RESUMO
During the last 10 to 15 yr, in vitro research to predict antral follicle growth and oocyte maturation has delivered interesting advances in the knowledge of processes regulating follicle growth and developmental competence of oocytes. This review discusses the contribution of cumulus and mural granulosa cells in the process of oocyte maturation and cumulus expansion in cumulus-oocyte complexes (COCs) from follicles of different sizes and shows that differences in gene expression in oocytes, granulosa, and theca cells of small and large follicles impact the success of in vitro blastocyst development. In addition, the molecular mechanisms by which COC metabolism and antioxidant defense provide oocyte competence are highlighted. Furthermore, new insights and perspectives on molecular and cellular regulation of in vitro oocyte maturation are emphasized.
Assuntos
Desenvolvimento Embrionário/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Folículo Ovariano/fisiologia , Animais , FemininoRESUMO
SummaryThis study aimed to investigate the effects of IL1ß and TNFα on growth and maturation of oocytes from small follicles (1-3 mm) during in vitro culture. To this end, cumulus-oocyte complexes (COCs) with diameters of ~110 µm were cultured in TCM-199 medium alone or supplemented with IL1ß (10 ng/ml), TNFα (10 ng/ml) or both for 48 h. The oocytes were measured at the beginning and at the end of the culture period. COCs were cultured for 20 h in pre-maturation medium and then half of the COCs of each group was destined for in vitro maturation and the remaining COCs were used to evaluate meiotic progression, mitochondrial distribution and the expression of mRNAs for GDF-9, c-Mos, Cyclin-B1 and H1foo. The results showed that COCs cultured with TNFα alone or together with IL1ß had higher diameters than those cultured in control medium alone or supplemented with IL1ß. Control oocytes isolated from large antral follicles (>5 mm) had heterogeneous distribution of mitochondria. Oocytes isolated from small antral follicles, that had been grown in vitro in TCM-199 alone or supplemented with TNFα had similar heterogeneous mitochondrial distribution before in vitro maturation (IVM). After IVM, mitochondria were heterogeneously distribution when cultured in TCM-199. However, when cultured with TNFα and/or IL1ß, mitochondria were homogeneously distributed. Presence of TNFα and/or IL1ß in TCM-199 culture medium did not influence the expression of mRNAs for GDF-9, c-Mos, Cyclin-B1 and H1foo. In conclusion, TNFα and a mixture of TNFα and IL1ß both stimulated the growth of bovine oocytes during their in vitro culture, but do not influence gene expression in grown oocytes.
Assuntos
Técnicas de Maturação in Vitro de Oócitos/métodos , Interleucina-1beta/farmacologia , Oócitos/fisiologia , Folículo Ovariano/citologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Bovinos , Células Cultivadas , Ciclina B1/genética , Feminino , Regulação da Expressão Gênica , Fator 9 de Diferenciação de Crescimento/genética , Mitocôndrias/efeitos dos fármacos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mos/genéticaRESUMO
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
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/citologiaRESUMO
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
Ovarian fragments were exposed to 0.5 M sucrose and 1 M ethylene glycol (freezing solution; FS) with or without selenium or Trolox. Histological and ultrastructural analyses showed that the percentages of normal follicles in control tissue and in tissue after exposure to FS + 50 µM Trolox were similar. Trolox prevented endoplasmic reticulum (ER)-related vacuolization, which is commonly observed in oocytes and stromal tissue after exposure to FS. From the evaluated stress markers, superoxide dismutase 1 (SOD1) was up-regulated in ovarian tissue exposed to FS + 10 ng/ml selenium. Ovarian fragments were subsequently frozen-thawed in the presence of FS with or without 50 µM Trolox, followed by in vitro culture (IVC). Antioxidant capacity in ovarian fragments decreased after freeze-thawing in Trolox-free FS compared with FS + 50 µM Trolox. Although freezing itself minimized the percentage of viable follicles in each solution, Trolox supplementation resulted in higher rates of viable follicles (67 %), even after IVC (61 %). Furthermore, stress markers SOD1 and ERp29 were up-regulated in ovarian tissue frozen-thawed in Trolox-free medium. Relative mRNA expression of growth factors markers was evaluated after freeze-thawing followed by IVC. BMP4, BMP5, CTGF, GDF9 and KL were down-regulated independently of the presence of Trolox in FS but down-regulation was less pronounced in the presence of Trolox. Thus, medium supplementation with 50 µM Trolox prevents ER stress and, consequently, protects ovarian tissue from ER-derived cytoplasmic vacuolization. ERp29 but not ERp60, appears to be a key marker linking stress caused by freezing-thawing and cell vacuolization.
