Seon-Ung Hwang

Effects of coculture with cumulus-derived somatic cells on in vitro maturation of porcine oocytes

In the process of IVM, cumulus-oocyte complexes (COCs) separate from the follicular microenvironment, leading to the loss of endocrine interactions between follicular mural somatic cells and COCs. To restore the microenvironment, a coculture system was established using cumulus-derived somatic cells (CSCs) for IVM. The CSCs were cultured in Dulbeccos modified Eagles medium for 48 hours with varying numbers of CSCs (0.0, 2.5 × 10(4), 5.0 × 10(4), and 10.0 × 10(4)) and then cultured in tissue culture medium 199 (TCM 199) for 4 hours before adding the oocytes. Cumulus-oocyte complexes from 3- to 6-mm follicles were matured in 500 μL of TCM 199 with eCG and hCG for 22 hours and then cultured in TCM 199 without hormones for 22 hours. After IVM, the group with 2.5 × 10(4) CSCs showed a significant increase in intracellular glutathione levels compared with the control group. In the evaluation of sperm penetration, efficient fertilization was increased in the groups with 2.5 × 10(4) and 5.0 × 10(4) CSCs compared with controls (44.9 and 46.5 vs. 32.1, respectively). The mRNA expression pattern analysis in matured COCs showed a significant upregulation of PCNA, COX-2, Has2, Ptx3, and Nrf2 in the 2.5 × 10(4) CSC group compared with controls. During COC maturation at 0, 11, 22, 33, and 44 hours, the 2.5 × 10(4) and 5.0 × 10(4) CSC groups showed a significantly altered mRNA expression of BMP15 and GDF9. The developmental competence of the matured oocytes in all groups was evaluated after IVF and parthenogenetic activation (PA). After IVF, the 2.5 × 10(4) CSC group showed significantly higher cleavage, blastocyst formation rate, and total cell numbers compared with controls (60.0%, 35.7%, and 127.3 vs. 43.2%, 21.1%, and 89.3, respectively). After PA, the 2.5 × 10(4) CSC group had significantly higher blastocyst formation rate and total cell number than the control group (52.0% and 120.4 vs. 35.4% and 90.9, respectively). In conclusion, these results suggest that the presence of a population of 2.5 × 10(4) CSCs during IVM synergistically improved the developmental potential of IVF- and PA-derived porcine embryos by increasing the intracellular glutathione level via changing of a specific gene expression pattern during oocyte maturation.

Supplementation of zinc on oocyte in vitro maturation improves preimplatation embryonic development in pigs

We investigated the effects of zinc supplementation during the IVM of porcine oocytes. Nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, subsequent embryonic development, and gene expression were evaluated. Zinc concentrations in porcine plasma and follicular fluid were 0.82 and 0.84 μg/mL, respectively. Zinc was not detected in IVM medium. After treatment with various zinc concentrations (0.0, 0.4, 0.8, 1.2, and 1.6 μg/mL), no significant difference in IVM was observed among groups (85.7%, 88.7%, 90.4%, 90.3%, and 87.2%, respectively). The effects of different zinc concentrations on porcine oocyte intracellular GSH and ROS levels were examined in mature oocytes. Intracellular GSH levels were significantly higher in the 0.8-, 1.2-, and 1.6-μg/mL groups than in the control (P < 0.05). Intracellular ROS levels of oocytes matured with 0.8, 1.2, and 1.6 μg/mL were reduced significantly (P < 0.05) compared with the control and 0.4-μg/mL groups. The developmental competence of oocytes matured with different zinc concentrations was evaluated after parthenogenetic activation (PA) and in vitro fertilization (IVF). Oocytes treated with zinc during IVM showed no significant difference in cleavage rate after PA. Oocytes treated with 0.8 and 1.2 μg/mL zinc during IVM had significantly higher blastocyst formation rates after PA (41.5% and 41.1%, respectively) than the control (27.2%). IVF embryos showed similar results. The blastocyst formation rate was significantly higher (28.2%) in the 0.8-μg/mL group. TNFAIP2 and Bax were decreased in zinc-treated cumulus cells. Increased POU5F1 and decreased Bax transcript levels were observed in zinc-treated oocytes. POU5F1 and Bcl-2 transcript levels were significantly higher in zinc-treated IVF blastocysts. These results indicate that treatment with adequate zinc concentrations during IVM improved the developmental potential of porcine embryos by regulating the intracellular GSH concentration, the ROS level, and transcription factor expression.

