Perfil em SDS-PAGE das proteínas do plasma seminal e sua relação com a congelabilidade do sêmen de touros doadores da raça gir

Foram utilizados dez animais doadores de sêmen em nível de Central de Inseminação Artificial, da raça Gir, divididos em dois grupos, de acordo com o grau de congelabilidade do sêmen de cada animal. Os animais com sêmen de alta congelabilidade foram aqueles cuja porcentagem de ejaculados viáveis pós-descongelação foi superior a 80%. O grupo de baixa congelabilidade tinha animais com porcentagem menor que 50% de ejaculados viáveis pós-descongelação. Os critérios de avaliação da viabilidade do sêmen e seleção dos animais foram definidos pelo controle de qualidade do Departamento de Produção da Central de Inseminação Artificial. Foram feitas quatro coletas semanais consecutivas, sendo que obtiveram-se as amostras de plasma seminal por centrifugação a 1.500 g por 15 a 20 minutos a 4°C, momentos após a coleta do sêmen em vagina artificial. O plasma seminal foi dialisado em membrana de celulose, em tampão Tris-Glicina pH-7,4 por 24 horas a 4°C, em agitação lenta e constante. As amostras foram padronizadas em 1,0 mg/ml de proteína total, por diluição em tampão Tris-HCl 62mM pH-6,2 mais 20% de glicerol e 4% de SDS. Através de eletroforese do tipo SDS-PAGE, foram feitas as corridas em gel a 13%. A corrida foi feita com a constante de 25 mA, por um período de 5 horas. A coloração do gel foi feita por Coomassie Brilliant Blue. Pelos resultados obtidos, verificou-se que existe uma banda no grupo de alta congelabilidade, cujo fragmento polipeptídico desta proteína tem Mr (mobilidade relativa) 20,3 e PM (peso molecular) aproximado de 61.800 Da. Esta banda não foi detectada nas amostras do grupo de baixa congelabilidade, o que sugere ser um possível marcador bioquímico quanto ao potencial de criopreservação do sêmen de bovinos.

Comparative cytogenetic and morphological analysis of Astyanax scabripinnis paranae (Pisces, Characidae, Tetragonopterinae)

Foram realizados estudos citogenéticos e morfológicos em nove populações locais de Astyanax scabripinnis paranae. Todas as populações apresentaram 2n = 50 cromossomos e conspícuas diferenças envolvendo a morfologia dos cromossomos, número e posição das NORs e quantidade e localização dos blocos de heterocromatina constitutiva. O estudo quantitativo dos dados citogenéticos, usando a análise das variáveis canônicas (CVA), mostraram que oito populações apresentaram diferentes constituições cariotípicas. Análises morfológicas baseadas em nove caracteres morfométricos e dois merísticos foram eficazes no estabelecimento de uma clara identificação de cinco populações. Análises comparativas dos dados citogenéticos e morfológicos sugerem que as modificações cromossômicas e morfológicas ocorreram em diferentes taxas, sendo as cromossômicas mais rápidas do que as morfológicas. A despeito da similaridade morfológica encontrada entre algumas populações, diferenças cromossômicas foram detectadas entre todas as populações estudadas.

Chromosome banding in three species of Brazilian toads (Amphibia-Bufonidae)

The chromosomes of Bufo crucifer, B. ictericus, and B. pamacnemis were studied by conventional staining as well as with C banding and NOR techniques. These species have a diploid number of 2n = 22 and identical karyotypes, composed of metacentric and submetacentric chromosomes. The C banding patterns and NOR data indicate that these species of Bufo are not differentiated by the distribution and amount of constitutive heterochromatin or the position of the nucleolar organizer regions.

HETEROCHROMATIN AND KARYOTYPE REORGANIZATION IN FISH OF THE FAMILY ANOSTOMIDAE (CHARACIFORMES)

Mitotic chromosomes of four fish species of the family Anostomidae, belonging to the genera Leporinus, Leporellus, and Schizodon, were studied. With 2n = 54 meta- and submetacentric chromosomes, this family appears to be characterized by marked karyotypic stability. Although perceptible differences exist, mainly in the amount of constitutive heterochromatin present in the chromosomes of these species, these differences do not affect the structure and/or size of these chromosomes. Chromatin substitutions and/or modifications may have led, in one direction, to an increase in heterochromatin in some species and, in the opposite direction, to heterochromatin reduction in others. Whether these changes are accompanied by changes in the amount of euchromatin in the chromosomes is an open question. The nucleolar organizer regions, which may be located on different chromosomes in the various species, may also be indicators of reorganization of these karyotypes.

