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

ABSTRACT

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.

KEYWORDS:

• Melatonin
• testis
• circadian rhythm
• Tph
• Aanat
• Asmt
• zebrafish

Acknowledgments

The authors are grateful to the Directors, IBSD, India, along with all the scientists, technicians, and administrative persons for their support. The authors are appreciative of Mr. W. Rahul for his support in conducting experiments and maintenance of animals in laboratory conditions. We are also grateful to the security personnel/of the Institute for their assistance during continuous experimentation during official holidays. Our sincere gratitude to every Indian taxpayer for their contribution towards the funding for research.

Author contributions

SDD acquisition of data, analysis/interpretation, statistical analysis, drafting of the manuscript. GM immunohistochemistry capture of image, formatting, sampling. ZAK critical analysis of the data, organization of figures. HKS planning and reviewing. AC, concept/design, manuscript preparation, and critical review of the definitive version

Disclosure of potential conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Additional information

Funding

Core grant of IBSD, Imphal, Unit of Excellence grant of Department of Biotechnology (DBT), (BT/407/NE/U-Excel/2013), Department of Science and Technology; Department of Biotechnology, Ministry of Science and Technology, India [BT/407/NE/U-Excel/2013];