A possible role of the ecdysone receptor in modulating gonad-inhibiting hormone gene expression in the black tiger prawn, Penaeus monodon

Abstract

Gonad-inhibiting hormone (GIH) is a neuronal eyestalk peptide that inhibits ovarian maturation in crustaceans. In this study, the aim was to investigate the transcriptional regulation of the GIH gene in the black tiger prawn (Penaeus monodon). An analysis of a 1158 bp DNA fragment 5′ upstream of the GIH gene revealed a putative half-site of the hormone response element (HRE) at −561 to −566 relative to the predicted transcription start site with an upstream imperfect palindromic sequence separated by one nucleotide. Deletion of the HRE from the GIH 5′ fragment increased the promoter activity in Sf9 cells, whereas the addition of 20-hydroxyecdysone (20E) reduced the promoter activity by approximately 60–70%. A candidate HRE-binding nuclear receptor, an ecdysone receptor (EcR) that serves to bind 20E in crustaceans, was therefore functionally characterized. EcR was expressed ubiquitously in female prawns, with the highest expression levels occurring in eyestalks, suggesting that EcR possibly stimulates vitellogenesis by binding to the HRE in the GIH 5′ regulatory sequence, thereby inhibiting GIH expression. This was verified by specific binding between a recombinant EcR-GST fusion protein and the HRE in the GIH 5′ DNA fragment. In addition, in vivo knockdown of EcR resulted in a 3-fold elevation of GIH expression levels in the eyestalks of adult female prawns. Taken together, the results obtained in this study indicate that EcR acts as an inhibitory factor in the regulation of the GIH gene.