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Annu. Rev. Plant PhysioL Plant Mol. Biol. 1991. 42:189-204
Copyright © 1991 by Annual Reviews Inc. All rights reserved
ISOLATION AND
CHARACTERIZATION OF SPERM
CELLS IN FLOWERINGPLANTS
Scott D. Russell
Department
homa
of Botany
and Microbiology,
University
of Oklahoma,
Norman,
Okla-
73019-0245
KEYWORDS:angiosperm,
gametogenesis,
fertilization,
male cytoplasmic
inheritance
CONTENTS
INTRODUCTION
.....................................................................................
189
DEVELOPMENTAL
CONTEXTOF SPERMCELLS ........................................
Structural Characterization.....................................................................
SpermDimorphismand Preferential Fertilization ..........................................
190
190
191
192
193
197
198
198
198
199
200
200
ISOLATION
OF SPERM
CELLS.................................................................
Technical Details and Protocols ...............................................................
Assessmentof SpermCell Quality .............................................................
PHYSIOLOGICALCHARACTERIZATION
OF SPERM CELLS ..........................
ImmunologicalCharacterization ...............................................................
Polypeptide/Protein Characterization .........................................................
Elemental Characterization.....................................................................
MolecularCharacterization .....................................................................
CONCLUSIONS
ANDOVERVIEW
..............................................................
INTRODUCTION
Considering
the crucialrole of the malegamete
in doublefertilization, it is
remarkablethat research on the physiology of sperm cells in flowering plants
got underwayonly recently. Accordingto Mendelian
genetics, offspring
receiveone nucleusof the malegameteandonefromthe femalegamete.The
effect of DNA-containing
cytoplasmicorganelles in the malegamete(namely
189
1040-2519/91/0601-0189502.00
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RUSSELL
mitochondria and plastids) on the cytoplasmic organization of the offspring
depends on the presence and quantity of these organelles in the sperm.
This review treats the function, isolation, and characterization of sperm
cells, therefore summarizingresearch conducted mainly since 1984. Literature concerning pollen development(26), physiology (31, 32), incompatibility (9, 45), and the isolation and characterization of generative cells (30,
has been reviewed elsewhere.
DEVELOPMENTALCONTEXT OF SPERM CELLS
The sperm of angiosperms are cellular descendants of meiotic divisions
occurring in the anther of the flower. Meiosis in each meiocyte forms four
genetically dissimilar microspores. Each microspore undergoes a mitotic
division to forma large vegetative cell and a smaller, reproductive generative
cell. Together these cells form the pollen grain or, strictly speaking, the
microgametophyte. Subsequently, the generative cell migrates into the
vegetative cell and becomesan elongated, spindle-shaped, or even filiform
cell, depending on the species; each generative cell retains its ownplasma
membraneand is, in turn, enveloped by the vegetative cell membrane(26).
The generative cell undergoes a second mitotic division to form the two
sperm.
Dependingon the timing of generative cell mitosis the two spermcells may
be formed before germination of the pollen (tricellular) or after pollen
germination(bicellular). Of 243 families surveyed(11), bicellular pollen
present in 137 families (56%), tricellular pollen in 55 families (23%),
both types of pollen in 51 families (21%). Bicellular pollen is typically
regarded as the ancestral condition in most families, tricellular pollen as the
derived condition (11). Most of the research reviewed here concerns sperm
cells isolated frompollen grains in tricellular species. Bicellular species must
be cultured in order to growpollen tubes, trigger mitosis, and obtain sperm
cells.
Structural Characterization
Floweringplant spermare structurally simple, apparently nonmotilecells that
contain a normal complementof cellular organelles, including heritable
cytoplasmic organelles such as mitochondria and, in some plants, plastids
(10, 19). In contrast to animal species, competition in angiosperms is
property of the gametophyte(pollen grain and tube) and not the gamete; once
this competitionis completed,both spermcells contribute to the formation of
the embryoduring double fertilization: Onesperm cell fuses with the egg to
produce the embryo, the other fuses with the central cell to produce the
nutritive endosperm.
The sperm cells are typically connected to one another and physically
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SPERM CELLS OF FLOWERING PLANTS
191
Table1 Summary
of the fertilization characteristics of the spermcells in angiosperms
Pollentype and species
Tricellularpollen:
Monocotyledons:
Hordeumvulgaris
Zea mays
Dicotyledons:
Brassica campestris
Brassicaoleracea
Euphorbiadulcis
Gerberajamesonii
Plumbagozeylanica
Spinaciaoleracea
Bicellularpollen:
Monocotyledons:
Gladiolusgandavensis
Dicotyledons:
Nicotiana tabacum
Petuniahybrida
Rhododendron
spp.
