Article Effect of ionomycin on oocyte activation and embryo development in mouse

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1 RBMOnline - Vol 17. No Reproductive BioMedicine Online; on web 21 October 2008 Article Effect of ionomycin on oocyte activation and embryo development in mouse Elke Heytens has obtained an MSc degree in biochemistry and a Master s degree in molecular medical biotechnology at Ghent University, Belgium. She joined the research team of the Centre for Reproductive Medicine at Ghent University Hospital in She is currently working on her PhD and her major interest is oocyte activation in assisted reproduction. Ms Elke Heytens Elke Heytens 1, Reza Soleimani 1, Sylvie Lierman 1, Simon De Meester 1, Jan Gerris 1, Marc Dhont 1, Josiane Van der Elst 1,2, Petra De Sutter 1 1 Centre for Reproductive Medicine, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium; 2 Centre for Reproductive Medicine, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium Correspondence: Elke.Heytens@UGent.be Abstract Artificial oocyte activation using the calcium ionophore ionomycin is applied successfully in assisted reproduction but some concern exists on the clinical use. The aims of the present study were to optimize the oocyte activation scheme and to address embryo toxicity in a mouse model. Efficiency of oocyte activation and subsequent development was evaluated and ionomycin was found to be an efficient activator at 10 µmol/l. An improved effect of a second exposure to 5 µmol/l ionomycin on blastocyst development was observed. Toxicity of ionomycin on embryos was then investigated by evaluating pre- and post-implantation development of in-vivo fertilized oocytes following exposure to ionomycin. Blastocyst development, blastocyst cell numbers in trophectoderm and inner cell mass were not different between treated and non-treated zygotes. Also implantation rates and fetal parameters such as length, weight and morphological parameters were similar between the fetuses originating from zygotes treated with ionomycin and non-treated zygotes. Furthermore, healthy offspring originating from ionomycin-treated zygotes was born. In conclusion, no adverse effects of ionomycin on in-vitro or in-vivo mouse embryo development were noticed, giving arguments in favour of the use of ionomycin, although negative long-term effects of this compound cannot be excluded at present. Keywords: calcium ionophore, embryo development, ionomycin, oocyte activation, zygote test Introduction 764 Calcium ion (Ca 2+ ) signalling plays a major role at fertilization, because a spermatozoon not only delivers its genetic material but also triggers rises in intracellular Ca 2+ concentration ([Ca 2+ ] i ), which are necessary for the completion of all the events of oocyte activation and initiation of embryonic development (Cuthbertson et al., 1981; Miyazaki et al., 1993). Rises of [Ca 2+ ] i are required to induce oocyte activation and are needed to provoke the cortical reaction, resumption of meiosis, maternal mrna recruitment, pronuclear development and mitotic cleavage (Ducibella et al., 2002). Ca 2+ oscillations are also observed after intracytoplasmic sperm injection (ICSI) and, although the pattern is somehow different from that after normal fertilization, ICSI is an efficient technique in male infertility (Palermo et al., 1992; Sato et al., 1999). However, an abnormal Ca 2+ pattern has been accounted for the unsuccessful oocyte activation after ICSI in bovine (Malcuit et al., 2006). The importance of successful triggering of oocyte activation is shown by the fact that an aberrant oocyte activation can be the cause of failed fertilization after ICSI in human (Sousa and Tesarik, 1994). This emphasizes the role of Ca 2+ at fertilization, both during natural fertilization and after assisted reproduction. Mammalian oocytes such as murine, rabbit, bovine, rat, porcine and also human oocytes can be activated parthenogenetically or following nuclear transfer just by increasing [Ca 2+ ] i using electrical activation, ethanol treatment or Ca 2+ ionophore challenge (Ware et al., 1989; Ozil, 1990; Kline and Kline, 1992; Collas et al., 1993; Mizutani et al., 2004; Im et al., 2006). This technique of artificially induced oocyte activation or assisted oocyte activation (AOA) at the time of ICSI is also proposed as an option to couples after repeated 2008 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

