Telomere biology of trypanosomatids: beginning to answer some questions

Studies of telomere structure and maintenance in trypanosomatids have provided insights into the evolutionary origin and conservation of some telomeric components shared by trypanosomes and vertebrates. For example, trypanosomatid telomeres are maintained by telomerase and consist of the canonical TTAGGG repeats, which in Trypanosoma brucei can form telomeric loops (t-loops). However, the telomeric chromatin of trypanosomatids is composed of organ ism-specific proteins and other proteins that share little sequence similarity with their vertebrate counterparts. Because telomere maintenance mechanisms are essential for genome stability, we propose that the particular features shown by the trypanosome telomeric chromatin hold the key for the design of antiparasitic drugs.

The Leishmania amazonensis TRF ( TTAGGG repeat-binding factor) homologue binds and co-localizes with telomeres

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evidence of epigenetic regulation of the tumor suppressor gene cluster flanking RASSF1 in breast cancer cell lines

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ultrastructure of spermiogenesis and spermatozoa of Gymnotus cf. anguillaris and Brachyhypopomus cf. pinnicaudatus (Teleostei : Gymnotiformes)

The ultrastructure of spermiogenic stages and spermatozoa of representatives of two gymnotiform families, Gymnotus cf. anguillaris (Gymnotidae) and Brachyhypopomus cf. pinnicaudatus (Hypopomidae) were studied. Spermiogenesis of both species is characterized by lateral development of the flagellum and formation of a nuclear fossa. Some differences were found between these species, such as whether (B. cf. pinnicaudatus) or not (G. cf. anguillaris) nuclear rotation occurs, permanence of the cytoplasmic channel, and type and localization of the nuclear fossa. In the G. cf anguillaris spermatozoon the nucleus is spherical with highly condensed chromatin. The nuclear fossa is shallow and lateral and is associated with the centriolar complex through stabilizing fibrils. The midpiece is short, with many vesicles, a cytoplasmic channel, and elongate mitochondria. In the B. cf. pinnicaudatus spermatozoon the ovoid nucleus is elongated lateral and posterior to the centriolar complex, and has highly condensed chromatin. The eccentric nuclear fossa is of the moderate type, and contains the entire centriolar complex. The midpiece is long, with numerous vesicles, elongate mitochondria, and no cytoplasmic channel. In both species the flagella are laterally disposed in relation to the nucleus and comprise of the classical 9 + 2 axoneme. Most of the characteristics found in the spermatozoa of these two species of Gymnotiformes are shared with species of Characiformes, whereas only a few are also found in Siluriformes. This suggests that Gymnotiformes and Characiformes may be more closely related than previously proposed. (C) 2007 Elsevier Ltd. All rights reserved.

Cytogenetic analysis of A- and B-chromosomes of Prochilodus lineatus (Teleostei, Prochilodontidae) using different restriction enzyme banding and staining methods

Different cytogenetic techniques were used to analyse the chromosomes of Prochilodus lineatus with the main objective of comparing the base composition of A- and B-chromosomes. The results of digestion of chromosomes with 10 different restriction endonucleases (REs), silver staining, CMA(3) staining and C-banding indicated the existence of different classes of highly repetitive DNA in the A-set and also suggested the existence of compositional differences between the chromatin of A- and B-chromosomes. The 5-BrdU incorporation technique showed a late replicating pattern in all B-chromosomes and in some heterochromatic pericentromeric regions of A-chromosomes. The cleavage with RE BamHI produced a band pattern in all chromosomes of P. lineatus which permitted the tentative pairing of homologues in the karyotype of this species. We concluded that the combined use of the above techniques can contribute to the correct identification of chromosomes and the karyotypic analysis in fishes. on the basis of the results, some aspects of chromosome structure and the origin of the B-chromosomes in P. lineatus are discussed.

