• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.83-90
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• 2007

Insulin-like growth factor II (IGF2) and H19 genes are mutually imprinted genes which may be responsible for abnormalities in the cloned fetuses and offspring. This study was performed to identify putative differentially methylated regions (DMRs) of porcine H19 locus and to explore its genomic imprinting in in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) embryos. Based on mice genomic data, we identified DMRs on H19 and found porcine H19 DMRs that included three CTCF binding sites. Methylation patterns in IVF and SCNT embryos at the 2-, 4-, $8{\sim}16$-cells and blastocyst stages were analyzed by BS (Bisulfite Sequencing)-PCR. The CpGs in CTCF1 was significantly unmethylated in the 2-cell stage IVF embryos. However, the 4- (29.1%) and $8{\sim}16$-cell (68.2%) and blastocyst (48.2%) stages showed higher methylation levels (p<0.01). On the other hand, SCNT embryos were unmethylayted ($0{\sim}2%$) at all stages of development. The CpGs in CTCF2 showed almost unmethylation levels at the 2-,4- and $8{\sim}16$-cell and blastocyst stages of development in both IVF ($0{\sim}14.1%$) and SCNT ($0{\sim}6.4%$) embryos. At all stages of development, CTCF3 was unmethylated in IVF ($0{\sim}17.3%$) and SCNT ($0{\sim}1.2%$) embryos except at the blastocyst stage (54.5%) of IVF embryos. In conclusion, porcine SCNT embryos showed an aberrant methylation pattern comprised to IVF embryos. Therefore, we suggest that the aberrant methylation pattern of H19 loci may be a reason for increased abnormal fetus after embryo transfer of porcine SCNT embryos.

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.139-147
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• 2018

Somatic cell nuclear transfer (SCNT) is a useful biotechnological tool for animal cloning. Until now, SCNT has been inefficient, especially in dog. It is believed that an embryo developmental block in SCNT embryos is cause of low production efficiency. However, no studies have been performed on canines for embryo developmental block. In this study, we attempted to evaluate the beneficial role of EDTA in canine parthenogenic (PA) embryos development to overcome embryo developmental block. The PA embryos were divided into 0.01 mM EDTA treated and non-treated groups. Embryo developmental efficiency was measured by activating chemically parthenote. After EDTA induction, PA embryos were evaluated for embryonic development, Reactive Oxygen Species (ROS) activity, mitochondrial integrity, ATP production and genomic activation. The EDTA treated PA embryos showed significantly higher survival rate and improved cavity formation compared to non-treated. Furthermore, cytoplasmic ROS level was mitigated and mitochondrial membrane potential was found significantly higher in EDTA treated group followed by higher ATP production. Moreover, major embryonic genomic activation specific markers/factors were also elevated in EDTA treated group. Conclusively, we elucidated that EDTA showed substantially positive effect to overcome embryo developmental block in canine.

• Journal of Embryo Transfer
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• v.32 no.4
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• pp.287-295
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• 2017

Mitochondrial dysfunction is found in oocytes and transmitted to offspring due to maternal obesity. Treatment of obese mothers with endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL) can reverse the mitochondrial dysfunction and result in normal embryonic development. Pig oocytes have also shown ER stress mostly in metaphase II stage. ER stress in oocytes may hinder the in vitro production of pig embryos. This study investigated the effect of ER stress inhibition by SAL treatment during in vitro maturation (IVM) of porcine oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we tested various concentrations of SAL. SAL at 10 nM showed higher (P < 0.05) developmental competence to the blastocyst stage (55.6%) after parthenogenesis (PA) than control (44.2%) while not different from other concentrations (49.2, 51.6, and 50.8% for 1, 50, and 100 nM, respectively). Secondly, we performed time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h of IVM significantly improved PA embryonic development to the blastocyst stage compared to control (40.5, 46.3, 51.7 and 60.2% for control, 0 to 22 h, 22 to 44 h and 0 to 44 h of IVM, respectively, P < 0.05). Glutathione (GSH) content is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on developmental competence of oocytes. For this, we determined the intraoocyte levels of GSH and ROS after 44 h of IVM. It was found that SAL increased intraoocyte GSH level and also decreased ROS level (P < 0.05). Finally, we performed somatic cell nuclear transfer (SCNT) after treating oocytes with 10 nM SAL during IVM. SAL treatment significantly improved blastocyst formation of SCNT embryos compared to control (39.6% vs. 24.7%, P < 0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development PA and cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.86-86
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• 2003

