CHARACTERIZATION OF ISOLATED EGG CELLS, IN-VITRO FUSION PRODUCTS AND ZYGOTES OF ZEA-MAYS L USING THE TECHNIQUE OF IMAGE-ANALYSIS AND CONFOCAL LASER-SCANNING MICROSCOPY

Changes in membrane Ca2+, calcium receptor protein calmodulin, endopla smic reticulum (ER), mitochondria and cellulose in unfixed, living, is olated egg cells and fusion products of pairs of one egg and one sperm cell of Zea mays L, have been investigated using chlorotetracycline, fluphenazine, immunocytochemical techniques, 3,3'-dihexyloxa-carbocyan ine iodide (DiOC6(3)) and calcofluor white in conjunction with compute r-controlled video image analysis. In addition, confocal laser scannin g microscopy has been used in conjunction with ethidium bromide to det ect the nature and location of the sperm cell nuclear chromatin before and after karyogamy. Digitised video images of chlorotetracycline (CT C) fluorescence reveal that egg cells contain high levels of membrane Ca2+ in organelles present around the nucleus while the cytosolic sign al is relatively low. Intense CTC fluorescence is invariably present j ust below the plasma membrane of egg cells and a certain degree of reg ionalised distribution of Ca2+ in cytoplasm is also discernible. Simil arly, the fluphenazine (FPZ)-detectable calmodulin (CaM) and that loca lised immunocytochemically using monoclonal anti-CaM antibodies reveal high levels of CaM in the vicinity of the nucleus in egg cells. Only a few ER profiles and mitochondria could be visualised in the egg cell and no calcofluor fluorescence could be detected. Following in vitro fertilisation of single isolated eggs substantial changes in the Ca2levels occur which include an increase in the membrane Ca2+ of the fus ion product, particularly in the cytosol and around the nucleus. Unlik e in the eggs the fine CTC fluorescence signal below the plasma membra ne is not detectable in the fusion products. Compared with isolated eg g cell protoplasts an increase in the CaM level in the cytoplasm was o bserved in the fusion products. There is a slight increase in the fluo rescence associated with ER profiles in the fusion products compared w ith egg cell protoplasts. A distinct calcofluor fluorescence around th e fusion product is visible after 16 h in culture. The sperm cell chro matin in the fusion product is highly condensed, unlike that of the eg g cell, and confocally imaged serial optical sections of the in vitro fusion product reveal the occurrence of karyogamy 35 min following gam ete fusion. First visual evidence for intermingling of sperm nuclear c hromatin in the zygotic nuclei is also provided.