RESUMO
This study evaluated the effects of frutalin (0.6, 6.0 or 60.0⯵g/mL) and doxorubicin (0.3⯵g/mL) on survival, growth and ultrastructure of in-vitro cultured goat secondary follicles. The effects of these substances on the levels of mRNA for Casp3, Casp6, Bax, and Bcl2 were also investigated. Results showed that, after 6â¯days of culture, frutalin or doxorubicin reduced the percentage of normal follicles (Pâ¯<â¯0.05), but doxorubicin had higher toxicity than frutalin. Except for follicles cultured with 0.6⯵g/mL frutalin, follicular growth rate was reduced after culture with doxorubicin or frutalin (Pâ¯<â¯0.05). The presence doxorubicin or 60.0⯵g/mL frutalin increased the levels of mRNA for Casp3, Casp6, Bax, and Bcl2 (Pâ¯<â¯0.05). Higher mRNA levels for Casp3, Casp6 and Bax were found in follicles cultured with doxorubicin, but higher levels of Bcl2 mRNA were found in follicles cultured with frutalin (Pâ¯<â¯0.05). In conclusion, frutalin has lower toxic effects than doxorubicin on secondary follicles cultured in vitro.
Assuntos
Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Galectinas/farmacologia , Cabras , Folículo Ovariano/efeitos dos fármacos , Animais , Antineoplásicos/administração & dosagem , Relação Dose-Resposta a Droga , Feminino , Galectinas/administração & dosagem , Regulação da Expressão Gênica/efeitos dos fármacos , Folículo Ovariano/ultraestrutura , RNA Mensageiro/genética , Técnicas de Cultura de TecidosRESUMO
This study was conducted to detect the protein expression of TNF-α system members (TNF-α/TNFR1/TNFR2) in bovine ovarian follicles and to evaluate the effects of TNF-α or dexamethasone on the survival and growth of primordial follicles in vitro, as well as on gene expression in cultured ovarian tissue. It was hypothesized that TNF-α induces follicular atresia in ovarian tissues cultured in vitro, and that dexamethasone suppresses the production of endogenous TNF-α, which can improve follicle viability in vitro. Ovarian fragments were cultured for 6days in α-MEM+ supplemented with TNF-α (0, 1, 10, 100 or 200ng/ml) or dexamethasone (0, 1, 10, 100 or 200ng/ml). After culture, the expression of mRNAs for BCL-2, BAX, P53, TNF-α, and CASP3 and CASP6 were evaluated. Immunohistochemical results showed that the TNF-α system members, were detected in bovine preantral and antral follicles. After 6days, the TNF-α (10ng/ml) treatment reduced the percentage of normal preantral follicles and increased the number of TUNEL-positive cells in cultured tissue. Dexamethasone (10ng/ml) during 6days of culture did maintain the percentage of normal follicles and the ultrastructure of follicles, while the presence of TNF-α or dexamethasone did not influence primordial follicle activation. However, TNF-α or dexamethasone had no effect on the levels of mRNA for P53, BCL-2, BAX and CASP6, in cultured tissues, but the presence of dexamethasone reduced the levels of CASP3 compared to ovarian slices cultured in control medium (α-MEM+). In conclusion, proteins of the TNF-α system are expressed at different bovine follicle stages. The addition of TNF-α in culture reduces follicle survival and increases the number of apoptotic cells in ovarian tissue, while the presence of dexamethasone maintains follicle ultrastructure in cultured tissue.
