S U Hwang’s research while affiliated with Chungbuk National University and other places

Pigs are one of the most suitable alternative laboratory models than other animals, because they have similar cardiovascular, renal and gastrointestinal organs with those of human. However, in the case of genetically engineered animals, early development of embryos is inhibited by expression of foreign genes, there are many cases of miscarriage or birth early mortality. To overcome these problems, we constructed pig glial fibrillary acidic protein (GFAP) promoter-Cre recombinase fused to a mutated ligand-binding domain of the human oestrogen receptor (CreERT2) and enhanced green fluorescent protein (EGFP)-LoxP transgenes for tamoxifen(TM)-inducible CreERT2-mediated recombination. We then established donor transgenic pig fibroblasts with pGFAP-CreERT2; LCMV-EGFPLoxP transgenes for somatic cell nuclear transfer (SCNT). We produced the SCNT embryos using a Cloud male #5 pGFAP-CreERT2+LCMV-EGFPLoxP donor cell line that was verified in vitro. It was transferred into a surrogate mother and then 5 pGFAP-CreERT2; EGFPLoxP TG piglets were born. By immunofluorescence staining and semi-nested PCR analysis, it was proved that CreER-mediated astrocytic-specific recombination system was operated in some cerebral astrocytic cells after TM-administration to TG pig #4. Additionally, we obtained brain magnetic resonance imaging (MRI) images using 3T-tesla MRI. Brain compartment volume (total brain, grey matter, white matter, cerebellum, brainstem, lateral ventricle, thalamus, midbrain, pons, medulla oblongata, hypophysis) was no significant differences between normal pig and pGFAP-CreERT2; EGFPLoxP transgenic (TG) pig. In summary, we verified the pGFAP promoter-driven CreERT2-LoxP recombination system in TG pig generated by SCNT depending on the TM administration. We suggest that this technology will be a useful tool for studying physiology of astrocytes and generating TG pig model of neurological disease such as Huntington’s disease, Alzheimer’s disease and brain tumour.

This work was supported, in part, by a grant from the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ011077, PJ011288), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean government (NRF-2013R1A2A2A04008751), Republic of Korea.

Zinc (Zn) is one of the abundant transition metals in biology and is an essential component of most cells. However, there are few reports about the effect of Zn in porcine oocytes. The objective was to investigate the effects of supplementary Zn during in vitro maturation (IVM) of porcine oocytes. We investigated nuclear maturation, intracellular glutathione (GSH) levels, reactive oxygen species (ROS) levels, and subsequent embryonic development after IVF. Before the experiment, Zn concentrations in IVM medium and body fluids were measured using inductively coupled plasma spectrophotometer (sensitivity: 1μM) and treatment concentrations were determined. Zinc concentration was 12.6μM in porcine plasma and 12.9μM in porcine follicular fluid. We confirmed that Zn was not detected in IVM medium. A total of 541 cumulus-oocyte complexes (COC) were used for the evaluation of nuclear maturation. The COC were matured in TCM-199 medium supplemented with various concentrations of Zn (0, 6, 12, 18, and 24μM). After 44h of IVM, no significant difference was observed in all groups (metaphase II rate: 85.7, 88.7, 90.4, 90.3, and 87.2%, respectively). A total of 100 matured oocytes were examined for the effects of different Zn concentrations (0, 6, 12, 18, and 24μM) on porcine oocyte intracellular GSH and ROS levels, which were measured through fluorescent staining and image analysis program. The groups of 12, 18, and 24μM showed a significant (P<0.05) increase in intracellular GSH levels (1.45, 1.67, and 1.78, respectively) compared with the control and 6μM group (1.00 and 1.08, respectively). The intracellular ROS level of oocytes matured with 12, 18, and 24μM (0.82, 0.68, and 0.55) were significantly (P<0.05) decreased compared with the control and 6μM groups (1.00 and 1.03, respectively). Finally, the developmental competence of oocytes matured with different concentrations of Zn (0, 6, 12, 18, and 24μM) was evaluated after IVF. There were no significantly different in cleavage rates. However, cleavage patterns and blastocyst (BL) formation were different. Fragmented embryo ratio of the 12μM group (14.9%) was significantly lower than that of the other groups (control, 6, 18, and 24μM: 26.4, 17.8, 18.4, and 18.0%, respectively). Oocytes treated with 12μM Zn during IVM had a significantly higher BL formation rate (28.2%) after IVF compared with the control (19.8%). In conclusion, these results indicate that Zn treatment as body fluid concentration during IVM improved the developmental potential of IVF in porcine embryos by increasing the intracellular GSH concentration and decreasing the ROS level.

