Testicular Dnmt3 expression and global DNA methylation are down-regulated by gonadotropin releasing hormones in the ricefield eel Monopterus albus

Yize Zhang,Weimin Zhang,Lihong Zhang,Xin Sun

doi:10.1038/srep43158

Abstract

In vertebrates, DNA methyltransferase 3 (Dnmt3) homologues are responsible for de novo DNA methylation and play important roles in germ cell development. In the present study, four dnmt3 genes, dnmt3aa, dnmt3ab, dnmt3ba and dnmt3bb.1, were identified in ricefield eels. Real-time quantitative PCR analysis showed that all four dnmt3 mRNAs were detected broadly in tissues examined, with testicular expression at relatively high levels. In the testis, immunostaining for all four Dnmt3 forms was mainly localized to spermatocytes, which also contained highly methylated DNA. All three forms of Gonadotropin-releasing hormone (Gnrh) in the ricefield eel were shown to decrease the expression of dnmt3 genes in the in vitro incubated testicular fragments through cAMP and IP3/Ca2+ pathways. Moreover, in vivo treatment of male fish with three forms of Gnrh decreased significantly the testicular Dnmt3 expression at both mRNA and protein levels, and the global DNA methylation levels. These results suggest that the expression of Dnmt3 and global DNA methylation in the testis of ricefield eels are potentially down-regulated by Gnrh, and reveal a novel regulatory mechanism of testicular Dnmt3 expression in vertebrates.

Highlights

Regulation of DNA methyltransferases (Dnmts)[3] expression[15]
Four forms of ricefield eel dnmt[3] cDNAs were obtained in the present study, which were designated as dnmt3aa (KX524491), dnmt3ab (KX524492), dnmt3ba (KX524493), and dnmt3bb.[1] (KX524494), respectively, based on the phylogenetic analysis (Supplementary Fig. S1) and by following the nomenclature of zebrafish dnmt[3] genes
All three forms of gonadotropin-releasing hormone (Gnrh) increased cAMP levels in the in vitro incubated testicular fragments (Supplementary Fig. S8). These results suggest that Gnrh down-regulates dnmt[3] expression in the testis of ricefield eels possibly through both cAMP and IP3/Ca2+ pathways but not the DAG/ PKC pathway

Summary

Introduction

Regulation of Dnmt[3] expression[15]. The zinc finger DNA-binding domain proteins Sp1 and Sp3 activate the transcription of human DNMT3A and DNMT3B16, and vascular endothelial zinc finger 1 (Vezf1) activates the transcription of mouse Dnmt3b17. We have demonstrated that DNA methylation of cyp19a1a promoter is increased in gonads of ricefield eels during sex change towards male[28], suggesting an important role for DNA methylation in testicular differentiation and development. Four de novo DNA methyltransferases, Dnmt3aa, Dnmt3ab, Dnmt3ba, and Dnmt3bb.[1], were identified in the ricefield eel, and their expression was characterized at both mRNA and protein levels, in the testis. It is demonstrated for the first time that Dnmt[3] expression and global DNA methylation are potentially down-regulated by Gnrh signals in the testis of a vertebrate, the ricefield eel Monopterus albus

Methods

Results

Conclusion