Exploring the Relationship between Spontaneous Sister Chromatid Exchange and Genome Instability in Two Cryptic Species of Non-Human Primates.

There are extensive studies on chromosome morphology and karyotype diversity in primates, yet we still lack insight into genomic instability as a key factor underlying the enormous interspecies chromosomal variability and its potential contribution to evolutionary dynamics. In this sense, the assessment of spontaneous sister chromatid exchange (SCE) frequencies represents a powerful tool for evaluating genome stability. Here, we employed G-banding, fluorescence plus Giemsa (FPG), and chromosome orientation fluorescence in situ hybridization (CO-FISH) methodologies to characterize both chromosome-specific frequencies of spontaneously occurring SCE throughout the genome (G-SCE) and telomere-specific SCE (T-SCE). We analyzed primary fibroblast cultures from two male species of Ateles living in captivity: Ateles paniscus (APA) and Ateles chamek (ACH). High frequencies of G-SCEs were observed in both species. Interestingly, G-SCEs clustered on evolutionary relevant chromosome pairs: ACH chromosomes 1, 2, 3, 4, and 7, and APA chromosomes 1, 2, 3, 4/12, 7, and 10. Furthermore, a statistically significant difference between the observed and expected G-SCE frequencies, not correlated with chromosome size, was also detected. CO-FISH analyses revealed the presence of telomere-specific recombination events in both species, which included T-SCE, as well as interstitial telomere signals and telomere duplications, with APA chromosomes displaying higher frequencies, compared to ACH. Our analyses support the hypothesis that regions of Ateles chromosomes susceptible to recombination events are fragile sites and evolutionary hot spots. Thus, we propose SCE analyses as a valuable indicator of genome instability in non-human primates.

Sperm Morphology in Neotropical Primates.

The morphological and morphometric characterization of spermatozoa has been used as a taxonomic and phylogenetic tool for different species of mammals. We evaluated and compared the sperm morphometry of five neotropical primate species: Alouatta caraya, Ateles belzebuth and Ateles chamek of family Atelidae; and Cebus cay (=Sapajus cay) and Cebus nigritus (=Sapajus nigritus) of family Cebidae. After the collection of semen samples, the following parameters were measured on 100 spermatozoa from each specimen: Head Length, Head Width, Acrosome Length, Midpiece Length, Midpiece Width and Tail Length. Considering the available literature on sperm morphometry, we gathered data of 75 individuals, from 20 species, 8 genera and 2 families. These data were superimposed on a phylogeny to infer the possible direction of evolutionary changes. Narrower and shorter spermatozoa seem to be the ancestral form for Cebidae, with a trend toward wider and larger heads in derived groups. The spermatozoa of Atelidae may show an increase in total length and midpiece length. Sperm heads would have become narrower in the more derived groups of Ateles. Sperm length may increase in the more derived species in both families. Our results are discussed in the context of sperm competition and sexual selection.

Cytogenetic Characterization of Brown Howler Monkeys, Alouatta guariba clamitans (Atelidae, Platyrrhini): Meiotic Confirmation of an X1X1X2X2X3X3/X1X2X3Y1Y2 Sex Chromosome System.

For brown howler monkeys (Alouatta guariba clamitans), diploid chromosome numbers varying from 2n = 45 to 2n = 52, with XX/XY, X1X1X2X2/X1X2Y, and X1X1X2X2X3X3/X1X2X3Y1Y2 sex chromosome systems have been described by mitotic studies but still await confirmation by meiotic analyses. We analyzed 3 male individuals sampled in the wild (in the municipality of Santa Maria, RS, Brazil) as well as 1 male and 1 female individual in captivity at the São Braz breeding center. Peripheral blood samples and testicular biopsies were taken. We found different diploid numbers for both sexes in somatic cells, 2n = 45,X1X2X3Y1Y2 in males and 2n = 46,X1X1X2X2X3X3 in females, with 4 metacentric (9-12), 7 submetacentric (1-6, 8), and 9 acrocentric autosomal chromosome pairs (13-20, 22). X1 and X2 were submetacentric chromosomes, while X3, Y1, and Y2 were acrocentric ones. Spermatocyte microspreads were examined for synaptonemal complexes. Pachytene spermatocyte analysis was done to verify the chromosome number and morphologies observed in mitotic karyotypes. Immunodetection was performed using anti-SMC3 and anti-CREST antibodies. The presence of a sex chromosome pentavalent X1X2X3Y1Y2 in the males was confirmed by C-banding in metaphase I and by immunodetection in prophase I by the clear identification of 5 centromeres. The G-banded karyotype corresponded to that previously described for A. g. clamitans in the south of Brazil (Curitiba, Parana State, and Blumenau, Santa Catarina State) and for the Misiones Province, Argentina.

