Steady-state levels of vasoactive intestinal peptide (VIP) mRNA in goat ovaries and the effect of VIP on the in vitro development of isolated preantral follicles

The aims of this study were to verify the steady-state level of vasoactive intestinal peptide (VIP) mRNA in goat follicles at various developmental stages and to investigate the influence of VIP on the survival, antrum formation and growth of secondary follicles cultured for 6 days. Primordial, primary and secondary goat follicles and small and large antral follicles were obtained to quantify VIP mRNA by real-time reverse transcription with the polymerase chain reaction. The influence of VIP and the presence or absence of follicle-stimulating hormone (FSH) on the development of secondary follicles and on mRNA expression for VIP and FSH receptor (FSHR) were determined after 6 days of culture. Survival, antrum formation and follicular diameter were evaluated every other day of culture. The levels of VIP mRNA in primary and secondary follicles were significantly higher than in primordial follicles. Cumulus–oocyte complexes (COCs) from both small and large antral follicles had significantly higher levels ofVIP mRNA than their respective granulosa/theca cells. During culture, the addition of VIP and/or FSH had o effect on follicular development. However, the presence of FSH and/or VIP in the culture medium significantly reduced VIP mRNA levels, but did not alter FSHR mRNA levels. In conclusion, VIP mRNA was detected in all goat follicular categories and cellular types, VIP and/or FSH did not affect the development of secondary follicles and reduce the expression of VIP mRNA levels.(AU)

Steady-state levels of vasoactive intestinal peptide (VIP) mRNA in goat ovaries and the effect of VIP on the in vitro development of isolated preantral follicles

The aims of this study were to verify the steady-state level of vasoactive intestinal peptide (VIP) mRNA in goat follicles at various developmental stages and to investigate the influence of VIP on the survival, antrum formation and growth of secondary follicles cultured for 6 days. Primordial, primary and secondary goat follicles and small and large antral follicles were obtained to quantify VIP mRNA by real-time reverse transcription with the polymerase chain reaction. The influence of VIP and the presence or absence of follicle-stimulating hormone (FSH) on the development of secondary follicles and on mRNA expression for VIP and FSH receptor (FSHR) were determined after 6 days of culture. Survival, antrum formation and follicular diameter were evaluated every other day of culture. The levels of VIP mRNA in primary and secondary follicles were significantly higher than in primordial follicles. Cumulus–oocyte complexes (COCs) from both small and large antral follicles had significantly higher levels ofVIP mRNA than their respective granulosa/theca cells. During culture, the addition of VIP and/or FSH had o effect on follicular development. However, the presence of FSH and/or VIP in the culture medium significantly reduced VIP mRNA levels, but did not alter FSHR mRNA levels. In conclusion, VIP mRNA was detected in all goat follicular categories and cellular types, VIP and/or FSH did not affect the development of secondary follicles and reduce the expression of VIP mRNA levels.

Papel dos antioxidantes no cultivo in vitro de células ovarianas / The role of antioxidants in the in vitro culture of ovarian cells

Radicais livres são moléculas que contêm elétrons desemparelhados na camada de valência atuando na defesa do organismo contra agentes estranhos, podendo ser importantes marcadores da remodelação dos tecidos. Entretanto, um desequilíbrio entre sua produção e neutralização pode levar a danos celulares, denominados estresse oxidativo. Os agentes neutralizantes dessas moléculas são conhecidos como antioxidantes e podem ser de natureza enzimática ou não. Tendo em vista que o metabolismo oxidativo é essencial para a produção de energia em gametas e embriões, a produção de radicais livres é inevitável. Alguns agentes antioxidantes têm sido adicionados aos meios de cultivo de células ovarianas a fim de reduzir o estresse oxidativo. Sendo assim, a presente revisão objetiva descrever os principais antioxidantes e suas funções em compartimentos celulares com ênfase nas células ovarianas.(AU)