Functional
Methoda Morphologyb ¢sperm
Sample
Recon I
Recon D
-B+(3, 55)
----P+ (60)
--
Reference
n=5
40
n = 1, 1, 1 37, 56, 57
n
n
n
n
n
n
=
=
=
=
=
=
5
3
1
1
11
7
36
36
43
48
59
78
Recon
Recon
Recon
Recon
Recon
Recon
MtD
MtD
MtD
MtD
MtPD
MtD
ImAn
D
--
n= 6
66
Recon
I
--
n= 9
Recon I
ImAn D
---
n=1
n= 7
H.-S. Yu,
personal
communication
77
66
almAn:
imageanalysisof nuclearfluorochromatic
patterns;Recon:serial TEM
and3-Dreconstruction
b I: isomorphic;
D:dimorphic
(conditionof organellarDNA
unknown);
MtD:
mitochondrial
dimorphism;
MtPD:
mitochondrial
andplastiddimorphism
CB+:spermcell containing
B-chromosomes;
P+:spermcell containingmostplastids
associated with the vegetative nucleus, transported within the pollen tube as a
"linked unit" (64) termed the "male germ unit" (14). The linkage of the sperm
cells increases the effectiveness of gamete delivery, reduces heterofertilization, and ensures nearly simultaneous transmission of the two sperm cells into
the receptive embryo sac (64). This association is present in most species
flowering plants studied to date (24) or is formed soon after pollen germination (41).
Sperm Dimorphism
and Preferential
Fertilization
Although the association between one sperm cell and the vegetative nucleus
imposes a degree of polarity, strong cellular differences may also occur
between the two sperm, including differences in the content of heritable
organelles termed cytoplasmic heterospermy, and differences in the nucleus
termed nuclear heterospermy (60). Although cytoplasmic heterospermy may
be relatively commonin flowering plants (Table 1), nuclear differences have
been reported only in Zea mays, in which B-chromosomesfrequently undergo
nondisjunction during generative cell mitosis (3). The most commonform
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RUSSELL
cytoplasmic dimorphismis mitochondrial inequality: Spermcells associated
with the vegetative nucleus usually contain more mitochondriathan the other
cell. The most extreme form examinedto date is found in Plumbagozeylanica, where both plastid and mitochondrial content differ (10, 59).
Preferential fertilization, in whichone spermcell has a greater likelihood of
fusing with the egg, mayalso occur in species where the sperm cells differ
(Table 1). In Plumbago,fusion between the egg and the plastid-rich sperm
cell occurs in 94%of the cases examined(60), selectively transmitting male
plastids into the zygote (58). Transmissionof male mitochondriainto both the
egg and central cell, however, occurs at a nearly constant 1:1000 ratio of
male:female mitochondria (62), because the differences in mitochondrial
content in the egg and central cell match those of the dimorphicspermcells.
Whether this ratio represents the maximumpermissible dose of sperm
mitochondria to prevent sperm transmission of mitochondrial DNAor the
minimumneeded to make recombination of mitochondrial DNApossible is
unclear (62).
Preferential fertilization (termedmeiotic drive by geneticists) also occurs
Zea, in which the sperm cell containing one set of extra B-chromosomes
fuses
with the egg about 65%of the time (2, 3, 55). Rather than simply providing
marker for identifying the spermcell destined to fuse with the egg, however,
B-chromosomes
appear to confer a selective advantage to the sperm cell that
contains them(2). It is interesting that preferentiality maybe eliminated
introducing excessive B-chromosomesinto the sperm or by introducing a
specific B-chromosome
(TB-9b) into the egg. This observation suggests that
maternal discrimination is active only under relatively strict conditions (2).
Maternalcontrol of fertilization is also suggestedin a mutantline of barley
in which seeds are produced containing normal endosperm but haploid
embryos(38). Onespermcell apparently fuses with the central cell regardless
of pollen source whereas the other remains unfused (38). Research on the
mechanism
of fertilization in normallines of barley indicates that the zygote
of plants with uniparental, maternal cytoplasmic inheritance are cytoplasmically restrictive: Male cytoplasmic organelles maybe shed by the separation
or pinching off of cellular folds during maturation of the sperm(40) and the
sperm cytoplasm excluded from the egg, remaining outside of the cell (39).
However,the central cell in the same plant may be cytoplasmically permissive, allowingspermorganelles to be transmitted into the central cell (39).
