Examination of the potential role of CHH in regulating the expression of IAGBP gene through the eyestalk-testis pathway

Abstract

Crustacean hyperglycemic hormone (CHH) is a peptide hormone of the CHH family produced by the X-organ/sinus gland complex and plays role in metabolism, osmoregulation, molting, and reproduction. However, the mechanism by which CHH regulates crustacean sexual behavior remains unclear. To elucidate the mechanism of CHH-mediated sexual regulation in this study, the giant freshwater prawn (Macrobrachium rosenbergii) CHH gene (MrCHH) was cloned and characterized. The MrCHH open reading frame was 408 bp in length and was found to encode a 135–amino acid putative protein of 15.42 kDa. In normal prawns, MrCHH mRNA transcripts were abundant in the examined tissues, as follows: eyestalk > intestine > androgenic gland > testis > ventral nerve cord > stomach > gills > heart > brain. In prawns injected with dsCHH RNA, MrCHH mRNA and protein expression were down-regulated at 24 h in the eyestalk and testis compared with prawns injected with negative control dsGFP. Moreover, MrCHH dsRNA increased mRNA and protein expression of M. rosenbergii insulin-like androgenic gland hormone–binding protein (MrIAGBP), indicating that MrCHH inhibits MrIAGBP expression. Additionally, after unilateral or bilateral eyestalk ablation, expression of both insulin-like androgenic gland hormone (IAG) and IAGBP in the prawn testis was significantly up-regulated. Immunohistochemical analyses showed that after unilateral or bilateral eyestalk ablation, MrIAGBP protein levels also increased in the testis, further suggesting that MrCHH is an inhibitor that negatively regulates the expression of genes that play roles in male gonadal development. Our findings thus suggest that MrCHH serves to enrich the “eyestalk-androgenic gland-testis” endocrine axis involved in IAG signaling, providing significant information for further explorations of the mechanism underlying sex regulation in other decapod crustaceans.