A natural triploid in Trichomycterus davisi (Siluriformes, Trichomycteridae): mitotic and meiotic characterization by chromosome banding and synaptonemal complex analyses

Within a total of 50 analyzed specimens a male individual of Trichomycterus davisi has been recorded with 81 chromosomes including 60 metacentric, 18 submetacentric and three subtelocentric chromosomes. When compared with diploid individuals (2n = 54) and the morphological standard of chromosomes, this male is a triploid with 3 = 81 chromosomes. Since staining with silver nitrate indicates three active nucleolar organizer regions (NORs), the three NOR- bearing chromosomes in this individual are genetically active. Analysis of the synaptonemal complex (SC) by electronic microscopy shows that there is an incomplete pairing of the third set of chromosomes in the triploid individual.

Cytogenetics of three Brazilian Goniosoma species: A new record for diploid number in Laniatores (Opiliones, Gonyleptidae, Goniosomatinae)

Currently, 60 species of harvestmen have been karyotyped and all of these are from the Nearctic and Palearctic regions. This work is the first cytogenetic report of three gonyleptid species of the suborder Laniatores: Goniosoina aff. badiuln, G. proxiinuni and G. spelaeum of the Neotropical region, from the southeastern region of Brazil. Conventional Giemsa stain chromosome preparations were obtained from embryonic cells and adult male testes. Embryo mitotic plates of G. aff. badium and G. proximum indicated 88 chromosomes, and mitotic spermatogonial plates of G. spelaeum males revealed intra- and interindividual variation of chromosome number, ranging from 92-109 chromosomes. In the three analyzed species, the mitotic chromosomes were meta- or subinetacentric with no obvious sex chromosomes being identified during mitosis. Prophase I spermatocytes of G. spelaeum also revealed intra- and interindividual bivalent number variation and furthermore indicated the presence of multivalence. The karyotypes of these three Goniosoina species exhibited the largest chromosome pair with a negative heteropycnosis in the distal region of the shortest arrn chromosomes of G. spelaeum submitted to silver impregnation evidenced this negative heteropycnotic region as nucleolus organizer region (NOR). These results, when compared with cytogenetic data of other Laniatores species from the Palearctic region, indicated that a new record for diploid chromosome number probably characterize the genus Goniosoma in the Neotropical region.

Chromosomal analysis of the leptodactylids Pleurodema diplolistris and Physalaemus nattereri (Amphibia, Anura)

Detailed characterizations of the karyotypes of the Brazilian leptodactylid frogs Pleurodema diplolistris, the only species of Pleurodema not studied cytogenetically so far, and Physalaemus nattereri, a species in the Ph. biligonigerus group, are presented. Both karyotypes had 2n = 22 and their chromosomes had a very similar morphology, except for pair 11, which was metacentric in Pl. diplolistris and telocentric in Ph. nattereri. The localization of nucleolar organizer regions (NORs) and heterochromatic bands allowed the differentiation of chromosomes that were morphologically indistinguishable between these species, such as pairs 1, 3 and 10, which showed interstitial C-bands in Ph. nattereri, and pair 8, that had an NOR and an adjacent C-band in Pl. diplolistris. Pair 8 also has NOR-bearing chromosomes in many other Pleurodema species. However, in these species, the NOR is located proximal to the centromere on the short arm, while in Pl. diplolistris it occurred distally on the long arm, a condition that may be considered a derived state. In Ph. nattereri, the NOR occurred on chromosome I 1 and differed from the other species of the Ph. biligonigerus group. In contrast, C-banding revealed a heterochromatic block near the centromere on the short arm of pair 3, a characteristic common to all members of this group of Physalaemus.