Spermiogenesis and introsperm ultrastructure of Scoloplax distolothrix (Ostariophysi : Siluriformes : Scoloplacidae)

Spermiogenesis in scoloplacids is characterized by initial lateral development of the flagellum, nuclear rotation, medial nuclear fossa formation, complex centriolar migration, and cytoplasmic channel formation. The scoloplacid spermiogenesis is similar to those found in Diplomystidae, the most primitive siluriform family. The scoloplacid spermatozoa have all the main characteristics of introsperm. They exhibit a conic head, a symmetric midpiece, a medial flagellum, and no acrosome. The conic forward-elongated nuclei contain homogeneous chromatin. The thin extremity of the nuclei is strongly curved and along its internal face there is a well-developed membranous compartment. The centrioles are completely inside the medial nuclear fossa, perpendicular to each other and with an electron-dense material between them. In a cross view of the midpiece, the mitochondria form a ring surrounding internally the cytoplasmic channel, and in a longitudinal view they are organized in a row along it. Several elongated vesicles are distributed peripherally, mainly concentrated in the mid-piece basal region. The flagellum contains the classical axoneme (9 + 2) and has two lateral projections or fins. The spermatozoa of scoloplacids share several characteristics with those of Auchenipteridae. Since these two families are not phylogenetically related this similarity seems to be due to convergence once both families are, until now, the only known siluriform families with introsperm.

Ultrastructure of spermiogenesis in Plagioscion squamosissimus (Teleostei, Perciformes, Sciaenidae)

Spermiogenesis in Plagioscion squamosissimus occurs in cysts. It involves a gradual differentiation process of spermatids that is characterized mainly by chromatin compaction in the nucleus and formation of the flagellum, resulting in the spermatozoa, the smallest germ cells. At the end of spermiogenesis, the cysts open and release the newly formed spermatozoa into the lumen of the seminiferous tubules, the spermatozoa do not have an acrosome and are divided into head, midpiece, and tail or flagellum, the spermatozoa of P. squamosissimus are of perciform type with the flagellum parallel to the nucleus and the centrioles located outside the nuclear notch. (C) 1999 Harcourt Publishers Ltd.

Ultrastructure of spermatogenic cells and spermatozoa in Hoplias malabaricus (teleostei, characiformes, erythrinidae)

The differentiation of spermatids in Hoplias malabaricus is characterized by chromatin compaction, flagellum development, nuclear rotation, nuclear fossa formation, and excess cytoplasm elimination. In the resulting spermatozoon, the head is round and the nucleus contains chromatin compacted in thick filaments, peripherically arranged, to a central electron-lucent area. The acrosome is absent. The nuclear fossa is eccentric but not pronounced. The proximal centriole penetrates it and is oblique to the flagellum. The long midpiece has several converging elongate vesicles, forming membranous hoops in the initial segment of the flagellum, but has no cytoplasmic channel. The mitochondria are elongate and branched or C-shaped and located around the initial segment of the axoneme. The lateral flagellum does not show lateral projections. The ultrastructural characteristics of H. malabaricus spermatozoa are similar to the Cypriniformes. (C) 2001 the Fisheries Society of the British Isles.

Spermatogonia and spermatocyte ultrastructure in Hoplias malabaricus (Teleostei, Characiformes : Erythrinidae)

The Hoplias malabaricus primary spermatogonium shows a large nucleus, central nucleolus, and low electron-dense cytoplasm containing nuages. In cysts, they undergo several mitotic divisions with incomplete cytokinesis, giving rise to secondary spermatogonia. These are smaller than the primary spermatogonia and their nuclei have one or two eccentric nucleoli. Spermatocytes I can be identified by the presence of synaptonemal complexes. Spermatocytes II are smaller than spermatocytes 1, displaying roughly compacted chromatin. All these cell types remain interconnected by thick-walled intercellular bridges, which have membranous reinforcements during mitosis and meiosis. These cell types show a well-developed endomembranous system, one of the centrioles anchored to the plasma membrane and small nuages. Their mitochondria are large and circular, with few cristae. In the last generations of spermatogonia, the mitochondria are smaller, elongate and have more cristae. In the spermatocytes, the mitochondria are small and round. Similarities found in relation to germ cells of other teleosts are discussed.

