돼지의 체세포 핵이식(Somatic cell nuclear transfer,SCNT)은 인간에게 약리적 효과가 있는 단백질, 이종 간 장기이식(xenotransplantation)에 사용되는 장기, 질병 연 구 목적의 모델 동물을 제공한다. 특히 형질전환 돼지를 활용한 심장 이식이 세계 최초로 성공한 후 형질전환 돼 지 생산의 안정화는 다음 연구를 위한 중요한 점으로 대 두되고 있으나, 미니돼지의 체세포 핵이식 배아의 생산 효율은 아직 낮은 실정이다. 형질전환의 성공은 양질의 SCNT 배아 생산에서 시작되어야 한다. 이러한 SCNT 배 아의 생산 효율을 향상할 수 있는 요인 중에는 공여 세포 의 형태가 있으며, 성공적인 공여 세포의 생산을 위해서 는 종축에 따른 세포의 특성을 파악하여야 하고, 혈액형 의 차이에서 발생하는 문제점 해결을 위해 OO 타입의 선 별이 필요하다. 본 연구에서는 지속적인 계대 배양을 통 하여 공여 세포로 사용되는 미니돼지의 태아섬유아세포의 계대 배양 조건을 확립하고자 한다. 또한 미니돼지의 혈 액형을 PCR 기반으로 분석하여 분류하고 OO 타입의 선 별을 통하여 이종 간 이식에 용이하게 공여 세포의 조건 을 확립하였다. 이후 sgRNA(single guide RNA)를 사용하 여 CRISPR-Cpf1로 GGTA1(α-1,3 galactosyl-transferase) 유전자를 knock-out 한 미니돼지의 생산으로, 급성면역반 응을 유발하는 Gal(1,3)Gal epitope이 제거된 미니돼지의 세포 주를 구축 및 체세포 핵이식을 통해 GGTA1 knock-out 미니돼지를 생산하였으며, 이러한 연구는 이후 체세포 핵이식 및 이종 간 장기이식에 중요한 기초자료로 사용될 것이라고 생각된다.

Somatic cell nuclear transfer (SCNT) in pigs has been used as a very important tool to produce transgenic for the pharmaceutical protein, xenotransplantation, and disease model and basic research of cloned animals. However, the production efficiency of SCNT embryos is very low in pigs and miniature pigs. The type of donor cell is an important factor influencing the production efficiency of these cloned pigs. Here, we investigated the developmental efficiency of SCNT embryos to blastocysts and full term development using fetal fibroblasts (FF) and mesenchymal stem cells (MSCs) to identify a suitable cell type as donor cell. We isolated each MSCs and FF from the femoral region and fetus. Cultured donor cell was injected into matured embryos for cloning. After that, we transferred cloned embryos into surrogate mothers. In term of in vitro development, the SCNT embryos that used MSCs had significantly higher in cleavage rates than those of FF (81.5% vs. 72%) (p<0.05), but the blastocyst formation rates and apoptotic cell ratio was similar (15.1%, 6.18% vs. 20.8%, 9.32%). After embryo transferred to surrogates, nine and nineteen clone piglets were obtained from the MSCs and FF group, respectively, without significant differences in pregnancy and birth rate (50%, 40% vs. 52.3%, 45.4%) (p>0.05). Moreover, there was no significant difference in the corpus hemorrhagicum numbers of ovary, according to pregnancy, abortion, and delivery of surrogate mothers between MSCs and FF groups. Therefore, the MSCs and FF are useful donor cells for production of clone piglets through SCNT, and can be used as important basic data for improving the efficiency of production of transgenic clone pigs in the future.

