Earticle

Alpha-linolenic acid (ALA) is one of n-3 polyunsaturated fatty acids and found mainly in the chloroplasts. Many studies have been reported that when intracellular reactive oxygen species (ROS) in mammalian oocytes was reduced by supplementation of ALA in in vitro maturation (IVM) medium. Based on these reports, we expected that ALA acts as an antioxidant during IVM of porcine oocytes. Therefore, the objective of this study was to investigate the antioxidant effect of ALA supplementation during IVM in porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated in IVM medium containing 200 μM H2O2 or H2O2 with 50 μM ALA for 44 h. Nuclear maturation stage of oocytes was evaluated using aceto-orcein method. For measurement of oxidative stress status, intracellular ROS and glutathione (GSH) levels were measured using carboxy-DCFDA and cell tracker red, respectively. In results, oocytes in metaphase-II stage were reduced by H2O2 treatment (control: 80.00%; H2O2 grop: 61.84%) and it was slightly recovered by treatment of ALA (69.76%) (p<0.05). The intracellular GSH levels decreased in H2O2 groups compared with control groups, but it was enhanced by ALA treatment (p<0.05). On the contrary, H2O2 treatment increased intracellular ROS level in oocytes and H2O2 induced ROS was decreased by treatment of ALA (p<0.05). Our findings shown that ALA treatment under oxidative stress environment recovered oocyte maturation via increase of GSH and decrease of ROS in oocytes. Therefore, these results suggest that ALA have an antioxidative ability and it could be used as antioxidant in in vitro production system of porcine embryo.

OPU (Ovum pick up) has been an important technique to increase genetic resources for early establishment of genetically important herd and in vitro embryo production system. This study compared relationship between OPU-derived COCs quality and embryo development. The recovery procedures of COCs were performed twice a week using ultrasound guided OPU tools. The 18 gauge disposable needle was connected into 50 mL conical tube by teflon tubing, The grade of COCs was evaluated by microscopic examination. Grade A had 3 or more layers of cumulus cell and compact cytoplasm. Grade B had 1~3 layers of cumulus cell and compact cytoplasm. Grade C had 1 layers cumulus cell and compact cytoplasm. Grade D was denuded oocyte and poor cytoplasm. The collected COCs were fertilized in vitro and developed for 7~8 day in mSOF media. The cleavage rate of grade A/B dominant pick-up group (above 60% of A/B grade rate from total recovered COCs) was 48.9% and that of poor quality was 51.9%. The rate of blastocyst rate in grade A/B dominant OPU procedures after IVF was 28.9%, but poor quality group (below 60% of A/B grade COCs) was 14.8%. The developed blastocysts were transferred into recipient or cryopreserved for further research. When total 11 blastocysts were transferred into surrogate cows with the pregnancy rate (4/11) of 36.3% and 4 calves are expected.

Survival depends on the cell type and its ability to endure stresses like of cooling and thawing. The aim of this study was to examine the survival rate of embryonic cell death and DNA fragmentation by controlling the time of equilibration exposure to equilibrium during freezing of in vitro matured porcine embryos. In vitro fertilization (IVF) embryos were exposed to Equilibration solution (HEPES within TCM199, 10% dimethylsulfoxide (DMSO), 10% ethylene glycol (EG)) and vitrification solution (20% DMSO, 20% EG, 0.5 mol/L sucrose) processing the three-step method. Embryos were loaded on open cryotop sheet and minimal volume of vitrification solution surrounding the embryo. Plunge the cryotop into liquid nitrogen quickly. For vitrification and warming steps were performed on a heated plate at 37°C. The experiment, different of vitrification solution exposure time used in the different protocols. The reagent composition of the three-step method used in the experiment was the same. A total of 82 in vitro fertilization embryo were randomly requested for fresh embryo group (control, n=27), conventional method group (Group 1, Equilibration time 15 min, n=27) or method of reducing time group (Group 2, Equilibration time 5 min and 5 min, n=28). Group 2 reduced the survival rate (10%), apoptosis (15%) and mitochondrial DNA fragmentation (17%) (p<0.05) compared with group 1. But there was no significant differences with cell number and pattern among three groups (control, group 1, group 2). These result suggest that time adjustment at the equilibration solution in vitrification may increase the quality of in vitro fertilization embryo.

Parthenogenetic embryo without contribution of paternal genome would be terminated during early gestation period in pigs. Therefore, the present study was designed to analyze the characteristics of parthenogenetic fetuses to identify the possible causes for fetal degeneration during early gestation period. In brief, the pig parthenogenetic fetuses were produced by embryo transfer of parthenogenetic embryos into surrogates. Then, the pig parthenogenetic fetuses were recovered at day 26 and 35 of gestation and conducted to analyze histological characteristics. In results, the parthenogenetic pig fetuses were generated and recovered at day 26 and 35 successfully. The size of parthenogenetic fetuses (n=15, 1.5 cm) recovered at day 26 of gestation were significantly smaller than normal fetuses (n=18, 2.0 cm) while the weights were comparable (0.36 vs. 0.51 g). The size and weight of parthenogenetic fetuses (n=9, 1.8 cm, 0.47 g) recovered at day 35 of gestation were significantly lower than normal fetuses (n=3, 3.67 cm, 3.71 g). The paraffin-embedded sections from parthenogenetic fetus at day 26 showed normal formation of major organs while parthenogenetic fetus at day 35 showed terminated and degenerated formation of major organs. Additionally, the apoptotic cell number of parthenogenetic fetuses (4.4) recovered at day 35 of gestation were significantly (p<0.05) higher than normal fetuses (2.0) while the result of day 26 of gestation showed comparable (1.8 vs. 2.0). Conclusively, our results can suggest that increased apoptosis in fetal tissues also leads to abnormal development of parthenogenetic pig fetus resulting delayed development until day 26 and then termination and degeneration from day 36.

