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Regular Article |
a Department of Gene Expression and Development, Roslin Institute, Roslin, Midlothian EH25 9PS, United Kingdom
b Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom
c Germplasm and Gamete Physiology Laboratory, U.S. Department of Agriculture, Beltsville, Maryland 20705
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, early development, embryo, reproductive technology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several factors are believed to influence an oocyte's response to electrical activation, including age, the applied voltage field strength, and the pulse number and duration. In addition, there probably are interactions between these factors. In most studies, the activation rate increases with oocyte age [4–8]. Although a single pulse is still more commonly used in activation protocols for pig oocytes [2, 8–11], better development may be obtained by the administration of multiple pulses. This approach is thought to more closely mimic an oocyte's response to a fertilizing sperm by eliciting repeated calcium transients [12].
Three interrelated series of experiments were carried out to investigate factors influencing the efficiency of electrical activation of pig oocytes. Our first objective was to investigate the effects of epidermal growth factor (EGF) and amino acids in the medium used for the in vitro maturation of pig oocytes, as judged by development to the blastocyst stage after activation. Next, the effect of oocyte age at activation, applied voltage field strength, and pulse number and duration were evaluated for their effect on parthenogenetic development to define an improved electrical activation protocol. Blastocysts produced by this improved protocol were then karyotyped to assess their potential for in vivo development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovary Collection and Maturation
Porcine gilt ovaries were collected from a local abattoir and stored at 25–30°C during transportation. When ovaries arrived at the laboratory, they were washed 3 times with warmed Dulbecco PBS (Oxoid Ltd., Basingstoke, Hampshire, U.K.) and then stored in a water bath at 25–30°C before use. Cumulus oocyte complexes (COCs) were aspirated from ovarian follicles 3–8 mm in diameter using a 10-ml syringe fitted with an 18-gauge needle. Follicular fluid was collected in 50-ml universal containers and left for 5 min at 25–30°C. Subsequently, COCs were washed 3 times with Hepes-buffered Tyrode albumin lactate pyruvate medium (TL-Hepes) containing 0.1% polyvinylalcohol (PVA). Only COCs with uniform cytoplasm and at least 3 layers of compact cumulus cells were selected for maturation. Oocytes were normally matured in groups of 50 per 500 µl of maturation medium at 39°C in an atmosphere of 5% CO2 in air. The base maturation medium was BSA-free NCSU 23 medium [13] supplemented with 10% (v/v) porcine follicular fluid and 0.6 mM cysteine. This medium was supplemented with 10 IU/ml eCG (Folligon; Intervet U.K. Ltd., Cambridge, U.K.) and 10 IU/ml hCG (Chorulon; Intervet) during the first 22 h of culture only, after which culture was hormone free. In study 2, the base maturation medium (i.e., with or without hormones) was further supplemented with 10 ng/ml EGF, 1% essential (B-6766; Sigma) and 0.5% nonessential (M-7145; Sigma) amino acids (AA), or both EGF and AA. For all other experiments, the base medium (i.e., with or without hormones) was supplemented with essential and nonessential AA only.
Oocyte Activation and Embryo Culture
Following in vitro maturation, oocytes were denuded of cumulus cells by repeated pipetting on a warm stage at 37°C. Denuded oocytes were washed 3 times in Ca2+-free TL-Hepes-PVA medium and then rinsed twice in activation medium (0.3 M mannitol, 0.1 mM Mg2+, and 0.05 mM Ca2+). For activation, oocytes were transferred between electrodes covered by activation medium in a chamber connected to an electrical pulsing machine (FC-150; BLS Ltd., Budapest, Hungary). The method of electrical stimulation was dependent on the experiment. In all experiments, oocytes were first aligned by administration of 0.25 kV/cm AC for 5 sec. In study 1, oocytes were activated 44 h postmaturation by one 80-µsec pulse of 1.0 kV/cm DC. In study 2, the method of activation was varied according to oocyte age, voltage field strength, and pulse number and duration. In study 3, oocytes were activated by an optimized protocol established in the second study consisting of 3 consecutive 80-µsec pulses of 1.0 kV/cm DC applied at 44 h postmaturation. NCSU 23 containing 0.4% BSA [13] was used for culturing activated oocytes and embryos at 39°C in 5% CO2 in air. In all experiments, activated oocytes were immediately transferred into embryo culture medium supplemented with 7.5 µg/ml cytochalasin B (C-6762; Sigma) and cultured for 6 h. Embryos were then cultured in groups of 30–40 per 500 µl of culture medium under mineral oil (M-8410; Sigma) for 6 or 7 days.
