Effect Of Wnt/PLC/Ca²⁺ Pathway On Apoptosis Of Porcine Follicular Granulosa Cells And Oocytes And Hormone Secretion Of Granulosa Cells

More than 99%of mammalian oocytes undergo atresia and non-ovulation during development.Under the action of gonadotropin,some of them can reach the pre-ovulation stage.In order to promote in vitro egg production of endangered species and genetically superior livestock and poultry during non-reproductive period,the mechanism of oogenesis and follicular development still needs further study.The hormones and growth factors secreted by granulosa cells play an important role in follicular development.At the same time,oocyte secretion of various growth factors can also regulate the proliferation and differentiation of granulosa cells.There are many different levels of steroid hormones in follicular fluid,such as estrogen,progesterone and androgen,which may come from blood circulation,granulosa cells or follicular membrane cells,or from environment and food.They can affect ovarian follicular development by acting on granulosa cell growth and follicular fluid formation.Wnt pathway plays an important role in the maintenance of ovarian function in mice and human.It can participate in the regulation of follicular atresia and other physiological processes.When granulosa cell Wnt5a loses its activity,it can lead to increased follicular atresia and decreased ovulation rate in mice.However,the role of Wnt pathway in porcine follicular development has not been systematically reported.In this paper,we investigated the effect of Wnt/PLC/Ca²⁺pathway on apoptosis and hormone secretion in porcine follicular development.In this experiment,follicular fluid of 2-5mm diameter follicles was obtained by aspiration,granulosa cells and oocytes were collected,and a certain dose of Wnt/Ca²⁺protein inhibitor or activator including Wnt protein inhibitor IWP-2,phospholipase C?PLC?protein inhibitor U73122 or activator m-3M3FBS,and PKC?inhibitor Enzastaurin was added into the culture medium in vitro.The excretion rate of the first polar body was observed by microscope,and the effect of IWP-2,U73122,m-3M3FBS or Enzastaurin on the viability of granulosa cells was observed by CCK8 test respectively.Calcium content in granulosa cells was monitored by fluoro-Ca²⁺indicator Fluo 3-AM,and calcium content in oocyte was measured by Ca²⁺indicator Fura-2AM.Real-time fluorescence quantitative analysis?q RT-PCR?was used to detect the expression of pathway-related genes,hormone secretion and apoptotic gene transcription levels.Annexin V-FITC was used to detect the effect of PLC inhibitors and activators on apoptosis of granulosa cells.Enzyme linked immunosorbent assay?ELISA?was used to detect the secretion of estradiol and progesterone in porcine granulosa cells.Western Blot assay was used to detect the relative expression of apoptosis-related proteins,PLC protein and calcium sensitive protein PKC?,CAMK II?and Caln A.The main results are as follows:1.0.05?M or 5?M Wht inhititor IWP-2 had no effect on the viability of porcine granulosa cells,and 0.1,0.5 and 2.5?M IWP-2 increased the viability of porcine granulosa cells?P<0.05?compared with the control group.This means that Wnt ligands could inhibit the viability of porcine granulosa cells to some degree.The activity of granulosa cells did not change when the concentration of PLC inhibitor U73122 was not more than 0.5?M,but decreased at 4 h,24h and 48 h when the concentration increased?P<0.05?,while the activity of granulosa cells increased first and then decreased before 12 hours with the increase of the concentration of activator m-3M3FBS.The activity of granulosa cells was the highest when the concentration of activator was 0.5?M?P<0.05?.This indicates that PLC protein participates in the viability of porcine granulosa cells and promotes the viability of granulosa cells to some extent.Enzastaurin of 0.5?M and 2?M could inhibit the viability of porcine granulosa cells?P<0.05?.2.0.1-5?M Wnt inhibitor IWP-2 could inhibit oocyte maturation in a dose-dependent manner,and 0.5?M IWP-2 inhibited the cleavage?P<0.01?and blastocyst?P<0.01?.U73122 of 0.5?M promotes the oocyte first polar body excretion rate?P<0.05?,but inhibites the cleavage?P<0.001?and blastocyst?P<0.01?.0.5?M m-3M3FBS reduces the oocyte first polar body excretion rate?P<0.05?,and promotes