Two cases of polydactyly in wild brown howler monkeys (Alouatta guariba clamitans).

We report the first two cases of polydactyly in an atelid species: (i) a wild ca. 16-week-old infant female presenting seven digits in both feet and other bone malformations and (ii) a wild newborn male presenting six digits in both feet with the extra digit fused to the hallux.

DNA repair kinetic of hydrogen peroxide and UVA/B induced lesions in peripheral blood leucocytes from xeroderma pigmentosum patients and healthy subjects.

The objective of the present work was to study the fine kinetics of DNA repair in xeroderma pigmentosum (XP) syndrome, a complex disorder linked to a deficiency in repair that increases cancer susceptibility. The repair process was evaluated by the comet assay (CA) in cells from 2 XP patients and 9 controls exposed to UVA/B (UVA 366/UVB 280 nm) and H2O2 (150 µM) at temperatures of 4, 15, and 37°C. Samples were taken at 2-min intervals during the first 10 min to analyze the "fine kinetics" repair during the initial phase of the curve, and then at 15, 20, 25, 30, 45, 60, and 120 min. CA evaluation of DNA repair activity points to BER/NER initiation in the first 30 min with both inductors at 37°C and 15°C, but final comet length showed differences according to treatment. Repair kinetics during 120 min showed a good correlation with clinical features in both XP patients. Differences in final comet length were less pronounced in XP cells treated with H2O2 than with UVA/B, probably because the peroxide produces mainly base oxidation but less bulky lesions; UVA/B generates a mixture of both. These findings reinforce the value of CA in testing in DNA repair ability or exposure monitoring.

Primates karyological diagnosis and management programs applications.

BACKGROUND: Captive primates are often maintained in groups without geographic origin or genetic heritage information. This could lead to an incorrect assignment of species, which could result in an inadequate management of the colonies. METHODS: We present a cytogenetic protocol adapted to be successfully used in an accurate taxonomic diagnosis of non-human primates (Platyrrhini), including lymphocyte culture, G- and C-banding, meiosis, and fluorescent in situ hybridization technique (FISH). RESULTS: Using classical cytogenetic diagnosis, the species status was determined in 541 Platyrrhini individuals. Of these, 99 were previously erroneously sexed or assigned to a different species using only morphological characteristics. CONCLUSIONS: The cytogenetic results highlight the relevance of the genetic characterization of primates both in captivity and in the wild. These techniques had been used in our research group for more than 30 years in different research projects, not only for characterizing hundreds of primates, but also different for topics regarding primates genomes and evolution.

Baseline values of micronuclei and comet assay in the lizard Tupinambis merianae (Teiidae, Squamata).

