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Animal Reproduction Science 63 2000 101–111 www.elsevier.comrlocateranireprosci
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 — UhrıneÕes, Czech Republic
ˇ´ ˇ
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 50 mM for 0.5,
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1, 3, 5 or 7 min and then cultured with cycloheximide 0 or 10 mgrml 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
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treatment of ionophore 25 mM with cycloheximide. The percentage of oocytes developing up to the morula and blastocyst stage was not significantly increased after cycloheximide treatment.
q 2000 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: jilekaf.czu.cz F. Jılek .
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0378-4320r00r - see front matter q 2000 Elsevier Science B.V. All rights reserved. Ž
. PII: S 0 3 7 8 - 4 3 2 0 0 0 0 0 1 5 0 - 0
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
