Molecular Mechanism Underlying Reproductive Development Of RFRP-3 In Female Mice

RFRP-3 inhibits the synthesis and release of gonadotropin by acting on pituitary gonadotropes and GnRH neurons,which is a unique inhibitor of HPG axis at the hypothalamic level in mammals.Despite its functional significance,the molecular mechanism of RFRP-3 action in the target cells has not been fully elucidated.Especially,there is little evidence showing that RFRP-3 plays a decisive role in initiating the puberty of mammals.Basing on RNA-seq sequencing and bioinformatics analysis,this study clarifies the molecular mechanism of RFRP-3 on regulating reproductive development of mammals by using molecular biological techniques in vivo and vitro.The detailed research is as follows:(1)Identified the effects of RFRP-3 on puberty development and onsetThe modulatory effects of the RFRP-3 on the pubertal development were explored by injecting exogenous RFRP-3 neuropeptide or RFRP-3 over-expression lentivirus into the lateral ventricles in this study.Quantitative real-time PCR and immunohistochemistry results showed that both RFRP-3 polypeptide and lentivirus injection suppressed the level of GnRH mRNA and protein,the kiss1/GPR54 genes exprssion as well.In addition,both RFRP-3 polypeptide and lentivirus injection significantly decreased the serum concentrations of luteinizing hormone and estradiol.However,ovariectomy eliminated the inhibitory role of RFRP-3 on plasma LH levels in prepubescent female mice,while this phenomenon has not been detected in adult female mice.This result indicated that the effects of RFRP-3 on the gonadotropin secretion may be associated with estrogen levels and developmental stages.Besides,anatomy results showed that RFRP-3 lentivirus injection significantly decreased endometrium thickness and the number of initial follicles.These results suggested that RFRP-3 could suppress the GnRH/LH surge,via acting on GnRH neurons directly or indirectly,thereby delaying the onset of puberty,then playing an important role in gonad development and maturation.(2)Explored the molecular mechanism of RFRP-3 on gonadotropin secretion in pituitary cells.Whether the inhibition of LH secretion by RFRP-3 occurs at the pituitary level or the hypothalamus level remains controversial.In the present study,the molecular mechanism of RFRP-3 on gonadotropin secretion was performed in pituitary L?T2 cell.The results showed that RFRP-3 polypeptide did not change the basal gonadotropin secretion,while it significantly suppressed GnRH-induced gonadotropin secretion,RFRP-3 gene overexpression as well.Through high-throughput sequencing data and bioinformatics analysis,the results showed that the protein kinase A and protein kinase C pathways of GnRH signaling pathway,obtained based on KEGG,were activated by GnRH.In addition,PKA inhibitor H89 and PKC pathway inhibitor GF pretreatment results showed that H89 and GF have weakened the inhibitory effect of RFRP-3 on GnRH-induced gonadotropin secretion,respectively.The above-mentioned results indicated that RFRP-3 may exert the physiological inhibitory effect by inhibiting protein kinase A and protein kinase C-mediated ERK activation.These results suggested that the inhibitory role of RFRP-3 on gonadotropin secretion was dependent on GnRH at pituitary level,by directly inhibiting GnRH-mediated PKA and PKCsignaling pathway.(3)Confirmed the molecular mechanism of RFRP-3 on GnRH release in hypothalamic cellsBy using the GT1-7 cell line,a model of GnRH-secreting neurons of the hypothalamus,the present study examined the potential signal transduction pathways involved in RFRP-3 action in GnRH neurons.It discovered that exogenous RFRP-3 and RFRP-3 gene overexpression significantly inhibited GnRH transcription and protein synthesis and secretion.According to high-throughput sequencing data,bioinformatics analysis showed that the activations of cAMP signaling pathways,MAPK signaling pathways,and GnRH signaling pathways were repressed.Based on the KEGG database,we speculated that RFRP-3 may inhibit GnRH secretion by inhibiting cAMP/PKA/ERA signaling pathway activation in GT1-7 cells.Combining different cascade signaling pathway inhibitors and RFRP-3,the hypothesis has been verified at the cellular level.Pretreatment H89 attenuated the inhibitory effect of RFRP-3 on GnRH secretion,while pretreatment H89 did not change the inhibition of RFRP-3.It was further comfirmed that RFRP-3 exerts its inhibitory effect on Gn RH transcriptions and translationby especifically acting on the cAMP/PKA/ERK pathway.In addition,the mutual interaction of RFRP-3,estrogen signaling pathway and kisspeptin signaling pathway were also confirmed in this study.The results showed RFRP-3,estrogen and kisspeptin were mutually enhanced,and co-regulated GnRH release in unique pathway.The molecular mechanism of RFRP-3 on modulating mammalian reproductive development in female mice was elaborated from multi-aspect and systematically in the present study.In mice,RFRP-3,binding to the principal receptor GPR147 that couple to Ga_i,reducing intracellular cAMP/PKA mediated ERK phosphorylation,thus inhibiting GnRH synthesis and secretion.GnRH secreted into the pituitary portal venous system and regulated the secretion of gonadotropins at the pituitary level,thereby regulating the gonads development and sex hormones synthesis and secretion,and controlling puberty development and onset,reproductive function as well.