RESUMO
OBJECTIVE: Folliculogenesis is a complex process involving various ovarian paracrine factors. During folliculogenesis, vitamin D3 and progesterone are significant for the proper development of follicles. This study aimed to investigate the effects of vitamin D3 and selective progesterone receptor modulator ulipristal acetate on ovarian paracrine factors. METHODS: In the study, 18 female Wistar-albino rats were randomly divided into three groups: control group (saline administration, n=6), vitamin D3 group (300 ng/day vitamin D3 oral administration, n=6), and UPA group (3 mg/kg/day ulipristal acetate oral administration, n=6). Ovarian tissue was analyzed by histochemistry and immunohistochemistry. For quantification of immunohistochemistry, the mean intensities of growth differentiation factor 9, bone morphogenetic protein 15, and forkhead box O3a expressions were measured by Image J and MATLAB. Blood samples were collected for the analysis of serum anti-Müllerian hormone levels by ELISA. RESULTS: Atretic follicles and hemorrhagic cystic structures were observed in the UPA group. After immunohistochemistry via folliculogenesis assessment markers, growth differentiation factor 9, bone morphogenetic protein 15, and cytoplasmic forkhead box O3a expressions decreased in the UPA group (p<0.05). Anti-Müllerian hormone level did not differ significantly between the experimental groups (p>0.05). CONCLUSION: Ulipristal acetate negatively affects folliculogenesis via ovarian paracrine factors. The recommended dietary vitamin D3 supplementation in healthy cases did not cause a significant change.
Assuntos
Hormônio Antimülleriano , Proteína Morfogenética Óssea 15 , Proteína Forkhead Box O3 , Fator 9 de Diferenciação de Crescimento , Ovário , Animais , Feminino , Ratos , Hormônio Antimülleriano/metabolismo , Proteína Morfogenética Óssea 15/metabolismo , Colecalciferol/farmacologia , Fator 9 de Diferenciação de Crescimento/metabolismo , Ratos Wistar , Proteína Forkhead Box O3/metabolismoRESUMO
Granulosa cells (GCs) play important roles in the regulation of ovarian functions, and in vitro culture is a relevant model for the study of steroidogenesis in ovarian follicles. Thus, growth factors secreted by the oocyte, like Growth and Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15), play an important part in the luteinization of granulosa cells. The aim of this work was to express GDF9 and BMP15 genes in bovine GCs in vitro and evaluate their effects on the luteinization process. Samples of culture medium and GCs transfected with GDF9 and BMP15 were obtained for 21 consecutive days to analyse the steroidogenic hormones' concentration (progesterone (P4 ) and estradiol (E2 )) and the expression of STAR, GDF9 and BMP15 and their respective receptors. The results demonstrated an inhibitory effect of GDF9 and BMPF15 on P4 secretion in bovine GCs cultured in vitro. Moreover, our study demonstrated the entire expression of their respective receptors (TGFBR1, BMPR1B and BMPR2) and the inhibition of the steroidogenic marker, STAR gene. This work sheds light on a novel biological function of BMP15 and GDF9 in bovine GCs physiology, which could elucidate a non-described biological role for GDF9 and BMP15 in bovine granulosa cells' metabolism.
Assuntos
Proteína Morfogenética Óssea 15/metabolismo , Estradiol/metabolismo , Fator 9 de Diferenciação de Crescimento/metabolismo , Progesterona/metabolismo , Animais , Proteína Morfogenética Óssea 15/genética , Bovinos , Células Cultivadas , Feminino , Células da Granulosa , Fator 9 de Diferenciação de Crescimento/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismoRESUMO
The objective of this study was to determine the effects of TNF-α and IL-1ß on development and survival of bovine secondary follicle culture in vitro for 18 days. Secondary follicles (~0.2 mm) were isolated from ovarian cortex and individually cultured at 38.5°C, with 5% CO2 in air, for 18 days, in TCM-199+ alone (cultured control) or supplemented with 10 ng/ml IL-1ß, 10 ng/ml TNF-α or both TNF-α and IL-1ß. The effects of these treatments on growth, follicular survival, antrum formation, viability, ultrastructure and mRNA levels for GDF-9, c-MOS, H1foo and Cyclin B1 were evaluated. The results showed that addition of TNF-α to culture medium increased follicular diameter and rate of antrum formation, whereas that of IL-1ß and a mixture of IL-1ß and TNF-α did not do so. Ultrastructural analysis showed that, among the tested cytokine treatments, follicles cultured in the presence of TNF-α had the best-preserved oocytes and granulosa cells. The presence of TNF-α, IL-1ß or both did not influence the expression of mRNAs analysed. In conclusion, in contrast to IL-1ß, TNF-α promotes growth of and antrum formation in in vitro cultured bovine secondary follicles, while their ultrastructure and viability were maintained.
