RESUMO
Context The overproduction of reactive oxygen species (ROS) during in vitro culture of ovarian tissues impairs follicular development and survival. Aims To evaluate the effects of punicalagin on the development and survival of primordial follicles, stromal cell and collagen fibres, as well as on the levels of mRNA for nuclear factor erythroid 2-related factor 2 (NRF2 ), superoxide dismutase 1 (SOD1 ), catalase (CAT ), glutathione peroxidase 1 (GPX1 ) and perirredoxin 6 (PRDX6 ), and activity of antioxidant enzymes in cultured bovine ovarian tissues. Methods Bovine ovarian cortical tissues were cultured for 6days in α-MEM+ alone or with 1.0, 10.0, or 100.0µM punicalagin at 38.5°C with 5% CO2 . Follicle morphology and growth, stromal cell density, and collagen fibres were evaluated by classical histology, while the expression of mRNA was evaluated by real-time PCR. The activity of enzymes was analysed by the Bradford method. Key results Punicalagin improved follicle survival and development, reduced mRNA expression for SOD1 and CAT , but did not influence stromal cells or collagen fibres. Punicalagin (10.0µM) increased the levels of thiol and activity of SOD1, CAT , and GPX1 enzymes. Conclusions Punicalagin (10.0µM) promotes follicle survival and development and activates SOD1, CAT , and GPX1 enzymes in bovine ovarian tissues. Implications Punicalagin improves follicle development and survival in cultured ovarian tissues.
Assuntos
Catalase , Glutationa Peroxidase GPX1 , Glutationa Peroxidase , Taninos Hidrolisáveis , Folículo Ovariano , Animais , Feminino , Bovinos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Folículo Ovariano/enzimologia , Taninos Hidrolisáveis/farmacologia , Glutationa Peroxidase/metabolismo , Glutationa Peroxidase/genética , Catalase/metabolismo , Catalase/genética , Ovário/efeitos dos fármacos , Ovário/enzimologia , Ovário/metabolismo , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase-1/genética , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Técnicas de Cultura de Tecidos , Superóxido Dismutase/metabolismoRESUMO
To assess the effect of biodentine (BD) and MTA-angelus (MTA) on biocompatibility, BMP2, BMP4, and osteocalcin (OC) expression. Subcutaneously implanted tubes of four groups (MTA, BD, Control, and Sham) were kept over 15, 30, and 60 days; histological analyses were performed using H&E and Von Kossa; ELISA quantified IL-1ß and IL-8 expression; and qRT-PCR verified gene expression of BMPs and OC. Sham showed slight changes in profile/intensity of inflammatory infiltrate in all periods. Control had an inflammatory score significantly higher than Sham at 15 days (p < .05). BD revealed a similar inflammatory response to Sham, without significant changes over periods. MTA group exhibited an increase in chronic inflammatory profile at 30 days, with significant reduction at 60 days, when compared to Sham (p < .05). At 30/60 days, experimental groups presented birefringent areas. At 30/60 days, BD and MTA significantly increase IL-1ß compared to Control, whereas an increase in IL-8 was observed only in BD. At 30/60 days, BD produces an expression of BMP2 whereas MTA influenced BMP4 and OC. Materials tested are biocompatible and they have osteoinductive activity; the materials influenced the expression of the tested mediators differently, suggesting different affinities with the substrate and the dental substrates.
Assuntos
Materiais Biocompatíveis/farmacologia , Bismuto/farmacologia , Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 4/biossíntese , Calcificação Fisiológica/efeitos dos fármacos , Compostos de Cálcio/farmacologia , Óxidos/farmacologia , Materiais Restauradores do Canal Radicular/farmacologia , Silicatos/farmacologia , Animais , Masculino , Ratos , Ratos WistarRESUMO
Infertility is the result of failure due to an organic disorder of the reproductive organs, especially their gametes. Recently, much progress has been made on generating germ cells, including oocytes, from various types of stem cells. This review focuses on advances in female germ cell differentiation from different kinds of stem cells, with emphasis on embryonic stem cells, adult stem cells, and induced pluripotent stem cells. The advantages and disadvantages of the derivation of female germ cells from several types of stem cells are also highlighted, as well as the ability of stem cells to generate mature and functional female gametes. This review shows that stem cell therapies have opened new frontiers in medicine, especially in the reproductive area, with the possibility of regenerating fertility.