Does alveolar soft-part sarcoma exhibit skeletal muscle differentiation? An immunocytochemical and biochemical study of myogenic regulatory protein expression

There has been persistent controversy regarding the nature of cell differentiation in alveolar soft-part sarcoma (ASPS) since its first description in 1952. Some studies suggest that ASPS might represent an unusual variant of skeletal muscle tumor, Given the availability of new monoclonal antibodies to probe for skeletal muscle differentiation and the rapid advance in immunocytochemical techniques for deparaffinized, formalin-fixed tissue sections, we wished to test the proposed hypothesis that ASPS might represent a new type of rhabdomyosarcoma Twelve archival samples of ASPS were retrieved, and we investigated the expression of two myogenic regulatory proteins, MyoD1 and myogenin, as rvell as other muscle-associated proteins, using sensitive immunocytochemical techniques. Despite the presence of desmin immunostaining in six ASPSs, no tumors were positive for either muscle actin or myoglobin Most importantly, no specimen showed nuclear expression of MyoD1 or myogenin, In 11 tumors, however, there was considerable granular immunostaining in the tumor cell cytoplasm with the anti-MyoD1 monoclonal antibody 5.8A, a phenomenon observed in various nonmuscle normal and neoplastic tissues with this antibody, To analyze the exact nature of immunostaining of MyoD1 and desmin in ASPS, biochemical analyses using available fresh frozen tumor tissue were performed, Although a 53-kDa band was noted with antidesmin antibody on Western blot analysis, no specific protein band that corresponds to the 45-kDa MyoD1 was detected with antibody 5.8A. These results confirm the presence of desmin in ASPS but argue against authentic expression of MyoD1, They also suggest that the cytoplasmic immunostaining observed with anti-MyoD1 antibody 5.8A most likely represents a nonspecific cross-reaction with an unknown cytoplasmic antigen, Considering the master role that MyoD1 and myogenin play in skeletal muscle commitment and differentiation and the lack of expression of these two proteins in ASPS as determined immunocytochemically and biochemically, we think that the histogenesis of ASPS remains unknown.

Cytogenetics of two species of Paratelmatobius (Anura: Leptodactylidae), with phylogenetic comments

In this paper we provide a cytogenetic analysis of Paratelmatobius cardosoi and Paratelmatobius poecilogaster. The karyotypes of both species showed a diploid number of 24 chromosomes and shared some similarity in the morphology of some pairs. On the other hand, pairs 4 and 6 widely differed between these complements. These karyotypes also differed in their NOR number and location. Size heteromorphism was seen in all NOR-bearing chromosomes of the two karyotypes. In addition, both karyotypes showed small centromeric C-bands and a conspicuous heterochromatic band in the short arm of chromosome 1, although with a different size in each species. The P. cardosoi complement also showed other strongly stained non-centromeric C-bands, with no counterparts in the P. cardosoi karyotype. Chromosome staining with fluorochromes revealed heterogeneity in the base composition of two of the non-centromeric C-bands of P. cardosoi. Comparison of the chromosomal morphology of these Paratelmatobius karyotypes with that of P. lutzii showed that the P. poecilogaster karyotype is more similar to that of P. lutzii than P. cardosoi. These cytogenetic results agree with the proposed species arrangements in the P. cardosoi and P. lutzii groups based on morphological and ecological data.

Karyotype variability in eight species of the subfamilies Loricariinae and Ancistrinae (Teleostei, Siluriformes, Loricariidae)

Loricariidae is one of the largest fish families of the world, with about 650 species separated into six subfamilies. To date, cytogenetic data on only 56 species of this family are available. In the present study, the karyotypes of three Ancistrinae species and five Loricariinae species were studied. The lowest diploid number, 2n=38, was observed in Ancistrus n.sp. 1 (Ancistrinae) and the highest diploid number, 2n=70, was observed in Rineloricaria n.sp. (Loricariinae). The nucleolar organizer regions (NORs) were seen at a terminal position in six species and at an interstitial position in two. The karyotypic analysis of Loricariinae and Ancistrinae species revealed that these groups exhibit a large diversity of diploid numbers, suggesting the occurrence of intense karyotypic evolution during their evolutionary history.

