Morphometric and ultrastructural characterization of Bos indicus preantral follicles

The aim of the present study was to characterize the ultrastructure of zebu cow preantral follicles (PAFs). Ovarian cortex samples were processed for light and transmission electron microscopy. Primordial follicles consisted of an oocyte surrounded by one layer of flattened or flattened-cuboidal granulosa cells. The oocyte contained a large and usually eccentric nucleus. Most organelles were located at the perinuclear ooplasm. Round shaped mitochondria, which contained electron-dense granules, smooth and rough endoplasma reticulum and a Golgi apparatus were also observed. Vesicles and coated pits were often observed in the cortical ooplasm. In primary follicles, the oocyte was surrounded by one layer of cuboidal granulosa cells. Short microvilli were observed on the oolema. Secondary follicles consisted of an oocyte surrounded by a variable number of layers of cuboidal granulosa cells. Small secondary follicles had an ultrastructure very similar to that observed in primary follicles. At this follicular stage, the zona pellucida was beginning to form around the oocyte. In large secondary follicles, the zona pellucida was totally developed around the oocyte. Several granulosa cell projections could be detected that were encroaching into the zona pellucida and protruding towards the oocyte, where gap junctions were observed between oocyte and granulosa cell membranes. Organelles within the oocyte were located at the periphery of the ooplasm, and clusters of cortical granules were observed. Round mitochondria were abundant in all developmental stages. In conclusion, this study described the ultrastructure of zebu cow PAFs, and some unique characteristics could be observed as compared with what has been reported for follicles of Bos taurus cattle

Ultrastructural and morphometric characterization of buffalo (Bubalus bubalis) ovarian preantral follicles

The main objective of the present study was to characterize buffalo preantral ovarian follicles. Parts of ovarian cortex, collected from postpubertal buffalo females that were having estrous cycles at regular intervals, were selected under stereomicroscopy and processed for optic and transmission electron microscopy. Primordial follicles were characterized as an oocyte encircled by one layer of flattened cells. The buffalo primordial follicle has a mean diameter of 35 μm and the oocyte diameter is 24.9 μm. The oocyte nucleus is relatively large and eccentric; and in the cytoplasm a large amount of mitochondria, vesicles and endoplasmic reticulum cistern, mainly of the smooth type is observed. The primordial follicles cells are rich in plasma membrane invaginations, which are observed within the cell and between the cell and the oocyte. The primary follicles (mean diameter of 41.8 μm) consist of an oocyte, with a medium diameter of 26.9 μm, surrounded by one layer of cubical granulosa cells. At this follicular stage, the beginning of zona pellucida deposition can also be seen in areas between the oocyte and follicular cells. The secondary follicles, which are surrounded by more than one layer of cubical cells, have a diameter of 53.3 μm, and the oocyte has a mean diameter of 29.4 μm. The ultrastructural analysis showed a large amount of coalescent vesicles, more evident in the oocyte periphery. The zona pellucida (ZP) is thicker at this stage and contains a large quantity of glycoproteins. In general, the ultrastructure of buffalo preantral follicles was similar to that of other mammalian species, but some differences were observed, which indicate species specific characteristics. The main differences observed were cytoplasmic vesicles quantity, mitochondria shape and inner content, ZP deposition and granulosa cell–oocyte junctions. In conclusion, the morphological differences described in this paper, could be responsible for some functional differences observed in Bubalus bubalis in vitro embryo production and follicular dynamics, when compared with Bos taurus or Bos indicus species

Effects of α-tocopherol and ternatin antioxidants on morphology and activation of goat preantral follicles in vitro cultured