The Micronucleus test (MN) and Comet assay (CA) are currently the most widely used methods that allow the characterization of DNA damage induced by physical and chemical agents in wild species. The continuous expansion of the cultivated areas in Argentina, since the introduction of transgenic crops, mainly soy, in association with the increased use of pesticides, transformed deeply the natural environments where the lizard Tupinambis merianae (tegu lizard) occurs. Despite the fact that reptiles have shown to be excellent bioindicators of environmental contaminants, there is no record of genotoxicity studies in T. merianae. The aim of the present study was to adjust the MN test and CA protocols to be applied in erythrocytes of T. merianae, and determine the baseline values of DNA damage in this species. We used 20 adult lizards (10 males: 10 females) from Estación Zoológica Experimental "Granja La Esmeralda" (Santa Fe, Argentina). Peripheral blood samples were collected from all animals and the MN test and CA applied according to the protocols established for other reptilian species. We test critical parameters of CA protocol (cell density, unwinding and electrophoresis times) using increasing concentrations of H2O2 (10, 25 and 50 µM) as a known genotoxic agent to induce DNA damage. Based on this, we determined the most suitable conditions for the CA in this species: a cell density of 4×10(3) erythrocytes per slide, 10 min of unwinding and 15 min of electrophoresis at 0.90 V/cm approximately. The baseline frequency of micronuclei (BFMN=MN/1000 erythrocytes counted) determined for this species was 0.95±0.27 and the basal damage index (BDI: calculated from 100 comet images classified in arbitrary units)=103.85±0.97. No differences were observed between sexes in the BFMN or BDI (p>0.05), and no relation was found between baseline values and length or weight of the analyzed animals (p>0.05). These results demonstrated the sensitivity of both biomarkers of genotoxicity to be applied in erythrocytes of this species, with baseline values comparable to those reported in other reptilian species. These results allow us to propose the tegu lizard for future in vivo studies to assess the genotoxicity of different agents, including those possibly affecting it in its natural geographic distribution.

Oxidative damage and antioxidant response of Allium cepa meristematic and elongation cells exposed to metronidazole.

The toxicity of metronidazole (MTZ) in meristematic and elongation zones of Allium cepa roots was analyzed for 30 h of exposition. Toxic effects were evaluated by lipid peroxidation (content of thiobarbituric-reactive substances [TBARS]), reduced glutathione (GSH) levels, ascorbate acid and dehydroascorbate acid content, and enzymatic activities of superoxide dismutase and catalase. The root zones showed differentiated susceptibility to MTZ. In the elongation zone, MTZ induced an increase of TBARS content and a significant rise in GSH levels, whereas in the meristematic zone, lipid peroxidation was not observed and all antioxidant defense parameters analyzed were significantly increased. These results indicate that MTZ exposure induced oxidative stress in A. cepa roots, and that the antioxidant defenses in the meristematic zone are more efficient compared with the elongation zone, which is probably related to higher oxidative metabolism of meristematic tissue.

Cytogenetic and microtubule array effects of the zineb-containing commercial fungicide formulation Azzurro(®) on meristematic root cells of Allium cepa L.

Zineb [ethylene bis(dithiocarbamate) zinc] is a widely employed foliar fungicide for agricultural and industrial applications. Allium cepa L. is a reliable model for the assessment of xenobiotic genotoxicity and cytotoxicity. We evaluated the effects of the zineb-containing commercial formulation Azzurro(®) (70% zineb) in cell cycle stages of the meristem root cells of A. cepa. The mitotic index (MI), chromosomal aberrations at anaphase/telophase (CAs), micronuclei (MN), and abnormalities in immunodetected microtubule structures, e.g., preprophasic band (PPB), mitotic spindle (MS), and phragmoplast (Phrag), were used as end-points. Azzurro(®) (1 and 10µg/ml) induced a significant increase in the frequency of CAs (P<0.05), and the higher concentration inhibited the MI (P<0.05) compared to control values. The frequency of MN did not differ from control values at any concentration. Treatment with 1µg/ml Azzurro(®) induced a significant increase in the frequency of abnormal PPB (P<0.01), MS (P<0.001), and Phrag (P<0.01) and, at 10µg/ml, enhancements in the frequencies of abnormal MS (P<0.05) and Phrag (P<0.05) were seen. A tubulin immunodetection assay showed that exposure to Azzurro(®) interferes with normal assembly of microtubule structures during mitosis.

