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.     

The reproductive cycle in the male gonads of Danio rerio (Teleostei, Cyprinidae). Stereological analysis

The studies in Danio rerio testis were performed during the 8 weeks between the first and second spawning. The testes were dissected from the animals and fixed after successive weeks. Semithin epon sections were analysed using stereological methods to assess the relative volumes of cysts containing successive stages of spermatogenesis. During the first three weeks the cysts with spermatogonia B have the highest relative volumes. At the 2nd week the first cysts with spermatocytes appear. At the 4th week the first cysts containing early spermatids are formed. During the 4th, 5th and the 6th weeks of the gonad cycle cysts containing spermatocytes I are the most numerous but one can also see volumes of cysts filled with late spermatids and sperm cells growing. In the 7th week of the cycle the volumes of cysts containing early spermatids as well as those containing late spermatids are very large. Many cysts have released completely formed sperm cells into the tubule lumen. In the 8th week of the gonad cycle the testis tubules are filled with sperm cells. In many places the tubule walls break off and join to make vast chambers filled with sperm. Therefore, the germinal epithelium of the D. rerio testis at this stage turns into a "discontinuous" type.     

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 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.     

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.     

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.     

Organization and quantification of the elements in the intertubular space in the adult jaguar testis (Panthera onca, LINNAEUS, 1758)

The endocrine portion of mammal testicle is represented by Leydig cells which, together with connective cells, leukocytes, blood and lymphatic vessels, form the intertubular space. The arrangement and proportion of these components vary in the different species of mammals and form mechanisms that keep the testosterone level – the main product of the Leydig cell – two to three times higher in the interstitial fluid than in the testicular blood vessels and 40–250 times higher in these than in the peripheral blood. Marked differences are observed among animal species regarding the abundance of Leydig cells, loose connective tissue, development degree and location of the lymphatic vessels and their topographical relationship with seminiferous tubules. In the jaguar about 13% of the testicular parenchyma is occupied by Leydig cells, 8.3% by connective tissue and 0.3% by lymphatic vessels. Although included in standard II, as described in the literature, concerning the arrangement of the intertubular space, the jaguar has grouped lymphatic vessels in the intertubular space instead of isolated ones. In the jaguar the average volume of the Leydig cell was 2386 μm3 and its average nuclear diameter was 7.7 μm. A great quantity of 2.3 μm diameter lipidic drops was observed in the Leydig cell cytoplasm of the jaguar. The Leydig cells in the jaguar occupy an average 0.0036% of the body weight and the average number per gram of testicle was within the range for most mammals: between 20 and 40 million.     

Structure and ultrastructure of the spermatozoa of Trypoxylon (Trypargilum) albitarse Fabricius 1804 (Hymenoptera: Apoidea: Crabronidae)

The ultrastructure of the spermatozoa of Trypoxylon (Trypargilum) albitarse is described here for the first time within this genus. Testes and seminal vesicles were dissected and processed for transmission electron microscopy. In the testicular follicles, the spermatids are arranged in a maximum number of 32 for each cyst. The spermatozoa are slender and they measure approximately 150 μm in length. The head is about 17 μm long and is formed by the acrosome and the nucleus. The flagellum consists in an axoneme, two mitochondrial derivatives, two accessory bodies and, at the nucleus–flagellum transition, a symmetric centriolar adjunct. The axoneme presents the typical 9 + 9 + 2 microtubule pattern. In the terminal region, the central microtubules and nine doublets finish first, followed by the accessory microtubules. Both mitochondrial derivatives begin together and are inserted in the base of the centriolar adjunct. Along the middle region, the larger derivative has almost twice the area of the smaller one and includes a discrete paracrystalline region. At the tip, the smaller derivative ends before the larger one and both before the axoneme. The characters derived from the ultrastructure of the spermatozoa of T. albitarse show synapomorphies shared with the Apoidea and present characters that are probably apomorphic for the Crabroninae subfamily.     

Cytogenetics of four Omophoita species (Coleoptera,Chrysomelidae, Alticinae): A comparative analysis using mitotic and meiotic cells submitted to the standard staining and C-banding technique

Omophoita belongs to the tribe Oedionychini and is endemic from Neotropical region. The species of the tribe Oedionychini have revealed certain singular chromosomal features, such as sex chromosomes with extremely large size, asynapsis, and synthelic or amphithelic orientation during meiosis. Additionally, some species also showed post-reductional segregation of the gigantic sex chromosomes in meiotic division. The purpose of this work was to characterize cytogenetically four Omophoita species (O. magniguttis, O. octoguttata, O. personata, and O. sexnotata) in relation to their diploid number, chromosomal morphology, type of sex chromosome system, and constitutive heterochromatin pattern in mitotic and meiotic cells, and compare the obtained data with those of related species to establish the mechanism involved in the chromosomal differentiation of these species during the evolutionary process. The diploid number, 2n = 22 = 20 + X + y, and meiotic formulae, 10II + X + y, observed in these species were similar to those of the same genus and other species related. The autosomal morphology was acrocentric in O. magniguttis and O. octoguttata, metacentric in O. personata, and predominantly metacentric in O. sexnotata. In all these species, the sex chromosomes were metacentric. The secondary constriction occurred in pair 6 and X chromosome of O. personata, and in pair 6 and y chromosome of O. sexnotata. The constitutive heterochromatin was pericentromeric in O. magniguttis and centromeric in O. sexnotata, with the exception of the mitotic sex chromosomes of O. sexnotata, in which centromeric C band was lacking. Additional C bands in the sex chromosomes of O. magniguttis and certain autosomes and sex chromosomes of O. sexnotata were observed. Collochores were indirectly identified in the spermatocytes of O. octoguttata, O. personata, and O. sexnotata. The main mechanisms involved in the karyotype evolution of these species were discussed.     

