Introduction
The development of somatic cell nuclear transfer (SCNT) technology to produce cloned animals opened a new era in reproductive biology and biomedical science (Tian et al., Reference Tian, Kubota, Enright and Yang2003). In the porcine research field, numerous experiments have been conducted in attempts to realise this potential, ever since the first successful cloning of pigs by SCNT (Polejaeva et al., Reference Polejaeva, Chen, Vaught, Page, Mullins, Ball, Dai, Boone, Walker, Ayares, Colman and Campbell2000). However, the overall developmental ability of porcine SCNT embryos to term is still low. The efficiency of SCNT is affected by many factors including the quality of recipient oocytes (Wakayama et al., Reference Wakayama and Yanagimachi2001), stage of donor cell cycle (De Sousa et al., Reference De Sousa, Dobrinsky, Zhu, Archibald, Ainslie, Bosma, Bowering, Bracken, Ferrier, Fletcher, Gasparrini, Harkness, Johnston, Ritchie, Ritchie, Travers, Albertini, Dinnyes, King and Wilmut2002), duration of exposure of the donor nucleus into inactivated oocytes (Akagi et al., Reference Akagi, Adachi, Matsukawa, Kubo and Takahashi2003), activation method (Yang et al., Reference Yang, Zhao, Li, Li, Liu, Huang and Zeng2005) and epigenetic and genetic status of the donor cell genome (Inoue et al., Reference Inoue, Noda, Ogonuki, Miki, Inoue, Katayama, Mekada, Miyoshi and Ogura2007). In particular, the mode of activation is regarded as one of the most critical factors directly affecting the developmental competence of SCNT embryos (Solter, Reference Solter2000).
As the first cloned piglets have been produced by electrical activation (Polejaeva et al., Reference Polejaeva, Chen, Vaught, Page, Mullins, Ball, Dai, Boone, Walker, Ayares, Colman and Campbell2000), electrical activation of oocytes has been frequently employed for the activation of porcine SCNT, and this approach resulted in a number of cloned piglets. Further research is needed to improve the oocyte activation step in porcine SCNT because electrical activation induces only a single transient rise in intracellular Ca2+ concentrations (Swann et al., Reference Swann and Ozil1994; Wang et al., Reference Wang, Abeydeera, Prather and Day1998), rather than the multiple rises called ‘calcium oscillations’ that are observed during fertilization. A single transient rise in intracellular Ca2+ is not enough to support high rates of development beyond resumption of the second meiotic division. Thus, several chemicals such as 6-dimethylaminopurine (6-DMAP) (Betthauser et al., Reference Betthauser, Forsberg, Augenstein, Childs, Eilertsen, Enos, Forsythe, Golueke, Jurgella, Koppang, Lesmeister, Mallon, Mell, Misica, Pace, Pfister-Genskow, Strelchenko, Voelker, Watt, Thompson and Bishop2000) or cytochalasin B (Onishi et al., Reference Onishi, Iwamoto, Akita, Mikawa, Takeda, Awata, Hanada and Perry2000) have been studied for supplementing successful electrical activation of porcine oocytes, and chemical treatments were found to improve developmental competence in combination with electrical pulses (Lee et al., Reference Lee, Kim, Nam, Hyun, Lee, Kim, Lee, Kang, Lee and Hwang2004). However, data were obtained only using long-term (3 to 6 h) chemical treatment. In previous reports, treatment with cytochalasin B (Onishi et al., Reference Onishi, Iwamoto, Akita, Mikawa, Takeda, Awata, Hanada and Perry2000), 6-DMAP (Loi et al., Reference Loi, Ledda, Fulka, Cappai and Moor1998), demecolcine (DE) (Russell et al., Reference Russell, Ibanez, Albertini and Overstrom2005) and cyclohexamide (Varga et al., Reference Varga, Pataki, Lorincz, Koltai and Papp2008) needed 2, 3, 4 and 6 h, respectively, for activation. It is still an open question as to whether such long-term treatment to cause sufficient activation is really necessary considering possible adverse effect on embryo quality (Campbell et al., Reference Campbell, Fisher, Chen, Choi, Kelly, Lee and Xhu2007). If short-term treatment triggers efficient activation, it would improve the technology of oocyte activation with practical advantages.