Assessment of Meiotic Maturation and Nuclear Counts
Cumulus cells were removed from IVM oocytes by incubation for 10 min in Hepes-buffered NCSU 23 medium containing 0.4% BSA and 300 units/ml hyaluronidase (Sigma). Oocytes were then repeatedly pipetted to remove remaining cumulus cells. Groups of completely denuded oocytes (10–30) were mounted on cleaned glass slides, and a coverslip was attached using a mixture of solid paraffin (4%) and Vaseline (96%) applied to 2 edges of the slide. When observing the oocytes, the coverslip was gently pressed down until the oocytes were not able to roll but remained intact. Mounted slides were fixed and stained in a 3:1 methanol:acetic acid solution containing 1% orcein (O-7380; Sigma) for a minimum of 72 h before evaluation. Day 6 or Day 7 blastocysts were washed twice in Hepes-buffered NCSU 23 with 0.4% BSA and treated for 10–15 min in the same medium containing 5 µg/ml Hoechst 33342. After incubation, blastocysts were mounted and viewed under ultraviolet light or stored at 4°C until evaluated.
Study 1: Effects of EGF and AA in Maturation Medium
Oocytes were matured in base maturation medium alone (control) or in medium supplemented with 10 ng/ml EGF, with 1% essential and 0.5% nonessential AA, or with both EGF and AA. Development to the blastocyst stage 6 days after activation was then evaluated. This experimental design was repeated 5 times, and the total number of oocytes allocated to each group ranged from 231 to 263. To determine the timing of metaphase II arrest, oocytes were matured in base maturation medium supplemented with AA and were fixed at 0, 22, 36, 37, 38, 39, 40, 41, 42, and 43 h.
Study 2: Optimization of Electrical Activation Parameters
Experiment A: interaction of oocyte age at activation and applied voltage field strength Oocytes were activated by a single 80-µsec pulse of 1.0, 1.25, or 1.5 kV/cm DC applied 36, 40, 44, or 48 h postmaturation (4 x 3 factorial design). Activated oocytes were then cultured for 6 days. This design was repeated 3 times, and the total number of oocytes per treatment group ranged from 89 to 200.
Experiment B: interaction of applied voltage field strength and number of pulses Oocytes were activated at 44 h postmaturation by 1, 3, or 5 consecutive 80-µsec pulses of 1.0, 1.25, or 1.5 kV/cm DC (3 x 3 factorial design). This design was repeated 3 times with over 100 oocytes per treatment group.
Experiment C: interaction of pulse number and duration At 44 h postmaturation, oocytes were activated by 1 or 3 consecutive pulses of 1.0 kV/cm DC of 20, 40, 60, 80, 100, 120, 140, or 160 µsec duration (2 x 8 factorial design). Development to the blastocyst stage was evaluated 7 days after activation. This design was repeated 7 times with a total of 233–260 oocytes per treatment group.
Study 3: Karyotype of Parthenogenetic Blastocysts
Day 6 and Day 7 blastocysts produced by implementing strategies optimized in studies 1 and 2 were cultured in embryo culture medium containing 0.2 µg/ml colcemid (Invitrogen Ltd., Paisley, U.K.) for at least 6 h before being treated for 5 min in a hypotonic solution of 0.6% sodium citrate (S-4641; Sigma). Subsequently, blastocysts were placed individually in a marked frame in the middle of a freshly cleaned glass slide onto which fresh fixative composed of 3:1 methanol:acetic acid at room temperature was dropped. After slides were dry, they were stained in 5% Giemsa at pH 6.8 for 10–15 min. Slides were then washed with distilled water and dried in air at room temperature. The chromosome number of each spread was determined by observation under a microscope (Microphot-SA; Nikon, Tokyo, Japan). As a control, in vivo-fertilized and in vitro-cultured blastocysts were also karyotyped in the same way.
