Ž then washed three times in a culture medium and cultured with cycloheximide 0 or 10
. mgrml for 6 h. The oocytes were then washed in a cycloheximide-free medium and
cultured in a cycloheximide-free medium for another 18 h. The aim of Experiment 2 was to investigate the development of pig oocytes activated
by a combined treatment of calcium ionophore and cycloheximide. For this experiment, we chose treatments that induced activation rates higher then 50 in Experiment 1.
The pig oocytes were cultured in vitro for 48 h and then exposed to calcium ionophore A 23187 at concentrations of 25 or 50 mM for 3, 5 or 7 min. The oocytes
Ž were washed three times in a culture medium and cultured with cycloheximide 0 or 10
. mgrml for 6 h. After that the oocytes were washed in a cycloheximide-free medium
and cultured in a cycloheximide-free medium for 6 days. The stage of development was checked in all oocytes at the end of the culture.
2.5. Statistical analysis Each experiment was carried out four times. The results were pooled for presentation
Ž .
and evaluated by chi-squared analysis Snedecor and Cochran, 1980 . The mean
percentage of oocytes reaching the given stage of development in all trials did not vary from the pooled percentage by more than 2.5. A P value of less than 0.05 was
considered significant.
3. Results
Under our culture conditions, more than 95 of the oocytes matured to the stage of Ž
metaphase II after 48 h of in vitro culture. In control experiments, no oocytes 0 of 100 .
Ž .
oocytes were activated after prolonged culture overall culture time 72 h without treatment with calcium ionophore A 23187 or cycloheximide. Similarly, we did not
Ž observe any activation of matured oocytes treated with only cycloheximide 0 of 100
. Ž .
oocytes Table 1 .
Table 1 Ž
. Ž
Effect of cycloheximide 10 mgrml on the activation of pig oocytes with calcium ionophore A 23187 10 .
Ž .
mM . In vitro matured pig oocytes ns120 for each treatment were exposed to ionophore and then cultured Ž
. Ž
in a cycloheximide-free medium for 24 h IONO 10qCHX 0 or cultured for 6 h with cycloheximide 10 .
Ž .
mgrml and then in a cycloheximide-free medium for 18 h IONO 10qCHX 10 . The activation rate was then Ž
. estimated number of activated eggsrtotal number of eggs
Ž .
Exposure of eggs to ionophore min 0.5
1 3
5 7
IONO 10qCHX 0 3
7 7
IONO 10qCHX 10 20
30 47
Ž .
Statistically significant differences P -0.05 between the group treated with cycloheximide and the
group cultured in cycloheximide-free medium are indicated by asterisks.
Table 2 Ž
. Ž
Effect of cycloheximide 10 mgrml on the activation of pig oocytes with calcium ionophore A 23187 25 .
Ž .
mM . In vitro matured pig oocytes ns120 for each treatment were exposed to ionophore and then cultured Ž
. Ž
in a cycloheximide-free medium for 24 h IONO 25qCHX 0 or cultured for 6 h with cycloheximide 10 .
Ž .
mgrml and then in a cycloheximide-free medium for 18 h IONO 25qCHX 10 . The activation rate was then Ž
. estimated number of activated eggsrtotal number of eggs
Ž .
Exposure of eggs to ionophore min 0.5
1 3
5 7
IONO 25qCHX 0 11
16 30
42 55
IONO 25qCHX 10 26
43 50
60 85
Ž .
Statistically significant differences P -0.05 between the group treated with cycloheximide and the
group cultured in a cycloheximide-free medium are indicated by asterisks.
In Experiment 1, activation was observed in oocytes matured in vitro and then treated with calcium ionophore. We can conclude that higher ionophore concentration and
prolonged treatment with ionophore increased the activation rate. Further increase in the activation rate was observed in oocytes exposed to a combined treatment of ionophore
and cycloheximide. A positive effect of cycloheximide on the activation rate was
Ž .
obvious especially after activation of the oocytes with 25 mM ionophore Table 2 . After treatment of the oocyte with 10 mM ionophore, a positive effect of cycloheximide on the
activation rate was obvious after longer exposures of the oocytes to ionophore. Shorter Ž
. exposure of the oocytes to 10 mM ionophore 0.5 and 1 min is probably insufficient for
oocyte activation. When oocytes were treated with ionophore at concentration of 50 mM Ž
. Table 3 , the cycloheximide treatment did not significantly increase the activation rate.
Ž .
Longer exposure to cycloheximide 24 h did not increase the activation rate of the Ž
. oocytes treated with ionophore results are not involved in this study .
For further experiments, we used treatments that induced an activation rate higher than 50 in Experiment 1.
Ž .
In the control experiment, only a few in vitro matured oocytes 3 of 100 oocytes cleaved or fragmented after a 6-day culture without treatment with calcium ionophore
A23187 or cycloheximide. Similarly, we observed very low percentage of cleavage or
Table 3 Ž
. Ž
Effect of cycloheximide 10 mgrml on the activation of pig oocytes with calcium ionophore A 23187 50 .
