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The Notch and transforming growth factor (TGF)-b 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-b signalling in the regulation of preantral granulosa cell (PAGC) proliferation. Activation of TGF-b 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-b signalling by Smad3 knockdown reduced Hey2 expression. Inhibition of the proliferation of PAGCs by N-[N-(3,5- difluorophenacetyl)-L-alanyl]-S-phenylglycine z-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-b 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article presents the study regarding cell density fermentation and on the effect of recombinant chicken interferon-gamma (ChIFN-γ) activity on chicken macrophages, which showed that DO-stat feeding method enhances final cell density.