Transgenic animals has been produced by using microinjection method for target gene editing. However, the producing efficiency of gene edited embryos in livestock is very low. Therefore, the objective of this study is production of high quality blastocyst and establishment of appropriate microinjection condition for gene editing embryos. Frist, we treated the cytochalasin B (CB) within injection medium for reduction of microinjection damage, and microinjected embryos were cultured in CR1aa medium supplemented with 0.1 μM melatonin. As a result, blastocysts development of microinjected embryos in CB treated group was significantly higher than that of non-treated group (p<0.05, CB+, 18.5±2.0% vs CB-, 11.2±3.5%). After microinjection under CB treated condition, blastocyst formation was significantly increased (p<0.05) in Mela treated group. Next, to investigate DNA damage in blastocyst stage embryos, fluorescence expressions of H2AX139ph (DNA damage protein) and RAD51 (DNA repairs protein) were observed by using immunofluorescence staining. DNA damage in developing blastocysts was reduced in the CB and Mela treated embryos. Finally, we identified the blastocyst development after microinjection by using CRISPR/Cas9 system according to protein or mRNA of Cas9. Green fluorescence expressions by GFP were increased in developing blastocysts derived from Cas9 protein microinjected embryos. Moreover, gene targeting rate in GFP expressed blastocysts was increased in Cas9 protein injected group (p<0.05; Cas9 protein, 30% vs Cas9 mRNA, 20%). These results demonstrated that positive effect of CB and Mela treatment improves the blastocysts qualities and their developmental competence via reduction of microinjection damage in gene editing bovine embryos. We also verified the improvement of knock-in efficiency in microinjected bovine embryos by using Cas9 protein.

The objective of this experiment was to explore the effects of cobalamin prior to in vitro maturation (IVM) of immature pig oocyte. Specifically, the effects of cobalamin exposure on nuclear and cytoplasmic maturation, diameter, intracellular glutathione (GSH) and reactive oxygen species (ROS), and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT embryos have been measured. Cumulus oocyte complexes (COCs) have been exposed in 200 pM of cobalamin for 22 h, and afterward cultured for 22 h without exposure of cobalamin. The COCs that were matured in the same IVM for 44 h without cobalamin used as control group. Diameter of cobalamin treated oocytes were significantly greater than control group. GSH was higher in cobalamin group than control oocytes group but reduction of ROS in cobalamin than control group of oocytes. In PA, exposure with cobalamin has been significantly greater than control group in rates of maturation, cleavage and blastocyst formation. Similarly, in SCNT embryos rates of maturation, cleavage and formation of blastocyst have been also significantly greater in cobalamin group than control group. SCNT embryos treated with cobalamin 22 h in IVM showed greater expression levels of POU5F1, DPPA2 and NDP52Il mRNA compared with control group. Our results demonstrate that cobalamin treatment prior to IVM improves the development competence of porcine oocyte.

Sperm treatment methods affect sperm fertility and developmental potential of in vitro fertilized(IVF) embryos. Confirmation of the developmental rate according to the information of donor cow will be helpful for the production of elite Jeju Black Cattle(JBC) embryos. To optimize the suitable JBC sperm treatment method, the following methods of percoll gradient, triladyl and percoll-triladyl combination(P, T and PT, respectively) were used to sperm treatment. At 2 days after IVF, there was no difference in the fertilization rate among all groups. The cleavage rate at day 4 was higher in PT and T groups than in P group. The blastocyst formation rate at day 8 was significantly increased in T and PT groups(p<0.05). In comparison of the in vitro development potential of donor and non-donor cows, there were no differences from IVF rate to blastocyst formation rate. According to JBC grade(1++, 1+, 1, 2), the development rate was the highest in 1++ grade cattle, followed by 1, 1+ and 2 grade. Also, in vitro developmental potential by carcass weight was the highest at the 300~350 kg, followed by 300 kg or less, 350~400 kg, and then 400 kg more over. While the T method can collect a small amount of viable sperm, the P method was present a little toxic to JBC sperm. Thus, we developed PT method combined two sperm treatment methods. In this study, the development of IVF embryos was more effective when the carcass grade was high and the carcass weight was about 350 kg. This result demonstrates that JBC sperm can be effectively recovered with simple and efficient PT method and using this technique, elite JBC IVF embryos can be produced in vitro for the mass production of JBC.

