Induction and activation of meiosis and subsequent parthenogenetic development of growing pig oocytes using calcium ionophore A23187

Theriogenology 60 (2003) 1609–1620

Induction and activation of meiosis and subsequent parthenogenetic development of growing pig oocytes using calcium ionophore A23187 Marke´ta Sedmı´kova´a,*, Jana Burdova´b, Jaroslav Petrc, Milan Etrycha, Jirˇ´ı Rozineka, Frantisˇek Jı´leka a

Department of Veterinary Science, Faculty of Agronomy, Czech University of Agriculture Prague, Prague, 6-Suchdol, 165 21, Czech Republic b Natural Science Faculty, Charles University in Prague, Prague 1, Czech Republic c Research Institute for Animal Production, 10-Uhrineves, Prague, Czech Republic Received 12 February 2002; accepted 13 November 2002

Abstract The pig ovary contains a large number of growing oocytes, which do not mature in vitro and cannot be readily used in various biotechnologies. This study was conducted to determine the possibility of inducing meiotic maturation in growing pig oocytes with an internal diameter of 110 mm, which had developed partial meiotic competence. Most of these oocytes spontaneously stopped maturation at the metaphase I stage (68%); a limited number proceeded to the metaphase II stage (26%). Treatment with calcium ionophore A23187 (50 mM for 5 or 10 min) after 24 h in vitro culture overcame the block at the metaphase I stage, and treated growing pig oocytes matured to the metaphase II stage (66%). Oocytes in which maturation had been induced by calcium ionophore were again treated with calcium ionophore. Up to 58% of the treated oocytes were activated. Parthenogenetic development in oocytes treated with ionophore for meiosis induction and activation was very limited. The portion which reached morula stage did not exceed 8% and at most 3% developed to the blastocyst stage. # 2003 Published by Elsevier Inc. Keywords: Pig oocyte; Meiotic competence; Parthenogenesis; Calcium ionophore

1. Introduction Meiotic maturation of oocytes starts during the fetal development of mammalian females, but is arrested at the late diplotene stage. The ability to resume meiosis and * Corresponding author. Tel.: þ420-224382933; fax: þ420-234381841. E-mail address: [email protected] (M. Sedmı´kova´).

0093-691X/$ – see front matter # 2003 Published by Elsevier Inc. doi:10.1016/S0093-691X(03)00079-7

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to continue in maturation beyond this stage is acquired gradually during the subsequent period of oocyte growth. In fully grown oocytes, meiosis continues after germinal vesicle breakdown (GVBD) through the stages of metaphase I, anaphase I and telophase I to the stage of metaphase II, when meiosis is again arrested. The ability to pass through all these stages is designated as full meiotic competence. During the growth period, oocytes pass through a stage in which meiotic competence is only partially developed [1]. These oocytes acquire the ability to undergo GVBD and to enter metaphase I stage. However, they are unable to exit from metaphase I stage, to reach metaphase II stage, and to complete meiotic maturation [2–5]. Growing pig oocytes enter this phase on reaching an internal diameter of about 110 mm [2,3,6,7]. Present methods for in vitro embryo production and cloning using nuclear transfer depend on the use of oocytes with full meiotic competence, which are present in the ovary in limited numbers. Numerous populations of follicles with growing oocytes that have partially developed meiotic competence cannot be used for these purposes. However, embryos produced from these oocytes could be used for pig breeding, production of cloned or transgenic pigs, or for preservation of endangered breeds. To this end, culture systems for in vitro growth and acquisition of full meiotic competence of pig oocytes have been developed [8,9]. However, the processes involved in the acquisition of full meiotic competence are not fully understood. In our previous work [7], we demonstrated that drugs elevating intracellular calcium levels can overcome the meiotic block in oocytes with partially developed meiotic competence and can induce their maturation to the metaphase II stage. To achieve these results we used cyclopiazonic acid, the inhibitor of calcium-dependent ATPases, which elevates intracellular levels of free calcium ions through the mobilization of intracellular calcium deposits [10,11]. The aim of this study was to determine the possibility of inducing meiosis in pig oocytes with partially developed meiotic competence using calcium ionophore A23187, which is known to bring calcium ions into the cells from extracellular sources. Another aim of the study was to verify the quality of oocytes matured after this treatment according to their activation rate and the rate of parthenogenetic development after activation using a second treatment with calcium ionophore A23187.