The purpose of this study was to investigate the effects of co-culture with cumulus-derived somatic cells (CSC) during porcine in vitro maturation (IVM) and subsequent embryonic development after IVF. The CSC were cultured in Dulbecco's modified Eagle medium for 48h with various numbers of cumulus-derived somatic cells (0.0, 2.5, 5.0, and 10.0×10(4)), and then cultured in TCM-199 for 4h before the oocytes were added. Cumulus-oocytes complexes from 3- to 6-mm follicles were matured in 500μL of TCM-199, with eCG and hCG, for 22h, and then cultured in M199 without hormones for 22h. Each experiment consisted of at least 4 replicates. Statistical analyses were carried out using SPSS 17.0 software (SPSS Inc., Chicago, IL). Percentage data were compared by one-way ANOVA, followed by Duncan's multiple range test. Data were presented as means±s.e.m. Differences were considered to be significant if the P-value was 0.05. After IVM, no significant difference (P<0.05) was observed in nuclear maturation rate among the 0.0, 2.5, 5.0, and 10.0×10(4) groups (88.0±2.37, 81.5±2.17, 87.0±1.98 and 86.0±1.93%, respectively). The 2.5×10(4) group showed a significant (P<0.05) increase in intracellular glutathione (GSH) levels compared with that of the other groups. Intracellular reactive oxygen species (ROS) levels of mature oocyte in all groups showed no significant differences. The developmental competence of matured oocytes in all groups was evaluated after IVF. The 2.5 and 5.0×10(4) groups showed significantly (P<0.05) high cleavage rates (60.0±4.7 and 64.52±5.9%, respectively) compared with the 0 and 10.0×10(4) groups (43.15±5.0 and 53.8±5.0%, respectively). The 2.5×10(4) group showed a significantly (P<0.05) higher BL formation rate (35.7±2.9) than control group (21.0±3.8%, respectively), and higher total cell number (127.25±7.7) compared with the 0 and 10×10(4) groups (89.3±4.0 and 92.6±3.7, respectively). In the analysis of gene expression, IVF-BL derived from the 2.5 and 5.0×10(4) groups showed higher (P<0.05) mRNA expression of PCNA, which is an essential component of the DNA replication and repair machinery and POU5F1 has been used to evaluate developmental potential in embryos. The 10.0×10(4) group showed higher (P<0.05) mRNA expression of caspase-3 and Bak as known pro-apoptotic factors, compared with the control group IVF-BL. The results of cortical granules distribution which leads digesting sperm receptor proteins ZP2 and ZP3 to block polyspermy, showed that the 2.5×10(4) group was increased significantly (P<0.05) compared with the other co-culture groups (13.7±6.1, 29.2±9.5, 18.3±0.8 and 19.52±5.3, respectively). In conclusion, co-culture with 2.5×10(4) cumulus-derived somatic cells during IVM improved the developmental potential of porcine IVF embryos by increasing the intracellular GSH level and distribution of cortical granules during oocyte maturation.

