ALTERAÇÕES HISTOPATOLÓGICAS TESTICULARES ASSOCIADAS A INFECÇÃO EXPERIMENTAL POR VÍRUS ZIKA EM MACACOS-DE-CHEIRO

The Zika virus belongs to the family Flaviviridae, genus Flavivirus. It is an arbovirus whose main vectors are mosquitoes of the genus Aedes. Studies in rodents have shown that the persistence of the virus in the testicles causes damage to the reproductive tissue. This work aimed to study the effect of experimental infection by the Zika virus on fertility in non-human primates of the species Saimiri collinsi. Five pre-pubertal males (≤2 years old) of the species Saimiri collinsi were used. Three animals were infected (infected group) with the strain of Zika virus BE H815744. Two other uninfected males were used as a negative control (uninfected group). Twenty-one days after infection, infected and uninfected males were euthanized. After euthanasia, they were referred for necroscopic examination for macroscopic and microscopic evaluation. During the necropsy, the testicles were collected and fixed in 10% formaldehyde. After fixation, the tissues were processed routinely and embedded in paraffin. The slides were stained with hematoxylin and eosin for histopathological evaluation. Histopathological changes were observed in the testis of three of the five animals. Different degrees of inflammation were identified, in addition to degeneration and/or necrosis. The three animals presented a reduced number of sperm cells, with no sperm and severe necrosis. The results obtained conclude that the Zika virus can cause pathological changes in the reproductive system of males of the species Saimiri collinsi.O vírus Zika pertence à família Flaviviridae, gênero Flavivirus. É uma arbovirose cujos principais vetores são mosquitos do gênero Aedes. Estudos em roedores mostraram que a persistência do vírus nos testículos causa danos ao tecido reprodutivo. Este trabalho teve como objetivo estudar o efeito da infecção experimental pelo vírus Zika na fertilidade em primatas não humanos da espécie Saimiri collinsi. Foram utilizados cinco machos pré-púberes (≤2 anos) da espécie Saimiri collinsi. Três animais foram infectados (grupo infectado) com a cepa do vírus Zika BE H815744. Dois outros machos não infectados foram usados como controle negativo (grupo não infectado). Vinte e um dias após a infecção, machos infectados e não infectados foram sacrificados. Após a eutanásia, foram encaminhados para exame necroscópico para avaliação macroscópica e microscópica. Durante a necropsia, os testículos foram coletados e fixados em formaldeído a 10%. Após a fixação, os tecidos foram processados rotineiramente e incluídos em parafina. As lâminas foram coradas com hematoxilina e eosina para avaliação histopatológica. Alterações histopatológicas foram observadas no testículo de três dos cinco animais. Diferentes graus de inflamação foram identificados, além de degeneração e/ou necrose. Os três animais apresentavam número reduzido de espermatozoides, ausência de espermatozoides e necrose severa. Os resultados obtidos concluem que o vírus Zika pode causar alterações patológicas no sistema reprodutor de machos da espécie Saimiri collinsi

Análise imunohistológica e desenvolvimento folicular após auto-transplante de tecido ovariano de macaca de cheiro Saimiri collinsi