Assuntos
Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Folículo Ovariano/metabolismo , Técnicas de Cultura de Tecidos/veterinária , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apoptose , Bovinos , Sobrevivência Celular , Feminino , Folículo Ovariano/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
This study evaluated (1) the effects of in vivo GnRH treatment on mRNA expression of TNF-α system (TNF-α, TNFR1 and TNFR2) in granulosa cells of bovine preovulatory follicles, (2) the in vitro influence of gonadotropins on mRNA expression of TNF-α system in cultured cumulus cells, (3) the protein expression of the TNF-α system in late antral follicles and, (4) the influence of TNF-α on cumulus cells expansion, ultrastructure and on expression of HAS2, CASP3 and CASP6 in follicular cells cultured for 24 h. An increased expression of TNF-α and TNFR1 was observed after 3, 6 and 12 h of GnRH treatment when compared to 0 and 24h. Higher TNFR2 mRNA levels were observed 3, 6 and 12 h after GnRH, when compared to 0 and 24 h. Proteins of TNF-α system were also expressed in late antral follicles. In vitro, TNF-α did not affect cumulus cells expansion, but reduced the HAS2, CASP3 and CASP6 mRNA levels in cumulus cells after 12 h. After 24 h of culture, TNF-α increased the mRNA levels for CASP6 in mural granulosa cells, while the TNF-α, TNFR1 and TNFR2 mRNA levels were increased in cumulus-oocyte complexes (COCs) cultured for 12 h with gonadotropins, but not after 24 h. Ultrastructural analysis confirmed the integrity of COCs cultured in presence of TNF-α. In conclusion, TNF-α system members are present in bovine antral follicles and expression of TNF-α is influenced by gonadotropins in vivo and in vitro. In vitro, TNF-α maintained cumulus cells ultrastructure during COC culture.
Assuntos
Hormônio Liberador de Gonadotropina/farmacologia , Hormônio Luteinizante/metabolismo , Folículo Ovariano/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Bovinos , Células Cultivadas , Células do Cúmulo/metabolismo , Células do Cúmulo/ultraestrutura , Feminino , Expressão Gênica , Hormônio Luteinizante/farmacologia , Oócitos/metabolismo , Oócitos/ultraestrutura , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
This study aimed to investigate the expression of interleukin 1 (IL-1) system members (proteins and messenger RNA of ligands and receptors) and its distribution in ovarian follicles of cyclic cows and to evaluate the effects of IL-1ß on the survival and activation of primordial follicles in vitro. The ovaries were processed for localization of IL-1 system in preantral and antral follicles by immunohistochemical, real-time polymerase chain reaction, and Western blot analysis. For in vitro studies, ovarian fragments were cultured in α-MEM(+) supplemented with IL-1ß (0, 1, 10, 50, or 100 ng/mL), and after 6 d, the cultured tissues were processed for histologic analysis. Immunohistochemical results showed that the IL-1 system proteins IL-1ß, IL-1RA, IL-1RI, and IL-1RII were detected in the cytoplasm of oocytes and granulosa cells from all follicular categories and theca cells of antral follicles. Variable levels of messenger RNA for the IL-1 system members were observed at different stages of development. After 6 d of culture, the presence of IL-1ß (10 or 50 ng/mL) was effective in maintaining the percentage of normal follicles and in promoting primordial follicle activation. In conclusion, IL-1 system members are differentially expressed in ovarian follicles according to their stage of development. Moreover, IL-1ß promotes the development of primordial follicles. These results indicate an important role of the IL-1 system in the regulation of bovine folliculogenesis.
