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Activation of pig oocytes using calcium ionophore:
effect of protein synthesis inhibitor cycloheximide
F. Jılek
a,), R. Huttelova
b, J. Petr
c, M. Holubova
a, J. Rozinek
a´
¨
´
´
a
Department of Veterinary Sciences, Czech UniÕersity of Agriculture, Kamycka 129,
165 21 Prague 6 — Suchdol, Czech Republic
b
Pronatal Sanatorium, IVF Unit, Prague 5 — Stodulky, Czech Republic˚
c
Department of ReproductiÕe Biology, Research Institute for Animal Production,
Prague 10 — Uhrıneˇ´ ˇÕes, Czech Republic
Received 12 November 1999; received in revised form 6 March 2000; accepted 28 March 2000
Abstract
In vitro matured pig oocytes were activated using a combined treatment of calcium ionophore
Ž .
A 23187 with cycloheximide. The oocytes were exposed to ionophore 10, 25 or 50mM for 0.5,
Ž .
1, 3, 5 or 7 min and then cultured with cycloheximide 0 or 10mgrml for 6 h. Cycloheximide treatment significantly increased the activation rate of oocytes and the percentage of oocytes that were able to develop after activation. The highest activation rate was observed after treatment with 50 mM ionophore. The highest percentage of developing eggs was observed after combined
Ž .
treatment of ionophore 25mM with cycloheximide. The percentage of oocytes developing up to the morula and blastocyst stage was not significantly increased after cycloheximide treatment.
q2000 Elsevier Science B.V. All rights reserved.
Keywords: Pig ovarium; Oocyte activation; Calcium ionophore; Cycloheximide
1. Introduction
The artificial activation of eggs induces the completion of oocyte meiosis and is linked to many important problems including that of nuclear transfer. For this reason, many studies are focused on the activation of oocytes in large domestic animals.
)Corresponding author. Tel.:q420-2-24382935; fax:q420-2-20921649.
Ž .
E-mail address: [email protected] F. Jılek .´
0378-4320r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved.
Ž .
Oocyte activation is induced under natural conditions by the sperm penetrating the egg and plays a key role in meiosis. Meiosis in the matured mammalian egg is blocked at the metaphase II stage, when the first polar body is extruded from the egg. Further progress of meiosis depends on the activating stimulus. This stimulus is brought into the egg by the sperm during fertilisation and it involves mechanisms inducing the oscillation
Ž .
of intracellular levels of free calcium ions Yanagimachi, 1988 . The elevated cytoplas-mic levels of calcium ions in activated eggs influences the activity of molecules of the
Ž .
M-phase promoting factor Hashimoto and Kishimoto, 1988 and the cytostatic factor
ŽMasui, 1991 , which are responsible for the meiotic block at the metaphase II stage..
Artificial stimuli elevating the cytoplasmic levels of calcium ions can induce activa-tion of the oocytes even without penetraactiva-tion of the sperm into the oocyte. A wide spectrum of activating stimuli, e.g. ionophore, ethanol or electrical pulses, are used to induce the artificial activation of mammalian eggs. However, the search for other suitable means of activation or their combinations is still going on.
It seems that many activation treatments did not provide the pig oocytes with an
Ž .
adequate or full-valued activating stimulus Wang et al., 1998a,b . It is suggested that these activation methods are unable to suppress the synthesis of proteins that are involved in the blockage of oocyte meiosis at the metaphase II stage. For this reason, activation treatments combining the activating stimulus with the effect of protein synthesis inhibitors, e.g. cycloheximide, were tested. These experiments resulted in a
Ž .
higher activation rate in cattle Pressicce and Yang, 1994a,b; Yang et al., 1994 and in
Ž .
pig oocytes Nussbaum and Prather, 1995; Petr et al., 1996 .
An increased activation rate in pig oocytes after combined treatment with calcium
ionophore A 23187 and cycloheximide was indicated in the studies of Grocholova et al.