Spermiogenesis and spermatozoa ultrastructure in Salminus and Brycon, two primitive genera in Characidae (Teleostei : Ostariophysi : Characiformes)

In Salminus, spermiogenesis is cystic and gives origin to a type I aquasperm. Spermatid differentiation is characterized by chromatin condensed into thick fibres, nuclear rotation, nuclear fossa formation, cytoplasmic channel formation, mitochondrial fusion producing long and ramified mitochondria, and the presence of several membranous concentric rings around the plasma membrane that encircles the cytoplasmic channel. In Salminus and Brycon, spermatozoa are very similar. They exhibit a spherical nucleus and chromatin condensed into fibre clusters, and a deep nuclear fossa. They show a long midpiece with few elongate mitochondria at the initial region and a cytoplasmic channel completely encircled by one or two membranous concentric rings. The flagellar axis is perpendicular to the nucleus and exhibits the classic axoneme (9 + 2). The very strong similarity observed between Salminus and Brycon spermatozoa supports the hypothesis that these subfamilies are likely to have a monophyletic origin.

Spermatozoa ultrastructure in Sciaenidae and Polynemidae (Teleostei : Perciformes) with some consideration on Percoidei spermatozoa ultrastructure

Spermatozoa ultrastructure was studied in five marines (Paralonchurus brasiliensis, Larimus breviceps, Cynoscion striatus, Micropogonias furnieri, Menticirrhus americanus, Umbrina coroides, Stellifer rastrifer), and one freshwater (Plagioscion squamosissimus) species of Sciaenidae and one species of Polynemidae (Polydactylus virginicus). The investigation revealed that, in all species, spermatozoa display a round head, a nucleus containing highly condensed, filamentous chromatin clusters, no acrosome, a short midpiece with a short cytoplasmic channel, and a flagellum showing the classic axoneme structure (9 + 2) and short irregular lateral fins. In Sciaenidae, the spermatozoa are type II, the flagellar axis is parallel to the nucleus, the lateral nuclear fossa is double arched, the centriolar complex is outside the nuclear fossa, the proximal centriole is anterior and perpendicular to the distal centriole, and no more than ten spherical (marine species) or elongate (freshwater species) mitochondria are observed. Polynemidae spermatozoa are of the intermediate type with the flagellar axis eccentric to the hemi-arc-shaped nucleus, and exhibit no nuclear fossa, the centriolar complex close to the upper nuclear end, the proximal centriole lateral and oblique to the distal centriole, and one large ring-shaped mitocondrion. The data available show that no characteristic is exclusively found in the spermatozoa of members of the Sciaenidae family when compared to other Percoidei with type II spermatozoa. However, three characteristics were exclusively found in Polynemidae: (1) the hemi-arched nucleus; the positioning of the centrioles; and (2) the ring-shaped mitocondrion. The interrelationships between Sciaenidae and Polynemidae as well as between these two families and other Percoidei are herein discussed. (c) 2005 Elsevier Ltd. All rights reserved.

The ultrastructure of Sorubim lima (Teleostei, Siluriformes, Pimelodidae) spermatogenesis: premeiotic and meiotic periods

The ultrastructure of Sorubim lima spermatogenesis during the premeiotic and meiotic periods was studied. Our observations showed that the germ cells in the cysts are connected by cytoplasmic bridges and the mitotic and meiotic divisions are slightly asynchronous, the first and the last spermatogonial generations differ in the cellular and nuclear volume, nucleolus, chromatin condensation, distribution, size, density, and shape of the mitochondria, presence of 'lamellae anulata', amount and dimension of the 'nuages', and movement of the centrioles. In addition to the nuclear prophase structures, the spermatocyte I shows changes in all other cellular organelles and elongated vesicles appear in the cytoplasm. The accentuated cytoplasmic density and thickened walled vesicles are morphological characteristics that differentiate spermatocytes II from the other germ cells in the cysts of Sorubim lima testis. (C) 1999 Harcourt Publishers Ltd.