Os efeitos do α-tocoferol e da ternatina sobre morfologia, ativação e crescimento de folículos pré-antrais caprinos cultivados in vitro, por um ou cinco dias, foram avaliados. Os fragmentos ovarianos foram imediatamente fixados (controle não-cultivado) ou cultivados in vitro, por um ou cinco dias, em Meio Essencial Mínimo (MEM) com ou sem suplementação com α-tocoferol ou ternatina nas concentrações de 5, 10 ou 15M, formando os tratamentos MEM, TOC5, TOC10, TOC 15, TER5, TER10, TER15. O percentual de folículos pré-antrais normais no controle não-cultivado foi de 73,2%, depois de cinco dias de cultivo, houve redução desse percentual em todos os tratamentos, quando comparados com o controle não-cultivado (P<0,05). O cultivo por cinco dias aumentou a ativação folicular em todos os tratamentos (P<0,05). A análise ultra-estrutural não mostrou folículos pré-antrais íntegros após cinco dias de cultivo em meio contendo antioxidantes. Concluiu-se que o α -tocoferol e a ternatina podem promover a ativação folicular, no entanto a adição desses antioxidantes nas concentrações testadas reduziu a viabilidade folicular após o cultivo in vitro. ______________________________________________________________________________________________________________ ABSTRACTThe effects of α-tocopherol and ternatin on the morphology, activation, and growth of goat preantral follicles in vitro cultured, for one or five days, were evaluated. Ovarian fragments were immediately fixed (non-cultured control) or in vitro cultured for one or five days in Minimum Essential Medium (MEM) with or without α-tocopherol or ternatin supplementation, both at concentrations of 5, 10, or 15M, corresponding to the following treatments: MEM, TOC5, TOC10, TOC 15, TER5, TER10, and TER15. The percentages of morphologically normal preantral follicles in non-cultured ovarian tissue (control) was 73.2% and after five days of culture, there was a decrease on these percentages in all treatments (P<0.05) when compared with non-cultured control. Culture of ovarian cortex for five days increased the percentages of follicular activation in all treatments (P<0.05). Ultrastructural analysis did not confirm the integrity of caprine preantral follicles cultured for five days in medium containing antioxidants. This study demonstrated that α-tocopherol and ternatin can promote follicular activation; however, addition of these antioxidants in the tested concentrations reduced the follicular viability after in vitro culture

Role of CD100 in the pathogenesis of atherosclerosis

A aterosclerose é uma doença degenerativa crônica dos vasos, com conseqüências clínicas agudas que incluem o infarto do miocárdio e o acidente vascular cerebral, resultantes geralmente da ruptura da placa e trombose. É atualmente reconhecida como de característica inflamatória, iniciada e propagada no contexto da hipercolesterolemia. Um trabalho de nosso grupo utilizou técnicas de phage display para comparar placas ateroscleróticas e carótidas normais objetivando a busca de proteínas alteradas potencialmente envolvidas na patogênese da doença. Diversas semaforinas e plexinas (receptores de semaforinas) foram identificadas dentre elas a plexina B1, que possui alta afinidade por CD100, sugerindo assim uma concentração aumentada de CD100 na placa aterosclerótica. CD100 foi a primeira semaforina descrita no sistema imune e a única até hoje descrita como possuidora de duas formas de funcionalidades distintas, sendo uma de membrana (mCD100) e outra solúvel (sCD100). Neste trabalho demonstramos a expressão da semaforina CD100 em macrófagos e células espumosas em placas ateroscleróticas humanas, assim como seu padrão de expressão ao longo da diferenciação monócito-macrófago-célula espumosa, e sob estímulos distintos. Além disso, identificamos pela primeira vez o receptor que medeia suas atividades nessas células, a plexina B2. Adicionalmente, detectamos também pela primeira vez detectamos a expressão de CD100 em células endoteliais teciduais e cultivadas in vitro, o que sugere um papel significativo da semaforina em fenômenos vasculares. Com base nessas observações e nos resultados de experimentos de bloqueio de adesão constatamos que CD100 pode atuar na fase mais precoce da aterosclerose, como uma molécula de adesão envolvida na ligação entre monócitos e células endoteliais. Verificamos ainda que CD100 diminui a captação de LDLox em macrófagos e células espumosas. Poucos estudos relatam a presença ou possível atividade biológica de CD100 tanto na aterosclerose quanto em macrófagos. Devido às já estabelecidas ações no sistema imune, acreditamos que a expressão diferencial dessa semaforina desempenha um papel amplificador na patogênese da aterosclerose. Posteriormente, essa proteína poderá servir como alvo de inibição da progressão da doença e de suas complicaçõesAtherosclerosis is a chronic degenerative disease affecting vessels, with acute clinical consequences that include myocardium infarction or stroke, generally resulting from plaque rupture and thrombosis. It is now recognized as an inflammatory disease, initiated and developed in a hipercholesterolemic context. A work in our lab has used phage display techniques to compare atherosclerotic plaques and normal carotids, searching for altered proteins potentially involved in the pathogenesis of the disease. Many semaphorins and plexins (semaphorin receptors) have been identified, among which plexin B1, a high affinity receptor for CD100, suggesting an augmented level of CD100 in the atherosclerotic plaques. CD100 is the first semaphorin described in the immune system, and the only to possess two forms with distinct functionalities, being one associated to the membrane, mCD100, and another soluble form, sCD100. In the present work we have demonstrated CD100 expression in macrophages and foam cells of human atherosclerotic plaques, as well as its pattern of expression along monocyte-macrophage-foam cell differentiation and under distinct stimuli. Furthermore, we have identified for the first time the receptor involved in CD100 activities in these cells, namely plexin B2. Aditionally, we have detected CD100 expression in tissue as well as in in vitro cultured endothelial cells, also for the first time. According to these informations and adhesion blockage experiments we have shown that CD100 may act in the earliest phase of the establishment of atherosclerosis, as an adhesion molecule involved in monocyte-endothelial cell association. We have also verified that CD100 diminishes the intake of oxLDL in macrophages and foam cells. Only a few studies describe the presence or possible biological activity of CD100 in atherosclerosis or macrophages. Since the molecule has been shown to participate in the immune system, we believe that the differential expression of this semaphorin plays an amplifying role in the pathogenesis of atherosclerosis. In the future, this protein could act as an inhibition target of the disease progression as well as its complication