Assuntos
Bovinos/fisiologia , Interleucina-1/análise , Interleucina-1beta/farmacologia , Folículo Ovariano/química , Folículo Ovariano/fisiologia , RNA Mensageiro/análise , Animais , Western Blotting , Feminino , Células da Granulosa/química , Imuno-Histoquímica/veterinária , Proteína Antagonista do Receptor de Interleucina 1/análise , Proteína Antagonista do Receptor de Interleucina 1/genética , Interleucina-1/genética , Interleucina-1beta/análise , Interleucina-1beta/genética , Oócitos/química , Folículo Ovariano/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Receptores Tipo II de Interleucina-1/análise , Receptores Tipo II de Interleucina-1/genética , Células Tecais/químicaRESUMO
This study evaluated the effect of different concentrations of bone morphogenetic protein-4 (BMP-4), as well as the interaction of BMP-4 and follicle stimulating hormone (FSH) on growth, ultrastructural integrity, and expression of mRNA for growth differentiation factor-9 (GDF-9), BMP-15, maternal antigen that the embryo requires (Mater) and nucleoplasmin-2 (Npm-2) in bovine secondary follicles cultured in vitro for 18 days. Follicles cultured in the presence of 50 ng/ml BMP-4 had a progressive increase in their diameters with the increase of culture period from 0 to 6 and 12 days, but no significant differences were observed among treatments. The presence of both FSH and BMP-4 in a culture medium did not stimulate follicle growth when compared to the control medium. After 12 days, the percentage of normal follicles was maintained similar to that of day 0 in the medium supplemented with both FSH and BMP-4, but no significant differences among treatments were observed after 18 days of culture. BMP-4 maintained the ultrastructural integrity of follicles after 18 days of culture, while follicles cultured in medium supplemented with FSH or both BMP-4 and FSH had oocyte with irregular zona pellucida, vesicular bodies, and an abundance of vacuoles. Follicles cultured in the presence of BMP-4 had an increase in the levels of BMP-15 mRNA, when compared to those cultured in medium supplemented with FSH alone. In conclusion, the addition of BMP-4 in culture medium contributes to preserve follicular ultrastructure, but BMP-4 did not interact positively with FSH. Regarding secondary follicles cultured in the presence of FSH, BMP-4 increases the expression of mRNA for BMP-15.
Assuntos
Feminino , Animais , Bovinos , Bovinos/anatomia & histologia , Bovinos/fisiologia , Hormônio Foliculoestimulante/análogos & derivados , Hormônio Foliculoestimulante/efeitos adversos , /administração & dosagem , /efeitos adversos , Oócitos/enzimologia , RNA Mensageiro/análise , RNA Mensageiro/químicaRESUMO
This study evaluated the effect of different concentrations of bone morphogenetic protein-4 (BMP-4), as well as the interaction of BMP-4 and follicle stimulating hormone (FSH) on growth, ultrastructural integrity, and expression of mRNA for growth differentiation factor-9 (GDF-9), BMP-15, maternal antigen that the embryo requires (Mater) and nucleoplasmin-2 (Npm-2) in bovine secondary follicles cultured in vitro for 18 days. Follicles cultured in the presence of 50 ng/ml BMP-4 had a progressive increase in their diameters with the increase of culture period from 0 to 6 and 12 days, but no significant differences were observed among treatments. The presence of both FSH and BMP-4 in a culture medium did not stimulate follicle growth when compared to the control medium. After 12 days, the percentage of normal follicles was maintained similar to that of day 0 in the medium supplemented with both FSH and BMP-4, but no significant differences among treatments were observed after 18 days of culture. BMP-4 maintained the ultrastructural integrity of follicles after 18 days of culture, while follicles cultured in medium supplemented with FSH or both BMP-4 and FSH had oocyte with irregular zona pellucida, vesicular bodies, and an abundance of vacuoles. Follicles cultured in the presence of BMP-4 had an increase in the levels of BMP-15 mRNA, when compared to those cultured in medium supplemented with FSH alone. In conclusion, the addition of BMP-4 in culture medium contributes to preserve follicular ultrastructure, but BMP-4 did not interact positively with FSH. Regarding secondary follicles cultured in the presence of FSH, BMP-4 increases the expression of mRNA for BMP-15.(AU)
Assuntos
Animais , Feminino , Bovinos , Bovinos/anatomia & histologia , Bovinos/fisiologia , Proteína Morfogenética Óssea 4/administração & dosagem , Proteína Morfogenética Óssea 4/efeitos adversos , Hormônio Foliculoestimulante/análogos & derivados , Hormônio Foliculoestimulante/efeitos adversos , Oócitos/enzimologia , RNA Mensageiro/análise , RNA Mensageiro/químicaRESUMO
The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (â¼0.2â mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200â µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1â µg/mL PHA (96.43%); 10â µg/mL PHA (84.85%); 50â µg/mL PHA (85.29%); 100â µg/mL PHA (88.57%), and 200â µg/mL PHA (87.50)], but the presence of 10â µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10â µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10â µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.
