Expressão gênica e viabilidade de folículos ovarianos pré-antrais de Sapajus apella congelados e cultivados in vitro

The aim of the present study was to develop a freezing protocol for the preservation of preantral follicles from Sapajus apella (capuchin monkeys). To this end, ovarian fragments were exposed to different cryoprotectant solutions, added or not by antioxidants (selenium or trolox), frozen and in vitro cultived by 24 hours. Morphology, ultrastructure, viability, oxidative stress and mocelular analyses were performed. The collection site was in the Primates Nacional Center (CENP) and nine healthy mature female capuchin monkeys were used. Ovarian biopsies of 1 mm3 were collected by laparoscopy. The follicles were classified accordingly to their developmental phase in primordial, primary or secondary. Follicular viability scored using fluorescent markers (propidium iodide and Hoechst). qRT-PCR was used to evaluate the expression of hormones and growth factors. TEAC was used to measure oxidative stress in the tissue. In cryoprotectant solution containing trolox did not affected follicular morphology and gene expression. Cryopreservation resulted in higher rates of follicular viability when trolox was present in the solution, but expression of genes encoding BMP4 and KL was negatively affected. Our findings show a favorable effect of adding trolox to a cryopreservation solution. However, follicular viability and gene expression was affected after in vitro culture.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorO objetivo do presente estudo foi desenvolver um protocolo de congelação para a preservação de folículos pré-antrais de Sapajus apella (macaco-prego). Para este fim, fragmentos ovarianos foram expostos à diferentes soluções crioprotetoras, adicionadas ou não por antioxidantes (selênio e trolox), congelados e cultivados in vitro por 24/horas. Análises morfológicas, ultraestruturais, de viabilidade e estresse oxidativo foram desenvolvidas. A coleta do material foi realizada no Centro Nacional de primatas (CENP) e nove macacos-prego maduras e saudáveis foram usadas. Biopsias ovarianas de 1 mm³ foram coletadas por laparoscopia exploratória. Os folículos coletados foram classificados de acordo com sua fase de desenvolvimento em primordial, primário ou secundário. A viabilidade folicular foi observada através da utilização de marcadores fluorescentes (Iodeto de propídeo e Hoechst) qRT-PCR foi usado para avaliar a expressão de hormônios e fatores de crescimento. O TEAC foi usado para mensurar o estresse oxidativo no tecido. Os resultados mostraram que a solução congelação contendo trolox não afetou a morfologia folicular e expressão gênica. A criopreservação resultou em elevadas taxas de viabilidade folicular quando o trolox estava presente na solução, porém a expressão de genes codificando BMP4 e KL foi negativamente afetada. Nossos achados mostraram um efeito favorável da adição do trolox à solução de congelação. Entretanto, a viabilidade folicular e expressão gênica foram afetados após cultivo in vitro

Vitrificação de tecido ovariano de gata doméstica (Felis catus): um modelo para a preservação da fertilidade em felinos silvestres