The main objective of the present work was to study ovarian folliculogenesis in Saimiri sciureus. Therefore, we evaluated the expression of intra-ovarian factors related with the follicular development, inhibition and cell proliferation, as well as investigation the effects of pretreatment with vitamin E analogue, Trolox, on the viability and follicular function, apoptosis index, cell proliferation, vascularization and fibrosis after auto-transplanted of fresh ovarian tissue. It was possible for the first time imuno localize proteins expressed as: growth and differentiation factor -9 (GDF -9), c- kit/Kit Ligand and Ki-67 and confirm the expression of anti-Mullerian Hormone (AMH) in different stages of follicular development in squirrel monkey ovary. Regarding pre-incubation with the Trolox antioxidant, it can improves follicular survival after autotransplantation, decrease apoptosis rates in stromal cells, although increase areas of fibrosis after graft. Our results suggest that fresh ovarian tissue can be grafted and incubated without major impact on early follicular growth and morphology after subcutaneous autotransplantation for a short-term. We believe that our results provide an important contribution to understanding the process of folliculogenesis in neotropical monkeys. This knowledge is an important tool to evaluate follicular viability and functionality after cryopreservation of ovarian tissue transplantation and in vitro culture of early follicles, especially for animals at risk of extinction and women that are undergoing to chemotherapy and radiation with risk of loss fertility.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorO objetivo deste trabalho foi estudar a foliculogênese ovariana de Saimiri sciureus. Para tanto, foi avaliada a expressão de fatores intra-ovarianos ligados ao desenvolvimento, inibição folicular e proliferação celular, além da investigação dos efeitos do pré-tratamento com o análogo da vitamina E, Trolox®, sobre a viabilidade e funcionalidade folicular, índice de apoptose, proliferação celular, vascularização e fibrose, após o auto-transplante do tecido ovariano fresco. Foi possível imunolocalizar pela primeira vez a expressão das proteínas: fator crescimento e diferenciação-9 (GDF-9), c-Kit/Kit Ligante e Ki-67 e confirmar a expressão do hormônio anti-mülleriano (AMH) em diferentes fases do desenvolvimento folicular de macaco de cheiro. No que diz respeito a pré-incubação com o antioxidante Trolox®, pôde-se observar uma melhora na sobrevivência folicular após o autotransplante, diminuição da taxa de apoptose em células do estroma, porém, aumento nas áreas de fibrose no tecido. Os nossos resultados sugerem que o tecido ovariano fresco pode ser incubado e enxertado sem grande impacto sobre o crescimento folicular precoce e a morfologia após auto-transplante subcutâneo por curto período. Acreditamos que nossos resultados oferecem uma contribuição importante para a compreensão do processo de foliculogênese em macacos neotropicais. Este conhecimento é uma ferramenta importante para avaliar a viabilidade folicular e funcionalidade após a criopreservação, transplante de tecido ovariano e cultivo in vitro de folículos pré-antrais, em especial para os animais com risco de extinção e às mulheres com risco de perda de fertilidade devido à quimioterapia e radioterapia

Theoretical study of quinolines derivatives’ redox mechanism in the antimalarial activity

Malaria is a serious public health problem worldwide, causing socioeconomic deficits and contributing to subdevelopment in affected countries. In this context is important to study electronic properties, quinoline derivatives antioxidant potential and antimalarial activity relationship to design effective antimalariais prototypes. In this dissertation are used molecular modeling methods to study antioxidant and antimalarial structure-activity relationships selecting moieties and eletronic and conformacional parameters to improve farmacological activity and decrease derivatives toxicity. The HOMO and PI values analysis indicates that imino-tautomer is, probably, better antioxidant than amino-tautomer. It also observed that tautomers equilibrium is favored to amino-quinoline in the gas phase, and in water and chloroform using PCM method, with energy barriers values to 10.78 Kcal/mol, 21.65 Kcal/mol and 22.04 Kcal/mol, respectively. Then, may be noted that in quinoline analogues derivatives the electron-donor groups decrease the ionization potential, as exemple of the amino group at 8-position replaced by an alkylamine group. In 4- 8- amino-quinoline derivatives association observed that presence of quinoline moiety second nitrogen decrease its antioxidant activity, except in the 5-position, representing the most prominent group in the reduction of ionization potential and probably high antioxidant activity.CNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoA malária é um sério problema de saúde pública mundial, acarretando perdas socioeconômicas e contribuindo para o subdesenvolvimento dos países afetados. Neste contexto, faz-se necessário estudar a relação entre as propriedades eletrônicas e a capacidade antioxidante de derivados quinolínicos na atividade antimalárica, o que servirá de subsídio para propor protótipos eficazes na terapêutica da doença. Nesta dissertação, foram utilizadas técnicas de modelagem molecular, no estudo da relação estrutura e atividade antioxidante correlacionada com a atividade antimalárica, no processo de seleção de grupamentos e parâmetros eletrônicos e conformacionais que permitam aperfeiçoar a atividade farmacológica e reduzir a toxicidade dos derivados. A análise dos valores de HOMO e PI indicou que o tautômero imino-quinolina é, provavelmente, melhor antioxidante que o tautômero amino-quinolina. Também se observou que o equilíbrio dos tautômeros é mais deslocalizado para a estrutura amino-quinolina na fase gasosa, e em água e clorofórmio no método PCM, apresentando valores de barreiras de energia da faixa de 10,78 Kcal/mol, 21,65 Kcal/mol e 22,04 Kcal/mol, respectivamente. Assim pôdese observar que nos derivados análogos de quinolina, os grupos elétrons-doadores mostraram destaque na redução do potencial de ionização, como os grupos amina na posição 8 substituído por um grupo alquilamina. Nos derivados da associação de 4- e 8-amino-quinolina notou-se que a presença de um segundo nitrogênio no grupo quinolina diminui seu potencial antioxidante, com exceção da posição 5, representando o grupo de maior destaque na redução do potencial de ionização e conseqüente provável elevada atividade antioxidante