Further contributions to the flora of lichens and lichenicolous fungi of the Azores.

Several lichens are reported new to the flora of the Azores. Mycoporum sparsellum, and the lichenicolous coelomycete Laeviomyces fallaciosus are reported for the first time in Europe. Arthothelium crozalsianum, Bacidia friesiana, Belonia incarnata, Julella sericea, Micarea assimilata, Mycomicrothelia confusa and Roselliniopsis ventosa are recorded for the first time in Laurimacaronesia. New for the Azores are Acarospora umbilicata, Buellia aethalea, B. subdisciformis, Byssoloma marginatum, Canoparmelia texana, Catillaria atomarioides, Chaenotheca furfuracea, Chromatochlamys muscorum, Cladonia cyathomorpha, C. pocillum, C. rangiformis var. gracillima, Cliostomom griffithii, Endocarpon pusillum, Hypotrachyna taylorensis, Opegrapha ochrocheila, O. vermicellifera, O. vulgata, Parmotrema mellissii, Peltula euploca, Pertusaria hymenea, Phaeophyscia hispidula, Porina aenea, P. borreri, Pyrenula acutispora, Ramalina lacera, R. subpusilla, Rinodina anomala, R. intermedia, Scoliciosporum umbrinum, Strigula taylorii, Toninia mesoidea, Xanthoria candelaria and X. fallax. Fourteen additional lichenicolous fungi double the list of species from the Azores

Resting and IFN-γ stimulated monocytes show increased levels of CD100 mRNA compared to macrophages and foam cells (A, B).

<p>Quantitative RT-PCR (qRT-PCR) for CD100 (A) or STAT-1 (B) in PB monocytes, macrophages and foam cells stimulated (+) or not (-) with IFN-γ. Folds relative to non stimulated monocytes (mono-), normalized with GAPDH. (a) * = significant <i>p</i> values (p≤0.05) in the comparisons in unstimulated and stimulated conditions.</p

Infiltrating cells of the intima in coronary and carotid plaques are CD100 positive.

<p>CD100 labeling (E-H) and negative controls (A-D) in artery tissue sections of carotid plaques (A, B, E, F) and coronary plaques (C, D, G, H) by immunohistochemistry. Positive CD100 staining was observed only in preserved endothelium (arrowhead), and in infiltrating cells in the intima (asterisks). Scale bars: 10µm (<i>A</i>, <i>E</i>, <i>C</i> and <i>G</i>), and 50µm (<i>B</i>, <i>F</i>, <i>D</i> and <i>H</i>).</p

CD100 is expressed only in endothelia of normal coronary and carotid arteries.

<p>CD100 labeling (E-H) and negative controls (A-D) in normal carotid (A, B, E, F) and coronary (C, D, G, H) artery tissue sections by immunohistochemistry. Positive CD100 staining was observed only in endothelia of normal arteries (arrowheads). Scale bars: 10µm (<i>A</i>, <i>E</i>, <i>C</i> and <i>G</i>) and 50µm (<i>B</i>, <i>F</i>, <i>D</i> and <i>H</i>).</p

Cultured monocytes express higher amounts of CD100 protein than <i>in vitro</i> differentiated macrophages and foam cells.

<p>CD100 and β-actin protein expression was evaluated in activated T lymphocytes (TL – positive control) and PB monocytes (Mono), macrophages (Mac) and foam cells (Foam) A. Western blot showing CD100 and β-actin bands B. Densitometry of CD100 (sum of 120 and 150kDa bands) and β-actin protein bands, showing CD100/β-actin. Mean ± SD of 3 independent experiments.</p