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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.

References

Bacon, J.P., Gonzalez, C.. & Hutchison, C.J.. (1991). Applications of confocal laser scanning microscopy. Trends in cell Biol. 1, 172–5.CrossRefGoogle ScholarPubMed
Berger, F.. & Brownlee, C.. (1993). Ratio confocal imaging of free cytoplasmic calcium gradients in ppolarising and polrised Fucus zygotes. Zygote 1, 9–15.CrossRefGoogle ScholarPubMed
Boss, W.F., Grimes, H.D.. & Brightman, A.. (1984). Calcium induced fusion of fusogenic wild carrot proplasts. Protoplasma 120, 209–15.CrossRefGoogle Scholar
Brawley, S.. (1991). The fast block against polyspermy in fucoid algae is an electrical block. Dev. Biol. 144, 94–106.CrossRefGoogle ScholarPubMed
Brawley, S.H.. & Roberts, D.M.. (1989). Calmodulin-binding proteins are developmentally-regulated in gamets and embroys of fucoid algae. Dev. Biol. 131, 313–20.CrossRefGoogle Scholar
Cass, D.D.. & Fabi, G.C.. (1988). Structure and properties of sperm cells isolated from pollen zea mays. Can. J. Bot. 66, 819–25.CrossRefGoogle Scholar
Conover, J.C.. & Gwatkin, RBL. (1988). Pre-loading of mouse oocytes with DNA specific fluorochrome (Hoechst 33342) permits detection of sperm-oocyte fusion. J. Reprod. Fertil. 82, 681–90.CrossRefGoogle ScholarPubMed
Cotton, G.. & Driessche, V.T.. (1987). Identification of calmodulin in Acetabularia: its distribution and physiological significance. J. Cell Sci. 87, 337–47.CrossRefGoogle Scholar
Diboll, A.G..(1968). Fine structural development of the megagametophyte of zea mays following fertilization. Am. J. Bot. 55, 787–806.CrossRefGoogle Scholar
Dumas, C.. & Mogensen, L.H.. (1993). Gametes and fertilization: maize as a model system for expermental embryogenesis in flowering plants. Plant Cell 5, 1337–48.CrossRefGoogle Scholar
Duzgunes, N., Wilschut, J., Fraley, R.. & Papahadjopulos, D.. (1981). Studies on the mechanism of membrance fusion: role of head-group composition in calcium- and magnesium-induced fusion of mixed phospholipid vesicles. Biochim. Biophys. Acts 642, 182–95.CrossRefGoogle Scholar
Eisen, A., Kiehardt, D.P., Wieland, S.J.. & Reynolds, G.T.. (1984). Temporal sequences and spatial distribution of early events of fertilization in single sea urchin eggs. J. Cell Biol. 99a, 1647–54.CrossRefGoogle ScholarPubMed
Faure, J.-E., Mogensen, H.L., Kranz, E., Diagonnet, C.. & Dumas, C.. (1992). Ultrastructural characterization and three-dimensional reconstruction of isolated maize (Zea mays L.) egg cell protoplasts. Protoplasma 171, 97–103.CrossRefGoogle Scholar
Faure, J.E., Mogensen, H.L., Dumas, C., Lorz, H.. & Kranz, E.. (1993). Karyogramy after electrofusion of single egg and sperm cell protoplasts from maize: cytological evidence and time cours. Plant Cell 5, 747–55.CrossRefGoogle Scholar
Faure, J.-E., Digonnet, C.. & Dumas, C.. (1994). An in vitro system for adhesion and fusion of maize gametes. Science. 263, 1598–600.CrossRefGoogle Scholar
Franklin-Tong, V.E., Ride, J.P., Read, N.D., Trewavas, A.J.. & Franklin, F.C.H.. (1993). The self-incompatibility response in papaver rhoeas is mediated by cytosolic free calcium. Plant. 4, 163–77.Google Scholar
Galbraith, D.W.. (1981). Microfluorimetric quantitation of cellulose biosynthesis by plant protoplasts using calcofluor white. Physiol. Plant. 53, 111–16.CrossRefGoogle Scholar
Goodner, B.. & Quatrano, R.S.. (1993). Fucus embryogenesis: a model to study the establishment of polarity. Plant cell 5, 1471–81.CrossRefGoogle Scholar
Grimes, H.D.. & Boss, W.F.. (1985). Intracellular calcium and calmodulin involvement in protoplast fusion. Plant Physiol. 79, 253–8.CrossRefGoogle ScholarPubMed
Harrington, B.J.. & Raper, K.B.. (1968). Use of a fluorescent brightener to demonstrate cellulose in cellular slime molds. Appl. Microbiol. 16, 106–13.CrossRefGoogle ScholarPubMed
Hausser, I., Herth, W.. & Reiss, H.-D.. (1984). Calmodulin in tip-growing plant cells, visualized by fluorescing calmodulin-binding phenothiazines. Planta 162, 33–9.CrossRefGoogle ScholarPubMed