Immunolocalization of Growth, Inhibitory, and Proliferative Factors Involved in Initial Ovarian Folliculogenesis From Adult Common Squirrel Monkey (Saimiri collinsi)

We performed an immunohistochemical (IHC) study to determine the follicular expression of growth differentiation factor 9 (GDF-9), anti-Müllerian hormone (AMH), Kit Ligand (KL), and c-Kit in squirrel monkey ovary. Ovarian tissue fragments from 4 squirrel monkeys were collected by laparotomy and processed for classical histology and IHC. Additionally, follicle development was assessed by Ki67 immunostaining to evaluate proliferative status of granulosa cells. A total of 4025 follicles were examined (1475 for classical histology and 2550 for immunohistochemistry). More than 80% of the evaluated follicles were morphologically normal. The GDF-9 protein was detectable in oocyte cytoplasm from primordial (100%), primary (99.1%), and secondary (100%) follicles. The AMH was not expressed in primordial follicles but just in few primary follicles (13.8%). On the other hand, it was highly expressed in granulosa cells from secondary follicles (67.9%). c-Kit, KL receptor, was found in the oolemma of primordial (100%), primary (100%), and secondary (100%) follicles. The KL expression was observed in oocytes and granulosa cells from primordial (94.9%), primary (91.6%) and secondary follicles (100%). Ki67 immunostaining was observed in granulosa cells from primary (5.7%) and secondary (54.8%) follicles but not in primordial follicles. In conclusion, we described the localization of GDF-9, KL, c-Kit, and Ki67 proteins and confirmed the presence of AMH protein in preantral follicles from squirrel monkey. Our results offer contribution for understanding of folliculogenesis in neotropical nonhuman primates. Moreover, these markers can be used to assess follicular viability and functionality after cryopreservation, transplantation, or in vitro culture of ovarian tissue

First step in developing a 3D biodegradable fibrin scaffold for an artificial ovary