Assuntos
Cebus/metabolismo , Cromanos/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Congelamento , Ovário/efeitos dos fármacos , Ovário/patologia , Vitamina E/análogos & derivados , Animais , Calreticulina/metabolismo , Crioprotetores/farmacologia , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Ovário/metabolismo , Ovário/ultraestrutura , Superóxido Dismutase/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 aim of this study was to evaluate whether an in vitro culture (IVC) medium containing either or not ß-mercaptoethanol (BME), bone morphogenetic protein 4 (BMP4), or pregnant mare serum gonadotrophin (PMSG) could be able to promote the development of capuchin monkeys' preantral follicles enclosed in ovarian cortical strips. Follicular viability after IVC was similar to control (89.32%). Primordial follicle recruitment to primary stage was not reached with IVC, but the rate of secondary follicle formation was increased in the medium supplemented with BME, BMP4, and PMSG (44.86%) when compared to IVC control (9.20%). In the medium supplemented with BME, BMP4, and PMSG, contrary to other media, anti-müllerian hormone-messenger RNA (mRNA) expression in ovarian tissue was upregulated (3.4-fold), while that of growth differentiation factor-9 was maintained. The BMP4-mRNA expression, however, appeared downregulated in all cultured tissues. Our findings show a favorable effect of BME, BMP4, and PMSG on the in vitro development of secondary follicles from capuchin monkeys.
Assuntos
Cebus/fisiologia , Técnicas de Maturação in Vitro de Oócitos , Folículo Ovariano/fisiologia , Ovário/fisiologia , Animais , Hormônio Antimülleriano/genética , Hormônio Antimülleriano/metabolismo , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 4/farmacologia , Cebus/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Gonadotropinas Equinas/farmacologia , Fator 9 de Diferenciação de Crescimento/genética , Fator 9 de Diferenciação de Crescimento/metabolismo , Técnicas de Maturação in Vitro de Oócitos/métodos , Mercaptoetanol/farmacologia , Folículo Ovariano/metabolismo , Ovário/metabolismo , RNA Mensageiro/metabolismo , Fatores de Tempo , Técnicas de Cultura de Tecidos , Sobrevivência de TecidosRESUMO
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.
Assuntos
Hormônio Foliculoestimulante/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator 9 de Diferenciação de Crescimento/metabolismo , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oogênese , Folículo Ovariano/metabolismo , RNA Mensageiro/metabolismo , Matadouros , Animais , Bovinos , Sobrevivência Celular , Feminino , Líquido Folicular/enzimologia , Líquido Folicular/metabolismo , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/biossíntese , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Hialuronan Sintases , Isoenzimas/antagonistas & inibidores , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Oócitos/citologia , Oócitos/enzimologia , Oócitos/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/crescimento & desenvolvimento , Antígeno Nuclear de Célula em Proliferação/biossíntese , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteoglicanas/antagonistas & inibidores , Proteoglicanas/biossíntese , Proteoglicanas/genética , Proteoglicanas/metabolismo , Receptores do FSH/antagonistas & inibidores , Receptores do FSH/biossíntese , Receptores do FSH/genética , Receptores do FSH/metabolismo , Técnicas de Cultura de Tecidos/veterináriaRESUMO
This study investigated the stability of housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase, ß-tubulin, ß-actin, phosphoglycerate kinase (PGK), 18S rRNA, ubiquitin and ribosomal protein 19) and the levels of mRNA for bone morphogenetic protein-2 (BMP-2), -4 (BMP-4), -6 (BMP-6), -7 (BMP-7) and -15 (BMP-15), their receptors (BMPR-IA, -IB and -II) and Similar to Mothers Against Decapentaplegic (SMADs) (-1, -5 and -8) in goat follicles of 0.2, 0.5 and 1.0mm, as well as in secondary follicles before and after culture for 18 days. ß-tubulin and PGK were the most stable housekeeping genes and the levels of mRNA for BMP-2 in follicles of 0.2mm were higher than in follicles of 0.5 and 1.0mm. For BMP-4, -6 and -7, the highest levels of mRNA were found in follicles of 1.0mm. The expression of BMPR-IB was higher in follicles of 0.2mm, whereas the levels of BMPR-II were higher in follicles of 0.5mm. The levels of mRNA for SMAD-5 were higher in follicles of 0.2mm, whereas SMAD-8 had higher levels in 0.5-mm follicles. After culture, follicles showed increased levels of mRNA for BMP-2 and reduced mRNA for BMP-4, BMP-7, BMPR-IA and SMAD-5. In conclusion, ß-tubulin and PGK are the most stable reference genes, and BMPs, their receptors and SMADs have variable levels of mRNA in the follicular size classes analysed.