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Somatic cell nuclear transfer (SCNT) has been widely used as an efficient tool in biomedical research for the generation of transgenic animals from somatic cells with genetic modifications. Although remarkable advances in SCNT techniques have been reported in a variety of mammals, the cloning efficiency in domestic animals is still low due to the developmental defects of SCNT embryos. In particular, recent evidence has revealed that mitochondrial dysfunction is detected during the early development of SCNT embryos. However, there have been relatively few or no studies regarding the development of a system for evaluating mitochondrial behavior or dynamics. For the first time, in mitochondria of bovine SCNT embryos, we developed a method for the visualization of mitochondria and expression of fluorescence proteins. To express red fluorescence in mitochondria of cloned embryos, bovine ear skin fibroblasts, nuclear donor, were stably transfected with a vector carrying mitochondria-targeting DsRed2 gene tagged with V5 epitope (mito-DsRed2-V5 tag) using lentivirus-mediated gene transfer because of its ability to integrate in the cell genome and the potential for long-term transgene expression in the transduced cells and their dividing cells. From western blotting analysis of V5 tag protein using mitochondrial fraction and confocal microscopy of red fluorescence using SCNT embryos, we found that the mitochondrial expression of the mito-DsRed2 protein was detected until the blastocyst stage. In addition, according to image analysis, it may be suggested possible use of the system for visualization of mitochondrial localization and evaluation of mitochondrial behaviors or dynamics in early development of bovine SCNT embryos.

While triclosan (TCS) exerts detrimental effects on female reproduction, the effect of TCS-derived toxins on porcine oocytes during in vitro maturation (IVM) is unclear. This study investigated the effects of TCS on mitochondrion-derived reactive oxygen species (ROS) production and apoptosis pathways during porcine oocyte maturation. Porcine oocytes were treated with TCS (1, 10, and 100 &mu, M) and triphenylphosphonium chloride (Mito-TEMPO, 0.1 &mu, M), and matured cumulus oocyte complexes (COCs) were stained with orcein, dichlorofluorescein diacetate (DCF-DA), and Mito-SOX. Proteins and mRNA levels of factors related to cumulus expansion and mitochondrion-mediated apoptosis and antioxidant enzymes were analyzed by western blotting and reverse-transcription polymerase chain reaction (RT-PCR), respectively. Meiotic maturation and cumulus cell expansion significantly decreased for COCs after TCS treatment along with an increase in mitochondrial superoxide levels at 44 h of IVM. Further, mitochondrion-related antioxidant enzymes and apoptosis markers were significantly elevated in porcine COCs following TCS-mediated oxidative damage. The protective effect of Mito-TEMPO as a specific superoxide scavenger from TCS toxin improved the maturation capacity of porcine COCs. Mito-TEMPO downregulated the mitochondrial apoptosis of TCS-exposed porcine COCs by reducing superoxide level. In conclusion, our data demonstrate that TCS mediates toxicity during porcine oocyte maturation through superoxide production and mitochondrion-mediated apoptosis.

Bisphenol A (BPA) is synthetic organic compound that exhibits estrogen-like properties and it induces mitochondrial superoxide production. Melatonin (Mela) protects against BPA-mediated cell damage and apoptosis. However, the antioxidative effects of Mela against BPA-induced superoxide production in porcine oocytes are still not known. In this study, we investigated the antioxidative effects of Mela against BPA-derived superoxide on oocyte maturation in pigs. To investigate the effects of the superoxide specific scavenger, Mito-TEMPO, on porcine oocyte maturation in response to BPA exposure apoptosis proteins, we treated the oocytes with Mito-TEMPO (0.1 &micro, M) after pre-treating them with BPA (75 &micro, M) for 22 h. As expected, the reduction in meiotic maturation and cumulus cell expansion of cumulus-oocyte-complexes (COCs) in the BPA (75 &micro, M) treated group was recovered (p &lt, 0.01) by treatment with Mito-TEMPO (0.1 &micro, M). An increase in the levels of mitochondrial apoptotic proteins (AIF, cleaved Cas 3 and cleaved Parp1) in response to BPA-induced damage was also reduced by Mito-TEMPO treatment in porcine COCs. Interestingly, we confirmed the positive effects of Mela with respect to superoxide production upon BPA exposure during oocyte maturation and also confirmed the reduction in mitochondrial apoptosis in Mela (0.1 &micro, M)-treated porcine COCs. These results provide evidence for the first time that antioxidative effects of Mela on BPA-derived superoxide improve porcine oocyte maturation.