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Diferenciação Celular/fisiologia , Células Germinativas/citologia , Oócitos/citologia , Células-Tronco/citologia , Animais , Feminino , HumanosRESUMO
Frutalin is a galactose-binding lectin that has an irreversible cytotoxic effect on HeLa cervical cancer cells, by inducing apoptosis and inhibiting cell proliferation. It was previously shown that after in vitro incubation, frutalin is internalized into HeLa cells nucleus, which indicates that frutalin apoptosis-inducing activity might be linked with its nuclear localization. Considering that drugs commonly used for cancer treatment have a deleterious effect on germ cells, the aim of this study was to evaluate the effect of frutalin on the activation, survival, ultrastructure and gene expression in follicles cultured within ovarian tissue. Goat ovarian fragments were cultured for 6 days in α-MEM⺠alone or supplemented with frutalin (1, 10, 50, 100 or 200 µg/ml). Non-culturad and cultured tissues were processed for histological and ultrastructural analysis and they were also stored to evaluate the expression of anti- and pro-apoptotic genes by quantitative polymerase chain reaction (qPCR). The results showed that the frutalin, at all concentrations tested, reduced follicular survival when compared with control medium. Higher concentrations of frutalin (50, 100 or 200 µg/ml) also reduced follicular survival when compared with those tissues cultured with 1 or 10 µg/ml of frutalin. The ultrastructural analysis showed that atretic cultured follicles had retracted oocytes and a large number of vacuoles spread throughout the cytoplasm. In addition, signs of damage of mitochondrial membranes and cristae were observed. Moreover, although a dose-response effect on gene expression has not been observed, when compared with tissues culture in control medium, the presence of frutalin increased in mRNA expression pro-apoptotic genes. In conclusion, frutalin reduces follicular survival at all concentrations tested, its effects being more pronounced when high concentrations of this lectin (50, 100 and 200 µg/ml) are used. Gene expression profile and ultrastrutural features of cultured follicles suggest that follicular death in goat ovarian tissue cultured in presence of frutalin occurs via necrosis.
Assuntos
Antineoplásicos Fitogênicos/toxicidade , Galectinas/toxicidade , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica , Cabras , Necrose , Folículo Ovariano/metabolismo , Folículo Ovariano/ultraestrutura , Ovário/metabolismo , Ovário/ultraestrutura , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
This study evaluates the effect of different concentrations (0, 10, 50 and 100ng/mL) of bone morphogenetic protein-2 (BMP-2) on primordial and secondary follicle development. It also investigates the effects of FSH and BMP-2 on the growth, morphology, ultrastructure and expression of mRNA for GDF9, NLRP5 and NPM2 genes in secondary follicles cultured for 18 days. The presence of BMP-2 at all tested concentrations increased the development of primordial follicles in vitro, but the highest concentration of BMP-2 (100 ng/mL) reduced the percentage of normal follicles when compared with tissues cultured with 10 ng/mL BMP-2. During culture of secondary follicles, in contrast to higher concentrations (50 or 100 ng/mL), 10 ng/mL BMP-2 kept the morphology of follicles during initial stages of in vitro culture. This concentration of BMP-2 also benefits maintenance of the ultrastructure of 18-day cultured follicles. The presence of both BMP-2 and FSH in culture medium resulted in a significant (P<0.05) increase in follicular diameter after 18 days of culture. However, both FSH and BMP-2 reduced follicular mRNA expression of GDF9 and NLRP5 when compared to follicles cultured in media containing only FSH. In combination with FSH, BMP-2 reduced the mRNA levels of NPM2, when compared to follicles cultured in control medium. It is concluded from these data that 10 ng/mL BMP-2 promotes the growth of primordial in vitro and it helps to maintain the ultrastructure of secondary follicles, while FSH is more important for better expression of follicular markers like GDF9 and NLRP5.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Folículo Ovariano/fisiologia , Animais , Proteína Morfogenética Óssea 2/farmacologia , Bovinos , Células Cultivadas , Feminino , Hormônio Foliculoestimulante/metabolismo , Hormônio Foliculoestimulante/farmacologia , Regulação da Expressão Gênica , Fator 9 de Diferenciação de Crescimento/biossíntese , Técnicas In Vitro , Nucleoplasminas/biossíntese , Oócitos , Folículo Ovariano/efeitos dos fármacos , RNA Mensageiro/análiseRESUMO
The bone morphogenetic protein (BMP) family consists of several growth factor proteins that belong to the transforming growth factor-ß (TGF-ß) superfamily. BMPs bind to type I and type II serine-threonine kinase receptors, and transduce signals through the Smad signalling pathway. BMPs have been identified in mammalian ovaries, and functional studies have shown that they are involved in the regulation of oogenesis and folliculogenesis. This review summarizes the role of the BMP system during formation, growth and maturation of ovarian follicles in mammals.