2i system is useful for maintenance of navie state of embryonic stem cells in various species. However, the role of 2i system in porcine preimplantation stage embryos unclear. In this study, we investigated the quality of blastocyst and pluripotent-related factor by 2i system. And, we also investigated about DNA and RNA epigenetic modification status by 2i system during porcine early embryo development. After treatment of MEK1/2 inhibitor PD0325901(4 μM), GSK-3 inhibitor CHIR99021(0.3 μM) and 2i (PD0325901+CHIR99021) during pre-implantation development, blastocyst formation rate had no significantly difference. However, blastocyst size increased in 2i treatment embryos. Blastocyst quality also increased after treatment of CHIR and 2i using TUNEL assay and Brdu assay. Interestingly, main transcription factor OCT4 and SOX2 positive cells in blastocyst was significantly increased in 2i groups. Especially, histone modification- relation proteins like h3k9me3 and h3k9ac changed after treatment of CHIR and 2i. Global gene expression patterns related with pluripotency and epigenetic also changed in inhibitor treatment groups. Furthermore, bi-sulfite sequencing results showed that 2i treatment groups demethylated in satellite I region. In conclusion, our findings strongly suggest that 2i system can be useful method for increasing of blastocyst quality by affecting of ICM/TE formation by regulating epigenetic modification during early embryo development.

In mammalian oocyte, acentriolar MTOC(Microtubule organizing center) surrounded by pericentriolar material (PCM) serve as the core structure of the centrosome. Acentriolar MTOC acts like a functional centriole in mouse oocyte. However, the underlying mechanisms remain poorly understood in mammalian germ cell. Here we show that Cep192, an evolutionarily conserved protein, is required for generation of a functional MTOC organizing spindle assembly in porcine oocyte. We found that Cep192 is recruited to the proximal acentriolar MTOC in metaphase I stage during porcine in vitro maturation. In particularly, Cep192 is expressed well in proximal regions of spindle at Anaphase I. Using injection of dsRNA against Cep192, Cep192 mRNA and proteins was decreased and depletion of Cep192 caused porcine oocyte maturation and impair spindle assembly in porcine oocyte. We also find that two cell cycle related kinase, polo-like kinase 1 (PLK1) and aurora kinase A (AURKA) are involved in the porcine oocyte maturation, indicating the common roles of cell cycle kinases in mammalian oocytes. Taken together, Cep192 is one of essential factor for the acentriolar spindle formations in porcine oocyte and indicating the conservation of roles of Cep192 in spindle assembly in mammalian meiotic cell division.

Meiotic oocytes lack classic centrosomes; therefore, bipolar-spindle assembly depends on the clustering of acentriolar microtubule-organizing centers (MTOCs) into two poles. Tumor suppressor P53-binding protein 1 (TP53BP1) is a known mediator in DNA damage response. Recent studies have revealed a new role of TP53BP1 in maintaining centrosome integrity in mitosis. However, the role of TP53BP1 in oocyte meiosis is unclear. Our results show that TP53BP1 participates in DNA damage responses in oocyte, as well as stablishment of chromosome alignment and spindle bipolarity by controlling the clustering of MTOCs during oocyte maturation. TP53BP1 was localized in the cytoplasm and concentrated around the spindle/chromosome region. Knockdown of TP53BP1 induced abnormal DNA damage response involving RAD51 and formation of γH2AX foci. TP53BP1 was also required for the clustering of MTOCs into two spindle poles during pro-meiosis I. Deletion of TP53BP1 induced perturbation in MTOC-localized Aurora Kinase A (AURKA). The knockdown of TP53BP1 induced microtubules to dis-attach from the kinetochores and increased the rate of aneuploidy. Taken together, our data show that TP53BP1 plays crucial roles in chromosome stability and spindle bipolarity during meiotic maturation by regulating the clustering of MTOCs.

LIM kinase(LIMK)s which includes LIMK1 and LIMK2, belongs to serine kinases and is discovered more than a decade ago, and LIMK is shown to be a regulator of actin dynamics. LIMKi3 is a specific inhibitor of LIMK including LIMK1 and LIMK2. Previous results show that LIMKi3 suppresses porcine oocyte maturation in vitro by affecting of actin distribution. However, the roles of LIMKi3 in porcine in early embryo stage embryos is unknown. In this study, we investigated the role of LIMK and LIMK inhibitor function in actin filament organization by affecting p-cofilin, And, we also investigated the cell junction assembly during early embryo development. Firstly, we confirmed LIMK and p-cofilin localization by immunocytochemistry and confocal microscopy. LIMK localized in cytoplasm and cell junction with F-actin after morula. p-Cofilin localized in cytoplasm during early embryo development. LIMKi3(10 uM) treatment with LIMKi3 after parthenogenetic activation, blastocyst rates was significantly decreased compared to the control group. After LIMKi3 treatment in morula, intensity levels of actin and p-cofilin, a downstream molecule of LIMK, were decreased in morula. Furthermore, cell junction related proteins were significantly decreased in LIMKi treatment. Thus, our result indicate that Limk interferes with porcine early embryo development and reduces actin formation by affecting cell junction assembly.

Zinc plays an essential role in mammalian oocyte maturation, fertilization, and early embryogenesis. The zinc concentration increases during oocyte maturation, but decreases soon after fertilization. Depletion of zinc impairs various stages of oocyte maturation, including cell cycle control, asymmetric division, and cytokinesis. However, the exact mechanisms by which zinc is involved in these processes is unknown. Here, we report that zinc, via the actin nucleator Spire, acts as an essential regulator of the actin cytoskeleton remodeling during mouse oocyte maturation and fertilization. Depletion of zinc in the mouse oocyte by either chemical chelation or knockdown of zinc transporters impaired cortical and cytoplasmic actin formation. Spire colocalized with zinccontaining vesicles via its FYVE domain, which contains zinc finger motifs that are essential for proper localization: mutation of zinc-binding cysteines in the FYVE domain abolished proper localization and actin mesh formation in oocytes. Expression of Spire truncation mutants revealed that all three major domains of the actin nucleator are required for correct localization and activity. After fertilization/parthenogenetic activation, Spire localization was dramatically altered following zinc release from the oocyte. Collectively, our data reveal novel roles for zinc in the regulation of the actin nucleator Spire and suggest a novel mechanism for the transition from asymmetric to symmetric cell division during mammalian oocyte maturation and fertilization.