2 cycles with failed fertilization after ICSI, before considering gamete donation. AOA by using Ca 2+ ionophores to increase [Ca 2+ ] i can restore normal fertilization rates in these cases (Moaz et al., 2006) and support further development as shown by successful pregnancies and deliveries reported by the research centre (Rybouchkin et al., 1997; Heindryckx et al., 2005) and also by others (Kim et al., 2001; Eldar-Geva et al., 2003; Chi et al., 2004; Ahmady and Michael, 2007; Nasr-Esfahani et al., 2008). Although it has been suggested that any stimulus that causes a sufficient amount of Ca 2+ release during activation is effective in stimulating early development (Toth et al., 2006), modifying the Ca 2+ pattern may also influence pre- and post-implantation development (Ozil, 1990; Ozil and Huneau, 2001; Ozil et al., 2006). Therefore, optimization of oocyte activation protocols is important in order to obtain a high activation efficiency and also optimal further embryonic development. Besides, little is known yet about the possible adverse effects of ionophores on embryo development and some concern exists about the use of a Ca 2+ ionophore as an oocyte activating agent in assisted reproduction (Rybouchkin et al., 1997; Moaz et al., 2006; Nasr-Esfahani et al., 2008). Since ionomycin is not uniquely selective for Ca 2+, it is probably advisable to treat the ionophore with caution regarding metabolic processes, because many ions are co-factors for enzymes and many membrane pumps are also ion dependent for correct function. Even though the exposure to Ca 2+ ionophore for the purpose of AOA is short, it is thus appropriate to examine possible embryo toxic effects of ionomycin. The goal of the present study was two-fold. Mouse oocytes and zygotes were used as test models for optimizing oocyte activation and for studying embryo toxic effects of ionomycin, respectively. Parthenogenetic activation of mouse oocytes was used to evaluate the efficiency of different ionomycin treatment protocols on blastocyst development. A protocol based on the currently used AOA method in the study centre was included. Embryo toxicity was assessed by determining the quality of the embryos resulting from zygotes exposed to ionomycin by differential staining of the inner cell mass (ICM) and the trophectoderm (TE). Furthermore, postimplantation development after transfer of embryos treated with ionomycin to foster mothers was evaluated. This is the first report in which the effect of ionomycin on in-vitro and in-vivo embryo development of mouse zygotes exposed to ionomycin has been investigated. Materials and methods Collection of mouse oocytes and embryos Seven- to 12-week-old female B6D2/F1 mice (Charles River Laboratories, Brussels, Belgium) were stimulated to superovulate with 5 IU equine chorionic gonadotrophin (Folligon, Intervet, Oss, The Netherlands) and 48 h later with 5 IU human chorionic gonadotrophin (HCG; Chorulon, Intervet). Oocytes were collected at 14 h post HCG administration, freed from cumulus cells by a short incubation in hyaluronidase (200 IU, 3 5 min, Sigma, Bornem, Belgium) and cultured in potassium simplex optimized medium (KSOM; Lawitts and Biggers, 1993) supplemented with 0.4% bovine serum albumin (KSOM-BSA, Calbiochem, Bierges, Belgium) at 37 C and 6% CO 2 until start of parthenogenetic activation. To obtain zygotes, the stimulated mice were paired with B6D2/F1 males and checked the morning after for a vaginal plug. Zygotes were collected 22 h post HCG administration and freed from cumulus cells by a short incubation in hyaluronidase and immediately used. All procedures involving live animal handling and killing were performed according to standard animal protocols approved by the Ghent University Hospital Ethical Committee for Laboratory Animals (ECP 06/03). Parthenogenetic activation At 17 h post HCG administration, the oocytes were activated with 1, 5 or 10 µmol/l ionomycin (Sigma) in KSOM-BSA at 37 C and 6% CO 2 for 5, 10, 30 min or twice for 10 min with a 30 min interval. The AOA protocol currently used in the study centre consists of incubating twice for 10 min with a 30 min interval with 10 µmol/l ionomycin 30 min after ICSI with CaCl 2 (5 pl of 0.1 M) (Heindryckx et al., 2005). After carrying out the activation protocols, the oocytes were transferred to KSOM-BSA supplemented with 2 µg/ml cytochalasin D (Sigma) for 3 h. As a positive control, oocytes were activated for 3 h with 10 mmol/l SrCl 2 (Sigma) in Ca 2+ - free KSOM-BSA supplemented with 2 µg/ml cytochalasin D. Non-treated zygotes were the positive control for culture conditions and non-treated oocytes were the negative control for spontaneous activation. These controls were included