Plants with biparental inheritance appear to be cytoplasmically permissive
during the entire double fertilization process (58, 60).
ISOLATION
OF SPERM CELLS
Spermcell isolation was first reported in detail by Cass, whoplaced pollen
grains of barley in a Brewbaker-Kwack
(BK) pollen germination medium(1)
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SPERMCELLS OF FLOWERINGPLANTS
193
with 20%sucrose (4), obtaining release of the sperm cells by osmotic shock
and observing the behavior of the living cells using Nomarskidifferential
interference contrast microscopy. Over 30 min, the originally paired sperm
cells separated, lost their spindle shape, and becameellipsoidal to spheroidal.
Altemativemedia, such as filtrates of ovular material, were also examinedbut
there were no specific benefits or effects on spermcell appearanceor behavior
(4).
Technical Details and Protocols
Numerousisolation protocols have emerged(Table 2), including somemodified for collection of spermcells en masse(Table 3). Spermcell release has
been conducted using two general techniques: osmotic shock, or direct
physical separation by grinding or applied pressure. The pollen cytoplasmand
gametes are usually incubated for an additional 10-20 min to allow the
gametesto separate from vegetative cell cytoplasm,and then pollen cell walls
are removedby filtration. A nylon meshtwo thirds to one half the average
diameterof the pollen efficiently captures the pollen walls. Enmasseisolation
of spermcells involves either centrifugation on a discontinuous gradient or
filtration using a polycarbonate (e.g. Nuclepore)filter. Details of the procedures used and results are given in Table 2.
Isolation of spermcells using osmoticshockrelies on the greater sensitivity
of the vegetative cell membrane
to changes in the milieu. In somecases, the
shock of entering an aqueous mediumfrom a nearly desiccated state is
sufficient to rupture pollen grains regardless of the osmoticconcentrationused
(29), but spermcells maybe surprisingly intact (61). After prehydration
example, exposure in a humidchamber), pollen is more gradually introduced
to a hydrated state, possibly enhancingviability of the isolated spermcells
(67). Pollen grains maythen be subjected to an abrupt change in osmotic
concentration (80) or pH (54) or lysed in other ways.
The isolation of sperm cells can also be accomplishedby using physical
stress to break the pollen plasma membrane.Methodsused include glass
tissue homogenizers(6, 67, 68) or the pressure of a glass roller applied to
smooth glass surface (71-73). Clearance height in the homogenizeris not
critical, becausethe resistant exines will set the minimum.
Isotonic or slightly
hypertonic mediaare best for the isolation of the cells (68).
The most frequently uscd mediafor spermisolation are modifications of the
BK medium(l)---e.g.
100 ppm H3BO3, 300 ppm Ca(NO3)2"4H20,200 ppm
MgSOn.7H20,100 ppmKNO3,and 10%sucrose. Other successfully applied
media include the Roberts medium,which is a Tris-buffered modification of
BKmedium(52); a modified K3 Kao protoplast culture mediumcontaining
BKmacronutrients, plus micronutrients, coenzymes,vitamins, and hormones
(44); and the RY-2protoplast mediumused for plantlet regeneration (79).
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RUSSELL
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SPERM CELLS OF FLOWERING PLANTS
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SPERMCELLS OF FLOWERINGPLANTS
197
Becausefew of the cited reports use parallel methodology,it is difficult to
conclude which, if any, of the mediais ideal for most angiosperms; but the
life expectancy of isolated sperm cells seems strongly improvedby complex
media(Tables 2 and 3) and particularly by the addition of vitamins (53).
protocols are refined, a distinction will likely be madebetween isolation
media(designed to optimize recovery) and storage or culture media(designed
to extend spermcell viability).
Assessment
of Sperm Cell
Quality
Spermcells have been assessed for (a) structural intactness, (b) membrane
integrity, (c) enzyme
activity, and (d) respiratory activity. All of these aspects
are appropriate; perhapsmostcrucial wouldbe an assay of the cell’s ability to
effect fertilization, whichis not currently feasible.
