Gnrh-like Peptide From The Brain Of Chinese Mitten Crab Eriocheir Sinensis: Isolation, Immunological Localization And Functional Characterization

Oocyte has accomplished yolk accumulation later, meiosis restrat, enter thefinal physiological mature stage （meiosis）. Only when achieve this stage oocyte canovulation and fertilization. Germinal vesicle break down （GVBD） was considered tothe clear symbol of meiotic resumption and the final stages of oocyte maturation.Gonadotropin releasing hormone （GnRH） is a core peptide that could stimulate therelease of gonadotropins,i.e.,follicle stimulating hormone （FSH） and luteinizinghormone （LH） form the pituitary, as a key factor in thehypothalamo-hypophysial-gonadal （HPG） axis to regulation of GVBD invertebrates but decapod existence of similar regulatory pathway is not clear.The chinese mitten crab, Eriocheir sinensis, is one of the most commerciallyimportant native species for aquaclture industry in China. In this study a GnRH likepeptide was partial isolated by reverse phase high performance liquidchromatography （RP-HPLC） from the brain extract of Eriocheir sinensis.Immunological dot blot analyses from RP-HPLC fractions using the anti-octGnRHrevealed that one of fractions is immune reaction positive, suggesting that thefractions of brain extract contains GnRH-like substance. This fraction was furtheridentified by using matrix-assisted laser desorption/ionization tandem time-of-flightmass spectrometry （MALDI-TOF/TOF MS）. Two GnRH-related peptides（EsGnRH-â… and EsGnRH-Ⅱ） were characterized by mass spectrometry fromextracts of Eriocheir sinensis. Amino acid sequences were QSYHFSHGWKP andQAYHFSHGWKP, molecular weight respectively1356.6Da and1373.6Da. GnRHanalogues were compared with other species of the30types, the results showed theexperimentally obtained analogs with GnRH family the same amino acid of conserved sites, including Q¹, H⁴, S⁶, G⁸, P¹¹five amino acid sites and vertebrateGnRH-type I, type II, type III identical, W9loci the same as the vertebrate GnRH-type II, III, IV. At the same time the analogs with invertebrate GnRH typicalstructure, and vertebrates analogues compared in the first amino acid is also morethan two amino acids, with invertebrates and consistent F5loci, one subtype andsoftware animal GnRH only in tenth amino acid differences. There results suggestthat the isolated peptides probably crab-GnRH. We demonstrated the existence ofcrab GnRH-liked immunoreactivity （ir-EsGnRH） cell bodies of various neurons oflocated in neuronal cluster number8,11the anterior part （protocerebrum） andlocated in neuronal cluster number10in the middle part of the supraesophagealganglion （brain）. Especially is the existence of ir-EsGnRH cell bodies on theperiphery of olfactory lobe （ON）. In the oesophageal connectives and antenna IInerve of tritocerebrum were found in the immunoreactive material. There resultssuggest that the GnRH secretion after through the two nerve channel transport intothe target cell and the GnRH analogues can not only regulation of gonadaldevelopment in Eriocheir sinensis. The cycle of ovarian maturation in Eriocheirsinensis administered with the two isoforms of GnRH was shorter than that of thecontrol animals. we identify EsGnRH-like peptie in significantly inducing oocytematuration, ovulation and GVBD. In experiment each injected groups could beenobservated GVBD, especially EsGnRH-â… injected groups with500ng/g statisticallyhighly significant（P<0.01）, the proportion of GVBD is40pre. In both EsGnRH-â… /-â…¡ did not vary significantly among EsGnRH-â…  and EsGnRH-â…¡ （P>0.05）.EsGnRH1-12did not vary significantly among treatments（P>0.05）. These findingssuggest that GnRHs may be highly conserved peptides that play an important rolein inducing the the final stages of oocyte maturation. These results suggest incrustacean exist the brain-gonad regulation pathway and as a key factor in thispathway to regulation of GVBD. In order to further reveal the crustacean GnRHneuroendocrine mechanism.