Mesenchymal stem cells (MSCs) have been widely used as donor cells for somatic cell nuclear transfer (SCNT) to increase the efficiency of embryo cloning. Since replicative senescence reduces the efficiency of embryo cloning in MSCs during in vitro expansion, transfection of telomerase reverse transcriptase (TERT) into MSCs has been used to suppress the replicative senescence. Here, TERT-transfected MSCs in comparison with early passage MSCs (eMSCs) and sham-transfected MSCs (sMSCs) were used to evaluate the effects of embryo cloning with SCNT in a porcine model. Cloned embryos from tMSC, eMSC, and sMSC groups were indistinguishable in their fusion rate, cleavage rate, total cell number, and gene expression levels of OCT4, SOX2 and NANOG during the blastocyst stage. The blastocyst formation rates of tMSC and sMSC groups were comparable but significantly lower than that of the eMSC group (p < 0.05). In contrast, tMSC and eMSC groups demonstrated significantly reduced apoptotic incidence (p < 0.05), and decreased BAX but increased BCL2 expression in the blastocyst stage compared to the sMSC group (p < 0.05). Therefore, MSCs transfected with telomerase reverse transcriptase do not affect the overall development of the cloned embryos in porcine SCNT, but enables to maintain embryo quality, similar to apoptotic events in SCNT embryos typically achieved by an early passage MSC. This finding offers a bioengineering strategy in improving the porcine cloned embryo quality.

The establishment of porcine embryonic stem cells (ESCs) from porcine somatic cell nuclear transfer (SCNT) blastocysts is influenced by in vitro culture day of porcine reconstructed embryo and feeder cell type. Therefore, the objective of the present study was to determine the optimal in vitro culture period for reconstructed porcine SCNT embryos and mouse embryonic fibroblast (MEF) feeder cell type for enhancing colony formation efficiency from the inner cell mass (ICM) of porcine SCNT blastocysts and their outgrowth. As the results, porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days showed significantly increased efficiency in the formation of colonies, compared to those for 7 days. Moreover, MEF feeder cells derived from outbred ICR mice showed numerically the highest efficiency of colony formation in blastocysts produced through in vitro culture of porcine SCNT embryos for 8 days and porcine ESCs with typical ESC morphology were maintained more successfully over Passage 2 on outbred ICR mice-derived MEF feeder cells than on MEF feeder cells derived from inbred C57BL/6 and hybrid B6CBAF1 mice. Overall, the harmonization of porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days and MEF feeder cells derived from outbred ICR mice will greatly contribute to the successful establishment of ESCs derived from porcine SCNT blastocysts.

Somatic cell nuclear transfer derived embryonic stem cells (NT-ESCs) have significant advantages in various fields such as genetics, embryology, stem cell science, and regenerative medicine. However, the poor establishment of NT-ESCs hinders various research. Here, we applied fasudil, a Rho-associated kinase (ROCK) inhibitor, to develop somatic cell nuclear transfer (SCNT) embryos and establish NT-ESCs. In the study, MII oocytes were isolated from female B6D2F1 mice and performed SCNT with mouse embryonic fibroblasts (MEFs). The reconstructed NT-oocytes were activated artificially, and cultured to blastocysts in KSOM supplemented with 10 μM fasudil. Further, the blastocysts were seeded on inactivated MEFs in embryonic stem cell medium supplemented with 10 μM fasudil. A total of 26% of embryos formed into blastocysts in the fasudil treated group, while this ratio was 44% in the fasudil free control group. On the other hand, 30% of blastocysts were established NT-ESCs after exposure of fasudil, which was significantly higher than the control group (10%). The results suggest that fasudil reduced blastocyst development after SCNT due to inhibition of 2 cell cleavage while improved the establishment of NT-ESCs through the anti-apoptotic pathway.

Although somatic cell nuclear transfer (SCNT)-derived embryonic stem cells (ESCs) in pigs have great potential, their use is limited because the establishment efficiency of ESCs is extremely low. Accordingly, we tried to develop in-vitro culture system stimulating production of SCNT blastocysts with high performance in the colony formation and formation of colonies derived from SCNT blastocysts for enhancing production efficiency of porcine ESCs. For these, SCNT blastocysts produced in various types of embryo culture medium were cultured in different ESC culture medium and optimal culture medium was determined by comparing colony formation efficiency. As the results, ICM of porcine SCNT blastocysts produced through sequential culture of porcine SCNT embryos in the modified porcine zygote medium (PZM)-5 and the PZM-5F showed the best formation efficiency of colonies in α-MEM-based medium. In conclusion, appropriate combination of the embryo culture medium and ESC culture medium will greatly contribute to successful establishment of ESCs derived from SCNT embryos.