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/genética , Cabras/genética , Folículo Ovariano/metabolismo , Proteínas Smad/genética , Animais , Receptores de Proteínas Morfogenéticas Ósseas/análise , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Morfogenéticas Ósseas/análise , Proteínas Morfogenéticas Ósseas/metabolismo , Tamanho Celular , Células Cultivadas , Feminino , Cabras/metabolismo , Cabras/fisiologia , Folículo Ovariano/citologia , Folículo Ovariano/fisiologia , Estabilidade Proteica , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Smad/análise , Proteínas Smad/metabolismo , Fatores de TempoRESUMO
This study verified the in vitro effects of IGF-1, FSH or both on caprine preantral follicle development and mRNA levels encoding IGF-1, IGFR-1 and FSHR. Secondary follicles were cultured for six days with FSH, IGF-1 or IGF-1+FSH. The results showed that IGF-1 and/or FSH addition did not influence follicular development for six days. The interaction between IGF-1 and FSH increased the mRNA levels of IGF-1 and FSHR, and FSH increased the expression of the IGFR-1 mRNA. Thus, IGF-1 and/or FSH increased IGF-1, IGFR-1 and FSHR mRNA levels in in vitro cultured caprine secondary follicles, but they did not influence their development after six days of in vitro culture.
Assuntos
Hormônio Foliculoestimulante/farmacologia , Cabras/fisiologia , Fator de Crescimento Insulin-Like I/farmacologia , Folículo Ovariano/fisiologia , Receptor IGF Tipo 1/metabolismo , Receptores do FSH/metabolismo , Animais , Feminino , Hormônio Foliculoestimulante/metabolismo , Regulação da Expressão Gênica/fisiologia , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor IGF Tipo 1/genética , Receptores do FSH/genéticaRESUMO
This study aimed to evaluate the effects of FSH and BMP - 7 on growth and on expression of FSH - R , BMP - 7 and BMP receptors in cultured secondary follicles. Goat secondary follicles (~200 μm) were isolated and cultured in vitro, with 5% CO 2 in air at 39°C, for 6 days in the presence of BMP - 7 (50 ng/m l ) supplemented or not with FSH (50 ng/m l ). Follicular diameter and the formation of the antrum were evaluated before and after culture. For each treatment, at the end of culture period, groups of 6 follicles were collected and, after extraction of total RNA and cDNA synthesis, the levels of mRNA for FSH - R, BMP - 7 and BMP receptors in cultured secondary follicles were quantified by real time PCR. The results showed that addition of BMP - 7 or F SH to culture medium stimulated growth of secondary follicles , while addition of both BMP - 7 and FSH was needed to significantly increase the percentage of follicles forming an antrum and the follicular levels of mRNA for both BMP - 7 and FSH - R. For BMP receptors, FSH reduced the levels of mRNA for BMPR - IA and BMP - RII in comparison with those follicles cultured in MEM alone and supplemented with BMP - 7, respectively. In conclusion , l ike FSH, BMP - 7 affect s in vitro growth of cultured secondary follicle s , but it stimulates antrum formation and expression of the mRNA s for BMP - 7 and FSH - R only in presence of both BMP - 7 and FSH . However, the levels of mRNA f or BMP - RIA and BMP - RII are reduced in follicles culture d in medium supplemented with FSH.(AU)
Assuntos
Animais , Folículo Ovariano/anatomia & histologia , Cabras/fisiologiaRESUMO
This study aimed to evaluate the effects of FSH and BMP - 7 on growth and on expression of FSH - R , BMP - 7 and BMP receptors in cultured secondary follicles. Goat secondary follicles (~200 μm) were isolated and cultured in vitro, with 5% CO 2 in air at 39°C, for 6 days in the presence of BMP - 7 (50 ng/m l ) supplemented or not with FSH (50 ng/m l ). Follicular diameter and the formation of the antrum were evaluated before and after culture. For each treatment, at the end of culture period, groups of 6 follicles were collected and, after extraction of total RNA and cDNA synthesis, the levels of mRNA for FSH - R, BMP - 7 and BMP receptors in cultured secondary