Techniquesto assay structural intactness include transmission (6, 15, 42,
68, 73, 74) and scanning electron microscopy(66, 68), which can be used
assess membrane
structure and cell shape; intramembraneparticle distribution
can be inferred from freeze-fracture replicas (74). Integrity of membranes
can
be assessed using the exclusion of polar dyes such as Evans blue (17)
propidium iodide (25), both of which are rapidly absorbed into dead cells
(Table 2). Enzymeactivity is readily demonstrated using the so-called
fluorochromatic reaction (21) in which fluorescein diactate is cleaved
cellular esterase. Fluorescein diacetate is easily absorbedby living cells but
becomesfluorescent only if esters are cleaved from the fluorescein molecule
and the cell is intact enoughto retain the polar dye. Respiratory activity has
been demonstrated using an assay for ATPinvolving cleavage by luciferin
yielding measurableluminescence. The results of this methodroughly correspondto those using fluorescein diacetate (54) but ATPis still evident even
whenthe cells are no longer viable according to the previous method.
During isolation, the sperm cells commonlyassume an ellipsoidal to
spheroidal shape and lose their microtubules(5, 15, 73). A comparableloss
microtubules has been described in detail using anti-tubulin
immunofluorescence
of freshly isolated generative cells in bicellular plants (70).
These changes appear comparable to those observed in isolated generative
cells (70). Anexceptionto this is the isolation of spermcells in Gerbera,in
which the native shape is retained (68). Spheroidal sperm cells are not
typically seen in young growing pollen tubes in vivo, but a significant
roundingof these cells seemscharacteristic as pollen tubes near the ovule (38,
58, 64, 65). Althoughspheroidal sperm cells obtained through isolation are
artifactual, this feature is common
in somatic plant protoplasts, typically
without impairing cell quality.
Isolated spermcells lose the plasma membrane
of the vegetative cell that
usually surrounds them in vivo (26). This exposes the true surface of the
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RUSSELL
sperm cells (15, 64) and makes it possible to characterize the plasma membrane. In vivo, the samemodification of the surface of the spermcell occurs
once sperm cells are discharged into the embryo sac (38, 58, 65) and
postulated to be a prerequisite for sperm fusion in angiosperms(58).
A significant remainingquestion about the condition of isolated spermcells
is whetherthey are completelymaturein the pollen grain or whetheressential
gene products remainto be transcribed or translated during pollen tube growth
prior to fertilization (66). Giventhe vast range of times betweenpollination
and gametic fusion in different angiosperms(20 min to several months), these
plants likely vary in this respect.
PHYSIOLOGICAL
CHARACTERIZATION
OF SPERM CELLS
Immunological
Characterization
Hybridomaantibodies have been elicited to an inoculumof intact sperm cells
of Brassica (23) and Plumbago(47) to generate antibody libraries to surface
epitopes. Intact cells were used because of the small numberof sperm cells
available and the technical problemsof preparing isolated plasmamembranes.
Depending on how long sperm cells remain intact in the inoculum, whole
cells wouldbe likely to elicit someantibodies to surface compounds.Initial
attempts werepartially successful, but techniquesfor retaining integrity of the
spermcells during inoculation and for increasing the sensitivity of screening
for surface-specific epitopes will be neededbefore it is possible to producea
useful monoclonalantibody library for recognition studies.
The only detailed report on antibodies elicited to isolated sperm, in Plumbago (47), indicates the feasibility of this approach. Spermantigens are
effective in eliciting antibody-producinghybridomalines to a wide spectrum
of different epitopes in whichpollen wall compounds,including allergens, are
not immunodominant.
The secreting lines were specific for epitopes in the
sperm-enriched(23%), cytoplasmic-particulate (4%), and water-soluble fractions (8%). Antibody-producinglines were mainly IgM(61%), reflecting
high glycoprotein content of the cells (47). Identification of a recognition
factor from this approachis complicatedby the need to develop an appropriate
functional assay. Further research in this area is warrantedif specific recognition compounds
occur as in pollen tube-pistil interactions (9, 45).
Polypeptide/Protein
Characterization
Biochemical characterization of the proteins of male gametes using SDSPAGEhas been conducted on a small number of species: Brassica, Gerbera
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SPERMCELLS OF FLOWERINGPLANTS
199
(27), Plumbago(18), and Zea (P. Roeckel, C. Dumas,personal communication). Theseresults indicate that uniquepolypeptidebands are readily identified in sperm fractions of higher plants. In order to reduce this effect,
presence/absence comparisons with possible contaminants were conducted in
Plumbago(18).
Two-dimensional gel electrophoresis (IEF/SDS-PAGE)
was conducted
comparepolypeptide heterogeneity in P. zeylanica (18). The sperm-enriched
fraction and two selected contaminant fractions were examined. Cytoplasmic
particulates pelleted at 100,000 × g constituted the first fraction; water
soluble molecules collected from the supernatant formedthe second fraction.