the cleavage?P<0.01?and blastocyst?P<0.01?.This indicates that Wnt protein in Wnt/Ca²⁺pathway may promote the maturation of porcine oocytes,and PLC protein has an inhibitory effect on the maturation of oocytes.3.0.5?M IWP-2 can down-regulate the concentration of Ca²⁺in granulosa cells?P<0.01?and oocyte?P<0.05?.The abundance of PLCB1?P<0.001?,PLCG1?P<0.01?and PLCZ1?P<0.001?m RNA was reduced treated with 0.5?M IWP-2 for 24 h in porcine granulosa cells.The expression of PLCB1?P<0.001?,PLCD3?P<0.001?,PLCG1?P<0.001?and PLCZ1?P<0.001?m RNA and the expression of PLCD3 protein?P<0.05?was decreased treated with 0.5?M IWP-2 for 44 h in porcine oocytes.The optimum time of action of inhibitor U73122 and activator m-3M3FBS on PLCB1m RNA was 4 h,and the optimum concentration of treatment was 0.5?M.The expression of PLCB1?P<0.05?,PLCD3?P<0.05?and PLCZ1?P<0.05?m RNA were decreased by 0.5?M U73122 treatment for 4 hours,the relative abundance of PLCB1?p=0.001?and P LCG1?P<0.05?proteins and the calcium concentration?P<0.001?were also decreased treated with 0.5?M U73122 for 4 h in porcine granulosa cells.The expression of PLCB1?P<0.05?,PLCD3?P<0.05?and PLCZ1?P<0.05?m RNA and the concentration of calcium ion?P<0.01?was increased treated with 0.5?M m-3M3FBS for 4 h in granulosa cells.Compared with the control group,the expression of PLCB1?P<0.001?,PLCD3?P<0.01?and PLCZ1?P<0.05?m RNA treated with 0.5?M U73122 for 44 h was decreased in porcine oocytes,while the concentration of calcium ion did not change much.The expression of PLCB1?P<0.01?,PLCD3?P<0.001?,PLCG1?P<0.05?and PLCZ1?P<0.05?m RNA was increased,and the abundance of PLCB1 protein?P<0.05?and calcium concentration?P<0.05?was increased significantly treated with 0.5?M m-3M3FBS for 44h in porcine oocytes.IWP-2 combined with U73122 decreased the level of calcium in granulosa cells?P<0.001?and oocyte?P<0.05?,while IWP-2 combined with m-3M3FBS had no effect on the level of calcium in granulosa cells and oocyte.The results showed that Wnt ligand and PLC protein could co-regulate the change of calcium level in follicles and increase the increase of Ca²⁺in cytoplasm.4. In granulosa cells,inhibitor U73122 decreased the expression abundance of calcium-sensitive protein PKC??P<0.05?,CAMKII??P<0.05?and Caln A?P<0.01?,while activator m-3M3FBS increased the expression of CAMKII??P<0.001?.In oocyte,PLC protein has positive regulation function on PKC?and CAMKII?protein?P<0.05?,but does not affect the expression of Caln A.In granulosa cells,inhibitor U73122 can down-regulate the expression of CDC42?P<0.01?,NFATc1?P<0.01?and NF?B?P<0.01?,and up-regulate CTNNB slightly?P>0.05?.Activator m-3M3FBS could up-regulate the expression of CDC42?P<0.05?,NFATc1?P<0.01?and NFkappa B?P<0.05?,and down-regulate the expression of CTNB?P<0.05?.In oocyte,PLC up-regulates downstream genes,but does not affect the expression of CTNNB?P>0.05?.The results showed that PLC protein could activate calcium sensitive protein and downstream genes of porcine granulosa cells and oocytes and inhibit CTNNB expression in granulosa cells.5.0.5?M IWP-2 up-regulates the expression of BAK?P<0.001?,BAX?P<0.01?,CASP8?P<0.05?and TP53?P<0.05?genes,while the expression of Bak,Bax and Cleaved caspase-3 protein increased significantly?P<0.05?in porcine granulosa cells.The expression of BAX?P<0.01?,CASP3?P<0.05?and TP53?P<0.01?m RNA increased,while the expression of BCL6?P<0.05?m RNA decreased,the expression of Bax?P<0.01?and Cleaved caspase-3?P<0.05?protein increased,and the expression of Bcl-6?P<0.01?protein decreased treated with 0.5?M IWP-2 for 44 h in porcine oocytes.The change of PLC activity can inhibit apoptotic regulatory genes of granulosa cells,and the effect on apoptotic rate varies at different time.0.5?M PLC inhibitor U73122 can up-regulate the m RNA expression levels of BAK?P<0.01?,BAX?P<0.05?and CASP3?P<0.01?.The early?P<0.01?and late apoptotic rates?P<0.05?of granulosa cells are the highest treated with 0.5?M U73122 for 4 hours.PLC activator m-3M3FBS can up-regulate the expression of anti-apoptotic gene BCL2?P<0.01?,and down-regulate the expression of BAX?P<0.05?and CASP3?P<0.05?,while the expression of Bax protein?P<0.01?was reduced and the expression of Bcl-2 protein?P<0.001?was increased.The early apoptotic rate of granulosa cells is the highest at 4 h?P<0.05?and the late apoptotic rate of