Statistical Analysis
Data from study 1 were analyzed using a Student t-test. Data from experiments A and B of study 2 were analyzed as binomial proportions on the logit scale. Oocyte age (experiment A only), pulse voltage, pulse number (experiment B only), replicate trials, and all 2-factor interactions were fitted as explanatory variables. The dependent variable was the proportion of oocytes reaching the blastocyst stage. The data from experiment C of study 2 were analyzed as percentage blastocyst rates. Fourier series of the form y = A + B sin {2
(x - E)/W} were fitted to the results for the groups receiving either 1 or 3 pulses, where x is the duration of the pulse(s) and y is the percentage of oocytes developing to the blastocyst stage. Nested models, incorporating both common and separate values for the parameters y, A, B, and W, were fitted to the 2 groups. The fit of the models was assessed by analysis of deviance. The estimators of the peak blastocyst rate and the associated pulse duration are A + B and (E + W)/4. A chi-square test was used to analyze the data in study 3.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Maturation medium containing AA alone gave the highest blastocyst rate of 32% (n = 249 oocytes). This value was significantly higher than that for the medium containing EGF alone (20%, n = 263) or for the medium with EGF and AA (26%, n = 231; P < 0.05, t-test) (Table 1). The results for the latter 2 treatments did not differ from those of the control group lacking both EGF and AA (26%, n = 233; P > 0.05). No differences across the treatments were observed in the mean number of nuclei/blastocyst. Based on these results, maturation medium containing AA alone was selected as the standard for all subsequent experiments. Characterization of meiotic maturation in this medium revealed that at the start of maturation 82% of oocytes (n = 50) were at the GV (germinal vesicle) stage, whereas 68% of oocytes (n = 44) reached the MI stage at 22 h of maturation. The percentage of MII oocytes was 75% at 36 h (n = 36), reaching over 90% by 37–43 h (n = 34, 35, 43, 37, 54, 58, and 45 at each hourly time point, respectively; Fig. 1).
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Study 2: Optimization of Electrical Activation Parameters
Three experiments were carried out to optimize electrical activation parameters, specifically with respect to oocyte age at activation, applied voltage field strength, and pulse number and duration. In the first experiment, 3 different voltage field strengths (1.0, 1.25, 1.5 kV/cm DC) were applied as a single pulse of 80 µsec duration to oocytes at 36–48 h postmaturation. The percentage of oocytes forming blastocysts at 36, 40, 44, and 48 h was 15% (n = 110), 19% (n = 180), 29% (n = 148), and 32% (n = 167), respectively, for 1.0 kV/cm DC; 21% (n = 86), 20% (n = 189), 35% (n = 200), and 18% (n = 172), respectively, for 1.25 kV/cm DC; and 17% (n = 98), 27% (n = 157), 41% (n = 163), and 20% (n = 119), respectively, for 1.5 kV/cm DC. There was no significant effect of voltage field strength on blastocyst development (P > 0.05). In addition, there were no differences in the mean number of nuclei/blastocyst across voltage field strength and oocyte age combinations (P > 0.05). However, there was a significant effect of oocyte age on blastocyst development (P < 0.05), which took the form of a curvilinear relationship peaking at 44 h postmaturation for 1.25 and 1.5 kV/cm (Fig. 2).