The aim o the present thesis was to develop an efficient vitrification protocol of the ovarian tissue from domestic cat (Felis catus). This study was divided: Phase I: Effect of different basis media during the vitrification of cat ovarian tissue; Phase II: Effect of different sugars (extracellular cryoprotectants) and the vitrification technique for the vitrification of feline ovarian tissue. In phase I, the morphology of preantral follicles was similar (p > 0.05) to fresh control when RPMI-1640 was used as basis medium for vitrification. RPMI-1640 does not contain phenol red, which was found to enhance ethylene glycol (EG) toxicity during vitrification. In phase 2, the percentage of morphologically normal preantral follicles was similar (p > 0.05) to fresh control only when the vitrification medium contained 0.1 or 0.5 M trehalose, instead of sucrose or raffinose at same concentrations. Furthermore, based on parameters such as morphology, cell proliferation and thickness of collagen fibers, it is possibe to assume that efficient vitrification of feline ovarian tissue can be performed by combining trehalose with EG, with or without dimethylsulfoxide (DMSO), applying the solid-surface vitrification (SSV) or ovarian tissue cryosystem (OTC) method. Although vitrification with OTC in the presence of EG did not differ from the other treatments, this protocol presented the highest percentages of preserved preantral follicles (56%), being similar to control (64%). Additionally, no effect on gene regulation was observed after vitrification when apoptosis markers (BAX – protein X associated to Bcl-2), endoplasmic reticulum (ER) stress (ER protein 29 – ERP29), water channels proteins like aquaporins 3 and 9 (AQP3 and AQP9), the membrane ABC transporters ABCB1 and ABCG2, except when the SSV method was applied using only EG as cryoprotectant followed by seven days in vitro culture, where ERP29 up-regulation (ER stress) and AQP9 down-regulation (impaired water transport) were observed. Based on this, it can be concluded that to efficiently preserve feline ovarian tissue, is is necessary the use of a vitrification protocol free of phenol red, supplemented with trehalose, as extracellular cryoprotectant, and EG alone or in combination with DMSO, as intracellular cryoprotectants. Both open (SSV) and closed (OTC) systems are equaly efficient to maintain follicular survival during the vitrification procedure.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorEsta tese tem como principal objetivo desenvolver um protocolo eficiente de vitrificação de tecido ovariano de gata doméstica (Felis catus). O estudo foi dividido em: Fase I: Efeito de diferentes tipos de meios-base durante a vitrificação de tecido ovariano de gata; Fase II: Efeito de diferentes crioprotetores extracelulares e técnicas de vitrificação em tecido ovariano de gata; Na fase I, a morfologia de folículos pré-antrais foi similar ao controle fresco (p > 0,05), quando o RPMI-1640 foi utilizado como meio-base. RPMI-1640 não contém vermelho fenol que, adicionado ao meio, intensificou a toxicidade do crioprotetor etileno glicol durante a vitrificação. Na fase II, a percentagem de folículos morfologicamente normais foi similar ao controle, apenas quando o meio de vitrificação foi suplementado com 0,1 M ou 0,5 M de trealose (p > 0,05). Além disso, através dos parâmetros como a morfologia, proliferação celular e espessura de fibras colágenas pode-se dizer que a combinação de trealose com etilenoglicol (EG) sozinho ou adicionado de dimetilsufóxido (DMSO), aplicando os métodos Solid-surface vitrification (SSV) ou Ovarian Tissue cryosystem (OTC), apresentaram sucesso na preservação do tecido ovariano vitrificado. Apesar do OTC com EG não apresentar diferença significativa dos demais tratamentos, uma vez que este protocolo apresentou o maior percentual de folículos morfologicamente normais (56%), sendo similar ao controle (64%). Adicionalmente, nenhum efeito sobre a regulação de expressão gênica foi observada nos grupos testados, quando foram avaliados marcadores de apoptose (BAX - proteína X associada ao Bcl-2), de estresse do retículo endoplasmático (ERP29 – proteína do retículo endoplasmático 29), de canais de água como as aquaporinas 3 e 9 (AQP3 e AQP9), e os transportadores de membrana ABC (ABCB1 e ABCG2), com exceção do método SSV com EG que apresentaram, após 7 dias de cultivo in vitro, um aumento da expressão da ERP29 (indica estresse no reticulo endoplasmático) e a diminuição da expressão da AQP9 (afeta canais de transporte de agua). Com isso, para a manutenção da preservação do tecido ovariano de gata é necessário o uso de um protocolo de vitrificação contendo meio-base livre de vermelho fenol, suplementado com trealose, como crioprotetor extracelular, e EG sozinho ou associado com DMSO, como crioprotetores intracelulares. Ambos os sistemas abertos (SSV) e fechados (OTC) são equivalentes na eficiencia em manter a sobrevivência folicular durante o processo de vitrificacao

The Lewis histo-blood group system: molecular analysis of the 59T>G, 508G>A, and 1067T>A polymorphisms in an Amazonian population.

