Expression of β-Catenin in Pig’s Ovary and the Effect of β-Catenin on Porcine Granulosa Cells Apotosis and Steroidogenesis Related Enzyme
|School||Nanjing Agricultural College|
|Course||Animal Genetic Breeding and Reproduction|
|Keywords||pig granulosa cell Wnt/β-catenin signaling pathway RNAi over-expression apoptosis steroid hormone synthesis|
P-catenin is a critical signal transduction molecules of the Wnt signaling pathway plays an vital role in the female during development, deletion or mutation ofβ-catenin will lead to Wnt/β-catenin signaling pathway disorder, and thus have impact on body tissues or cells. Wnt4 knockout mice before birth can lead to some of the sex reversal and the prostration of oocytes, P-catenin knockout mouse develop follicular containing pleomorphic follicular granulosa cells, which lead to the occurrence of granular cell carcinoma. Recently found thatβ-catenin involved in transcriptional regulation network of gonadotropin-releasing hormone, plays an important role in hormone formation.In this study, Erhualian pig, Pietrain Pig, commercial pigs and pig ovarian granulosa cells were taken as experimental material, real time RT-PCR were utilized to detect the different expression ofβ-catenin within the pig ovary and ovarian tissue;β-catenin was knocked down by RNAi methods, and based on the principle that LiCl can antagonize the GSK-3βto phosphorylate of P-catenin thus degradation, which will lead to the accumulation of P-catenin in nucleus.granulosa cells were continuously treated with LiCl to over-express P-catenin in GCs. Flow cytometry, real time PCR, western blot and other methods were used to examine the effect of P-catenin on granulosa cell apoptosis and steroid hormone synthesis from silencing and over-expressing ofβ-catenin gene in pig ovarian granulosa cell.The main results are as follows:1 Real time PCR was explored to detect the expression ofβ-catenin, the key signal transduction molecule of Wnt/β-catenin signaling pathway,in different ovary tissues and in ovaries of different varieties of porcine.The results show that:β-catenin in porcine of different fertility differentially expressed in ovary, the expression ofβ-catenin in erhualian pig is over 3 times of that in commercial pig ovary. While the expression ofβ-catenin in Pietrain pig is of 40% of that in commercial pig ovary.β-catenin is differently expressed in different development stage,the expression ofβ-catenin in large follicles was significantly higher than in the medium follicles and small follicles (p<0.05).β-catenin in ovarian granulosa cells was also is nearly 2.5 times of that in oocytes(p<0.05).2 To demonstrate the effects of Wnt/β-catenin signal on cell apoptosis and steroid hormone synthesis of porcine GCs, granulosa cells were isolated from 3-5mm antral follicles in diameter and cultured in vitro. Based onβ-catenin in the core molecular of the pathway, A strategy of RNAi-mediated’gene silencing’ofβ-catenin were used to "silence" pigβ-catenin gene, inhibit Wnt/β-catenin signaling pathway, while LiCl was used to activate the Wnt/β-catenin signaling pathway, to detect the effect ofβ-catenin on porcine granulosa cell apoptosis and steroid hormone synthesis.The results showed that:(1) P-catenin gene successfully "knocked down" by using the RNAi technology, mRNA levels ofβ-catenin was reduced by 59.93%(p<0.05), protein levels were also decreased. Knocked down P-catenin significantly increased granulosa cell apoptosis rate, by up-regulating Bax by 32.76%(p<0.05)and down-regulated BCL-2 by 44.28%(p<0.05), the expression of P450arom mRNA was reduced by 47.72%(p<0.05), but the expression of P450scc was not affected. (2) LiC1 treatment successfully activated Wnt/β-catenin pathway,β-catenin mRNA levels was up-regulated by 95.15%(p<0.05), protein levels was increased. Over-expression ofβ-catenin was significantly increased apoptosis rate,by down-regulating Bax mRNA by 25.60%(p<0.05) and increasing BCL-2 mRNA by 12.58%(p>0.05), and P450arom mRNA was up-regulated by 151.6%(p<0.05), P450scc mRNA was upregulated by 124.3%(p<0.05). Indicate that the Wnt/β-catenin signaling pathway have important role in porcine granulosa cells.