Algunas certeza entre las tantas preguntas de os últimos diez años acerca dell genoma de los primaess / Some Certainties Among the Manyquestions of the Last 10 Years about the Primate Genome

En el año 2000 se presentó lo que se dio en llamar nuestro libro de la vida, el primer borrador del genoma humano. Aquello generó grandes expectativas por sus potenciales en beneficio de las ciencias biológicas. ¿Qué ha sucedido diez años después? Se conoce el número de genes que forman parte de nuestro genoma y se determinó la función de algunos de ellos. Se conocen las secuencias de tres genomas completos de mamíferos, Mus musculus, Pan troglodytes y Sus scrofa y genomas completos o borradores de otros numerosos eucariota (otros animales, plantas, hongos y protistas) y procariota (Archea y Bacterias), ver: http://ncbi.nlm.nih.gov/genomes. Sin embargo, el estudio del genoma no se limita a la mera descripción de las secuencias que lo componen. Las respuestas que se elaboren tendrán enfoques muy diversos, desde evolución y conservación de la biodiversidad hasta terapia génica y transformación maligna, donde el estudio de las particularidades individuales y poblacionales requiere fuentes de información tanto pasadas como actuales sobre estos genomas en estudio. Así, los avances en ciencia siempre son provisorios y por tanto, plausible de continuarse, completarse e incluso reinterpretarse ya que, conforme avanzamos en el conocimiento van surgiendo nuevos interrogantes.

In 2000 the first draft of the human genome, what became known as our book of life, was presented. It generated high expectations for its potential applications to the benefit of the biological sciences. What happened 10 years later? We know how many genes we have in our genome and analyzed the function of some of them. Nowadays, we know the sequences of 3 mammalians genomes: M. musculus, P. troglodytes y S. scrofa and the genomes or borradores from other eucaryotes (other animals, plants, fungi and protists) and procaryotes (Archea and Bacterias). However, the study of the genome is not merely a description of the sequences that compose it. The answers provided will have very different approaches from evolution and conservation of biodiversity to gene therapy and malignant transformation, where the study of individual and population particularities requires sources of information both past and present on these genomes under survey. Thus, advances in science are always provisional and therefore liable to be continued, completed and even reinterpreted as we advance in knowledge, new questions arise.

Analysis of the heterochromatin of Cebus (Primates, Platyrrhini) by micro-FISH and banding pattern comparisons.

The karyotype of the neotropical primate genus Cebus (Platyrrhini: Cebidae), considered the most ancestral one, shows the greatest amount of heterochromatin described among Platyrrhini genera. Banding techniques and restriction enzyme digestion have previously revealed great variability of quantity and composition of heterochromatin in this genus. In this context, we use fluorescence in situ hybridization (FISH) to analyse this genomic region and discuss its possible role in the diversification of Cebus.We used a heterochromatin probe for chromosome 11 of Cebus libidinosus (11qHe+ CLI probe), obtained by chromosome microdissection. Twenty-six specimens belonging to the families Atelidae, Cebidae, Callitrichidae and Pithecidae (Platyrrhini) were studied. Fourteen out of 26 specimens were Cebus (Cebidae) individuals of C. libidinosus, C. xanthosternos, C. apella, C. nigritus, C. albifrons, C. kaapori and C. olivaceus. In Cebus specimens, we found 6 to 22 positive signals located in interstitial and telomeric positions along the different species. No hybridization signal was observed among the remaining Ceboidea species, thus reinforcing the idea of a Cebus-specific heterochromatin composed of a complex system of repetitive sequences.

Genetic, enzymatic and developmental alterations observed in Caiman latirostris exposed in ovo to pesticide formulations and mixtures in an experiment simulating environmental exposure.

In South America, economic interests in last years have produced a constant increase in transgenic soybean cropping, with the corresponding rise in pesticide formulated products. The aim of this study was to determine the effects of pesticides formulations and mixtures on a South American caiman, Caiman latirostris, after in ovo exposure. We conducted a field-like experiment which simulates the environmental exposure that a caiman nest can receive in neighbouring croplands habitats. Experimental groups were Control group, Treatment 1: sprayed with a glyphosate herbicide formulation, and Treatment 2: sprayed with a pesticide mixture of glyphosate, endosulfan and cypermethrin formulations. Results demonstrated genotoxicity, enzymatic and metabolic alterations, as well as growth delay in caimans exposed in ovo to Treatments 1 and 2, showing a higher toxicity for the mixture. Integral evaluation through biomarkers of different biological meaning is highly informative as early indicators of contamination with pesticides and mixtures in this wildlife species.