C-phycocyanin (C-PC) is a biliprotein enriched in blue-green algae that is known to possess antioxidant, antiapoptosis, anti-inflammatory, and radical-scavenging properties in somatic cells. But the protective effect of C-PC on porcine embryo developmental competence in in vitro is little known. In the present study, we investigated the effect of C-PC on the development of porcine early embryos as well as the underlying its mechanisms. Different concentrations of C-CP (1, 2, 5, 8, 10 μg/mL) was added to the porcine zygote medium 5 (PZM-5) during in vitro culture. The results showed that 5 μg/mL C-PC significantly increased blastocyst formation. Blastocyst formation and its quality were significantly increased in 50 μM H2O2 treatment group following 5μg/mL C-PC addition. C-PC prevented H2O2-induced compromise of mitochondrial membrane potential, release of cytochrome C from the mitochondria and reactive oxygen species generation. Furthermore, apoptosis, DNA damage level and autophagy in the blastocysts were attenuated by supplement of C-PC in H2O2-induce doxidativ injury group compared with control.Taken together, these results suggest that C-PC has beneficial effects on the development of porcine parthenotes by attenuating mitochondrial dysfunction and oxidative stress.

Cyclin E1 (CCNE1) is a core component of cell cycle regulation that drives the transition into the S phase. CCNE1 plays critical roles in cell cycle, cell proliferation, and cellular functions. However, the function of CCNE1 in early embryonic development is limited. In the present study, the function and expression of Ccne1 in porcine early parthenotes were examined. Immunostaining experiments showed that CCNE1 localized in the nucleus, starting at the four-cell stage. Knockdown of Ccne1 by double-stranded RNA resulted in the failure of blastocyst formation and induced blastocyst apoptosis. Ccne1 depletion increased expression of the pro-apoptotic gene Bax, and decreased the expression of Oct4 and the rate of inner cell mass (ICM)/trophectoderm formation. The results indicated that CCNE1 affects blastocyst formation by inducing cell apoptosis and ICM formation during porcine embryonic development.

Bisphenol A (BPA) is an environmental contaminant widely used in the plastic industry. BPA has been demonstrated to be an endocrine disruptor and has an adverse effect on the embryonic development of mammals. However, the mechanism of action of BPA is limited. In this study, we investigated the role and mechanism of BPA in porcine embryonic development. First, the parthenotes were treated with different concentrations of BPA. We found that blastocyst formation was impaired and the parthenotes were arrested at the 4-cell stage after treatment with 100 μm BPA. Second, ROS increased following the addition of BPA, which further caused mitochondrial damage, and cytochrome c was released from the mitochondria to induce apoptosis. The adaptive response was demonstrated through LC3 immunofluorescence staining and by assessing autophagy-related gene expression. In addition, BPA caused DNA damage through the p53-p21 signaling pathway. Thus, our results indicate that BPA displays an adverse effect on porcine early embryonic development through mitochondrial and DNA damage.

The acetyltransferase TIP60 (also known as Kat5) is a member of the MYST family of histone acetyltransferases and was initially identified as a cellular protein. TIP60 acetylates histone and nonhistone proteins and is involved in diverse biological processes, including apoptosis, cell cycle, and DNA damage responses. In this study, a specific inhibitor of TIP60 was used to detect the function of TIP60 in porcine parthenogenetic embryos. The results showed that TIP60 inhibition impaired porcine parthenogenetic embryonic development. The mechanism of TIP60 was also determined. We found that the TIP60 inhibition impaired embryonic development by ROS induced DNA damage, as demonstrated by the number of γH2A in the nuclei. TIP60 inhibition triggered DNA damage through the regualetion of p53-p21 pathway and TIP60 played a role in DNA repair. TIP60 inhibition decreased the efficiency of DNA repair by regulating 53BP1-dependent repair after DNA damage. Inhibition of TIP60 also increased the adaptive response, autophagy, by modulating LC3. Therefore, TIP60 plays a role in early porcine parthenogenetic embryonic development by regulating DNA damage and repair.

Alpha lipoic acid (ALA) is a biological membranes compound. as the antioxidant, it decreases the oxidized forms of other antioxidant substances such as vitamin C, vitamin E and glutathione (GSH). To examine the effect of ALA on the in vitro maturation (IVM) of porcine oocytes, we investigated intracellular GSH and reactive oxygen species (ROS) levels, and subsequent embryonic development after parthenogenetic activation (PA). Intracellular GSH levels in oocytes treated with 50uM of ALA increased significantly (p<0.05) and exhibited a significant (p<0.05) decrease in intracellular ROS levels compared with the control group. Oocytes matured with 50uM during IVM displayed significantly higher cleavage rates (67.8% vs. 83.4%, respectively), and higher blastocyst formation rates and total cell number of blastocysts after PA (31.6%, 58.49 vs. 46.8%, 68.58, respectively) than the control group. In conclusion, these results suggest that treatment with ALA during IVM improves the nuclear maturation of porcine oocytes by increasing the intracellular GSH levels, thereby decreasing the intracellular ROS levels and subsequent embryonic developmental potential of PA.

