Regulation Of Porcine Oocyte Maturation By RFRP-3 Via WNT/β-catenin Pathway

Mammalian RFamide-related peptide-3（RFRP-3）can inhibit the activity of hypothalamic Gn RH neurons.In turn,it can inhibit the synthesis and secretion of gonadotropins in the anterior pituitary and can directly participate in the regulation of ovarian growth and follicular development of female animals through paracrine or autocrine methods.It has been proven that the main research study about RFRP-3 is mainly focused on the regulation of granulosa cells and hormone secretions,but the function and mechanism of RFRP-3 on porcine oocytes have not been elucidated.In this study,the oocytes of commercial pigs were used as the experimental objects.Firstly,the expression of RFRP-3 in oocytes was determined,and the effect of RFRP-3 on oocyte maturation rate and cumulus expansion was observed.Secondly,the effect of RFRP-3on the factors related to oocyte maturation was detected.Finally,whether RFRP-3 can regulate oocyte maturation through Wnt/β-Catenin pathway was detected.Effects of RFRP-3on porcine oocyte maturation and the mechanism of action were discussed.Results show as follows:1.Amplification of RFRP-3 gene fragment was showed in the porcine oocytes by PCR.Different concentrations of RFRP-3(10^-6 mol/L,10^-8mol/L,10^-10 mol/L,10^-12 mol/L)were added to the culture medium to analyze its effect on the maturation of cumulus oocyte complex（COCs）in vitro.The result of the first polar body excretion rate was significantly reduced by adding 10^-8 mol/L and 10^-10 mol/L RFRP-3 to the oocyte in vitro culture system.What’s more,the expression of cumulus expansion factors such as Has2,Ptgs2 and Ptx3genes were significantly reduced by real-time fluorescence quantitative PCR（qRT-PCR）detection.2.ELISA was used to detect the effect of RFRP-3 at different concentrations on the secretion levels of MPF and cAMP.It was found that 10^-8 mol/L and 10^-10 mol/L RFRP-3could significantly reduce the concentration of MPF in oocytes,but had no significant effect on cAMP.Secondly,the effect of RFRP-3 on the hormone secretion of COCs was detected by radioimmunoassay（RIA）.The results have showed that RFRP-3 can significantly reduce the concentration of estradiol（E₂）and progesterone（P₄）.The expression level of mature marker factor（GDF9、BMP15）gene and maternal gene（CDK1、cyclin B1）mRNA was significantly decreased by 10-10 mol/L RFRP-3.The above results demonstrate that 10^-10mol/L RFRP-3 exhibit the most significant inhibitory effect on porcine oocyte maturation.3.qRT-PCR and Western-blot verification showed that 10^-10mol/L RFRP-3significantly up-regulated the expression of gene and protein levels of the transcription factorβ-catenin,a kind of protein related to WNT/β-catenin pathway,but had no significant effect on both gene and protein levels of the ligand Frizzled5.It is also found that the addition of WNT/β-catenin pathway inhibitor FH535 can significantly reverse the inhibitory effect of10^-10 mol/L RFRP-3 on the porcine oocyte maturation.The results showed that RFRP-3could inhibit oocyte maturation through Wnt/β-catenin signaling pathway.In summary,RFRP-3 is expressed on oocytes,and 10^-10 mol/L RFRP-3 can inhibit the maturation of oocytes by regulating levels of cumulus expansion factor,down-regulating MPF and E₂,P levels.Reduced expression of oocyte maturation marker（GDF9、BMP15）gene and maternal gene CDK1 and cyclin B1 inhibits oocyte maturation.Furthermore,WNT/β-catenin pathway is activated by accumulation of increased levels ofβ-catenin,which is involved in the regulation of oocyte maturation.