Research has been in progress for more than a decade to production of useful proteins by genetic modification in cattle. However, the levels of protein production in transgenic cattle have been reported very low. To enhance protein production in transgenic animal, we tried homologous recombination to donor cells for production of transgenic clone cattle through nuclear transfer procedure. Thus, we constructed the two targeting vectors of human thrombopoietin (TPO) at bovine $\beta$-casein locus using homologous recombination with 13.6 kb and 9.6 kb homology. In two targeting vectors, positive selection was through the neomycin resistance gene and negative selection was by the diphtheria toxin (DT). Gene targeting was attempted in bovine embryonic fibroblasts (bEF) and bovine ear skin fibroblasts (bESF). To determine the most appropriate concentration of neomycin for bEF and bESF, G4l8 resistance was confirmed by culturing the cells in various concentrations of the drug and both of the cells were optimally selected at $900 \mu g/ml$ of neomycin. The transfected bEF and bESF by the targeting vectors were colonized efficiently at the ratio of DNA to transfection reagent such as $4 \mu g$:2 ${mu}ell$ and $1 \mu g$:$2 \mu l$. Comparing number of healthy clones from passage 4 to passage 8, bESF (17%) persist in culture for much longer than bEF (6%). The two gene-targeted bESF clones of 30 random-integrated clones with 9.6 kb homology length were confirmed, however, nothing was out of 72 random integration clones with 13.6 kb homology length, The DT also worked more efficiently in clones transfected with the vector of 9.6 kb homology length. Our data suggests that the choice of donor cell for long culture period should be considered to obtain targeted cell clone, and the gene-targeting frequency and the DT working efficiency are dependent on the length of target homology.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.53-58
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• 2006

The study was conducted to evaluate the effects of culture period and fusion method on the development of somatic cell nuclear transfer (SCNT) embryos reconstituted with Korean bovine fetal fibroblast cells (KbFF) and Korean bovine adult ear skin fibroblast cells (KbESF). KbFF were isolated from a day 51 Korean cattle (Hanwoo) fetus, and KbESF were isolated from a 28 month old Hanwoo calf. The cells were cultured up to 15 weeks (passage 15) in vitro for SCNT. Chamber and electrode needles were used for comparing fusion of reconstituted eggs. The doubling times of KbFF and KbESF were 17.3 hr and 24.3 hr, respectively. The fusion and cleavage rates were significantly higher in needle group (76.1 and 81.2% respectively, P<0.05) than those in chamber group. However, the blastocyst development rate was not different between both groups. Fusion and cleavage rates of NT eggs reconstituted with KbESF did not affected by passage number, however, blastocyst rates were lower in passage $1{\sim}4$ group (21.3%) than passage $5{\sim}8$ (39.4%) and $13{\sim}15$ groups (40.4%, P<0.05). Whereas, fusion rate was lower in passage $1{\sim}4$ group (61.5%) than those of passage $5{\sim}8$(75.0%) and $13{\sim}15$ (76.8%) groups, but cleavage and blastocyst rates were similar regardless of passage number in the KbFF. The results suggest that fusion method can affect the development of SCNT embryos, whereas the long term culture up to 15 passages may not affect the development of SCNT embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.1
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• pp.44-51
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• 2012

Calcium ions play an important role in the establishment and maintenance of pregnancy, but molecular and cellular regulatory mechanisms of calcium ion action in the uterine endometrium are not fully understood in pigs. Previously, we have shown that calcium regulatory molecules, transient receptor potential vanilloid type 5 (TRPV6) and calbindin-D9k (S100G), are expressed in the uterine endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-specific manner, and that estrogen of conceptus origin increases endometrial TRPV6 expression. However, regulation of S100G expression in the uterine endometrium and conceptus expression of S100G has been not determined during early pregnancy. Thus, we investigated regulation of S100G expression by estrogen and interleukin-$1{\beta}$ (IL1B) in the uterine endometrium and conceptus expression of S100G during early pregnancy in pigs. We obtained uterine endometrial tissues from day (D) 12 of the estrous cycle and treated with combinations of steroid hormones, estradiol-$17{\beta}$ ($E_2$) and progesterone ($P_4$), and increasing doses of IL1B. Real-time RT-PCR analysis showed that $E_2$ and IL1B increased S100G mRNA levels in the uterine endometrium, and conceptuses expressed S100G mRNA during early pregnancy, as determined by RT-PCR analysis. To determine if endometrial expression of S100G mRNA during the implantation period was affected by the somatic cell nuclear transfer (SCNT) procedure, we compared S100G mRNA levels in the uterine endometrium from gilts with SCNT-derived conceptuses with those from gilts with conceptuses derived from natural mating on D12 of pregnancy. Real-time RT-PCR analysis showed that levels of S100G mRNA in the uterine endometrium from gilts carrying SCNT-derived conceptuses was significantly lower than those from gilts carrying conceptuses derived from natural mating. These results showed that S100G expression in the uterine endometrium was regulated by estrogen and IL1B of conceptus origin, and affected by the SCNT procedure during early pregnancy. These suggest that conceptus signals regulate S100G, an intracellular calcium transport protein, for the establishment of pregnancy in pigs.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.199-203
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• 2008