Growth differentiation factor8 (GDF8) is a member of the transforming growth factor-β that has been identified as a robust physiological regulator. According to recent studies, the GDF8 is detected in oviduct fluid and uterus which led us to suggest that the GDF8 may effect on preimplantation embryonic development and act paracrine role to correlate with successful late-blastocyst implantation in in vivo. We investigated the effect of GDF8 supplement during in vitro culture (IVC) of porcine embryos derived from in vitro fertilization (IVF) on cleavage and blastocyst (BL) formation rate, and gene transcription level analysis in BL. Data were analyzed by one way ANOVA, followed by Tukey’s range test. Respectively 0.2, 2 and 20 ng/mL of GDF8 were added during IVC, and experimental groups were as follows; control (0), 0.2, 2, and 20 GDF8 supplement groups. At IVC 48hr, there was no significant difference on cleavage rate from the different concentration of GDF8 supplement groups (65.7%, 66.0%, 66.3%, and 65.8%, respectively). After additional 120hr of embryo culture, 0.2 group was shown significantly (p<0.05) higher than control in blastocyst formation rate and total cell number (32.5% and 88.0±7.3 VS 40.4% and 118.4±12.7, respectively). Using the achieved IVF BLs, the specific gene expression pattern were evaluated. The embryo development competence marker Pcna, Pou5f1 and Sox2 (1.81, 2.85 and 2.09 times, respectively), and cell junction assembly regulator Adam10, Adam17, Tjp1, Cdh1 (1.40, 1.98, 1.79 and 1.80 times, respectively) genes mRNA transcript levels in 0.2 group were significantly increased in 0.2 group compared with control. Moreover, pro-apoptotic factor Cas3 gene mRNA transcript levels was significantly decreased in 0.2 group than control (0.62 times). In conclusion, the supplementation of 0.2 ng/mL GDF8 during IVC significantly improved embryonic developmental potential via regulating embryo developmental competence markers and cell junction assembly regulator transcriptional levels.

Recent decade, the studies for generation of chimeric organs have been advanced remarkably with the production of chimeric animals using by pluripotent stem cells (PSCs). However, the distribution rate of injected cells into an embryo is still low and it is considered as one of the major reasons of low efficiency of chimera production. The purpose of this study is to investigate the effect of growth differentiation factor 8 (GDF8) on porcine chimeric embryo development. In first, we produced mCherry-marked porcine iPSCs (mCh-iPSCs). The mCh-iPSCs were injected into 8 cells or morula stage of in vitro fertilized (IVF) embryos at day 2 under a microscope with the micromanipulator. These chimeric embryos were then cultured porcine embryonic stem cell medium (DEME F10/low glucose with 15% FBS) for 18hr and then transferred to fresh porcine zygote medium 5 (PZM5 with or without 150 pg/mL of GDF8). We investigated the effect of supplemented GDF8 during in vitro culture (IVC) of these chimeric embryos on their cleavage patterns, blastocyst formation ratio, and injected cells’ distributions. Data were analyzed by ANOVA followed by Tukey’s range test using SPSS (Statistical Package for Social Science). After day 5 of in vitro chimeric embryo culture, the GDF8 supplement group was shown significantly higher Using the chimeric embryos of the blastocyst stage, we evaluated distributions of the injected cells by immune staining of SOX2 as porcine inner cell mass (ICM) marker. The GDF8 supplement group was shown significantly increased mCherry and SOX2 per SOX2 expressing cells ratio than control (49.2% VS 20.7, respectively). In conclusion, the supplementation of GDF8 during IVC of porcine chimeric embryos improved embryonic developmental competence and enhanced the distributions of injected cells into ICM of chimeric embryos at the pre-implantation stage.

Ferulic Acid (FA) is a metabolite of phenylalanine and tyrosine, a phenolic compound commonly found in fruits and vegetables. Several studies have shown that FA has various functions such as antioxidant effect, prevention of cell damage from irradiation, protection from cell damage caused by oxygen deficiency, anti-inflammatory action, anti- aging action, liver protective effect and anti-cancer action. In this study, we investigated the maturation rate, intracellular glutathione (GSH) and reactive oxygen species (ROS) of porcine oocytes by adding FA to the in vitro maturation (IVM) medium and examined subsequent embryonic developmental competence at 5% oxygen through parthenogenesis. There is no significant difference between the the control group (0 μM) and treatment groups (5 μM, 10 μM, 20 μM) on maturation rates. Intracellular GSH levels in oocyte treated with 5 μM of FA significantly increased (p<0.05), and 20 μM of FA revealed significant decrease (p<0.05) in intracellular ROS levels compared with the control group. Oocytes treated with FA exhibited significantly higher cleavage rates (79.01% vs 89.19%, 92.20%, 90.89%, respectively) than the control group. Oocytes treated with 10 μM showed significantly higher blastocyst formation rates (28.3% vs 40.3%, respectively) after PA than the control group. Total cell numbers in blastocyst of 10 μM FA displayed significantly higher (39.4 vs 51.9, respectively) than the control group. In conclusion, our results suggested that treatment with FA during IVM improved the developmental potential of porcine embryos by increasing cleavage rate, blastocyst formation and total cell numbers in blastocysts.

