Novel Approaches To Localize And Quantify Glucose-6-Phosphate Dehydrogenase During Fertilization Of Sea Urchin (Psammechinus Miliaris L.)

Immunocytochemical methods are widely used modern techniques, which are essential for the identification of proteins and their quantitative localization in cells and tissues. Nowadays, there are plenty of immunocytochemical methods. Each of them has their own characteristic and limitation. So it is important to compare the advantages and disadvantages among these methods to find out their unique application at different research levels.In the present study, the model organism– sea urchin is used as the experimental material. Novel approaches, Immunofluorescence technique and Immunogold Silver Staining (IGSS) combined with semithin cryosectioning, are newly developed to localize enzyme protein under light microscope. The key enzyme protein during fertilization process of sea urchin, glucose-6-phosphate dehydrogenase (G6PDH), is localized and quantified. In addition, based on the in vivo image analysis technique and"Grey Value - Absorbance"conversion, a method for measuring in vivo enzyme activity during fertilization is developed and it is used to analyze the activity of glucose-6-phosphate dehydrogenase.(1) Data of these two novel techniques show their own unique applicable condition. Due to the stable labeling signal, Immunogold Silver Staining combined with semithin cryosection is good for visualizing enzyme protein especially for enzyme amount and localization in combination with activity studies with other histochemical approaches. On account of the easier detected labeling signal, Immunofluorescence technique is an appropriate tool for localizing enzyme protein at site of their action. (2) Furthermore, localization pattern of G6PDH during sea urchin fertilization is detected by immunofluorescence labeling technique. Data shows that: the G6PDH is accumulated in cortical granules and bounded in endoplasmic membranes in unfertilized eggs. After fertilization, G6PDH is released from endoplasmic reticulum and cortical granules. Moreover, quantitative determination of G6PDH amount during fertilization is conducted by IGSS. A decreasing trend of G6PDH amount is found. The results of one-way ANOVA and homogeneous subset analysis demonstrate that the variance of G6PDH amount before and after fertilization is significant (p<0.05). (3) Based on the in vivo image analysis technique, the activity of G6PDH during sea urchin fertilization is studied. The experimental methods are designed from the combination of biochemistry, physical optics and photographic technique. It carries out the in vivo quantitative measurement of complete egg during fertilization. The results indicate that, G6PDH is inactive in unfertilized eggs. After fertilization G6PDH is active, and the activity is increasing during fertilization. Furthermore, the activity of G6PDH is higher in cortical region than that in cytoplasm.In conclusion, both of immunofluorescence labeling technique and IGSS technique are sensitive, specific and reliable, and will become widely used tools in immunohistochemical study. The novel in vivo image analysis and"Grey Value - Absorbance"conversion will be more developed and used widely in the future. Moreover, during the fertilization process, G6PDH is transformed from insoluble and inactive enzyme to soluble and active enzyme, and the activity increases in the following fertilization time.