| RFRP-3 is a kind of neuropeptide synthesized mainly by the hypothalamus,which negatively affects the hypothalamic-pituitary-gonad axis.RFRP-3 regulates mammalian reproductive function by affecting synthesis and secretion of Gn RH,follicle-stimulating hormone（FSH）and luteinizing hormone（LH）in mammals.In female mammals,current studies mainly focus on the effects of RFRP-3 on gonadotropin secretion,granulococcus cell proliferation and differentiation,and estrus cycle changes,but there are few reports on the regulatory effect and mechanism of RFRP-3 on oocyte maturation in vitro.The Wnt/β-catenin signaling pathway is a highly conserved pathway that regulates cell proliferation,cell differentiation,cell apoptosis,stem cell self-renewal,tissue homeostasis and wound healing.It has been shown that Wnt/β-catenin signaling pathway is involved in follicular growth,embryo development and oocyte maturation.However,whether Wnt/β-catenin signaling pathway is involved in the regulation of RFRP-3 on oocyte maturation remains unknown.In order to explore whether RFRP-3 can regulate oocyte maturation in vitro through Wnt/β-catenin signaling pathway,oocytes in the mice were used as experimental subjects to preliminarily explore the regulatory effect of RFRP-3 on oocyte maturation in vitro,laying a foundation for the subsequent exploration of the mechanism of RFRP-3 regulating economic animal（pig）oocyte maturation.The results of this study are as follows:1.Effects of RFRP-3 on in vitro maturation of mouse oocytes（1）The results showed that compared with the control group,the supplementation of 10-8 M RFRP-3 significantly reduced the naked egg maturation rate（P<0.01）,the addition of 10-10 M RFRP-3 significantly reduced the naked egg maturation rate of mice（P<0.05）,adding 10-6 M and 10-12 M RFRP-3 had no significant effect on the maturation rate of nude eggs in mice（P<0.05）.（2）That the expression of GDF9 and BMP15 was significantly decreased by adding 10-8 M RFRP-3 compared with the control group（P<0.01）,and adding 10-10M RFRP-3 can significantly down-regulate the expression of BMP15（P<0.05）,but had no significant effect on GDF9（P<0.05）,adding 10-6 and 10-12 M RFRP-3 had no significant effect on GDF9 and BMP15（P<0.05）.（3）That adding 10-8 M RFRP-3 could significantly increase the content of c AMP（P<0.01）,adding 10-6,10-10,10-12 M RFRP-3 could significantly increase the content of c AMP（P<0.05）;All the concentrations of RFRP-3 had no significant effect on MPF secretion（P<0.05）.2.Effects of RFRP-3 on in vitro maturation of porcine oocytes through WNT/β-catenin signaling pathway（1）Effects of RFRP-3 on in vitro maturation rate of porcine oocytes through WNT/β-catenin signaling pathwayCompared with DMSO group,when FH535 was added to culture COCs,the in vitro maturation rate was significantly improved（P<0.05）;The addition of RFRP-3could significantly reduce the in vitro maturation rate of COCs（P<0.05）,when RFRP-3 and FH535 were added together,FH535 could significantly antagonize the inhibitory effect of RFRP-3 on in vitro maturation of pig COCs（P<0.05）.Compared with the control group,when Li Cl was added to the cultured COCs,the maturation rate was significantly reduced（P<0.01）;When RFRP-3 and Li Cl were added together,the inhibition of in vitro maturation rate of pig COCs was more significant than that of RFRP-3 added alone（P<0.01）.（2）Effects of RFRP-3 on cumulus expansion and cumulus expansion factor expression of porcine COCs through WNT/β-catenin signaling pathwayThere was no significant change in cumulus expansion in all treatment groups compared with DMSO group.The results showed that the addition of FH535significantly increased the cumulus expansion factor PTGS2 compared with DMSO group（P<0.01）,HAS2（P<0.01）and PTX3（P<0.01）;RFRP-3 significantly reduced the cumulus expansion factor PTGS2（P<0.01）,HAS2（P<0.01）and PTX3（P<0.01）;When RFRP-3 and FH535 were combined with COCs,FH535 could antagonize RFRP-3 against PTGS2（P<0.01）,HAS2（P<0.01）and PTX3（P<0.01）.Microscopically,whether the addition of Li Cl alone or RFRP-3 combined with Li Cl inhibited cumulus expansion compared with the control group.The expression of cumulus expansion factor of COCs was significantly reduced when added Li Cl COCs alone significantly reduced the expression of cumulus expansion factor（P<0.01）,the inhibitory effect of RFRP-3 and Li Cl on the cumulus expansion factor of pig COCs was more significant than that of RFRP-3 alone（P<0.01）.