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Proteínas Morfogenéticas Ósseas/fisiologia , Oogênese/fisiologia , Ovário/crescimento & desenvolvimento , Animais , Receptores de Proteínas Morfogenéticas Ósseas/genética , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Feminino , Mamíferos , Ovário/fisiologia , Transdução de SinaisRESUMO
This study aims to investigate the effects of jacalin and follicle-stimulating hormone (FSH) on activation and survival of goat primordial follicles, as well as on gene expression in cultured ovarian tissue. Ovarian fragments were cultured for 6 days in minimum essential medium (MEM) supplemented with jacalin (10, 25, 50 or 100 µg/ml - Experiment 1) or in MEM supplemented with jacalin (50 µg/ml), FSH (50 ng/ml) or both (Experiment 2). Non-cultured and cultured tissues were processed for histological and ultrastructural analysis. Cultured tissues from Experiment 2 were also stored to evaluate the expression of BMP-15, KL (Kit ligand), c-kit, GDF-9 and proliferating cell nuclear antigen (PCNA) by real-time polymerase chain reaction (PCR). The results of Experiment 1 showed that, compared with tissue that was cultured in control medium, the presence of 50 µg/ml of jacalin increased both the percentages of developing follicles and viability. In Experiment 2, after 6 days, higher percentages of normal follicles were observed in tissue cultured in presence of FSH, jacalin or both, but no synergistic interaction between FSH and jacalin was observed. These substances had no significant effect on the levels of mRNA for BMP-15 and KL, but FSH increased significantly the levels of mRNA for PCNA and c-kit. On the other hand, jacalin reduced the levels of mRNA for GDF-9. In conclusion, jacalin and FSH are able to improve primordial follicle activation and survival after 6 days of culture. Furthermore, presence of FSH increases the expression of mRNA for PCNA and c-kit, but jacalin resulted in lower GDF-9 mRNA expression.
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Hormônio Foliculoestimulante/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Lectinas de Plantas/farmacologia , Animais , Proteína Morfogenética Óssea 15/genética , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Cabras , Fator 9 de Diferenciação de Crescimento/genética , Folículo Ovariano/citologia , Folículo Ovariano/fisiologia , Antígeno Nuclear de Célula em Proliferação/genética , Fator de Células-Tronco/genética , Técnicas de Cultura de TecidosRESUMO
This study investigated mRNA levels for insulin-like growth factors (IGFs) IGF1 (IGF-I) and IGF2 (IGF-II), IGF receptors (IGF1R and IGF2R), and binding proteins (IGFBP-1, IGFBP-2. IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6) in bovine follicles of 0.2, 0.5 or 1.0 mm in diameter. mRNA expression levels in in vitro cultured follicles that reached approximately 0.5 mm were compared with that of in vivo grown follicles. IGF1R and IGF2R expression levels in 0.5 mm in vivo follicles were higher than in 1.0 or 0.2 mm follicles, respectively. IGFBP-1, IGFBP-2. IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6 showed variable expression in the follicular size classes analyzed. In vitro grown follicles had significantly reduced expression levels for IGF1, IGF1R, IGFBP-3, IGFBP-5 and IGFBP-6 mRNA when compared with 0.2 mm follicles, but, when compared with in vivo grown follicles (0.5 mm), only IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-6 showed a reduction in their expression. In conclusion, IGFs, their receptors and IGFBPs showed variable expression of mRNA levels in the follicular size classes analyzed.
