Transcript Cell cycle synchronization in male and female gametes of

Cell cycle synchronization in male and female gametes
of tobacco (Nicotiana tabacum)
Introduction
Scott D. Russell1, Hui Qiao Tian1,2, Tong Yuan1
1Department
of Botany and Microbiology, University of Oklahoma, Norman, OK 73019 USA
2School of Life Science, Xiamen University, Xiamen 361005, Fujian, CHINA
Abstract
Nuclear DNA content of male and female gametes of tobacco was determined using 4',6-diamindino-2-phenylindole (DAPI) using quantitative microfluorimetry. Pollen grains are released
with generative cells in G2, with a 2C DNA complement. Generative cell mitosis occurs in the pollen tube 8-12 h after germination, resulting in sperm cells with a 1C DNA content. This
1C DNA complement persists throughout pollen tube elongation in the style. Sperm cells deposited in the degenerated synergid have a DNA content between 1C and 2C, indicating that
once in the synergid, sperm cells proceed through S-phase. Concomitant with pollen tube arrival, egg cells increase in DNA quantity from 1C (at pollination) to between 1 and 2C in egg
cells at 48 h after pollination. Without pollination, S-phase in the egg cell is delayed by over a day compared to pollinated ovules. Newly-formed zygotes contain nuclear DNA
concentrations between 3C and 4C at 52 h after pollination as nuclei near karyogamy. The zygote approaches 4C, at 84 h after pollination, long before zygote division. Tobacco displays
cell fusion after completion of S-phase occurring at G2. Failure to achieve an optimized system for in vitro fertilization in Nicotiana despite sustained long-term experimentation may
reflect the challenges of achieving cell cycle synchrony in gametes isolated during protracted S-phases. Such male and female gametes may only reach cell cycle congruity moments before
fusion.
Results
The bicellular pollen of tobacco at anthesis contains a vegetative cell and a generative cell, with generative cell mitosis occurring after 9h of pollen tube elongation.
Newly-formed sperm cells serve as a control for determining the 1C condition, which
appears to persist throughout pollen tube elongation in the style (Figure 2).
Egg cells in vivo progress from a 1C DNA complement to a 2C complement
simultaneously and possibly in synchrony with the progression of the cell cycle in
sperm cells. Newly-formed zygotes display a 4C complement of DNA, indicating that
gamete fusion in tobacco occurs at G2 (Figure 5).
12 hr after pollination (eggs)
30
25
DNA content of generative & sperm nuclei
250
Relative DNA Content
(RFU)
Regulation of the cell cycle is under strict control in eukaryotic nuclei, serving as
a major mechanism controlling growth and development through mitosis. Evidence
of the importance of the cell cycle is reflected in cell cycle control molecules, which
display complex interactions and are regulated by molecules that are highly
conserved (7). However, synchrony of the cell cycle within the mature tissues of a
plant or animal rarely occurs and is rarely favored as a growth strategy. In sexual
reproduction, including male and female gametes in particular, there is a special need
for synchrony, particularly as some cells develop in a shared cytoplasmic
background in which asynchrony cannot be sustained. Asynchronous cell cycle
progression in nuclei located in the same cytoplasm seems unlikely to occur (4).
Gamete fusion in essentially all animals and other eukaryotes involves gametes
in G1 phase, containing one copy of the DNA complement (C1). Egg and zygote
activation ensues, with protracted cellular activity involving the completion of Sphase, and concludes with zygotic division. Flowering plants, in contrast, display
remarkable heterogeneity in their mode of nuclear fusion. Some species with tricellular
pollen, such as Zea (13), and Hordeum (1, 12), have sperm cells that do not progress
beyond G1 prior to fusion. Other species with tricellular pollen may have sperm
cells progress through S-phase and fuse at G2 phase. For example, Crepis, Elytrigria
(2) and other cultivars of Hordeum (10, 11) release sperm cells at G2 phase, with G2
fusion. In contrast, Chlorophytum, Ligularia (2) and Arabidopsis (4) have tricellular
pollen that is disseminated when sperm cells are in mid-S-phase, with a 1.4 to 1.5 C
DNA; these progress to G2 phase during pollen tube elongation. Potentially, other
variants also occur that have not yet been documented (Fig. 1). The only other
bicellular species studied before, Tradescantia (22), revealed fusion at G1 phase. Few
studies report coordinated data on the cell cycle in both the egg and sperm cell.
