| The tiger shrimp, penaeus monodon, is an important traditional shrimp for farming. Generally, the practical technique for the induction ofovarian maturation and spawning, is eyestalk ablation. Although this method provides a reliable way to obtain oocyte maturation and spawning, the regulatory mechanism is not clear, so, exploring the regularity of the secretion of reproduction-related hormones is significant meaningful. Gn RH is a well-known decapeptide to dodecapeptide that play an important role in the propagation of animals. For understanding the secretion regulation, function and mechanism of Gn RH in penaeus monodon nervous system, in this study, tissue sections, immunohistochemistry, RP-HPLC, tissue cultivation and other methods are used to obtain the distribution and function of Gn RH, the effect of Gn RH-induced ovarian maturity as well as initially established the method for the separation and purification of Gn RH. the structure of this study are as follows(1)the observation of nervous system and ovarian tissue in Penaeus monodonIn this study, the detailed descriptions of the histological observation of the nervous system and ovarian tissue in Penaeus monodon were reported using paraffin sections, HE staining. the nervous system includes the brain, subesophageal ganglion, thoracic nerve and abdominal ganglion,the result show that the nervous system has the structural characteristics of highly healing. The brain was located between the bases of two eyestalks, and consisted of protocerebrum,deutocerebrum,tritocerebrum. A variety of neuropiles and 12 types of cell clusters were observed in brain sections. The thoracic ganglion and abdominal ganglion was observed,different types of cell clusters could also be observed. Penaeus monodon ovaries had natural autofluorescence. The fluorescence focus on the region including the follicular cells, the cytoplasm of developing oocyte and mature oocyte which contain amount of yolk globules. The fluorescence intensity is positive correlated to ovarian developmental stages(P <0.01). By observing the characteristics of the ovary in each period, we can accurately determine the period of sample collection.(2) The distribution of Gn RH of the nervous system and ovary during the reproductive developmentIn the present study, we demonstrated the existence of Gn RH-like peptides in the central nervous system(CNS) and ovary of each stage in Penaeus monodon using immunocytochemistry and quantitative real-time PCR of vitellogenin expression quantity in ovary and hepatopancreas. The immunoreactivity(ir) of Gn RH was detected in the soma of medium-sized neurons located in deutocerebrum, both medium-sized and large-sized neurons in thoracic ganglia and abdominal ganglion. In addition, ir-Gn RH was observed in the cytoplasm of late previtellogenic oocyte and early vitellogenic oocyte. Fluorescence quantitative detection of Vg expression quantity show highest expression in stage V, different to the expression in hepatopancreas. This ?nding provides supporting data that ir-Gn RH-I like peptide may exist in Penaeus monodon, as well as may has a direct and indirect role in promoting ovarian maturation. In the process, vitellogenin in hepatopancreas turn to the ovaries.(3) In vitro the influence of Gn RH on ovarian development in Penaeus monodonThis study investigated the treatment of Eriocheir sinensis Gn RH(Es Gn RH-I), Procambarus clarkii Gn RH(Pc Gn RH), domperidone(DOM) and nerve tissues effects on ovarian development in Penaeus monodon. Then we analyzed the three type of hormones effected on the ovarian development by ovarian tissues cultured in vitro. The results showed that 50 ng?ml-1 Es Gn RH-I, 500 ng?ml-1 Pc Gn RH, 0.3 μg?ml-1 DOM can significantly increase the diameter of the oocytes(P <0.01), similar to the treatment of brain and thoracic nerve. Combined use of Pc Gn RH and DOM can significantly increase the diameter of the oocytes(P <0.01). Real time quantitative PCR showed that the treatment of Gn RH and DOM can obviously induce the accumulation of vitellogenin. All the result indicated that crustaceans Gn RH and DOM can promote oocyte maturation of Penaeus monodon, and combined using of them had a synergistic effect.(4) Preliminary separation and purification of Gn RH in brain tissueIn the present study, we preliminary separate Gn RH analogues from the brain tissue by the method of reversed phase liquid chromatography(RP-HPLC). By adjusting the parameters of the initial separation and purification, we obtain the desired separation profiles, then, combined with the results of dot immunobinding assay(DIBA) to find the location of Gn RH analogues. the results of isolation and identification showed that the early development of Penaeus monodon, stage II and III crude extracts of brain tissue can separate more the components, and under optimized conditions, The immunoreactivity(ir) of Gn RH was detected in the collected components of the 22-26 min. All the result indicated that the present of Gn RH analogues in the brain, as well as the optimized conditions provide a reference for the the further research of purification and identification of Gn RH amino acid structure and sequence. |
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