Ž .
mM . In vitro matured pig oocytes ns120 for each treatment were exposed to ionophore and then cultured Ž
. Ž
in a cycloheximide-free medium for 24 h IONO 50qCHX 0 or cultured for 6 h with cycloheximide 10 .
Ž .
mgrml and then in a cycloheximide-free medium for 18 h IONO 50qCHX 10 . The activation rate was then Ž
. estimated number of activated eggsrtotal number of eggs
Ž .
Exposure of eggs to ionophore min 0.5
1 3
5 7
IONO 50qCHX 0 26
32 46
60 70
IONO 50qCHX 10 31
42 56
66 83
Ž .
Statistically significant differences P -0.05 between the group treated with cycloheximide and the
group cultured in cycloheximide-free medium are indicated by asterisks.
Table 4 Ž
. Effect of cycloheximide 10 mgrml on the cleavage of pig oocytes after their activation with calcium
Ž .
Ž .
ionophore A 23187 25 mM . In vitro matured pig oocytes ns100 for each treatment were exposed to Ž
. Ž .
ionophore and then cultured in a cycloheximide-free medium for 6 days 144 h IONO 25qCHX 0 or
Ž .
Ž cultured for 6 h with cycloheximide 10 mgrml and then in cycloheximide-free medium for 138 h IONO
. Ž
25qCHX 10 . The percentage of developing eggs was then estimated number of cleaving eggsrtotal number .
of eggs Ž
. Exposure of eggs to ionophore min
3 5
7 IONO 25qCHX 0
32 42
50 IONO 25qCHX 10
49 67
62 Ž
. Statistically significant differences
P -0.05 between the group treated with cycloheximide and the group cultured in cycloheximide-free medium are indicated by asterisks.
Ž .
fragmentation of in vitro matured oocytes 5 of 100 oocytes
treated with only cycloheximide and then cultured in cycloheximide-free medium for 6 days.
In Experiment 2, we observed a positive effect of the cycloheximide treatment on the cleavage of oocytes activated with calcium ionophore. This effect is significant after
Ž .
oocyte treatment with 25 mM ionophore for 3 and 5 min Table 4 . After oocyte treatment with 50 mM ionophore, the percentage of oocytes developing to 2-cell stage or
greater is significantly lower then that of oocytes activated with 25 mM ionophore. Ž
. Cycloheximide treatment partially counteracted this decrease Table 5 . The rate of
Ž .
cleavage in oocytes treated with ionophore 50 mM and cycloheximide is higher then Ž
. the cleavage percentage of oocytes treated only with ionophore 50 mM . But this
percentage of cleavage is still lower than in oocytes activated with 25 mM ionophore alone.
The percentage of individual developmental stages of parthenogenetic embryos is shown in Table 6. After activation with 25 mM, ionophore some oocytes developed up
to the stage of morula and blastocyst, but the percentage of these developmental stages Ž
. was very low up to 7 for morula and up to 3 for blastocyst . A similar percentage of
Table 5 Ž
. Effect of cycloheximide 10 mgrml on the cleavage of pig oocytes after their activation with calcium
Ž .
Ž .
ionophore A 23187 50 mM . In vitro matured pig oocytes ns100 for each treatment were exposed to Ž
. Ž .
ionophore and then cultured in a cycloheximide-free medium for 6 days 144 h IONO 50qCHX 0 or
Ž .
Ž cultured for 6 h with cycloheximide 10 mgrml and then in a cycloheximide-free medium for 138 h IONO
. Ž
50qCHX 10 . The percentage of developing eggs was then estimated number of developing eggsrtotal .
number of eggs Ž
. Exposure of eggs to ionophore min
3 5
7 IONO 50qCHX 0
22 21
14 IONO 50qCHX 10
36 33
33 Ž
. Statistically significant differences
P -0.05 between the group treated with cycloheximide and the group cultured in cycloheximide-free medium are indicated by asterisks.
Table 6 The percentage of individual developmental to cleavage stages of pig oocytes activated by combined treatment
Ž .
with calcium ionophore and cycloheximide. Oocytes were treated with ionophore 25 mM or 50 mM for Ž
different times 3, 5 or 7 min — data for different exposure times to ionophore were pooled for presentation .
and only data about different ionophore and cycloheximide treatment are presented and then cultured for 144 Ž
. h in a cycloheximide-enriched 10 mgrml or a cycloheximide-free medium
Treatment of eggs developing to cleavage stages:
Total no. Ionophore
Cycloheximide 2 cells
3–4 cells 5–16 cells
Morula Blasto.
Ž .
Ž .
mM mgrml
25 11
31 7
3 1
300 25
10 13
31 9
5 1
300 50
7 8
4 300
50 10
10 10
13 1
300
morula and blastocyst was observed in oocytes treated with 25 mM ionophore and cycloheximide.
4. Discussion