On the other hand, it was suggested that combined treatment with thimerosal/dithiothreitol (Thi/DTT) treatment induced efficient activation in porcine embryos (Machaty et al., Reference Machaty, Wang, Day and Prather1997) without electrical pulses. In a previous study of porcine oocyte activation, short-term treatment (40 min) with Thi/DTT increased not only the developmental competence of SCNT embryos but also the mean cell number with an increased ratio of inner cell mass (ICM) to trophectoderm (TE) (Im et al., Reference Im, Seo, Hwang, Kim, Kim, Yang, Yang, Lai and Prather2006). Thimerosal triggers a series of Ca2+ spikes in the oocytes, moreover, if followed by incubation with dithiothreitol, it can stimulate pronucleus formation. The combined Thi/DTT treatment also causes cortical granule exocytosis, subsequent zona pellucida hardening and development of the activated oocytes to the blastocyst stage (Machaty et al., Reference Machaty, Wang, Day and Prather1997). As there have been many successful efforts to improve developmental competence using a combination of electric pulses and different chemicals, it was expected that the combination of Thi/DTT with other chemicals would enhance the developmental competence of parthenogenetic porcine embryos.
Accordingly, as to determine the optimal activation protocol of parthenogenetic porcine embryos, we examined whether short-term treatment with 6-DMAP and DE improves the developmental competence of electrically or Thi/DTT-activated porcine parthenogenetic embryos, then investigated the optimal activation protocol for activation of porcine embryos.
Materials and Methods
Chemicals and care of animals
All chemicals and reagents used for this study were purchased from Sigma-Aldrich Co., unless otherwise stated. Gyeonggido Veterinary Service was responsible for breeding of the pigs in compliance with the Gyeonggido Veterinary Service Institutional Animal Care and Use Committee, administered by the National Veterinary Research & Quarantine Service.
Collection of oocytes and in vitro maturation
Ovaries were collected at a local abattoir and stored in sterile physical saline at 30–35°C during transportation. Cumulus–oocyte complexes (COCs) were aspirated from antral follicles (3–6 mm) with an 18-gauge needle attached to a 10 ml disposable syringe. COCs with several layers of cumulus cells and uniform cytoplasm were chosen and cultured in tissue culture medium (TCM)-199 (Invitrogen) supplemented with 10 ng/ml EGF, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 5 μg/ml insulin, 1% (v/v) penicillin–streptomycin (Invitrogen), 1 mM dibutyryl adenosine 3,′5′-cyclic monophosphate (dbcAMP), 0.5 μg/ml follicle stimulating hormone, 0.5 μg/ml luteinizing hormone and 10% porcine follicular fluid at 39°C in a humidified atmosphere of 5% CO2, first, with GnRH and dbcAMP for 22 h and then without them for a further 22 h. COCs were washed at each step. After a total of 44 h maturation culture, oocytes were denuded by pipetting with 0.1% hyaluronidase in Dulbecco's PBS (DPBS) (Invitrogen) supplemented with 0.1% polyvinyl alcohol. Denuded oocytes with evenly-granulated and homogeneous cytoplasm were selected and then assigned randomly among different activation groups.
Electrical and Thi/DTT activation of porcine oocytes
After denuding, oocytes were divided into four groups for electrical and/or Thi/DTT activation.
For electrical activation, the first group was electric pulse (EP) control. Oocytes were equilibrated in pulsing medium then transferred to a chamber containing two electrodes overlaid with the pulsing medium. The pulsing medium was 0.26 M mannitol solution containing 0.5 mM HEPES, 0.1 mM CaCl2 and 0.1 mM MgSO4. Oocytes were activated with a single DC pulse of 1.5 kV/cm for 60 μs utilizing BTX electro-cell Manipulator 2001 (BTX, Inc.). The other three groups were exposed to 2 mM 6-DMAP (EP + 6-DMAP), 0.4 μg/ml demecolcine (EP + DE) or both together (EP + 6-DMAP + DE) for 40 min at 39°C in a humidified atmosphere of 5% CO2 after EP treatment.