The present paper reports the progression of the cell cycle in the egg and sperm
cells during development in Nicotiana, apparently indicating a critical need for
gametic synchrony at the time of cell fusion. These data suggest that development of
in vitro fertilization systems may depend on cell cycle synchrony in the gametes,
which may be critical in predicting the success of gamete fusion under in vitro
conditions.
Discussion
20
GC
Sua
Svn
200
15
10
150
5
100
0
48 hr after pollination (eggs)
50
10
0
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0.5
1
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3
Length of style (cm )
4
6
4
2
Fig. 2. Epifluorescence micrograph (left) of tube nucleus (longer diffuse nucleus) and sperm nuclei (two
bright ellipsoidal nuclei) in two representative tobacco pollen tubes in the style, as visualized using
DAPI. Chart (right) indicates relative DNA content of generative and sperm nuclei at five different stages
of in vivo/in vitro grown pollen tubes. Sperm nuclei remain in G1 throughout pollen tube elongation in
the style. Bars indicate standard error. 100 RFU  1C DNA content.
0
52 hr after pollination (zygotes)
15
10
Method of culture used during pollen tube elongation has no effect on observed
DNA quantities as measured in pollen tubes cultured using either the in vitro method,
or in vivo/in vitro (Figure 3).
5
0
84 hr after pollination (zygotes)
30
120
25
Sua
100
20
Svn
80
15
60
10
References
5
40
405-420
375-390
345-360
315-330
285-300
255-270
225-240
195-210
In Vivo/In Vitro
165-180
In Vitro
135-150
0
105-120
0
20
75-90
Fig. 3. Comparison of relative DNA of
sperm nuclei using in vitro- and in vivo/in
vitro-cultured pollen tubes. 100 RFU 
1C DNA content.
Relative DNA content (RFU)
Culture method & sperm nuclear DNA
Fig. 1. Cell cycle variability of sperm cells during fertilization in flowering plants (Friedman, 1999).
Increases in DNA content are not observed until the pollen tube enters the ovary.
Once sperm cells are deposited in the receptive synergid, these cells appear to progress
from G1 to S-phase, with DNA quantities increasing dramatically. A wide range of
DNA concentrations, from 1.2C to nearly 2C is observed, indicating that sperm cell
cycle progression occurs within the receptive synergid (Figure 4).
Fig. 5. Epifluorescence microscopy of sperm nucleus fusing with nuclei of egg (top left) and central cell
(bottom left). Charts on the right indicate relative DNA fluorescence in the egg cell at different times
before and after fertilization. Immature egg cells are 1C at pollination and 12h afterwards, increasing in
DNA content to ~2C prior to fertilization (48 h). After fertilization, DNA content approaches 4C (52h),
reaching 4C at 84h after pollination.
DNA content of sperm in tube & synergid
Relative DNA content (RFU)
250
200
150
Sua
Svn
100
Sp 1
Sp 2
50
Emasculated flowers in which pollination has been withheld display an unchanged
amount of DNA (1C) from 0 to 48 h after anthesis. Egg cells approach a 2C DNA
complement at 84 h after anthesis (Figure 6). These results indicate that although egg
cells progress to G2 without pollination, this process is delayed. That synchrony in cell
cycle progression of male and female gametes occurs in pollinated flowers suggests
that gametes and/or gametophytes participate in long distance signaling.
2.
Tube
5.
6.
7.
8.
9.
10.
11.