The osmolarity of a medium that is commonly used for in vitro culture (IVC) of oocytes and embryos is lower than that of oviductal fluid in pigs. In vivo oocytes and embryos can resist high osmolarities to some extent due to the presence of organic osmolytes such as glycine and alanine. These amino acids act as a protective shield to maintain the shape and viability in high osmotic environments. The aim of this study was to determine the effects of glycine or/and alanine in medium with two different osmolarities (280 and 320 mOsm) during IVC on embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. To this end, IVC was divided into two stages; the 0-2 and 3-7 days of IVC. In each stage, embryos were cultured in medium with 280, 320, or 360 mOsm and their combinations with or without glycine or/and alanine according to the experimental design. Treatment groups were termed as, for example, "T(osmolarity of a medium used in 0-2 days of IVC)-(osmolarity of a medium used in 3-7 days of IVC)" T280-280 was served as control. When PA embryos were cultured in medium with various osmolarities, T320-280 showed a significantly higher blastocyst formation (29.0%) than control (22.2%) and T360-360 groups (6.9%). Glycine treatment in T320-280 significantly increased blastocyst formation (50.4%) compared to T320-280 only (36.5%) while no synergistic was observed after treatment with glycine and alanine together in T320-280 (45.7%). In contrast to PA embryonic development, the stimulating effect by the culture in T320-280 was not observed in SCNT blastocyst development (27.6% and 23.7% in T280-280 and T320-280, respectively) whereas the number of inner cell mass cells was significantly increased in T320-280 (6.1 cells vs. 9.6 cells). Glycine treatment significantly improved blastocyst formation of SCNT embryos in both T280-280 (27.6% vs. 38.0%) and T320-280 (23.7% vs. 35.3%). Our results demonstrate that IVC in T320-280 and treatment with glycine improves blastocyst formation of PA and SCNT embryos in pigs.

Nitric oxide (NO) has an important role in oocyte maturation and embryonic development in mammals. This study examined the effect of exogenous NO donor S-nitroso-N-acetylpenicillamine (SNAP) in a maturation medium on meiotic progression and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. When oocytes were exposed to 0.1 μM SNAP for first 22 h of in vitro maturation (IVM) in Experiment 1, SNAP significantly improved blastocyst development in both defined and standard follicular fluid-supplemented media compared to untreated control (48.4 vs. 31.7-42.5%). SNAP treatment significantly arrested meiotic progression of oocytes at the germinal vesicle stage at 11 h of IVM (61.2 vs. 38.7%). However, there was no effect on meiotic progression at 22 h of IVM (Experiment 2). In Experiment 3, when oocytes were treated with SNAP at 0.001, 0.1 and 10 μM during the first 22 h of IVM to determine a suitable concentration, 0.1 μM SNAP (54.2%) exhibited a higher blastocyst formation than 0 and 10 μM SNAP (36.6 and 36.6%, respectively). Time-dependent effect of SNAP treatment was evaluated in Experiment 4. It was observed that SNAP treatment for the first 22 h of IVM significantly increased blastocyst formation compared to no treatment (57.1% vs. 46.2%). Antioxidant effect of SNAP was compared with that of cysteine. SNAP treatment significantly improved embryonic development to the blastocyst stage (49.1-51.5% vs. 34.4-37.5%) irrespective of the presence or absence of cysteine (Experiment 5). Moreover, SNAP significantly increased glutathione (GSH) content and inversely decreased the reactive oxygen species (ROS) level and mitochondrial oxidative activity in IVM oocytes. SNAP treatment during IVM showed a stimulating effect on in vitro development of SCNT embryos (Experiment 7). These results demonstrates that SNAP improves developmental competence of PA and SCNT embryos probably by maintaining the redox homeostasis through increasing GSH content and mitochondrial quality and decreasing ROS in IVM oocytes.