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In the second experiment, the interaction of applied voltage field strength with the number of consecutive pulses (1, 3, or 5) was examined in oocytes activated at 44 h postmaturation, with pulse duration set at 80 µsec. Mean blastocyst rates obtained with 1.0, 1.25, and 1.5 kV/cm voltage strengths were 32% (n = 135), 37% (n = 128), and 34% (n = 135), respectively, for a single pulse; 55% (n = 138), 26% (n = 114), and 35% (n = 115), respectively, for 3 pulses; and 41% (n = 166), 27% (n = 103), and 21% (n = 159), respectively, for 5 pulses. Overall, the relationship between blastocyst yields and pulse number was quadratic for 1.0 kV/cm DC and approximately linear for 1.25 and 1.5 kV/cm (Fig. 3). At higher field strengths (1.25 and 1.5 kV/cm), multiple pulses (3 or 5) were generally inferior to a single pulse for blastocyst yield. However, the blastocyst rate obtained with 3 consecutive pulses of 1.0 kV/cm (55%) was significantly higher than that of all of the other groups (P < 0.05). No differences across the treatments were observed in the mean number of nuclei/blastocyst (P > 0.05; data not shown).
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In the third experiment, a voltage field strength of 1.0 kV/cm DC was used to compare blastocyst development following activation with 1 or 3 consecutive pulses of different pulse duration (i.e., 20, 40, 60, 80, 100, 120, 140, and 160 µsec). Data from 7 replicate experiments were variable. However, the large numbers of oocytes sampled in this study (233–260/treatment group) was sufficient to calculate the overall pattern of the results by fitting Fourier (sine) series curves with separate coefficients for the 2 different numbers of pulses applied (Fig. 4). Blastocyst development was generally superior using 3 pulses, with a predicted optimal pulse duration of 65 µsec associated with a mean blastocyst rate of 35%. In contrast, the predicted optimal duration for 1 pulse was 114 µsec, with a mean blastocyst rate of 28%. Differences in the location and size of the peaks for the 2 numbers of pulses were significant (P < 0.05). The observed optimum pulse duration for 1 and 3 pulses was 100 and 80 µsec, with blastocyst rates of 33% and 36%, respectively. These values were within 1 SD of predicted optimal pulse duration and blastocyst proportions (data not shown).
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Study 3: Karyotype of Parthenogenetic Blastocysts
Day 6 and Day 7 blastocysts were produced by activation with 3 consecutive 80-µsec pulses of 1.0 kV/cm DC applied to oocytes matured for 44 h in vitro (Fig. 5). A total of 130 blastocysts were karyotyped, although only 73 had 1–9 cells with spread chromosomes. Of these 73, 61 (84%) were diploid (2N), 7 (9%) were tetraploid (4N), and 5 (7%) were of mixed ploidy. Nine of 21 in vivo-fertilized and in vitro-cultured blastocysts had spread chromosomes, all of which were diploid. There was no significant difference between the 2 groups in the percentage of diploid blastocysts (P > 0.05, 
2 test; Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effect of Oocyte Age
The developmental competence of electrically activated oocytes was dependent on the age of oocytes at activation and peaked at 44 h of maturation, 7 h after 90% of IVM oocytes had arrested at the MII stage. An optimum window of time to activate oocytes for development may depend on both the time required for completion of meiotic and cytoplasmic maturation and the time by which mature oocytes begin to deteriorate, both of which may differ among species. Cytoplasmic maturation is likely to include changes in the properties, size, and density of cytoplasmic Ca2+ channels necessary for an oocyte to elicit Ca2+ oscillations in response to an activation stimulus and subsequent development [12]. Conversely, oocyte deterioration may include diminished capacity to maintain meiotic arrest at MII of meiosis, reflected by an increased likelihood of spontaneous activation. In IVM pig oocytes, both oocyte aging and ability to be parthenogenetically activated is correlated with a gradual decrease in histone H1 kinase activity required for maintenance of chromatin condensation at MII [4]. Although an age-dependent activation response has been described previously in several species, including the mouse [14], cow [15, 16], and pig [17, 18], the focus of such studies was on pronuclear formation and not blastocyst development. However, our results for the pig are distinct from those for cattle, in which an increase in developmental competence in oocytes between 24 and 29 h of activation has been reported [19].