Porcine uterine ex vivo model is a useful tool for investigating gamete maturation, activation, fertilization and early embryonic development. This study was to evaluate fertilization ability of in vitro matured (IVM) porcine oocyte (MII) using porcine uterine ex vivo model. IVM-MII oocytes were fertilized with 7.5×107, 15×107 and 30×107 sperm for 20min in porcine uterine ex vivo model. And, the oocytes were then flushed and performed in vitro culture (IVC). IVM-MII oocytes used for in vitro fertilization (IVF) served as control 1. Before IVF, IVM-MII oocytes cultured in porcine uterine ex vivo model for 20min served as control 2. In the results, it was found that the sperm penetration rate in the control 2 (73.0±4.2) was significantly higher than the other treatment group: 7.5×107 (29.7±4.4), 15×107 (34.3±3.2) and 30×107 (44.3±7.4). But there was no significant difference between control 1 and control 2. Monospermic penetration rate in all treatment groups was significantly higher than control groups, and polyspermy rate in all treatment group was also significantly lower than control groups. These results suggested that porcine uterine ex vivo model improved monospermic penetration by reducing sperm penetration ratio. In the further study, it is required to evaluate the embryonic development, GSH and ROS accumulation levels of MII oocytes fertilized using porcine uterine ex vivo model.

GWAS (Genome wide association study) strategy has been applied with the purpose to identity chromosomal regions and SNPs that control relevant traits. The development of SNP panel with the medium density (PorcineSNP60 BeadChip) allows the detection of QTL (Quantitative trait loci) and candidate genes near QTL with a higher resolution compared to using microsatellites in the past. The total number born (TNB) and the number of piglets weaned (NPW) are the most economical traits in pigs due to a huge impact on the efficiency of pig production. Therefore, the aim of this study was to detect regions (1-Mb windows) and SNPs associated with litter size (total number born, TNB) and the number of piglets weaned (NPW) for Yorkshire pigs. Reference population of 1,978 Yorkshire pigs were genotyped using Illumina Porcine SNP60 v2 SNP panel which were then imputed for missing genotypes with FImpute v2.2. BayesB with set to π (0.99) method was used to identify the informative window regions and deregressed EPD and their reliabilities were used as a response variable and a weighting factor in GWAS model. In total, 8 and 6 genome regions explaining over 0.75% of the genetic variance and 9 and 6 SNPs were identified for TNB and NPW traits, respectively. Those identified genome regions were located on SSC1, SSC6, SSC7, SSC11, SSC14 and SSC16 for TNB and SSC1, SSC4, SSC7 and SSC14 for NPW. No overlapping significant chromosome areas or QTL between TNB and NPW traits were detected. Our results suggested that it is feasible to improving the accuracy of the genomic selection by detecting QTLs for TNB and NPW traits.

GWAS (Genome wide association study) strategy has been applied with the purpose to identity chromosomal regions and SNPs that control relevant traits. The development of SNP panel with the medium density (PorcineSNP60 BeadChip) allows the detection of QTL (Quantitative trait loci) and candidate genes near QTL with a higher resolution compared to using microsatellites in the past. The minimization of weaning to estrus interval (WEI) is the most economical trait in pigs due to a huge impact on the efficiency of pig production. Therefore, the aim of this study was to detect regions (1-Mb windows) and SNPs associated with weaning to estrus interval (WEI) for Yorkshire pigs. Reference population of 1,978 Yorkshire pigs were genotyped using Illumina PorcineSNP60 v2 SNP panel which were then imputed for missing genotypes with FImpute v2.2. BayesB with set to (0.99) method was used to identify the informative window regions and deregressed EPD and their reliabilities were used as a response variable and a weighting factor in GWAS model. In total, 6 genome regions explaining over 0.75% of the genetic variance and 6 SNPs were identified for WEI trait. A 1 Mb regions on chromosome 1 (SSC1), 4 (SSC4), 7 (SSC7) and 14 (SSC14), which showed strong evidence of linkage disequilibrium with QTL, explained from 0.75% to 1.52% for genetic variation for weaning to estrus interval trait. Our results suggested that it is feasible to improving the accuracy of the genomic selection by detecting the siginificant QTL regions for WEI trait.

The naked mole rat (NMR, Heterocephalus glaber) is an emerging model animal for researching on aging and cancer due to their exceptionally long life span and cancer-resistance. It has been proven that carcinogenesis and obtaining pluripotency from normal cells undergo similar cellular and molecular mechanisms. Here, we tried to reprogram fibroblasts for investigating cellular reprogramming process in NMR having anti-cancer property. Firstly, we investigated which culture conditions and reprogramming factors were adequate for reprogramming of NMR cells. Mouse reprogramming factors and high glucose culture conditions efficiently facilitated pluripotent genes of NMR fibroblasts. However, as tumor-repressor genes such as NF2 and Rb1 were highly expressed, expression of pluripotent genes were gradually decreased during reprogramming, and no primary colonies of iPSCs were derived. Next, tumor suppressor genes were inhibited by using RNA interference technique to improve reprogramming efficiency. As a result, HAS2-knocked down cells were converted into AP-positive iPSCs by ectopic expression of reprogramming factors, and NMR iPSCs could be differentiated into beating cardiomyocytes and endodermal cells. Overall, we derived NMR iPSCs through downregulating tumor suppression mechanism, which could be applied for researches of ageing and cancer.