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.     

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.     

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.     

Cytotoxic evaluation of N-isopropylacrylamide monomers and temperature-sensitive poly(N-isopropylacrylamide) nanoparticles

The objective of this research project is to investigate the biocompatibility of N-isopropylacrylamide (NIPAAm) monomers and poly(N-isopropylacrylamide) (PNIPAAm) nanoparticles in vitro. PNIPAAm nanoparticles of different sizes were synthesized and characterized by transmission electron microscopy and dynamic light scattering. Cytotoxicity studies using MTS assays were conducted on fibroblasts, smooth muscle cells, and endothelial cells. In addition, the concentration of NIPAAm monomers remaining on PNIPAAm nanoparticles was determined using bromination and spectrophotometry. The cytotoxicity results did not show a significant difference in cell survival when cells were exposed to different particle sizes (100, 300, and 500 nm). Dose studies showed that all three cell types exposed to 100 nm PNIPAAm nanoparticles at concentrations less than or equal to 5 mg/mL were compatible, while cells exposed to NIPAAm monomers exhibited toxicity even at very low concentrations. We also found that 1 mg/mL concentration of 100 nm PNIPAAm nanoparticles was cytocompatible for 4 days, whereas NIPAAm monomers were cytotoxic after 24 h of exposure. Photomicrographs showed altered morphology in cells exposed to NIPAAm monomers, while cells exposed to PNIPAAm nanoparticles maintained their normal morphology. Finally, a very low concentration of NIPAAm monomers remained on the PNIPAAm nanoparticles after synthesis and dialysis. Our results demonstrate that NIPAAm monomers are cytotoxic, whereas PNIPAAm nanoparticles are compatible at 5 mg/mL concentration or below for fibrobasts, smooth muscle cells, and endothelial cells.     

Structural and ultrastructural characterization of male reproductive tracts and spermatozoa in fig wasps of the genus Pegoscapus (Hymenoptera, Chalcidoidea)

The three Pegoscapus species present the same internal reproductive tract features comprising testes with a single testicular tubule, seminal vesicles, vasa deferentia, accessory glands and an ejaculatory duct. The seminal vesicle shows two morphologically distinct portions although they do not resemble the separate chambers found in other Chalcidoidea. The anterior portion of the seminal vesicle shows a prominent epithelium and stores the mature spermatozoa, while the posterior region is formed by a thicker muscular sheath that participates on ejaculation. The sexual maturation in Pegoscapus is achieved at emergence, when the testicular degeneration occurs. The spermatozoa of Pegoscapus reveal a basic structure similar to that of other Chalcidoidea. In Pegoscapus sp1. and Pegoscapus sp2. they present the same features, whereas Pegoscapus tonduzi comprises some different characteristics. It measures approximately 160 μm in Pegoscapus sp1. and Pegoscapus sp2., while in P. tonduzi the spermatozoa measure about 360 μm. The extracellular sheath thickness is another difference among the species. While Pegoscapus sp1. and Pegoscapus sp2. show a thick extracellular sheath, in P. tonduzi this sheath is very thin resulting in a large space intervening between the extracellular sheath and the nucleus. Despite these differences, the three species analyzed share some characteristics that allow the establishment of an identity to the spermatozoon of the genus Pegoscapus: the seminal vesicle not divided in chambers; the absence of acrosomal structures in the spermatozoa; the length of the extracellular sheath; the central microtubules being the firsts to terminate in the sequence of microtubular cutoff at the final axonemal portion.     

Electro-acupuncture promotes survival, differentiation of the bone marrow mesenchymal stem cells as well as functional recovery in the spinal cord-transected rats

Background  

Bone marrow mesenchymal stem cells (MSCs) are one of the potential tools for treatment of the spinal cord injury; however, the survival and differentiation of MSCs in an injured spinal cord still need to be improved. In the present study, we investigated whether Governor Vessel electro-acupuncture (EA) could efficiently promote bone marrow mesenchymal stem cells (MSCs) survival and differentiation, axonal regeneration and finally, functional recovery in the transected spinal cord.     

An ultrastructural study of testes permeability in sea urchins,Strongylocentrotus intermedius

Permeability of testes in sea urchins, Strongylocentrotus intermedius, was investigated by using an electron-opaque tracer, lanthanum nitrate. This tracer is able to enter the basal compartment of germinative epithelium, where developing germ cells are located. However, its ability to penetrate the gonadal lumen was reduced. An incomplete permeability barrier between the basal compartment and the gonadal lumen is supposed to exist in testes of S. intermedius.     

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.