´
Ž1997 and Cha et al. 1997 . However, these studies did not test various concentrations. Ž .
of ionophore and the length of ionophore treatments. Similar to other studies performed
Ž
on activated pig oocytes Hagen et al., 1991; Petr et al., 1996; Prochazka et al., 1992;
´
.
Prather et al., 1991 , these studies focused mainly on the development of pronuclei after parthenogenetic activation and the parthenogenetic development of activated oocytes was not investigated. The aim of this study was to test the combined treatment of pig oocytes with calcium ionophore A 23187 and cycloheximide and to follow their further development in vitro.
2. Material and methods
2.1. Oocyte collection and inÕitro maturation
Pig ovaries were obtained from a local slaughterhouse and transported to the
Ž .
laboratory in a saline solution 0.9% sodium chloride at 388C. Fully grown oocytes
Ž .
Oocytes were cultured in a modified M199 medium USOL, Prague, Czech Republic
Ž .
containing sodium bicarbonate 0.039 ml of 7% solution per millilitre of medium ,
Ž . Ž . Ž .
calcium lactate 0.6 mgrml , gentamicin 0.025 mgrml , HEPES 1.5 mgrml , 13.4 iu
Ž .
eCG:6.6 iu hCGrml P.G. 600, Intervet, Boxmeer, Holland and 10% of bovine serum
ŽZD Hustopece, Czech Republic . The oocytes were cultured in 3.5-cm diameter Petri
ˇ
.Ž .
dishes Nunc, Roskilde, Denmark containing 3 ml of culture medium at 398C in a
mixture of 5% CO in air.2
2.2. ActiÕation of oocytes
Fully grown pig oocytes were cultured in vitro for 48 h. The oocytes were stripped of cumulus cells by repeated pipetting through a narrow glass pipette and were then activated by exposure to ionophore A 23187.
2.3. EÕaluation of oocyte maturation
At the end of the culture, the oocytes were mounted on slides, fixed with acetic
Ž .
alcohol 1:3 vrv for at least 24 h and stained with 1% orcein. The oocytes were
examined under a phase contrast microscope. Activation was considered to have occurred if the oocytes were in the pronuclear stage. Oocytes remaining at metaphase II or arrested at anaphase II or at telophase II were not considered to be activated.
2.4. Arrangement of experiments
2.4.1. Control experiments
These experiments were carried out to evaluate the rate of spontaneous activation of pig oocytes that could be due to a prolonged in vitro culture and to test the activation rate of oocytes after treatment with cycloheximide without calcium ionophore.
One group of oocytes was cultured in vitro for 48 h, stripped of cumulus cells and then cultured for a further 24 h.
Another group of oocytes was cultured in vitro for 48 h, stripped of cumulus cells
Ž .
and then cultured with cycloheximide 10 mgrml for 6 h. Subsequently, the oocytes
were carefully washed in a culture medium and then cultured in a cycloheximide-free medium for another 18 h.
In experiments designed to control the spontaneous cleavage or fragmentation, the oocytes were cultured in vitro for 48 h, stripped of cumulus cells and then exposed to cycloheximide or cycloheximid-free medium for 6 h more. Subsequently, the oocytes were carefully washed in a culture medium and then cultured in cycloheximide-free medium for 6 days.
Experiment 1 was performed to test the activation rate of pig oocytes after combined treatment with calcium ionophore A 23187 and cycloheximide.
The oocytes were cultured in vitro for 48 h and then exposed to calcium ionophore A
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 10mgrml 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 0 0 3 7 7
) ) )
IONO 10qCHX 10 0 0 20 30 47
Table 2
Ž . Ž
Effect of cycloheximide 10mgrml 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
)Statistically significant differencesŽP-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 25mM ionophore Table 2 . After treatment of the oocyte with 10mM 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 10mM ionophore 0.5 and 1 min is probably insufficient for
oocyte activation. When oocytes were treated with ionophore at concentration of 50mM
ŽTable 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 10mgrml 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
Table 4
Ž .