Assuntos
Hormônio Foliculoestimulante/metabolismo , Mitógenos/farmacologia , Folículo Ovariano/efeitos dos fármacos , Fito-Hemaglutininas/farmacologia , Animais , Feminino , Hormônio Foliculoestimulante/genética , Cabras , Técnicas In Vitro , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/ultraestrutura , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Mensageiro/metabolismoRESUMO
The objective of the present study was to determine the effects of bone morphogenetic protein (BMP)-15 and FSH on the growth, viability, and expression of mRNA for FSH (FSH-R) and BMP-15 (BMPR-IB and BMPR-II) receptors in cultured bovine secondary follicles. Secondary follicles were microdissected and cultured for 12 days in minimum essential medium-α alone or supplemented with BMP-15, sequential FSH, both BMP-15 and FSH, or BMP-15 from days 0 to 6, and FSH from days 7 to 12. Thereafter, the effect of these treatments on the follicular volume, viability, and antrum formation and the levels of mRNA for BMPR-IB, BMPR-II, and FSH-R were assessed. Compared with day 0, the follicles cultured with FSH or BMP-15, or both, had a significant and progressive increase in volume (P < 0.05). However, the follicles cultured for 12 days with both BMP-15 and FSH had the greatest volume and a greater rate of antrum formation than those in control medium, but results similar to those cultured with FSH (days 0 to 12) or BMP-15 (days 0 to 6) and FSH (days 7 to 12). Together with their accelerating effect on in vitro follicle growth, the combination of FSH and BMP-15 induced ultrastructural changes in the cultured follicles and increased atresia. However, adding either BMP-15 or FSH to the culture medium, not only promoted follicular growth and follicular antrum formation, but also maintained follicular viability during culture. Except for follicles cultured in minimal essential medium-α, the levels of mRNA for BMPR-IB were reduced, and the levels of mRNA for FSH-R were significantly greater in follicles cultured in medium supplemented with BMP-15. In conclusion, all in vitro follicle treatments supported growth of bovine preantral follicles; however, adding both BMP-15 and FSH to the culture medium (minimal essential medium-α) for 12 days provided the greatest stimulation. Furthermore, the viability and ultrastructural integrity of cultured follicles were only maintained when only BMP-15 or FSH was added to the culture medium.
Assuntos
Proteína Morfogenética Óssea 15/farmacologia , Bovinos , Hormônio Foliculoestimulante/farmacologia , Atresia Folicular/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Animais , Receptores de Proteínas Morfogenéticas Ósseas/genética , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Feminino , Regulação da Expressão Gênica , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do FSH/genética , Receptores do FSH/metabolismoRESUMO
The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (∼0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1 µg/mL PHA (96.43%); 10 µg/mL PHA (84.85%); 50 µg/mL PHA (85.29%); 100 µg/mL PHA (88.57%), and 200 µg/mL PHA (87.50)], but the presence of 10 µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10 µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10 µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.
Assuntos
Animais , Feminino , Hormônio Foliculoestimulante/metabolismo , Mitógenos/farmacologia , Folículo Ovariano/efeitos dos fármacos , Fito-Hemaglutininas/farmacologia , Hormônio Foliculoestimulante/genética , Cabras , Técnicas In Vitro , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/ultraestrutura , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Mensageiro/metabolismoRESUMO
The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e., hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL⻹) from Days 0 to 6 and 500 ng mL⻹ from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P<0.05). Alone, 200 ng mL⻹ GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL⻹ bovine serum albumin, 10 µg mL⻹ insulin, 5.5 µg mL⻹ transferrin, 5 ng mL⻹ selenium, 2 mM glutamine, 2mM hypoxanthine and 50 µg mL⻹ ascorbic acid (α-MEMâº). Comparisons of uncultured (0.2 mm) and α-MEM⺠cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P<0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.