Free radical is any molecule containing unpaired electrons in the valence shell, which protects the body against foreign agents and can act as important markers in tissue remodelation. Nevertheless, an imbalance between production and neutralization of these factors can damage cells in a process named oxidative stress. Neutralizing agents for these molecules are known as antioxidants, which can be enzymatic in nature or not. Since oxidative metabolism is essential for energy production in gametes and embryos, free radicals generation is unavoidable. Some antioxidant agents have been employed in culture media for ovarian cells. In this context, the aim of present review is to describe the main antioxidants and their functions within cellular compartments, with focus on ovarian cells.(AU)

Papel dos antioxidantes no cultivo in vitro de células ovarianas / The role of antioxidants in the in vitro culture of ovarian cells

Radicais livres são moléculas que contêm elétrons desemparelhados na camada de valência atuando na defesa do organismo contra agentes estranhos, podendo ser importantes marcadores da remodelação dos tecidos. Entretanto, um desequilíbrio entre sua produção e neutralização pode levar a danos celulares, denominados estresse oxidativo. Os agentes neutralizantes dessas moléculas são conhecidos como antioxidantes e podem ser de natureza enzimática ou não. Tendo em vista que o metabolismo oxidativo é essencial para a produção de energia em gametas e embriões, a produção de radicais livres é inevitável. Alguns agentes antioxidantes têm sido adicionados aos meios de cultivo de células ovarianas a fim de reduzir o estresse oxidativo. Sendo assim, a presente revisão objetiva descrever os principais antioxidantes e suas funções em compartimentos celulares com ênfase nas células ovarianas.

Free radical is any molecule containing unpaired electrons in the valence shell, which protects the body against foreign agents and can act as important markers in tissue remodelation. Nevertheless, an imbalance between production and neutralization of these factors can damage cells in a process named oxidative stress. Neutralizing agents for these molecules are known as antioxidants, which can be enzymatic in nature or not. Since oxidative metabolism is essential for energy production in gametes and embryos, free radicals generation is unavoidable. Some antioxidant agents have been employed in culture media for ovarian cells. In this context, the aim of present review is to describe the main antioxidants and their functions within cellular compartments, with focus on ovarian cells.

Ascorbic acid improve the survival and in vitro growth of isolated caprine preantral follicles

The present study aims to investigate the influence of two concentrations of ascorbic acid on the survival, growth , antral formation and m RNA expression of the matrix metalloproteinases - 9 (MMP - 9) and their tissue inhibitor - 2 (TIMP - 2) on caprine preantral follicles during long - term in vitro culture. Isolated preantral follicles were individ ually cultured without or with ascorbic acid at 50 μ g/ml (AA50) or 100 μ g/ml (AA100) during 18 days. The parameters evaluated were follicular viability, growth, antrum formation and extruded oocytes. The genes MMP - 9 and TIMP - 2 were quantified by real - time polymer ase chain reaction (qPCR) after 18 days of culture in the control medium (MEM + ) or ascorbic acid (50 or 100 μ g/ml) and in fresh control (non cultured) . At the end of culture, AA50 significantly increased the percentage of viable follicles compared w ith other treatments . Moreover, mean daily increase in follicular diameter (μm/day) was significantly higher in the presence of both concentrations of ascorbic acid than in MEM + alone. Higher rates of antral formation and lower percentages of extruded oocy tes were observed in medium containing AA50 compared with control medium. Real Time RT - PCR assays showed that AA50 increase s MMP - 9 expression significantly compared with fresh control and MEM + alone. In conclusion, ascorbic acid at 50 μ g/ml was very import ant for the maintenance of caprine preantral follicle viability and development after in vitro culture and influences in vitro the enzymes involved with basement membrane remodeling.

Ascorbic acid improve the survival and in vitro growth of isolated caprine preantral follicles

