G. A. Kim

Supplementation with spermine during in vitro maturation of porcine oocytes improves early embryonic development after parthenogenetic activation and somatic cell nuclear transfer

Spermine plays an important role in protection from reactive oxygen species (ROS) in bacteria, yeast, and mammalian cells, but there are few studies on the effects of spermine on porcine oocyte maturation and subsequent embryo development. The aim of this study was to determine the effects of spermine on in vitro maturation (IVM) of porcine oocytes and their developmental competence after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). We evaluated nuclear maturation, intracellular glutathione (GSH), and ROS levels in oocytes, and their subsequent embryonic development, as well as gene expression in mature oocytes, cumulus cells, and PA blastocysts. After treatment with various concentrations of spermine in IVM culture medium, there was no significant difference in nuclear maturation rate. However, spermine treatment groups (10- 500 µM) showed significantly increased intracellular GSH levels and decreased ROS levels compared to the control ( < 0.05). Furthermore, 10 µM spermine supported significantly higher blastocyst formation rates after PA than the control group ( < 0.05). According to the optimal condition from the PA results, we investigated the effects of 10 µM spermine on SCNT, and it also significantly improved blastocyst formation rates compared with the control group ( < 0.05). In evaluating the effects of 10 µM spermine on gene expression, there was significantly lower expression of a proapoptotic gene () and higher expression of an antiapoptotic gene () in cumulus cells ( < 0.05). was increased in spermine-treated oocytes. Levels of transcription for and were significantly increased in PA blastocysts. In conclusion, 10 µM spermine supplementation during IVM improved the development of porcine PA and SCNT embryos by increasing intracellular GSH, scavenging ROS levels, and regulating gene expression.

37 NORMALITY OF NEONATAL REFLEX IN CLONED DOGS

Since the birth of the worlds first cloned dog, Snuppy, somatic cell nuclear transfer (SCNT) has been a useful tool to propagate the dogs with identical genetic information. However, it is known that cloned animals sometimes exhibit phenotypic instability or abnormality. There have been few investigations about the normality of the neonatal reflex in cloned animals. Therefore, the objective of this study was to evaluate the neonatal reflex in 3 breeds of cloned dogs including shepherd, retriever, and beagle from birth to 28 days of age. Through SCNT, 8 cloned dogs were produced. After birth, 3 types of neonatal reflexes were examined and scored. For examining the flexor dominance reflex, neonatal cloned dogs were held upright and the flexor position of the limb was scored. To evaluate the withdrawal and crossed extensor reflexes, neonates were placed in lateral recumbence and their forelimbs were allowed to relax. Then, the distal forelimbs were pinched and responses were scored according to the frequency and intensity (strong=score 2, variable=score 1, and absent=score 0). The standard responses of neonates were referred from Lindsay et al. (2000 Handbook of Applied Dog Behavior and Training 1, 31-47). Descriptive analysis was used, which was based on the scores from 3 referees who evaluated the videos. The flexor dominance reflex could not be observed (score 0.0) in shepherd by Day 8, in beagle by Day 14 and in retriever by Day 16. Withdrawal reflex began to decrease on Day 22 with score 1.8 for beagle and retriever but decreased in shepherd starting on Day 24 with score 1.8. Crossed extensor reflex for shepherd started to disappear on Day 14 with score 1.5 and completely disappeared (score 0.0) on Day 22; for beagle started to disappear on Day 16 with score 1.8 and was still found until Day 28 with score 1.1; for retriever started to disappear on Day 20 and 28 with score 1.7 and 0.7, respectively. Flexor dominance reflex disappeared in cloned shepherd at a similar time to standard but beagle and retriever seem delayed 6 to 8 days compared with the reference. Withdrawal reflex in all breeds showed normal changes that should persist until adulthood. Cross extensor reflex in shepherd was close to reference but in beagle and retriever was delayed beyond Day 28; this reflex should disappear before adulthood. This study demonstrated that normal neonatal reflexes were identified in the cloned dogs, with some variations among breed. To adapt neonatal reflex as a marker to confirm phenotypic normality in cloned dogs, further investigation using various breeds of cloned dogs and greater numbers of subjects is needed.

