The Ultrastructure Of Hatching Gland And Expression Of Hatching Enzyme MRNA During The Embryonic And Larval Development Of Odontobutis Potamophila

The Odontobutis potamophila belongs to Perciformes, Odontobutidae. It is an important freshwater commercial fish in China. As far as we known, the biology, embryonic development and organ development of the Odontobutis potamophila had been well-studied. In this study, the hatching gland (HG) of the Odontobutis potamophila were examined with transmission electron microscopy (TEM) during its embryonic development; Subsequently, in order to investigation the space-time connection between hatching gland (HG) and hatching enzyme (HE) of the Odontobutis potamophila, the part sequence of hatching enzyme (HCE) were isolated and the quantities of mRNA expression of HE gene during the embryonic and larval development were analysed with the method of the Real time PCR.The HG of the Odontobutis potamophila is unicellular gland, and was detected first at the stage of lens formation. The hatching gland cells (HGCs) were located in the ventral of the head, the joint region between the head and yolk sac, and the ventral of the yolk sac. At the blood circulation stage , the number and the volume of HGCs increased significantly, and HGCs spread more widely with the development of the embryo; When the embryo reached the eye pigmentation stage,zymogen granules were detected, and every one of the zymogen granule was wrapped by membrane, so that the limit between others can be distinguished significantly; When the embryo reached the pre-hatching stage, the hatching enzyme granules were secreted into the perivitelline space from the top of HGCs. The secreted granules were spherical and 0.25– 1.00μm in diameter. Some of the secreted granules were monomer and the others got together. The hatching enzyme was dissolved in the perivitelline fluid ,where it gained access to the egg envelope and digested the inner layer of egg envelope;At the larval fish just after hatching stage, the secretion of HGCs have finished and HGCs gradually disappeared from epidermis. The results showed that the hatching enzyme granules of the Odontobutis potamophila gradually maturated during its embryonic and larval development, and the mature hatching enzyme granules were excreted from the open of the HGCs to the perivitelline space for the embryo hatching.The total RNA of the Odontobutis potamophila was extracted with TRIZOL reagent, and the ratio of A280 / A260 was detected. The results showed that all of the rations were between 1.8 and 2.0. The cDNA was obtained by AMV reverse transcription systems. The cDNA sequence of the HE of the Odontobutis potamophila were amplified with primers designed based on the homologous sequence of relative fish. And the cDNA fragment of 84bp base pairs was obtained.Then the homologies of the cDNA have been compared with other fishes. The results showed that the cDNAs which we obtained were the HE expressing genes.For further study the quantities of mRNA expression of HE gene during the embryonic and larval development of the Odontobutis potamophila, the Real-time PCR method was used with SYBR Green as reporter dye and the product of PCR as standard curve template. The result showed that the expression of HE mRNA was low from fertilized egg stage to lens formation stage; Subsequent the expression of HE mRNA increased rapidly, and the expression of HE mRNA riched the highest level at the eye pigmentation stage. After the eye pigmentation stage, the expression of HE mRNA decreased. However, the expression of HE mRNA increased a few at one day after hatching. After that, the expression of HE mRNA also decreased. The results suggested that the expression of HE mRNA was a dynamic course during its embryonic and larval development, and there was close relationship of the HE gene expression and the development of HG.