Abstract
Animal Cells and Systems, 2014 Vol. 18, No. 5, 324–332, http://dx.doi.org/10.1080/19768354.2014.948488 a,b a* Bo-Woong Sim and Kwan-Sik Min Animal Biotechnology, Graduate School of Bio & Information Technology, Institute of Genetic Engineering, Hankyong National University, Anseong 456-749, Republic of Korea; National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang 363-883, Republic of Korea (Received 31 March 2014; received in revised form 23 June 2014; accepted 21 July 2014) Mouse chimeras can also successfully be produced using tetraploid host embryos. This study was conducted to optimize the efficiency of cloning and to produce cloned mice using tetraploid host embryos. Six hours of activation with strontium (SrCl ) was optimal for somatic cell nuclear transfer (SCNT) embryos. Cytochalasin B (CB) concentration (5 µg/ml) during enucleation was evaluated in the efficiency of implantation sites and fetus offspring. Continuous exposure to 5–50 nM trichostatin A (TSA), a histone-deacetylase inhibitor (HDACi), for 10 h is recommended for production of clone mice. Aggregated SCNTs were constructed by aggregation of SCNT embryos with tetraploid embryos to reduce epigenetic errors in the placenta. The pregnancy and implantation rates of aggregated SCNT were significantly higher than those of SCNT alone. The full-term developmental rate of aggregated embryos was also higher than that of SCNT (3.57 vs. 1.16). The placental weight of SCNT clones was significantly higher than that from in vitro fertilization (IVF). However, the placenta weight of aggregated SCNT clones was nearly the same as that of embryos in the IVF group. The placentas of SCNT-only clones appeared to have the hyperplastic histology typical of mouse clones. We produced a total of 36 clone mice, including nine heads derived from aggregated SCNT. One-half of clones derived from aggregated SCNT survived to adulthood, and 14-clones derived from SCNT grew into healthy adults. The aggregated SCNT method was useful for significantly reducing the placental weight of cloned mice and improving the efficiency of SCNT. Keywords: cloning; mice; aggregation; tetraploid; placenta Introduction 1999), goats (Baguisi et al. 1999), pigs (Onish et al. 2000; Polejaeva et al. 2000), cats (Shin et al. 2002), and mice The success rate of somatic cell nuclear transfer (SCNT) (Wakayama et al. 1998). Mice have been cloned using a cloning depends on the technical skills of those creating piezo-actuated NT method with cumulus cells (Wakayama the clone and the biological material used. This study is et al. 1998), tail-tip cells (Wakayama et al. 1999), sertoli subject to increase the efficiency of cloning and produce cells (Ogura et al. 2000), embryonic stem (ES) cells cloned mice, using the aggregated SCNT method with (Wakayama et al. 1999), and germ cells (Miki et al. 2005) tetraploid embryos to reduce placental weight. The as nucleus donors. The success of SCNT cloning has led majority of cloned mammals derived by nuclear transfer to applications such as species preservation, livestock (NT) die during gestation, display neonatal phenotypes propagation, and medical cell therapy. However, the resembling large offspring syndrome (Young et al. 1998), cloning of animals is inefficient. often with respiratory and metabolic abnormalities, and The use of donor cells in SCNT generally requires the have enlarged and dysfunctional placentas (Wakayama & inhibition of cytokinesis in order to prevent chromosome Yanagimachi 1999). SCNT in animals is remarkably loss via the extrusion of a second polar body (Kono et al. inefficient with reported rates (number of live offspring/ 1993;Cheong et al. 1994;Wakayamaet al. 1998). To transferred cloned embryos) of 5% in mice, 3% in cattle, improve development of SCNT embryos, cytochalasin (C) and 0.5% in pigs (Wilmut et al. 1997; Cibelli et al. 1998; should be applied at a concentration that varies with the Baguisi et al. 1999; Wakayama 2007). degree of spindle formation after SCNT. Cytochalasin D, like The first clone was produced in sheep and was generated by cell fusion (Willadsen 1986). The most Cytochalasin B (CB), is a microfilament-disrupting reagent that has been used to induce polyploidy in preimplantation important stage in clone study is the developmental embryos (Snow 1973; Siracusa et al. 1980; Bos-Mikich et al. reprogramming of the donor nucleus after injecting into 1995). Mouse cloning is typically achieved by activation of an enucleated recipient oocyte or zygote. A breakthrough in the study of cloning was the birth of the first clone from somatic cell nucleus transferred embryos using strontium 2+ a somatic cell (Wilmut et al. 1997). Successful somatic (Sr ) ions (Wakayama et al. 1998), although activation by cell cloning has been reported in various animals, such as ethanol, an electric pulse, or even spermatozoa, may result in cows (Cibelli et al. 1998; Kato et al. 1998; Wells et al. equivalent cloning efficiencies (Kishikawa et al. 1999; *Corresponding author. E-mail: ksmin@hknu.ac.kr © 2014 Korean Society for Integrative Biology DEVELOPMENTAL BIOLOGY Animal Cells and Systems 325 Amano et al. 2001). Previous reports indicate that co-transfer embryos, which may increase the contribution of tetra- of parthenogenetically activated (PA) embryos served better ploid derivatives to the placental tissues. for SCNT embryo pregnancy maintenance in pigs than timed administration of estradiol or equine chorionic gonadotropin Methods (eCG), and a piglet has been cloned using a PA embryo (De Sousa et al. 2002). Pregnancy was established in pigs by Animals co-transfer of a low number of PA (King et al. 2002)and B6D2F1 