The objective was to investigate the effects of zinc (Zn) insufficiency during in vitro maturation (IVM) of porcine oocytes. Zinc insufficiency was induced by treatment of Zn chelator, N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylendiamine (TPEN). In experiment 1, we investigated the effect of duration of Zn insufficiency in IVM on oocytes maturation and subsequent embryonic development after parthenogenetic activation (PA). First, 10μM TPEN was added to the IVM medium for 0, 7, 15, or 22h. After TPEN treatment, 10μM Zn were supplemented on IVM medium except in the 0h group. Reductions in the nuclear maturation rates were dependent on TPEN duration. The 0-h-treated oocytes showed 83.9±3.9% metaphase II (MII) rate; the 7-h-treated oocytes had significantly lower MII rate (44.8±3.0%) than 0-h-treated oocytes. The majority of 15- and 22-h-treated oocytes were arrested at metaphase I (MI rate: 98.0±1.0 and 97.2±1.7%, MII rate: 0 and 0%, respectively). Embryonic developmental competence was similar to maturation results. Reduction in cleavage and blastocyst (BL) rates were also dependent on duration of TPEN treatment (cleavage rate: 65.3±1.4, 42.6±4.8, 2.6±0.1, and 3.0±1.6%; BL formation rate: 29.3±2.8, 9.2±1.5, 0, and 0% for 0, 7, 15, and 22h). Total cell number of BL was also significantly different. Total cell number of BL in the 0-h-treated group (51.4±4.5) was significantly higher than that in the 7-h-treated group (23.2±1.6). In experiment 2, to confirm that the Zn insufficiency caused oocyte immaturities and loss of developmental competence in TPEN-treated oocytes, we investigated nuclear maturation and subsequent embryonic development following 3 groups: (1) non treatment (control); (2) 10μM TPEN treatment during 22h of IVM; (3) 10μM TPEN+10μM Zn treatment during 22h of IVM. Only TPEN-treated oocytes and TPEN+Zn-treated oocytes showed contrasting results. Oocyte maturation rates and subsequent embryonic development competence in TPEN with Zn-treated oocytes were similar to control (MII rate: 93.0±1.2 and 92.7±1.8%, BL formation rate: 42.0±6.7 and 40.0±7.5% for TPEN+Zn-treated oocytes and control). These results were significantly different compared with only TPEN-treated oocytes' results (MII rate: 0.61±0.61%, BL formation rate: 0%). In conclusion, Zn is an essential element for successful oocyte maturation and embryo development in porcine. Zinc insufficiency caused meiotic block and had lasting effects on early embryo development.

The aims of this study were to collect oocytes from ovaries of Delphinus capensis (long-beaked common dolphin) and to examine the morphological characteristics of their reproductive organ. Female genital organs were collected from 2 incidentally caught Delphinus capensis (ID-96: estimated 5-6 years, and ID-97: estimated 7-8 years). Collected ovaries were transported to the laboratory over 3.5h in 0.9% saline at 39°C. To perform assisted reproductive techniques in wild animals, it is important to maximize the number of oocyte recovered per ovary. In this study, Delphinus capensis oocytes were collected by 2 methods: (1) aspiration of visible follicles (2 to 3mm in diameter) and (2) remnant dissection, in which the ovary is finely dissected. The collected cumulus-oocyte complexes (COC) were separated by ovary of origin and collection method. Oocytes were subjected to in vitro maturation (IVM). Immature oocytes were placed into IVM medium [TCM-199 supplemented with 10% (vol/vol) fetal bovine serum, 0.5IUmL(-1) LH, and 0.5IUmL(-1) porcine FSH] in the 4-well dish for 120h at 37°C in 5% CO2 in a humidified atmosphere. The uterus type was observed to be a uterus bicornis. Morphology of ovary was similar to the bovine ovary among land animals. In total, 114 oocytes were obtained. From the ovary of ID-97, the number of oocytes recovered by aspiration and ovary dissection were 15 and 57, respectively. However, only 42 oocytes were recovered by ovary dissection from the ovary of ID-96. The number of oocytes recovered per ovary by dissection (49.5±7.5) was higher than that by the aspiration (7.5±7.5) method, but not significantly different (P=0.06). For accurate statistical analysis, additional trials will be performed. Most oocytes had either no cumulus cells or had only 1 to 2 layers attached to the zona pellucida enclosing a homogeneous cytoplasm. After IVM, all oocytes were at the metaphase I stage, with even fewer attached cumulus cells than at recovery. To summarise, we describe the gross morphology of the female reproductive tract of the long-beaked common dolphin and determined that dissection of the ovary is required for maximal recovery of oocytes.

