RESUMO

This study describes the effect of sphingosine 1-phosphate (S1P) for development of preantral follicle, therefore the activation and follicular viability of caprine follicles cultured in vitro. Ovarian fragments were cultured for 1 or 7 days in Minimum Essential Medium with different S1P concentrations (0, 1, 10, 50, 100 or 200ng/mL). All ovarian fragments were processed for histological analysis in optical microscopy, transmission electron microscopy and fluorescence analysis. The treatment using 1ng/mL of S1P was able to maintain the percentage of normal follicles with the progression of the culture from day 1 to 7. At end of the 7-day culture period there was a significant reduction (P<0.05) in the percentage of primordial follicles in all groups treated with S1P, compared with fresh control (FC) and Control Culture (CC), which was followed by an increase of activated follicles (intermediary, primary and secondary). In addition, the culture for 7 days with media supplemented with S1P with 1ng/mL preserved the ultrastructure of organelles and kept the preantral follicular viability when evaluated by fluorescence microscopy. In conclusion, after 7 days of culture, the 1ng/mL of S1P activates the development of preantral caprine follicles, cultured in situ and maintains the oocitary and follicular viability.(AU)

Este estudo descreve o efeito da esfingosina 1-fosfato (S1P) no desenvolvimento de folículos pré-antrais, portanto da ativação e viabilidade de folículos caprinos cultivados in vitro. Fragmentos de ovários foram cultivados por um ou sete dias em meio essencial mínimo com diferentes concentrações de S1P (0, 1, 10, 50, 100 ou 200ng/mL). Os fragmentos de ovário foram processados para análise histológica em microscopia óptica, microscopia eletrônica e microscopia de fluorescência. O tratamento usando 1ng/mL de S1P foi capaz de manter a porcentagem de folículos normais durante o período de cultivo de sete dias. Ao final do período de cultivo, houve uma redução significativa (p<0,05) na porcentagem de folículos primordiais em todos os grupos tratados com S1P, comparados com os grupos controle (FC e CC), seguida por um aumento do número de folículos ativados (intermediários, primários e secundários). Adicionalmente, na cultura por sete dias com meio suplementado com S1P (1ng/mL), houve preservação da ultraestrutura das organelas e manteve-se a viabilidade dos folículos pré-antrais avaliados por microscopia de fluorescência. Em conclusão, após sete dias de cultura, o meio suplementado com 1ng/mL de S1P ativa o desenvolvimento de folículos pré-antrais de caprino, cultivados in situ e mantém as viabilidades oocitária e folicular.(AU)

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RESUMO

This study describes the effect of sphingosine 1-phosphate (S1P) for development of preantral follicle, therefore the activation and follicular viability of caprine follicles cultured in vitro. Ovarian fragments were cultured for 1 or 7 days in Minimum Essential Medium with different S1P concentrations (0, 1, 10, 50, 100 or 200ng/mL). All ovarian fragments were processed for histological analysis in optical microscopy, transmission electron microscopy and fluorescence analysis. The treatment using 1ng/mL of S1P was able to maintain the percentage of normal follicles with the progression of the culture from day 1 to 7. At end of the 7-day culture period there was a significant reduction (P<0.05) in the percentage of primordial follicles in all groups treated with S1P, compared with fresh control (FC) and Control Culture (CC), which was followed by an increase of activated follicles (intermediary, primary and secondary). In addition, the culture for 7 days with media supplemented with S1P with 1ng/mL preserved the ultrastructure of organelles and kept the preantral follicular viability when evaluated by fluorescence microscopy. In conclusion, after 7 days of culture, the 1ng/mL of S1P activates the development of preantral caprine follicles, cultured in situ and maintains the oocitary and follicular viability...

Este estudo descreve o efeito da esfingosina 1-fosfato (S1P) no desenvolvimento de folículos pré-antrais, portanto da ativação e viabilidade de folículos caprinos cultivados in vitro. Fragmentos de ovários foram cultivados por um ou sete dias em meio essencial mínimo com diferentes concentrações de S1P (0, 1, 10, 50, 100 ou 200ng/mL). Os fragmentos de ovário foram processados para análise histológica em microscopia óptica, microscopia eletrônica e microscopia de fluorescência. O tratamento usando 1ng/mL de S1P foi capaz de manter a porcentagem de folículos normais durante o período de cultivo de sete dias. Ao final do período de cultivo, houve uma redução significativa (p<0,05) na porcentagem de folículos primordiais em todos os grupos tratados com S1P, comparados com os grupos controle (FC e CC), seguida por um aumento do número de folículos ativados (intermediários, primários e secundários). Adicionalmente, na cultura por sete dias com meio suplementado com S1P (1ng/mL), houve preservação da ultraestrutura das organelas e manteve-se a viabilidade dos folículos pré-antrais avaliados por microscopia de fluorescência. Em conclusão, após sete dias de cultura, o meio suplementado com 1ng/mL de S1P ativa o desenvolvimento de folículos pré-antrais de caprino, cultivados in situ e mantém as viabilidades oocitária e folicular...