Polo-like kinase 1 (Plk1) has multiple roles in somatic cell and mammalian oocyte division. In mice, Plk1 distributes to the centromeres from prophase to anaphase and compose spindle apparatus in mitosis stages. Somatic cell nuclear transfer (SCNT) has diverse advantages. However, low cloning efficiency of SCNT procedure causes difficulty to application. The causes of this low efficiency are still unclear. However, they are attributed to the cumulative results of several biological and technical factors. In this study, Plk1, a biological factor, was investigated. B6D2F1 mice (7 weeks old) were superovulated with 10 IU of pregnant mare’s serum gonadotropin and 9 U of human chorionic gonadotropin (HCG) 48 hr later. The oocytes were collected 14 hr after HCG injection and cultured on potassium simplex optimized medium. The BI2536, Plk1-specific inhibitor, was used to understand the influence of Plk1. Also, the embryos were assessed by immunofluorescence. All BI2536-treated embryos failed to the first mitotic division. It showed Plk1 has a critical role in the first mitotic division of the mouse embryo. Moreover, there were significant differences between the control and SCNT embryos in the patterns of Plk1. All SCNT embryos which failed 2-cell development presented incorrect positioning and low expression of Plk1. On the other hand, the control embryos which failed to 2-cell division showed only low expression of Plk1. Taken together, this results demonstrate that Plk1 is critical for successful mitotic division of mouse embryos. Also, correct localization of Plk1 has crucial effect in the development of murine SCNT embryos.

Interferon tau (IFNT), has known as a key signal molecule for a period of pregnancy in ruminants owing to the need on maternal recognition of pregnancy. It is generated in trophectoderm cells of the elongation bovine conceptus at day 13-21 and a peak output is at day 15-17 of pregnancy period. Moreover, other studies indicated that it can be effective in the embryonic development and quality. In previous study, there were 8 bovine IFNT, but only 2 forms of IFNTs, IFNT2 and IFN-tau-c1, were expressed by the conceptuses during the peri-implantation. In this study, we target the one between the two, IFN-tau-c1 and then the effect of IFNT knockout in donor cells to bovine cloned embryonic development by somatic cell nuclear transfer (SCNT) was investigated. In order to proceed this study, the immature oocytes from the ovaries at local slaughterhouse have been matured in vitro for 22 hours. For preparing the donor cell that have a mutation on IFNT gene, somatic cells were transiently transfected with Cas9 protein and single guide RNA targeting IFNT, and various single derived colonies with high proliferation were isolated and confirm the mutation by PCR. Finally, one colony had mono-allelic mutation (4bps deletion) was picked out and applied as the donor cell to SCNT. A donor cell was injected into an oocyte that nucleus was removed. Reconstructed oocytes with the donor cell were fused by electrical shock, activated by chemical stimulation and cultured for 7 days in chemically defined medium. In this study, control (n=199) and IFNT knockout-group (n=219) were compared with four replications. As results, there was no significant difference between control-and IFNT-knockout group not only in cleavage rate, but also blastocyst formation rate (Control: 12.3% ± 9.2, IFNT knockout-group: 20.1 ± 11%). In addition, the number of blastocyst cell was not different between control (91.7 ± 26.2) and IFNT knockout group (83.5 ± 21.3). Some IFNT mutated blastocysts from SCNT were randomly selected for confirmation of the deletion of IFNT and all samples were positive for mutation. In conclusion, these data indicated that the interruption of IFNT did not influence the embryonic development. In future study, we will transfer these mutated embryos toto test the effect of IFNT for pregnancy period. This work was supported by BK21 PLUS Program for Creative Veterinary Science, the National Research Foundation of Korea (2017R1A2B3004972) and the Technology Development Program (S2566872) by MSS.

