• Proceedings of the Korean Society of Developmental Biology Conference
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• 2005.10a
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• pp.87-88
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• 2005

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2005.10a
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• pp.89-90
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• 2005

• Reproductive and Developmental Biology
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• v.36 no.2
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• pp.115-120
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• 2012

This study was conducted to examine the optimal concentration and treatment time of antioxidants for inhibition of the ROS generation in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine oocytes were activated parthenogenetically, during which oocytes were treated with various antioxidants to determine the optimal concentrations and kind of antioxidants. Determined antioxidants were applied to oocytes during in vitro maturation (IVM) and/or SCNT procedures. Finally, antioxidant-treated SCNT embryos were compared with in vitro fertilized (IVF) embryos. $H_2O_2$ levels were analyzed in embryos at 20 h of activation, fusion or insemination by staining of embryos in $10{\mu}M$ 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA) dye, followed by fluorescence microscopy. $H_2O_2$ levels of parthenogenetic embryos were significantly lower in $25{\mu}M$ ${\beta}$-mercaptoethanol (${\beta}$-ME), $50{\mu}M$ L-ascorbic acid (Vit. C), and $50{\mu}M$ L-glutathione (GSH) treatment groups than each control group ($24.0{\pm}1.5$ vs $39.0{\pm}1.1$, $29.7{\pm}1.0$ vs $37.0{\pm}1.2$, and $32.9{\pm}0.8$ vs $36.3{\pm}0.8$ pixels/embryo, p<0.05). There were no differences among above concentration of antioxidants in direct comparison ($33.6{\pm}0.9{\sim}35.2{\pm}1.1$ pixels/embryo). Thus, an antioxidant of $50{\mu}M$ Vit. C was selected for SCNT. $H_2O_2$ levels of bovine SCNT embryos were significantly lower in embryos treated with Vit. C during only SCNT procedure ($26.4{\pm}1.1$ pixels/embryo, p<0.05) than the treatment group during IVM ($29.9{\pm}1.1$ pixels/embryo) and non-treated control ($34.3{\pm}1.0$ pixels/embryo). Moreover, $H_2O_2$ level of SCNT embryos treated with Vit. C during SCNT procedure was similar to that of IVF embryos. These results suggest that the antioxidant treatment during SCNT procedures can reduce the ROS generation level of SCNT bovine embryos.

• Journal of Embryo Transfer
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• v.24 no.2
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• pp.97-104
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• 2009

The objective of this study was to examine the effect of macromolecule in a maturation medium on nuclear maturation, intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were cultured in maturation medium that was supplemented with each polyvinyl alcohol (PVA), pig follicular fluid (pFF) or newborn calf serum (NBCS) during the first 22 h and the second 22 h. Oocyte maturation was not influenced by the source of macromolecules during in vitro maturation (IVM). Embryo cleavage and cell number in blastocyst after PA was altered by the source of macromolecule but no difference was observed in blastocyst formation among treatments. Oocytes matured in PVA-PVA medium showed lower rates of oocyte-cell fusion (70.4% vs. 77${\sim}$82%) and embryo cleavage (75% vs. 86${\sim}$90%) after SCNT than those matured in other media but blastocyst formation was not altered (13${\sim}$27%) by different macromolecules. pFF added to IVM medium significantly increased the intracellular GSH level of oocytes compared to PVA and NBCS, particularly when pFF was supplemented during the first 22 h of IVM. Our results demonstrate that source of macromolecule in IVM medium influences developmental competence of oocytes after PA and SCNT, and that pFF supplementation during the early period (first 22 h) of IVM increases intracellular GSH level of oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.9
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• pp.1255-1261
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• 2013

