Study On The Role Of Phoenixin In The Growth And Reproduction Of Spotted Scat, Scatophagus Argus

Phoenixin(Pnx)is an endogenous peptide known to be involved in reproduction and food intake in rats,with two active isoforms,phoenixin-14(Pnx-14)and phoenixin-20(Pnx-20).However,little is known about the functions of Pnx in teleost.In order to elucidate the role and mechanism of pnx in growth and reproduction of Scatophagus argus,in the present study,we cloned the c DNA fragment of S.argus containing pnx open reading frame(ORF).The expression of pnx in 11 tissues(hypothalamus;pituitary,ovary,testis,heart,liver,kidney,spleen,head-kidney,intestine,muscle)in males and females were detected by semi-quantitative reverse transcription PCR(RT-PCR).The fine localization of Pnx in hypothalamus was analyzed by immunohistochemical technique.The expression of pnx in the hypothalamus and pituitary gland during different developmental stages(stages II,II and IV)were measured by real-time PCR(q PCR).The effects of Pnx-14 and Pnx-20 on Ghrhr(growth hormone releasing hormone receptor)and Gh(growth hormone)in the pituitary and Ghrs(growth hormone receptor)in the liver after in vivo injection(10 ng/g body weight and 100 ng/gbw)and in vitro incubation(10 n M and 100 n M)were detected by q PCR.The effects of Pnx-14 and Pnx-20 on Gn RHs(gonadotropin-releasing hormone)in the hypothalamus and Gn RHR(gonadotropin-releasing hormone receptor),Lh(luteinizing hormone)and Fsh(follicular stimulating hormone)in the pituitary were measured after injection(10 ng/gbw and 100 ng/gbw)and in vitro incubation(10 n M and 100 n M)by q PCR.The main results are as follows:1.Molecular cloning,characterization and temporal and spatial expression of pnx in S.argus.The ORF of pnx was 210 bp which encoded 69 amino acids.Homology comparison revealed that S.argus Pnx was highly homologous to Larimichthys crocea,Oreochromis niloticus and Fundulus heteroclitus(>80%).pnx was detected in all 11 tissues(hypothalamus;pituitary,ovary,testis,heart,liver,spleen,kidney,head-kidney,intestine,muscle)of both male and female in spotted scat(Scatophagus argus),including growth axis(hypothalamus,pituitary and liver)and reproduction axis(hypothalamus,pituitary and gonad).Immunohistochemical results showed that Pnx positive signals were detected in the paraventricular nucleus(PVN),arcuate nucleus(ARC),and lateral hypothalamic area(LH).During ovary development,the expression of pnx in phase IV was higher than in phase II and III in the hypothalamus.In the pituitary,pnx expression was highest in phase IV,moderate in phase III,and lowest in phase II.2.Effects of Pnx-14 and Pnx-20 on related to growth expression in S.argus In vivo,the expression of Ghrhr and Gh in the pituitary,as well as Ghr1 and Ghr2 in the liver,increased significantly after injecting S.argus with Pnx-14 and Pnx-20(10 ng/g and 100 ng/g bodyweight).In vitro,when pituitary and liver fragments were cultured in vitro with Pnx-14 and Pnx-20(10 n M and 100 n M)for 6 h,the expression of Ghrhr(growth hormone-releasing hormone receptor)and Gh(growth hormone)in the pituitary,and Ghr1(growth hormone receptor 1)in the liver significantly increased,except Ghr2(growth hormone receptor 2)incubated with 10 n M and 100 n M Pnx-20 and Ghr1 incubated with 10 n M Pnx-20.The results of in vivo and in vitro tend to be consistent,indicating that Pnx-14 and Pnx-20 play an important role in the growth activities of S.argus.3.Effects of Pnx-14 and Pnx-20 on related to reproduction expression in S.argus In vivo,The expression of Gn RHR(gonadotropin releasing hormone receptor),Lh(luteinizing hormone)and Fsh(follicular stimulating hormone)in the pituitary increased significantly after injecting S.argus with Pnx-14 and Pnx-20(10ng/g and 100 ng/g body weight),except Gn RHR and Fsh treated with 10 ng/gbw Pnx-20 in the pituitary and Gn RHs(gonadotropin releasing hormone)in the hypothalamus in vivo.When hypothalamus and pituitary fragments were cultured in vitro with Pnx-14 and Pnx-20(10 n M and 100 n M)for 6 h,the expression of Gn RHR,Lh and Fsh in the pituitary increased significantly,except Gn RHs in the hypothalamus.The results of in vivo and in vitro tend to be consistent,indicating that Pnx-14 and Pnx-20 play an important role in the reproduction activities of S.argus.Pnx is likely to be involved in the regulation of the growth in S.argus by increasing Ghrhr and Gh expression in the pituitary,Ghr1 and Ghr2 in the liver,and Ghr1 directly in the liver.In addition,the expression of pnx increased gradually in the II phase,III phase and IV phase during hypothalamus and pituitary ovary development.The results indicate that Pnx can increase the expression of Gh RHR in the pituitary,thereby promoting the secretion of Lh and Fsh,and also directly participate in the reproductive regulation of S.argus.by promoting the expression of Lh in the pituitary.In conclusion,pnx may play an important role in the growth and reproductive regulation of S.argus.