in each experiment. Two pronuclei (2PN) and two-cell formation were scored respectively 4 and 24 h after the start of the activation. Blastocyst development was evaluated at 120 h post HCG administration. Ionomycin treatment and culture of zygotes After collection, zygotes were randomly divided into two groups: a control group and an ionomycin group. In the ionomycin group, the zygotes were treated with the AOA protocol that was shown to be the most effective from among the above parthenogenetic activation experiments. Because previous results demonstrated that the two-step sequential culture in KSOM and Cook blastocyst media (Cook Belgium, Strombeek-Bever, Belgium) is superior for culture of mouse zygotes (Heindryckx et al., 2003), zygotes were preferably cultured in KSOM-BSA and then transferred to Cook blastocyst medium at h post HCG administration. Cell counting of blastocysts To evaluate the preimplantation development, differential staining of ICM and TE cells of blastocysts was performed at 120 h post HCG as described previously (Van Soom et al., 2001), based on the method of Hardy et al. (1989). Briefly, zona pellucida of expanded and hatching blastocysts were removed by incubation for 1 min in pronase (0.5% Protease, Sigma). Zona-free and completely hatched blastocysts were incubated in 10 mmol/l trinitrobenzene-sulphonic acid 765

3 766 (Sigma) in phosphate-buffered saline containing 3 mg/ml polyvinylpyrrolidone (Sigma), ph 7.4 at 2 8 C for 10 min. Then they were incubated in a 30:70 dilution of rabbit antidinitrophenol BSA (Sigma) in HEPES-buffered medium supplemented with 10 µg/ml propidium iodide (Sigma) for 30 min at 37 C. After finishing complement-mediated cell lysis and the staining of lysed cells by propidium iodide, the blastocysts were fixed in ice-cold absolute ethanol and stained with 10 µg/ml bisbenzimide (Hoechst 33342; Sigma) in absolute ethanol at 4 C for at least 2 h. Finally, the blastocysts were transferred to a drop of glycerol on a microscopic slide and covered with a cover slip. ICM nuclei labelled with bisbenzimide only appeared blue and TE nuclei labelled with both bisbenzimide and propidium iodide appeared pink-tored. Numbers of ICM and TE nuclei were counted directly under a fluorescence microscope ( 100 magnification) and expressed as mean ± SD. Embryo transfer Recipient B6D2 female mice were stimulated as described above and paired with vasectomised CD1 males (Charles River Laboratories) to induce a state of pseudopregnancy. Females were checked for a vaginal plug the morning after, indicating that mating had occurred and that the female was at day 1 of pseudopregnancy. Ten 2-cell embryos were transferred to the left oviduct of pseudopregnant mice on day 1 of pseudopregnancy. Foster mothers were killed on day 17 of gestation. The concepti were removed from the uterus, and the number of implantation sites, resorbing concepti and live fetuses were noted. The fetuses were examined for major external anomalies, weight and length (crown to rump). The developmental age was determined according to Wahlsten and Wainwright (1977). In attempt to obtain live offspring, foster mothers from each group were left to carry to term and deliver the pups. The pups were weighed at day 7 and from then twice a week until they were 8 weeks old and compared with pups obtained after mating. Development was scored by the time of pinna unfolding, generalized hair growth and eye opening. At the age of 12 weeks, the mice were mated to evaluate their fertility. Post-natal development was compared with pups born after natural mating. Statistical analysis Chi-squared tests and Fisher s Exact tests were used for statistical analysis of oocyte activation and pre- and post-implantation embryo development. A Bonferroni adjustment was applied for multiple comparisons of counts. Parameters of blastocyst quality (cell numbers for ICM, TE, TCN [total cell number] and ICM/ TCN) and fetal development (estimated age) were analysed by two-sided t-test or non-parametric Mann Whitney U-test. The estimated age based on weight and length was corrected for the number of fetuses/foster mother by analysis of variance (ANOVA) with number of fetuses as random factor. Levene s test for equality of variances (α = 0.01) and normality tests (Kolmogorov Smirnov, α = 0.01) were performed to choose the appropriate test. A significance level of α = 0.05 was used throughout, unless otherwise mentioned. Results Effect of ionomycin concentration and time of incubation on parthenogenetic oocyte activation and development Three replicates