Assuntos
Bovinos/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-1beta/farmacologia , Folículo Ovariano/crescimento & desenvolvimento , Fator de Necrose Tumoral alfa/farmacologia , Animais , Ciclina B1/genética , Ciclina B1/metabolismo , Feminino , Fator 9 de Diferenciação de Crescimento/genética , Fator 9 de Diferenciação de Crescimento/metabolismo , Histonas/genética , Histonas/metabolismo , Interleucina-1beta/administração & dosagem , Folículo Ovariano/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mos/genética , Proteínas Proto-Oncogênicas c-mos/metabolismo , Técnicas de Cultura de Tecidos/veterinária , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
CONTEXT: Bone morphogenetic protein (BMP)15 is an oocyte-specific growth factor, which, together with growth differentiation factor (GDF) 9, regulates folliculogenesis and ovulation rate. Multiple mutations in BMP15 have been identified in women with primary ovarian insufficiency (POI), supporting a pathogenic role; however, the underlying biological mechanism of many of these mutants remains unresolved. OBJECTIVES: To determine how mutations associated with ovarian dysfunction alter the biological activity of human BMP15. DESIGN: The effects of 10 mutations in BMP15 on protein production, activation of granulosa cells, and synergy with GDF9 were assessed. RESULTS: Sequencing of 35 patients with POI identified both an unrecognized BMP15 variant (c.986G>A, R329H) and a variant (c.581T>C, F194S) previously associated with the condition. Assessing expression and activity of these and 8 other BMP15 mutants identified: (1) multiple variants, including L148P, F194S, and Y235C, with reduced mature protein production; (2) three variants (R138H, A180T, and R329H) with â¼fourfold lower activity than wild-type BMP15; and (3) 3 variants (R68W, F194S, and N196K) with a significantly reduced ability to synergize with GDF9. CONCLUSIONS: Mutations in BMP15 associated with POI reduce mature protein production, activity, or synergy with GDF9. The latter effect is perhaps most interesting given that interactions with GDF9 most likely underlie the physiology of BMP15 in the human ovary.
Assuntos
Proteína Morfogenética Óssea 15/genética , Fator 9 de Diferenciação de Crescimento/metabolismo , Insuficiência Ovariana Primária/genética , Adulto , Proteína Morfogenética Óssea 15/metabolismo , Proteína Morfogenética Óssea 15/farmacologia , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Humanos , Modelos Moleculares , Mutação , Insuficiência Ovariana Primária/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) play important functions in follicular and oocyte development in many species. This study evaluated the dynamic expression of GDF-9 and BMP-15 in canine follicles cells using flow cytometry analysis. Follicular cells were removed from three sizes of antral follicles (small, medium and large) from ovaries of bitches throughout the estrus cycle. Cells were incubated with anti-human GDF-9 polyclonal and anti-mouse BMP-15 monoclonal antibodies. A size and complexity discriminatory gate was used for the cytometryc analysis in the initial dot plot and, additionally, a CD45 marker for leukocyte and propidium iodide (PI) were used for erythrocyte and debris discrimination. The evidence corroborated the presence of both proteins in canine follicle cells, but these proteins were not expressed equally during follicular development. The results analyzed by ANOVA showed that GDF-9 expression decreased (P<0.05) during follicular growth in anestrus and proestrous/estrous, but increased in diestrus (P<0.05). The expression levels of BMP-15 rose (P<0.05) from small to medium sizes in anestrous without changing at diestrus. Small antral follicles expressed the highest values of GDF-9 at anestrus while only BMP-15 showed higher value in small antral follicles at proestrous-estrus compared to diestrus and anestrus. Both proteins decreased in proestrous/estrous (P<0.05) with increasing follicle size, registering the lowest levels in large follicles. The flow cytometric assay was able to assess GDF-9 and BMP-15 expression in canine follicular cells, showing that these proteins were differentially expressed during follicular development, possibly related to the special features of canine reproduction.