The sperm-enriched fraction contained the most polypeptide spots (515 from
Mr 33,000 to 205,000). The cytoplasmic-particulate fraction yielded 427
spots, and the water-soluble fraction had 285. One quarter of the spots were
foundin all fractions, about half were foundin two fractions, and one quarter
werefound in a single fraction. Of the latter group, 51.9%of the polypeptides
were unique to the sperm-enriched fraction, 40.4% to the water-soluble
fraction, and 2%to the cytoplasmic-particulate fraction. Polypeptides of
sufficient molecular weight to be involved in recognition (Mr 70,000 to
>200,000) were present in the sperm-enriched fraction (18). Someof
differences betweenpolypeptides undoubtedlyresult from generational differences of gene expression between the sporophyte and gametophyte(32).
Althoughpollen wall proteins are synthesized by the sporophyte, gametophytic gene products maycontrol gametic recognition, fusion, and fertilization.
Nostudies of developmentalpolypeptide changes in the male gametic line
are currently available. Polypeptidechanges in developing pollen and microspores of Zea (16), Triticum (76), and Brassica (13) indicate that developmental changes do occur. In Triticum, 11 new bands were detected in
SDS-PAGE
and IEF gels and 4 bands were lost during pollen maturation,
indicating a small numberof stage-specific changes. Changesduring anther
developmentin Zea (12) reflect similar stage-specific changesin the proteins
and activities of specific enzymes.With further research, considerable progress in identifying sperm-specific polypeptides maybe expected.
Elemental
Characterization
Energy-dispersive X-ray analysis of pollen componentsindicates that sperm
cells contain significant quantities of carbon, oxygen, phosphorus, calcium,
and potassium, with smaller amountsof magnesium,silicon, manganese,and
detectable levels of chromium.Althoughmostly similar, the dimorphicsperm
of Plumbago
exhibit small differences in Kc~lines of calcium, potassium, and
phosphorus(63). Other studies of pollen have indicated similar elemental
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200
RUSSELL
composition in the pollen tube tip (50). The surfaces of pollen grains
different species, however,display significant elemental differences (7).
Molecular
Characterization
Conflicting information is available regarding the synthesis of RNA
in sperm
cells. In the tricellular pollen of Secale cereale, [5-3H]uridine was incorporated into both the sperm and vegetative nuclei during in vitro pollen
tube growth (20). The types of RNAsynthesized by the sperm nuclei were not
determined. However,in the bicellular pollen of Hyoscyamusniger, exposure
to short pulses of [5-3H]uridine did not detect incorporation (51). Determination of whetherRNA
synthesis is related to the timing of spermcell formation
will require additional research.
Translational products within sperm cells have been obtained by meansof
in vitro translation of isolated RNA
species using [35S]methionine,combined
with electrophoresis and radioautography (P. Roeckel and C. Dumas,personal communication). These studies demonstrate that translatable mRNA
pools exist for a numberof polypeptides in the sperm cells of corn; their
identity has not yet been fully determined. The use of stage-specific promoters (e.g. 73a) and reporter genes in combinationwith particle-gun transformation to elicit transgenic plants seems to be a promising approach to understanding the developmental processes underlying sperm differentiation.
Adaptation and application of existing molecular biological methodsto sperm
cell biology are critically needed.
CONCLUSIONS
AND OVERVIEW
Spermcells have only recently been isolated and used in basic and applied
science. Living spermcells have been isolated in manyspecies of flowering
plants; nowkey methodological improvementswill be required if we are to
extend the useful lifespan of these cells. Further research involving immunologicalcharacterization, cell sorting, cell culture, radiolabeling, gene
expression, and molecular biology of the sperm cells will require basic
knowledgeabout the maintenance and storage of sperm cells.
After these technical problemsare solved, spermcells could play a unique
role in the developing field of reproductive-cell engineering. Because the
cytoplasm of somesperm cells lacks plastids, it could be used to produce
fusion hybrids with selected cytoplasmicconstitutions. Spermcells are naturally produced, haploid cells that maybe a logical substitute for somatic
protoplasts in specific instances or in germ plasm storage. Recently, Kranz
and coworkers have combinedthe isolated egg and sperm cells of Zea using
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SPERM CELLS OF FLOWERING PLANTS
201
electrofusion
to produce a microcallus (28). As problems coping with gametic
cells are resolved, in vitro fusion of male and female gametes of higher plants
ex ovulo may help
angiosperms.
Literature
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understand
fertilization
in
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2. Carlson, W. R. 1969. Factors affecting
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3. Carlson,
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4. Cass, D. D. 1973. Anultrastructural and
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