Ultrastructural characterization of porcine oocytes and adjacent follicular cells during follicle development : lipid component evolution

The objective of this study was to characterize the morphometry and ultrastructure of porcine preantral and antral follicles, especially the lipid component evolution. Ovarian tissue was processed for light microscopy. Ovarian tissue and dissected antral follicles (< 2, 2–4, and 4–6 mm) were also processed for transmission electron microscopy using routine methods and using an osmium-imidazole method for lipid detection. Primordial follicles (34 ± 5 μm in diameter, mean ± SD) had one layer of flattened-cuboidal granulosa cells around the oocyte, primary follicles (40 ± 7 μm) had a single layer of cuboidal granulosa cells around the oocyte, and secondary follicles (102 ± 58 μm) had two or more layers of cuboidal granulosa cells around the oocyte. Preantral follicle oocytes had many round mitochondria and both rough and smooth endoplasmic reticulum. In oocytes of primordial and primary follicles, lipid droplets were abundant and were mostly located at the cell poles. In secondary and antral follicles, the zona pellucida completely surrounded the oocyte, whereas some microvilli and granulosa cells projected through it. Numerous electron-lucent vesicles and vacuoles were present in the oolemma of secondary and antral follicles. Based on osmium-imidazole staining, most of these structures were shown to be lipid droplets. As the follicle developed, the appearance of the lipid droplets changed from small and black to large and gray, dark or dark with light streaks, suggesting that their nature may change over time. In summary, although porcine follicles and oocytes had many similarities to those of other mammalian species, they were rich in lipids, with lipid droplets with varying morphological patterns as the follicle developed

Evaluation of ovarian structures using computerized microtomography

Visualization and clear understanding of the ovarian structures are important in determining the stage of oestrus, helping to diagnose several pathologies and supporting advances in reproductive technologies. In this research, computerized microtomography (microCT) was used to explore and characterize the ovarian structure of seven mammalian species. Ovaries of rats, female dog, queens, cows, mares, sows and a female donkey were used. After microCT scanning, the same samples were prepared for histologic evaluation, used here as a validation criterion. It was possible to distinguish regions of the cortex and medulla, visualize the morphology and distribution of blood vessels, clearly observe corpus luteum and antral follicles, and visualize oocytes inside some antral follicles. This is the first report using microCT to explore and compare ovarian structures in several domestic mammals. MicroCT revealed great potential for the evaluation of ovarian structures. This research open prospects for the use of computerized tomography (CT) as a non-invasive approach to studying ovarian structures in live animals, which may be especially attractive for scientific study of development of ovarian structures and/or ovarian pathologies in small animals’ models

Cryopreservation and characterization of canine preantral follicles

The aim of this study was to define the population, morphological and ultrastructural characteristics of bitch preantral follicles (PAFs) and to compare the effects on the morphology of PAF of two cryopreservation techniques - slow freezing (SF) and vitrification (V) - of bitches' ovarian tissue. The average population (number per ovary) of PAFs was 48,541 ± 18,366, where 94.25% were primordial (45,145 ± 16,076). The average diameter of the primordial follicles was 27.5 ± 4.2 μm. The overall percentage of morphologically normal PAFs was 93.66 ± 6.81% for the control group, 86.16 ± 11.05% after SF and 68.14 ± 12.75% after V. The percentage of normal primordial follicles was 96.69 ± 4.72% in control, 89.51 ± 10.39% in SF and 75.32 ± 9.23% in V. There was no significant difference in the overall percentage of normal PAFs among SF and the control. However, slow frozen follicles presented ultrastructural damage, while vitrified primordial and primary follicles were well preserved. In conclusion, although slow freezing seemed to be a good preserving method, vitrification was more effective than slow freezing in preserving the ultrastructure of primordial and primary follicles of bitches

Estudo da população e criopreservação de folículos ovarianos pré-antrais de cadelas

