tion of nDNA andor cytDNA synthesis is closely involved in the induction of zygote formation.
We noticed that the pyrenoids in C. ehrenbergii were greatly enlarged during sexual reproduction
compared with those during asexual reproduction. This would suggest that regulation of pyrenoid
function occurs during the conversion from asex- ual to sexual reproduction in this alga. In the
present
study, we
investigated the
ratio of
pyrenoidcell number, and the pyrenoidal sizes of the alga throughout the asexual and sexual stages
of reproduction, and found that the modulation of pyrenoid production and function accompanied
the formation of pregametes pregametogenesis.
The present study investigated whether the inhi- bition of nDNA andor cytDNA synthesis is in-
volved in the induction of zygote formation in C. ehrenbergii and if so, which form of inhibition is an
initial signal for the induction. To this end, the effect of selective inhibitors of cytDNA or nDNA
synthesis on the frequency of zygote formation in the alga, as well as on the modulation of pyrenoid
production and pyrenoidal size during pregameto- genesis, was examined.
2. Materials and methods
2
.
1
. Strains and culture conditions Previously described [3] heterothallic strains of
C. ehrenbergii, M-16-4a mating-type plus: m
+
and M-16-4b mating-type minus: m
−
were used in the present study. They were cultured in C
medium [9] at 23°C, and illuminated with fluores- cent lamps of 2.25 × 10
− 1
Wm
2
1500 lx with a 16-h per day photoperiod.
2
.
2
. Chemicals For staining pyrenoids, carmine Merck, C.I.
No. 75470 was purchased from Nacalai Tesque Kyoto, Japan. As DNA synthesis inhibitors, the
following chemicals were used. EB and Mitomycin C MC were purchased from Aldrich Chemical
Co. Inc. Milwaukee, USA and Kyowa Hakko Co. Tokyo, Japan, respectively. Acridine orange
AO, novobiocin NB, sodium salt and ara- binosyl cytosine Ara-C were from Nacalai
Tesque. Aphidicolin APC and hydroxyurea HU were from Sigma Chemical Co. St. Louis, USA.
2
.
3
. Experimental procedures To investigate the frequency of zygote forma-
tion, vegetative cells of each strain were grown for 17 days in C medium and collected, and approxi-
mately 10
4
cells of each strain were mixed together. The mixed cells were centrifuged at 72 × g for 1
min, washed twice with a nitrogen-deficient conju- gation medium MIH medium [10], and suspended
in the same medium 2 × 10
3
cells per ml. Subse- quently, a 0.1-ml aliquot of the suspension was
inoculated per well of a Linbro tissue culture multi-well plate ICN Biomedicals, Inc. contain-
ing 0.7 ml of MIH medium with or without a nitrate source final concentrations of 16.9 mgl of
CaNO
3 2
· 4H
2
O and 13.1 mgl of KNO
3
. To examine the effect of DNA synthesis inhibitors on
zygote formation, the DNA inhibitors above were added to the MIH medium at various concentra-
tions. The cells were incubated for 5 days under the conditions described above, except that the light
intensity was elevated to 3000 lx to induce zygote formation. Counting of zygotes, gametes and vege-
tative cells was achieved under a stereomicroscope at 10 × magnification. A visual field was selected
for each test well, and a minimum of 100 cells were counted. The frequency of zygote formation
was calculated from the equation: 2Nz × 100 2Nz + Ng + 2Nv, where Nz is the number of
zygotes normal plus abnormal zygotes, Ng is the number of gametes and Nv is the number of
vegetative cells.
To test the effect of DNA synthesis inhibitors on growth asexual reproduction, vegetative cells of
the m
+
strain were grown for 17 days in C medium, collected by centrifugation and suspended
in fresh C medium 10
3
cells per ml. Subsequently, 10-ml aliquots of the suspension were inoculated
into wells containing 790 ml of C medium together with the test chemicals at various concentrations.
The culture was maintained for 5 days under illumination of 2000 lx, and then the growth rate
per day was calculated.
To investigate the number of pyrenoids per cell and pyrenoidal size during asexual and sexual
reproduction, cells were collected in logarithmic growth log and stationary phases and at
pregamete and gamete stages. To obtain cells in log and stationary phases, vegetative cells of each
strain, which were grown for 22 days in C medium, were collected and suspended separately
in fresh C medium 2.5 × 10
3
cells per ml. Four 0.1-ml aliquots containing about 250 cells each
of each cell suspension were separately inoculated into respective petri dishes which contained 25 ml
of C medium. Subsequently, the inocula were in- cubated for 4, 11, 17 or 22 days under illumination
of 2000 lx, and at the end of each culture period, cells of each strain were harvested. To obtain cells
at pregamete and gamete stages, vegetative cells of each strain grown for 17 days in C medium were
collected and mixed together in the cell number noted above. The mixed cells were washed twice
with MIH medium, and then inoculated into a petri dish containing 25 ml of MIH medium 300
cells per ml. Subsequently, the inoculum was in- cubated for 4 days with illumination of 3000 lx
and then harvested. To count the number of pyrenoidscell, pyrenoids were stained according
to Rosowski and Hoshaw [11] Fig. 1L and M, with the exception that 50 propionic acid which
contained 4 ferric ammonium sulfate was used as the mordant, followed by 3-D counting under
an Olympus BX50 microscope. To determine pyrenoidal size, ten or more cells were selected for
each stage and photographed under the micro- scope at 100 × . For each stage, the diameters of
500 pyrenoids were measured on microphoto- graphs enlarged at a final magnification of 400 × .
To investigate the effects of DNA synthesis inhibitors on pyrenoid production and pyrenoidal
size, vegetative cells of each strain were grown for 17 days, collected and inoculated separately into
petri dishes which contained 25 ml of C medium 300 cells per ml with or without the test chemi-
cals. The cells were incubated for 5 days with illumination of 3000 lx and then harvested. This
was followed by measurement of pyrenoidscell number and pyrenoid diameter.
2
.
4
. Statistical analysis Experiments to investigate the frequency of
zygote formation and growth rate per day were performed in triplicate. Experiments on pyrenoid
number and size were repeated more than twice, and the same tendencies were observed. To com-
pare treatment means with a control mean, statis- tical analyses were carried out using Student’s
t-test, and the degree of significance was expressed in terms of P-values.
3. Results