mice (C57BL/6 × DBA/2) were used to prepare SCNT embryos (Ohgane et al. 2004), but the pregnancy oocytes and cumulus cells. Surrogate females were females terminated at Day 69 (Lai et al. 2002). Cotransfer of SCNT imprinting control regions (ICR) mated with vasectomized embryos with a large number of fertilized embryos resulted males of the same strain. All experiments were conducted in the birth of only fertilized piglets (Onish et al. 2000). according to the Guidelines for the Care and Use of Recently, PA co-transfer was also used to maintain pregnan- Animals, Hankyong National University. The protocol was cies of rat embryonic NT embryos (Popova et al. 2006). approved by the Committee on the Ethics of Animal However, there are few studies of co-transfer with cloned Experiments of the Hankyong National University (Permit embryos, and even fewer of the use of this technique in mice. Number: 2012-10). One of the most critical factors for the success of SCNT is the successful epigenetic reprogramming of the Collection of oocytes donor nucleus. Molecular analysis of cloned embryos reveals abnormal epigenetic modification, such as DNA MII oocytes were collected from the oviducts of 6 to 12- methylation and histone modification (Dean et al. 2001; week-old females that had been induced to superovulate Kang et al. 2001; Ohgane et al. 2001; Rybouchkin et al. by injection of pregnant mare serum gonadotropin (PMSG) (7.5 IU) followed by human chorionic gonado- 2006). Tichostatin A (TSA) is a histone-deacetylase inhi- tropin (hCG) (7.5 IU) 48 h later. Oocytes were collected bitor that enhances the pool of acetylated histones and from oviducts 13–14 h after hCG injection, placed DNA demethylation (Yoshida et al. 1990; Hattori et al. in (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)- 2004). TSA treatment improves full-term development of buffered-CZB medium (HEPES-buffered CZB medium), mouse embryos obtained by transfer of cumulus cell and treated with 300 unit/ml hyaluronidase until the nuclei (Kishigami, Mizutani, et al. 2006). cumulus cells dispersed. The oocytes were transferred to The common finding of placental defects in SCNT fresh droplets of HEPES-buffered CZB medium and were mice, cows, and sheep suggests placental pathology as a denuded of almost all cumulus cells by gentle pipetting. major cause of complications in pregnancies involving The oocytes were then placed in synthetic oviductal SCNT embryos (Yang et al. 2007). In cloned mice, the medium enriched with potassium medium (KSOM; Sum- placenta at term is characterized by a type of hyperplasia mers et al. 1995), covered with paraffin oil (Nacalai caused by enlargement of the spongiotrophoblast (ST) Tesque, Kyoto, Japan), and stored at 37°C in a 5% CO layer (junctional zone) and an increase in glycogen-bearing atmosphere until use. trophoblastic cells (Wakayama et al. 1999; Tanaka et al. 2001). Recently, chimeric offspring were produced follow- ing aggregation of totipotent cells of four-cell rhesus Preparation of donor cells for nucleus transfer monkeys embryos (Tachibana et al. 2012). Many steps Cumulus cells of B6D2F1 mice were removed from are necessary to produce a cloned mammal. The process oocytes using hyaluronidase as described above. Cumulus includes the removal of the maternal chromosomes, the cells were transferred and maintained in HEPES-buffered injection or fusion of somatic nuclei into enucleated CZB medium at 4°C. Immediately prior to use, cumulus oocytes, the reconstruction of the somatic chromosomes, cells were resuspended in 10% polyvinylpyrrolidine oocyte activation, treatment with CB to produce a diploid (PVP) in HEPES-buffered CZB medium. cloned embryos, and in vitro culture. Many of these steps result in a decrease in the developmental rate of blastocyst Enucleation of MII oocytes and high levels of abnormalities in the offspring. Therefore, SCNT protocols should be optimized. In Enucleation was performed in HEPES-buffered CZB the present study, we examined improvements of the medium supplemented with 3 or 5 µg/ml CB. After each activation and subsequent embryonic development of oocyte was immobilized in an oocyte-holding pipette, its SCNT embryos by comparing the duration and concen- zona pellucida was ‘drilled’ by applying several piezo- tration of activation reagents, including SrCl , CB, and pulses at the tip of an enucleation pipette (∼12-µm outside TSA. We also improved the efficiency of SCNT progeny diameter). The MII chromosome-meiotic spindle complex production using co-transfer with PA embryos and was visualized as a translucent spot in the ooplasm using aggregation of SCNT embryos with tetraploid host differential interference contrast optics and was drawn 326 B.-W. Sim and K.-S. Min into the pipette with a small amount of accompanying in KSOM at 37°C under 5% CO to obtain two-cell ooplasm. The chromosome-meiotic spindle complex was embryos. Two-cell embryos were electrofused to produce then gently pulled away from the oocyte until the cyto- one-cell tetraploid embryos. Tetraploid were placed between plasmic bridge was severed (Kono et al. 1993) Enucleated 1-mm-apart wire chambers in modified Dulbecco’sPhos- oocytes were washed three times in CB-free CZB phate Buffered Saline (D-PBS) medium that was supple- medium, transferred into CB-free KSOM, and stored for mented with 3 mg/ml BSA. Electrofusion was induced by up to 1 h at 37°C under 5% CO until nucleus transfer. single 45 µs DC pulses of 0.7 KV/cm and delivered by a BTX Electro Cell Manipulator 200 (BTX, San Diego, CA). Fused embryos were cultured in KSOM at 37°C under 5% Nucleus transfer CO . One 4 to 8 cell-stage SCNT embryos was aggregated SCNT embryos