71 MELATONIN IMPROVES PORCINE IN VITRO MATURATION VIA SONIC HEDGEHOG SIGNALLING

Melatonin (N-acetyl-5-methoxytryptamine) is the hormone synthesised from the mammalian pineal gland, which has an antioxidant property and regulates physiological processes such as cellular metabolism. It is well known that melatonin affects in vitro maturation of oocytes and embryonic development in many species. However, limited information is available on the underlying beneficial effects of melatonin. Sonic Hedgehog (Shh) signalling is important for follicular development, oocyte maturation, and embryo development. To elucidate the relationship between melatonin and Shh signalling, we designed an experiment with the following three groups: (1) control, (2) melatonin, and (3) melatonin with cyclopamine (smoothened inhibitor) during porcine in vitro maturation. Porcine ovaries were collected from prepubertal gilts at a local slaughterhouse and transported to the laboratory at 28 to 32°C. The contents of follicles 3 to 6mm in diameter were recovered by aspiration with an 18G needle. Cumulus-oocyte complexes were pooled and cultured in TCM-199 medium for 44h. The aim of this study was to evaluate the effects of melatonin (10-9 M) with or without cyclopamine (2μM) on cumulus cell expansion (a total of 432 cumulus-oocyte complexes were used in 3 replicates), embryo development after parthenogenetic activation (a total of 432 oocytes were used in 4 replicates). Moreover, we detected gene expression related to cumulus expansion, oocyte maturation, and hedgehog signalling in cumulus cells and oocyte. Results indicated that melatonin treatment significantly increased cumulus expansion index (3.75±0.02v. 3.51±0.03 and 3.59±0.05, respectively; P<0.05) and blastocyst formation rates (30.4±2.4v. 21.9±2.2 and 20.0±2.2, respectively; P<0.05) compared with control and melatonin with cyclopamine. In addition, the expression of cumulus expansion-related genes (Ptgs1, Ptgs2, Has2, Ptx-3, and Tnfaip6) and hedgehog signalling-related genes (Shh, Pthc1, Smo, and Gli-1) in cumulus cells were up-regulated in melatonin treatment compared with control and melatonin with cyclopamine. Similarly, the expressions of oocyte maturation-related genes (GDF9 and BMP15) in porcine oocytes were up-regulated in melatonin treatment compared with control and melatonin with cyclopamine. In conclusion, Shh signalling mediated melatonin to improve porcine cumulus cell expansion, oocyte maturation, and subsequent embryo development. Further studies are needed to evaluate the effect of melatonin on protein levels of Shh signalling. Statistical analyses were performed using SPSS 22.0 (SPSS Inc., Chicago, IL, USA). All data were tested for normality and homoscedasticity and then subjected to one-way ANOVA, followed by Duncans multiple range test (when the variances were assumed to be equal) or Dunnets T3 test (when the variances were assumed to be unequal) to determine differences among experimental groups. All results are expressed as means±SEM; P-values<0.05 were considered to be statistically significant.

27 OXIDATIVE STRESS OF LIVER IN TRANSGENIC PIGLETS WITH MULTIPLE COPIES OF TRANSGENES SOLUBLE HUMAN TUMOUR NECROSIS FACTOR RECEPTOR TYPE Ig-Fc AND HUMAN HEME OXYGENASE-1

It has been demonstrated that transgene expression is associated with copy number in transgenic animals. Here in, we generated 7 genetically modified pigs expressing both soluble human tumour necrosis factor receptor type Ig-Fc (shTNFRI-Fc) and human heme oxygenase-1 (HO-1). 1 day after Caesarean section, all transgenic cloned piglets showed postnatal death. In the present study, the transgene copy number, H2O2 and superoxide dismutase (SOD) levels in cloned piglet liver were examined to identify the relationship between transgene copy number and oxidative stress of postnatal liver. In this study, 2,209 cloned embryos using somatic cells with 15 copies of shTNFRI-Fc and HO-1 were produced by somatic cell nuclear transfer, and transferred into 6 synchronized recipient sows. Among them, pregnancies were identified in 4 recipients using ultrasonography and only 1 recipient was maintained until full term. In total, 7 cloned piglets were delivered by the Caesarean section. On the next day, they showed postnatal death with clinical symptoms such as dyspnea (Group A). As control group, 292 cloned embryos produced from the cells with at least 4 copies of 2 transgenes shTNFRI-Fc and HO-1 were transferred into a synchronized recipient and pregnancy was identified. Two cloned piglets were delivered normally and maintained healthy. The liver of a live cloned piglet with at least 4 copies (Group B) at 2 days after the Caesarean section was isolated and compared with those of dead 7 cloned piglets (Group A) for HO-1, shTNFRI-Fc, H2O2, and SOD by ELISA analysis. The transgene copy number and expression of shTNFRI-Fc and HO-1 were confirmed by genomic DNA PCR, quantitative real-time PCR (qRT-PCR) and ELISA with appropriate antibodies. Statistical analysis was performed using Graphpad Prism. Level of HO-1, shTNFRI-Fc, H2O2, and SOD ELISA results of each piglets were analysed by unpaired t-test with Welchs correction. While a transgenic piglet (Group B) had at least 4 copy numbers, all dead cloned piglets (Group A) showed 15 copy numbers. A high level of transgene HO-1 and shTNFRI-Fc expression of liver-derived cells in cloned piglets (Group A) was significantly identified compared with those of a transgenic piglet (Group B) by qRT-PCR and ELISA. While the H2O2 level in cloned piglet liver with 15 copy numbers (Group A) was significantly higher (P<0.05), the SOD level was lower than those of a cloned pig (Group B; P<0.05). These results demonstrated that multiple copy numbers could affect the level of oxidative stress in cloned piglet liver. It also affected the transgene expression levels and mortality of cloned piglets.