Spermiogenesis and sperm ultrastructure in Cichla intermedia with some considerations about Labroidei spermatozoa (Teleostei, Perciformes, Cichlidae)

Spermiogenesis and spermatozoal structure were studied in Cichla intermedia, a primitive species of Neotropical cichlids. The analysis shows that spermiogenesis is characterized by chromatin compaction, flagellum development, nuclear rotation, nuclear fossa formation and residual cytoplasm elimination. In the spermatozoa, the head is round, the nucleus contains highly condensed filamentous clusters of chromatin and an acrosome is absent. The nuclear fossa is slightly eccentric and shows a projection that penetrates into the nuclear outline. The proximal centriole is located in the initial segment of the nuclear fossa. The midpiece and the cytoplasmic channel are long. The mitochondria, about 10 in number, are round or slightly elongated, disposed in two layers around the initial segment of the flagellum. The flagellum has a classical 9 + 2 axoneme and two lateral fins. The data available show that no characteristics of spermiogenesis or spermatozoa are exclusively found in members of the suborder Labroidei. However, three characteristics seem to be exclusively observed in Cichlidae: (1) compact filamentous clusters of chromatin; (2) slightly eccentric nuclear fossa; and, (3) number of mitochondria. (C) 2003 Elsevier Ltd. All rights reserved.

Fertility of rat epididymal sperm after chemically and surgically induced sympathectomy

Guanethidine, a chemical that selectively blocks sympathetic noradrenergic neurons, was used to investigate the role of sympathetic innervation in the fertility of rat epididymal sperm, using both natural mating and in utero insemination protocols. This animal model correlates, at least in part, with spinal cord injury (SCI) in men. Adult male rats were treated daily by i.p. injections, for 21 or 42 days, with 0 or 6.25 mg/kg guanethidine. To compare the effects of guanethidine-induced sympathectomy with those following surgically induced sympathectomy, the inferior mesenteric ganglion and the proximal hypogastric nerves were removed in another group of rats. Both chemically and surgically induced sympathectomy increased the weight of the epididymis and seminal vesicles/coagulating glands as well as the number and the transit time of cauda epididymal sperm. Neither serum testosterone levels nor LH was affected by treatment with guanethidine. Using natural mating, no litters were produced by guanethidine-treated rats. Chemically denervated rats failed to produce copulatory plugs or ejaculate into the uterus. However, distal cauda epididymal sperm from chemically or surgically denervated rats displayed normal fertilization ability (80%) using in utero inseminations. In addition, the sperm of denervated rats did not show abnormal sperm chromatin structure using an assay that detects DNA damage. We conclude that sympathectomy delays the transit of sperm through the cauda epididymidis and produces ejaculatory dysfunction but does not compromise sperm quality in the distal cauda epididymidis. Moreover, these data provide compelling evidence that there is no association between the prolonged transit time of sperm within the epididymis, i.e., pre-ejaculatory sperm aging, and the fertility of those sperm, which has important implications for artificial insemination using sperm from men with SCI.

The ultrastructure of spermiogenesis and spermatozoa in Diplomystes mesembrinus

Spermiogenesis in Diplomystes mesembrinus, one of the most primitive species from the Siluriformes, occurs in cysts. Differentiation of spermatids is characterized by chromatin compaction, flagellum development, nuclear fossa formation, rotation of the nucleus, and excess cytoplasm elimination. The spermatozoon head is round, the nucleus contains highly condensed chromatin clusters, the midpiece is short, the axoneme shows a 9 + 2 pattern with two discrete lateral projections, and the acrosome is absent. The nuclear fossa penetrates deeply into the nucleus, including the centriolar complex and the start of the axoneme. The single large C-shaped mitochondrion sur rounds the: initial segment of the axoneme. The structural features of D. mesembrinus spermatozoon are similar to the Clupeiformes. (C) 2001 the Fisheries Society of the British Isles.