Toxicity of waterborne Copper in premetamorphic tadpoles of Lithobates catesbeianus (Shaw, 1802).

Toxicity parameters of Copper to pre-metamorphic larvae of Lithobates catesbeianus were evaluated in laboratory conditions. The acute toxicity (as LC-50 96 h) was 3.96 mg Cu(2+) L(-1) (95% confidence interval: 3.21-4.89); the bioconcentration of the metal after 96 h exposure followed an exponential increase. The potential genotoxicity effect evaluated with Micronucleus Test showed a reduced sensitivity of the animals to the assayed concentrations of the metal, exhibiting only a modest increase in the frequency of erythrocytes micronuclei, meanwhile larvae exposed to cyclophosphamide (positive control) showed significant increases. The Condition Factor was significantly reduced while the Hepatosomatic Index remained unaltered.

Pairing and recombination features during meiosis in Cebus paraguayanus (Primates: Platyrrhini).

BACKGROUND: Among neotropical Primates, the Cai monkey Cebus paraguayanus (CPA) presents long, conserved chromosome syntenies with the human karyotype (HSA) as well as numerous C+ blocks in different chromosome pairs.In this study, immunofluorescence (IF) against two proteins of the Synaptonemal Complex (SC), namely REC8 and SYCP1, two recombination protein markers (RPA and MLH1), and one protein involved in the pachytene checkpoint machinery (BRCA1) was performed in CPA spermatocytes in order to analyze chromosome meiotic behavior in detail. RESULTS: Although in the vast majority of pachytene cells all autosomes were paired and synapsed, in a small number of nuclei the heterochromatic C-positive terminal region of bivalent 11 remained unpaired. The analysis of 75 CPA cells at pachytene revealed a mean of 43.22 MLH1 foci per nucleus and 1.07 MLH1 foci in each CPA bivalent 11, always positioned in the region homologous to HSA chromosome 21. CONCLUSION: Our results suggest that C blocks undergo delayed pairing and synapsis, although they do not interfere with the general progress of pairing and synapsis.

Meiotic characterization and sex determination system of neotropical primates: Bolivian squirrel monkey Saimiri boliviensis (primates: Cebidae).

The chromosomal sex determination system differs among platyrrhine monkeys more than any other group of primates. Although a number of studies have investigated mitotic chromosomes across platyrrhine species, the meiotic chromosomes of many genera have not yet been described. The goal of this study was to characterize the sex determination system of Saimiri boliviensis. We described for the first time the meiotic cycle, confirming the sexual system in germ cells from testicular biopsies of four adult male S. boliviensis. All specimens were weighed and testicular volume was measured. We observed 22 bivalents corresponding to 2N = 44, and a "human-like" XY bivalent was found in diakinesis/metaphase I. In addition, mitotic studies from blood samples of both sexes were performed and G- and C-banding patterns agreed with previously reported karylogy of S. boliviensis boliviensis. Further meiotic studies should be performed in New World primates based on the great value of those studies for systematic evolutionary biology and conservation programs.

Metronidazole-induced alterations in murine spermatozoa morphology.

The aim of this work was to assess the effect of metronidazole (MTZ) on the stages of the seminiferous epithelial cycle and spermatozoa morphology when the drug is administered in human therapeutic doses to 60-day-old CFW male mice. The frequency of the stages was established by counting spermatocytes in pachytene and spermatids. Abnormalities in the flagellum or the head, lack of maturity and multiple malformations, were considered in the morphological analysis. Murine control strain was compared with MTZ treated group (v.ip 130 mg/kg/bw) both kept in standard captivity conditions. Cellular composition or number of stages in the seminiferous tubules were not altered in MTZ exposed animals, though the number of cells in stages I, V and XII was increased. The sperm cell morphology was severely affected by the treatment with potentially serious consequences on the normal fertilization process. Thus, the MTZ has to be considered as a conceivable thread regarding male fertility.