were produced by injecting a cumulus cell with two 2 to 4-stage cell tetraploid embryos. The zona nucleus into an enucleated B6D2F mature oocyte. Single pellucida of embryos was removed by treatment with 0.5% cumulus cells in 10% PVP medium were drawn in and out pronase for 5 min. These embryos were nestled in a hole in of an injection pipette (6–7 µm inner diameter) until the a plastic dish that was made using a tack. Aggregated plasma membranes were broke. After a nucleus was embryos were cultured in KSOM at 37°C under 5% CO to drawn deep into the pipette, the pipette was then passed obtain morula/blastocyst-stage embryos. through the zona pellucida of an enucleated oocyte by applying piezo pulses. The nucleus was then injected into Embryo transfer and examination of fetuses and an enucleated oocyte at room temperature using the piezo- placentas driven pipette, as described previously (Kimura & Yana- gimachi 1995). Injected oocytes were kept in KSOM for Some of two-cell embryos (24 h after the onset of 1–2 h before activation treatment at 37°C under 5% CO activation) were transferred to the oviducts of foster in air and transferred. mothers (ICR, albino) that had been mated with vasecto- mized ICR males 1 day previously. SCNT embryos and aggregated SCNT morula/blastocysts were transferred to Activation and culture of injected oocytes the uteri of foster mothers (ICR, albino) that had been The injected oocytes were activated by 5, 7.5, or 10 mM mated with vasectomized ICR males 3 days previously. 2+ SrCl in Ca -free CZB supplemented with 2, 2.5, 3, 4, or Each recipient surrogate female received 10–20 SCNT and 5 mg/ml CB and 5 or 50 nM TSA for 0.5, 1, or 6 h at aggregated SCNT embryos. In the co-transfer experiment, 37°C under 5% CO . Oocytes activated for 0.5 or 1 h PA embryos were transferred into one side of the uterus of were cultured for an additional 5.5 h or 5 h, respectively, each foster mother, and SCNT embryos were transferred in the presence of 2.5 µg/ml CB (for a total of 6 h) to into the other side. Some of the recipient females were prevent the extrusion of polar bodies that contained donor euthanized at 9.5 dpc and examined for the presence or chromosomes. Reconstructed oocytes were then incubated absence of fetuses and implantation sites. All other in 5 or 50 nM TSA in KSOM for 10 h at 37°C under 5% recipient females were euthanized at 19.5 dpc and their CO . Oocytes with at least one pseudo-pronucleus and uteri examined for the presence of fetuses and implanta- without a second polar body were considered normally tion sites. Live fetuses were raised by lactating foster activated and were washed and cultured in KSOM at mothers (ICR). Full-term offspring and placenta were 37°C, 5% CO until they reached the two-cell or morula/ individually weighed. blastocyst stage. Statistical analysis Production of PA embryos Statistical analyses were performed using Minitab soft- MII oocytes were collected from superovulated B6D2F1 ware. Data were analyzed using one-way analysis of female mice 14 h after hCG injection. The oocytes were variance (ANOVA). Differences were considered signific- incubated in KSOM at 37°C under 5% CO for 3–4h ant when p < 0.05, p < 0.01. 2+ prior to PA by 10 mM SrCl in Ca -free CZB supple- mented with 2, 3, 4, or 5 µg/ml CB for 6 h. After activation, the PA embryos were cultured in KSOM at Results 37°C under 5% CO until the morula and blastocyst stage. Development of SCNT embryos after SrCl activation To compare the developmental effects of activation of Production of tetraploid embryos and aggregation adult cumulus cell clones by different concentrations of Twenty hours after hCG injection, fertilized zygotes were SrCl on SCNT embryos, SCNT oocytes were activated by collected from superovulated ICR female mice that were 5, 7.5, or 10 mM SrCl for 6 h. Among SCNT embryos mated with ICR males. Collected zygotes were cultured that underwent the first cleavage, fewer embryos were Animal Cells and Systems 327 Table 1. Development of SNCT embryos activated by 5, 7.5, and 10 mM SrCl No. of embryos developed to each No. of stages (%) activated No. of No. of No. of clone Concentration No. of embryos Morula/ transferred No. of implanted fetuses at 9.5 of SrCl (mM) injected (%) Two-cell Four-cell blastocyst (recipients) pregnancies sites (%) dpc (%) d c 10 102 91 (89.2) 88 (96.7) 69 (75.8) 63 (69.2) 63 (4) 3 33 (52.4) 1 (1.6) c bd 7.5 144 126 (87.5) 106 (84.1) 81 (64.3) 57 (45.2) 57 (3) 2 26 (45.6) 2 (3.5) 5 51 48 (94.1) 44 (91.7) 39 (81.3) 33 (68.8) 33 (2) 1 12 (36.4) 0 (0.0) Note: Values with different superscripts are significantly different (a vs. b, p < 0.05; c vs. d, p < 0.01). Effects of SrCl activation time To optimize the duration of SrCl activation (10 mM), SCNT oocytes were activated with SrCl for 0.5, 1, or 6 h. Those oocytes activated for 0.5 h had a significantly higher rate of second polar body formation than those activated for 1 or 6 h (6.6% vs. 1.7% and 0.0%, respectively; p < 0.01; data now shown). However, the rate of development to morula/blastocysts was not differ- ent between groups (35.8 vs. 43.0 and 43.5%; Table 2). Similarly, the implantation rates in surrogate mothers were not different between groups. The 6 h group had a slightly higher production rate of live fetuses than those of the 0.5 and 1 h groups, but the difference was not statistically Figure 1. Fetuses derived from a SCNT embryo 9.5 dpc. Two significant (3.8, 1.4, and 1.5%, 6 h, 1 h, and 0.5 h, fetuses had beating heart. respectively; Table 2). activated by 7.5 mM SrCl developed to the morula/ blastocyst than those activated by 5 or 10 mM SrCl Influence of CB concentration in the enucleation (45.2% vs. 68.8% and 69.2%, respectively, p < 0.05). To determine the optimal concentration of CB in enucle- There was no significant difference between groups in the ation medium for development of SCNT embryos, we implantation rate of morula/blastocyst-stage SCNT calculated the developmental rate of reconstructed embryos 9.5 dpc (36.4%, 45.6%, and 52.4% for embryos embryos enucleated in 3 or 5 µg/ml CB medium. SCNT activated by 5, 7.5, and 10 mM SrCl , respectively), and embryo activation rates did not differ between these two one or two live fetuses were obtained from the 10 and groups. There was no statistically different significance at 7.5 mM SrCl groups (Table 1). All clone fetuses were the activation rate of PA oocytes for 6 h by CB morphologically normal at 9.5 dpc. Heartbeats were concentration (2, 3, 4, 5 ug/ml; data not shown). However, observed in these fetuses (Figure 1). a greater percentage of SCNT embryos treated with 5 µg/ml Table 2. Full-term development of SCNT embryos activated by SrCl treatment for 0.5, 1, and 6 h. No. of embryos developed to each stages (%) Exposure No. of Morula/blastocyst No. of No. of Time No. of activated (/activated), transferred No. of implanted No. live clone (hour) injected (%) Two-cell (/injected) (recipient) pregnancies sites (%) offspring (%) 0.5 201 123 (61.2) 114 (92.7) 72 (58.5), (35.8) 65 (4) 3 27 (41.5) 1 (1.5) a d 0.5 h 115 77 (67.0) 72 (93.5) – 67 (3) 2 25 (37.3) 1 (1.5) 1 h 181 156 (86.2) 144 (92.3) 78 (50.0), (43.0) 72 (5) 4 21 (29.2) 1 (1.4) 6 h 168 145 (86.3) 133 (91.7) 73 (50.3), (43.5) 73 (4) 3 28 (38.4) 2 (2.7) a c 6h 41 39 (95.1) 35 (89.7) – 33 (2) 1 13 (39.4) 2 (6.1) Note: Values with different superscripts are significantly different (p < 0.01). Embryos were transferred to recipient females at the two-cell stage. 328 B.-W. Sim and K.-S. Min Table 3. Full-term development of somatic cell nucleus transfer embryos enucleated in media containing 5 or 3 µg/ml CB. No. embryos of developed to each stages (%) No. of No. of No. of Concentration No. of activated Morula/ transferred No. of implanted No. of live clone of CB (µg/ml) injected (%) Two-cell blastocyst (recipient) pregnancies sites (%) offspring (%) d d 3 71 60 (84.5) 54 (90.0) 19 (31.7) 19 (1) 1 5 (26.3) 0 ( 0.0) c c 5 62 54 (87.1) 49 (90.7) 30 (55.6) 20 (1) 1 11 (55.0) 2 (10.0) Note: Values with different superscripts are significantly different (p < 0.01). CB developed into morula/blastocyst than did those treated Implantation site and offspring by cotransfer with PA embryos with 3 µg/ml CB (55.6% vs. 31.7%; p < 0.01). The SCNT embryos that developed into morula/blastocysts were To improve the overall efficiency of SCNT pregnancy and transferred to recipient females. There was significant full-term development, we cotransferred PA embryos with difference in implantation rate between the 3 and 5 µg/ml SCNT embryos. There was a small, but significant differ- CB groups (26.3% vs. 55.0%), but only those SCNT ence in the implantation rates between the cotransferred embryos in the 5 µg/ml CB group developed to full term, group and the SCNT-only group (29.6% vs. 19.6%), but resulting in two live offspring (Table 3). there was no difference between groups in birth rate (2.60 vs. 2.95%). None of the PA embryos developed to term (Table 5). All full-term fetuses were located on the side of Effects of TSA treatment the uterus into which the SCNT embryos were transferred. To examine the effects of TSA concentration on adult cumulus cell clones, SCNT oocytes were treated with 5 Consequences of aggregation of SCNT embryos with tetraploid embryos and 50 nM TSA for 10 h. A larger percentage of SCNT embryos that were continuously exposed to 50 nM TSA Aggregated SCNT embryos were constructed by aggre- developed into morula/blastocysts than did those exposed gating individual SCNT embryos (4–6 cell stage) with two to 5 nM TSA (61.0% vs. 49.3%; p < 0.05). However, tetraploid embryos (2–4 cell stage). We used this approach there was no difference between 5 and 50 nM TSA in the because diploid cells have a strong tendency to preferen- production of clone mice. TSA concentrations of 5–50 nM tially colonize the epiblast and its derivatives. In contrast, are recommended for production of clone mice (Table 4). tetraploid cells are restricted to extraembryonic tissues. As Table 4. Full-term development of SCNT embryos treated with 5 and 50 nM TSA for 10 h. No. of embryo developed to each stages (%) No. of No. of Concentration No. of Morula/ transferred No. of implanted No. of live clone of TSA (nM) cultured Two-cell Four-cell blastocyst (recipients) pragnancies sites (%) offspring (%) 5 148 139 (93.9) 101 (68.2) 70 (47.3) 70 (4) 3 31 (44.3) 2 (2.9) 50 146 140 (95.9) 103 (70.5) 89 (61.0) 89 (5) 3 28 (31.5) 2 (2.2) Note: Values with different superscripts are significantly different (p < 0.05). Table 5. Full-term development of SCNT embryos transferred alone and cotransferred with PA embryos. Type of No. of embryos No. of embryo transferred No. of No. of recipients pregnant implanted No. of implanted No. of live clone transfer (SCNT + PA) recipients at 19.5 dpc (%) sites (%) SCNT sites (%) offspring (%) 1 b Cotransfer 271 + 256 30 8 (26.7) 127 (24.1) 53 (19.6) 8 (2.95) SCNT 115 6 3 (50.0) – 34 (29.6) 3 (2.60) Note: Values with different superscripts are significantly different (p < 0.05). PA embryos and SCNT embryos were transferred to opposite oviducts in the same recipient. Animal Cells and Systems 329 Table 6. Full-term development of SCNT embryos and aggregated SCNT embryos. No. of embryos No. of No. of pregnant recipients at No. of implanted No. of live clone Type of Embryo transferred recipients 19.5 dpc (%) sites (%) offspring (%) a c Aggregated SCNT 252 14 10 (71.4) 100 (39.7) 9 (3.57) SCNT 258 16 8 (50.0) 63 (24.4) 3 (1.16) Note: Values with different superscripts are significantly different (p < 0.01). Aggregated embryos were SCNT embryos aggregated with tetraploid embryos. shown in Table 6, there was a greater implantation rate for clone group and the IVF group (Figure 2). There was no aggregated embryos than for SCNT embryos alone (39.7% significant difference in birth weight, between the SCNT, aggregated SCNT, and IVF groups (1.487, 1.492, and vs. 24.4%; p < 0.01). The production rate of live fetuses 1.429 g, respectively; Figure 3). was slightly higher for aggregated SCNT than for SCNT alone, but the difference was not statistically significant. To compare the birth weight and placenta weight of Discussion aggregated SCNT and SCNT clones, a total of 27 SCNT fetuses and placenta were weighed and 9 aggregated We demonstrated that the aggregated SCNT method with SCNT fetuses and placenta were weighed. SCNT placen- tetraploid embryos significantly reduced placental weight. tas were significantly heavier than aggregated SCNT and High placental weight is generally known as a problem in vitro fertilization (IVF) placentas (0.287 vs. 0.215 and in cloned mice. Our results also suggest that the rates of 0.147 g, respectively; p < 0.05). There was no significant implantation and live offspring production are increased difference in placenta weight between the aggregated by the aggregate SCNT method. There was a slight increase in the rate of implantation when SCNT embryos were cotransferred with PA embryos, but there was no such 0.287 (n = 27) increase in birth rates. The efficiency of SCNT cloning 0.4 in mice is still low, and most losses of SCNT embryos 0.215 occurred during pregnancy and in the peri- or post- 0.3 (n = 9) implantation periods. The loss of chromosomes by 0.147 pseudo-polar body formation is thought to be one of the 0.2 (n = 8) causes of SCNT embryo developmental failure, and CB treatment during activation can inhibit polar body extrusion 0.1 and potential chromosome loss (Wakayama et al. 1998). In mouse oocytes, SrCl induces repetitive intracellu- 0 2 Control Aggregated SCNT lar calcium releases, similar to those that occur during fertilization (Kline & Kline 1992; Bos-Mikich et al. 1995). SCNT It has therefore been used as a reagent for mouse oocyte Figure 2. Comparison of full-term placental weight for in vitro activation in both physiological (Cuthbertson et al. 1981; fertilized (control), aggregated SCNT and SCNT embryos. *p < O’Neill et al. 1991) and cloning studies (Ono et al. 2001; 0.05 vs. control and aggregated SCNT. Kono et al. 2004). SrCl concentrations and the duration of treatment, however, vary greatly between studies, ranging from 1–100 mM and from 10 min to 6 h (Marcus 1.42 1.49 1.49 2.00 1990; Otaegui et al. 1999; Ono et al. 2001; Kono et al. (n = 8) (n = 9) (n = 27) 2004). Usually, 10 mM SrCl is included in activation 1.50 media used for cloning (Boiani et al. 2003; Kono et al. 2004; Kishigami, Wakayama, et al. 2006). Clone mouse 1.00 was produced from a hematopoietic stem cell using 3 mM SrCl (Inoue et al. 2006). In the present study, concentra- 0.50 tions of 7.5–10 mM SrCl and an exposure time of 6 h 0.00 increased the production rate of live fetuses. Control Aggregated SCNT Most of the two-cell stage embryos treated with CB SCNT (5 µg/ml) during activation had a small degree of frag- mentation between blastomeres (data not shown). Similarly, Figure 3. Comparison of birth weight of full-term fetuses reconstructed embryos derived from aged fertilization- derived from in vitro fertilized (control), aggregated SCNT, and SCNT embryos. failure oocytes had small fragments between blastomeres Weight (g) Weight (g) 330 B.-W. Sim and K.-S. Min (Wakayama et al. 2007). This fragmentation may be 1990). The cell number of trophectoderm (TE) cells especially harmful to the subsequent development of enhances the implantation ability (Ohat et al. 2008). The SCNT embryos. The most commonly used reagents for use of tetraploid embryos in the trophoblast would be useful activation are CB and CD (Bos-Mikich et al. 1997), which for a more in-depth evaluation of the developmental disrupted actin filaments. In contrast to reconstructed potential of epiblast derivatives at later (fetal) stages of NT zygotes activated in the presence of CB, reconstructed embryo the development (Jouneau et al. 2006). Mouse zygotes activated with nocodazole have many small ESCs can develop into whole stem cell-derived offspring pseudo-pronuclei (Wakayama & Yanagimachi 2001). For when aggregated with tetraploid host embryos (Nagy et al. cloning, 5 µg/ml CB is usually included in strontium- 1993). Chimera studies with 4n–2n embryos have shown containing activation media (Ono et al. 2001; Inoue et al. that tetraploid cells can contribute to functional TE and 2002). In the present study, a larger percentage of the primitive endoderm lineages, but not to the epiblast (James SCNT embryos exposed to 5 µg/ml CB developed into et al. 1996). However, the aggregation of three or more morula/blastocysts and resulted in full-term fetuses than did cleaving monkey embryos results in chimeric offspring with SCNT embryos treated with 3 µg/ml CB. Therefore, we extensive contributions to the embryo proper and extraem- suggest that 5 µg/ml CB is useful for reducing physical bryonic lineages by tetraploid cells. In the resultant fetuses, damage from micromanipulation during the enucleation. chimerism was present in all tissues and organs (Tachibana It was the first to report that TSA treatment improves et al. 2012). In the present study, the implantation rate and full-term development of mouse embryos obtained by the pregnancy rate at 19.5 dpc were significantly greater for transfer of cumulus cell nuclei (Kishigami, Mizutani, et al. aggregated SCNT embryos than for SCNT-only embryos. 