Heizmann, C.W.. & Hunziker, W.. (1991). Intracellular calciun-binding proteins: more sites than insights. Trends Biochem. Sci. 16, 98–103.CrossRefGoogle ScholarPubMed
Helper, P.K.. (1989). Calcium transients during mitosis observations in flux. J. Cell biol. 109, 2576–83.Google Scholar
Hinkey, R.E., Wright, D.B.. & Lynn, J.W.. (1986). Rapid visual detection of sperm–egg fusion using the DNA specific flurochrome Hoechst 33342. Dev. Biol. 118, 148–54.CrossRefGoogle Scholar
Huang, B.Q.. & Russell, S.D.. (1992). Female germ unit: organization, isolation, and function. Int. Rev. Cytol. 140, 223–93.Google Scholar
Jablonsky, P.P., Grolig, F., Perkin, J.L.. & Williamson, R.E.. (1991). Properties of monoclonal antibodies to plant calmodulin. Plant Sci. 76, 175–84.CrossRefGoogle Scholar
Jaffe, L.F.. (1983). Sources of calcium in egg activation: a review and hypothesis. Dev. Biol. 99, 256–76.CrossRefGoogle ScholarPubMed
Jaffe, L.F.. (1990). The roles of intermembrane calcium in polarizing and activating eggs. In Mechanism of Fertilization, ed. B., Dale, NATO ASI Series vol. H 45, pp. 389–417. Berlin: Springer–Verlag.Google Scholar
Kovacs, M., Barnabas, B.. & Kranz, E.. (1994). The isolation of viable egg cells of wheat (Triticum aestivum L.). Sex. Plant Reprod. 7, 311–12.CrossRefGoogle Scholar
Kranz, E.. (1992). In vitro fertilization of maize mediated by electrrfusion of single gametes. In Plant Tissue Culture Manual, Suppl. 2.ed. K., Lindsey, pp. 1–12. Dordrecht: Kluwer.Google Scholar
Kranz, E.. & Lorz, H. (1993). In vitro fertilization with isolated, single gametes results in zygotic embryogenesis and fertile maize plants. Plant cells 5, 739–46.CrossRefGoogle ScholarPubMed
Kranz, E.. & Lorz, H. (1994). In vitro fertilization of single maize gametes mediated by high calcium and high pH. Zygote. 2, 125–8.CrossRefGoogle ScholarPubMed
Kranz, E., Bautor, J.. & Lorz, H. (1991a). In vitro fertilization of single, isolated gametes of maize mediated by electrofusion. Plant Reprod. 4, 12–16.Google Scholar
Kranz, E., Bautor, J.. & Lorz, H. (1991b). Electrofusionmediated transmission of cytoplasmic organelles through the in vitro fertilization process, fusion of sperm cells with synergids and central cells, and reconstitution in maize. Sex. Plant Reprod. 4, 17–21.CrossRefGoogle Scholar
Kranz, E., Lorz, H., Digonnet, C.. & Faure, J.-E.. (1992)In vitro fusion of gametes and production of zygotes. Int. Rev. Cytol. 140, 407–23.CrossRefGoogle Scholar
Kropf, D.L.. & Quatrano, R.S.. (1987). Localization of membrane-associated calcium during development of fucoid algae using chlorotetracycline. Planta 171, 158–70.CrossRefGoogle ScholarPubMed
Kropf, D.L., Jordan, J.R., Allen, V.W.. & Gibbon, B.C.. (1992). Cellular polarity in pelvetia zygotes: studies of intracellular pH and division alignment. Curr. Top. Plant Biochem. Mol. Biol. Physiol. 11, 143–52.Google Scholar
Kubota, H.Y., Yoshimoto, Y., Yoneda, Y.. & Hiramoto, Y.. (1987). Free calcium wave upon activation in Xenopus eggs. Dev. Biol. 119, 126–36.CrossRefGoogle ScholarPubMed
Levin, R.M.. & Weiss, B.. (1975). Mechanism by which psychotropic drugs inhibit cyclic AMP-phosphodiesterase in brain. Mol. Pharmacol. 12, 581–9.Google Scholar
McCulloh, D.H.. & Chambers, E.L.. (1991). A localized zone of increased conductance progresses over the surface of the sea urchin egg during fertilization. J. Gen. Physiol. 97, 579–604.CrossRefGoogle ScholarPubMed
Murashige, T.. & Skoog, F.. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–97.CrossRefGoogle Scholar
Nelson, G.A., Andrews, M.L.. & Karnovsky, M.J.. (1982). Participation of calmodulin in immunogloubin capping. J. Cell Biol. 95, 771–80.CrossRefGoogle Scholar
Ogura, A., Yanagimachi, R.. & Usui, N.. (1993). Behaviour of hamster and mouse round spermatid nuclei incorporated into mature oocytes by electrofusion. Zygote. 1, 1–8.CrossRefGoogle ScholarPubMed
