Effects Of FSH And Wnt Signaling Pathway On Function Of Sheep Granulosa Cells In Vitro

Wnt signaling pathway is widely present in mammals, three main Wnt signaling pathways have been found today, which control various process, such as the development of the nervous system?tissues?organs and the occurrence of mammalian tumorigenesis. Besides, the signaling pathways regulate follicular development. IWR-1 is an inhibitor of Wnt/?-catenin signaling pathways. Though changing the stability of the protein complexes, IWR-1 ultimately has effect of inhibiting the classic signaling pathways. There were a lot of researches on the Wnt. However, no study was found about the effect of Wnt signaling pathway on proliferation and estrogen secretion of sheep granulosa cells and to explore whether the effect was related to FSH.In order to further study about regulating mechanism of the classic Wnt signaling pathway in sheep follicular granulosa cells, we chose several genes of Wnt signaling pathway which plays the important roles. We test the relative expression in sheep ovarian follicles in different sizes, in order to preliminary understanding the expression of Wnt signaling pathways in the follicular development process. Subsequently, the granular cells were collected and cultured in serum-free culture system with different concentration of IWR-1 and FSH in vitro:0 ng·mL^-1 FSH,0 ?mol·L^-1 IWR-1; 0 ng·mL^-1 FSH,0.1 ?mol·L^-1 IWR-1; 0 ng·mL^-1 FSH,1 ?mol·L^-1 IWR-1; 0 ng·mL^-1 FSH,10 ?mol·L^-1 IWR-1; 5 ng·mL^-1 FSH,0 ?mol·L^-1 IWR-1; 5 ng·mL^-1 FSH,0.1 ?mol·L^-1 IWR-1; 5 ng·mL^-1 FSH,1 ?mol·L^-1 IWR-1; 5 ng·mL^-1 FSH, 10 ?mol·L^-1 IWR-1. Each concentration set was designed as 6 duplications, and the medium was changed once each 48 h until 168 h, after which the cultured granulosa cells and medium were collected respectively. The cell viability was measured using Guava ViaCount??? Reagent and the estradiol concentrations were measured using sheep ELISA kit. The relative expressions of CYP19 and CCND2, which are the target genes of FSH, were analyzed by qRT-PCR.The main results were as follows:1. In the big follicles, relative abundance of CTNNB1 mRNA was greater and AXIN2 mRNA was lower than in medium follicles and small follicles. Furthermore, abundance of mRNA for FZD6 was significantly greater in big follicles and medium follicles compared to small follicles. However, mRNA abundance for DVL1 showed no significantly variation in expression among those follicles in different sizes. The results suggest that with the continuous development of follicle, the concentration of the Wnt signaling pathways also will increase. So we speculate that Wnt signaling pathway might play an important role in regulation of follicle growth.2. In terms of the number of cells, when the granulosa cells cultured in the absence of FSH, the inhibitory dose of IWR-1 of 0.1 ?mol·L^-1 andl ?mol·L^-1 could cause a significant ?P<0.05? reduction; However, when the granulosa cells cultured in the presence of FSH, all of inhibitory dose of IWR-1 could cause a significant ?P<0.05? reduction. With the presence of FSH, the addition of IWR-1 could significantly ?.P<0.05? decrease cell number. In terms of estrogen concentration, only under the stimulation IWR-1 treatment could significantly ?P<0.05? decrease estrogen concentration. When the concentration of IWR-1 was 1 ?mol·L^-1, its inhibitory effect on granulosa cells was of most obvious, and this function may be related to FSH.3. IWR-1 decreased the abundance of CYP19 mRNA and CCND2 mRNA in sheep follicular granulosa cells.In conclusion, Wnt signaling pathway could improve the biological function of sheep granulosa cells, and this function may be related to FSH.