2. Materials and methods 2.1. Isolation, culture, and evaluation of oocytes Pig ovaries were obtained from a local slaughterhouse from gilts at an unknown stage of the estrous cycle and transported to the laboratory within 1 h in a saline solution (0.9% of natrium chloride) at 39 8C. Fully grown oocytes were collected from follicles 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 maturation culture medium. Growing oocytes of different size categories were obtained from thin strips (10–15 mm long, 1–2 mm wide) dissected from the surface of the ovaries using a scalpel. The strips of

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ovarian tissue were placed in Petri dishes containing a culture medium. The oocytes were released from their follicles by opening the follicular wall using the tip of a 25-gauge needle. The internal diameter of the oocytes (without zona pellucida) was measured with an ocular micrometer mounted on a microscope. Only oocytes surrounded by several layers of cumulus cells were chosen for further experiments. The numbers of fully grown or growing oocytes collected per ovary varied widely, although the number of good quality, fully grown oocytes usually did not exceed 8 or 10 per ovary. We did not try to harvest all growing oocytes from the ovary, but we were able to collect several dozen growing oocytes per ovary, with 20–30 oocytes with an internal diameter of 110 mm. Before culture the oocytes were washed three times in the culture medium. The oocytes were cultured in a modified M199 medium (GibcoBRL, Life Technologies, Paisley, Scotland) containing sodium bicarbonate (0.039 ml of a 7% solution per milliliter of medium), calcium lactate (0.6 mg/ml), sodium pyruvate (0.25 mg/ml), gentamicin (0.025 mg/ml), HEPES (1.5 mg/ml), 13.5 IU eCG:6.6 IU hCG/ml (P.G.600 Intervet, Boxmeer, Holland) and 10% fetal calf serum (GibcoBRL, Life Technologies, Karlsruhe, Germany, Lot No. 40F2190F). The oocytes were cultured in 3.5 cm diameter Petri dishes (Nunc, Roskilde, Denmark) containing 3 ml of the culture medium at 39 8C in a mixture of 5% CO2 in air. At the end of the culture period, the oocytes were relieved of cumulus cells by repeated pipetting through a narrow glass pipette, mounted on slides, fixed with acetic alcohol (1:3, v:v) for at least 24 h and stained with 1% orcein. The oocytes were examined under a phase contrast microscope. The stages of maturation were determined according to the criteria published by Motlı´k and Fulka [2]. 2.2. Culture and evaluation of parthenogenetic embryos Activated oocytes were examined after aceto-orcein staining under a phase contrast microscope. Activation was considered to occur if the oocytes were in the pronuclear stage and a polar body was visible. We observed oocytes either with two pronuclei and with one polar body extruded, or oocytes with one pronucleus and with two polar bodies extruded. Rarely (2%) we observed oocytes with one pronuclear structure and one polar body, which could represent syngamy of two different pronuclei. Oocytes with any different configurations of chromatin were not considered as activated. Activated oocytes were cultured in a modified M199 medium (see above) without hormonal supplementation (i.e. without P.G.600) in 4-well Petri dishes (Nunc, Roskilde, Denmark), each well containing 1 ml of culture medium. The oocytes were cultured at 39 8C in a mixture of 5% CO2 in air for 7 days. The number of cells in parthenogenetic embryos was determined after mounting the embryos on slides, fixation with acetic alcohol for at least 24 h, and staining with 1% orcein. 2.3. Experimental design In Experiment 1, we investigated the meiotic competence of porcine oocytes with different internal diameters. Fully grown oocytes (internal diameter of 120 mm) or growing