Metabolic syndrome shows a complication at risk for cardiovascular disease and diabetes. With the high prevalence of obesity globally, the incidence of metabolic syndrome is increased. However, the basic mechanisms of metabolic syndrome are not completely known yet. Therefore, we attempted to develop large-animal model for the study of metabolic syndrome disease. Some studies have shown that constitutive overexpression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in mice leads to metabolic syndrome. Therefore, we tried to produce the 11β-HSD1 gene overexpressed pig by somatic cell nuclear transfer (SCNT). First, we established 11β-HSD1 overexpressed cells for the preparation of the transgenic (TG) donor cells. Porcine fetal fibroblasts were transfected with cytomegalovirus vector that included the porcine 11β-HSD1 gene. The 11β-HSD1-TG cell was injected into the enucleated ooplasm to produce 11β-HSD1-TG cloned embryos. In total, 833 TG porcine SCNT embryos (TG-SCNT embryos) were made. Of these, 155 TG-SCNT embryos were cultured in procine zygote medium-3 to evaluate the in vitro developmental potential of TG-SCNT embryos. Among these porcine SCNT-TG embryos, 109 embryos (70.3%) were cleaved at 48h. On Day 7, 31 transgenic porcine SCNT embryos (20.0%) developed to the blastocyst stage. The remaining 678 TG-SCNT embryos were transferred to 6 surrogates (average 113 embryos per surrogate). On 25 days after embryo transfer, 2 surrogates were diagnosed as pregnant (pregnancy rate, 33.3%). On Day 114, we obtained 9 live piglets and 3 stillborn piglets from 2 surrogates. By PCR analysis, we confirmed that 1 live piglet and 2 stillborn piglets were integrated with 11β-HSD1 gene. We succeeded to obtain TG piglets at sixth trials, and for the re-cloning by SCNT, a stable cell line transfected with the 11β-HSD1 gene was established from a TG cloned piglet. This study presents new possibilities for large-animal model development for the study of metabolic syndrome disease.

Evaluation of morphology of first polar body (1st PB) could be a method for the oocyte's quality and developmental competence. The developmental potential of oocyte with fragmented PB after in vitro maturation (IVM) is a controversial issue. The aim of this study is to investigate the effects of PB morphology type on oocyte quality and developmental competence after IVF. Porcine ovaries were obtained from prepubertal gilts at a local slaughterhouse and transported to the laboratory within 2h in physiological saline supplemented with 100IUmL(-1) penicillin G and 100mgmL(-1) streptomycin sulfate. The cumulus-oocyte complexes (COC) were aspirated using an 18-gauge needle attached to a 10-mL disposable syringe from superficial follicles 3 to 6mm in diameter followed by IVM. After IVM, oocytes were classified into 3 types as follows, oocytes with normal PB (A type), oocytes with a little of fragmented PB (B type), and oocytes with separated 2 PBs (C type), respectively. As classification of PB types, we analysed the distribution ratio of each PB type after IVM, and then performed IVF for analysis of fertilization rate and developmental potential. The ratio of oocyte with A type (73%) was significantly (P<0.05) higher than that of B type (24.5%) or C type (2.5%) after IVM. Only mature oocytes were selected from A and B type and were subjected to IVF because of a small number of oocytes with C type. In the IVF experiment, the efficiency of monospermy and fertilization were significantly higher in oocytes of A type (46.7%) than those of type B (20.0%). The cleavage rate of oocytes with A type (63.9%) was significantly (P<0.05) higher than the oocytes with B type (43.8%). Embryonic developmental competence to the blastocyst stage after IVF was significantly (P<0.05) higher in the A-type oocytes (26.3%) than in the B-type oocytes (16.9%). The levels of glutathione and reactive oxygen species were not affected by the morphological classification of the PB. In summary, these results suggest that polar body morphology could be a marker of oocyte quality after IVM. We are currently studying gene expression of each oocytes and blastocysts.