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Leydig cells play a critical role in male reproductive physiology, and their dysfunction is usually associated with male infertility. Melatonin has an important protective and regulatory role in these cells. However, the lack of suitable animal models impedes us from addressing the impact of endogenous melatonin on these cells. In the current study, by using arylalkylamine N-acetyltransferase (AANAT) overexpression transgenic sheep and AANAT knockout mice, we confirmed the regulatory effects of endogenously occurring melatonin on Leydig cells as well as its beneficial effects on male reproductive performance. The results showed that the endogenously elevated melatonin level was correlated with decreased Leydig cell apoptosis, increased testosterone production, and improved quality of sperm in melatonin-enriched transgenic mammals. Signal transduction analysis indicated that melatonin targeted the …