Effects Of N-carbamylglutamate,Arginine And Estrogen-receptor Inhibitor On GnRH Secretion And Relative Gene Expression In GT1-7 Cells

N-Carbamylglutamate（NCG）,which had been identified as an endogenous activator of arginine（Arg）,has been proved to be effective and safety on shortening the onset of puberty and improving animal reproduction,and has been widely applied in animal production,but there are few study on its mechanisms of action.This study was conducted to investigate the effects of NCG,Arg and estrogen-receptor inhibitor on GnRH secretion and genes expression related with GnRH production and secretion using GT1-7 cells,a model of mouse immortalized hypothalamus GnRH neuron in vitro.Experiment one: Effects on GT1-7 cell proliferation,GnRH secretion and relative gene（GnRH,Kiss-1,GPR54,ERα,c-fos and nNOS）mRNA expression treated with NCG or Arg were detected in different concentrations at different time points.GT1-7 cells were cultured in CO₂ incubator,then inoculated into 12-well culture plates.In group one,experimental groups were treated with NCG in different concentrations（10μM,100μM,1m M）at different time points（12,24h）,while control group was treated with dissolvent of NCG（1M NaOH）only.In group two,experimental groups were treated with Arg in different concentrations（0.4m M,2m M,4m M）at different time points（12,24h）,control group was treated with GT1-7 cells culture solution.After incubating 12 or 24 h,cell culture supernatant and cell pellet were collected respectively.GnRH concentration in supernatants were determined by mouse GnRH enzyme-linked immunosorbent assay（ELISA）kit.The mRNA expression of those group that affect GnRH seretion significantly were determined by relative fluorescence quantitative method.The results showed: comparing with control group,NCG or Arg treatments had no effects on cell number（P>0.05）;NCG treatments（10μM,100μM,1m M）12h and Arg treatment（4m M）24h decreased GnRH secretion in GT1-7 cells（P<0.05）.NCG treatments can decrease GnRH,Kiss-1,GPR54 and nNOS mRNA expressions（P<0.05）,while Arg treatment can decrease GnRH,Kiss-1,GPR54,ERα,and nNOS mRNA expressions（P<0.05）.Either NCG or Arg can suppress GnRH secretion through down-regulating mRNA expressions of GnRH,Kiss-1,GPR54 and nNOS.Experiment two: The regulation model of GnRH secretion induced by estrogen in GT1-7 cells had been builted in our previous research.GT1-7 cells were treated with 100 p M 17-β estrogen at 12 h with 3 differernt concentrations of estrogen-receptor inhibitor ICI182,780（10 p M,100 p M,1 n M）to study its effect to GnRH secretion and GnRH-related gene expressions.The results show that 100 p M 17-β estrogen increased GnRH secretion（P<0.05）.1 n M estrogen-receptor inhibitor ICI182,780 can suppress GnRH secretion（P<0.05）induced by estrogen,and decrease GnRH,Kiss-1,ERα,and nNOS mRNA expressions（P<0.05）as well.This experiment indicated that estrogen-receptor inhibitor decrease GnRH secretion by down-regulating GnRH,Kiss-1,ERα,and nNOS mRNA expression.In brief,NCG,Arg and ICI182,780 can decrease GnRH secretion through down-regulating GnRH,ERα,Kiss-1 and nNOS mRNA expression,which imply nNOS could be anothor signal molecule to regulate GnRH synthesis and release besides Kiss-1.