Characterisation 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.

Abstract

Changes in membrane Ca2+, calcium receptor protein calmodulin, endoplasmic reticulum (ER), mitochondria and cellulose in unfixed, living, isolated 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-carbocyanine iodide (DiOC6(3)) and calcofluor white in conjunction with computer-controlled video image analysis. In addition, confocal laser scanning microscopy has been used in conjunction with ethidium bromide to detect the nature and location of the sperm cell nuclear chromatin before and after karyogamy. Digitised video images of chlorotetracycline (CTC) fluorescence reveal that egg cells contain high levels of membrane Ca2+ in organelles present around the nucleus while the cytosolic signal is relatively low. Intense CTC fluorescence is invariably present just below the plasma membrane of egg cells and a certain degree of regionalised distribution of Ca2+ in cytoplasm is also discernible. Similarly, the fluphenazine (FPZ)-detectable calmodulin (CaM) and that localised 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 Ca2+ levels occur which include an increase in the membrane Ca2+ of the fusion product, particularly in the cytosol and around the nucleus. Unlike in the eggs the fine CTC fluorescence signal below the plasma membrane is not detectable in the fusion products.(ABSTRACT TRUNCATED AT 250 WORDS)