The present study aims to investigate the influence of two concentrations of ascorbic acid on the survival, growth , antral formation and m RNA expression of the matrix metalloproteinases - 9 (MMP - 9) and their tissue inhibitor - 2 (TIMP - 2) on caprine preantral follicles during long - term in vitro culture. Isolated preantral follicles were individ ually cultured without or with ascorbic acid at 50 μ g/ml (AA50) or 100 μ g/ml (AA100) during 18 days. The parameters evaluated were follicular viability, growth, antrum formation and extruded oocytes. The genes MMP - 9 and TIMP - 2 were quantified by real - time polymer ase chain reaction (qPCR) after 18 days of culture in the control medium (MEM + ) or ascorbic acid (50 or 100 μ g/ml) and in fresh control (non cultured) . At the end of culture, AA50 significantly increased the percentage of viable follicles compared w ith other treatments . Moreover, mean daily increase in follicular diameter (μm/day) was significantly higher in the presence of both concentrations of ascorbic acid than in MEM + alone. Higher rates of antral formation and lower percentages of extruded oocy tes were observed in medium containing AA50 compared with control medium. Real Time RT - PCR assays showed that AA50 increase s MMP - 9 expression significantly compared with fresh control and MEM + alone. In conclusion, ascorbic acid at 50 μ g/ml was very import ant for the maintenance of caprine preantral follicle viability and development after in vitro culture and influences in vitro the enzymes involved with basement membrane remodeling.(AU)

Androstenedione and follicle stimulating hormone involvement in the viability and development of goat preantral follicles in vitro

This study investigated the effects of androstenedione and follicle-stimulating hormone (FSH) on the viability and growth of caprine preantral follicles. Ovarian tissues were cultured for 1 or 7 days in Minimum. Essential Medium (MEM) containing androstenedione (0, 1, 10, 50 or 100 ng/ml). FSH (50 ng/ml), or a combination of these two hormones. Cultured and non-cultured control tissues were processed for histological and fluorescence analysis. In comparison with non-cultured control, a sinfnificant reduction was noted in the percentage of normal follicles in all treatments after 1 and 7 days of culture (except in all treatment with 1 ng/ml of androstenedione for 1 day). As the culture period progressed from 1 to 7 days, treatments with 10 ng/ml of androstenedione + FSH or 50 ng/ml of androstenedione alone maintained the percentage of normal follicles. After 1 day, treatments with 10, 50, or 100 ng/ml of androstenedione + FSH, or with 50 ng/ml of androstenedione alone had more developing follicles than fresh control tissue. When comparing the culture periods, treatments with 1, 10 or 100 ng/ml of androstenedione alone, or FSH alone, or FSH with 1 ng/ml of androstenedione, showed an increase in the percentage of developing follicles. After 1 and 7 days, there were no differences in oocyte and follicular diameter among the treated samples and non-cultured control or MEM+ cultured tissue. Fluorescence analysis demonstrated that only fragments cultured in 50 or 100 ng/ml of androstenedione + FSH had viable preantral follicles similar to those observed in MEM+ alone. In conclusion, androstenedione at 50 or 100 ng/ml, either associated with FSH or at 50 ng/ml alone, plays an important role in the maintenance of caprine preantral follicle viability and activation after only a short in vitro culture period. In addition, after 7 days MEM+ alone was efficient in the maintenance of viability and in follicular activation, showing the importance of basic medium composition.(AU)

Androstenedione and follicle stimulating hormone involvement in the viability and development of goat preantral follicles in vitro

This study investigated the effects of androstenedione and follicle-stimulating hormone (FSH) on the viability and growth of caprine preantral follicles. Ovarian tissues were cultured for 1 or 7 days in Minimum. Essential Medium (MEM) containing androstenedione (0, 1, 10, 50 or 100 ng/ml). FSH (50 ng/ml), or a combination of these two hormones. Cultured and non-cultured control tissues were processed for histological and fluorescence analysis. In comparison with non-cultured control, a sinfnificant reduction was noted in the percentage of normal follicles in all treatments after 1 and 7 days of culture (except in all treatment with 1 ng/ml of androstenedione for 1 day). As the culture period progressed from 1 to 7 days, treatments with 10 ng/ml of androstenedione + FSH or 50 ng/ml of androstenedione alone maintained the percentage of normal follicles. After 1 day, treatments with 10, 50, or 100 ng/ml of androstenedione + FSH, or with 50 ng/ml of androstenedione alone had more developing follicles than fresh control tissue. When comparing the culture periods, treatments with 1, 10 or 100 ng/ml of androstenedione alone, or FSH alone, or FSH with 1 ng/ml of androstenedione, showed an increase in the percentage of developing follicles. After 1 and 7 days, there were no differences in oocyte and follicular diameter among the treated samples and non-cultured control or MEM+ cultured tissue. Fluorescence analysis demonstrated that only fragments cultured in 50 or 100 ng/ml of androstenedione + FSH had viable preantral follicles similar to those observed in MEM+ alone. In conclusion, androstenedione at 50 or 100 ng/ml, either associated with FSH or at 50 ng/ml alone, plays an important role in the maintenance of caprine preantral follicle viability and activation after only a short in vitro culture period. In addition, after 7 days MEM+ alone was efficient in the maintenance of viability and in follicular activation, showing the importance of basic medium composition.