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oocytes (internal diameter of 80, 90, 100 or 110 mm) were cultured in vitro for 48 h and their maturation was evaluated at the end of the culture. Oocytes with an internal diameter of 110 mm were chosen for further experiments. Experiment 2 was conducted to investigate the possibility of stimulating meiotic maturation in growing pig oocytes with partially developed meiotic competence. Growing oocytes (internal oocyte diameter 110 mm) were cultured in vitro for 24 h. They were then relieved of cumulus cells by repeated pipetting through a narrow glass pipette and exposed to calcium ionophore A23187 (Sigma–Aldrich, St. Louis, MO, USA) (10, 25 or 50 mM) for 2, 5 or 10 min. Calcium ionophore was dissolved in DMSO and diluted to a final concentration in a culture medium not supplemented with serum. The concentration of DMSO in the culture medium never exceeded 1%. Subsequently, the oocytes were carefully washed in an ionophore-free medium with serum and then cultured for another 24 h. Control oocytes were exposed to an ionophore-free medium containing the same amount of DMSO for the same time. Our preliminary experiments showed that exposure of the oocytes to a serum-free culture medium supplemented with DMSO had no significant effect when compared with the maturation of oocytes cultured without this intervention. In Experiment 3, we investigated the possibility of parthenogenetically activating the oocytes in which meiosis had been stimulated under the conditions described in the previous experiment. Growing oocytes (internal oocyte diameter 110 mm) were cultured for 24 h and then exposed to calcium ionophore under conditions identical to those in Experiment 2. In Experiment 3, we stimulated meiosis with calcium ionophore (25 or 50 mM) for 5 or 10 min. After careful washing, the oocytes were cultured for another 24 h and exposed to a second ionophore treatment (25 or 50 mM) for 10 min. After washing, the oocytes were cultured for another 24 h in an ionophore-free medium. In control experiments for Experiment 3, we cultured growing oocytes for 72 h without ionophore treatment (i.e. without stimulation of meiosis or parthenogenetic activation) to evaluate the number of spontaneously activated oocytes. To estimate the rate of spontaneous activation in oocytes after stimulation of meiosis, we treated the oocytes with ionophore (50 mM for 10 min) after 24 h of in vitro culture and then cultured them for 48 h without any further ionophore treatment. We also examined the activation rate in the oocytes that were matured in vitro without meiosis stimulation. For this purpose, we cultured the oocytes for 48 h, and subsequently treated them with calcium ionophore (25 or 50 mM for 10 min) and then cultured them for another 24 h. We compared the activation rate of growing oocytes after meiosis stimulation with the activation rate of fully grown oocytes matured spontaneously in vitro and treated with calcium ionophore (25 or 50 mM for 10 min). In Experiment 4, we investigated the parthenogenetic development of growing oocytes with partially developed meiotic competence after meiosis stimulation and subsequent activation. The oocytes were cultured for 24 h in vitro under conditions identical to those in Experiment 2. After another 24 h of in vitro culture, the oocytes were activated under conditions identical to those in Experiment 3. For meiosis stimulation, we used calcium ionophore at a concentration of 25 or 50 mM for 5 or 10 min. For activation, we used calcium ionophore at concentrations of 25 or 50 mM. This treatment lasted for 10 min. After activation the oocytes were cultured for 7 days as above.

