Characterisation of isolated egg cells,in vitrofusion products and zygotes ofZea maysL. using the technique of image analysis and confocal laser scanning microscopy

Abstract

Changes in membrane Ca 2+ , 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 Ca 2 + 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 Ca 2+ 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 Ca 2 + levels occur which include an increase in the membrane Ca 2+ 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. Compared with isolated egg cell protoplasts an increase in the CaM level in the cytoplasm was observed in the fusion products. There is a slight increase in the fluorescence associated with ER profiles in the fusion products compared with egg cell protoplasts. A distinct calcofluor fluorescence around the fusion product is visible after 16 h in culture. The sperm cell chromatin in the fusion product is highly condensed, unlike that of the egg cell, and confocally imaged serial optical sections of the in vitro fusion product reveal the occurrence of karyogamy 35 min following gamete fusion. First visual evidence for intermingling of sperm nuclear chromatin in the zygotic nuclei is also provided.

Egg activation in flowering plants

“…However, it has been shown that an artificial cyt [Ca 2+ ] elevation (induced by Ca 2+ ionophore treatment) induces some manifestations of egg activation (Antoine et al 2000). Taken together with the raised levels of the calcium-calmodulin complex observed in in vitro zygotes (Tirlapur et al 1995), this result strongly suggests a role for Ca 2+ in egg activation in flowering plants, possibly via a calmodulin-mediated Ca 2+ signaling pathway. However, a demonstration that inhibition of the cyt [Ca 2+ ] elevation prevents egg activation is still lacking.…”

In vitro fertilization and expression of transgenes in gametes and zygotes

“…Synthesis of a new cell wall starts as early as 30 s after fusion of the gametes . Fusion of the two parental nuclei can take place 35 min after fusion (Tirlapur et al 1995), but generally karyogamy occurs between 45 and 120 min after in vitro fertilization of egg cells (Faure et al 1993;Kranz et al 1998). Two nucleoli were observed in zygotes 18 h after in vitro fertilization .…”

Isolation of a full-length cDNA encoding calreticulin from a PCR library of in vitro zygotes of maize

“…Ca 2 + plays a very important role at fertilization in animal systems [for review see 13], where Ca 2+ waves cross the egg cell after egg-sperm contact [15]. Electrofusion of the maize egg with the sperm cell also leads to a significant increase in Ca 2 + levels throughout the cytoplasm [43]. Further experiments are needed to determine whether calreticulin does have a role during fertilization as a Ca 2 + storage protein such as calsequestrin and calbindin [28,6] or other roles as suggested by [4,11,31].…”