were performed for each combination of concentration and time. A minimum of 40 oocytes was included in each group. The results on 2PN, 2-cell and blastocyst formation are presented in Table 1. Oocyte activation, as judged by 2PN formation, was overall significantly lower when the oocytes were activated with 1 µmol/l in comparison with 10 µmol/l ionomycin (range 37 49% versus 82 90%). When oocytes were exposed to 5 µmol/l ionomycin for 5 or 10 min, the oocyte activation rate was significantly lower than for oocytes exposed to 10 µmol/l ionomycin (60 68% versus 82 90%). After longer or a second incubation in 5 µmol/l ionomycin, similar activation percentages were reached in comparison to incubation with 10 µmol/l ionomycin. Significantly less oocytes developed to the two-cell stage when activated with 1 µmol/l ionomycin compared with 5 and 10 µmol/l (23 33% versus 60 84% and 64 73% respectively, P < 0.001). No significant differences in 2-cell formation could be detected between the activation protocols with 5 or 10 µmol/l ionomycin. Although, a second exposure to 5 µmol/l ionomycin improved 2-cell formation in comparison with a single exposure for 10 min (84% versus 69% respectively). A very low percentage (0 5%) of blastocysts was obtained after parthenogenetic activation with 1 µmol/l ionomycin. A lower blastocyst development rate was observed when oocytes were incubated once with 5 µmol/l ionomycin as compared with oocytes treated once with 10 µmol/l ionomycin (4 8% versus 25 30% respectively, P < 0.001). This difference was not seen when oocytes were exposed twice to 5 or 10 µmol/l ionomycin for 10 min. Oocytes treated twice with 5 µmol/l ionomycin developed better then the oocytes exposed only once to 5 µmol/l ionomycin (20% versus 5% respectively, P < 0.01). No significant differences in blastocyst development rates could be detected between the different incubation durations when the oocytes were exposed to 10 µmol/l ionomycin. Parthenogenetic activation with 5 or 10 µmol/l ionomycin twice for 10 min with a 30 min interval resulted in 20 and 24% blastocyst formation, respectively. When compared with the positive activation control with strontium, only the 10 µmol/l set of activation schemes and the exposure twice to 5 µmol/l ionomycin resulted in a similar level of activation as judged by 2PN formation. Two-cell formation and blastocyst development were significantly lower for all tested activation schemes with 10 µmol/l ionomycin and the exposure twice to 5 µmol/l protocol compared with activation with strontium (64 84% versus 94% and 20 30% versus 65%, P < 0.01 and P < 0.001). The protocol based on the AOA method currently used in the study centre was among the best and therefore possible embryo toxicity of ionomycin treatment was tested accordingly (10 µmol/l ionomycin twice for 10 min with a 30 min interval).

4 Development and cell counting of blastocysts after ionomycin treatment of zygotes In five replicate experiments, a total of 292 fertilized oocytes were selected and randomized between the control and the ionomycin group. In the ionomycin group, the zygotes were incubated twice for 10 min in 10 µmol/l ionomycin with an interval of 30 min. All the zygotes from both groups divided to the two-cell stage. Analysis of the 151 embryos in the control group and 141 in the treated group showed no significant differences in blastocyst development (96.7 and 97.9%). The percentages of expanded, hatching and completely hatched blastocysts at 120 h post HCG were not significantly different between the two groups (Figure 1). Differential staining was performed successfully on 107 blastocysts of the control group and 95 blastocysts of the ionomycin group and the results are presented in Figure 2. Only the blastocysts that were completely labelled and that were not damaged during the staining procedure were included. The percentage of blastocysts analysed was not significantly different between both groups (69% and 73% for control and ionomycin group respectively). The ICM, TE and total cell numbers (TCN) reached by the blastocysts obtained from zygotes treated with ionomycin (21 ± 5, 85 ± 24 and 106 ± 28) were not significantly different from the control group (23 ± 7, 90 ± 26 and 113 ± 29). Blastocysts in the ionomycin group showed an ICM/TCN ratio of 0.20 ± 0.34 and this ratio did not differ significantly from the control group (0.21 ± 0.05). Evaluation of post-implantation development At day 17 of gestation, the implantation sites were evaluated and the fetuses were weighed, measured and examined for