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We also compared the development of growing oocytes after meiosis stimulation and activation with the development of fully grown oocytes matured spontaneously (without stimulation) in vitro and treated with calcium ionophore (25 or 50 mM for 10 min). 2.4. Statistical analysis Data from all experiments were subjected to statistical analysis. Each experiment was carried out four times. The results were pooled and evaluated by chi-square analysis [12]. The mean percentage of oocytes or embryos reaching the given stage of maturation or development in all experiments 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 3.1. Experiment 1: verification of meiotic competence in growing pig oocytes during their in vitro culture Fully grown oocytes (internal diameter 120 mm) matured to the metaphase II stage (92%); only 2% of these oocytes remained at the germinal vesicle stage. The remaining oocytes (6%) were observed at stages from anaphase I to telophase I (mainly telophase I). The growing oocytes did not demonstrate full meiotic competence. When growing oocytes with an internal diameter of 110 mm were cultured in vitro for 48 h, only 26% of the oocytes reached the metaphase II stage; the majority (68%) were arrested at the metaphase I stage, and 6% remained at the germinal vesicle stage. Only 39% of oocytes with an internal diameter of 100 mm underwent germinal vesicle breakdown and no oocytes reached the metaphase II stage. Similarly, only 19% of oocytes with an internal diameter of 90 mm underwent germinal vesicle breakdown and no oocyte in this size category was observed at the stage of metaphase II after 48 h in vitro culture. Under our culture conditions, oocytes with an internal diameter of less than 80 mm were unable to undergo germinal vesicle breakdown during 48 h in vitro culture. For further experiments we used oocytes with an internal diameter of 110 mm, which have limited ability to complete meiotic maturation up to the metaphase II stage; maturation stopped spontaneously at metaphase I stage in the majority of these oocytes. 3.2. Experiment 2: the effect of calcium ionophore A23187 on meiotic maturation in growing oocytes Ionophore treatment of growing oocytes (internal diameter 110 mm) with partially developed meiotic competence, after 24 h in vitro culture and subsequent in vitro culture for another 24 h, resulted in a significant increase in the number of oocytes maturing to the metaphase II stage. The most important data are shown in Table 1. Under our culture conditions we observed the highest percentage of oocytes at the metaphase II stage after ionophore treatment with 25 or 50 mM for 5 or 10 min. These treatments were used for meiosis stimulation in further experiments.

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Table 1 Effects of ionophore A23187 on the maturation of growing pig oocytes with partially developed meiotic competence Oocytes (n)

120 120 120 120 120 120

Stimulation treatment

Stage of meiotic maturation reached after stimulation (%)

Concentration of ionophore (mM)

Duration of treatment (min)

GV

Metaphase I

Metaphase II

0 10 25 25 50 50

0 10 5 10 5 10

6a 9a 3a 3a 4a 4a

68a 60a 40b,c 40b,c 30c 30c

26a 30a 57b 57b 66b 66b

Statistically significant (P < 0:05) differences in the rates of respective stages of meiotic maturation (within columns) between different treatments (between different rows) are indicated by different superscripts. Oocytes (internal diameter of 110 mm) were cultured in vitro for 24 h, then treated with ionophore and subsequently cultured for another 24 h.

3.3. Experiment 3: parthenogenetic activation of oocytes in which maturation was stimulated using calcium ionophore We were able to induce parthenogenetic activation in oocytes in which meiosis had been stimulated after 24 h of in vitro culture (see Table 2). Activation with 25 mM ionophore for 10 min resulted in the highest activation rate in oocytes in which meiosis had been previously stimulated with 25 mM ionophore for 5 min (activation rate 58%) or with 50 mM ionophore for 10 min (activation rate 42%). Activation with 50 mM (10 min) resulted in the highest activation rate after meiosis stimulation with 50 mM ionophore for 5 min (activation rate 43%) or with 25 mM ionophore for 10 min (activation rate 52%). These treatments were used in further experiments. The pronuclear configuration did not differ Table 2 Parthenogenetic activation of growing pig oocytes after stimulation of meiosis Oocytes (n)

120 120 120 120 120

Stimulation treatment

Activation treatment

Activation rate (%)

Concentration of ionophore (mM)

Duration of treatment (min)

Concentration of ionophore (mM)

Duration of treatment (min)

0 25 25 50 50

0 5 10 5 10

50 25 50 50 25

10 10 10 10 10

7a 58c 52b,c 43b 42b

Statistically significant (P < 0:05) differences in the activation rates between different treatments (between different rows) are indicated by different superscripts. Oocytes were cultured for 24 h in vitro, subsequently exposed to calcium ionophore (stimulation treatment), and cultured in vitro for another 24 h. Oocytes were then exposed to a second ionophore treatment (activation treatment) and cultured for 24 h in vitro.

