Effect Of Notch Signaling Pathway Blocked With DAPT On Function Of Sheep Granulosa Cells In Vitro

DAPT is an inhibitor of the y-secretase complex. Notch is a key target of y-secretase, therefore DAPT indirectly inhibits the Notch pathway by affecting the activity of y-secretase. Notch signaling is an evolutionarily conserved signaling pathway which is widely present in vertebrates and invertebrates. Based on contact of neighboring cells, the signaling pathway regulates the differentiation and development of cells, tissues and organs. However, study about Notch signaling pathway on sheep follicle granulosa cells has not been reported.To better understand the role of Notch signal pathway in sheep follicle development, Jinzhong sheep were chosen for the present study, sheep ovaries and follicles were separated and granulosa cells were collected and cultured in serum-free culture system adding different concentrations of DAPT (0 μM,5 μM, 10 μM,20 μM). Each concentration was set up 6 duplications, and the medium was changed once every 48 h until 168 h, after which the cultured granulosa cells and culture medium were collected respectively. The cell viability was measured using Guava ViaCount(?) Reagent and the concentration of estradiol was measured using Sheep Elisa kit.Using qRT-PCR to investigate the relative expression level of the cell cycle genes (CyclinD2 and P21), those genes related to apoptosis (Bax, Bcl-2, P53, c-Myc) and estradiol biosynthesis gene(CYP19) of sheep granulosa cells.Results and analysis:(1) The results showed that Notch signaling pathway played an important role in regulating the proliferation and function of sheep granulosa cells. Compared with DAPT treatment groups, the control group had the maximum number of granulosa cells and the highest production of estradiol, and there were significant differences (P<0.05). In DAPT treatment groups, the number of granulosa cells and the amount of estradiol decreased at different degrees, indicating the proliferation and the estradiol production of sheep granulosa cells were regulated by the Notch signaling pathway.(2) After blocking Notch signaling pathway with different concentrations of DAPT, the relative expression levels of Bax, Bcl-2, c-Myc and CYP19 of sheep granulosa cells were significantly decreased (P<0.05), and CyclinD2, P21 and P53 had no remarkable change. The relative expression levels of Bcl-2, c-Myc gene had significant differences between the control group and the treatment group with 5 μM DAPT (P<0.05); Compared with the control group, the relative expression level of Bax in 20 μM DAPT treatment group was significantly lower (P<0.05), which indicated that the negative effect of Notch signaling pathway blocked with DAPT on proliferation and estradiol production of sheep granulose cells may result from the down regulation of Bax, Bcl-2 and CYP19.Blocking Notch signaling pathway with DAPT had a negative regulation role in proliferation and estradiol production of sheep granulosa cells, and the negative regulation of cell proliferation may be by regulating the expression of Bax and Bcl-2 gene and that of estradiol production by regulating the expression of estradiol synthesis-related gene CYP19. This study investigated the effect of Notch signaling pathway blocked with DAPT on proliferation and estradiol production of sheep granulosa cells in vitro, which provided a theoretical basis for further study about function and functional mechanism of Notch signaling pathway in female animal ovaries.