Mito-TEMPO is a well-known mitochondria-specific superoxide scavenger. However, the effect of Mito-TEMPO on porcine embryo development, to our knowledge, has not been studied yet. In the present study, porcine embryos were classified into two groups (G1 and G2) based on the cytoplasm lipid contents at the zygote stage. The development of blastocysts derived from G2 zygotes was reduced (G2:16.2 ± 7.9% vs G1: 26.5 ± 5.9%; 1.6-fold, p in vitro.

Gangliosides exist in glycosphingolipid-enriched domains on the cell membrane and regulate various functions such as adhesion, differentiation, and receptor signaling. Ganglioside GM3 by ST3GAL5 enzyme provides an essential function in the biosynthesis of more complex ganglio-series gangliosides. However, the role of gangliosides GM3 in porcine oocytes during in vitro maturation and early embryo development stage has not yet understood clear. Therefore, we examined ganglioside GM3 expression patterns under apoptosis stress during maturation and preimplantation development of porcine oocytes and embryos. First, porcine oocytes cultured in the NCSU-23 medium for 44 h after H2O2 treated groups (0.01, 0.1, 1 mM). After completion of meiotic maturation, the proportion MII (44 h) was significantly different among control and the H2O2 treated groups (76.8±0.3 vs 69.1±0.4; 0.01 mM, 55.7±1.0; 0.1 mM, 38.2±1.6%; 1 mM, P

Edaravone (Eda) is a potent scavenger of inhibiting free radicals including hydroxyl radicals (H2O2). Reactive oxygen species (ROS) such as H2O2 can alter most kinds of cellular molecules such as lipids, proteins and nucleic acids, cellular apoptosis. In addition, oxidative stress from over-production of ROS is involved in the defective embryo development of porcine. Previous study reported that Eda has protective effects against oxidative stress-like cellular damage. However, the effect of Eda on the preimplantation porcine embryos development under oxidative stress is unclear. Therefore, in this study, the effects of Eda on blastocyst development, expression levels of ROS, and apoptotic index were first investigated in preimplantation porcine embryos. After in vitro fertilization, porcine embryos were cultured for 6 days in PZM medium with Eda (10 μM), H2O2 (200 μM), and Eda+H2O2 treated group, respectively. Rate of blastocyst development was significantly increased (P

Catalpol, an iridoid glucoside, isolated from the root of Rehmannia glutinosa Libosch. It possesses a broad range of biological and pharmacological activity including anti-tumor, anti-inflammation and anti-oxidant by acting as a free radical scavenger. Therefore, in this study, the effects of catalpol on blastocyst development, expression levels of reactive oxygen species (ROS) and apoptotic index were investigated in porcine embryos. After in vitro maturation and fertilization, porcine embryos were cultured for 6 days in porcine zygote medium 3 (PZM-3) supplemented with catalpol (0, 100, 200 and 400 μM, respectively). Blastocyst development not significantly improved in the catalpol treated group when compared with control group. Otherwise, the intracelluar levels of ROS were decreased and the numbers of apoptotic nuclei were reduced in the catalpol (100 μM) treated porcine blastocysts (P

Objective: Phellodendron amurense (P. amurense) and Humulus japonicus (H. japonicus) are closely involved in anti-oxidative response and increasing antioxidant enzymes activities. However, the effects of their extracts on development of preimplantation bovine embryos have not been investigated. Therefore, we investigated the effects of P. amurense and H. japonicus extracts on developmental competence and quality of preimplantation bovine embryos. Methods: After in vitro fertilization, bovine embryos were cultured for 7 days in Charles Rosenkrans amino acid medium supplemented with P. amurense (0.01 mu g/mL) and H. japonicus (0.01 mu g/mL). The effect of this supplementation during in vitro culture on development competence and antioxidant was investigated. Results: We observed that the blastocysts rate was significantly increased (p