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between treatments. We observed 76% of activated oocytes with two pronuclei and one polar body and 22% of activated oocytes with one pronucleus and two polar bodies. Only 2% of activated oocytes exhibited one pronucleus and one polar body, which could represent syngamy of two pronuclei. Activation of growing oocytes without previous stimulation of meiosis yielded very low numbers of activated oocytes; this probably reflects the very small portion of oocytes able to mature to the metaphase II stage without ionophore treatment. We did not observe any activation in growing oocytes cultured for 72 h without ionophore treatment. Sixty-three percent of fully grown oocytes (i.e. oocytes with fully developed meiotic competence) were activated when treated with calcium ionophore (25 mM for 10 min) after 48 h of in vitro culture and subsequent 24 h culture. This is similar to the activation rate in growing oocytes in which meiosis had been stimulated with 25 mM ionophore for 5 min. The meiosis stimulation used before the most effective activation treatment induced maturation to the stage of metaphase II in 57% of growing oocytes. Therefore, we can speculate that almost all the oocytes with successfully induced meiosis were activated. When in vitro matured, fully grown oocytes were activated with 50 mM ionophore for 10 min, we observed an activation rate of 74%. This is a significantly higher activation rate than in growing oocytes activated after meiosis stimulation. The meiosis stimulation used before the most effective activation treatment induced maturation to the metaphase II stage in 66% of oocytes, and we can speculate that about 80% of the oocytes stimulated to mature to the metaphase II stage were successfully activated. 3.4. Experiment 4: parthenogenetic development of growing oocytes activated after meiosis stimulation with calcium ionophore In control experiments, a limited portion of fully grown oocytes (internal diameter of 120 mm) treated with 25 or 50 mM ionophore for 10 min was able to develop to the stage of morula or blastocyst. Very poor parthenogenetic development (only to the 4-cell stage) was observed in growing oocytes with partially developed meiotic competence after in vitro culture for 48 h (without the stimulation treatment) and subsequent activation treatment with ionophore. This low percentage of developing oocytes reflects a low percentage of growing oocytes maturing to the metaphase II stage (see Table 3). Table 3 Parthenogenetic development of fully grown pig oocytes with full meiotic competence Oocytes (n) Activation treatment

100 100

Stage of parthenogenetic development (%)

Concentration of ionophore (mM)

Duration of treatment (min)

2 cells

3–4 cells

5–8 cells

9–16 cells

Morula

Blastocyst

25 50

10 10

16a 27a

32a 27a

16a 4a

8a 6a

0a 8a

0a 3a

Statistically significant (P < 0:05) differences in the rates of respective stages of development (within columns) between different treatments (different rows) are indicated by different superscripts. Fully grown oocytes were cultured in vitro for 48 h treated with calcium ionophore (activation treatment) and then cultured in vitro for 7 days.

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Table 4 Parthenogenetic development of growing pig oocytes with partially developed meiotic competence Oocytes (n) Stimulation treatment Activation treatment

Stage of parthenogenetic development (%)

Ionophore Duration Ionophore Duration 2 cells (mM) (min) (mM) (min) 100 100 100

0 25 50

0 10 10

10 10 10

50 50 25

18a 8a 18a

3–4 cells

5–8 cells

9–16 Morula Blastocyst cells

8a 15a 18a

2a 8a 0a

0a 11a 0a

0a 8a 0a

0a 3a 0a

Statistically significant (P < 0:05) differences in the rates of respective stages of development (within columns) between different treatments (different rows) are indicated by different superscripts. Growing oocytes with partially developed meiotic competence were cultured in vitro for 24 h, exposed to calcium ionophore (stimulation treatment), cultured in vitro for another 24 h, treated with ionophore for a second time (activation treatment) and then cultured in vitro for 7 days.

Growing oocytes with partially developed meiotic competence, stimulated for maturation with ionophore after 24 h culture and exposed to activation treatment after another 24 h culture, also exhibited very low parthenogenetic development (see Table 4). However, stimulation treatment with 25 mM ionophore for 10 min followed by activation treatment with 50 mM ionophore for 10 min induced parthenogenetic development in 53% of the oocytes, with 8% of them reaching the morula stage and 3% reaching the blastocyst stage. These developmental rates are similar to the rates observed after activation of fully grown oocytes.