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The aim of this study was to investigate the effects of melatonin and follicle-stimulating hormone (FSH) on the in vitro culture of goat preantral follicles. Ovarian fragments were cultured for 7 d in α-minimum essential medium (α-MEM(+)) containing melatonin (100, 250, 500, or 1,000 pM), FSH (50 ng/mL), or a combination of the 2 hormones and further analyzed by histology and transmission electron and fluorescent microscopy. The results showed that after 7 d of culture, tissues cultured in α-MEM(+) alone or supplemented with FSH alone, melatonin (500 and 1,000 pM), or the combination of FSH and melatonin (1,000 pM) maintained percentages of normal preantral follicles similar to the fresh control. In contrast to the noncultured tissues, the percentage of developing follicles was increased under all culture conditions after 7 d (P < 0.05). The addition of 1,000 pM melatonin associated with FSH to the culture medium increased follicular and oocyte diameters compared with α-MEM(+) alone after 7 d of culture (P < 0.05). Ultrastructural and fluorescent analyses confirmed the integrity of follicles cultured with 1,000 pM of melatonin plus FSH for 7 d. In conclusion, this study demonstrated that the interaction between melatonin and FSH maintains ultrastructural integrity and stimulates further growth of cultured caprine preantral follicles.

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This paper aimed to evaluate the improvement of burn wounds healing by sodium alginate/chitosan-based films and laser therapy. Natural polymers with different biological activities are widely used as film dressings to improve wound healing. Lasers arrays accelerate the healing repair of soft tissue injuries. Burn procedures were performed on the backs of 60 male rats assigned into six groups: untreated (CTR), dressed with cellulose films (CL), dressed with sodium alginate/chitosan-based films (SC), laser-irradiated undressed wounds (LT), laser-irradiated wounds with cellulose (CLLT) and sodium alginate/chitosan-based films (SCLT). Laser therapy was applied for 7 days. Animals of each group were euthanised 8 and 14 days after the burn procedures. The inflammatory reaction was significantly more intense in the CTR group than in the irradiated groups after 8 and 14 days. Laser therapy stimulated myofibroblastic differentiation in 8 days, with or without dressing films. Combined laser therapy and both dressings improved epithelisation, blood vessels formation and collagenization, promoted rapid replacement of type III for type I collagen and favored the better arrangement of the newly formed collagen fibres. The combination of laser therapy and sodium alginate/chitosan-based dressing improves burn healing, apparently by modulating the epithelisation, blood vessels formation and collagenization processes.

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This study investigates steady-state level of bone morphogenetic protein-15 (BMP-15) mRNA in caprine follicles, and the effects of BMP-15 on in vitro development of preantral follicles. Ovarian fragments were cultured for one or seven days in Minimal Essential Medium (MEM(+)) with BMP-15 (0, 1, 10, 50, 100 or 200 ng/mL), and further analyzed by histology, transmission electron and fluorescent microscopy. BMP-15 mRNA in secondary follicles was higher than in primordial and primary follicles. After seven days, 10, 50 or 100 ng/mL of BMP-15 maintained the percentage of normal follicles similar to the control (non-cultured), and increased the oocyte and follicle diameters when compared to the control and MEM(+). BMP-15 at 100 ng/mL increased the secondary follicles and maintained their ultrastructural integrity. In conclusion, the BMP-15 mRNAs were detected in all follicular categories. BMP-15 (100 ng/mL) maintained the integrity and promoted the growth of caprine preantral follicles cultured for seven days.

