| Gonad-inhibiting hormone (GIH) is one of an important neuropeptide familyhormone, which synthesized and released in X-organ-sinus gland (XO-SG) complexof crustacean eyestalks, and inhibited the gonad maturation of males and femalescrustacean. In this study, cDNA encoding GIH (lvGIH) was cloned from the eyestalkof Litopenaeus vannamei by SMARTer RACE. The sequence and structure of lvGIHwere analyzed and predicted by bioinformatics.Immunocytochemical were carried outto determine the expressions and distribution of GIH in eyestalk. Expressions ofgonad development related genes(GIH, DMC1, VASA and VTG)were examined usingReal time fluorescent quantitative PCR after dealt with the estrogen and eyestalkextract respectively. Main results as follows:1Sequence analysis of GIHThe GIH cDNA full-length was865bp which containing56bp5’UTR,521bp3’UTR(poly(A) tail included), and288bp ORF, which encoded96amino acidsincluding a17amino acids signal peptide. Sequence and phylogenetic tree analysisshowed that lvGIH shared a shortest evolutional distance and the shared96%identitywith the Penaeus monodon GIH,63%identity with the Metapenaeus ensis GIH, butbelow45%with Homarus gammarus GIH, Homarus americanus GIH, Scyllaparamamosain GIH, Macrobrachium nipponense GIH and Armadillidium vulgareGIH, However, lvGIH shared the77%,93%and89%identity with the Penaeusjaponicus MIH, Litopenaeus stylirostris MIH and Eriocheir sinensis MIHrespectively.Structure analysis indicate that the lvGIH precursor molecular weight is11158.05Da, and the isoelectric point is9.15. Amino acid analysis showed that thelvGIH contained13.5%Leu (L),11.5%Arg (R) and8.3%Asn (N), respectively.Inaddition, lvGIH is a kind of transmembrane protein which included two possibletransmembrane structure, one is located at3-20amino acids alpha helix and from inner to outer, and another is located at1-20amino acids alpha helix and from outer toinner.The first17amino acids were signal peptide. lvGIH subcellular localizationshowed that77.8%subcellular organelles located outside of the cell(includingcytoderm),11.1%in vacuole and endoplasmic reticulum. Three-dimensional (3-D)modeling showed that lvGIH protein consists of five alpha-helices, which is simpleand highly similar to lvVIH.2Immunohistochemical of lvGIHGIH secretory cells were mainly located in the XO-SG, rarely in the axonal tract,GIH secretory cells in XO located in the medulla terminalis and medulla interna，wichin SG located in the medulla externa, Additionally, the fluorescence intensity of GIHsecretory cells in XO and SG were similar.3Effects of17β-estradiol on GIH expressionslvGIH expressions gradually decreased with the development of ovary in femalesL. vannamei.17β-estradiol inhibited the expressions of GIH in a dose-dependent andtime-dependent manner (P<0.05).4Effects of the eyestalk extract and eyestalk ablation on DMC1, VASA and VTGexpreesionsDMC1and VASA only expressed in the testis and ovary in L. vannamei. DMC1mRNA expressions decreased significantly in testis and ovry after eyestalk ablation(P<0.05), while increased significantly after eyestalk hormone injection (P<0.05). Onthe contrary, VTG expressions in ovaries, as well as VASA expressions in testis andovaries increased significantly after eyestalk ablation, but which decreased aftereyestalk hormone injection.Results suggested that lvGIH belongs to the class II CHH family peptidehormones, and shared the highest homology with Penaeus monodon. GIH proteinmainly located in the XO-SG, and the fluorescence intensity in XO and SG wassimilar; The GIH secretory cells scattered in the MT and ME.17β-estradiol inhibitedthe GIH mRNA expressions in a dose-dependent and time-dependent manner.Hormones in the eyestalk promoted the DMC1mRNA expressions but inhibited theexpressions of VASA and VTG. |
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