For Thi/DTT activation, the first group was thimerosal/ dithiothreitol (Thi/DTT) control. Oocytes were treated with 0.2 mM Thi for 10 min, followed by 8 mM DTT for 30 min. The other three groups were Thi/DTT + 6-DMAP, Thi/DTT + DE and Thi/DTT + 6-DMAP + DE. (i) 6-DMAP, (ii) DE, (iii) both 6-DMAP and DE were added to Thi/DTT respectively.
In vitro culture
Embryos were washed and transferred into 500 μl of porcine zygote medium-3 (PZM-3) covered with mineral oil. The culture medium was 39°C, 5% CO2, 5% O2 and 90% N2. Embryos were evaluated for cleavage on day 2. On day 4, 10% fetal bovine serum was added to the IVC medium. Blastocyst formation and the number of nuclei were determined at day 7.
Total cell count in blastocyst
Briefly, blastocysts were fixed in absolute alcohol then nuclei were stained with 25 μg/ml bisbenzamide (Hoechst 33342) overnight at 4°C. Fixed and stained blastocysts were mounted on a glass slide in a drop of glycerol, gently flattened with a cover glass and visualized for cell counting with a fluorescence microscope using a 346 nm excitation filter.
Somatic cell nuclear transfer
For SCNT, a micromanipulation system (NT-88, Nikon-Narishinge) attached to an inverted microscope (TE-2000, Nikon Instruments) was used. A cumulus-free oocyte was held with a holding micropipette and the zona pellucida was partially dissected with a fine glass needle to make a slit near the adjacent cytoplasm, presumably containing the metaphase-II chromosomes, were extruded by aspiration with the same needle. Enucleation was confirmed by staining with Hoechst 33342 during manipulation. Single fibroblast cells with smooth surface were selected under a microscope and transferred into the perivitelline space of enucleated oocytes. These couplets were placed in a pulsing medium for 4 min and transferred to a chamber consisting of two electrodes overlaid with pulsing medium. The pulsing medium was 0.26 M mannitol solution containing 0.5 mM HEPES and 0.1 mM MgSO4. Couplets were fused with a single 1.2 kV/cm DC pulse for 30 μs using a BTX Electro-Cell Manipulator 2001 (BTX, Inc.) After fusion, couplets were activated by Thi for 10 min and DTT 30 min with 6-DMAP and DE. Fused couplets were used for embryo transfer within 2 h without additional in vitro culture.
Embryo transfer and pregnancy diagnosis
Embryo transfer was followed as the previous report (Koo et al., Reference Koo, Park, Kwon, Kang, Jang and Lee2009). As briefly described, in the laboratory, 120 to 150 of fused SCNT embryos were loaded into a sterilized 0.25 ml straw (Minitüb) and kept in a portable incubator (Minitüb) during transportation to the embryo transfer facility. An oestrus-synchronized recipient was anaesthetized by a combination of ketamine (2.3 mg/ml; Yuhan ketamine®, Yuhan Yanghang) and xylazine (0.6 mg/kg; Celactal®, Bayer Korea) through IV for induction and 3% of isoflurane (Ifran®, Hana Parm Co., Ltd) for maintenance. One oviduct was exposed by laparotomy. The straw containing the embryos was put directly into the oviduct of the recipient and embryos were expelled from the straw using a 1 ml syringe (Becton Dickinson). Recipients were checked for pregnancy by transabdominal ultrasound examination in day 30 after embryo transfer.
Experimental design
Effect of short-term treatment with DE on inhibition of polar body extrusion
As DE maintains diploid DNA content by inhibiting second polar body (PB2) extrusion and thereby indirectly affects embryo development ability, we needed to confirm whether short-term treatment with DE could sufficiently disturb PB2 extrusion. Hoechst 33342 staining was performed to evaluate polar body extrusion in parthenogenetic embryos. At 6 h after activation, embryos were fixed for 24 h 4°C in 4% formaldehyde in D-PBS. The fixed embryos were stained with 25 μg/ml Hoechst 33342 and then mounted on slides. Extrusion of PB2 was examined under UV light using a fluorescence microscope with a 346 nm excitation filter.