The purpose of this study is to examined the electrofusion and activation conditions for the production of porcine somatic cell nuclear transfer (SCNT) embryos. In this study, immature oocytes were cultured in TCM-199 with and without hormones for 22 hours. Skin fibroblasts cells of porcine were transferred into the perivitelline space of enucleated in vitro matured oocytes. Cell fusion was performed with two different pulses that each one pulse (DC) of 1.1 kV/cm or 1.5 kV/cm for 30 μsec. After fusion subsequent activation were divided into three groups; non-treatment (control) and treatment with 2 mM 6-DMAP or 7.5 μg/ml cytochalasin B for 4 hours. Transferred embryos were cultured in PZM-3 (Porcine Zygote Medium-3) in 5% CO₂ and 95% air at 39℃ for 7 day. Apoptosis-related genes (Caspase-3, BCL-2, mTOR, and MMP-2) were analyzed by immunofluorescence staining. There was no significant difference between two different electrofusion stimuli in the cleavage rate; 64.9±4.8% in 1.1 kV/cm and 62.7±4.0% in 1.5 kV/cm. However, blastocyst formation rate (%) was significantly different among three different activation groups (no treatment, 2 mM 6-DMAP or 7.5 μg/ml cytochalasin B) combined with electrofusion of 1.1 kV/cm. The blastocyst formation rate was 12.6±2.5, 20.0±5.0, and 34.9±4.3% in control, 2 mM 6-DMAP, and 7.5 μg/ml cytochalasin B, respectively. Immunofluorescence data showed that expression levels of caspase-3 in SCNT embryos undeveloped to blastocyst stage were higher than those in the blastocyst stage embryos. Expression levels of Bcl-2 in blastocyst stage embryos were higher than those in the arrested SCNT embryos. These results showed that the combination of an electric pulse (1.1 kV/cm for 30 μsec) and 7.5 μg/ml cytochalasin B treatment was effective for production of the porcine SCNT embryos.

This study was conducted to establish the optimal chemical post-activation conditions in porcine embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) using 4 different chemical compositions (cytochalasin B (CB), cyclohexamide (CHX), demecolcine (DC), 6-dimethylaminopurine (DMAP). Porcine embryos were produced by PA and SCNT and then, cultured for post-activation with CB (7.5 μg/mL), CB (7.5 μg/mL) + CHX (10 μg/mL), CB (7.5 μg/mL) +DC (0.4 μg/mL), and CB (7.5 μg/mL) + DMAP (2 mM). In PA embryonic development, cleavage rates have been significantly higher in CB group (94.7%) and CB+DMAP group (94.1%) than that of CB+CHX and CB+DC group (88.1 and 84.3%, respectively). There have been no significant differences in blastocyst formation rates among the four groups. In cell number of blastocyst was shown in CB group (42.3%) significantly higher than CB+CHX and CB+DC group (40.6 and 40.6%, respectively). In SCNT embryonic development, CB+DMAP group (89.7%) significant differences were found on embryo cleavage rates when compared with other three groups. Blastocyst formation rates in CB+DMAP group (26.9%) were significantly higher when compared with CB, CB+CHX, and CB+DC groups (25.5, 20.2, and 22.1%, respectively). In blastocyst cell number, CB+DMAP group (41.4%) was found higher significant difference compared with other three groups. Additionally, we have investigated survivin expression in early development stages of porcine SCNT embryos for more confirmation. Our results establish that CB group and CB+DMAP group for 4 h during post-activation improves pre-implantation improvement of PA and SCNT embryos.

The Somatic cell nuclear transfer (SCNT) method can be applied to various fields such as species conservation, regenerative medicine, farming industries and drug production. However, the efficiency using SCNT is very low for many reasons. One of the troubles of SCNT is that it is highly dependent on the researcher’s competence. For that reason, four somatic cell nuclear injection methods were compared to evaluate the effect of hole-sealing process and existence of cytochalasin B (CB) on efficiency of murine SCNT protocol. As a results, the microinjection with the hole-sealing process, the oocyte plasma membrane is inhaled with injection pipette, in HCZB with CB was presented to be the most efficient for the reconstructed in SCNT process. In addition, we demonstrated that the oocytes manipulated in Hepes-CZB medium (HCZB) with CB does not affect the developmental rate and the morphology of the blastocyst during the pre-implantation stage. For this reason, we suggest the microinjection involving hole-sealing in HCZB with CB could improve SCNT process efficiency.

