Receptor Identification And Expression Regulatory Mechanism Study Of Insulin-Like Androgenic Gland Hormone In Prawn

The endocrine regulatory system unique to crustaceans regulates various physiological processes,such as sexual differentiation,growth and molting.Previous studies reveal that the “sinus gland-AG-testicular” endocrine axis plays a key role in sexual differentiation of crustaceans.However,the key factors are still unclear except for the androgenic gland hormone(IAG)which is secreted by AG and regulates male sexual differentiation.The molecular mechanism of sexual differentiation in crustacean needs to be further studied.Meanwhile,crustaceans generally display sex dimorphism in growth,which show significantly different in size between male and female individuals.Therefore,as a representative species in crustacean,the study on sexual differentiation in penaeid shrimp has great theoretical significance and economical value.In this study,several key nodes in the “sinus gland-AG-testicular” endocrine axis in shrimp were studied.We identified the receptor of IAG,the CHH family neuropeptides and their receptors that regulated IAG expression and systematically studied their function in male differentiation.These results not only enriched the basic theory of the regulation process of “sinus gland-AG-testicular” endocrine axis and male sexual differentiation mechanism,but also provided new clues in developing monosex aquaculture technologies in shrimp.The major progresses are as follows:1.A putative IAG receptor gene was identified in Fenneropenaeus chinensis,designated as FcIAGR.The deduced amino acid sequence of Fc IAGR contained several conserved domains of insulin-like receptor proteins,including two L(L1 and L2)domains,a cysteine-rich(CR)domain,three fibronectin III(FN-III)domains,a transmembrane domain and an intracellular tyrosine kinase domain.FcIAGR was predominantly detected in the androgenic gland(AG)and testis.In situ hybridization analysis showed that FcIAGR transcripts were exclusively expressed in the cells of the AG,spermatocytes and sperm cells in the testis.Protein co-localization analysis in HEK293 cells showed that FcIAGR could co-localize with both Fc IAG1 and FcIAG2,respectively.Yeast two-hybrid assay further confirmed the interactions between FcIAGR and FcIAGs.After a long-term silencing of FcIAGR with dsRNA,the development of spermatocytes from the secondary spermatocytes was arrested,indicating that FcIAGR was involved in spermatogenesis.The results laid a foundation for further study on male sexual differentiation in shrimp.2.Two putative crustacean hyperglycemic hormone(CHH)genes,which were differentially expressed in male and female individuals,were identified in Litopenaeus vannamei,designated as LvCHH4751 and LvCHH4752.The deduced amino acid sequences of LvCHH4751 and LvCHH4752 belonged to type I CHH family neuropeptides,both containing a signal peptide,a CHH-precursor-related peptide,a mature peptide,a processing signal KR and an amidation signal GK.LvCHH4751 and LvCHH4752 was predominantly detected in eyestalk and gonad of both sexes,and the expression level was significantly higher in males than in females.In situ hybridization analysis showed they were expressed in endocrine cells of eyestalk.After silencing with dsCHH4751 or dsCHH4752,the expression level of LvIAG was significantly upregulated,consistent with the result after unilateral eyestalk ablation.The expression level of LvIAG was apparently down-regulated after injection with the recombinant protein of LvCHH4751 and LvCHH4752,respectively.The data indicated that LvCHH4751 and LvCHH4752 participated in the negatively regulation of LvIAG expression.3.A guanylate cyclase gene,designated as LvGC,was identified in L.vannamei.The deduced amino acid sequences of LvGC belonged to single-pass transmembrane protein,containing an extracellular binding domain,a transmembrane domain,an intracellular protein kinase-like domain and an intracellular guanylate cyclase catalytic domain.LvGC was predominantly detected in nerve tissues of both sexes and the expression level was significantly higher in males than in females.After silencing with dsCHH4751 or dsCHH4752,the relative expression level of LvGC was significantly down-regulated.When injection with the recombinant protein of LvCHH4751 or LvCHH4752,the relative expression level of Lv GC was significantly up-regulated.Yeast two-hybrid assay showed that LvGC could interact with LvCHH4751 and LvCHH4752.After silencing of LvGC with dsRNA,the relative expression level of LvIAG was significantly up-regulated.The data indicated that LvGC participated in the regulation of male sexual differentiation probably through interacting with LvCHHs.The present study will provide important evidence for understanding the molecular mechanism that regulates the male sexual differentiation in crustacean.