Papel do Hormônio Folículo Estimulante na foliculogênese in vivo e in vitro / Role of Follicle Stimulating Hormone in folliculogenesis in vivo and in vitro

A foliculogênese é controlada por diversos hormônios e fatores de crescimento, que sinergicamente agem regulando os eventos envolvidos na fisiologia da reprodução. Dentre esses hormônios, destaca-se o Hormônio Folículo Estimulante (FSH), que é uma gonadotrofina presente em todas as fases da foliculogênese, atuando de forma direta ou indireta para promover o desenvolvimento folicular in vivo e in vitro. Nesse contexto, a revisão irá abordar o papel do FSH na regulação da foliculogênese de mamíferos, bem como a influência das diferentes origens de FSH sobre o crescimento folicular in vivo e in vitro e o seu papel na reprodução assistida.(AU)

The foliculogenesis is controlled by various hormones and growth factors, which act synergistically to regulate the events involved in the physiology of reproduction. Among these hormones, the Follicle Stimulating Hormone (FSH) is a gonadotropin which is present in all stages of folliculogenesis, acting directly or indirectly to promote the follicular development in vivo and in vitro. In this context, the review will contribute to a better understanding of the role of FSH in the regulation of folliculogenesis of mammals as well as the influence of different sources of FSH on the follicular growth in vivo and in vitro and its role in assisted reproduction.(AU)

Papel do Hormônio Folículo Estimulante na foliculogênese in vivo e in vitro / Role of Follicle Stimulating Hormone in folliculogenesis in vivo and in vitro

A foliculogênese é controlada por diversos hormônios e fatores de crescimento, que sinergicamente agem regulando os eventos envolvidos na fisiologia da reprodução. Dentre esses hormônios, destaca-se o Hormônio Folículo Estimulante (FSH), que é uma gonadotrofina presente em todas as fases da foliculogênese, atuando de forma direta ou indireta para promover o desenvolvimento folicular in vivo e in vitro. Nesse contexto, a revisão irá abordar o papel do FSH na regulação da foliculogênese de mamíferos, bem como a influência das diferentes origens de FSH sobre o crescimento folicular in vivo e in vitro e o seu papel na reprodução assistida.

The foliculogenesis is controlled by various hormones and growth factors, which act synergistically to regulate the events involved in the physiology of reproduction. Among these hormones, the Follicle Stimulating Hormone (FSH) is a gonadotropin which is present in all stages of folliculogenesis, acting directly or indirectly to promote the follicular development in vivo and in vitro. In this context, the review will contribute to a better understanding of the role of FSH in the regulation of folliculogenesis of mammals as well as the influence of different sources of FSH on the follicular growth in vivo and in vitro and its role in assisted reproduction.

Angiogenic factors and ovarian follicle development

Ovarian follicles require an adequate blood supply for oxygen, nutrients and hormones, in addition to eliminating CO2 and other metabolites. Acquisition of an adequate vascular supply is probably a limiting step in the selection and maturation of the dominant follicle. In this way, there is a progressive interest in the study of the growth factors involved in the angiogenic process. In addition, a better understanding about the mechanisms that regulate the expression and action of these factors could be a key point to increase the reproductive performance in females. Therefore, this review aims to summarize current data on the importance of the pro- and anti-angiogenic growth factors which regulate angiogenesis in ovarian follicle development.(AU)

Angiogenic factors and ovarian follicle development

Ovarian follicles require an adequate blood supply for oxygen, nutrients and hormones, in addition to eliminating CO2 and other metabolites. Acquisition of an adequate vascular supply is probably a limiting step in the selection and maturation of the dominant follicle. In this way, there is a progressive interest in the study of the growth factors involved in the angiogenic process. In addition, a better understanding about the mechanisms that regulate the expression and action of these factors could be a key point to increase the reproductive performance in females. Therefore, this review aims to summarize current data on the importance of the pro- and anti-angiogenic growth factors which regulate angiogenesis in ovarian follicle development.