Canine induced Pluripotent Stem Cells (iPSCs) can provide great potential for regenerative veterinary medicine and may assist in the development of new therapeutics and pre-clinical studies for both dogs and human. To date, there have been several reports on the generation of canine iPSCs using retroviral or lentiviral transduction of Yamanaka’s factors. However, there is no report of canine iPSCs generated by genomic integration-free methods. According to previous studies, a polycistronic and synthetic self-replicating RNA system was developed for generating human iPSCs by the RNA replicon of Venezuelan Equine Encephalitis (VEE) virus. The VEE replicon is a positive-sense and single-stranded RNA which is similar with cellular mRNA containing a 5’ cap and poly (A) tail. It is no potential for genomic DNA integration problems because it does not use a DNA intermediate. Here, we investigated to generate canine iPSCs by a single transfection of the VEE-reprogramming factor (VEE-RF) RNA. To generate integration- free canine iPSCs, the VEE-OKS-iG RNA that expresses four reprogramming ORFs (hOct4, hKlf4, hSox2 and hGlis1) was transfected. Also, B18R mRNA was co-transfected to reduce immune response by VEE replicon. Putative canine iPSC colonies first appeared between day 15-25. Interestingly, they have two distinct types of initial canine iPSC colonies. They were identified by immunohistochemistry of live cells using TRA-1-60 antibody and also showed clear alkaline phosphatase (AP) activity. Further study will be required to analyze the characterization for a clinical application, and the non-integrating and self-replicating VEE RNA replicon system has the potential to make a great contribution to generating clinically applicable canine iPSCs.

The ability to generate patient’s induced pluripotent stem cells (iPSCs) could provide tremendous promises for regenerative medicine. It has reported that choice of reprogramming tools is essential for the safety and the increasing efficiency of reprogramming process and transduction. Non-integrating techniques as like mRNA based reprogramming protocols are available for the production of genetically stable iPSCs while avoiding the risks of genomic integration. In this study, to derive brain cancer patient’s iPSCs by mRNA’s reprogramming, we obtained patient’s somatic tissues at brain tumor surgery that informed consent obtained according to institutionally- approved protocols. Patient's primary somatic cells were isolated, and they used as the cell source for iPSCs generation under xeno-free conditions. Reprogramming produced by non-integration methods utilized the combination of reprogramming mRNAs (OSKMNL) with evasion mRNAs (EKB) (Stemgent) during four days. Reprogrammed cells showed the colonies like iPSCs after six days of transduction, and the colonies were expanded until pick-up at day 14. These colonies were stained for pluripotency-associated genes using TRA-1-60 and TRA-1-81 by immunocytochemistry. Established colonies were also expanded without feeder condition and stained with Alkaline Phosphatase. In the further study, generated patient's iPSCs will be required to investigate their availability of the clinical application under xeno-free conditions which could provide a path to GMP applicability that should facilitate the clinical implementation of patient- or disease-specific iPSCs therapies.

Since the first report of the successful preservation of sperm, cryopreservation has been applied as a routine technique for processing bovine sperm in artificial insemination (AI), and numerous studies have been carried out to evaluate fundamental biological properties. Although fertility with frozen–thawed bull semen was generally acceptable for AI, the cryopreservation techniques at the time still resulted in the loss of 40 ～50% of viable sperm during the freezing–thawing process with little improvement over the last several decades. Cold shock, osmotic stress, ice crystal formation or oxidative damage were the main sources of sperm cryoinjury, and finally caused the loss of sperm viability and fertility. This study was designed to compare fresh and insemination egg yolk in an extender for cryopreservation of Hanwoo bull semen. Sperm motility, plasma membrane integrity and viability were assessed at different stages of cryopreservation (post-dilution, pre-freezing and post-thawing). Sperm plasma membrane integrity remained similar at all the stages of cryopreservation. Sperm motility and viability were significantly higher after thawing in the extender containing insemination egg yolk. In conclusion, insemination egg yolk may be used in an extender for the cryopreservation of Hanwoo bull spermatozoa.