The bone marrow micronucleus test and metronidazole genotoxicity in different strains of mice (Mus musculus)

The mouse (Mus musculus) bone marrow micronucleus test was carried out using 24 outbred National Institutes of Health (NIH) mice, 24 inbred Swiss Webster (CFW) mice and 20 inbred Bagg albino/color locus Jackson (BALB/cJ) mice. The mice in the experimental group (n = 32) were injected intraperitoneally with 133 mg kg-1 of metronidazole parenteral solution and the control group consisted of mice (n = 36) which had not been injected with metronidazole. There was no significant difference (p > 0.05) between the sexes regarding the micronucleus frequency in either the experimental or the control group. When the Mn frequencies of the three strains were compared, the results for the CFW and BALB/cJ strains did not differ statistically (p > 0.05) for either the experimental or control groups but there were significant (p < 0.05) differences between the CFW and NIH strains and the NIH and BALB/cJ strains for the experimental and control groups, with the NIH strain always showing the highest micronucleus frequency. Our results also show that metronidazole was possible genotoxic agent because it produced a significant increase (p < 0.05) in the micronucleus frequency of the experimental group as compared to the control group for all the three mouse strains tested.

Exposure of bovine oocytes to the endogenous metabolite 2-methoxyestradiol during in vitro maturation inhibits early embryonic development.

Catecholestrogens are endogenous metabolites that have been shown to modulate granulosa, theca, and luteal cell function in some species. The present study was aimed at determining the possible role of these steroids on oocyte maturation. Cumulus-enclosed bovine oocytes were matured for 24 h, fertilized, and then cultured for 8 days. Whereas estradiol was without effect, addition of catecholestrogens (2-hydroxyestradiol, 4-hydroxyestradiol, and 2-methoxyestradiol [2-MOE2]) to the maturation medium did not affect the cleavage rate but was associated with a decrease in blastocyst production on Day 8. Although 2-MOE2 was also able to inhibit blastocyst formation when added during embryo culture, the effects were less pronounced than those seen when the steroid was added only during maturation. In agreement with the known ability of 2-MOE2 to bind tubulin at the colchicine site, marked alterations were observed in the spindle assembly of oocytes exposed to 2-MOE2 during maturation, which lead to gross chromosomal aberrations after fertilization and consequent developmental arrest at the morula stage. Moreover, that the blastocyst rate was not affected when meiosis was blocked with roscovitine during 2-MOE2 exposure is consistent with the idea that altered nuclear maturation is the cause of the low developmental competence. Because 2-MOE2 could be increased in follicular fluid in response to aryl hydrocarbon-receptor ligands, such as some environmental contaminants, our results show that abnormally high intraovarian levels of catecholestrogens could have a deleterious effect on oocyte maturation and early embryonic development arising from the alterations in the meiotic spindle.

Description of the cytochrome c oxidase subunit II gene in some genera of New World monkeys (primates, Platyrrhini).

Nucleotide sequence variation at the mitochondrial cytochrome c oxidase subunit II gene (COII) was analyzed in 27 New World monkey specimens, nine newly reported herein. The study involved comparisons among platyrrhines and also between platyrrhines and catarrhines. The analysis of the frequencies of transitions and transversions at each codon position showed transitional saturation at third codon position. Neighbor-Joining trees obtained from genetic distances estimated by means of Kimura's (1980) two-parameter model showed poor resolution of phylogenetic relationships among platyrrhine genera. Rates of nucleotide substitutions were largely homogeneous except in the genus Saimiri that showed low numbers of unique substitutions suggesting the maintenance of ancestral or plesiomorphic states of platyrrhine mt DNA nucleotide characters probably due to its large population sizes as compared to other platyrrhines.