RESUMO

This study evaluated the effects of frutalin (0.6, 6.0 or 60.0 µg/mL) and doxorubicin (0.3 µg/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 6 days of culture, frutalin or doxorubicin reduced the percentage of normal follicles (P < 0.05), but doxorubicin had higher toxicity than frutalin. Except for follicles cultured with 0.6 µg/mL frutalin, follicular growth rate was reduced after culture with doxorubicin or frutalin (P < 0.05). The presence doxorubicin or 60.0 µg/mL frutalin increased the levels of mRNA for Casp3, Casp6, Bax, and Bcl2 (P < 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 (P < 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 Tecidos

RESUMO

This study evaluated the effect of bone morphogenetic proteins 2 (BMP2) and 4 (BMP2) on follicle development and mRNA expression for GDF9, Cyclin B1, BMPR1A, BMPR1B, BMPRII, FSHR and SMAD1 in bovine secondary follicles cultured in vitro. Isolated secondary follicles were cultured for 18 days in TCM199+ medium alone or supplemented with BMP2 (10 ng/mL), BMP4 (100 ng/mL) or combination of both BMP2 and 4. Real-time PCR was used to analyze mRNA levels in fresh and cultured follicles. After 18 days of culture, follicles cultured with BMP2 alone or with BMP4 alone had larger diameters when compared to control (P < .05). In addition, all treatments promoted antrum formation and maintained a high viability rate through the growing period. The presence of BMP2, BMP4 or both together did not influence mRNA expression for the tested genes. However, the in vitro culture induces down-regulation for mRNA expression of BMPR1A. In conclusion, the addition of BMP2 or BMP4 alone in cultured medium promotes follicular growth and antrum formation in bovine follicles after 18 days of in vitro culture.

Assuntos

Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Folículo Ovariano/efeitos dos fármacos , Animais , Receptores de Proteínas Morfogenéticas Ósseas/genética , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Oogênese/efeitos dos fármacos , Oogênese/genética , Folículo Ovariano/fisiologia

RESUMO

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/farmacologia

RESUMO

Stem cells have been isolated from ovaries, and their ability to differentiate into oocytes in vitro has been demonstrated for mice and human, but not for bovine species. The aims of this study were to isolate germline stem cells from bovine ovaries and to evaluate the effects of bone morphogenetic proteins (BMPs) 2 and 4, and follicular fluid on the differentiation of these stem cells into oocyte-like structures. The ovarian stem cells were isolated and cultured in α-MEM+ supplemented with BMP2, BMP4 or follicular fluid. On days 0 and 14, cells were evaluated for their morphological appearance, viability, expression of alkaline phosphatase and for markers of germ cell formation (VASA and DAZL) and oocyte development (GDF9, ZPA and SCP3) by qPCR. Levels of mRNA were analysed using ANOVA and Bonferroni test (p < .05). The results showed that at day 0, ovarian stem cells expressed specific markers of pluripotency (OCT4, SOX). In addition, these cells were positive for alkaline phosphatase, which is a marker commonly used to identify primordial germ cells (PGCs). After the period of differentiation, cells had morphological features that resemble PGCs and oocyte-like cells (OLCs). An increase, ranging from five to 14 times, in the expression of VASA was observed in cells cultured in medium supplemented with BMPs and follicular fluid, while the increase in DAZL expression ranged from four to six times. In addition, OLCs had an increase in expression of mRNAs for GDF9, ZPA and SCP3 that ranged from two to eight times. In conclusion, OLCs can be differentiated in vitro from ovarian stem cells and BMPs and follicular fluid are effective in stimulating the expression of mRNAs for germ cell and oocyte markers.

Assuntos

Técnicas de Cultura de Células/veterinária , Diferenciação Celular/fisiologia , Ovário/citologia , Células-Tronco/fisiologia , Animais , Bovinos , Células Cultivadas , Feminino , Células Germinativas/citologia , Oócitos/citologia

RESUMO

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/química

RESUMO

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/química

RESUMO

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/química

RESUMO

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

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/metabolismo

RESUMO

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/metabolismo

RESUMO

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/metabolismo