2006). TSA itself is teratogenic (Svensson et al. 1998). Therefore, we suggest that the aggregated SCNT method High TSA concentrations and longer exposure times lead to may increase the efficiency of live SCNT offspring. a reduction in cloning success rates, suggesting that TSA High placental weights have been reported in cloned overdose may cause developmental defects after implanta- farm animals and mice (Young et al. 1998; Ono et al. 2001). tion (Kishigami, Wakayama, et al. 2006). As in previous SCNT clones commonly have a high rate of placental studies, we confirmed that 5 and 50 nM TSA treatments defects (Yang et al. 2007).In the present study, placenta have a similar effect on the efficiency of full-term overgrowth was only observed in SCNT clones and not in development, as reflected by similar live birth rate for the aggregated SCNT clones. It is possible that, relative to two treatments (2.9% vs. 2.2%). SCNT-only embryos, aggregated SCNT embryos were able In the present study, we confirmed that co-transfer to implant in the endometrium more efficiently, and trigger a with PA embryos did not improve the implantation and more receptive status, thereby maintaining pregnancy. pregnancy rate of SCNT embryos at 19.5 dpc. PA embryos Previous studies have also shown that aggregation of a single SCNT embryo and three tetraploid embryos reduces only develop to about 10 dpc (Surani & Barton 1983). As placental weight of clone mice (Miki et al. 2009). Previous expected, none of the PA embryos developed to the full term in the present experiment. SCNT mouse embryos reports also indicate that clone fetuses from ES cells have a have lower developmental capacity, at least until the mean placental weight of 0.32 g: significantly heavier than blastocyst stage, than either PA or fertilized embryos the placentas of ES cell-tetraploid pups and pups derived (Meng et al. 2008). In porcine studies, cotransferred from normal embryos cultured in vitro to the blastocyst fertilized embryos might have to compete with the stage (Eggan et al. 2001). In the present study, the placental developmentally inferior SCNT embryos, as, of several weight of aggregated SCNT embryos was significantly hundreds of SCNT embryos that were cotransferred with reduced in comparison to that of SCNT-only embryos. The fertilized embryos, only one was derived from a trans- reason that aggregated SCNT embryos had less hyperplastic ferred SCNT embryo (Onish et al. 2000; Verma et al. placentas than SCNT-only embryos was not identified and 2000). In rats, co-transfer of 76 NT-cloned embryo neither were the genetic and molecular factors that are blastomeres and 36 fertilized embryos into two recipients responsible for the development of hyperplastic placentas in resulted in full-term pregnancy in both recipients; how- cloned mice. Hyperplastic placentation is preceded by the ever, of the 19 pups born, only one was from a cloned hypomorphic development of diploid trophoblastic tissues embryo (Popova et al. 2006). These results indicate that shortly after implantation (Nagy et al. 1990; Jouneau et al. while cotransferred embryos might help SCNT embryos to 2006; Wakisaka-Saito et al. 2006). implant, they also might compete with them. Our results We produced a total of 36 clone mice, including nine suggest that none of the embryos migrated. heads derived from aggregated SCNT. One-half of clones Tetraploid embryos are not capable of independently derived from aggregated SCNT survived to adulthood and completing normal development (Caufman & Webb 1990). 14-clones derived from SCNT grew into healthy adults. Of They develop into conceptus where embryonic lineages are the clone mice derived from aggregated SCNT that reached derived entirely from the ES cells and extraembryonic adulthood, three of four were proven fertile by mating lineages arise largely from the 4n component (Nagy et al. with general males. Similarly, 13 of 14 SCNT-only clones Animal Cells and Systems 331 derived by nuclear cloning and tetraploid embryo comple- that grew to adulthood were fertile (data not shown). The mentation. Proc Natl Acad Sci USA. 98:6209–6214. aggregated SCNT method significantly reduced placental Hattori H, Nishino Y, Ko YG, Hattori N, Ohgane J, Tanaka S, weight relative to that obtained using the SCNT-only Shiota K. 2004. Epigenetic control of mouse Oct-4 gene method and improved the efficiency of SCNT. expression in embryonic stem cells and trophoblast stem cells. J Biol Chem. 279:17063–17069. Inoue K, Kohda T, Lee J, Ogonuki N, Mochida K, Noguchi Y, Acknowledgments Tanemura K, Kaneko-Ishino T, Ishino F, Ogura A. 2002. We would like to thank Dr HH Seong (Institute of Animal Faithful expression of imprinted genes in cloned mice. Science) for critical reading of this manuscript and Mrs YS Kang Science. 295:297. for valuable technical assistance. Inoue K, Ogonuki N, Hirose M, Noda S, Kim JM, Aoki F, Miyoshi H, Ogura A. 2006. Inefficient reprogramming of the hematopoietic stem cell genome following nuclear transfer. J Funding Cell Sci. 119:1985–1991. James DE, Kreutz FT, Biggs DF, Suresh MR. 1996. Production This work was supported by a grant from the Next-Generation of murine/guinea pig and rat/guinea pig heterohybridomas. BioGreen 21 Program [grant number PJ0095042014], Rural Hybridoma. 