In vitro development of primordial follicles after long-term culture of goat ovarian tissue

This study aims to investigate the effects of follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on the survival and growth of caprine preantral follicles. Ovarian tissues were cultured for 1, 7, 14, 21 or 28 days in medium supplemented with FSH (FSH-2d or FSH-7d, i.e., with replacement of the culture medium every 2 or 7 days, respectively) or FSH + FGF-2 (replacement of the medium every 2 days). Non-cultured (control) and cultured ovarian fragments were processed for histological and ultrastructural analysis. After 28 days of culture, the media supplemented with FSH-2d was the most effective in maintaining the percentage of normal follicles and in promoting follicular growth. Furthermore, both treatments with FSH increased the percentage of the primary follicles. However, ultrastructural studies did not confirm follicular integrity from 14 days of culture onward. In conclusion, culturing tissue for up to 7 days in medium containing FSH alone or combined with FGF-2 maintains caprine preantral follicle integrity and promotes their growth in vitro

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

Histological and ultrastructural feature and nitrite production of caprine preantral follicles in vitro cultured in the presence or absence of serum

Avaliou-se o efeito da adição de diferentes tipos e concentrações de soro sobre o desenvolvimento e a sobrevivência de folículos ovarianos pré-antrais (FOPA) caprinos in vitro. Além disso, verificou-se a relação entre as concentrações de nitrito presentes no meio de cultivo e a viabilidade folicular. Cada par ovariano foi dividido em 29 fragmentos, sendo um destinado ao controle. Os fragmentos foram cultivados por um ou sete dias em meio essencial mínimo suplementado (MEM+) ou MEM+ com diferentes concentrações (10 ou 20%) de soro fetal bovino (SFB), soro de cabra em estro (SCE) ou soro de cabra em diestro (SCD). Na análise morfológica após sete dias, apenas o tratamento com 10% de SFB apresentou percentual de FOPA normais similar ao MEM+ (P>0,05). A análise ultra-estrutural dos folículos cultivados por sete dias com MEM+ ou MEM+ com 10% de SFB mostrou danos oocitários, porém células da granulosa normais. A análise do meio de cultivo revelou correlação positiva entre a viabilidade folicular e a produção de nitrito. A suplementação com soro não melhorou a viabilidade de FOPA e a concentração de nitrito no meio de cultivo funcionou como um indicador da viabilidade das células da granulosa de FOPA caprinos cultivados in vitro. ______________________________________________________________________________________________________________ ABSTRACTThe effect of the addition of different types and concentrations of sera on the viability and development of caprine preantal follicles (PAF) in vitro cultured was analyzed. In addition, it was evaluated the correlation between nitrite concentrations in culture medium and folicular viability. Each ovarian pair was divided in 29 fragments and one was used as control. The fragments were cultured for one or seven days in minimal essential medium (MEM+) or MEM+ with different concentrations of (10 or 20%) bovine fetal serum (BFS), estrous goat serum (EGS), or diestrous goat serum (DGS). After seven days, the morphological analysis showed that only the treatment with 10% BFS maintained the percentage of normal PAF similar to MEM+ (P>0.05). The ultrastructural analysis of follicles cultured for seven days in MEM+ or MEM+ with 10% BFS showed some oocyte damage, although the granulosa cells were normal. Analysis of culture medium revealed a positive correlation between follicular viability and nitrite production. Supplementation with serum did not improve the viability of PAF and nitrite levels in culture medium served as an indicator of viability of granulose cells from caprine PAF in vitro cultured

Tissue patrol by resident memory CD8+ T cells in human skin

Emerging data show that tissue-resident memory T (TRM) cells play an important protective role at murine and human barrier sites. TRM cells in the epidermis of mouse skin patrol their surroundings and rapidly respond when antigens are encountered. However, whether a similar migratory behavior is performed by human TRM cells is unclear, as technology to longitudinally follow them in situ has been lacking. To address this issue, we developed an ex vivo culture system to label and track T cells in fresh skin samples. We validated this system by comparing in vivo and ex vivo properties of murine TRM cells. Using nanobody labeling, we subsequently demonstrated in human ex vivo skin that CD8+ TRM cells migrated through the papillary dermis and the epidermis, below sessile Langerhans cells. Collectively, this work allows the dynamic study of resident immune cells in human skin and provides evidence of tissue patrol by human CD8+ TRM cells.Toxicolog

A multi-integrated approach on toxicity effects of engineered TiO2 nanoparticles

The new properties of engineered nanoparticles drive the need for new knowledge on the safety, fate, behavior and biologic effects of these particles on organisms and ecosystems. Titanium dioxide nanoparticles have been used extensively for a wide range of applications, e.g, self-cleaning surface coatings, solar cells, water treatment agents, topical sunscreens. Within this scenario increased environmental exposure can be expected but data on the ecotoxicological evaluation of nanoparticles are still scarce. The main purpose of this work was the evaluation of effects of TiO2 nanoparticles in several organisms, covering different trophic levels, using a battery of aquatic assays. Using fish as a vertebrate model organism tissue histological and ultrastructural observations and the stress enzyme activity were also studied. TiO2 nanoparticles (Aeroxide® P25), two phase composition of anatase (65%) and rutile (35%) with an average particle size value of 27.6±11 nm were used. Results on the EC50 for the tested aquatic organisms showed toxicity for the bacteria, the algae and the crustacean, being the algae the most sensitive tested organism. The aquatic plant Lemna minor showed no effect on growth. The fish Carassius auratus showed no effect on a 21 day survival test, though at a biochemical level the cytosolic Glutathione-S-Transferase total activity, in intestines, showed a general significant decrease (p<0.05) after 14 days of exposure for all tested concentrations. The presence of TiO2 nanoparticles aggregates were observed in the intestine lumen but their internalization by intestine cells could not be confirmed