Interaction of Voltage Field Strength and Pulse Number
Our results indicated that better development could be obtained using multiple pulses only when they were combined with lower field strength. Multiple pulses of electrical stimulation are beneficial to oocyte activation and the developmental competence of activated oocytes in several species, including the mouse [20], rabbit [21, 22], cow [23], and pig [24]. However, in those studies, pulses were spaced by intervals of several minutes up to 30 min to mimic the Ca2+ oscillations that occur during fertilization. Because pulses in our study were administered consecutively, the mechanism responsible for the benefit we observed was likely different. One possibility is that the combination of multiple pulses of lower field strength would be less damaging to the oocyte plasma membrane than would higher field strengths applied as a single pulse. Parthenogenetic fetuses have recently been described following stimulation of oocytes with a single pulse of higher field strength (1.5 kV/cm DC), and more degenerate oocytes (4–15%) were observed in that study [25] than in our study, in which application of multiple pulses of 1 kV/cm infrequently resulted in oocyte loss (data not shown). A single high-field strength (1.5 kV/cm DC) electrical pulse was recently used by Onishi et al. [2] in a somatic cell pig cloning experiment. Although 31% of ovulated oocytes activated by this method developed to the blastocyst stage, less than 3% of IVM oocytes did so when using the same method. In contrast, our method resulted in over 40% of IVM oocytes developing to the blastocyst stage.
Interaction of Pulse Number and Pulse Duration
For electrical activation of pig oocytes, pulse number and duration have interactive effects on blastocyst development. With multiple pulses, the pulse duration required for optimal development is shortened. These findings are consistent with the first report describing the cloning of a pig from embryonic blastomeres, wherein 2 pulses of 1.25 kV/cm and 30–60 µsec were used [26]. Previously, in studies focusing on rabbit oocyte activation pulse duration was suggested to be less important than oocyte age, voltage field strength, and pulse number in determining optimal developmental response [11]. Although it is difficult to calculate the relative impact of multiple parameters, our results suggest that the effect of pulse duration cannot be overlooked.
Effect of AA and EGF
The addition of AA to the maturation medium promoted development of parthenogenetically activated porcine oocytes to the blastocyst stage, whereas addition of EGF had no positive effect. Both EGF and essential and nonessential AA improve nuclear maturation and male pronuclear formation following fertilization [27]. However, during in vitro maturation of pig oocytes the addition of AA has been suggested to result in nondisjunction of chromosomes, leading to aneuploidy in fertilized embryos [28]. The metabolic requirements of parthenogenetically activated oocytes and embryos differ from those of fertilized embryos [29–33], which may account for our observation of a differential effect of EGF + AA versus AA alone on subsequent development. Differential embryo metabolism may in turn stem from differences in mechanisms of oocyte activation. Parthenogenetic blastocysts derived from oocytes matured in the presence of amino acids in our study were karyotypically normal. Thus, at least for oocytes activated electrically, there was no significant evidence of chromosome nondisjunction.
In summary, factors affecting the electrical activation of pig oocytes were assessed by evaluating development to the blastocyst stage. In vitro maturation in the presence of AA yielded karyotypically normal parthenogenetic blastocysts, and optimal development was obtained by activation during a defined window of oocyte age using multiple electrical pulses. The effect of pulse number was interactive with both voltage field strength and pulse duration. These results should contribute to improved protocols for oocyte activation such as would be required for animal cloning by nuclear transfer. In our experiments, an optimized protocol consisting of 3 consecutive 80-µsec pulses of 1.0 kV/cm DC applied during a set time in maturation has subsequently been used to produce parthenogenetic fetuses for pregnancy maintenance and to clone embryos by somatic cell nuclear transfer [34].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 These experiments were funded by Roslin Bio-Med and subsequently by Geron Bio-Med. 
2 Correspondence. FAX: 44 0131 527 4493; ian.wilmut{at}bbsrc.ac.uk
Accepted: October 22, 2001.
Received: May 31, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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