Effects Of WNT Signaling Pathway On Maturation Of Porcine Oocyte In Vitro

Different from male gametes, oocytes can be cultured in vitro and develop to maturation states. Meanwhile, they are studied as important part in reproductive medicine, cell biology, species protection, and other fields. However, when the oocytes were cultured in vitro and out of the follicle environment their nucleus would mature spontaneously, and the resulting desynchrony of nuclear maturation and cytoplast maturation would cause the poorer quality and quantity of oocyte matured in vitro than that matured in vivo. WNT signaling is a complicated pathway that regulates many kinds of cell activities such as cell fate determination, differentiation, vitality, proliferation and apoptosis, even the embryogenesis. At present, there is little research about the effect and mechanism of WNT signaling pathway on porcine oocyte maturation. To demonstrate the role of WNT signaling pathway in porcine oocyte maturation, this study optimized the in vitro maturation system of porcine oocyte, and added two WNT inhibitors FH535, SB202190 to treat the COCs or the granulosa cells and to reveal the effects of WNT signal pathway on porcine oocyte maturation in vitro. Results were:1. The optimal culture system of porcine oocyte maturation in vitro withthe maturation rate of65%, the cleavage rate of 45.5%, the blastocyst rate of 15.1%, was determined finally. Thespecific componentsof the system are: TCM199 supplemented with 10% fetal bovineserum(FBS), 10 % porcine follicle fluid(PFF), 100 IU/m L penicillin-streptomycin, 10ng/m L epidermal growth factor(EGF), 0.57 m M L-cysteine(L-Cys), 2.5 IU/m Lfollicle-stimulating hormone(FSH), 10 IU/m L pregnant mare serum gonadotropin(PMSG),10 IU/m L human chorionic gonadotropin(HCG), 0.91 m M Sodium pyruvate, 3.05 m Mglucose and 0.1 g/L polyvinyl akohol(PVA). The glass culture dish with groove belongingto open-type and the continuous effect of hormones with 44 h were chosen.2. Complexes of porcine oocyte and cumulus cells were cultured in vitro and the culturemedia supplemented with WNT/β-catenin signal inhibitor FH535. The addition of FH535improved the percentage of porcine oocytes maturation to metaphase II(P<0.05), improved the potential of developing to early embryosand the number of nuclei in the resulting blastocyst stage was increased significantly(P<0.05). Though q RT-PCR and Western Blot, we found that the inhibition of WNT could improve the gene expression of Cdc-2, Bmp-15, and Mos, with decrease of Gdf-9(P<0.05). The expression of β-catenin had not changed on m RNA level but the protein abundance decreased(P<0.05). Therefore, the inhibition of FH535 on WNT signaling pathway could promote the maturation of porcine oocytes in vitro.3. Complexes of porcine oocyte and cumulus cells were cultured in vitro and the culturemedia supplemented with p38 MAPK inhibitor SB202190. Via the observation ofstereoscopic microscope and the detection of q RT-PCR, results showed that with theaddition of 20 μM SB202190 in vitrothe extension of cumulus cells was significantlysuppressed, and the gene expression of β-catenin,Cdc-2, Bmp-15, Cyclin B1,Mos and p34were increased after the addition of SB202190(P<0.05), with no difference of theexpression of Gdf-9(P>0.05). The Western Blot results showed, after the supplement ofSB202190, the abundance of p38 MAPK were declined, also with the phosphorylation ofp38MAPK.4. Porcine granulosa cells were cultured in vitro and the culture media supplemented witdifferent concentrations(0, 1.0, 10, 20 μM) of SB202190 which is the specific inhibitor op38MAPK. The viability of porcine granulosa cells was assessed by CCK-8 kit, theporcine granulosa cell apoptosis and cell cycle retardation were determined by kit, therelative m RNA levels of apoptotic-related and cell cycle related genes were detected byreal-time q PCR. The results showed that the addition of SB202190 inhibited the growthactivity of porcine granulosa cell and the concentration of 20 μM was significant(P<0.05).However, it could not significantly influenced the apoptosis of porcine granulosa cells, andmost cells were retarded at G1/G0 phase with significant difference between this group andcontrol group(P<0.05). q PCR results showed that after SB202190 was added, thexpression of Bax., Bcl-2, p27 were promoted(P<0.05) with no significant change of theratio of Bcl-2 and Bax(P>0.05), and the expression of Fas, Fasl and Cdk-4 weresuppressed(P<0.05). Though the Western Blot assay, after the supplement of SB202190,the abundance of p38 MAPK in porcine oocyte was significantly declined(P< 0.05), butthe phosphorylation of p38 MAPK was not changed.In conclusion, the system of porcine oocyte maturation in vitro established in the experiement could obtain high maturation ratio and percentage of cleavage. When culturing the porcine oocyte in vitro, the addition of 1 μM FH535 which is the specific inhibitor ofWNT/β-catenin could promote porcine oocyte maturation and improve the potential of developing to early embryos, while the addition of 20 μM SB202190 could inhibit the p38 MAPK of WNT signaling to suppress porcine oocyte maturation. With the supplement of 20 μM SB202190, the cell cycle activity of cultured porcine granulosa cells were retarded, and the growth activity was inhibited.