In vitro fertilization and expression of transgenes in gametes and zygotes
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Abstract The in vitro fertilization system of maize isthe well characterized model system for the fertilizationprocess and early zygotic embryogenesis of higherplants. Application of molecular methods to the in vitrofertilization system led to the isolation of new genes anduncovered specific expression patterns of cell cycleregulators. Recent studies showed that expression oftransgenes is possible in gametes and zygotes, thus trans-genic approaches might offer an opportunity to unravelthe roles of genes during fertilization and early develop-ment. The competence of gametes and zygotes to expresstransgenes will also enable the expression of GFP basedreporter genes for the visualization of subcellular compo-nents in these cells in vivo. This review focuses on thedata concerning the expression of transgenes in gametesand zygotes and describes some examples of recentdevelopments in transgenic technology illustratingthe emerging possibilities in experimental design bycombining this technology with in vitro fertilization.
Keywords In vitro fertilization, egg cell, central cell,zygote, transgene expression
Fertilization is the central event in the life cycle of higherorganisms. In higher plants two fertilization processesoccur (Nawaschin 1898): one sperm cell of a pollengrain fuses with the egg cell forming the zygote whichdevelops into the embryo, whereas the second sperm cellfuses with the central cell forming the primary endospermcell which develops into the endosperm.
These events take place deeply embedded in maternaltissue. Therefore the isolation of gametes is necessary toperform fertilization in vitro. The fusion of isolatedgametes in angiosperms in vitro can be mediated electri-
cally and chemically by calcium or polyethylene glycol(recently reviewed by Kranz and Kumlehn 1999). Inmaize, both fertilization processes are possible in vitroby electrofusion of isolated gametes and central cells.These in vitro fertilized egg and central cells are able todevelop in culture and are capable of self-organization ina typical manner independently from maternal tissue(Kranz et al. 1991; Kranz et al. 1998).
Besides leading to new insights, cytological studiesshow that development of the fusion products in cultureis characteristic and comparable with the situation inplanta. In vitro produced zygotes are highly metabolicallyactive. Synthesis of a new cell wall starts as early as 30 safter fusion of the gametes (Kranz et al. 1995). Fusion ofthe two parental nuclei can take place 35 min afterfusion (Tirlapur et al. 1995), but generally karyogamyoccurs between 45 and 120 min after in vitro fertilizationof egg cells (Faure et al. 1993; Kranz et al. 1998). Twonucleoli were observed in zygotes 18 h after in vitrofertilization (Kranz et al. 1995). In planta maize zygotesare dividing about 16 h after karyogamy (Mol et al.1994). In vitro produced zygotes displayed an unequalfirst cell division as in planta, depending on the cultureconditions between 29 and 46 h after fertilization, andthey could be regenerated to phenotypically normal andfertile plants (Kranz and Lrz 1993). Karyogamy of invitro fertilized central cells takes place within 120 minafter fusion. The in vitro produced primary endospermcells develop into oblong structures. Early nucleardivisions are not followed by cell wall formation, conse-quently generating a syncytium. Cellularization occurs 3to 5 days after in vitro fertilization and further develop-ment leads to formation of a white compact tissue. Thedevelopment of in vitro produced endosperm is concludedas in planta (Kranz et al. 1998).
In vitro fertilization systems provide the basis formolecular studies of the fertilization process, gametes,zygotes and early developmental stages without theinfluence of maternal tissue. To explore gene expressionduring fertilization and early development, cDNA librariesof egg cells and zygotes were generated (Dresselhaus et
S. Scholten E. Kranz ()Institut fr Allgemeine Botanik, AMPII, Universitt Hamburg,Ohnhorststrae 18, 22609 Hamburg, Germanye-mail: firstname.lastname@example.org
Sex Plant Reprod (2001) 14:3540 Springer-Verlag 2001
R E V I E W
Stefan Scholten Erhard Kranz
In vitro fertilization and expression of transgenesin gametes and zygotes
Received: 20 December 2001 / Revision accepted: 6 June 2001
al. 1994, 1996). Differential analysis of these librariesshowed that expression of several genes is up- or down-regulated after fertilization (Dresselhaus et al. 1999).Screening these libraries and a cDNA library of maturepollen for clones encoding transcription factors led to theisolation of novel cDNAs encoding MADS box proteins(Heuer 1999; Heuer et al. 2000). Transgenic approachesdemonstrate the homeotic function of MADS box genesduring flower development (reviewed by Riechmann andMeyerowitz 1997). The presence of transcripts of two ofthe novel MADS box genes in egg cells (Heuer 1999)implicate a role of these genes during fertilization and/orzygote development.