4. Discussion In the present study, we demonstrated the possibility of inducing meiosis in growing pig oocytes with partially developed meiotic competence. Oocytes treated with ionophore after 24 h culture in vitro were able to reach metaphase II stage when cultured in vitro for another 24 h after ionophore treatment. Oocytes which completed meiotic maturation after this stimulation treatment were activated and parthenogenetic development was observed. In our study, we confirmed previous observations on the gradual acquisition of meiotic competence during the growth period of porcine oocytes [2,6]. We used growing pig oocytes which developed only partial meiotic competence. These oocytes were able to resume meiosis, but the majority did not reach metaphase II stage, and maturation was stopped at the metaphase I stage. It is known that this spontaneous blocking of meiosis is induced by a permanent increase in maturation promoting factor (MPF). Oocytes blocked at metaphase I stage are unable to decrease the activity of histone H1 kinase as maturing fully grown oocytes with full meiotic competence do during their progression from metaphase I to metaphase II [13]. Fully grown oocytes with full meiotic competence spontaneously stop meiotic maturation at the metaphase II stage, and their exit from the metaphase II stage involves calcium-dependent events [1]. In further experiments [7], we proved that growing oocytes exhibited similar calcium-dependent exit from arrest at the stage of metaphase I.

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To realize the first aim of the study, i.e. induction of meiosis, the growing pig oocytes were treated with calcium ionophore A23187, and exit from the spontaneous arrest of meiosis at the stage of metaphase I was stimulated. This treatment was able to induce meiosis in up to 66% of growing oocytes, which reached the stage of metaphase II. This is significantly higher (P < 0:05) than in growing oocytes maturing without ionophore treatment (26% in metaphase II) but significantly lower than in fully grown oocytes maturing spontaneously to metaphase II (92% in M II). Calcium ionophore A23187 is known to elevate intracellular calcium levels in the cytoplasm of pig oocytes through the influx of calcium from extracellular spaces [14,15]. Therefore, the observed meiosis stimulation is in agreement with the results of our previous study [7], which demonstrated a similar calcium-dependent mechanism for exit from the meiotic metaphase in growing pig oocytes with partially developed meiotic competence, in which maturation is spontaneously arrested at the metaphase I stage. However, elevation of the intracellular pH in the cytoplasm of fully grown pig oocytes matured to the metaphase II stage by calcium ionophore A23187 has also been described [16–18]. Unfortunately, we do not know to what extent calcium ionophore A23187 influences intracellular pH in growing pig oocytes with partially developed meiotic competence after spontaneous maturation block at metaphase I. We can assume that the observed effect of A23187 on exit from the spontaneous arrest of meiosis at the metaphase I stage in growing oocytes with partially developed meiotic competence is due to both the calcium-dependent processes and the increase of intracellular pH in the oocyte cytoplasm. All these processes are able to lower the activity of MPF, which is necessary for transition from metaphase I to metaphase II stage in mammalian oocytes [13], and which was observed during spontaneous meiotic maturation in fully grown pig oocytes with full meiotic competence [19]. Another aim of our study was to verify the quality of matured oocytes resulting from the stimulation treatment. This was performed using an activation treatment with ionophore. Up to 58% of growing oocytes were activated by 25 mM ionophore treatment. This is similar to the activation rate in fully grown oocytes activated under the same conditions (activation rate 63%). Activation with 50 mM ionophore resulted in a maximum 43% activation rate in growing oocytes. This is significantly lower than the activation rate in fully grown oocytes activated under the same conditions (activation rate 74%), which in turn was similar to the activation rate of in vitro matured fully grown oocytes activated using calcium ionophore A23187 as described by other authors [18]. This could indicate a higher sensitivity of growing oocytes to the ionophore activation treatment. In some experimental groups, the activation rate in growing oocytes was very close to that of oocytes reaching metaphase II stage after meiosis stimulation. For example, stimulation with 25 mM of ionophore for 5 min resulted in 57% of growing oocytes matured to the stage of metaphase II, and the activation treatment of these oocytes with 25 mM of ionophore for 10 min resulted in a 58% activation rate. This suggests that almost all oocytes stimulated to complete meiotic maturation had been activated. It could indicate that growing oocytes forced to complete maturation are very sensitive to activation using ionophore treatment, perhaps due to lower levels of MPF or lower stability of MPF. On the other hand, it is possible that activation had also been induced in oocytes that did not complete meiotic maturation at the metaphase II stage. Fully grown pig oocytes can even