174 EFFECT OF HUMAN ENDOTHELIAL PROGENITOR CELLS ON IN VITRO MATURATION OF PORCINE OOCYTES AND PARTHENOGENETIC EMBRYO DEVELOPMENT COMPETENCE

In oocyte maturation, hepatocyte growth factor and vascular endothelial growth factor (VEGF) contribute to promote granulosa cell proliferation and cumulus cell expansion. It is well known that human endothelial progenitor cells (hEPC), which are isolated from monocytes and macrophages, secrete a variety of growth factors, such as hepatocyte growth factor and VEGF, and improve the process of angiogenesis. Therefore, the aim of this study was to investigate the effects of hEPC on in vitro oocyte maturation and subsequent embryo development in pigs. To isolate and culture hEPC, human peripheral blood sample was collected from a healthy donor and peripheral blood mononuclear cells were separated. The peripheral blood mononuclear cells were seeded into flask with defined Keratinocyte-SFM-based medium and incubated at 37°C, 5% CO2. The hEPC were cultured and cryopreserved until use for co-culturing with porcine oocytes obtained from a local slaughterhouse ovaries. Cumulus-oocyte complexes were randomly cultured in 2 groups; 1) co-culturing with hEPC and 2) culturing without hEPC. Cumulus-oocyte complexes were cultured in the in vitro maturation (IVM) medium containing TCM-199 supplemented with 0.57mM cysteine, 0.91mM sodium pyruvate, 5μLmL-1 of insulin-transferrin-selenium solution 100X (Invitrogen, Seoul, South Korea), 10% porcine follicular fluid, 10IUmL-1 of eCG, and 10IUmL-1 of hCG. After IVM, the first polar body extrusion was observed under the microscope. To evaluate embryo development competence, the matured oocytes were activated with electrical stimulus and cultured in porcine zygote medium-5 for 7 days. The cleavage and blastocyst formation rates were observed on Day 2 and 7, respectively. Also, blastocysts were stained with Hoechst 33342 and total blastocyst cell numbers were evaluated under a fluorescence microscope. As a result, the oocyte maturation rate or first polar body extrusion rate of the hEPC co-culture group (90.06±0.75) was significantly higher than the control group (90.06±0.75v. 85.79±0.59; P<0.05). There was no significant difference between the hEPC co-cultured and the control groups in cleavage rate. However, a significant difference in blastocyst formation rate was observed between the hEPC co-cultured and the control groups (28.45±4.92v. 15.87±2.27; P<0.05), whereas total blastocyst cell numbers did not show significant difference between the 2 groups. The all data were analysed by unpaired t-test using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Values are means±standard error of mean. In conclusion, the results in the present study demonstrated that co-culturing with hEPC improved the in vitro oocyte maturation and blastocyst formation rate. Also, we are underway in analysing the concentration of VEGF families in the hEPC co-culture medium after IVM. For further study, we will analyse the genes of the VEGF signaling pathway in the cumulus cells and matured oocytes derived from the 2 groups.

29 POSITRON EMISSION TOMOGRAPHY IMAGING OF BRAIN METABOLISM AND DOPAMINERGIC NEURON DESTRUCTION IN PARKINSON'S DISEASE MODEL PIG

Due to similarities between human and porcine, pigs have been proposed as an excellent experimental animal for human medical research. Especially in paediatric brain research, piglets share similarities with human infants in the extent of peak brain growth at the time of birth and the growth pattern of brain. Thus, these findings have supported the wider use of pigs rather than rodents in neuroscience research. Previously, we reported the production of porcine model of Parkinsons disease (PD) by nuclear transfer using donor cell that had been stably infected with lentivirus containing the human α-synuclein gene. The purpose of this study was to determine the alternation of brain metabolism and dopaminergic neuron destruction using noninvasive method in a 2-yr-old PD model and a control pig. The positron emission tomography (PET) scan was done using Biograph TruePoint40 with a TrueV (Siemens, Munich, Germany). The [18F]N-(3-fluoropropyl)-2β-carbomethoxy-3β-(4-iodophenyl) nortropane (FP-CIT) was administrated via the ear vein. Static images of the brain for 15min were acquired from 2h after injection. The 18F-fluorodeoxy-D-glucose PET (18F-FDG PET) images of the brain were obtained for 15min at 45min post-injection. Computed tomography (CT) scan and magnetic resonance imaging (MRI) were performed at the same location of the brain. In both MRI and CT images, there was no difference in brain regions between PD model and control pigs. However, administration of [18F]FP-CIT was markedly decreased in the bilateral putamen of the PD model pig compared with the control pigs. Moreover, [18F]FP-CIT administration was asymmetrical in the PD model pig but it was symmetrical in control pigs. Regional brain metabolism was also assessed and there was no significant difference in cortical metabolism of PD model and control pigs. We demonstrated that PET imaging could provide a foundation for translational Parkinson neuroimaging in transgenic pigs. In the present study, a 2-yr-old PD model pig showed dopaminergic neuron destruction in brain regions. Therefore, PD model pig expressing human α-synuclein gene would be an efficient model for human PD patients.