15:387–389. Development Administration Republic of Korea (http://atis.rda. Jouneau A, Zhou Q, Camus A, Brochard V, Maulny L, go.kr). However, the funders had no role in study design, data Collignon J, Renard JP. 2006. Developmental abnormalities collection and analysis, decision to publish, or preparation of the of NT mouse embryos appear early after implantation. manuscript. Development. 133:1597–1607. Kang YK, Koo DB, Park JS, Choi YH, Chung AS, Lee KK, Han References YM. 2001. Aberrant methylation of donor genome in cloned Amano T, Tani T, Kato Y, Tsunoda Y. 2001. Mouse cloned from bovine embryos. Nat Genet. 28:173–177. embryonic stem cells synchronized in metaphase with Kato Y, Tani T, Sotomaru Y, Kurokawa K, Kato J, Doguchi H, nocodazole. J Exp Zool. 289:139–145. Yasue H, Tsunoda Y. 1998. Eight calves cloned from Baguisi A, Behboodi E, Melican DT, Rollock JS, Destrempes somatic cells of a single adult. Science. 282:2095–2098. MM. 1999. Production of goats by somatic cell nucleus Kimura Y, Yanagimachi R. 1995. Intracytoplasmic sperm injec- transfer. Nat Biotechnol. 17:456–461. tion in the mouse. Biol Rerpod. 52:709–720. Boiani M, Eckardt S, Leu NA, Scholer HR, McLaughlin KJ. King TJ, Dobrinsky JR, Zhu J, Finlayson HA, Bosma W. 2002. 2003. Pluripotency deficit in clones overcome by clone Embryo development and establishment of pregnancy after embryo transfer in pigs: coping with limitations in the aggregation: epigenetic complementation. EMBO J. availability of viable embryos. Reproduction. 123:507–515. 22:5304–5312. Kishigami S, Mizutani E, Ohta H, Hikichi T, Thuan NV, Bos-Mikich A, Swann K, Whittingham DG. 1995. Calcium Wakayama S, Wakayama T. 2006. Significant improvement oscillations and protein synthesis inhibition synergistically of mouse cloning technique by treatment with trichostatin A activate mouse oocytes. Mol Reprod Dev. 41:84–90. after somatic nuclear transfer. Biochem Biophys Res Com- Bos-Mikich A, Whittingham DG, Jones KT. 1997. Meiotic and 2+ mun. 340: 83–89. mitotic Ca oscillations affect cell composition in resulting Kishigami S, Wakayama S, Thuan NV, Ohta H, Mizutani E, blastocysts. Dev Biol. 182:172–179. Caufman MH, Webb S. 1990. Postimplantation development of Hikichi T, Bui HT, Balbach S, Ogura A, Boiani M, tetraploid mouse embryos produced by electrofusion. Devel- Wakayama T. 2006. Production of cloned mice by somatic opment. 110:1121–1132. cell nuclear transfer. Nat Protocols. 1:125–138. Cheong HT, Takahashi Y, Kanagawa H. 1994. Relationship Kishikawa H, Wakayama T, Yanagimachi R. 1999. Comparison between nuclear remodeling and subsequent development of of oocyte-activating agents for mouse cloning. Cloning. mouse embryonic nuclei transferred to enucleated oocytes. 1:153–159. Mol Reprod Dev. 37:138–145. Kline D, Kline JP. 1992. Repetitive calcium transients and the Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, role of calcium in exocytosis and cell cycle activation in the Ponce de Leon FA, Robl JM. 1998. Cloned transgenic calves mouse egg. Dev Biol. 149:80–89. produced from nonquiescent fetal fibroblasts. Science. Kono T, Obata Y, Wu Q, Niwa K, Ono Y, Yamamoto Y, Park ES, 280:1256–1258. Seo JS, Ogawa H. 2004. Birth of parthenogenetic mice that 2+ Cuthbertson KS, Whittingham DG, Cobbold PH. 1981. Ca can develop to adulthood. Nature. 428:860–864. increases in exponential phases during mouse oocyte activa- Kono T, Sotomaru Y, Sato Y, NAkahara T. 1993. Development tion. Nature. 294:754–757. of androgenetic mouse embryos produced by in vitro fertil- Dean W, Santos F, Stojkovic M, Zakhartchenko V, Walter J, ization of enucleated oocytes. Mol Reprod Dev. 34:43–46. Wolf E, Reik W. 2001. Conservation of methylation Lai L, Park KW, Cheong HT, Kuhhölzer B, Samuel M, Bonk A, reprogramming in mammalian development: aberrant repro- Im GS, Rieke A, Day BN, Murphy CN, et al. 2002. gramming in cloned embryos. Proc Natl Acad Sci USA. Transgenic pig expressing the enhanced green fluorescent 98:13734–13738. protein produced by nuclear transfer using colchicine-treated fibroblasts as donor cells. Mol Reprod Dev. 62:300–306. De Sousa PA, Dobrinsky JR, Zhu J, Archibald AL, Ainslie A. Marcus GJ. 1990. Activation of cumulus-free mouse oocytes. 2002. Somatic cell nuclear transfer in the pig: control of Mol Reprod Dev. 26:159–162. pronuclear formation and integration with improved methods Meng Q, Wang M, Stanca CA, Bodo S, Dinnyes A. 2008. for activation and maintenance of pregnancy. Biol Reprod. Cotransfer of parthenogenetic embryos improves the preg- 66:642–650. nancy and implantation of nuclear transfer embryos in Eggan K, Akutsu H, Loring J, Jackson-Grusby L, Klemm M. mouse. Cloning Stem Cells. 10:429–434. 2001. Hybrid vigor, fetal overgrowth, and viability of mice 332 B.-W. Sim and K.-S. Min Miki H, Inoue K, Kohda T, Honda A, Ogonuki N, Yuzuriha M, Surani MA, Barton SC. 1983. Development of gynogenetic eggs Mise N, Matsui Y, Baba T, Abe K, et al. 2005. Birth of mice in the mouse: implications for parthenogenetic embryos. produced by germ cell nuclear transfer. Genesis. 41:81–86. Science. 222:1034–1036. Miki H, Wakisaka N, Inoue K, Ogonuki N, Mori M, Kim JM, Svensson K, Mattsson R, James TC, Wentzel P, Pilartz M, Ohta A, Ogura A. 2009. Embryonic rather extraembryonic MacLaughlin J, Miller SJ, Olsson T, Eriksson UJ, Ohlsson tissues have more impact on the development of placental R. 1998. The paternal allele of the H19 gene is progressively hyperplasia in cloned mice. Placenta. 30:543–546. silenced during early mouse development; the acetylation Nagy A, Gocza E, Diaz EM, Prideaux VR, Ivanyi E, Markkula status of histones may be involved in the generation of M, Rossant J. 1990. Embryonic stem cells alone are able to variegated expression patterns. Development. 