Effects of short-term treatment with 6-DMAP and/or DE on either electrical or Thi/DTT activation of oocytes
This experiment was to find out whether short-term treatment with 6-DMAP and DE improved developmental competence of electrically or Thi/DTT-activated parthenogenetic porcine embryos. As a control, EP was compared with other groups with or without 6-DMAP and DE (EP vs. EP + 6-DMAP, EP + DE and EP + 6-DMAP + DE). Evaluation of development competence, cleavage rate, rate of blastocyst formation and total blastocyst cell number were checked on days 2 and 7. Then, results with Thi/DTT were compared with other groups with or without 6-DMAP and DE (Thi/DTT vs. Thi/DTT + 6-DMAP, Thi/DTT + DE and Thi/DTT + 6-DMAP + DE).
Comparison of short-term and long-term treatment with 6-DMAP and DE on electrical or Thi/DTT activation of oocytes
Previous experiments showed which combination of short-term treatments improved embryo development ability with electrical or Thi/DTT activation. Therefore, we compared these combinations of treatments on both electrical and Thi/DTT activation to validate the effect of short-term treatment with 6-DMAP and DE.
Optimization of short-term treatment with 6-DMAP and DE on electrical and Thi/DTT activation of oocytes
Finally, to optimise the activation protocol, the best conditions in electrically- and Thi/DTT-activated groups were compared.
In vivo capacity of short-term treatment with 6-DMAP and DE on Thi/DTT activation of oocytes
The capacity of embryos activated by the optimized activation system to develop after transfer was investigated in this experiment. SCNT embryos were activated by Thi/DTT + 6-DMAP + DE which are the optimized protocol in Experiment 4.
Statistical analysis
All data were subjected to one-way ANOVA followed by Tukey's test using Prism version 4.0 (GraphPad Software) to determine differences among experimental groups. Statistical significance was determined when p-value was less than 0.05.
Results
Effect of short-term treatment with DE on inhibition of polar body extrusion
Inhibition of PB2 extrusion after oocyte activation would be consistent with maintenance of diploid DNA content in parthenogenetic embryos. The number of embryos showing only 1PB was higher in both EP and Thi/DTT groups treated with demecolcine 6 h after activation (39.0 ± 4.5, 46.8 ± 4.5 vs. 20.5 ± 3.8, 24.1 ± 4.0%, respectively, p < 0.05) (Table 1).
Table 1 Effect of short-term treatment with DE on inhibition of polar body extrusion in porcine oocytes.
DE, demecolcine; EP, electric pulse; PB, polar body; Thi/DTT, thimerosal/dithiothreitol.
a,b Values for different superscripts in the same column are significantly different (p < 0.05).
Experiments were repeated at least four times.
Effects of short-term treatment with 6-DMAP and/or DE on either electrical or Thi/DTT activation of oocytes
Cleavage rates and blastocyst formation rate of EP + 6-DMAP and EP + 6-DMAP + DE were significantly different among treatments (69.1 ± 3.2, 75.6 ± 3.0% and 31.4 ± 3.2, 33.8 ± 3.2%, respectively, p < 0.05) (Table 2). Blastocysts activated with 6-DMAP and DE showed significantly more cell number compared with others (46.3 ± 1.0). Similar to short-term electrical activation treatment, embryos Thi/DTT activated with 6-DMAP and DE showed the highest cleavage rate (73.9 ± 3.1%, p < 0.05), blastocyst formation (32.9 ± 3.3%, p < 0.05) and total cell number (47.3 ± 0.4, p < 0.05) (Table 3).
Table 2 Effects of short- term treatment with 6-DMAP and DE on electrical activation of porcine oocytes.
6-DMAP, 6-dimethylaminopurine; DE, demecolcine; EP, electric pulse.
a–cValues for different superscripts in the same column are significantly different (p < 0.05).
Experiments were repeated at least six times.