morphological features. The data are presented in Table 2 and Figure 3. The pregnancy and implantation rates were not significantly different between the ionomycin group and controls (47.1 and 21.1% versus 33.3 and 24.2%). Also the fetal survival was not different between both groups (69.4% versus 72.4% for ionomycin and control group respectively). The fetuses obtained from embryos treated with ionomycin were examined and showed an average weight of 0.86 ± 0.16 g and a crown rump length of 19.8 ± 1.9 mm, which corresponds to an estimated developmental age of 17.0 ± 0.4 days. Evaluation of morphological features revealed an estimated developmental age of 16.2 ± 0.5 days. This was not significantly different from control fetuses, which showed an average weight of 0.71 ± 0.13 g and a crown rump length of 17.7 ± 2.0 mm corresponding with an estimated developmental age of 16.7 ± 0.4 and 16.1 ± 0.9 days respectively. Evaluation of morphological features revealed an estimated developmental age of 16.1 ± 0.9 days. Two foster mothers in the control group and four in the ionomycin group were left to carry to term and deliver the pups. The mothers cannibalized the pups and only two pups developed from zygotes treated with ionomycin were raised by the mother (ionomycin pups; one male and one female). No pups from the control group could be obtained. The ionomycin pups did not show any abnormalities and their post-natal growth (body Table 1. Effect of ionomycin concentration and incubation time on parthenogenetic mouse oocyte activation and development in comparison with strontium. Ionomycin Timing No. of 2PN Two-cell Blastocysts concentration (min) treated embryos (µmol/l) oocytes (49) b 10 (24) b 2 (5) b (49) b 12 (28) b 2 (5) b (43) b 14 (33) b 0 (0) b a (37) b 10 (23) b 0 (0) b (60) b,c 46 (60) c 3 (4) b (68) b,c 53 (69) c 4 (5) b (75) c,d 52 (69) c 6 (8) b a (72) c,d 63 (84) d 15 (20) c (90) e 55 (71) c,d 20 (26) c (82) d,e 47 (64) c 22 (30) c (86) d,e 61 (79) c,d 19 (25) c a (87) d,e 55 (73) c,d 18 (24) c Strontium (10 mmol/l) (87) e 140 (93) e 98 (65) d Values are number of oocytes (percentage of treated oocytes); 2PN = 2 pronuclei. a 10 min incubation twice with a 30 min interval. b e Values with a common letter in the same column are not significantly different (Bonferroni adjusted a, P = 0.037, Fisher s Exact test). 767

5 Figure 1. Distribution of blastocysts in relation to their developmental stage on day 5. Solid bars show embryos obtained from zygotes treated with ionomycin and open bars embryos obtained from non-treated zygotes. In the ionomycin group 138 and in the control group 146 blastocysts were evaluated. *Not fully expanded blastocysts. No significant differences were detected (chi-squared test). Figure 2. Total cell number (TCN) and number of cells in inner cell mass (ICM) and trophectoderm (TE) of blastocysts originating from zygotes treated with ionomycin (solid bars, n = 95) compared with non-treated zygotes (open bars, n = 107). Bars represent mean ± SD. No significant differences were detected (two-sided t-test). Table 2. Implantation and fetal development of control mouse embryos and embryos treated with 10 µmol/l ionomycin following transfer to pseudopregnant mice. Control embryos Embryos from zygotes treated with ionomycin Pregnancy rate a 4/12 (33.3) 8/17 (47.1) Implantation rate b 29/120 (24.2) 36/170 (21.2) Fetal survival c 21/29 (72.4) 25/36 (69.4) No significant differences could be detected (Fisher s Exact test). a Number of mice having at least one implantation site/number of mice (percentage). b Number of implantation sites/embryos transferred (percentage). c Number of viable fetuses/number of implantation sites (percentage). Weight (g) Figure 3. Developmental characteristics of fetuses on gestational day 17 originating from control mouse embryos (open bars, n = 21) and embryos treated with 10 µmol/l ionomycin (solid bars, n = 25) following transfer to pseudopregnant mice. Estimated age determined according to Wahlsten and Wainwright (1977) based on length, weight and morphology (Morph). Bars represent mean ± SD. No significant differences could be detected (Mann Whitney U-test; estimated age based on length and weight was corrected for fetuses/foster mother by analysis of variance) Age (weeks) Female ionomycin Male ionomycin Female in vivo Male in vivo 8 10 Figure 4. Post-natal growth rate during first 8 weeks after birth of the female (n = 1, circles) and male (n = 1, triangles) ionomycin pup and female (n = 4, squares) and male (n = 6, crosses) in-vivo controls. Data are mean ± SD.