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, 2012.O objetivo desse trabalho foi definir as características populacionais, morfológicas e ultraestruturais de folículos ovarianos pré-antrais (FOPA) e comparar o efeito das técnicas de criopreservação de congelamento lento (CL) e vitrificação (V) de tecido ovariano de cadela sobre a morfologia dos FOPA. A média da estimativa da população de FOPA de cadelas foi de 48541±18366, onde 94,25% eram primordiais (45145±16076), 4,92% primários (2358±1253) e 0,83% secundários (397±351). A média de diâmetro do folículo, ovócito e núcleo do ovócito e número de células da granulosa de folículos primordiais foram 27,5±4,2μm, 21,7±2,7μm e 11,3±1,6μm e 6,0±1,8 respectivamente. Para folículos primários foram, respectivamente, 42,6±12,5μm, 27,8±7,5μm, 13,7±2,6μm e 15,0±7,0. Para folículos secundários, foi 101,6±62,9μm para diâmetro do folículo, 48,0±12,6μm para diâmetro do ovócito, 18,7±4,2μm para diâmetro do núcleo do ovócito e 61,5±63,5 células da granulosa. A porcentagem total de FOPA morfologicamente normais foi de 93,66±6,81% para o grupo controle, 86,16±11,05% para o CL e 68,14±12,75% para a V. Para folículos primordiais a porcentagem de FOPA normais foi 96,69±4,72% no controle, 89,51±10,39% no CL e 75,32±9,23% na V. Para folículos primários e secundários os valores foram, respectivamente, 94,80±6,91% e 87,62±17,12% para o controle, 86,8±12,15% e 76,35±26,34% para CL e 61,53±14,78 e 52,25±22,13% para a V. Não houve diferença significativa na porcentagem de FOPA morfologicamente normais entre o CL e o grupo controle. Já na V essa porcentagem foi significativamente inferior aos outros 2 grupos (P<0,05). A ultraestrutura de folículos pré-antrais de cadela mostrou ser semelhante à descrita para outras espécies. Após criopreservação por CL os folículos apresentaram danos ultraestruturais, enquanto após vitrificação os folículos primordiais e primários estavam bem preservados. No entanto, os folículos secundários foram muito danificados em ambos os tratamentos. Em conclusão, este trabalho descreveu as características morfométricas, ultraestruturais e populacionais dos FOPA de cadela. A vitrificação foi mais eficiente em preservar a ultraestrutura dos folículos primordiais e primários que o congelamento lento. Os dois métodos de criopreservação testados apresentam resultados promissores, mas ainda precisam de aperfeiçoamento, especialmente em relação à preservação de folículos secundários. _________________________________________________________________________________ ABSTRACTThe aim of this study was to define the population, morphological and ultrastructural characteristics of bitch preantral follicles (PAF) and to compare the effects of two cryopreservation techniques - slow freezing (SF) and vitrification (V) - of bitches ovarian tissue on the morphology of PAF. The average population of PAF was 48,541 ± 18,366, where 94.25% were primordial (45145 ± 16076), 4.92% primary (2358 ± 1253) and 0.83% secondary (397 ± 351). The average diameter of the follicle, oocyte and the oocyte nucleus and the number of granulosa cells for primordial follicles were 27.5 ± 4.2μm, 21.7 ± 2.7μm, 11.3 ± 1.6μm and 6.0 ± 1.8, respectively. For primary follicles the values were, respectively, 42.6 ± 12.5μm, 27.8 ± 7.5μm, 13.7 ± 2.6μm and 15.0 ± 7.0. For secondary follicles follicle diameter was 101.6 ± 62.9μm, oocyte diameter was 48.0 ± 12.6μm, nucleus diameter was 18.7 ± 4.2μm, and the number of granulosa cells was 61.5 ± 63.5. The overall percentage of morphologically normal PAF was 93.66 ± 6.81% for the control group, 86.16 ± 11.05% for SF and 68.14 ± 12.75% for V. The percentage of normal primordial follicles was 96.69 ± 4.72% in control, 89.51 ± 10.39% in SF and 75.32 ± 9.23% in V. For primary and secondary follicles values were respectively 94.80 ± 6.91% and 87.62 ± 17.12% for control, 86.8 ± 12.15% and 76.35 ± 26.34% for SF and 61.53 ± 14.78 and 52.25 ± 22.13% for V. There was no significant difference on the percentage of normal PAF among SF and the control. However, V presented a significantly lower percentage of normal PAF than the other two groups (P<0.05). The ultrastructure of bitch PAF was similar to that described for other species. After, SF follicles presented ultrastructural damage, while after V, primordial and primary follicles were well preserved. Secondary follicles, however, suffered many damages in both cryopreservation techniques. In conclusion, this work described the morphometric and ultrastructural characteristics and the population of bitch PAF. Vitrification was more effective in preserving the ultrastructure of primordial and primary follicles than slow freezing. Both methods are promising, but need improvement, especially concerning secondary follicles preservation

Avaliação dos efeitos de dois fluidos magnéticos na reprodução de ratos Wistar machos

