Expression And Function Of Wnt2 In Sheep Follicular Granulosa Cells

The Wnts are an extracellular signal molecule,which regulates cell fate,proliferation,differentiation and apoptosis by combining with FZD family.As an important member of the Wnts family,Wnt2 participates in ovarian development and tumorigenesis.Studies have shown that the signal pathways in which Wnt2 involved play an important role in follicular development.However,there are no reports on the study of Wnt2 on sheep follicles.In order to further understand the role of Wnt2 in follicular development,in this experiment,the Wnt2 protein of sheep was analyzed by bioinformatics.The structure and biological function of Wnt2 protein in sheep were preliminarily recognized.And then the expression and localization of Wnt2 protein in follicular granulosa cells were studied.The expression difference of Wnt2 m RNA and Wnt2 proteins in follicles of different sizes was detected by q RT-PCR and Western blot.The role of Wnt2 in follicular granulosa cells of sheep at different developmental stages was explored.Finally,the follicle granulosa cells of sheep were cultured in vitro.After the Wnt2 gene was silenced,the relative expression of Wnt2 and the key gene CTNNB1 involved in the Wnt/?-catenin signaling pathway was measured.After gene was silenced,apoptosis of granulosa cells was detected.The results and conclusions of the experiments were as follows:(1)The results of bioinformatics analysis showed that the Wnt2 protein in sheep was a stable protein with signal peptide,composed of 360 amino acids and with glycosylation and phosphorylation sites.The isoelectric point was 9.21.Wnt2 protein was extracellular protein,involved in signal transduction and transcription regulation.The sequences alignment results of the Wnt2 protein of 10 species,including sheep,were basically the same as that of Cluster construction of phylogenetic tree,suggesting that the gene was highly conserved in evolution.(2)The results of immunohistochemistry showed that Wnt2 protein was expressed in Thecal cells,granulosa cells and cumulus cells of sheep.The results of q RT-PCR and Western blot werebasically the same,which indicated that the expression of Wnt2 m RNA and Wnt2 protein in granulosa cells of different sizes was significantly different(P(27)0.05),and the expression in large follicles was significantly higher than that in medium follicles(P(27)0.05),and the expression in medium follicles was significantly higher than that in small follicles(P(27)0.05).This indicated that Wnt2 was expressed in granulosa cells and played an important role in the proliferation of follicular granulosa cells in sheep.(3)After Wnt2 gene was blocked,the apoptosis test results showed that the number of apoptosis in Wnt2 si RNA group was significantly higher than that in nonsense sequence si RNA group(NC group)and blank control group(P(27)0.05),suggesting that Wnt2 could inhibit apoptosis of follicular granulosa cells in sheep,and improve proliferation.The results of q RT-PCR showed that the expressions of Wnt2 gene and ?-catenin(CTNNB1)gene in Wnt2 si RNA group were significantly lower than those in nonsense sequence si RNA group(NC group)and blank control group(P(27)0.05).This indicated that Wnt2 promoted the biological function of ovaries granulosa cells in sheep by Wnt/?-catenin signaling pathway.