follicles were quantified by real time PCR. The results showed that addition of BMP - 7 or F SH to culture medium stimulated growth of secondary follicles , while addition of both BMP - 7 and FSH was needed to significantly increase the percentage of follicles forming an antrum and the follicular levels of mRNA for both BMP - 7 and FSH - R. For BMP receptors, FSH reduced the levels of mRNA for BMPR - IA and BMP - RII in comparison with those follicles cultured in MEM alone and supplemented with BMP - 7, respectively. In conclusion , l ike FSH, BMP - 7 affect s in vitro growth of cultured secondary follicle s , but it stimulates antrum formation and expression of the mRNA s for BMP - 7 and FSH - R only in presence of both BMP - 7 and FSH . However, the levels of mRNA f or BMP - RIA and BMP - RII are reduced in follicles culture d in medium supplemented with FSH.
Assuntos
Animais , Folículo Ovariano/anatomia & histologia , Cabras/fisiologiaRESUMO
During the last decade, involvement of growth hormone (GH), insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in ovarian folliculogenesis has been extensively studied. This review provides an update on the GH, IGF system and their role in ovarian follicular development. In vitro studies and knockout experiments demonstrated an important role of GH in preantral follicle growth and differentiation through their binding with GH receptors, which are located both in the oocyte and follicular somatic tissues. Furthermore, GH stimulates the development of small antral follicles to gonadotrophin-dependent stages, as well as maturation of oocytes. With regard to the IGF system, IGF-I has no effects on primordial follicle development, but both IGF-I and IGF-II stimulate growth of secondary follicles. Depending on the species studies and method used, these proteins have been detected in oocytes and/or somatic cells. In antral follicles, these IGFs stimulate granulosa cell proliferation and steroidogenesis in most mammals. The bioavailability of IGFs is regulated by a family of intrafollicular expressed IGF binding proteins (IGFBPs). Facilitation of IGF can be increased through the activity of specific IGFBP proteases, which degrade the IGF/IGFBP complex, resulting in the production of IGFBP fragments and release of attached IGF.
Assuntos
Hormônio do Crescimento/fisiologia , Folículo Ovariano/fisiologia , Somatomedinas/fisiologia , Animais , Feminino , Hormônio do Crescimento/farmacologia , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/fisiologia , Folículo Ovariano/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
The aims of the present study were to evaluate the effects of fibroblast growth factor-2 (FGF-2) on survival, activation and growth of caprine early-staged (preantral) follicles using histological and ultrastructural studies. Fragments of caprine ovarian cortex were cultured for 1 or 5 days in an enriched minimum essential medium, supplemented or not with different concentrations of FGF-2 (10, 50 or 100 ng/ml). Fragments from non-cultured ovarian tissue (control) and from tissues cultured for 1 or 5 days in a specific medium were processed for transmission electron microscopy (TEM) or classical histology to evaluate the morphological quality of caprine preantral follicles and to calculate the percentages of normal follicles. Additionally, effects of FGF-2 on oocyte and follicle diameter of cultured preantral follicles were investigated. Our results showed that, although the percentages of histologically normal follicles were lower in cultured than in non-cultured ovarian tissue fragments, there were no differences in this regard among treatments, neither on day 1 nor on day 5 of culture. After 1 and 5 days of culture, a significantly higher percentage of growing follicles was observed in the medium supplemented with 50 ng/ml of FGF-2. This FGF-2 treatment furthermore resulted in an increase in diameter of both oocytes and follicles that were cultured for 5 days. TEM showed that the ultrastructural integrity of caprine preantral follicles was maintained during their 5-day culture in the presence of 50 ng/ml FGF-2. In conclusion, this study demonstrated that at a concentration of 50 ng/ml FGF-2 not only maintains the morphological integrity of caprine preantral follicles cultured for 5 days, but also stimulates the activation of primordial follicles and the growth of activated follicles.