Effect of cycloheximide 10 mgrml on the cleavage of pig oocytes after their activation with calcium
Ž . Ž .
ionophore A 23187 25mM . 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 10mgrml 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
)Statistically significant differencesŽP-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 50mM 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 25mM ionophore
alone.
The percentage of individual developmental stages of parthenogenetic embryos is
shown in Table 6. After activation with 25mM, 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 50mM . 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 10mgrml 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
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 25mM 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 10mgrml 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.
ŽmM. Žmgrml.
25 0 11 31 7 3 1 300
25 10 13 31 9 5 1 300
50 0 7 8 4 0 0 300
50 10 10 10 13 1 0 300
morula and blastocyst was observed in oocytes treated with 25 mM ionophore and
cycloheximide.
4. Discussion
In our study, we have shown the positive effect of the protein synthesis inhibitor cycloheximide on the activation and subsequent parthenogenetic development of in vitro matured pig oocytes activated by the use of calcium ionophore A 23187. In oocytes treated with calcium ionophore A 23187 and then exposed to the inhibitor of protein synthesis cycloheximide for 6 h, the activation rate is significantly increased. This
increase is especially significant after oocyte treatment with 10 and 25mM ionophore.
After treatment of oocytes with 50 mM ionophore, cycloheximide did not further
increase the activation rate. We suggest that 50mM ionophore provides the maximum
activation stimulus, and this cannot be further boosted using cycloheximide treatment. After combined treatment with ionophore and cycloheximide, we also observed an increased rate of parthenogenetic development in activated oocytes. However, the rate of oocytes reaching the stage of morula and blastocyst was not significantly increased.
During oocyte activation, the blockage of meiosis is overcome. This meiotic arrest occurred at the stage of metaphase II in matured mammalian eggs and is established by
cdc2 Ž
the stabilisation of cyclin B-p34 through the effects of the cytostatic factor Masui,
.
1991 . The elevation of intracellular calcium levels during fertilization or partheno-genetic activation is known to trigger exit from the metaphase II stage in matured
Ž .
mammalian eggs Parrington et al., 1996; Yanagimachi, 1988; Miyazaki et al., 1993
Ž .
through the calcium-dependent inactivation of CSF Zernicka-Goetz et al., 1995
Ž
probably due to the destruction of the product of proto-oncogene c-mos Watanabe et
. cdc2
al., 1989; Weber et al., 1991 and the subsequent inactivation of the cyclin B-p34
Ž .
complex Collas et al., 1995 . The increase of H1 kinase activity during the entry of the oocyte in the metaphase II stage and its subsequent decrease after activation and
Ž
fertilization was also demonstrated in pig oocytes Kikuchi et al., 1995a,b; Naito et al.,
.
Calcium ionophore A 23187 elevates the levels of frees calcium ions in the cytoplasm
Ž .
of various types of cell Pressman, 1976 . This property of calcium ionophore A 23187 is exploited for the parthenogenetic activation of mammalian oocytes includes those of
Ž .
the pig Hagen et al., 1991; Wang et al., 1998a,b . However, after oocyte activation using calcium ionophore, the activation rate is usually lower than after other activating
Ž .
stimuli, especially after electrical pulses Hagen et al., 1991; Prather et al., 1991 . Calcium ionophore A 23187 is probably unable to induce all processes necessary for full-valued activation of the pig oocyte.
One possibility is that the events induced by calcium ionophore do not suppress synthesis of the proteins responsible for maintenance of the oocyte in meiotic arrest at the metaphase II stage. Inhibitors of protein synthesis can be used to suppress these metaphase-arresting proteins. The positive effect of protein synthesis inhibitors was
Ž
observed after activation of cattle oocytes Pressicce and Yang, 1994a,b; Yang et al.,
.
