| Previous studies demonstrated that different wavelengths of monochromatic lights could influence the growth and reproduction of chicks.Hypothalamic GnRH-Ⅰ and GHRH are important factors in regulating of reproduction and growth,but the effects of light color on secretions of GnRH-Ⅰand GHRH in the chick hypothalamus and its pathway are still unclear.In this study newly hatched chicks were divided into intact,sham-operated and pinealectomy groups and exposed to white light(WL,400-760 nm),red light(RL,660 nm),green light(GL,560 nn)or blue light(BL,480 nm)using a light-emitting diode(LED)system for two weeks,respectively.We explore the effects of different monochromatic lights on chick hypothalamic GnRH-Ⅰ and GHRH expression and its pathway.The main results are as follows:Ⅰ:Role of melatonin in monochromatic light affected GnRH-Ⅰ expression in chick hypothalamusImmunohistochemical staining showed that GnRH-Ⅰ-ir neurons were observed in the POA,POP,SM,SL and nCPa of the hypothalamus,and GnRH-Ⅰ-ir neurons were most abundant in the nCPa.In the GL intact group,the mRNA and protein levels of GnRH-Ⅰ in the hypothalamus,the percent cell area and mean cell optical density(OD)of the GnRH-Ⅰ-ir cells of the nCPa were decreased by 13.18%-34.46%(P=0.013-0.046)and 19.59%-39.08%(P=0.002-0.018)compared to those in the chicks exposed to the WL and RL,respectively.In addition,similar to GnRH-Ⅰ expression,the plasma testosterone level in the GL was decreased by 19.04%,21.66%and 10.41%compared to the WL(P=0.009),RL(P=0.003)and BL(P=0.183)intact groups,respectively.After pinealectomy,with the decreased of melatonin level,GnRH-Ⅰ expression and plasma testosterone level increased,and there were no significant differences among the WL,RL,GL and BL groups(P=0.185-0.986).Meanwhile,the results in vitro showed that melatonin(250 pg/mL)significantly reduced the expression of hypothalamic GnRH-Ⅰ mRNA level.These results indicated that melatonin effected on monochromatic light-induced GnRH-Ⅰ expression in chick hypothalamusⅡ:Role of melatonin in monochromatic light influenced GnIH expression in chick hypothalamusGnIH-ir neurons were mostly observed in the PVN of the hypothalamus.The mRNA and protein levels of hypothalamic GnIH,the percent cell area and mean cell OD of the GnIH-ir cells of the PVN in the chicks exposed to GL were increased by 19.62%-34.46%(P=0.013-0.046),23.12%-39.08%(P=0.006-0.018)and 9.81%-17.33%(P=0.006-0.400)compared to the WL,RL and BL intact groups,respectively.However,after pinealectomy,the GnIH mRNA and protein levels decreased by 22.05%-24.25%,22.58%-25.04%,26.39%-26.94%and 24.55%-24.99%in the WL(P=0.001-0.005),RL(P=0.005-0.007),GL(P=0.000)and BL(P=0.001)groups compared to the corresponding sham-operated groups.The differences in GnIH levels according to light color were not significant in the pinealectomy groups.The results in vitro showed that melatonin(250 pg/mL)significantly enhanced the expression of GnIH in the hypothalamus.These results demonstrated that melatonin plays a critical role in monochromatic light-induced GnIH expression in chick hypothalamus.Ⅲ:Effect of melatonin receptor on monochromatic light-induced GnIH expression in chick hypothalamusMelatonin receptor subtype Mel1a-ir cells were distributed in the POA,POP and SCN,Mel1b-ir cells were distributed in the POA and SCN,and Mel1c-ir cells were distributed in the PVN of the chick hypothalamus.In the GL intact group,the mRNA and protein levels of Mel 1a,Mel 1b and Mel1c in the hypothalamus,and the plasma melatonin concentration were increased by 10.72%-31.28%,9.18%-36.91%and 7.58%-25.25%compared to the WL(P=0.000-0.008),RL(P=0.000-0.022)and BL(P=0.001-0.170)intact groups,respectively.In addition,the plasma melatonin concentration showed a positive correlation with GnIH protein.In vitro showed that melatonin effected the hypothalamic CGnIH expression by Mel1c.Double-labeled immunofluorescence showed that GnIH neurons coexpressed with Mellc.These results suggested that melatonin regulates the expression of GnIH induced by monochromatic light via Mel1c in the chick hypothalamus.Ⅳ:Role of GnIH in melatonin mediated monochromatic light-induced GnRH-I expression of chick hypothalamusThe mRNA and protein levels of GnIHR expression in the GL group reflected a significant increase by 21.61%-22.83%,26.69%-26.79%and 8.62%-12.12%compared with the WL(P=0.005-0.032),RL(P=0.002-0.020)and BL(P=0.075-0.296)groups.Furthermore,after pinealectomy,GnIHR expression decreased,and there were no significant differences among the WL,RL,GL and BL groups.The plasma melatonin concentration showed a negative correlation with GnRH-Ⅰ protein and a positive correlation with GnIHR protein.Both protein expressions of GnRH-Ⅰand GnIHR showed a negative correlation in the hypothalamus.The results in vitro also showed that melatonin suppressed the expression of GnRH-Ⅰ by Mel1c.Double-labeled immunofluorescence showed that GnIH axon terminals were near GnRH-Ⅰ neurons,some GnRH-I neurons coexpressed with GnIHR.These results demonstrated that GnIH take part in the effect of monochromatic light-induced GnRH-I expression by melatonin--receptor Me1lc pathway in the chick hypothalamus.Ⅴ:Role of melatonin in monochromatic light affected GHRH expression in chick hypothalamusImmunohistochemical staining results showed that the GHRH-ir neurons were distributed in the IN of the chick hypothalamus.The mRNA and protein levels of GHRH in the hypothalamus and the plasma GH concentrations in the chicks exposed to GL were increased by 9.10%-21.75%,9.19%-34.51%and 6.76%-8.71%compared to those in the chicks exposed to WL(P=0.010-0.050),RL(P=0.002-0.013)and BL(P=0.051-0.473)in the intact group,respectively.The plasma melatonin concentrations showed a positive correlation with the expression of GHRH and the plasma GH concentrations after the various monochromatic light treatments.After pinealectomy,however,these parameters decreased and there were no significant differences between GL and the other monochromatic light treatments.These results indicated that melatonin effected on monochromatic light-induced hypothalamus GHRH expression and GH secretion in chicks.Conclusions:Melatonin plays a critical role in the mechanism by which monochromatic green light affects GnRH-I expression by regulating the feedback of GnIHR and GnIH,which may be involved in signaling Mel1c in the hypothalamus of chicks,then reduced plasma testosterone level.On the other hand,melatonin mediated the monochromatic green illumination-enhanced GHRH expression in the hypothalamus and plasma GH concentrations in chicks. |
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