12.
Synergid
DNA content during egg development
Sperm in synergid
16
12
Fig. 6. Relative DNA content of egg nuclei in
emasculated, unpollinated flowers at three
different stages of maturation after anthesis.
Newly-formed sperm = 100 RFU  1C DNA.
8
4
0
120
4.
13.
0
130
140
150
160
170
180
190
200
Relative DNA content (RFU)
Nicotiana tabacum L. plants were grown in a growth chamber at 20-27C with 16 h daylight.
Flowers were emasculated 0.5 day before anthesis and pollinated at anthesis. Pollen tubes were grown
using the in vivo/in vitro technique (19) to obtain generative and sperm cell DNA measurements.
Pollinated styles were cut just beyond the region containing growing pollen tube tips and the cut stylar
end was immersed in pollen tube culture medium (0.01% [w/v] H3BO3, 0.01% [w/v] KH2PO4, 0.01%
[w/v] CaCl2·2H2O with 15% [w/v] sucrose added) for several hours until pollen tubes emerged from
the cut tip (20). The normal duration of pollen tube growth through the 4-cm style of tobacco is 2 d
from pollination to fertilization. Five stylar lengths were sampled (0.5, 1, 2, 3 and 4 cm, at 5, 13, 20,
27 and 34 h after pollination, respectively). Excised styles were floated vertically on culture medium
for 6 to 12 h, until pollen tubes emerged. Styles with emergent pollen tubes were fixed in 3 parts
ethanol to 1 part acetic acid for 24 h and stored in 70% ethanol at 4C. To compare potential effects
of in vitro growth on sperm DNA content, in vivo styles were also sectioned using paraffin technique.
Ovules from pollinated and unpollinated flowers were sampled at 0.5, 2 and 3.5 d after anthesis, fixed
as above, infiltrated and embedded using paraffin technique, serially sectioned at 7–8 µm and mounted
on glass slides. After removing paraffin, slides were labeled in a solution containing 0.25 µg/ml 4',6diamidino-2-phenylindole (DAPI) with 0.1 mg/ml ρ-phenylenediamine dihydrochloride in 0.05 M
Trizma buffer (pH 7.2) for 1 h. Slides were examined using a Zeiss epifluorescence microscope.
For microspectrofluorimetry, a UV filter set with a 365-nm excitation filter was used. A circular
detection aperture of 9.84 µm diameter was used to reduce scattered light. Background fluorescence
from the cytoplasm and embedding medium was subtracted from the nucleus, yielding a net
photometric value of relative fluorescence units (RFU) corresponding to nuclear DNA. RFU were
standardized by using a DAPI-labeled sample of newly-formed sperm or egg nuclei, and was adjusted
to a fluorescence value of 100 RFU, representing 1C DNA. Each stage was measured using at least
50 individuals (except for sperm nuclei in the synergid) and was repeated three times. To assure
microfluorimeter stability, all measurements made for a given stage were conducted at one sitting,
with controls measured at the beginning and end of measurements at each sample stage.
1.
3.
Num ber of nuclei
Materials and methods
Among eukaryotes, generally the fusion of male and female gametes occurs at G1
phase. Rarely has nuclear fusion at G2 phase been reported, although G2 fusion is not
uncommon in sea urchins and does occur in flowering plants (4). In angiosperms, not
only is there species-level cell cycle variability, but variability also in the number of
cells in pollen when it is released: bicellular pollen contains a generative cell (and
vegetative cell), whereas tricellular pollen contains the two sperm already formed. In
bicellular pollen at anthesis, the generative nucleus may be in G1, G2 or early Mphase, as in tobacco. In tricellular pollen, the sperm may be in G1, G2 or S phase (4).