6 weight at 1 week up to 2 months, Figure 4) and development, as assessed by time of pinna unfolding, generalized hair growth and eye opening (data not shown), was normal compared with pups delivered after natural mating. The male ionomycin mouse successfully mated and eight normal pups were born. Also, the female ionomycin mouse was fertile as proven by the delivery of five normal pups. Discussion Assisted oocyte activation by artificially raising the intracellular Ca 2+ concentration is applied successfully in human assisted reproduction (Rybouchkin et al., 1997; Kim et al., 2001; Eldar- Geva et al., 2003; Chi et al., 2004; Heindryckx et al., 2005; Ahmady and Michael, 2007). Since some studies have shown that the Ca 2+ transients during activation may influence the number of embryos reaching the blastocyst stage (Ozil, 1990; Ozil and Huneau, 2001), there may be a need to optimize the activation procedure to enhance embryo development. Ionomycin has already been used successfully for human assisted reproduction in the study centre (Heindryckx et al., 2005), and therefore it was preferred not to change the activating agent but to further optimize the activating scheme and evaluate the possible embryo toxicity induced by ionomycin. The scarcity of human oocytes available for research and ethical challenges associated with their use make it necessary to use a mouse model for this purpose. Although the mouse model may not be the best to compare with humans for reproductive studies (Ménézo and Herubel, 2002), it is frequently used in the development of assisted reproductive techniques (Edwards, 2005) and in the quality improvement of human assisted reproduction (McDowell et al., 1988; Lierman et al., 2007). In the present study, an effective oocyte activation protocol using ionomycin has been identified by comparing activation and development rates of mouse oocytes exposed to different concentrations of ionomycin during various time intervals. In addition, this was the first study evaluating the effect of two repetitive exposures to ionomycin. An enhanced effect of a second exposure to ionomycin at 5 µmol/l on mouse preimplantation development was observed. The important issue of possible adverse effects of ionomycin on in-vitro and in-vivo embryo development was addressed and importantly no negative impact on further pre- and post-implantation embryonic development of mouse zygotes was noted. The data presented here indicate that at low concentration (1 µmol/l) ionomycin was not an efficient oocyte activator and resulted in poor blastocyst development. However, activation rates were enhanced with higher concentrations of ionomycin (5 and 10 µmol/l), plausibly because a higher ionophore concentration caused release of more Ca 2+ inside the cell (Vincent et al., 1992). A single exposure to 5 µmol/l ionomycin resulted in a low percentage of blastocysts. Interestingly, when the oocytes were incubated twice with 5 µmol/l ionomycin, two-cell formation and blastocyst development were improved significantly and a blastocyst percentage similar to activation with 10 µmol/l ionomycin was obtained. In contrast, the [Ca 2+ ] i rise caused by a single exposure to 10 µmol/l ionomycin was as efficient for successful embryo development as the [Ca 2+ ] i rises induced by incubating twice with 10 µmol/l ionomycin. This suggests that the second incubation with ionomycin at 10 µmol/l was not necessary to obtain comparable blastocyst development. Ca 2+ transients may influence embryo development, implantation rate or even post-natal development (Ozil et al., 2006). The effect of the second exposure to ionomycin on post-implantation development should be further investigated, but since post-implantation development from parthenogenetically created embryos cannot be studied (McGrath and Solter, 1984), this could not be evaluated in the current model. Therefore, the original AOA protocol successfully applied in human assisted reproduction (10 µmol/l ionomycin twice for 10 min with a 30 min interval) was not altered for now. Strontium, an effective activation agent for mouse oocytes (O Neill et al., 1991; Otaegui et al., 1999), was used here as a control to compare the efficiency of ionomycin. Although ionomycin at 10 µmol/l was as effective as strontium in terms of oocyte activation, treatment with strontium resulted in markedly better blastocyst development. Others have also reported that activating mouse oocytes with strontium leads to improved blastocyst composition (Bos-Mikich et al., 1997) and superior preimplantation development (Loren and Lacham- Kaplan, 2006). The better embryo development of mouse oocytes exposed to strontium can probably be explained by a different mode of action in the oocytes. Strontium acts like a surrogate of Ca 2+ in mouse oocytes and causes a series of Ca 2+ oscillations similar to those observed at fertilization (Kline and Kline, 1992; Zhang et al., 2005). In contrast, ionomycin induces a single transient in [Ca 2+ ] i in mouse and human oocytes (Kline and Kline, 1992; Rinaudo et al., 1997). The data emphasize that although any stimulus that causes a sufficient amount of Ca 2+ release may activate the murine oocyte, it may not be optimal to promote further embryonic development (Ozil, 1990; Ducibella et al., 2002; Toth et al., 2006). Since the data presented here confirm