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This study evaluated the expression of FSH receptors (FSHR) in the different stages of goat follicle development and investigated whether the addition of increasing concentrations of FSH throughout the culture period influences the survival, growth and antral formation of in vitro-cultured caprine preantral follicles. The expression of FSHR was analysed before and after culturing follicles using real-time RT-PCR. For the culture, preantral follicles (≥150 µm) were isolated from ovarian fragments and cultured for 18 days in α-MEM+ alone or associated with recombinant FSH (rFSH: 100 or 1000 ng/ml), or in α-MEM+ supplemented with increasing concentrations of FSH throughout culture periods as follows: (a) sequential medium 1: FSH 100 ng/ml (from day 0 to 6), FSH 500 ng/ml (from day 6 to 12) and FSH 1000 ng/ml (from day 12 to 18); and (b) sequential medium 2: FSH 500 ng/ml (from day 0 to 9) and 1000 ng/ml (from day 9 to 18). Follicle development was evaluated on the basis of antral cavity formation, follicular and oocyte growth, and cumulus-oocyte complex health. The expression of FSHR in isolated caprine follicles increased from the preantral to antral phase. Regarding the culture, after 18 days, sequential medium 1 promoted follicular survival, antrum formation and a reduction in oocyte extrusion. Both sequential media promoted a higher rate of meiotic resumption compared with the other treatments. In conclusion, the addition of increased concentrations of FSH (sequential medium) has a significant impact on the in vitro development of caprine preantral follicles.

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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.

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Os hormônios esteroides estão diretamente relacionados com o processo de foliculogênese, o qual é bastante complexo e influenciado também por vários fatores de crescimento e por hormônios. Diversos estudos demonstraram a importância dos esteroides no desenvolvimento folicular, na maturação oocitária, bem como na formação do corpo lúteo. Desta forma, o objetivo da presente revisão foi sintetizar o processo de formação e o mecanismo de ação dos hormônios esteroides na foliculogênese, com ênfase no estradiol, na progesterona e nos andrógenos.

Steroid hormones are directly related to the folliculogenesis process, which is very complex and also influenced by several growth factors and hormones. Several studies have demonstrated the importance of steroids in follicular development in mature oocytes as well as corpus luteum formation. The objective of this review is to address the process of synthesis and mechanism of action of steroid hormones in folliculogenesis, with emphasis on estradiol, progesterone and androgens.

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RESUMO

Os hormônios esteroides estão diretamente relacionados com o processo de foliculogênese, o qual é bastante complexo e influenciado também por vários fatores de crescimento e por hormônios. Diversos estudos demonstraram a importância dos esteroides no desenvolvimento folicular, na maturação oocitária, bem como na formação do corpo lúteo. Desta forma, o objetivo da presente revisão foi sintetizar o processo de formação e o mecanismo de ação dos hormônios esteroides na foliculogênese, com ênfase no estradiol, na progesterona e nos andrógenos.(AU)

Steroid hormones are directly related to the folliculogenesis process, which is very complex and also influenced by several growth factors and hormones. Several studies have demonstrated the importance of steroids in follicular development in mature oocytes as well as corpus luteum formation. The objective of this review is to address the process of synthesis and mechanism of action of steroid hormones in folliculogenesis, with emphasis on estradiol, progesterone and androgens.(AU)

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The aim of the present study was to investigate the effects of fibroblast growth factor-10 (FGF-10) on the survival, activation (transition from primordial to primary follicles), and growth of goat preantral follicles cultured in vitro. Pieces of ovarian cortex were cultured for 1 and 7 d in the absence or presence of FGF-10 (0, 1, 10, 50, 100, and 200 ng/mL). Noncultured and cultured tissues were processed and analyzed by histology, transmission electron microscopy, and viability testing. Results showed that after 7 d, a greater percentage (79.9%) of morphologically normal follicles (containing an oocyte with regular shape and uniform cytoplasm, and organized layers of granulosa cells without a pyknotic nucleus) was observed when cultured with 50 ng/mL of FGF-10 when compared with other concentrations of FGF-10 (0 ng/mL, 67.3%; 1 ng/mL, 68.2%; 10 ng/mL, 63.3%; 100 ng/mL, 64.4%; 200 ng/mL, 52.7%). Ultrastructural analyses and viability testing using fluorescent markers confirmed the follicular integrity of FGF-10 (50 ng/mL)-treated fragments after 7 d of culture. After 7 d, all FGF-10 concentrations reduced the percentage of primordial follicles and increased the percentage of developing follicles. In the presence of 50 ng/mL of FGF-10, follicles increased in diameter after 7 d of culture when compared with other concentrations tested. In conclusion, this study demonstrates that FGF-10 maintains the morphological integrity of goat preantral follicles and stimulates the growth of activated follicles in culture. The culture conditions identified here contribute to the understanding of the factors involved in goat early follicular development.

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