Table 3 Effects of short-term treatment with 6-DMAP and DE on Thi/DTT activation of porcine oocytes.
6-DMAP, 6-dimethylaminopurine; DE, demecolcine; Thi/DTT, thimerosal + dithiothreitol.
a–dValues for different superscripts in the same column are significantly different (p < 0.05).
Experiments were repeated at least six times.
Comparison of short-term and long-term treatment with 6-DMAP and DE on electrical or Thi/DTT activation of oocytes
Together with short- and long-term treatment with 6-DMAP and DE on electrical activation, cleavage or blastocyst formation rates and total cell number were not significantly different (67.4 ± 3.5 vs. 72.7 ± 3.3, 32.7 ± 3.3 vs. 30.0 ± 3.4% and 41.9 ± 1.2 vs. 40.3 ± 2.4, respectively) (Table 4). Same as electrical activation, cleavage rate, blastocyst formation rate and total cell number of short- and long-term treatment with 6-DMAP and DE on Thi/DTT activation were not considerably different (76.0 ± 3.2 vs. 82.8 ± 2.8, 36.1 ± 3.6 vs. 38.3 ± 3.6% and 47.2 ± 1.7 vs. 42.2 ± 3.3, respectively) (Table 4).
Table 4 Comparison of short-term and long-term treatment with 6-DMAP and DE on electrical or Thi/DTT activation of porcine oocytes.
6-DMAP, 6-dimethylaminopurine; DE, demecolcine; EP, electric pulse; Thi/DTT, thimerosal/dithiothreitol.
There were no statistically significant differences among the groups (p > 0.05).
Experiments were repeated at least six times.
Optimization of short-term treatment of 6-DMAP and DE together with electrical and Thi/DTT activation
Thi/DTT combined with 6-DMAP and DE induced more effective activation of oocytes than did both EP + 6-DMAP + DE and EP + Thi/DTT + 6-DMAP + DE.B: blastocyst formation rate was significantly higher (36.1 ± 3.5 vs. 23.3 ± 3.0, 17.2 ± 2.7%, p < 0.05) and blastocyst cell numbers increased (46.9 ± 1.0 vs. 42.2 ± 1.1, 36.7 ± 1.5, p < 0.05) (Table 5).
Table 5 Optimization of short-term treatment with 6-DMAP and DE on electrical and Thi/DTT activation of porcine oocytes.
6-DMAP, 6-dimethylaminopurine; DE, demecolcine; EP, electric pulse; Thi/DTT, thimerosal/dithiothreitol.
a–cMeans with different superscripts in same column were significantly difference (p < 0.05).
Experiments were repeated at least six times.
In vivo capacity of short-term treatment with 6-DMAP and DE on Thi/DTT activation of oocytes
We transferred embryos which were activated with short-term treatment of Thi/DTT + 6-DMAP + DE to 30 surrogate pigs. As a result, four pigs were pregnant. Among them, 16 fetuses were collected as samples for another experiment from two surrogate pigs and one fetus was born from one. At time of writing another sow was pregnant. Pregnancy rate was approximately 13.3%.
Discussion
This study demonstrated that short-term treatment with 6-DMAP and DE on electrical and Thi/DTT activation of porcine embryos (EP + 6-DMAP + DE, Thi/DTT + 6-DMAP + DE) substantially improved their developmental competence. In addition, comparison of EP + 6-DMAP + DE, Thi/DTT + 6-DMAP + DE and EP + Thi/DTT + 6-DMAP + DE reveals that Thi/DTT + 6-DMAP + DE was the optimal protocol for activation of porcine embryo.
