Inhibitory effect of melatonin on testosterone synthesis is mediated via GATA-4/SF-1 transcription factors  Fenju Qin, Jie Zhang, Linsen Zan, Weiqiang Guo, Jin Wang, Lili Chen, Yi Cao, Ouxi Shen, Jian Tong  Reproductive BioMedicine Online  Volume 31, Issue 5, Pages 638-646 (November 2015) DOI: 10.1016/j.rbmo.2015.07.009 Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Reduced levels of cyclic adenosine monophosphate (a) and testosterone (b) in TM3 Leydig cells after the addition of various doses of melatonin, and restoration of levels when treated with the melatonin antagonist, N-acetyl-2-benzyltryptamin. The melatonin group was significantly different to controls: *P < 0.05, **P < 0.01; as was the N-acetyl-2-benzyltryptamin 1 µmol/l plus melatonin group compared with the melatonin group at the same dose: #P < 0.05, ##P < 0.01. cAMP = cyclic adenosine monophosphate. Reproductive BioMedicine Online 2015 31, 638-646DOI: (10.1016/j.rbmo.2015.07.009) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Reduced expression of several genes using real-time polymerase chain reaction and western blot analyses: GATA-4 (a, f), SF-1 (b, f), StAR (c, f), P450SCC (d, f) and 3β-HSD (e, f), following the addition of various doses of Mel to TM3 Leydig cells and restoration of levels with melatonin antagonist (N-acetyl-2-benzyltryptamin) treatment. The melatonin group was significantly different to the controls: *P < 0.05, **P < 0.01; as was the N-acetyl-2-benzyltryptamin 1 µmol/l plus melatonin group compared with the melatonin group at the same dose: #P < 0.05, ##P < 0.01. Reproductive BioMedicine Online 2015 31, 638-646DOI: (10.1016/j.rbmo.2015.07.009) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Time-dependent reduction in the concentrations of testosterone in the plasma of mice (up to 2 h) after the injection of melatonin into the testes of mice, and restoration to the 0-h level at 4 h. Other time-period groups were also compared with 0 h (the moment before injection): *P < 0 .05; **P < 0.01; n = 6 for all times groups. Reproductive BioMedicine Online 2015 31, 638-646DOI: (10.1016/j.rbmo.2015.07.009) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 (a–e) Immunohistochemical staining of GATA-4 protein (brown) and nuclear counterstain hematoxylin (blue) after 0 h, 0.5 h, 1 h, 2 h or 4 h of melatonin injection. Arrowheads denote L (Leydig cells, purple), S (Seminiferous tubules, green). Scale bars: 50 µm. (f) Changes in mean signal intensity of GATA-4 expression in the interstitial compartment. The other time-period groups were also compared with the group at 0 h (the moment before injection), **P < 0.01; n = 6 for all times groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Reproductive BioMedicine Online 2015 31, 638-646DOI: (10.1016/j.rbmo.2015.07.009) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 (a–e) Immunofluorescence staining of SF-1 protein (green) and nuclear counterstain DAPI (blue) after 0 h, 0.5 h, 1 h, 2 h or 4 h of melatonin injection. Arrowheads indicate L (Leydig cells, red), S (seminiferous tubules, brown). Scale bars: 50 µm. (f) Changes in mean fluorescence intensity of SF-1 expression in the interstitial compartment. The other time-period groups were compared with the group at 0 h (the moment before injection); **P < 0.01; n = 6 for all times groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Reproductive BioMedicine Online 2015 31, 638-646DOI: (10.1016/j.rbmo.2015.07.009) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 Gene and protein expression of GATA-4 (by real-time polymerase chain reaction and western blot analyses) in mice testes after 0 h, 0.5 h, 1 h, 2 h or 4 h of melatonin injection. The other time-period groups were compared with group 0 h (the moment before injection), **P < 0.01; n = 6 at all times groups. Reproductive BioMedicine Online 2015 31, 638-646DOI: (10.1016/j.rbmo.2015.07.009) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions

Figure 7 Gene and protein expression of SF-1 (by real-time polymerase chain reaction and western blot analyses) in mice testes after 0 h, 0.5 h, 1 h, 2 h or 4 h of melatonin injection. The other time-period groups were compared with the group at 0 h (the moment before injection); **P < 0.01; n = 6 for all time-period groups. Reproductive BioMedicine Online 2015 31, 638-646DOI: (10.1016/j.rbmo.2015.07.009) Copyright © 2015 Reproductive Healthcare Ltd. Terms and Conditions