Evaluation of miRNAs regulation of BDNF and IGF1 genes in T2DM insulin resistance in experimental models: bioinformatics based approach / Avaliação da regulação por miRNAs dos genes BDNF e IGF1 na resistência a insulina no diabetes do tipo 2 em modelos experimentais: in silico

microRNAs (miRNAs) are recognized as diabetes mellitus type 2 (T2DM) biomarkers useful for disease metabolism comprehension and have great potential as therapeutics targets. BDNF and IGF1 increased expression are highly involved in the benefits of insulin and glucose paths, however, they are down-regulated in insulin resistance conditions, while their expression increase is correlated to the improvement of glucose and insulin metabolism. Studies suggest the microRNA regulation of these genes in several different contexts, providing a novel investigation approach for comprehending T2DM metabolism and revealing potential therapeutic targets. In the present study, we investigate in different animal models (human, rat, and mouse) miRNAs that target BDNF and IGF1 in skeletal muscle tissue with T2DM physiological conditions. Bioinformatics tools and databases were used to miRNA prediction, molecular homology, experimental validation of interactions, expression in the studied physiological condition, and network interaction. The findings showed three miRNAs candidates for IGF1(miR-29a, miR-29b, and miR-29c) and one for BDNF (miR-206). The experimental evaluations and the search for the expression in skeletal muscle from T2DM subjects confirmed the predicted interaction between miRNA-mRNA for miR-29b and miR-206 through human, rat, and mouse models. This interaction was reaffirmed in multiple network analyses. In conclusion, our results show the regulation relationship between miR-29b and miR-206 with the investigated genes, in several tissues, suggesting an inhibition pattern. Nevertheless, these data show a large number of possible interaction physiological processes, for future biotechnological prospects.

Os microRNAs (miRNAs) são reconhecidos como biomarcadores do diabetes mellitus tipo 2 (DM2), úteis para a compreensão do metabolismo da doença, e possuem grande potencial como alvos terapêuticos. O aumento da expressão de BDNF e IGF1 está altamente envolvido nos benefícios as vias de insulina e glicose, porém, são regulados negativamente em condições de resistência à insulina, enquanto seu aumento de expressão está correlacionado com a melhora do metabolismo da glicose e da insulina. Estudos sugerem a regulação desses genes por microRNA em vários contextos diferentes, proporcionando uma nova abordagem de investigação para compreender o metabolismo do DM2 e revelar potenciais alvos terapêuticos. No presente estudo, investigamos em diferentes modelos animais (humanos, ratos e camundongos) miRNAs que têm como alvo BDNF e IGF1 em tecido muscular esquelético com condições fisiológicas de DM2. As análises foram realizadas utilizando ferramentas de bioinformática e bancos de dados para predição de miRNA, homologia molecular, validação experimental de interações, expressão na condição fisiológica estudada e interação em rede. Os resultados mostraram três candidatos a miRNAs para IGF1 (miR-29a, miR-29b e miR-29c) e um para BDNF (miR-206). As avaliações experimentais e a busca pela expressão no músculo esquelético de indivíduos com DM2 confirmaram a interação prevista entre miRNA-mRNA para miR-29b e miR-206 através de modelos humanos, ratos e camundongos. Essa interação foi reafirmada em múltiplas análises de rede. Em conclusão, nossos resultados mostram a relação de regulação entre miR-29b e miR-206 com os genes investigados, em diversos tecidos, sugerindo um padrão de inibição. Contudo, esses dados mostram um grande número de possíveis processos fisiológicos de interação para perspectivas biotecnológicas.