Study On Gpr173 In The Regulation Of Reproduction By Phoenixin In Spotted Scat,Scatophagus Argus

Gpr173?orphan Gprotein-coupledreceptor:Gpr173?is a super conserved receptor expressed in brain in mammals.Phoenixin?Pnx?,a novel neuropeptide,has been confirmed as a Pnx receptor in rat.And both of them were involved in reproductive regulation.It has been reported that the Pnx can increase the expression of Gn RHR,LH and Fsh and other reproductive related genes in the pituitary gland.However,the roles and mechanisms of Gpr173 in reproductive regulation remain unclear in Scatophagus argus.To clarify the roles of Gpr173 in Pnx signaling pathway and the relationship between Gpr173 and Pnx,the Gpr173 was isolated and characterized.Tissues distributaion,expression at different gonad development,expression after treatment by E₂,Pnx and DHA,EPA and oleic acid were analysed.The signal transduction pathway of Pnx,mediated by Gpr173,was also observed.The results will help to better understand the mechanism of reproductive regulation system by neuropeptide in fish.And it also can enrich and improve the reproductive endocrine regulation network in fish and even in vertebrates.The specific research results are as follows:1.Cloning,sequence analysis,and spatiotemporal expression of Gpr173Two gene subtypes?Gpr173-1 and Gpr173-2?were obtained and characterized.Tissue distribution revealed that Gpr173-1 and Gpr173-2 were mainly expressed in the hypothalamus and pituitary by q PCR.In females,Gpr173-1 was mainly expressed in stage?and?.Gpr173-2 was high at stage?.And both Gpr173-1 and Gpr173-2expressed highly in stage?in the hypothalamus during ovarian development.Both Gpr173-1 and Gpr173-2 reached the highest at stage?during ovary development in the pituitaries.There was no significant difference in the expression of Gpr173-1 and Gpr173-2 in the pituitary during ovarian development.In males,both Gpr173-1 and Gpr173-2 expressed highly in stage?in the hypothalamus during testis development.However,there was no significant difference of the Gpr173-1 and Gpr173-2 in the pituitaries during testis development.Western Blotting showed that the Gpr173 were detected in the whole brain and testis,but not in the ovary.2.Expression of Gpr173 after treatment by Pnx,E₂,DHA,EPA and oleic acidQPCR showed that Pnx-14 significantly increased the expression of Gpr173-1after injection of Pnx-14 in the hypothalamus,and significantly inhibited the expression of Gpr173-2.Injection of Pnx-20 also significantly promoted the expression of Gpr173-1.Pnx-20?10ng/gbw?decreased the expression of Gpr173-2.It showed the expression of Gpr173-1 significantly promoted after incubation with Pnx-14 primary hypothalamic cells.However,the level of Gpr173-2 was not changed.At 6h,injection of E₂ significantly increased the expression of Gpr173-1 in the hypothalamus in the female.However,the expression of Gpr173-2 was significantly decreased.Gpr173-1 and Gpr173-2 were significantly reduced in the pituitary.At 12h,the expression of Gpr173-1 and Gpr173-2 E₂were significantly promoted after incubation with E₂ in the primary hypothalamus cells in Scatophagus argus.QPCR showed that the expression of Gpr173-1 were significantly increased after incubation with DHA and EPA,while oleic acid can not change the expression of Gpr173-1 in the primary hypothalamus cells of spotted scat.EPA downregulated significantly Gpr173-2.But the expressions of Gpr173-2 were not changed after incubation with DHA and oleic acid.The expression of Pnx was increased significantly after incubation with Oleic acid.However,DHA and EPA can not change the exression of Pnx in the primary hypothalamus cells of spotted scat.3.Gene expression and signaling pathways of Pnx mediated by Gpr173To analyze the effect of Pnx on the activation of the signaling pathway of Gpr173,the dual-Luciferase Reporter Assay System was used.To block the regulatory pathway,the PKA inhibitors were used for verification.Pnx-14 can significantly enhance the activity of luciferase driven by CRE promoter and inhibit the activity of luciferase driven by c-fos and SRE promoter.Pnx-20 can significantly enhance the activity of luciferase driven by c-fos and SRE promoters.But no effect was observed by CRE promoter.As for Gpr173-2,Pnx-14 can significantly enhance the activity of luciferase driven by the c-fos promoter,without affecting the CRE and SRE promoters.Pnx-20 can significantly enhance the luciferase activity driven by the SRE promoter.But no effect on the c-fos and CRE promoters was observed.At 3h,q PCR showed that there was no significant change of Gpr173-1 in primary hypothalamic cells after incubation with Pnx-14.At 6h,Pnx-14 could significantly increase the expression of Gpr173-1.But the increasing of Gpr173-1 by Pnx-14 was inhibited when H89,PKA inhibitor,was added.At 3h,there was no significant change of Gpr173-2 in primary hypothalamic cells after incubation with Pnx-14.But the expression of Gpr173-2 significantly inhibited after addition of H89.At 6h,no significant change was observed in all groups.At 3h,the expression of sb Gn RH and Gn RHR were significantly decreased after incubation with Pnx-14 in primary hypothalamic cells.However,the levels of sb Gn RH and Gn RHR significantly increased after addition of H89.At 6h,the expression of sb Gn RH and Gn RHR was constant in all groups.In summary,PNX can activate Gpr173-1,through c AMP/PKA pathway.And the Pnx may be participated in the reproductive regulation by the reproductive related genes such as sb Gn RH and Gn RHR in spotted scat.In addition,Pnx and Gpr173 are regulated by DHA,EPA,and oleic acid,indicating that these two are involved in the regulation reproduction by nutritional levels in spotted scat.