In most mammals, metaphase II (MII) oocytes having high maturation promoting factor (MPF) activity have been considered as good oocytes and then used for assisted reproductive technologies including somatic cell nuclear transfer (SCNT). Caffeine increases MPF activity in mammalian oocytes by inhibiting p34cdc2 phosphorylation. The objective of this study was to investigate the effects of caffeine treatment during in Vitro maturation (IVM) on oocyte maturation and embryonic development after SCNT in pigs. To this end, morphologically good (MGCOCs) and poor oocytes (MPCOCs) based on the thickness of cumulus cell layer were untreated or treated with 2.5 mM caffeine during 22-42, 34-42, or 38-42 h of IVM according to the experimental design. Caffeine treatment for 20 h during 22-42 h of IVM significantly inhibited nuclear maturation compared to no treatment. Blastocyst formation of SCNT embryos was not influenced by the caffeine treatment during 38-42 h of IVM in MGCOCs (41.1-42.1%) but was significantly improved in MPCOCs compared to no treatment (43.4 vs. 30.1%, P<0.05). No significant effects of caffeine treatment was observed in embryo cleavage (78.7-88.0%) and mean cell number in blastocyst (38.7-43.5 cells). The MPF activity of MII oocytes in terms of p34cdc2 kinase activity was not influenced by the caffeine treatment in MGCOCs (160.4 vs. 194.3 pg/ml) but significantly increased in MPCOCs (133.9 vs. 204.8 pg/ml). Our results demonstrate that caffeine treatment during 38-42 h of IVM improves developmental competence of SCNT embryos derived from MPCOCs by influencing cytoplasmic maturation including increased MPF activity in IVM oocytes in pigs.

Somatic cell nuclear transfer (SCNT) is the technique which generates embryos by transferring diploid nucleus into an enucleated oocyte, it has produced specific animals successfully in a variety of species. However, the developmental capacity of SCNT embryos is still relatively lower than that of embryos produced in vivo. Oocyte is a kind of lipid rich cells, its quality limits the efficiency of embryo production. L-carnitine is a co-enzyme facilitating the transportation of long chain fatty acids across the inner mitochondria membrane where fatty acids are used for generating adenosine triphosphate (ATP) via beta-oxidation. It also has antioxidant actions which may protect mitochondrial membranes and DNA against damage induced by reactive oxygen species (ROS). Whether L-carnitine is functional in bovine SCNT embryos are unknown. Therefore, the objective of this study was to examine the effects of L-carnitine on oocyte maturation and developmental competence of subsequent SCNT embryos. L-carnitine was supplemented during IVM, then intracellular ROS and GSH levels, mitochondrial activity, gene expression of COCs were analyzed at the end of IVM. SCNT embryos were produced subsequently, apoptosis detection and gene expression evaluation were performed in blastocysts. In the results, treatments with 1.5 mM and 3 mM L-carnitine significantly improved maturation rates (P<0.05). Treatments with 3 mM L-carnitine effectively induced improvement in nuclear maturation, intracellular GSH levels and mitochondrial activity, as well as a reduction in intracellular ROS levels (P<0.05). mRNA levels of CPT1A, ACAA1, ACAA2, AREG, EREG, SOD1, GPX4, GLUT1 and CDC2 transcripts were effectively up-regulated by 3 mM L-carnitine treatments (P < 0.05). Similarly, 3mM L-carnitine induced an increase in blastocyst developmental rates and an improvement in blastocyst quality (P<0.05). Our study indicates that L-carnitine treatment during IVM improves oocyte nuclear maturation and subsequent SCNT embryo development.

