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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

Published online by Cambridge University Press:  26 September 2008

Uday K. Tirlapur ,
Erhard Kranz  and
Mauro Cresti
Uday K. Tirlapur
Affiliation:
Dipartimento di Biologia Ambientale, Università di Siena, Italy, and Institür Allgemeine Botanik, Hamburg, Germany.
Erhard Kranz
Affiliation:
Dipartimento di Biologia Ambientale, Università di Siena, Italy, and Institür Allgemeine Botanik, Hamburg, Germany.
Mauro Cresti*
Affiliation:
Dipartimento di Biologia Ambientale, Università di Siena, Italy, and Institür Allgemeine Botanik, Hamburg, Germany.
*
M. Cresti, Dipartimento di Biologia Ambientale, Università di Siena, via P.A. Mattioli 4, I-53100 Siena, Italy. Telephone: 0039 577 298854. Fax: 0039 577 298860.
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Summary

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 conjuction with computer-controlled video image analysis. In addition, confocal laser scannig microscopy has been used in conjuction with ethidium bromide to detect the nature and location of the sperm cell nuclear chromatin before and after karyogamy. Digitised video images of chlorotetra cycline (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 discrnible. 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. Compared with isolated egg cell protolasts an increase in the CaM level in the cytoplasm was observed in the fusion products. There is a slight increase in the CaM level in the cytoplasm was observed in the fusion products. There is a slight increase in the fluorescene around the fusion product is visible after 16 h in 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.

Keywords

CalciumCalmodulinConfocal microscopyEgg CellsElectrofusionImage analysisZygotes
Type
Article
Information
Zygote , Volume 3 , Issue 1 , February 1995 , pp. 57 - 64
Copyright
Copyright © Cambridge University Press 1995

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Footnotes

1Dipartimento di Biologia Ambientale, Università di Siena, Italy.
2Present address: Department of Biology, University of Utah, 201 Biology Building, Salt Lake City, UT 84112, USA.
3Institüt fü Allggemeine Botanik, Universitä Hamburg, Ohnhorststrasse 18, D-22609 Hamburg, Germany.

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