BACKGROUND: Although transplantation of cryopreserved ovarian tissue is a promising approach to restore fertility in cancer patients, it is not advisable for women at risk of ovarian involvement due to the threat of reintroducing malignant cells. The aim of this study was therefore to find an alternative for these patients by development of an artificial ovary. METHODS: For construction of the artificial ovary matrix, we used a central composite design to investigate nine combinations of fibrinogen (mg/ml) and thrombin (IU/mL) (F/T): F1/T4, F12.5/T1, F12.5/T20, F25/T0.1, F25/T4, F25/T500, F50/T1, F50/T20 and F100/T4. From the first qualitative analyses (handling and matrix size), five combinations (F12.5/T1, F25/T4, F50/T20, F50/T1 and F100/T4) yielded positive results. They were further evaluated in order to assess fibrin matrix degradation and homogeneous cell encapsulation (density), survival and proliferation (Ki67), and atresia (TUNEL) before and after 7 days of in vitro culture. To determine the best compromise between maximizing the dynamic density (Y1) and minimizing the apoptosis rate (Y2), we used the desirability function approach. RESULTS: Two combinations (F12.5/T1 and F25/T4) showed greater distribution of cells before in vitro culture, reproducible degradation of the fibrin network and adequate support for isolated human ovarian stromal cells, with a high proportion of Ki67-positive cells. SEM analysis revealed a network of fibers with regular pores and healthy stromal cells after in vitro culture with both F/T combinations. CONCLUSION: This study reports two optimal F/T combinations that allow survival and proliferation of isolated human ovarian cells. Further studies are required to determine if such a scaffold will also be a suitable environment for isolated ovarian follicles

Immunolocalization of growth, inhibitory, and proliferative factors involved in initial ovarian folliculogenesis from adult common squirrel monkey (Saimiri collinsi)

CapesFederal University of Pará. Laboratory of Wild Animal Biology and Medicine. Castanhal, PA, Brazil / Federal University of Pará. Animal Sciences PhD Program. Belém, PA, Brazil / Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Centro Nacional de Primatas. Ananindeua, PA, Brasil.Federal University of Pará. Laboratory of Wild Animal Biology and Medicine. Castanhal, PA, Brazil / Federal University of Pará. Animal Sciences PhD Program. Belém, PA, Brazil.Federal University of Pará. Laboratory of Wild Animal Biology and Medicine. Castanhal, PA, Brazil / Federal University of Pará. Animal Sciences PhD Program. Belém, PA, Brazil.Federal University of Pará. Laboratory of Wild Animal Biology and Medicine. Castanhal, PA, Brazil / Federal University of Pará. Animal Sciences PhD Program. Belém, PA, Brazil / Utrecht University. Faculty of Veterinary Medicine. Utrecht, the Netherlands.Université´ Catholique de Louvain. Institut de Recherche Expérimentale et Clinique. Pôle de Recherche en Gynécologie. Brussels, Belgium.We performed an immunohistochemical (IHC) study to determine the follicular expression of growth differentiation factor 9 (GDF-9), anti-Mu¨llerian hormone (AMH), Kit Ligand (KL), and c-Kit in squirrel monkey ovary. Ovarian tissue fragments from 4 squirrel monkeys were collected by laparotomy and processed for classical histology and IHC. Additionally, follicle development was assessed by Ki67 immunostaining to evaluate proliferative status of granulosa cells. A total of 4025 follicles were examined (1475 for classical histology and 2550 for immunohistochemistry). More than 80% of the evaluated follicles were morphologically normal. The GDF-9 protein was detectable in oocyte cytoplasm from primordial (100%), primary (99.1%), and secondary (100%) follicles. The AMH was not expressed in primordial follicles but just in few primary follicles (13.8%). On the other hand, it was highly expressed in granulosa cells from secondary follicles (67.9%). c-Kit, KL receptor, was found in the oolemma of primordial (100%), primary (100%), and secondary (100%) follicles. The KL expression was observed in oocytes and granulosa cells from primordial (94.9%), primary (91.6%) and secondary follicles (100%). Ki67 immunostaining was observed in granulosa cells from primary (5.7%) and secondary (54.8%) follicles but not in primordial follicles. In conclusion, we described the localization of GDF-9, KL, c-Kit, and Ki67 proteins and confirmed the presence of AMH protein in preantral follicles from squirrel monkey. Our results offer contribution for understanding of folliculogenesis in neotropical nonhuman primates. Moreover, these markers can be used to assess follicular viability and functionality after cryopreservation, transplantation, or in vitro culture of ovarian tissue

Evaluation of cryopreserved ovarian tissue from prepubertal patients after long-term xenografting and exogenous stimulation.