1994 . The positive effect of inhibitors of protein synthesis was also seen in pig oocytes
Ž .
after their activation using electrical pulses Nussbaum and Prather, 1995 or ethanol
ŽPetr et al., 1996 . The results including those of the present study also indicate that the.
activation of pig oocytes using calcium ionophore does not sufficiently suppress synthesis of the proteins that are responsible for blocking meiosis at the metaphase II stage, and that is why activation with calcium ionophore did not provide a full-valued activating stimulus to the egg. In our experiment, we observed the highest activation rate
Ž
in oocytes exposed to a combined treatment of calcium ionophore concentration of 25
.
or 50mM for 7 min and cycloheximide.
The concentration of cycloheximide used in this study is able to suppress germinal
Ž .
vesicle breakdown in pig oocytes Fulka et al., 1986 and is also able to induce the
Ž
formation of the interphase nucleus in pig oocytes at the metaphase I stage Ding et al.,
.
1992; Mattioli et al., 1991; Rozinek et al., 1996 . However, the treatment of in vitro matured pig oocytes with cycloheximide alone is unable to induce the exit of pig eggs from the meiotic arrest occurring spontaneously at the stage of metaphase II. The concentration of cycloheximide used in the present study is probably unable to
com-Ž .
pletely suppress protein synthesis in the pig oocyte Ding et al., 1992 but higher concentrations of this inhibitor have an adverse effect on the transport functions of
Ž .
membranes in the pig oocyte Ding et al., 1992 . For this reason we did not use higher concentrations of cycloheximide in our study.
In pig oocytes matured in vitro, the effect of cycloheximide on protein synthesis is performed mainly during the first hours of exposure of the oocyte to this protein
Ž .
synthesis inhibitor Nussbaum and Prather, 1995 . In agreement with this observation, we did not observe any increase in the activation rate after prolonged exposure of oocytes to cycloheximide. Activation and the formation of pronuclei occurred in pig
Ž
oocytes approximately 6 h after the activating stimulus Funahashi et al., 1995; our
.
unpublished observations . For this reason we decided to expose pig oocytes to cycloheximide for 6 h.
The conditions of oocyte activation determine not only the activation itself but also
Ž
the further parthenogenetic development of activated eggs Niemann and Reichelt, 1993;
.
Ozil, 1990 . This fact was manifested also in our study. Oocyte activation with 50mM
cycloheximide but the parthenogenetic development of these oocytes was substantially reduced. A much higher percentage of developing oocytes was observed after activation
of the oocytes with 25 mM. Based on our observations, we can conclude that the
activation rate, used as the criterion indicating the success of various activating methods, does not provide a universal and objective criterion and the percentage of developing oocytes has to be taken into account when various activating procedures are evaluated. Parthenogenetic development was tested in a modified M199 cultured medium. It has
Ž .
been suggested by several authors Hagen et al., 1991; Miyano et al., 1994 that this medium could have an adverse effect on the development of pig embryos and special
Ž
culture media were constructed for embryonic development in this species Petters and
. Ž .
Wells, 1993; Reed et al., 1992 . However, Kure-bayashi et al. 1996 reported successful development of parthenogenetic pig embryos up to the blastocyst stage in a M199 medium. In our laboratory, too, we observed embryonic development up to the morula
Ž .
or blastocyst stage in the M199 medium Petr, unpublished results after parthenogenetic
Ž
activation of pig oocytes using a combined treatment of ethanol and cycloheximide Petr
.
et al., 1996 .
Based on the results of our study, we can conclude that combined treatment of in vitro matured pig oocytes with calcium ionophore A 23187 and cycloheximide signifi-cantly increased the activation rate of oocytes and also increased the percentage of oocytes that developed after parthenogenetic activation.
Acknowledgements
We thank Mrs. Lucy Westcott for the editorial assistance with this manuscript. The
ˇ
study was supported by grants EP 0960006203 from MZe CR, 524r99r0301 from GA
ˇ
CR and VZJ 03r98:412100003.
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