Marking spherulous cell in marine sponge Axinella sp. using a 3,3′- diaminobenzidine (DAB) staining method

Abstract

Debromohymenialdisine(DBH) and hymenialdisine(HD) are two bioactive alkaloids which has been isolated from marine sponge Axinella sp.living in the South China Sea,and these two alkaloids are located particularly in spherulous cells in Axinella sp..Establishment of a specific marker for this type of cells by cytochemical staining can provide an indication method for cell differentiation,and can be applied in the metabolism regulation in in vitro cell culture.The 3,3′-diaminobenzidine(DAB) staining is commonly used to detect peroxidase activity.In this study,sponge cells were fractionated and stained by DAB.A distinguishing feature of dark brown granules in cytoplasm was observed in spherulous cells.Native-PAGE was carried out with protein extracts from fresh tissue and different cell fractions of marine sponge to examine the peroxidase responsible for DAB staining.By transmission electron microscope(TEM),two kinds of inclusions were found inside the spherulous cells,the larger ones of which showed consistency in dimen-sion and amount with the positive stained granules under light microscopy.In addition,this staining method for spheru-lous cell identification was also applied in in vitro cell culture(primmorph culture).The proportion of both granularly positive-stained cells and the spherulous cells synchronously varied during a 20-day culture period,and the ratio of positive-stained spherulous cells was kept above 93%.The above results suggested that the cytochemical method of DAB staining was reliable and could be applied in both fresh-tissue dissociation and in vitro cell culture,with the dis-tinctive characteristic of positive stained granules in cytoplasm marking spherulous cells in sponge Axinella sp..