granulosa cells is the highest at 8 h?P<0.05?treated with 0.5?M m-3M3FBS.After incubating porcine oocytes with 0.5?M U73122 for 44 h,the relative abundance of BAK?P<0.001?,BAX?P<0.01?,CASP3?P=0.001?,CASP8?P<0.001?and TP53?P<0.01?m RNA was increased,while the relative expression of BCL6?P<0.001?m RNA was decreased?Fig.4-6A?,the expression of Bak?P<0.05?,Cleaved caspase-3?P<0.05?and Caspase 8?P<0.05?proteins was increased compared with the control group.After incubating porcine oocytes with 0.5?M m-3M3FBS for 44 h,the relative m RNA abundance of BAK?P<0.01?,BAX?P<0.01?,CASP3?P<0.01?,CASP8?P<0.001?and TP53?P=0.001?was decreased,while that of BCL6?P<0.01?was increased,the relative protein abundance of Bcl-6?P<0.05?was increased,and the relative protein expression of P53?P<0.05?was decreased.Compared with the control group,the expression of BAK?P<0.01?,BAX?P<0.001?,CASP3?P<0.01?,CASP8?P<0.001?and TP53?P<0.05?m RNA were up-regulated,and the expression of BCL2?P<0.05?m RNA was down-regulated cultured with 0.5?M IWP-2and 0.5?M U73122 in porcine granulosa cells.Compared with the control group,the expression of BAK?P<0.001?and CASP8?P<0.001?m RNA was decreased when 0.5?M IWP-2 and 0.5?M m-3M3FBS were added to the culture medium of porcine granulosa cells.Compared with the control group,the expression of BAK?P<0.001?,BAX?P<0.001?and CASP3?P<0.01?m RNA were up-regulated,and the expression of BCL2 m RNA?P<0.001?was down-regulated treated with 2?M Enzastaurin,the expression of BAK?P<0.001?and BAX?P<0.05?m RNA were up-regulated,and the expression of BCL2 m RNA?P<0.01?was down-regulated treated with 0.5?M U73122 and 2?M Enzastaurin.The relative protein abundance of Bax?P<0.05?was increased,and the relative protein expression of Bcl-2?P<0.01?was decreased cultured with 2?M Enzastaurin,the relative protein abundance of Bcl-2?P<0.001?was decreased cultured with 0.5?M U73122 and 2?M Enzastaurin.The results showed that the Wnt ligand and PLC protein in Wnt/Ca²⁺pathway can synergistically inhibit the apoptosis process of porcine granulosa cells and oocytes.0.5?M U73122 and 2?M Enzastaurin could synergistically promote the apoptosis of porcine granulosa cells.6. The expression of estradiol-related genes CYP17A1?P<0.01?,CYP19A1?P<0.01?,ER1?P<0.001?and ER2?P<0.01?was up-regulated treated with 0.5?M IWP-2.The 0.5?M PLC inhibitor U73122 can up regulate the CYP11A1 gene?P<0.05?in porcine granulosa cells,while the 0.5?M m-3M3FBS has little effect on the regulation of hormones.The secretion of estradiol decreased after 2 h,8 h,12 h,24 h,48 h?P<0.05?treated with 0.5?M U73122 in granulosa cells,and the secretion of estradiol decreased?P<0.05?after treatment with 0.5?M m-3M3FBS in porcine granulosa cells.The secretion of progesterone increased?P<0.05?after treatment with 0.5?M U73122 for 4 h,and increased?P<0.05?treated with 0.5?M m-3M3FBS for 2 h and 8 h in porcine granulosa cells.The ratio of estradiol and progesterone decreased?P<0.05?at all time points except 8 h after treatment with 0.5?M U73122 in porcine granulosa cells.The ratio of estradiol to progesterone decreased after 2 hours?P<0.05?treated with 0.5?M m-3M3FBS in porcine granulosa cells.Treatment of IWP-2 and U73122 increased the expression level of genes involved in regulating E 2 and P4.Treatment of IWP-2 and m-3M3FBS reduced the expression level of CYP11A1?P<0.01?and increased the expression levels of CYP17A1?P<0.05?,CYP19A1?P<0.01?and ER1?P<0.05?.Compared with the control group,the expression of CYP11A1 m RNA?P<0.01?was increased and the expression of CYP17A1?P<0.001?m RNA was decreased treated with 2?M Enzastaurin or 0.5?M U73122 and 2?M Enzastaurin.The expression of CYP17A1m RNA was down-regulated more cultured with 0.5?M U73122 and 2?M Enzastaurin than that with 2?M Enzastaurin.Studies have shown that Wnt ligand and PLC protein in Wnt/Ca²⁺pathway can co-regulate the levels of estradiol?E2?and progesterone?P4?in porcine granulosa cells and oocytes.Three hormone related genes,including CYP11A1?CYP17A1 and CYP19A1,could be synergistically regulated by 0.5?M U73122 and 2?M Enzastaurin.In conclusion,Wnt/Ca²⁺signaling pathway has the function of inhibiting cell apoptosis and regulating hormone secretion in the process of porcine follicular development,and may be mediated by regulating the calcium sensitive protein and downstream genes of the pathway.