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be activated very early after meiosis resumption, including in the germinal vesicle stage [20]. However, the growing oocytes activated in our study exhibited the extruded polar body, which was not seen in fully grown pig oocytes activated early after meiosis resumption [20]. Moreover, in our previous studies [21], fully grown oocytes with full meiotic competence exhibited meiosis accelerated exit from metaphase I and a very fast entry into the metaphase II stage, but showed no activation after treatment with a drug elevating intracellular calcium levels at the metaphase I stage. Kikuchi et al. [22] described the presence of one pronucleus and the extrusion of one polar body after the activation of MI-arrested, fully grown pig oocytes. On the other hand, induction of interphase-like chromatin configuration in maturing pig oocytes at the stage of metaphase I is not accompanied by extrusion of a polar body [23–25]. In our study, we observed only 2% of activated oocytes with one pronucleus and one polar body; some of these could result from the syngamy of two pronuclei. On the basis of these data, we suggest that activated growing oocytes were mainly recruited from those forced to reach the stage of metaphase II by the first (stimulating) ionophore treatment. To further verify the quality of matured oocytes resulting from stimulation treatment, we evaluated their parthenogenetic division after activation treatment. We observed a very limited percentage of oocytes reaching the morula or blastocyst stage after the activation of fully grown oocytes with full meiotic competence using ionophore. Similarly, we observed limited parthenogenetic development in growing oocytes with partially developed meiotic competence after meiosis stimulation and activation with ionophore. The percentage of growing oocytes which divided was very low, and development was usually blocked at very early stages. This could be due to insufficient activation treatment. Wang et al. [18] described lower parthenogenetic development in fully grown oocytes activated using ionophore when compared with activation using boar sperm or an electrical stimulus. The low rate of division in growing oocytes could also be due to their lower developmental status because pig oocytes with partially developed meiotic competence also have lower competence for embryonic development after in vitro fertilization [26,27]. The division of activated oocytes can be negatively affected by culture in a modified M199 culture medium. It has been suggested that this medium could have an adverse effect on the development of the pig embryo [28,29], and special culture media were formulated for in vitro embryonic development in this species [30–32]. However, Kure-bayashi et al. [33] reported successful development of parthenogenetic pig embryos up to the blastocyst stage in M199 medium. Under our culture conditions, NCSU is not significantly superior to the M199 medium, and we use the M199 medium for the culture of parthenogenetic embryos [34,35]. In our study, one combination of stimulation and activation treatment of growing oocytes resulted in limited development to the morula and blastocysts stages. The percentage of oocytes reaching these stages was similar to the percentage of growing oocytes which are capable of completing meiosis and maturing to the metaphase II stage even without meiosis stimulation (i.e. without ionophore treatment after 24 h of culture). Therefore, it is impossible to distinguish whether the morula or blastocysts developed from oocytes whose exit from arrest at the metaphase I stage was induced by ionophore treatment, or from oocytes which were able to mature spontaneously without ionophore treatment. Based on our results, we can conclude that growing pig oocytes with partially developed meiotic competence can overcome the spontaneous arrest of meiosis after treatment with

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calcium ionophore A23187. These oocytes are capable of completing meiotic maturation by reaching the metaphase II stage and, after subsequent activation treatment, undergoing parthenogenetic division. The number of fully grown oocytes which can be harvested from an individual sow is limited, and the possibility of using oocytes with partially developed meiotic competence may be welcomed by those who wish to take further advantage of the genotype of individual excellent sows by in vitro fertilization, or wish to have a rich source of porcine oocytes for nuclear transfer. Further studies, however, especially experiments dealing with in vitro fertilization of growing oocytes stimulated to maturity, are needed for a more thorough evaluation of the quality of growing pig oocytes after meiosis stimulation.

Acknowledgements We thank Mrs. Lucy Westcott and Mrs. Lois Russell for editorial help with the manuscript. The study was supported by Grant MSM 412100003 and grants from MZe e`r QD0085 and MO2-99-01.

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