Somatic cell nuclear transfer (SCNT) is a useful tool for animal cloning, but the efficiency of producing viable offspring by SCNT is very low. To improve this efficiency in the production of cloned pigs, it is critical to understand the interactions between uterine function and cloned embryos during implantation. Lysophosphatidic acid (LPA) is a lipid mediator that plays an important role in the establishment of pregnancy in pigs; however, LPA production in the uterine endometrium of pigs carrying SCNT-cloned conceptuses has not been determined. Therefore, we investigated expression of ENPP2, an LPA-generating enzyme, in the uterine endometrium of gilts with conceptuses derived from SCNT during the implantation period. Uterine endometrial tissue and uterine flushing were obtained from gilts carrying SCNT-derived conceptuses and from gilts carrying conceptuses resulting from natural mating on d 12 of pregnancy. Our results demonstrated no difference in the level of ENPP2 mRNA expression in the uterine endometrium between gilts carrying SCNT-derived conceptuses and gilts carrying naturally-conceived conceptuses, but secretion of ENPP2 protein into the uterine lumen did decrease significantly in pigs with SCNT-derived conceptuses. These results indicate that expression and secretion of ENPP2, which are critical for appropriate LPA production and successful pregnancy, are dysregulated in the uterine endometrium of pigs carrying SCNT-derived conceptuses.

• International Journal of Oral Biology
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• v.30 no.4
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• pp.131-134
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• 2005

In the present study, performances of several in vitro maturation (IVM) systems for porcine follicular oocytes were evaluated, and an efficient chemically defined IVM system for porcine oocytes was proposed. The proposed one-step culture system supplemented with polyvinylalcohol (PVA) gave competitive efficiencies in terms of oocyte maturation and blastocyst development after parthenogenetic activation and in vitro culture, compared with the conventional two-step culture system by a supplementation of porcine follicular fluid (pFF). Additionally, it is identified that the proposed chemically defined one-step culture system yielded the comparable level of blastocyst production to the conventional maturation system in porcine somatic cell nuclear transfer (SCNT). Therefore, one can eliminate un-expected effects accompanied by supplementation of pFF. No medium replacement during whole maturation period is an additional benefit by applying this new system. Thus, these data support that the developed PVA supplemented chemically defined one-step IVM system for porcine follicular oocyte might be used in porcine SCNT program.

• Journal of Embryo Transfer
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• v.23 no.3
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• pp.133-139
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• 2008

DNA methylation is one of the reasons for poor survival of clone animals. The OCT-4 gene is essential for maintaining pluripotency of embryonic stem (ES) cells and early embryos. We previously reported that the 5'-promoter region of Oct-4 gene was a target of DNA methylation and the methylation status was changed variously during embryonic development in bovine. The study conducted to examine the expression and methylation pattern of tissue-dependent differentially methylated region (T-DMR) of Oct-4 gene in bovine somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) blastocysts. The Oct-4 gene expression was evaluated by RT-PCR and fluorescence immunocytochemistry. The methylation pattern of T-DMR was analyzed using restriction mapping and bisulfite sequencing methods. The Oct-4 transcripts were highly expressed in IVF, while they were not expressed in SCNT. The Oct-4 protein was not detected or expressed at very low level in SCNT, the intensity of Oct-4 protein, however, was strong in IVF. On the other hand, the T-DMR of Oct-4 gene was hypermethylated in SCNT compared to that of IVF. These results suggested that expression and the failure of demethylation of Oct-4 gene was closely associated with incomplete development of SCNT embryos.

• Journal of Veterinary Clinics
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• v.23 no.2
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• pp.123-128
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• 2006

Production of cloned pigs by somatic cell nuclear transfer (SCNT) has unlimited value for developing critical biotechnology such as xenotransplantation. Various efforts have been made to establish this technology, and several litters of live piglets have been produced after transfer of SCNT embryos. However, the efficiency is very low compared to piglet production by artificial insemination or natural mating. So far, most studies have been limited to in vitro production of SCNT embryos. This study was conducted to standardize a surrogate recipient (gilts) for transfer of SCNT embryos to improve pregnancy rate. Potential surrogate gilts over 7 months of age were checked for their estrous status by observing external signs; vaginal fluid, vulva redness, vulva swelling, and standing response to back pressure. Viscosity of vaginal fluid was evaluated and classified as none (0), medium (1), and strong (2). Vulva redness and swelling was respectively assessed by none or shrink (0), medium (1), strong (2). Back pressure was estimated by an immediate move (0), standing less than 10 sec (1), and standing over 10 see (2). And then ovulation status of each surrogate was classified as pre-ovulation (PO-17 surrogates), just prior to ovulation (JPO-20 surrogates), in ovulation (IO-12 surrogates), just after ovulation (JAO-14 surrogates) and after ovulation (AO-24 surrogates) at the time of surgery for embryo transfer (ET). Real-time ultrasonographic scanners have been used for pregnancy diagnosis by observing amniotic vesicles. The first pregnancy diagnosis was done on Day 30 after ET and then repeated 2-week interval. In the results, SCNT embryos transferred into JPO surrogates gave better pregnancy rates (45%) than others (4% to 11%) on Day 30 after ET. These result indicates that surrogate gilts in a status just prior to ovulation can offer optimal condition to establish pregnancy by transfer of SCNT pig embryos.