125:61–69. support fetal development in the mouse. Development. Tachibana M, Sparman M, Ramsey C, Ma H, Lee HS, Penedo 110:815–821. MCT, Mitalipov S. 2012. Generation of chimeric rhesus Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Tilghman monkeys. Cell. 148:285–295. SM. 1993. Derivation of completely cell culture-derived Tanaka S, Oda M, Toyoshima Y, Wakayama T, Tanaka M, mice from early-passage embryonic stem cells. Proc Natl Yoshida N, Hattori N, Ohgane J, Yanagimachi R, Shiota K. Acad Sci USA. 90:8424–8428. 2001. Placentomegaly in cloned mouse concepti caused by Ogura A, Inoue K, Ogonuki N, Noguchi A, Takano K, Nagano expansion of the spongiotrophoblast layer. Biol Reprod. R, Suzuki O, Lee J, Ishino F, Matsuda J. 2000. Production of 65:1813–1821. male cloned mice from fresh, cultured, and cryopreserved Verma PJ, Du ZT, Crocker L, Faast R, Grupen CG, McIlfatrick immature sertoli cells. Biol Reprod. 62:1579–1584. SM, Ashman RJ, Lyons IG, Nottle MB. 2000. In vitro Ohat H, Sakaide Y, Yamagata K, Wakayama T. 2008. Increasing development of porcine nuclear transfer embryos constructed the cell number of host tetraploid embryos can improve the using fetal fibroblasts. Mol Reprod Dev. 57:262–269. production of mice derived from embryonic stem cells. Biol Wakayama S, Suetsugu R, Thuan NV, Ohta H, Kishigami S, Rerpod. 79:486–492. Wakayama T. 2007. Establishment of mouse embryonic stem Ohgane J, Wakayama T, Kogo Y, Senda S, Hattori N, Tanaka S, cell lines from somatic cell nuclei by nuclear transfer into Yanagimachi R, Shiota K. 2001. DNA methylation variation aged, fertilization-failure mouse oocytes. Current Biol. in cloned mice. Genesis. 30:45–50. 17:120–121. Ohgane J, Wakayama T, Senda S, Yamazaki Y, Inoue K, Ogura Wakayama T. 2007. Production of cloned mice and ES cells from A, Marth J, Tanaka S, Yanagimachi R, Shiota K. 2004. The adult somatic cells by nuclear transfer: how to improve Sall3 locus is an epigenetic hotspot of aberrant DNA cloning efficiency? J Reprod Dev. 53:13–26. methylation associated with placentomegaly of cloned Wakayama T, Perry AC, Zuccotti M, Johnsom KR, Yanagimachi mice. Genes Cells. 9:253–260. R. 1998. Full-term development of mice from enucleated O’Neill GT, Rolfe LR, Kaufman MH. 1991. Developmental oocytes injected with cumulus cell nuclei. Nature. potential and chromosome constitution of strontium-induced 394:369–374. mouse parthenogenomes. Mol Reprod Dev. 30:214–219. Wakayama T, Rodriguez I, Perry AC, Yanagimachi R, Mom- Onish A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, baerts P. 1999. Mice cloned from embryonic stem cells. Proc Hanada H, Perry ACF. 2000. Pig cloning by microinjection Natl Acad Sci. 96:14984–14989. of fetal fibroblast nuclei. Science. 289:1188–1190. Wakayama T, Yanagimachi R. 1999. Cloning of male mice from Ono Y, Shimozawa N, Ito M, Kono T. 2001. Cloned mice from adult tail-tip cells. Nat Genet. 22:127–128. fetal fibroblast cells arrested at metaphase by a serial nuclear Wakayama T, Yanagimachi R. 2001. Effect of cytokinesis transfer. Biol Reprod. 64:44–50. inhibitors, DMSO and the timing of oocyte activation on Otaegui PJ, O’neill GT, Wilmut I. 1999. Parthenogenetic mouse cloning using cumulus cell nuclei. Reproduction. activation of mouse oocytes by exposure to strontium as a 122:49–60. source of cytoplasts for nuclear transfer. Cloning. 1:111–117. Wakisaka-Saito N, Kohda T, Inoue K, Ogonuki N, Miki H, Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Hikichi T, Mizutani E, Wakayama T, Kaneko-Ishino T, Dai Y, Boone J, Walker S, Ayares DL, et al. 2000. Cloned Ogura A, Ishino F. 2006. Chorioallantoic placenta defects pigs produced by nuclear transfer from adult somatic cells. in cloned mice. Biochem Biophys Res Commun. Nature. 407:86–90. 349:106–114. Popova E, Bader M, Krivokharchenko A. 2006. Full-term Wells DN, Misica PM, Tervit HR. 1999. Production of cloned development of rat after transfer of nuclei from two-cell calves following nuclear transfer with cultured adult mural stage embryos. Biol Reprod. 75:524–530. granulosa cells. Biol Reprod. 60:996–1005. Rybouchkin A, Kato Y, Tsunoda Y. 2006. Role of histone Willadsen SM. 1986. Nuclear transplantation in sheep embryos. acetylation in reprogramming of somatic nuclei following Nature. 320:63–65. nuclear transfer. Biol Reprod. 74:1083–1089. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH. Snow MH. 1973. Tetraploid mouse embryos produced by 1997. Viable offspring derived from fetal and adult mam- cytochalasin B during cleavage. Nature. 244:513–515. malian cells. Nature. 385:810–813. Siracusa G, Whittingham DG, De Felici M. 1980. The effect of Yang X, Smith SL, Tian XC, Lewin HA, Renard JP, Wakayama microtubule- and microfilament-disrupting drugs on preimplan- T. 2007. Nuclear reprogramming of cloned embryos and its tation mouse embryos. J Embryol Exp Morphol. 60:71–82. implications for therapeutic cloning. Nat Genet. 39:295–302. Shin T, Kraemer D, Pryor J, Liu L, Rugila J, Howe L, Buck S, Yoshida M, Kijima M, Akita M, Beppu T. 1990. Potent and Murphy K, Lyons L, Weshusin MA. 2002. Cell biology: A specific inhibition of mammalian histone deacetylase both cat cloned by nuclear transplantation. Nature. 415:859. in vivo and in vitro by trichostatin A. J Biol Chem. Summers MC, Bhatnager PR, Lawitts JA, Biggers JD. 1995. 265:17174–17179. Fertilization in vitro of mouse ova from inbred and outbred Young LE, Sinclair KD, Wilmut I. 1998. Large offspring strains: complete preimplantation embryo development in syndrome in cattle and sheep. Rev Reprod. 3:155–163. glucose-supplemented KSOM. Biol Reprod. 53:431–437.
Journal
Animal Cells and Systems
– Taylor & Francis
Published: Sep 3, 2014
Keywords: cloning; mice; aggregation; tetraploid; placenta