Recently, studies have focused on the improvement of oocyte activation protocols by combining electrical stimulation with administration of chemicals. Additional administration of 6-DMAP is one of the most widely used activation protocols for reconstructed oocytes (Jiang et al., Reference Jiang, Mizuno, Mizutani, Sasada and Sato2002; Alexander et al., Reference Alexander, Coppola, Di Berardino, Rho, St John, Betts and King2006; Meo et al., Reference Meo, Yamazaki, Ferreira, Perecin, Saraiva, Leal and Garcia2007). Such a strategy was applied successfully to produce cloned piglets (Holker et al., Reference Holker, Petersen, Hassel, Kues, Lemme, Lucas-Hahn and Niemann2005). Consistent with previous work, the present results show that treatment with 6-DMAP remarkably accelerated embryo development compared with EP or Thi/DTT only. Interestingly, oocytes exposed to 6-DMAP for a short time showed similar developmental ability compared with long-term treatment with respect to cleavage, blastocyst formation rate and total cell numbers (Table 4). We ascertained that short vs. long exposure to 6-DMAP did not produce significantly different results.
6-DMAP, a serine protease (phosphorylation) inhibitor, initiates pronuclear formation via complex mechanisms that involve blocking activity of key cell cycle regulatory proteins such as mitogen activated protein kinase (MAPK), myosin light chain kinase and the cyclin dependent p34cdc2. In oocytes that were treated with two electrical pulses (1.2 kV/cm for 60 μs), MAPK activity dropped markedly after 1 h and reached its lowest value 3 h after electroporation (Nanassy et al., Reference Nanassy, Lee, Javor and Machaty2007). However, by 4 h the activity increased again and remained at elevated levels until the time of pronuclear formation. This finding seems to explain the commonly used duration of treatment time with 6-DMAP for 3–4 h. 6-DMAP may suppress the MAPK activity faster and maintain the low level longer, by complementing the effect of EP on MAPK.
Our experiments suggest that 40 min exposure to 6-DMAP sufficiently dephosphorylates MAPK or serine/threonine kinase p90RSK within the MAPK pathway. More potent electrical stimuli (1.5 kV/cm for 60 μs) and Thi may downregulate MPF activity faster, which prevents maternal c-mos translation. This outcome reinforces the effect of 6-DMAP on MAPK inhibition. Thereby, responses to short-term treatment with 6-DMAP on electrically or Thi/DTT activated porcine embryos were equivalent to established long-term treatment. Besides, there have been several reports that long-term treatment with 6-DMAP can do harm to the embryo. The first report claimed that 6-DMAP may cause alteration in the cell's DNA content owing to an abnormal pattern of karyokinesis (De La Fuente et al., Reference De La Fuente and King1998) without cytokinesis (Campbell et al., Reference Campbell, Fisher, Chen, Choi, Kelly, Lee and Xhu2007), although longer exposure to 6-DMAP increased pronuclear formation, cleavage rate and blastocyst formation (Szollosi et al., Reference Szollosi, Kubiak, Debey, de Pennart, Szollosi and Maro1993). 6-DMAP is also a mutagenic agent that may influence the genetic background (Katoh et al., Reference Katoh, Araki, Ogura and Valdivia2004). Therefore, it is necessary to minimise adverse effects and maximise the efficiency of activation. If short-term treatment with 6-DMAP is sufficient to activate oocytes, there is no reason to administer it for a long time and risk severe side effects on embryo development.
On the other hand, synergistic effect was observed in this study with 6-DMAP and DE together, while there was no significant improvement in embryo development using DE alone. Retention of the diploid DNA complement has influences on the developmental competence of SCNT embryos receiving donor cells in the G0/G1 phase (Sugimura et al., Reference Sugimura, Kawahara, Wakai, Yamanaka, Sasada and Sato2008), because the DNA contents affect the subsequent development of SCNT embryos during the first cell cycle (Wakayama et al., Reference Wakayama, Perry, Zuccotti, Johnson and Yanagimachi1998). Unless SCNT embryos injected with donor nuclei in the G0/G1 phase are treated with cytoskeletal inhibitors such as DE or cytochalasin, they exclude some chromosomes as a pseudo-polar body after artificial activation, resulting in aneuploidy (Lai et al., Reference Lai, Tao, Machaty, Kuhholzer, Sun, Park, Day and Prather2001). DE impairs second polar body extrusion completely by the change of microtubules (Ibanez et al., Reference Ibanez, Albertini and Overstrom2003). DE was generally used for 0.4 μg/ml after EP from 1.5–2.5 h and 1.5–3.5 h, because the second polar body is extruded in the majority of oocytes 4 h after activation (Tian et al., Reference Tian, Wu, Liu, Cai, Zeng, Zhu, Liu, Li and Wu2006). However, DE assisted enucleation during the SCNT, which changes microtubule and facilitates removal of chromatin, only taking 30–45 min to impair microtubule (Li et al., Reference Li, Villemoes, Zhang, Du, Kragh, Purup, Xue, Pedersen, Jorgensen, Jakobsen, Bolund, Yang and Vajta2009; Saraiva et al., Reference Saraiva, Perecin, Meo, Ferreira, Tetzner and Garcia2009). Thus we would expect that brief exposure to DE sufficiently perturbs microtubules. Not surprisingly, it was observed that DE effectively disturbs second polar body extrusion and thus maintains diploid DNA content (Table 1). DE administration itself did not support efficient development, but the combined treatment of DE together with 6-DMAP did improve developmental competence of activated oocytes (Table 3). This finding indicates that DE advances development ability indirectly via inhibition of second polar body extrusion.