In tobacco, generative cell division occurs at ~10 h after pollination. Sperm cells
remain in G1 throughout subsequent pollen tube elongation in the style. When pollen
tubes reach the embryo sac, sperm cells are discharged into the receptive degenerate
synergid (14). DNA synthesis and the completion of S-phase in the sperm nuclei
occur within the unique environment of the synergid (5). The trigger for the onset of Sphase in the sperm cells is yet unknown, but induction of DNA synthesis may relate to
their proximity to and arrival in the synergid.
The receptive synergid presents a unique, purportedly apoptotic behavior that may
expose sperm cells to a stressful and oxygen-deprived environment (17) that in some
systems has been reported to trigger cell cycle progression (15). In tobacco, apparently
S-phase in the sperm cells largely coincides with their presence in the synergid. In
contrast with previous observations suggesting that sperm cell passage in the synergid
was transient (6), current research suggests that it may be quite protracted (21). This
feature may also occur in other plants with G2 fertilization in which sperm cells
complete S-phase within the receptive synergid.
An apparently critical feature of the cell cycle of gametes is the need to achieve
phase synchrony between the sexes. In tobacco, egg cells responded to the presence
of pollen tubes in the style and ovary by completing S-phase nearly simultaneously
with the sperm cells at ~52 h after pollination. In contrast, egg nuclei in unpollinated
flowers appeared to complete S-phase 1 d later, providing evidence of gametophytic
crosstalk. Since gametes in tobacco appear to fuse only at the completion of S-phase,
it seems possible that specific cell surface determinants may appear at the completion
of S-phase that are required for fusion.
Synchrony of gametes may also be required for gamete receptivity and successful
fertilization in vitro. To date, the greatest successes of in vitro fertilization have been
achieved using maize (3), which is a tricellular pollen species that releases pollen
with G1 gametes; these gametes are known to fuse naturally without further cell cycle
progression (13). Wheat has also been used successfully, which if consistent with
barley (12), also fuses at G1. Using tricellular G1 plants appears to be a propitious
choice for in vitro fertilization, as gametes are: i) fully formed at anthesis, ii)
synchronized relative to the cell cycle, and iii) gametes are at the appropriate stage of
the cell cycle for fusion.
In tobacco, studies of in vitro fertilization using isolated male and female gametes
collected at G1 have achieved limited success. Strongly fusigenic conditions, such as
polyethylene glycol, were required to initiate fusion, and fusion products tended to
arrest without any further cell division occurring (18,19). Although the gametes were
synchronized, they appear according to this study to be in the wrong phase for fusion.
Thus, it is possible that the gametes lack necessary surface epitopes for fusion and
once fused, were unable to divide. Similar arrest may also be expected if
unsynchronized gametes are fused.
Cell cycle congruity appears to be an important factor in predicting success during
in vitro fertilization (4). Plumbago, a plant in which pollen is released with sperm
cells in S-phase, likely also fuses at G2. Similar to tobacco, gametes of Plumbago also
have difficulty fusing in vitro (unpublished data). Success in obtaining fusion during
in vitro fertilization using maize gametes (3) may owe a significant amount of its
success to cell cycle phase congruency and phase appropriate fusion (13).
250
15.
200
150
16.
17.
100
18.
50
19.
0
Sperm
Fig. 4. Epifluorescence microscopy (left) of receptive synergids containing sperm cells, as visualized
using DAPI. The chart in the upper right indicates a dramatic increase in DNA content occurs in sperm
cells discharged from the pollen tube into the receptive synergid. Chart to the lower right displays the
range of DNA levels observed in individual sperm cells within synergids. Sperm DNA appears to nearly
double from 120 to 200 RFU in the synergid. Bars indicate standard error. 100 RFU  1C DNA content.
14.
12 hr Egg
48 hr Egg
84 hr Egg
20.
Egg m aturation relative to anthesis (hr)
21.
Abbreviations: Svn=sperm associated with the vegetative nucleus; Sua=sperm unassociated with the vegetative
nucleus; Sp1=larger of two sperm cells (when Svn and Sua cannot be identified); Sp2=smaller of two sperm cells.
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