that parthenogenetic activation using strontium results in better preimplantation development in the mouse, an AOA protocol using strontium may be considered for clinical application. In mice, applying strontium after ICSI of round spermatids resulted in livebirth results similar to those obtained with ICSI of mature spermatozoa (Loren and Lacham-Kaplan, 2006). Recently, a case report described pregnancies and childbirth achieved after AOA using strontium (Yanagida et al., 2006). However, the observation that no Ca 2+ oscillations were triggered in human oocytes treated with strontium (Rogers et al., 2004) suggests that other mechanisms than described for mouse oocytes are involved (Zhang et al., 2005) and that an activation protocol based on strontium should still be treated with caution. Likewise, there is some concern about the use of ionomycin in assisted reproduction. Up to now, no toxicity tests whatsoever have been performed on AOA using Ca 2+ ionophores. As a first step, the impact of the oocyte activating agent ionomycin itself on embryo development was assessed. For this purpose, zygotes were exposed to ionomycin, using the incubation timing and ionomycin concentration of the AOA protocol used in the study centre. Since it has been shown that Ca 2+ oscillations cease during entry into the interphase, at the time when the male and female pronuclei are forming (Jones et al., 1995), PN stage embryos were used. Therefore, the ionomycin treatment will not interfere with the Ca 2+ signalling induced by fertilization and the possible adverse effect of the ionophore 769

7 770 through other unknown mechanisms on embryo development is studied. So, the toxicity of ionomycin itself and not the described oocyte activation protocol is studied. Although it has to be acknowledged that exposing zygotes is not the same as incubating at the time of fertilization, embryo toxic effects of ionomycin might be detected in this model. The influence of ionomycin exposure on embryo preimplantation development was analysed by evaluating blastocyst development and by counting the number of cells in the ICM and in the TE, both being well-accepted parameters to evaluate blastocyst quality. The allocation of a sufficient number of cells to both TE and ICM is essential for normal preimplantation development and alterations in this process may lead to embryos that are unable to implant and further develop (Van Soom et al., 2001). Zygotes treated with ionomycin were not impaired in their blastocyst development and the proportion of completely hatched, hatching and expanded blastocysts was similar. The quality of the blastocysts from the ionomycin group was equal to that of nonexposed blastocysts. No significant differences in cell counts were detected between blastocysts obtained from zygotes treated with ionomycin in comparison with control blastocysts. Cell counts of blastocysts and especially the ICM population size are related to the implantation potential (Leppens et al., 1996). So, similar implantation rates and further embryonic and fetal growth can be expected between both blastocyst populations. Post-implantation development was evaluated after transfer of embryos originating from zygotes treated with ionomycin and non-treated embryos to foster mothers. To analyse possible adverse effects of ionomycin on post-implantation development fetuses were evaluated on day 17 of gestation. The pregnancy and implantation rates were not significantly different between the two groups, and were overall rather low. This can be caused by a negative effect of ovarian stimulation on embryo development competence as well as changes in the uterine milieu in the hormonal stimulated recipient mice (Ertzeid and Storeng, 2001). In this study, exposure of embryos at the zygote stage to ionomycin did not negatively influence or accelerate the further post-implantation development in comparison with the control group. Even more, healthy, normal, developing and fertile pups originating from zygotes treated were ionomycin were born. In summary, ionomycin at a concentration of 10 µmol/l can effectively activate oocytes and results in acceptable blastocyst development. Additionally, it was observed that incubating twice with 5 µmol/l ionomycin resulted in improved oocyte activation and further development in comparison with incubating once with 5 µmol/l ionomycin. Most importantly, no adverse effect of ionomycin exposure on mouse zygote development was noticed. Although this study offers arguments in favour of the safety of the use of ionomycin in the AOA protocol applied here, more work is necessary. Evaluation of the Ca 2+ pattern induced by the AOA protocol, which consists of CaCl 2 injection and ionomycin exposure, and studying the influence on post-implantation embryo development after ICSI and AOA is sensible. Also, these studies were performed in an animal model and appropriate clinical investigations are still needed. Therefore, follow-up of the children born after AOA remains necessary, whatever protocol is used. Acknowledgements EH and RS are supported by the Special Research Foundation (BOF, nr. 011/090/04 and 01W03306) of Ghent University. PDS is holder of a fundamental clinical research mandate by the Flemish Foundation of Scientific Research (FWO- Vlaanderen). 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