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.119-127
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• 2018

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 $30{\mu}sec$. After fusion subsequent activation were divided into three groups; non-treatment (control) and treatment with 2 mM 6-DMAP or $7.5{\mu}g/ml$ cytochalasin B for 4 hours. Transferred embryos were cultured in PZM-3 (Porcine Zygote Medium-3) in $5%\;CO_2$ and 95% air at $39^{\circ}C$ 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; $64.9{\pm}4.8%$ in 1.1 kV/cm and $62.7{\pm}4.0%$ in 1.5 kV/cm. However, blastocyst formation rate (%) was significantly different among three different activation groups (no treatment, 2 mM 6-DMAP or $7.5{\mu}g/ml$ cytochalasin B) combined with electrofusion of 1.1 kV/cm. The blastocyst formation rate was $12.6{\pm}2.5$, $20.0{\pm}5.0$, and $34.9{\pm}4.3%$ in control, 2 mM 6-DMAP, and $7.5{\mu}g/ml$ 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 $30{\mu}sec$) and $7.5{\mu}g/ml$ cytochalasin B treatment was effective for production of the porcine SCNT embryos.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.17-22
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• 2011

This study was designed to determine the effect of electric field strength, duration and fusion buffer in fusion parameters on the rate of membrane fusion between the somatic cell and cytoplast for Korean cattle (HanWoo) somatic cell nuclear transfer (SCNT) procedure. Following electrofusion, effect of 5 or $10\;{\mu}M$ $Ca^{2+}$-ionophore of activation treatment on subsequent development was also evaluated. Cell fusion rates were significantly increased from 23.1% at 20 V/mm to 59.7% at 26 V/mm and 52.9% at 27 V/mm (p<0.05). Due to higher cytoplasmic membrane rupture or cellular lysis, overall efficiency was decreased when the strength was increased to 30 V/mm (18.5%) and 40 V/mm (6.3%) and the fusion rate was also decreased when the strength was at 25 V/mm or below. The optimal duration of electric stimulation was significantly higher in $25\;{\mu}s$ than 20 and $30\;{\mu}s$ (18.5% versus 9.3% and 6.3%, respectively, p<0.05). Two nonelectrolyte fusion buffers, Zimmermann's (0.28 M sucrose) and 0.28 M mannitol solution for cell fusion, were used for donor cell and ooplast fusion and the fusion rate was significantly higher in Zimmermann's cell fusion buffer than in 0.28 M mannitol (91.1% versus 48.4%, respectively, p<0.05). The cleavage and blastocyst formation rates of SCNT bovine embryos activated by $5\;{\mu}M$ $Ca^{2+}$-ionophore was significantly higher than the rates of the embryos activated with $10\;{\mu}M$ of $Ca^{2+}$-ionophore (70.0% versus 42.9% and 22.5% versus 14.3%, respectively; p<0.05). This result is the reverse to that of parthenotes which shows significantly higher cleavage and blastocyst rates in $10\;{\mu}M$ $Ca^{2+}$-ionophore than $5\;{\mu}M$ counterpart (65.6% versus 40.3% and 19.5% versus 9.7%, respectively; p<0.05). In conclusion, SCNT couplet fusion by single pulse of 26 V/mm for $25\;{\mu}s$ in Zimmermann's fusion buffer followed by artificial activation with $5\;{\mu}M$ $Ca^{2+}$-ionophore are suggested as optimal fusion and activation methods in Korean cattle SCNT protocol.