Effects Of WNT Signaling Pathway Receptor Family Frizzleds On Porcine Oocyte Maturation In Vitro

In vitro maturation(IVM)of oocytes is the basic of in vitro fertilization,embryo engineering,transgenic cloning and other technologies.IVM can explore ovary abundant oocyte resources,and improve the efficiency of embryo in vitro production.However,IVM of porcine oocytes is immature because the embryonic development is not as good as the development of mature oocytes in vivo after in vitro fertilization and nuclear transplant.The culture system of IVM remains to be further improved.The WNT signaling pathway is highly conserved in evolution and plays an important role in cell proliferation,differentiation,apoptosis,stem cell maintenance,and embryonic development.Studies have shown that WNT signaling plays a key role in female gonadal development.At present,there is little research about the effect and mechanism of WNT signaling pathway on porcine oocyte maturation.In this study,we investigated the expression and localization of WNT signaling pathway receptors Frizzleds family in porcine ovary and oocyte,and screened the highest expression receptor in the ovary.In addition,we added the WNT inhibitor WNT-C59 to block WNT upstream pathway and revealed the effects of the Frizzled receptor family on IVM of porcine oocytes.The study may contribute exploring the specific function and mechanism of WNT / Frizzleds signaling pathway of porcine oocytes in vitro maturation.The main results are as follows: 1.Expression of the WNT pathway receptor Frizzleds family in the porcine ovaryAccording to the different Frizzleds family gene sequences of porcine preserved in GenBank,we analyzed the relative expression of Frizzled2-Frizzled7,Frizzled9 and Frizzled10 mRNA by real-time quantitative PCR(RT-QPCR).The results showed that the expression of Frizzleds family mRNA in ovarian tissue was Frizzled5>Frizzled7> Frizzled4≥Frizzled3≥Frizzled2≥Frizzled6≥Frizzled9≥Frizzled10(p<0.05).The expression of Frizzled5 was significantly higher than that of other Frizzled family members(p <0.05);The expression of Frizzled7 was significantly higher than Frizzled9 and Frizzled10.In addition,other family members expressed no significant difference(p> 0.05).Frizzled10 was almost unexpressed.The experiment revealed that the Frizzleds family was commonly expressed in porcine ovary and screened out that the Frizzled5 expressed highest in the receptor family.2.Expression of Frizzled5 in different size follicles and their localization on porcine ovaryWe detected the expression of Frizzled5 gene in different size follicles by RT-QPCR,as well as the localization of Frizzled5 protein in ovary by immunohistochemistry.The results showed that the expression of Frizzled5 in small follicles(<2mm)was significantly lower than that of medium follicles(2mm <d <5mm)and large follicles(> 5mm)(p <0.05),while there are no significant change between the medium and the large follicles.Immunohistochemical staining was used to detect the localization of Frizzled5 protein in porcine ovary tissues.Studies have shown that Frizzled5 protein is overexpressed in the oocyte membrane and granule cells,while the expression signal is medium in the ovary stroma.These results suggest that the WNT / Frizzleds signaling pathway may play a role in follicular development and oocyte maturation.3.Expression of Frizzleds receptors in porcine oocytes at different stages of maturation in vitroPorcine oocytes were cultured in vitro for 38-41 h,44-46 h and 48-50 h,respectively,and their maturation rate and cleavage rate were observed.We found that the maturation rate of culturing 44-46 h was better than that of 38-41 h and 48-50 h(p <0.05),and the cleavage rate was significantly higher than that of 38-41 h(p <0.05).However,there was no significant difference between the 44-46 h and 48-50 h in cleavage rate(p> 0.05).That is,too short or too long culture time will affect the quality of oocytes in vitro maturation.The relative expression levels of Frizzled2,Frizzled3,Frizzled4,Frizzled5 and Frizzled7 mRNA in oocytes were also analyzed by RT-QPCR.It demonstrated that the mRNA expression of Frizzleds family changed with the culture time,and the trend was inconsistent.The expression level of Frizzled5 in oocytes cultured in vitro for 22 h was significantly higher than that in culture 0 h and cultured for 44 h(p<0.05).The expression of Frizzled3 cultured in 22 h was significantly higher than that in 0 h(p <0.05),but there was no significant difference compared with that in 44 h(p> 0.05).The expression levels of Frizzled2 and Frizzled4 of oocytes cultured in vitro for 44 h were significantly lower than those in the other two periods(p <0.05).The expression level of Frizzled7 was higher than that of other two periods at 44 h(p> 0.05).The dynamic changes in the Frizzled family of genes in oocytes indicate that the WNT signaling pathway may rely on different receptors to play a role in oocyte maturation.4.Effects of WNT-C59 inhibitor on porcine oocytes maturation in vitroWNT-C59 Inhibitor was added in the maturation culture media.And the COCs was cultured for 44 h.Moreover,the expression of Frizzled5 and β-catenin treated by WNT-C59 was measured by RT-QPCR.The results showed that addition of WNT-C59 in vitro could significantly increase the percentage of oocytes to MII(p <0.05),and the cleavage rate of oocytes was also increased.And the maturation rate was highest at 1μmol concentration.And Frizzled5 and β-catenin both were downregulated in WNT-C59 treatment group(p <0.05).Therefore,WNT-C59 could promote the porcine oocytes maturation by inhibiting WNT upstream pathway.In conclusion,we investigated the expression of WNT signaling pathway receptors Frizzled family in porcine ovary,and screened the highest expression receptor frizzled5 in the ovary.The gene expresses dynamically in porcine follicular development,and the results showed high expression of frizzled5 in granulosa cells and oocytes,indicating that the WNT pathway plays an important role in follicular development and oocyte maturation.The Frizzled family was differentially expressed in different developmental stages of oocytes,suggesting that the WNT signaling pathway may rely on different receptors to play a role in oocyte maturation.The WNT signal inhibitor WNT-C59,when its concentration was 1μmol,can downregulate the expression of Frizzleds and β-catenin,and effectively promote the proliferation of cumulus oocyte cumulus and the maturation of porcine oocytes in vitro,as well as improving the early embryonic development.