Introduction Porcine embryonic stem cells (pESCs) derived from cloned embryos might be a useful animal model in biomedical research, however, establishment of cloned pESCs is difficult by its incomplete nuclear reprogramming. Here, we report the improved development competence of porcine cloned embryos by vitamin C (VC) supplement to establish the pESCs. Materials and Methods Slaughterhouse-derived oocytes were in vitro matured for 44h and parthenogenetic and cloned embryos were produced using matured oocytes. Both of embryos were cultured for 6 days in PZM-5 media and development rates were examined. Four different concentration of VC (0, 25, 50, 100, and 200 μg/ml) was supplemented in IVM and IVC media and preimplantation developments in the 5 groups were compared in both of embryos Results and Discussion In the cleavage rates of IVM group, significantly higher rate was shown in 50 mg/ml group than other groups (84.5 ± 0.6% vs. 69.8 ± 5.5, 75.7 ± 1.8, 80.4 ± 0.2, 72.4 ± 0.1%; P<0.05), respectively. Significantly higher rates of blastocyst development also were shown in 50 mg/ml group than other groups (27.0 ± 2.0% vs. 20.4 ± 1.4, 22.1 ± 1.3, 23.7 ± 1.2, 19.6 ± 1.3%; P<0.05), respectively. In the cleavage rate of IVC group, non-significantly different with each group (84.0 ± 1.3, 86.7 ± 1.0, 88.4 ± 1.4, 76.7 ± 3.0, 64.6 ± 4.4; P<0.05). In the blastocyst rate of IVC group, significantly higher rate was shown in 25mg/ml and 50 mg/ml group than other groups (22.3 ± 1.7, 23.8 ± 1.7% vs. 19.1 ± 1.3, 15.9 ± 1.0, 5.8 ± 1.5%; P<0.05) In conclusion, supplement of 50μg/ml of VC in IVM and IVC media enhanced the development of porcine parthenogenetic embryos and these results will be a helpful information in the development of porcine cloned embryos and derivation of its embryonic stem cells.

Polo-like kinase 1 (plk1) shows multiple events of somatic cell and mammalian oocyte division. In mice, Plk1 distributes to the centromeres from prophase to anaphase and compose spindle apparatus at different stages of mitosis in spindle organization. Somatic cell nuclear transfer (SCNT) has a number of advantages however it is difficult to apply to basic or translational researches due to its low cloning efficiency. The causes of this low cloning efficiency are unclear. However, they are attributed to the cumulative results of several biological and technical factors. In this study, a biological factor plk1 was investigated. B6D2F1 mice (7–8 weeks old) were superovulated with 10 IU of pregnant mare’s serum gonadotropin and 9 U of human chorionic gonadotropin (HCG) 48 hr later. The oocytes were then collected 14 hr after HCG injection and cultured on potassium simplex optimized medium (KSOM). The plk1-specific inhibitor BI2536 was used to understand the influence of plk1. The 2-cell stage embryos were assessed by fluorescence immunoassay. In consequence, all BI2536-treated embryos failed in the first mitotic division which showed plk1 have critical role in the first mitotic division of the mouse embryo. SCNT requires enucleation of oocyte and injecting a donor cell into the enucleated cytoplast. In this process, a respectable amount of plk1 that co-localize with nucleus may be removed together. Fluorescence immunoassay and qPCR were used to monitor the change of plk1 level during SCNT. There was significant difference between the control and enucleated embryos in the level of plk1. In all division-failure 2-cell embryos, incorrect positioning of plk1 was found. Taken together, this results demonstrate that plk1 is critical for successful mitotic division of mouse SCNT 1-cell embryos.