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Animal, 2016.A expansão da nanotecnologia aumentou o uso de nanomateriais nos procedimentos e pesquisas biomédicas e, consequentemente, a sua interação com organismos vivos. Modelos in vivo e in vitro já foram propostos para o estudo da interação de nanomateriais com os sistemas biológicos. Nanopartículas de óxido de ferro são conhecidas por terem baixa toxicidade. Informações sobre os efeitos tóxicos in vivo de nanopartículas metálicas no aparelho reprodutor masculino e na fisiologia espermática ainda são escassos. O desenvolvimento de novas tecnologias e abordagens com nanopartículas para o controle populacional de animais errantes é uma nova frente de pesquisa em que a nanotecnologia pode atuar. Este trabalho descreve os efeitos de dois fluidos magnéticos no sistema reprodutor de ratos Wistar machos. Uma primeira abordagem investiga os possíveis efeitos tóxicos de nanopartículas de maghemita funcionalizadas com citrato administrada por via endovenosa nos espermatozóides. Em uma segunda abordagem, foi feita uma tentativa de desenvolver uma nova técnica, utilizando nanopartículas magnéticas, com o intuito de promover a esterilidade em ratos machos.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).The expansion of nanotechnology has increased the use of nanomaterials in biomedical procedures and research, and therefore its interaction with living organisms. In vivo and in vitro models have been proposed to study the interaction of nanomaterials with biological systems. Iron oxide nanoparticles are known to present low toxicity. The information on toxic effects in vivo of metal nanoparticles in the male reproductive system and sperm physiology are still scarce. The development of new technologies and approaches with nanoparticles to control the population of stray animals is a new line of research that nanotechnology can provide. This study describes the effect of two magnetic fluids on the reproductive system of male Wistar Rats. The first approach was to evaluate the possible toxic effects of maghemite nanoparticles functionalized with citrate injected intravenously on the sperm cells. The second approach was to develop a new technique, using magnetic nanoparticles, for induction of infertility in male rats

Therapeutic Effects of <i>Zymomonas mobilis</i> on Experimental DSS-Induced Colitis Mouse Model

Zymomonas mobilis, a Gram-negative bacteria observed in some popular beverages, is considered safe and has been studied for its potential therapeutic benefits. In this study, we explored its effects on the inflammatory process, tissue integrity, differential gene expression, and microbiota composition in an experimental dextran sulfate sodium (DSS)-induced colitis model in mice. As a result, Z. mobilis alleviated the symptoms caused by DSS administration, as indicated by reduced weight loss, disease activity index, a significant reduction in the colon weight/length ratio, and histopathological improvement. Also, Z. mobilis could restore the mucosal barrier as well as increase the expression of Muc3 and Ocln genes. An analysis of 16S rRNA sequences showed that Z. mobilis alters gut microbiota, increasing Akkermansia muciniphila abundance and decreasing Escherichia coli. Furthermore, Z. mobilis seems to be involved in potentiating a regulatory phenotype by inducing immunomodulatory genes like Tgfb, Il5, Il10, and Foxp3 and reducing the relative mRNA expression of proinflammatory cytokines TNF, Il6, and Il17. Our data suggest that Z. mobilis could alleviate disease progression and be considered a possible probiotic adjuvant for pathologies of the bowel

Experimental orthotopic breast cancer as a model for investigation of mechanisms in malignancy and metastasis to the lymph nodes

To understand the fundamental mechanisms behind malignancy of breast tumors and also contribute to the discovery of improved methods for prevention, diagnosis and treatment, animal cancer models remain essential. We aimed to establish an optimal orthotopic cancer model for breast cancer in the immunocompetent Swiss mouse, describing the detailed microanatomy of the mammary glands, the sentinel lymph node and lymphatic mapping, evaluating histopathological changes and characterizing the tumor by computed microtomography and interleukins expression. The inoculation of fresh Ehrlich tumor cells led to a detectable tumor as early as 24 hours later; after 7 days, mammary, muscular, dermal vascular and lymphatic invasion were observed and also micrometastases in mammary adipose tissue, sentinel lymph node and contralateral lymph node. From the inoculation site the tumor invaded the host mammary gland structures, the dermis and endomysium of skeletal muscle tissue. Type 1 T helper cytokine levels (IL-1β and IL-17) were significantly higher than anti-inflammatory Type 2 T helper (IL-4) after inoculation of fresh tumor cells. Differently, frozen tumor cells induced tumor development only 14 days after the inoculation while presenting expression of distinct interleukins. The set of findings indicates that orthotopic transplantation provides the microenvironment critical for cell interactions involved in the development of cancer and subsequent metastasis. In this regard, the anatomical and physiological knowledge of the mammary glands and ductal networks favor studies, diagnoses and therapies related to breast cancer and metastasis. Moreover, this study provides anatomical support for the understanding of the lymphatic spread process of cancer cells.</p