This study was performed to analyze the characterization of plasma hormonal levels during pregnancy in the Hanwoo recipients pregnant by artificial insemination (AI) or somatic cell nuclear transfer (SCNT) embryos. The synchronized recipients pregnant by SCNT embryos produced by Hanwoo fetal fibroblast cells (n=8) and by AI (control, n=5) were used. The plasma hormonal levels were measured by RIA (P4 and E2) and ELISA (cortisol), respectively. In control, the increase of E2 and the decrease of P4 were occurred immediately before the initiation of parturition. The expression pattern of plasma P4 was similar in both groups from 50 to 10 days before parturition, however, it did not decrease even at the expected date of labor in the SCNT recipients. The plasma cortisol was expressed a lower level during pregnancy in the SCNT recipients. But, the cortisol was increased in the cow aborted around 100 days of pregnancy (n=1). Based on these results, it can be postulated that the failure of the hormonal changes immediately before parturition in the SCNT recipients may be one of the most important reasons for a delayed parturition in clone calving.

• Journal of Veterinary Clinics
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• v.32 no.4
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• pp.301-307
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• 2015

The purpose of this study was to compare echocardiographic parameters of cloned beagle dogs with the previously reported reference range. Seven cloned dogs were assessed for anatomical features and cardiac function through left- and right-sided heart and right ventricle outflow tract from M-mode, 2D-mode, pulsed wave Doppler and tissue Doppler imaging. In all the cloned dogs, there were no abnormalities in anatomical structure and measurements were within the normal reference range. In addition, left- and right-sided myocardial function was within the normal reference range. Especially, pulmonary hypertension and right-sided heart failure frequently encountered in cloned animals were not recognized in cloned dogs. In conclusion, no evidence of cardiovascular dysfunction in mature cloned dogs could be identified either at birth or the growing stage in this study. Therefore, serious adverse effects of somatic cell nuclear transfer technology including transgenesis on cardiac morphology and function were not found in cloned dogs.

• Journal of Veterinary Clinics
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• v.31 no.4
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• pp.267-271
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• 2014

Accurate determination of in vivo oocyte maturation is particularly critical for dog cloning compared to other assisted reproductive technologies because oocytes in metaphase II stage have to be recovered in order to undergo somatic cell nuclear transfer right after its recovery. The aim of present study was to evaluate the reliability and to set a reference range of a chemiluminescence enzyme immunoassay (CLEIA) compared to radioimmunoassay (RIA) method to retrieve in vivo matured oocytes. Serum progesterone concentration during proestrus and estrus was analyzed by RIA and CLEIA to determine ovulation day (Day 0). On Day 3, in vivo oocytes were recovered surgically and evaluated microscopically maturation status after staining nucleus with bisbenzimidazole dye. Mean progesterone concentration by CLEIA ($7.64{\pm}0.06ng/ml$) was significantly higher than by RIA ($6.46{\pm}0.04ng/ml$, P < 0.0001). It was not different between CLEIA ($10.01{\pm}0.34ng/ml$) and RIA values ($7.91{\pm}0.14ng/ml$, P < 0.05) on Day 0, but significantly higher CLEIA level on Day -1 and Day 1 ($6.41{\pm}0.15$ and $14.25{\pm}0.44ng/ml$) was assessed compared to RIA ($4.95{\pm}0.10$ and $11.29{\pm}0.34ng/ml$). However, with both methods, progesterone level was significantly increased from Day -1 to Day 2. To determine oocyte maturation with CLEIA method, a wider and higher reference range has to be considered.