Somatic cell nuclear transfer (SCNT) has been considered for preserving genetically valuable or endangered animals. Sapsaree is a Natianal Monument in Korea to maintian a pure pedigree. The aim of this study was to produce azoospermia Sapsaree using SCNT and identify normal reproductive ability of cloned azoospermia Sapsaree. Ear skin biopsy was performed on a thirteen-year-old azoospermia Sapsaree and ear skin fibroblasts were isolated for SCNT as donor cells. The fibroblasts were injected into enucleated in vivo matured oocytes, the couplets were electrical fused by two pulse of direct current (55 V for 15 μs) using titanium and platinum fusion needle and activated by calcium ionophore. Cloned embryos were surgically transferred into oviducts of natuarally estrus cycle synchronized recipient dogs. The fusion rate of platinum needle was 70%, which was higher than those of titanium needle (64.1%). Developmental rate to the 8 cells and 10 cell stages was higher in platinum needle group (24% and 16%, respectively) than those of platinum needle group (14.8% and 3.1%, respectively). Total 35 SCNT embryos were transferred into oviducts of 3 recipient dogs and one recipient finally delivered a puppy by caesarean section. As results, this study demonstrated that platinum fusion needle could be successfully make the reconstructed embryos and improve the efficiency of canine SCNT. Cloning azoospermia Sapsaree may contribute to conserve genetically valuable and unique pedigree. And further study should be confirm whether cloned live dog is azoospermia.

In mature oocytes, maturation promoting factor (MPF) activity is playing important roles in arrest at M-phase and its continuous phenomenon, oocyte aging. In most mammals, metaphase II oocytes show high MPF activity and have been used as ooplasts in somatic cell nuclear transfer (SCNT). Caffeine has been found to regulate MPF activity in mammalian oocytes. Caffeine inhibits p34cdc2 phosphorylation and increases MPF activity. The present study investigated the effects of caffeine treatment during last 4 hours of in vitro maturation (IVM) on oocyte maturation and embryonic development after parthenogenesis (PA) and SCNT. The IVM medium was medium-199, 10% (v/v) PFF, cysteine, pyruvate, epidermal growth factor, kanamycin, insulin, and hormones. Immature oocytes were matured in IVM medium without or with 2.5 mM caffeine during the last 4 hours of IVM. The in vitro culture medium for embryonic development was porcine zygote medium-3 containing 0.3% (w/v) bovine serum albumin. Nuclear maturation (83.6–87.2%) and intraoocyte glutathione contents (0.9–1.0 pixels/oocyte) of oocytes were not influenced by the caffeine treatment. The membrane fusion of cell-cytoplast couplets (75.5–76.5%) and cleavage (85.4–86.2%) were also not altered by the caffeine treatment. However, caffeine-treated oocytes showed higher (P<0.05) blastocyst formation after SCNT (47.5 vs. 34.3%) than untreated oocytes. Our results demonstrate that caffeine treatment during last 4 hour of IVM improves the developmental competence of SCNT embryos probably by influencing MPF activity.

Somatic cell nuclear transfer (SCNT) technique is a key point of producing transgenic animal disease models. During in vitro production of SCNT embryo, the quality of matured oocytes are one of the important factors that regulate embryo developmental capacity. In preliminary test, we confirmed the effect of fibroblast growth factor 10 (FGF10) on porcine oocyte maturation. In this study, we investigated the developmental potential of SCNT embryos treated with the 10 ng/ml FGF10 (10 F) during in vitro maturation of recipient oocytes. The polar body emission rate was significantly higher in the 10 F treated group than control group. After SCNT, although the rate of fusion was no significant difference, the rate of cleavage and blastocyst formation was significantly increased in the 10 F treated group (p<0.05). In 10 F treated group, the total cell number was increased and the percentage of apoptotic cell was decreased in the blastocyst stage at day 7 (p<0.1). The transcription level of apoptosis relative gene, Casp3 was significantly decreased, while anti-apoptosis gene BCL2l1 was increased in the 10 F treated group compared to control group. The 10 F treated group was highly expressed the reprogramming related genes, Sox2 and POU5f1. Also, the first cleaving time was more faster and the percentage of cell block was significantly lower in 10 F treated group than in control group. In this study, we confirmed that 10 ng/ml FGF10 has effect on enhance the oocyte maturation and developmental capacity. These results demonstrate that FGF10 treatment can be used for in vitro development of porcine SCNT embryos and subsequent production of transgenic animal model.