Carboxyethylgermanium sesquioxide (Ge-132) treatment during in vitro culture protects fertilized porcine embryos against oxidative stress induced apoptosis

Compared with the in vivo environment, porcine in vitro embryo-culture systems are suboptimal, as they induce oxidative stress via the accumulation of reactive oxygen species (ROS). High ROS levels during early embryonic development cause negative effects, such as apoptosis. In this study, we examined the effects of the antioxidant carboxyethylgermanium sesquioxide (Ge-132) during in vitro culture (IVC) on embryonic development in porcine in vitro fertilization (IVF) embryos. Zygotes were treated with different concentrations of Ge-132 (0, 100, 200 and 400 μg/ml). All of the Ge-132 treatment groups displayed greater total cell numbers after IVC (98.1, 98.5 and 103.4, respectively) compared with the control group (73.9). The 200 μg/ml Ge-132 treatment group exhibited significantly increased intracellular GSH levels compared with the control group, whereas the ROS generation levels decreased in Ge-132 dose-dependent manner (P < 0.05). The mRNA expression levels of the KEAP1 gene and proapoptotic genes BAX and CASPASE3 were lower in the Ge-132 treated blastocysts compared with the control group (P < 0.05). The percentages of apoptotic and necrotic cells in the Ge-132 treated embryos on day 2 (48 h) were significantly lower than the untreated embryos (9.1 vs. 17.1% and 0 vs. 2.7%, respectively). In the day 7 blastocysts, the percentages of apoptotic cells in 200 µg/ml Ge-132 treated group were lower compared to controls (1.6 vs. 2.5%). More KEAP1 protein was found to be localized in cytoplasm of the 200 μg/ml Ge-132 treated blastocysts, whereas KEAP1 protein was predominantly nuclei in the control blastocysts. These results indicate that the developmental competence of embryos cultured under Ge-132 treatment may be associated with KEAP1 signaling cascades involved in oxidative stress and apoptosis during porcine preimplantation embryo development.

25 PRODUCTION OF TRANSGENIC PIGS WITH CreER-MEDIATED ASTROCYTIC-SPECIFIC RECOMBINATION SYSTEM FOR NEUROLOGICAL DISEASE MODELS

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 Huntingtons disease, Alzheimers disease and brain tumour.

An Improved System for Generation of Diploid Cloned Porcine Embryos Using Induced Pluripotent Stem Cells Synchronized to Metaphase.

Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently.

Zinc supplementation during in vitro maturation increases the production efficiency of cloned pigs

Zinc supplementation (0.8 µg/ml) in in vitro maturation (IVM) medium significantly enhances oocyte quality. In this study, we compared the development of somatic cell nuclear transfer (SCNT) embryos produced from conventional IVM (control) and zinc-supplemented IVM oocytes. A total of 1206 and 890 SCNT embryos were produced using control and zinc-supplemented oocytes, respectively, and then were transferred to 11 and 8 recipients, respectively. Five control recipients and three zinc-supplemented recipients became pregnant. Two live piglets and eight mummies were born from two control recipients, and ten live piglets and six stillborn piglets were born from three zinc-supplemented recipients. The production efficiency significantly increased in the zinc-supplemented group (0.33% vs. 3.02%). This report suggests that zinc supplementation in IVM medium improved the production efficiency of cloned pigs.