Assuntos
Proteína Morfogenética Óssea 15/metabolismo , Cães/fisiologia , Fator 9 de Diferenciação de Crescimento/metabolismo , Folículo Ovariano/fisiologia , Animais , Proteína Morfogenética Óssea 15/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Fator 9 de Diferenciação de Crescimento/genética , Folículo Ovariano/metabolismoRESUMO
BACKGROUND: During fish oocyte maturation, specific molecules are expressed and accumulated within oocyte until fertilization and embryo development. Special attention have been paid in members of the transforming growth factor (TGF-ß) superfamily; growth differentiation factor 9 (GDF9/gdf9) and bone morphogenetic protein 15 (BMP15/bmp15), which exert regulatory functions during oocyte maturation and follicle development. However, little attention has been paid to the involvement of these molecules during embryogenesis considering its importance for the formation of a good quality egg and subsequent embryo survival. The purpose of this study was to analyze the expression of gdf9 and bmp15 in previtellogenic oocytes and during early embryonic development in Seriola lalandi, a pelagic fish with increasing prospect for its aquaculture development, which however, show high mortality at embryo and larval stages. RESULTS: Through RT-qPCR it was found that gdf9 expression was higher in previtellogenic oocytes decreasing after ovulation. This expression profile agrees with its participation in early stages of the follicular development. The transcripts for bmp15 also showed the highest levels in previtellogenic oocytes, however this expression was lower than obtained with gdf9. Conversely, in recently spawned oocytes mRNA bmp15 levels were highest than observed to gdf9. This, is consequent with the main role proposed for this growth factor at the final fish oocyte maturation: avoid the ovulation of an immature oocyte. During embryo development, low levels of mRNA were detected to gdf9, with an increase in 48 H post-fertilization embryos. The bmp15 expression did not change throughout development and was higher than gdf9 at 16 cells, blastula and appearance embryos stages. CONCLUSIONS: Both (gdf9 and bmp15) expression profiles in previtellogenic oocytes and newly spawned eggs are consistent with the described functions for these growth factors in vertebrate ovarian physiology in early and late stages of the follicular development. So, these genes could be considered as quality biomarkers at these stages. However, further studies of these proteins throughout folliculogenesis, are necessaries to fully understand their functions during the oocyte formation. In addition, the persistent expression of these growth factors during development, allows us to speculate possible roles in embryonic processes, which must also be addressed.