（3）Effects of RFRP-3 on GDF9 and BMP15 expression in porcine oocytes via WNT/β-catenin signaling pathwayThe addition of FH535 significantly increased GDF9（P<0.01）and BMP15（P<0.01）gene and protein expression compared with DMSO group,adding RFRP-3 can significantly reduce GDF9（P<0.05）and BMP15（P<0.05）gene expression,significantly decreased GDF9（P<0.01）and BMP15（P<0.01）protein expression,When RFRP-3 and FH535 were combined with COCs,FH535 could significantly antagonize RFRP-3 against GDF9（P<0.01）and BMP15（P<0.05）.Compared with the control group,Li Cl alone significantly reduced GDF9（P<0.01）and BMP15（P<0.01）;RFRP-3 and Li Cl combined with cultured porcine COCs increased the expression of GDF9（P<0.05）and BMP15（P<0.01）and the inhibitory effect was more significant than RFRP-3 alone.（4）Effects of RFRP-3 on c AMP and MPF contents in porcine oocytes through WNT/β-catenin signaling pathwayCompared with DMSO group,the addition of FH535 significantly reduced the content of c AMP（P<0.01）,significantly increased the content of MPF（P<0.01）,RFRP-3 could significantly increase the content of c AMP（P<0.01）,significantly reduced the content of MPF（P<0.01）,when both RFRP-3 and FH535 were added to COCs,FH535 could significantly antagonize RFRP-3 against c AMP（P<0.05）and MPF（P<0.01）.Compared with the control group,Li Cl supplementation significantly increased the content of c AMP（P<0.01）,significantly reduced the content of MPF（P<0.01）,when RFRP-3 combined with Li Cl was added to cultured porcine COCs,whether the promotion of c AMP in porcine oocytes（P<0.01）or the,inhibitory effect of MPF on MPF（P<0.05）was more significant than RFRP-3 alone.（5）Effects of RFRP-3 on secretion of progesterone and estradiol in COCs of pigs through WNT/β-catenin signaling pathwayCompared with DMSO group,progesterone was significantly increased in both the fluid I（P<0.01）and the liquidⅡ（P<0.01）,after the addition of FH535,the concentration of estradiol in liquid I（P<0.05）,was significantly increased,the concentration of estradiol was extremely significantly increased inⅡliquid（P<0.01）;The concentration of progesterone in solution I was extremely decreased by adding RFRP-3（P<0.01）,significantly decreased the concentration of estradiol in liquid I（P<0.05）,significantly decreased the concentration of progesterone and estradiol in solutionⅡ（P<0.05）,when RFRP-3 and FH535 were added to cultured pig COCs,FH535 highly antagonized the inhibition of RFRP-3 on progesterone secretion in liquid I（P<0.01）,significantly antagonized the inhibitory effect of RFRP-3 on progesterone secretion in solutionⅡ（P<0.05）,significantly antagonized the inhibitory effect of RFRP-3 on estradiol secretion in liquid I and liquidⅡ（P<0.05）.Compared with the control group,Li Cl alone significantly reduced the secretion of progesterone and estradiol in fluid I andⅡ（P<0.01）.When RFRP-3 and Li Cl were added to cultured pig COCs,the inhibition effect of RFRP-3,whether progesterone（P<0.01）and estradiol（P<0.05）in liquid I or progesterone（P<0.05）and estradiol（P<0.05）in liquidⅡwas more significant than that of RFRP-3 alone.In conclusion,different concentrations of RFRP-3(10-6,10-8,10-10 and 10-12 M RFRP-3)can inhibit the in vitro maturation of mouse oocytes,and 10-8 M RFRP-3 has the most significant inhibitory effect on the maturation of mouse oocytes.The WNT/β-catenin signaling pathway is involved in the regulation of RFRP-3 on cumulus expansion,meiosis and hormone secretion of porcine oocytes,and thus affects in vitro maturation of porcine oocytes,suggesting that RFRP-3 can regulate in vitro maturation of porcine oocytes through WNT/β-catenin signaling pathway. |
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