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
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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
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M-phase promoting factor Hashimoto and Kishimoto, 1988 and the cytostatic factor Ž
. Masui, 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 penetration 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
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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.
´
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1997 and Cha et al. 1997 . However, these studies did not test various concentrations 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 in Õitro maturation Pig ovaries were obtained from a local slaughterhouse and transported to the
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laboratory in a saline solution 0.9 sodium chloride at 388C. Fully grown oocytes were collected by aspirating follicles that were 2–5 mm in diameter with a 20-gauge
needle. Only oocytes with compact cumuli were chosen for further studies. Before culture the oocytes were washed three times in a culture medium.
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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 ,
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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
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ZD Hustopece, Czech Republic . The oocytes were cultured in 3.5-cm diameter Petri
ˇ
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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
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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
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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 23187 at concentrations of 10, 25 or 50 mM for 0.5, 1, 3, 5 or 7 min. The oocytes were
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
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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