Assuntos
Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like I/genética , Folículo Ovariano/fisiologia , Receptores de Somatomedina/genética , Animais , Bovinos , Feminino , Regulação da Expressão Gênica , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 6 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Folículo Ovariano/citologia , RNA Mensageiro , Receptor IGF Tipo 2/genética , Técnicas de Cultura de TecidosRESUMO
Expression of BMP-6 mRNA was quantified by real-time polymerase chain reaction (PCR) and the BMP-6 protein was demonstrated by immunohistochemistry in the primordial, primary, secondary, small and large antral follicles of goat. Furthermore, the influence of BMP-6 on increase in diameter, antrum formation and expression of BMP-6 and FSH-R in in vitro cultured secondary follicles was studied. Therefore, goat primordial, primary and secondary follicles, as well as small and large antral follicles were obtained and the mRNA levels of BMP-6 were quantified by PCR in real time. Expression of BMP-6 protein in goat follicles was demonstrated by immunohistochemistry. The influence of BMP-6 in the presence or absence of follicle-stimulating hormone (FSH) on both the development of secondary follicles and the expression of mRNA for BMP-6 and FSH-R was evaluated after 6 days of culture. Furthermore, the follicular diameter and the formation of the antrum were evaluated before and after 6 days of culture and compared by Kruskal-Wallis and chi-squared tests (P < 0.05), respectively. The results show that the level of mRNA for BMP-6 in primary and secondary follicles was significantly higher than in the primordial follicles (P < 0.05). Similar levels of BMP-6 mRNA were observed in cumulus-oocyte complexes and mural granulosa/theca cells from small and large antral follicles, respectively. BMP-6 protein was expressed in oocytes of all categories of follicles and in granulosa cells from secondary follicles onwards. Addition of BMP-6 to the culture medium increased the diameter of secondary follicles mainly by antrum formation after 6 days' culture, in the presence or absence of FSH (P < 0.05). Furthermore, addition of FSH resulted in increased levels of BMP-6 mRNA in these follicles (P < 0.05). Simultaneous administration of FSH and BMP-6 enhanced the levels of FSH receptor (FSH-R) mRNA (P < 0.05). It is concluded that BMP-6 mRNA is increased during transition from primordial to primary/secondary follicles in the goat ovaries and that BMP-6 enhances the growth of cultured secondary follicles.
Assuntos
Proteína Morfogenética Óssea 6/genética , Folículo Ovariano/fisiologia , Animais , Proteína Morfogenética Óssea 6/metabolismo , Proteína Morfogenética Óssea 6/farmacologia , Meios de Cultura/química , Meios de Cultura/farmacologia , Células do Cúmulo/metabolismo , Feminino , Hormônio Foliculoestimulante/metabolismo , Hormônio Foliculoestimulante/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/fisiologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Ovário/fisiologia , RNA Mensageiro/metabolismo , Receptores do FSH/genética , Técnicas de Cultura de TecidosRESUMO
The aim of the present study was to investigate the stability of six housekeeping genes, and the relative expression of growth factors (EGF, GDF-9, BMP-15, VEGF, FGF-2, BMP-6, IGF-1 and KL) and hormone receptors (FSH, LH and GH) in goat preantral follicles. To evaluate to stability of housekeeping genes micro-dissected fresh follicles (150-200 µm) as well as follicles that have been in vitro cultured for 12 days were used. In addition, isolated fresh follicles were used to compare expression of various growth factors and hormone receptors before culture. Both fresh and cultured follicles were subjected to total RNA extraction and synthesis of cDNA. After amplification of cDNA by real-time PCR, the geNorm software program was used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), ß-tubulin, ß-actin, phosphoglycerokinase (PGK), 18S rRNA, ubiquitin (UBQ) and ribosomal protein 19 (RPL-19). In addition, follicular steady-state levels of mRNA from the various growth factors under study were compared. Results demonstrated that, in goat preantral follicles, UBQ and ß-actin were the most suitable reference genes and thus could be used as parameters to normalize data from future in vitro studies. In contrast, 18S RNA appeared the least stable gene among the tested housekeeping genes. Analysis of mRNA for several hypophyseal hormone receptors in fresh preantral follicles showed significantly higher FSH-R mRNA levels than those of LH-R and GH-R, and no difference between GH-R and LH-R mRNA levels. In regard growth factor mRNA expression in goat preantral follicles, EGF mRNA levels appeared significantly lower than those of the other studied growth factors. Increasingly higher relative mRNA levels were observed for GDF-9, BMP-15, BMP-6, FGF-2, VEGF, Kl and IGF-1, successively. In conclusion, UBQ and ß-actin are the most stable housekeeping genes in fresh and 12-days cultured caprine preantral follicles. Furthermore, in fresh follicles, high levels of FSH-R mRNA are detected while among eight growth factors, IGF-1 is the most highly expressed and EGF the weakest expressed compound.