A Preliminary Study Of Notch Signaling Pathway Genes Expression In Bovine Ovarian Follicle Development

Objective: Notch signaling pathway is a highly conserved signaling pathway in evolution, which can be conducted signaling molecules in between cells. So Notch signaling pathway can influence cell proliferation, differentiation and apoptosis. However, nowadays people are mainly focused on the regulation of Notch signaling pathway in mammalian follicular development of the formation and recruitment of primordial follicles. The purpose of this experiment is to study the genes expression change of Notch signaling pathway in follicular growth、development and dominance. To explore which gene can help the follicular dominance of Bos taurus. The study will provide theory basis for understand the regulation of Notch signaling pathway in follicular development.Methods: In this study, we use the granulosa cells(GCs)/cumulus granulosa cells(CGCs) in growing follicles and dominant follicles of Bos taurus as experimental materials. And we use PT-PCR screening、 real-time quantitative PCR to detect gene expression changes about Notch2, Jagged1, RBP/Jκ, hes1 and c-myc in Notch signaling pathway. We culture grown follicle’ granulosa cells in vitro. And we detect these cells by immunofluorescence, MTT and cell transfection in order to observe the relationship between Notch signaling pathway and granulosa cells proliferation. In the days which followed we haved screened some Marker molecules of Bos taurus dominant follicles which as a sign of dominance granule cells. Then we stimulate or inhibit the Notch signaling to observe the change of Marker molecules.Results: we screened some Notch family members in bovine granulosa cells by RT-PCR. It includes: Notch2, Jagged1, RBP/Jκ, Hes1 and c-myc. The Notch2 maybe downregulated in dominant follicles. And it will affect its target genes(hes1 and c-myc) downregulation. DAPT(inhibitor of Notch signaling pathway) inhibits proliferation of granulosa cells, and N2ICD’s overexpression may promote granulosa cells proliferation. At the same time we also find granulosa cells proliferation rate is slowed in dominant follicles. We get a eukaryotic recombinant plasmid CD513B1-c-myc. And in vitro experiments, we find that Notch signaling pathway may stimulate granulosa cells proliferation by c-myc. But neither stimulating nor inhibiting can not make these Marker molecules up-regulation.Conclusion: Notch signaling pathway and granulosa cells proliferation are positively correlated. In vitro experiments, we find that Notch signaling pathway may stimulate granulosa cells proliferation by c-myc. But when the bovine follicles dominance, their granulosa cells have a lower proliferating rate. Neither stimulating nor inhibiting can not make these marker molecules up-regulation.