RESUMO
The Notch and transforming growth factor (TGF)-ß signalling pathways play an important role in granulosa cell proliferation. However, the mechanisms underlying the cross-talk between these two signalling pathways are unknown. Herein we demonstrated a functional synergism between Notch and TGF-ß signalling in the regulation of preantral granulosa cell (PAGC) proliferation. Activation of TGF-ß signalling increased hairy/enhancer-of-split related with YRPW motif 2 gene (Hey2) expression (one of the target genes of the Notch pathway) in PAGCs, and suppression of TGF-ß signalling by Smad3 knockdown reduced Hey2 expression. Inhibition of the proliferation of PAGCs by N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester (DAPT), an inhibitor of Notch signalling, was rescued by both the addition of ActA and overexpression of Smad3, indicating an interaction between the TGF-ß and Notch signalling pathways. Co-immunoprecipitation (CoIP) and chromatin immunoprecipitation (ChIP) assays were performed to identify the point of interaction between the two signalling pathways. CoIP showed direct protein-protein interaction between Smad3 and Notch2 intracellular domain (NICD2), whereas ChIP showed that Smad3 could be recruited to the promoter regions of Notch target genes as a transcription factor. Therefore, the findings of the present study support the idea that nuclear Smad3 protein can integrate with NICD2 to form a complex that acts as a transcription factor to bind specific DNA motifs in Notch target genes, such as Hey1 and Hey2, and thus participates in the transcriptional regulation of Notch target genes, as well as regulation of the proliferation of PAGCs.
Assuntos
Proliferação de Células , Células da Granulosa/citologia , Receptores Notch/fisiologia , Transdução de Sinais , Fator de Crescimento Transformador beta/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Camundongos , Regiões Promotoras Genéticas , Proteínas Repressoras/fisiologia , Proteína Smad3/fisiologiaRESUMO
The growth of oocytes and the development of follicles require certain pathways involved in cell proliferation and survival, such as the phosphatidylinositol 3-kinase (PI3K) pathway and the Notch signalling pathway. The aim of the present study was to investigate the interaction between Notch and the PI3K/AKT signalling pathways and their effects on primordial follicle recruitment. When the Notch pathway was inhibited by L-685,458 or N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester (DAPT) in vitro, the expression of genes in the pathway and the percentage of oocytes in growing follicles decreased significantly in mouse ovaries. By 2 days postpartum, ovaries exposed to DAPT, short interference (si) RNA against Notch1 or siRNA against Hairy and enhancer of split-1 (Hes1) had significantly decreased expression of HES1, the target protein of the Notch signalling pathway. In contrast, expression of phosphatase and tensin homologue (Pten), a negative regulator of the AKT signalling pathway, was increased significantly. Co immunoprecipitation (Co-IP) revealed an interaction between HES1 and PTEN. In addition, inhibition of the Notch signalling pathway suppressed AKT phosphorylation and the proliferation of granulosa cells. In conclusion, the recruitment of primordial follicles was affected by the proliferation of granulosa cells and regulation of the interaction between the Notch and PI3K/AKT signalling pathways.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Oogênese , Folículo Ovariano/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Animais , Comunicação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Camundongos , Oogênese/efeitos dos fármacos , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , PTEN Fosfo-Hidrolase/genética , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Transdução de Sinais/efeitos dos fármacos , Técnicas de Cultura de Tecidos , Fatores de Transcrição HES-1/antagonistas & inibidores , Fatores de Transcrição HES-1/genética , Fatores de Transcrição HES-1/metabolismoRESUMO
Diethylhexyl phthalate (DEHP) is an estrogen-like compound widely used as a plasticizer in commercial products and is present in medical devices, and common household items. It is considered an endocrine disruptor since studies on experimental animals clearly show that exposure to DEHP can alter epigenetics of germ cells. This study was designed to assess the effects of DEHP on DNA methylation of imprinting genes in germ cells from fetal and adult mouse. Pregnant mice were treated with DEHP at doses of 0 and 40 µg DEHP/kg body weight/day from 0.5 to 18.5 day post coitum. The data revealed DEHP exposure significantly reduced the percentage of methylated CpG sites in Igf2r and Peg3 differentially methylated regions (DMRs) in primordial germ cells from female and male fetal mouse, particularly, in the oocytes of 21 dpp mice (F1), which were produced by the pregnant micetreated with DEHP. More surprisingly, the modification of the DNA methylation of imprinted genes in F1 mouse oocytes was heritable to F2 offspring which exhibit lower percentages of methylated CpG sites in imprinted genes DMRs. In conclusion, DEHP exposure can affect the DNA methylation of imprinting genes not only in fetal mouse germ cells and growing oocytes, but also in offspring's oocytes.
Assuntos
Metilação de DNA/efeitos dos fármacos , Dietilexilftalato/toxicidade , Impressão Genômica/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Animais , Metilação de DNA/imunologia , Feminino , Impressão Genômica/genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Oogênese/genética , Gravidez , Receptor IGF Tipo 2/genéticaRESUMO
Notch signaling pathway, a highly conserved cell signaling system, exists in most multicellular organisms. The objective of this study was to examine Notch signaling pathway in germ cell cyst breakdown and primordial follicle formation. The receptor and ligand genes of Notch pathway (Notch1, Notch2, Jagged1, Jagged2 and Hes1) were extremely down-regulated after newborn mouse ovaries were cultured then exposed to DAPT or L-685,458 in vitro (P < 0.01). Since DAPT or L-685,548 inhibits Notch signaling pathway, the expression of protein LHX8 and NOBOX was significantly reduced during the formation of the primordial follicles. Down-regulated mRNA expression of specific genes including Lhx8, Figla, Sohlh2 and Nobox, were also observed. The percentages of female germ cells in germ cell cysts and primordial follicles were counted after culture of newborn ovaries for 3 days in vitro. The result showed female germ cells in cysts was remarkably up-regulated while as the oocytes in primordial follicles was significantly down-regulated (P < 0.05). In conclusion, Notch signaling pathway may regulate the formation of primordial follicle in mice.