First chromosome characterization in the neotropical eel, Gymnothorax ocellatus (Pisces, Muraenidae)

Cytogenetics studies in 12 specimens of Gymnothorax ocellatus reveled a diploid chromosome number of 2n=42 (16 metacentrics, 18 submetacentrics and 8 acrocentrics). The nucleolar organizer regions were located in a terminal position on the long arm of the chromosome pair number fifteen. Conspicuous blocks of constitutive heterochromatin were observed in the centromeric and pericentromeric regions of some chromosome pairs. The results obtained are similar to those previously described for others species of this family. However, the cytogenetic informations may be useful in the identification of a possible variety of this species in Brazilian coast and contribute to the understanding of relationships among the species and the process of diversification which occurred in this group. © 2005 The Japan Mendel Society.

Cytogenetic analysis of three sympatric Gymnotus species (Teleostei: Gymnotidae) from the Fundo Stream, MG, Brazil

Three sympatric species of Gymnotus from the Fundo stream, a small tributary of the Sapucai river, Minas Gerais State, Brazil, were studied in relation to their karyology. Gymnotus sylvius presented 2n=40 chromosomes (36 m/sm+4 st/a), Gymnotus sp. presented 2n=50 (26 m/sm+ 24 st/a) and Gymnotus paraguensis had 2n=54 (50 m/sm+4 st/a). C-banding demonstrated positively stained heterochromatic blocks in the centromeric position of few chromosomes on G. sylvius and in the centromeric region of all chromosomes on G. paraguensis samples. The nucleolus organizer region (NOR) was located on the short arm of one st chromosome pair in G. sylvius and Gymnotus sp., and in the interstitial position on the short arm of the pair number one and below the centromere on a third chromosome on G. paraguensis. The cytogenetic data obtained indicate that Gymnotus sp. represent a new Gymnotus specie with a karyotypic constitution never observed on others species from this genus. Some aspects related to the chromosome diversification of these Gymnotus are discussed. © 2007 The Japan Mendel Society.

Cytogenetical analyses in three Astyanax scabripinnis populations (Pisces, Characidae) from Minas Gerais State, Brazil

Cytogenetical data in 3 populations of characid fish assigned to the complex of Astyanax scabripinnis from São Francisco river basin and Grande river basin, Minas Gerais State, Brazil, are presented for the first time. The same diploid number, 2n=50, was detected in the 3 populations, which has conspicuous differences involving karyotype morphology: 8M, 20SM, 6ST and 16A (Cambeba stream), 6M, 28SM, 6ST and 10A (Machado headwater), 6M, 24SM, 8ST and 12A (Pedra Branca stream). Differences involving amount and/or locations of heterochromatin blocks, number and position of nucleolar organizer regions (NORs) and CMA3 positive signals were also observed. Some aspects related to the chromosome diversification of Astyanax scabripinnis are discussed. © 2007 The Japan Mendel Society.

Cytogenetic and random amplified polymorphic DNA analysis of Leptodactylus species from rural and urban environments (Anura, Amphibia)

Cytogenetic and random amplified polymorphic DNA analyses carried out in the species Leptodactylus podicipinus, L. ocellatus, L. labyrinthicus, and L. fuscus from rural and urban habitats of the northwest region of São Paulo State, Brazil, showed that the karyotypes (2n = 22), constitutive heterochromatin distribution and nucleolus organizer region (NOR) location did not differ between the populations from the two environments. The in situ hybridization with an rDNA probe confirmed the location of the NORs on chromosome 8 revealing an in tandem duplication of that region in one of the chromosomes of L. fuscus. DAPI showed that part of the C-band-positive heterochromatin is rich in AT, including that in the proximity the NORs in L. podicipinus and L. ocellatus. The molecular analyses showed that the two populations (urban and rural) of L. podicipinus and L. fuscus are similar from a genetic point of view. The urban and rural populations of species L. ocellatus and L. labyrinthicus showed differences in genetic structures, probably due to urbanization which interferes with the dispersion of those frogs. The marked differences observed between the two populations of L. ocellatus can be representing the cryptic condition of the species. Unweighted pair-group method of analysis and genetic distance analysis detected the genetic proximity between L. ocellatus and L. fuscus. The results indicate that there was no reduction in the genetic diversity in the populations from the urban environment; however, the survival of these frogs would not be guaranteed in the case of an increase in human impact especially for populations of L. labyrinthicus and L. ocellatus. ©FUNPEC-RP.