explosion waves with a short positive phase were performed for the first time. The results can be used in analyzing experiments aimed at modeling the initiation of combustion by the waves generated by a primary source in closed volumes with complex geometry.