Assuntos
Proteína Morfogenética Óssea 15/metabolismo , Fator 9 de Diferenciação de Crescimento/metabolismo , Oócitos/metabolismo , Perciformes/embriologia , Vitelogênese/fisiologia , Animais , Biomarcadores/análise , Primers do DNA , DNA Complementar/análise , Desenvolvimento Embrionário/genética , Peixes/embriologia , Perciformes/classificação , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Gênica/fisiologiaRESUMO
BACKGROUND: During fish oocyte maturation, specific molecules are expressed and accumulated within oocyte until fertilization and embryo development. Special attention have been paid in members of the transforming growth factor (TGF-ß) superfamily; growth differentiation factor 9 (GDF9/gdf9) and bone morphogenetic protein 15 (BMP15/bmp15), which exert regulatory functions during oocyte maturation and follicle development. However, little attention has been paid to the involvement of these molecules during embryogenesis considering its importance for the formation of a good quality egg and subsequent embryo survival. The purpose of this study was to analyze the expression of gdf9 andbmp15 in previtellogenic oocytes and during early embryonic development in Seriola lalandi, a pelagic fish with increasing prospect for its aquaculture development, which however, show high mortality at embryo and larval stages. RESULTS: Through RT-qPCR it was found that gdf9 expression was higher in previtellogenic oocytes decreasing after ovulation. This expression profile agrees with its participation in early stages of the follicular development. The transcripts for bmp15 also showed the highest levels in previtellogenic oocytes, however this expression was lower than obtained with gdf9. Conversely, in recently spawned oocytes mRNA bmp15 levels were highest than observed to gdf9. This, is consequent with the main role proposed for this growth factor at the final fish oocyte maturation: avoid the ovulation of an immature oocyte. During embryo development, low levels of mRNA were detected to gdf9, with an increase in 48 H post-fertilization embryos. The bmp15 expression did not change throughout development and was higher than gdf9 at 16 cells, blastula and appearance embryos stages. CONCLUSIONS: Both (gdf9 and bmp15) expression profiles in previtellogenic oocytes and newly spawned eggs are consistent with the described functions for these growth factors in vertebrate ovarian physiology in early and late stages of the follicular development. So, these genes could be considered as quality biomarkers at these stages. However, further studies of these proteins throughout folliculogenesis, are necessaries to fully understand their functions during the oocyte formation. In addition, the persistent expression of these growth factors during development, allows us to speculate possible roles in embryonic processes, which must also be addressed.
Assuntos
Animais , Oócitos/metabolismo , Vitelogênese/fisiologia , Perciformes/embriologia , Proteína Morfogenética Óssea 15/metabolismo , Fator 9 de Diferenciação de Crescimento/metabolismo , Transcrição Gênica/fisiologia , Perciformes/classificação , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Biomarcadores/análise , DNA Complementar/análise , Primers do DNA , Desenvolvimento Embrionário/genética , Reação em Cadeia da Polimerase em Tempo Real , Peixes/embriologiaRESUMO
The aim of this study was to characterize in canine oocytes and cumulus cells the dynamic expression of growth differentiation factor 9 (GDF-9) in relation to meiotic development and cumulus expansion throughout in vitro maturation (IVM). Cumulus oocytes complexes (COCs) from ovaries of adult bitches were cultured intact for IVM during 0, 48, 72, and 96 hours. At 0 hours or after IVM, COCs were divided into two groups: one group remained with their cumulus cells and in the other group the cumulus cells were extracted. The expression levels of GDF-9 were determined in both groups using indirect immunofluorescence and Western blot analysis. For immunofluorescence assay, in vivo-matured oocytes collected from oviducts were also used as a positive control. The nuclear stage was analyzed in parallel with 4'-6-diamidino-2-phenylindole staining in denuded oocytes from all maturing groups. The intensity of fluorescence, indicative of GDF-9 expression level, decreased with time (P < 0.05). High expression was observed only in germinal vesicle nonmature oocytes; in contrast, second metaphase oocytes showed only low expression. Western blot analysis showed bands of approximately 56 kd and a split band of approximately 20 kd representing the proprotein and possibly two mature protein forms of GDF-9, respectively. The proprotein was detected in all samples, and it was highly expressed before IVM and in a lesser degree, during the first 48 hours, declining thereafter in coincidence with the expansion of the cumulus cell (P < 0.05). There was a negative correlation (r = -0.97; P < 0.05) between the expression level of GDF-9 and mucification. Mature forms were evident only in COCs, before culture and up to 48 hours of IVM. It was concluded that GDF-9 is expressed in canine oocytes and cumulus cells, mainly in the early developmental states, with low levels in mature oocytes in vitro and in vivo, representing the first approach of GDF-9 dynamic in dog oocyte maturation.
Assuntos
Cães , Fator 9 de Diferenciação de Crescimento/metabolismo , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , Animais , Células Cultivadas , Células do Cúmulo/fisiologia , Feminino , Imunofluorescência , Oócitos/ultraestrutura , Oogênese/fisiologiaRESUMO
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.