일반적인 바이러스 치료제의 경우 바이러스의 life cycle과 관련된 유전자를 저해 하거 나 억제시킴으로서 항바이러스 효과를 갖는 방면, 재조합 가수분해 항체 3D8 scFv는 바 이러스 유전자를 직접 분해하는 작용으로 바이러스에 대한 저항성을 갖게 된다. 이전의 연구에서 3D8 scFv 형질 전환돼지를 생산한 바 있다. 본 연구에서는 3D8 scFv 형질전환 돼지의 후대 번식을 위하여 Computer Assessment of Sperm Analysis (CASA) system으 로 정액의 운동성을 분석하였다. 정액은 수압법을 이용하여 채취 하였고, 채취된 정액은 Androhep plus를 이용하여 1:1로 희석하였다. 희석된 정액은 CASA를 이용하여 전진 운 동성, 총 운동성, VCL, VAP, VSL을 일반돼지와 비교 분석하였다(3D8 scFv : 일반돼지). 정자 운동성은 0시간에 전진 운동성(79.8±4.6, 87.3±1.5), 총 운동성(98.5±0.3, 93.5±1.2), VCL (64±5.7, 85.9±1.9), VAP (41.5±2.9, 73.3±1.7), VSL (25.2±1.4, 36.8±1.5)으로 나타 났고, 24시간에는 전진 운동성 (60.4±2.6, 85.1± 1.0), 총 운동성 (81.6±1.8, 90.0±1.7), VCL (62.4±1.3, 76.1±1.8), VAP (44.3±0.4, 65.5±1.5), VSL (30.4±0.7, 37.4±0.9), 96시간 에는 전진 운동성 (66.5±3.4, 71.5±2.6), 총 운동성 (84.2±3.3, 82.2±1.5), VCL (91.3±4.8, 93.4±3.5), VAP (49.5±3.1, 61.7±1.8), VSL (19.1±1.3, 17.5±0.9). 120시간에는 전진 운동 성 (69.3±3.4, 68.3±3.1), 총 운동성 (91.1±2.7, 80.8±3.5), VCL (81.7±3.6, 83.5±3.9), VAP (49.1±2.2, 58.0±3.3), VSL (22.6±0.3, 21.5±0.9)로 조사되었다. 이상 결과는 형질전환 돼 지에 도입된 3D8 scFv 유전자는 돼지 정자의 운동성에 관한한 기능적 이상을 유도하지 않는 것으로 판단되며, 향후 항바이러스 효과 분석을 위한 후대번식에 이용할 것이다.

The Jeju Black Cattle (JBC) are a type of traditional Korean native cattle with a characteristic black fur that covers the entire body. Jeju black cattle are smaller than other breeds and their disposition is very calm. The breed's qualities and genetic uniqueness helped it to be named as a protected, state-designated national monument in 2013. However, they are endangered crisis. Therefore, in vitro fertilization (IVF) technology is needed to preserve these endangered animals and to further improve their traits. In this study, we compared the sperm fertility and developmental potential of IVF embryos using Hanwoo sperm supplied by Korea Animal Improvement Association and JBC sperm made by Jeju Special Self-Governing Province Livestock bull semen Promotion Institute. [Hanwoo A, B (KPN) grade and JBC A, B two type]. Sperm was inseminated in 44 μL IVF drop contained 10 oocytes with sperm concentration of 1× 106 cells/mL, and then 2 μL heparin and 2 μL PHE (20 μM penicillamine, 10 μM hypotaurine, 2 μM epinephrine) were added. At 2 days after IVF, the fertilization rate was higher in Hanwoo sperm group (80.9±1.6) than in JBC sperm group (72.0±5.2). The IVF rate was significantly higher in Hanwoo-B sperm group (82.7±1.1) and JBC-B sperm group (84.5) than in JBC-A sperm group (67.0±5.1)(p<0.05). The blastocyst formation rate was the highest in Hanwoo-B sperm group (43.0±3.9) among four treatment sperm groups(p<0.05). The trophectoderm cell number was higher in Hanwoo sperm group (122±7.8) than in JBC sperm group (73±12.6). The inner cell mass cell number of Hanwoo sperm group (46±2.5) was similar to that of JBC sperm group (46±4.0). Thus, the total cell number of day 8 blastocyst was higher in Hanwoo sperm group (168±8.9) than in JBC sperm group (119±15.9). These results demonstrate that there were different in sperm quality (viability and motility) between Hanwoo sperm group (70∼80%) and JBC sperm group (60∼70%), and their in vitro development capacity was also presented as difference. We need to more sperm fertility study using JBC sperm.

Bisphenol A (BPA) is an industrial chemical widely used to manufacture certain plastics. As a strong endocrine disruptor, BPA is capable of altering cell functions. Exposure to BPA correlated with reduction of motility and fertilization ability of spermatozoa by increasing oxidative stress. Since antioxidants are capable to protect cells from oxidative damage, it may minimize BPA-induced stress to cells. Here, we examine whether exposure of spermatozoa to antioxidants such as glutathione (GSH), vitamin C (Vit C), and vitamin E (Vit E) has any protective effects of BPA-exposed spermatozoa. In this in vitro trials, collected spermatozoa from sexually mature male mice were incubated with BPA and different antioxidants (Control, BPA, BPA+GSH, BPA+Vit C, BPA+Vit E, GSH, Vit C, Vit E) for 6 hours. The doses of BPA and antioxidants were chosen based on preliminary studies (BPA: 100 μM, GSH: 5 mM, Vit C: 100 μM, and Vit E: 2 mM). Following incubation, we detect the levels of reactive oxygen species (ROS) in spermatozoa as a measure of oxidative stress to the cell. Our results showed that antioxidants are capable to protect oxidative stress by ROS in BPA-exposed spermatozoa (p<0.05). Simultaneously, we examine motility parameters and capacitation status of spermatozoa by CASA, and CTC staining, respectively. Strikingly, antioxidants treatment of BPA-exposed spermatozoa was capable to restore sperm motility (p<0.05). However, hyperactivated sperm motility, motion kinematics parameters, and capacitation status of spermatozoa showed no difference between treatments groups. This findings evidence that antioxidants are capable to restore sperm functions from BPA mediated toxicity. Therefore, antioxidants can be considered as therapeutic agents to treat BPA toxicity. However, further studies are needed to confirm our preliminary findings.