This is the first description of transplantation of human cryopreserved prepubertal ovarian tissue to mice, demonstrating that a very high number of follicles survive after transplantation and a large pool of primordial follicles remains dormant. Growing follicles were observed, proving the responsiveness of prepubertal ovarian tissue to gonadotropins

Ovarian Transplantation: Highlight to Non-Human Primates Reproduction

Background: Ovarian transplantation in nonhuman primates (NHP) has been used as a strategy for the development of experimental models for biomedical research in the reproductive area. The prospects for application of this technique range from the restoration of female fertility to the conservation of endangered wild animals. However, studies with NHP were performed mostly focusing on the ovarian transplantation of cryopreserved tissue, in order to simulate the reality of human species, as well as aiming to obtain experimental models suitable for comparative studies. On the other hand, ovarian transplantation could be applied also in NHP species preservation. Review: According to the last census (2012-2014) of the International Union for Conservation of Nature (IUCN), currently, more than half of the 633 types of primates known around the world are in danger of disappearing forever due to the continued destruction of their natural habitat by human activities. Thus, there is an interest to expose the possible methods of ovarian preservation followed by transplantation that can be employed to promote the conservation of endangered NHP. Despite the positive results obtained with avascular autograft of fresh ovarian tissue in NHP, it is important to bring in mind the significant loss of follicles as a result of this procedure due to ischemia and reperfusion during the first days after grafting. This phenomenon leads to the production of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide anion and hydroxyl radical, which are responsible by lipid peroxidation, cell membrane damage and subsequent follicular atresia. An alternative to counteract this oxidative stress consists in the application/administration of different sources of antioxidants previously or during grafting, in the grafted tissue or in the animal receiving the transplant. The most used compounds with a claimed radical scavenger activity are catalase, trolox and some vitamins. In some cases, antioxidants are combined with angiogenic factors, e.g. vascular endothelial growth factor (VEGF) to stimulate revascularization. Another option to avoid the damage caused by ischemia is the whole ovarian transplantation with vascular anastomosis. Therefore, a greater proportion of the pool of follicles survives the procedure and the complete ovary is grafted and not only tissue fragments. However, the vascular microsurgical procedure is complex and, when it is associated with cryopreservation the risks of follicular loss and tissue damage due to improper perfusion are larger. In this context, the aim of this review is to cover important concepts about ovarian transplantation and its current situation in NHP, showing its importance and its advances to reproduction, especially to preserve those endangered ones. Conclusion: Despite advances in tissue response to various applications of the ovarian tissue transplantation reported in this review, it is clear that this technique has been used with paucity as a tool for the conservation of species of PNH endangered. The need for optimization of transplantation techniques, so as to reduce the loss of the follicles during the grafting processes and revascularization, determining the ideal location for the graft, the graft longevity and where in the amount of ovarian tissue required to preserve fertility still are points to be clarified. In this sense, research is ongoing and the perspectives are positive not only to use NHP as a model for human, but also for those endangered species

Antimicrobial sensitivity profile by components of bacterial oral and retal microbiota of non-human primates

Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Centro Nacional de Primatas. Ananindeua, PA, Brasil.Universidade Federal do Pará. Belém, PA, Brasil.Universidade Federal Rural da Amazônia. Belém, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Centro Nacional de Primatas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Centro Nacional de Primatas. Ananindeua, PA, Brasil.Universidade Federal do Pará. Belém, PA, Brasil.Os objetivos desta pesquisa foram identificar bactérias isoladas da cavidade oral e da ampola retal de Saimiri collinsi e Callithrix jacchus e determinar a sensibilidade a 16 antimicrobianos. Trinta indivíduos de cada espécie foram analisados e foram isoladas 136 bactérias em C. jacchus e 84 em S. collinsi. As bactérias isoladas em maior número em S. collinsi foram Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Raoutella ornithinolytica, Staphylococcus xylosus e Proteus mirabilis. As bactérias isoladas em C. jacchus foram K. pneumoniae, E. cloacae, E. coli, Serratia marcescens e S. xylosus na cavidade oral e ampola retal. O teste de sensibilidade mostrou que, dentre as cepas isoladas, os maiores percentuais de resistência foram observados para ampicilina, amoxicilina, cefalotina e nitrofurantoína. Na cavidade oral de ambas as espécies as cepas foram sensíveis à ceftazidima, ceftriaxona, meropenem, amicacina, levofloxacina e a sulfametoxazol/ trimetoprim. Na ampola retal, as isoladas foram sensíveis à cefoxitina, ceftazidima, ceftriaxona, ertapenem, meropenem, amicacina e levofloxacina. Conclui-se que as espécies de S. collinsi e C. jacchus apresentam sua microbiota oral e retal composta por várias espécies bacterianas e que a resistência pode ser um problema no criatório, uma vez que as cepas mostraram percentuais elevados de resistência a diferentes antimicrobianos

Ultrassonografia Doppler ovariana e uterina em cadelas

This paper aims to describe the uterine and ovarian ultrasonographic characteristics and Doppler velocimetric features of their arteries in bitches during the periovulatory period. Fifteen estrous cycles in 10 animals were evaluated. The ultrasonographic characteristics, resistance indices (RI) and pulsatility indices (PI) of the uterus and ovaries in each animal were recorded 5 days before and after ovulation (D0). The data were statistically analyzed, and the results were expressed as the mean ± standard error of mean (P<0.05). In results the ultrasonographic features of the uterus were the same on all of the cycles and evaluated days. The uterus had an average diameter of 0.85±0.02cm. An increase in the volume of the ovaries and the diameter of the ovarian follicles were measured. Ovaries had a volume of 0.64±0.06cm3, and the follicles cavities had a diameter of 0.46 ± 0.01 cm on the day of ovulation. After ovulation, it was observed that some follicles not collapse in some cycles. Two days prior to ovulation, the uterine blood perfusion decreased. This decrease remained unchanged until ovulation. Following ovulation, we measured a gradual increase in the uterine perfusion and in the ovarian artery. This artery directed blood flow to the ovaries and increased the intra-ovarian perfusion on the day after ovulation. In conclusion, specific features are observed in the uterus and ovarian ultrasound image and Doppler values of their arteries presented on the periovulatory days and when associated allow to estimate more accurately the date of ovulation.Este trabalho teve como objetivo descrever as características ultrassonográficas uterinas e ovarianas, e dopplervelocimétricas das suas artérias nos dias periovulatórios em cadelas. Quinze ciclos estrais em 10 animais foram avaliados. As características ultrassonográficas, índices de resistência (IR) e índices de pulsatilidade (IP) do útero e dos ovários em cada animal foram registrados 5 dias antes e depois da ovulação (D0). Os dados foram analisados estatisticamente e os resultados foram expressos em média ± erro padrão da média (P<0,05). Como resultados as características ultrassonográficas uterinas foram semelhantes em todos os ciclos e dias avaliados, tendo o corpo uterino um diâmetro médio de 0,85±0,02cm. Foi observado um aumento no volume dos ovários e nos diâmetros dos folículos, tendo os ovários um volume de 0,64±0,06cm3, e a cavidade dos folículos um diâmetro de 0,46±0,01cm no dia da ovulação. Após a ovulação, foi observado colapso dos folículos somente em alguns ciclos. A perfusão sanguínea uterina diminuiu dois dias antes da ovulação e permaneceu inalterada até a ovulação. Após a ovulação, houve um aumento gradual na perfusão das artérias uterinas e ovarianas, direcionando o fluxo de sangue para os ovários e aumentando a perfusão da artéria intra-ovariana um dia após a ovulação. Conclui-se que características específicas são observadas na imagem ultrassonográfica uterina e ovariana, e dopplervelocimétricas de suas artérias nos dias periovulatórios, e quando associadas permitem estimar com mais precisão o dia da ovulação