RT-PCR allows expression analysis of known genes.Applying this method, adapted to the analysis of singlecells by Richert et al. (1996), to the in vitro fertilizationsystem, Sauter et al. (1998) showed the differentialexpression pattern of cell cycle regulatory genes duringfertilization and the first embryonic cell cycle. Incontrast to the cdc2 protein kinase, which is constitutivelyexpressed before and after fertilization, cyclin genes aretranscribed de novo after fertilization and display adiverse expression pattern during fertilization and zygotedevelopment.
As discussed, in vitro fertilization systems led to theisolation of new genes and revealed particular expressionpatterns of cell cycle regulators, both of which mighthave important roles during fertilization and zygotedevelopment. Studies on functions of these genes inearly developmental stages of the plant life cycle are ofhigh interest.
Transgene technology provides a way to gain insightsinto gene function by altering the expression level of agiven gene with overexpression or expression of antisenseRNA. Many studies show that these techniques are aswell suited to uncover the role of genes important fordevelopment, like transcription factors, as to raise under-standing of the regulation of developmental processes(Ramachandran et al. 1994; Meisel and Lam 1997).
Moreover, the novel marker green fluorescent protein(GFP), isolated from Aequorea victoria, extends thepossibilities of transgenic technology. Due to its non-toxicnature and the non-invasive visualization by fluores-cence microscopy GFP permits real-time observations ofdynamic changes in living cells. GFP fusion proteins canbe used to study subcellular localization and movementsof proteins and organelles in vivo (Grebenok et al. 1997;Khler 1998). Other GFP-based approaches, of specialinterest for the application to the in vitro fertilizationsystem, enable visualization of cytoskeleton proteins ormeasurement of intracellular calcium concentrations. Amicrotubule reporter gene (gfp-mbd) was constructed byfusing the gfp gene to the microtubule binding domainof the mammalian microtubule-associated protein 4(MAP4) gene. GFP-MBD labels cortical microtubulesafter transient expression of the reporter gene in livingepidermal cells of fava bean (Marc et al. 1998). Grangerand Cyr (2000) showed that constitutive expressionof the microtubule reporter gene in stable transformed
tobacco BY-2 cells allows spatial and temporal resolutionof microtubule arrays as they reorganize throughout thecell cycle. By using GFP fusion proteins, which bind toactin, the visualization of dynamic changes of thiscomponent of the cytoskeleton could be achieved (Kostet al. 1998).
Calcium ions play a central role in the regulation ofmetabolic processes and signal transduction (reviewedby Zhang and Cass 1997) and this ion might have a rolein egg activation (Antoine et al. 2000 this volume).Digonnet et al. (1997) reported an increase in Ca2+concentration in egg cells after fertilization. An influx ofextracellular Ca2+ induced by gamete fusion was measuredwith the use of an extracellular Ca2+ selective vibratingprobe by Antoine et al. (2000). Starting in the vicinity ofthe sperm cell fusion side, the Ca2+ influx spread sub-sequently through the whole egg cell plasma membraneas a wavefront. The GFP based cameleon calciumindicator, developed by Miyawaki et al. (1997) couldpossibly be used to further characterize the spatial andtemporal distribution of calcium ions during fertilizationand early embryonic development in vivo. Recently, thefunction of this indicator was shown in guard cells of Arabidopsis (Allen et al. 1999).
These examples show that expression of transgenes inisolated gametes and zygotes produced in vitro willbecome a valuable tool for analysis of these develop-mental stages, for both functional analysis and cytologicalstudies.
So far, there are two strategies to study expression offoreign genes in gametes and zygotes. One is directdelivery of DNA into these cells via microinjection.With the second method transgene expression in gametesand zygotes can be analyzed after regeneration of plantstransformed via particle bombardment.
Transient expression of transgenes after microinjectionof plasmid DNA in zygotes was reported by Leduc et al.(1996). In this study the GUS gene under control of themaize histone H3C4 promoter followed by an actinintron and two anthocyanin regulatory genes under controlof the 35S promoter were used as reporter genes. Thesevectors were injected in zygotes of maize that wereisolated 24 h after pollination. Transient expression ofthe reporter genes, with a frequency of 3.5% on average,was reported in zygotes 4 days after injection. Pnya etal. (1999) demonstrated transient expression of reportergenes after microinjection of