Function And Mechanism Of RFRP-3 In Mouse Primordial Follicle Formation

In female,primordial follicles pool is considered as the only source of follicle and oocyte development during the whole lifetime,which is the basis of ensuring normal ovarian development and maintaining female reproductive performance.Previous studies have demonstrated that Arg(R)-Phe(F)-amide related peptide-3(RFRP-3)was involved in prepuberty and adult animal reproductive processes by regulating cell proliferation,apoptosis,steroidogenesis,and the key factors expression of signaling pathways.Nevertheless,the effect of RFRP-3 on primordial follicle formation in neonatal animal is poorly understood.The ubiquitin-proteasome system(UPS)is one of the predominant pathways of protein degradation in mammalian cells,which is participated in oocyte maturation,embryonic development,granulosa cell,and preantral follicle growth.CNOT4,one of the members of CCR4-NOT complex with ubiquitin E3 ligase activity,contributes to m RNA transcription,metabolism,and proteasome assembly in cells.However,the role of the CNOT4 in primordial follicle formation remains to be elucidated.Firstly,we detected the expression of RFRP-3 specific receptor GPR147 at different stages in mouse ovaries,and investigated the role of RFRP-3 in primordial follicle formation through an in vitro culture system.Subsequently,the underlying mechanism of RFRP-3 mediated by UPS in primordial follicle formation was explored.The results are as follows:1.Effect of RFRP-3 on primordial follicle formation in mouse.RFRP-3 specific receptor GPR147 was mainly expressed in the cytoplasm of oocytes in the 19.0 dpc fetal mouse ovary.During in vitro culture of 19.0 dpc embryonic mouse ovaries,the amounts of primordial follicles and growing follicles in the RFRP-3 group were increased in a dose-dependent manner(P<0.05).The expression levels of essential factors(Figla,BMP15,GDF9,and Sohlh2)related to oocyte development and pregranulosa cell proliferation markers(Ki67 and PCNA)were increased(P<0.05),while the percentage of TUNEL positive signals(4.25%±0.42 vs.1.48%±0.06)and proapoptosis factors(P53 and Caspase3)were decreased after 100 ng/m L RFRP-3treatment compared with the control(P<0.05).2.Effect of the UPS on primordial follicle formation in mouse.During in vitro culture of 19.0 dpc embryonic mouse ovaries,proteasome inhibitor MG132 treatment for 12 h significantly reduced the amounts of primordial follicles(P<0.05)and growing follicles(P<0.01).In addition,the expression levels of oocyte-specific genes(Figla,BMP15,GDF9,and Sohlh2)and pre-granulosa cell proliferation markers(Ki67 and PCNA)were decreased(P<0.01),while the percentage of TUNEL positive signals(2.98%± 0.42 vs.10.63% ± 1.58)and pro-apoptosis factors(P53 and Caspase3)were increased after proteasome inhibitor MG132 treatment for 12 h(P<0.01).However,proteasome inhibitor MG132 did not affect the protein content of PCNA(P>0.05).3.The role of RFRP-3 mediated by the UPS in mouse primordial follicle formation.19.0 dpc fetal mouse ovaries were pretreated with proteasome inhibitor MG132 at 12 h before 100 ng/m L RFRP-3 treatment,the effects on primordial follicle formation were observed.Compared with the RFRP-3 group,the number of primordial follicles and growing follicles in the MG132+RFRP-3 group were obviously reduced(P<0.05).The expression levels of Figla,BMP15,GDF9,and Sohlh2 gene,which were involved in oocyte growth,were markedly down-regulated in the MG132+RFRP-3group than that in the RFRP-3 group(P<0.05).Meanwhile,the percentage of TUNEL positive signals(1.48%±0.33 vs.3.65%±0.46)and the expression of pro-apoptosis factor P53 were increased in MG132+RFRP-3 treated ovaries compared to RFRP-3treated ovaries(P<0.05).To further investigate whether the role of UPS mediated RFRP-3 in primordial follicle is related to PI3K/Akt/m TOR signaling pathway,the protein levels and phosphorylation forms of Akt,m TOR and rp S6 in treatment groups are detected by western blotting(WB).The expression levels of p-Akt and p-rp S6 in the RFRP-3 treated ovaries were significantly higher than those in the control ovaries(P<0.01),while the expression level of p-m TOR showed a slight increase(P>0.05).Compared with RFRP-3 group,the expression levels of p-Akt and p-rp S6 in MG132+RFRP-3 group were decreased(P<0.05).Nevertheless,no significant changes were observed in the expression level of p-m TOR between RFRP-3 group and MG132+RFRP-3 group(P>0.05).4.The role of CNOT4 in RFRP-3 regulating primordial follicle formation in mouse.The results revealed that the CNOT4 was dominantly located in the cytoplasm of oocytes in the 19.0 dpc fetal mouse ovary.To further understand whether the role of RFRP-3 mediated by the UPS in primordial follicle formation is related to CNOT4,WB analysis is carried out to detect the CNOT4 protein concentration in proteasome inhibitor MG132 combined with RFRP-3 treated ovaries.Quantification of WB revealed that an increased protein concentration of CNOT4 in RFRP-3 group compared with the control group(P<0.05).Proteasome inhibitor MG132 reversed the increased concentration of CNOT4 caused by the RFRP-3(P<0.05).To analyze the possible regulatory role of CNOT4 during primordial follicle formation in mouse,a total of 852 proteins interacting with CNOT4 protein were screened by liquid chromatographytandem mass spectrometry analyses in 3 dpp neonatal mouse ovary.GO enrichment analysis disclosed that these proteins were dominantly participated in biological processes,cell component,and molecular function.KEGG signal pathways analysis showed that the signal pathways involved in these proteins were mainly related to ribosome metabolism,spliceosome,proteasome,and endocytosis.Network prediction of protein-protein interactions demonstrated that a total of 5 proteins(ABCE1,ZFP36L1,UBE2 M,CNOT7,and LSM4)were found to have a direct link with CNOT4.In conclusion,RFRP-3 is capable of accelerating the formation of primordial follicle in mouse by activating the PI3K/Akt/m TOR signaling pathway.The UPS mediates the role of RFRP-3 in primordial follicle formation through CNOT4.This study has uncovered a new regulatory mechanism of RFRP-3 during early ovarian follicular development,providing a new perspective to further explore the ovarian dysfunction and assisted reproduction.