BACKGROUND: The Lewis (FUT3) gene is responsible for the expression of the Le(a) and Le(b) blood group antigens. The individuals, who not synthesize these antigens have the phenotype Lewis negative, due to the presence of some single nucleotide polymorphisms (SNPs), such as 59T>G, 508G>A and 1067T>A, whose distribution is different in various ethnic groups. Our aim was to verify the frequencies of these SNPs in an admixed population of Belém-Pará-Brazil. MATERIALS AND METHODS: Polymerase chain reaction/restriction enzyme method were used to detect these SNPs in the FUT3 gene, whereas Lewis phenotypes were defined by the direct hemagglutination and in saliva by Dot-Elisa assay in a random sample of 150 individuals from admixed population of Belém in the northeast Brazilian Amazon region. RESULTS: The frequency of these SNPs was detected as 47.6% (59T>G), 17.3% (508G>A) and 5.3% (1067T>A).The discrepancies between blood and salivary Lewis phenotypes are related to the relatively high frequencies of 59T>G and the null allele 508G>A. Whereas 38.6% of the individuals were Lewis negative based on blood, only 17.24% also tested negative when their saliva were analyzed. CONCLUSION: We have found a marked consistency between the phenotypes and genotypes of the Lewis blood group system. Furthermore, our obtained FST values reveal distinct frequencies of the FUT3 SNPs between the present sample and its representative ancestral populations. These observations will help to evaluate the Lewis antigens impact as susceptibility markers, in genetic association studies to certain diseases

The F_ST values for pairwise comparisons of haplotype frequencies of the <i>FUT3</i> gene between the present study and other populations.

*<p>Soejima <i>et al</i>.; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069908#pone.0069908-Soejima1" target="_blank">[11]</a>.</p

Allele and genotype frequencies for the <i>FUT3</i> loci analyzed in the sample from the population of Belém in the northeast Brazilian Amazon region.

<p>Allele and genotype frequencies for the <i>FUT3</i> loci analyzed in the sample from the population of Belém in the northeast Brazilian Amazon region.</p

Lewis and ABO blood group phenotypes and <i>FUT3</i> genotypes recorded for the individuals from Belém in the northeast Brazilian Amazon region.

<p>le*: 59/508 and/or 59/1067.</p

Genotype frequencies for the Lewis (<i>FUT3</i>) gene and non-O blood groups in the positive and non-genuine Lewis-negative individuals.

<p>Genotype frequencies for the Lewis (<i>FUT3</i>) gene and non-O blood groups in the positive and non-genuine Lewis-negative individuals.</p

Amplification conditions for the <i>FUT3</i> gene and its restriction sites for the different endonucleases analyzed in the present study.

*<p>Kudo<i>et al.,</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069908#pone.0069908-Kudo1" target="_blank">[21]</a>;</p>**<p>Francez <i>et al.</i>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069908#pone.0069908-Francez1" target="_blank">[22]</a>.</p

Equol: A Microbiota Metabolite Able to Alleviate the Negative Effects of Zearalenone during In Vitro Culture of Ovine Preantral Follicles

The impact of zearalenone (ZEN) on female reproduction remains an issue, since its effects may differ among exposed cell types. Besides the use of decontaminants in animal diet, other approaches should be considered to minimise ZEN effects after exposure. Since the first organ in contact with ZEN is the gastrointestinal tract, we hypothesise that products of microbiota metabolism may play a role in ZEN detoxification. We aimed to evaluate the effect of 1 &micro;mol/L ZEN and 1 &micro;mol/L equol (a microbial metabolite), alone or in combination, on the survival and morphology of in vitro cultured ovarian preantral follicles. Ovaries from 12 sheep were collected at a local abattoir and fragmented, and the ovarian pieces were submitted to in vitro culture for three days in the presence or absence of the test compounds. The follicular morphology was impaired by ZEN, but equol could alleviate the observed degeneration rates. While ZEN decreased cell proliferation in primary and secondary follicles, as well as induced DNA double-strand breaks in primordial follicles, all these observations disappeared when equol was added to a culture medium containing ZEN. In the present culture conditions, equol was able to counteract the negative effects of ZEN on ovarian preantral follicles