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This study for the first time showed the presence of melatonin biosynthesizing genes ( Tph1a, Aanat1, Aanat2 and Asmt)  in the zebrafish ( Danio rerio ) testis. Melatonin is reported to be produced mainly in the pineal organ, but there is also evidence for the presence of melatonin in several extra-pineal sites. The presence of a single gene-specific band in agarose gel electrophoresis after end-point PCR and a single peak in qRT-PCR melting curve analysis indicates that the testis of zebrafish expresses the mRNA of enzymes for melatonin biosynthesizing machinery. Furthermore, we tried to demonstrate the presence of melatonin biosynthesizing enzymes at the translational level by immunohistochemistry, which confirms the localization of both Aanat and Asmt proteins in the testis of zebrafish. Moreover, melatonin ELISA reveals a higher level in the testis than the serum at noon, but a similar concentration is observed during the midnight. Moreover, testes display a rhythmic mRNA expression of  Tph1, Aanat1 , and  Aanat2 , while  Asmt  is arrhythmic. These results support an almost ubiquitous biosynthesis of melatonin in the peripheral organs of zebrafish, which can be related to a local role of this hormone as an autocrine or paracrine manner.

biological rhythm research

Differential gene expression and immunohistochemical localization of the key melatonin biosynthesizing enzymes in the testis of zebrafish ( Danio rerio )