O'Kane, D.J., Lenz, R.W., Schmidt, G.W., Palevitz, B.A.. & Cormier, M.J.. (1980). Binding of photo-oxidized stellazine derivatives to calmodulin. J. Cell Biol. 87, 199.Google Scholar
Papahadjopoulos, D.. (1978). Calcium-induced phase changes and fusion in natural and model membranes. In Membrane Fusion, ed. G., Poste. & G.L., Nicolson. pp. 765–90. New York: Elsevier.Google Scholar
Roux, S.J.. (1992). Calcium regulated nuclear enzymes: potential mediators of phytochrome-induced changes in nuclear metabolism? Photochem. Photobiol. 56, 811–14.CrossRefGoogle ScholarPubMed
Russell, S.D.. (1986). Isolation of sperm cells from the pollen of plumbango zelanica. Plant Physiol. 81, 317–19.CrossRefGoogle Scholar
Russell, S.D.. (1991). Isolation and characterization of sperm cells in flowering plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 189–204.CrossRefGoogle Scholar
Russell, S.D.. (1992). Double fertilization. Int. Rev. Cytol. 140, 357–88.CrossRefGoogle Scholar
Russell, S.D.. (1993). The egg cell: development and role in fertilization and early embryogenesis. Plant Cell. 5, 1349–59.CrossRefGoogle ScholarPubMed
Santhananatham, A.H., Ng, S.C., Trounson, A.O., Rathnam, S.S.. & bongso, T.A.. (1990). Human sperm-oocyte fusion. In Mechanism of Fertilization, ed, B., Dale, NATO ASI Series vol. H45, pp. 329–50. Berlin: Springer–Verlag.Google Scholar
Scali, M., Cai, G., DelCasino, C., Santucci, A., Tirlapur, U.K., Moscatelli, A., Cresti, M.. & Tiezzi, A.. (1994). Purification and biochemical characterization of calmodulin from corylus avellana L. pollen. Plant Physiol. 32, 831–8.Google Scholar
Schackmann, R.W., Eddy, E.M.. & Shapiro, B.M.. (1978). The acrosome reaction of strongylocentrotus purpuratus sperm: ion requirements and movements. Dev. Biol. 65, 483–95.CrossRefGoogle ScholarPubMed
Timmers, A.C.J.. (1993). Imaging of polarity during zygotic and somatic embroyogenesis of carrot (Daucus carota L.). PhD thesis, submitted to Wageningen Agricultural University, Wageningen, The Netherlands.Google Scholar
Timmers, A.C.J.., DeVeries, S.C.. & Schel, J.H.N.. (1989). Distribution of membrane-bound calcium and activated calmodulin during somatic embryogenesis of carrot (Daucus carota L.). Protoplasma. 153, 24–9.CrossRefGoogle Scholar
Timmers, A.C.J.., Reiss, H.-D.. & Schel, J.H.N.. (1991). Digitonin-aided loading of Fluo-3 into embryogenic plant cells. Cell Calcium. 12, 515–21.CrossRefGoogle ScholarPubMed
Terasaki, M.. (1989). Fluorscent labeling of endoplasmic reticulum. Methods Cells Biol. 29, 129–35.Google Scholar
Tirlapur, U.K.. & Cresti, M.. (1992). Computer-assisted video image analysis of spatial variations in membrane-associated Ca2+ and calmodulin during pollen hydration, germination and tip growth in Nictiana tabacum L. Ann. Bot. 69, 503–8.CrossRefGoogle Scholar
Tirlapur, U.K., Hader, D.P.. & Scheuerlein, R.. (1992). UV-B mediated damage in the photosynthetic flagellate Euglena gracilis studied by image analysis. Beitr. Biol. Pflanzen. 67, 305–17.Google Scholar
Tirlapur, U.K., VanWent, J.L.. & Cresti, M.. (1993). Visualization of membrane calcium and calmodulin in embryo sacs in situ and isolated from petunis hybrida L and Nicotiana tabacum L. Ann. Bot. 71, 161–7.CrossRefGoogle Scholar
Tirlapur, U.K., Scali, M., Del Casino, C., Cai, G., Moscatelli, A., Tiezzi, A.. & Cresti, M.. (1994). Confocal image analysis of spatial variations in immunocytochemically identified calmodulin during pollen hydration, germination and pollen tube tip growth in Nicotiana tabacum L. Zygote. 2, 63–8.CrossRefGoogle ScholarPubMed
Van Der Maas, H.M., Zaal, M.A.C.M.., De Tong, E.R., Krens, F.A.. & Van Went, J.L.. (1993). Isolation of viable egg cell of perennial ryegrass (Lolium prenun L.). Protoplasma. 173, 86–9.CrossRefGoogle Scholar
Wolniak, S.M., Hepler, P.K.. & Jackson, W.T.. (1980). Detection of membrane-calcium distribution during mitosis in Haemanthus endosperm with chlorotetracycline. J. Cell Biol. 87, 23–32.CrossRefGoogle ScholarPubMed
Whitaker, M.. & Swann, K.. (1993). Lighting the fuse at fertilization. Development. 117, 1–12.CrossRefGoogle Scholar