To further investigate the optimal activation protocol, we compared effects of short-term treatment with several combinations of 6-DMAP and DE on electrical activation and Thi/DTT activation. Development potential induced by Thi/DTT activation surpassed that of electrical activation (Table 5). Peculiar mechanisms of action of Thi/DTT can support this result. Thi, a sulfhydryl reagent, induces repetitive Ca2+ spikes in oocytes by oxidizing critical sulfhydryl groups on intracellular Ca2+ release proteins which are suspected to be either inositol-1,4,5-triphosphate receptors (Miyazaki et al., Reference Miyazaki, Shirakawa, Nakada, Honda, Yuzaki, Nakade and Mikoshiba1992) or ryanodine receptors (Swann, Reference Swann1992). It also oxidizes the tubulin sulphydryl groups and thus causes disassembly of the meiotic spindle which is required for transition from the M phase to interphase. However, this latter effect can be reversed by the reducing agent DTT to complete the subsequent series of mitotic divisions (Machaty et al., Reference Machaty, Wang, Day and Prather1997). The Ca2+ oscillation profile generated by thimerosal exceeds the single Ca2+ spike induced by an electric pulse. Contrary to expectations that electrical and Thi/DTT activation combined would cause optimal consequences, it was noted that EP + Thi/DTT + 6-DMAP + DE treatment did not result in better development than the other treatments (EP + 6-DMAP + DE and Thi/DTT + 6-DMAP + DE). It might be deduced that excessively increased Ca2+ levels deteriorate development (Collas et al., Reference Collas, Fissore, Robl, Sullivan and Barnes1993), hence developmental ability would be impaired.
Until now, these experiments showed that short-term treatment of Thi/DTT + 6-DMAP + DE activates oocytes effectively in vitro. To know the capacity of short-term treatment of Thi/DTT + 6-DMAP + DE in vivo, we transferred embryos that were activated with it to 30 surrogate pigs. As a result, four pigs are pregnant. Pregnancy rate is approximately 13.3%. This result showed that short-term treatment of Thi/DTT + 6-DMAP + DE can fully support in vivo embryo development and this activation protocol can be used in vivo.
Taking our results together, we verified that short-term treatment with 6-DMAP and DE can induce efficient activation of oocytes and concluded that the optimal activation protocol is Thi/DTT + 6-DMAP + DE. This combined activation method was highly effective in supporting blastocyst formation (Table 5) and increasing total blastocyst cell number, and when transferred to recipients, embryos were able to achieve full term development. Thus it may be the part of a successful oocyte-activating scheme during porcine SCNT procedures. Furthermore, embryo transfer to sow after short-term activation can reduce the damage of embryo quality and save both time and labour.
Acknowledgements
This study was financially supported by MKE (grant #2009-67-10033839, #2009-67-10033805), NRF (#M10625030005-508-10N250300510), IPET (#109023-05-1-CG000), the BK21 programme for Research Insitute for Veterinary Science, and Hanwha L&C. We thank Kyonggido Livestock and Veterinary Service for valuable contributions in discussions and animal husbandry.