Mitochondria are highly dynamic organelles that undergo constant fusion/fission as well as activities orchestrated by large dynamin-related GTPases. These dynamic mitochondrial processes influence mitochondrial morphology, size and function. Therefore, this study was conducted to evaluate the effects of mitochondrial fission inhibitor, mdivi-1, on developmental competence and mitochondrial function of porcine embryos and primary cells. Presumptive porcine embryos were cultured in PZM-3 medium supplemented with mdivi-1 (0, 10 and 50 μM) for 6 days. Porcine fibroblast cells were cultured in growth medium with mdivi-1 (0 and 50 μM) for 2 days. Our results showed that the rate of blastocyst production and cell growth in the mdivi-1 (50 μM) treated group was lower than that of the control group (P < 0.05). Moreover, loss of mitochondrial membrane potential in the mdivi-1 (50 μM) treated group was increased relative to the control group (P < 0.05). Subsequent evaluation revealed that the intracellular levels of reactive oxygen species (ROS) and the apoptotic index were increased by mdivi-1 (50 μM) treatment (P < 0.05). Finally, the expression of mitochondrial fission-related protein (Drp 1) was lower in the embryos and cells in the mdivi-1-treated group than the control group. Taken together, these results indicate that mdivi-1 treatment may inhibit developmental competence and mitochondrial function in porcine embryos and primary cells.

Humulus japonicus is an ornamental plant in the Cannabaceae family. Although the mode of action of Humulus japonicus is not fully understood, a strong relationship was observed between anti-inflammatory and anticancer in some types of cells. Recent studies also have shown that Humulus japonicus possesses anti-inflammatory activities and may significantly improve antioxidant potential in Raw 264.7 macrophage cells. Thus, the aim of this study was evaluated the effect of Humulus japonicus extract on sperm motility and subsequent preimplantation developmental competence of the bovine embryos. After in vitro maturation, the oocytes with sperms were exposed in in vitro fertilization (IVF) medium supplemented with Humulus japonicus extract (0.01, 0.05, 0.1 μg/mL, respectively) for 1 day. In our results, exposure of IVF medium to Humulus japonicus extract did not affect sperm motility and percentage of penetrated oocytes but ROS intensity was significantly decreased by 0.01 μg/mL compared with other groups (p< 0.05). Moreover, treatment with 0.01 μg/mL of Humulus japonicus extract was higher the frequency of blastocyst formation than the any other groups (p

Aims We examined whether the three unfolded protein response (UPR) signaling pathways, which are activated in response to endoplasmic reticulum (ER)-stress, are involved in progesterone production in the luteal cells of the corpus luteum (CL) during the mouse estrous cycle. Main methods The luteal phase of C57BL/6 female mice (8 weeks old) was divided into two stages: the functional stage (16, 24, and 48 h) and the regression stage (72 and 96 h). Western blotting and reverse transcription (RT)-PCR were performed to analyze UPR protein/gene expression levels in each stage. We investigated whether ER stress affects the progesterone production by using Tm (0.5 μg/g BW) or TUDCA (0.5 μg/g BW) through intra-peritoneal injection. Key findings Our results indicate that expressions of Grp78/Bip, p-eIF2α/ATF4, p50ATF6, and p-IRE1/sXBP1 induced by UPR activation were predominantly maintained in functional and early regression stages of the CL. Furthermore, the expression of p-JNK, CHOP, and cleaved caspase3 as ER-stress mediated apoptotic factors increased during the regression stage. Cleaved caspase3 levels increased in the late-regression stage after p-JNK and CHOP expression in the early-regression stage. Additionally, although progesterone secretion and levels of steroidogenic enzymes decreased following intra-peritoneal injection of Tunicamycin, an ER stress inducer, the expression of Grp78/Bip, p50ATF6, and CHOP dramatically increased. Significance These results suggest that the UPR signaling pathways activated in response to ER stress may play important roles in the regulation of the CL function. Furthermore, our findings enhance the understanding of the basic mechanisms affecting the CL life span.

Bovine somatic cell nuclear transfer (SCNT) is an important and powerful tool for basic research and biomedical and agricultural applications, however, the efficiency of SCNT has remained extremely low. In this study, we investigated the effects of cathepsin B inhibitor (E-64) supplementation of culture medium on in vitro development of bovine SCNT embryos. We initially used three concentrations of E-64 (0.1, 0.5, 1.0 μm), among which 0.5 μm resulted in the highest rate of blastocysts production after in vitro fertilization (IVF), and was therefore used for further experiments. Blastocyst development of SCNT embryos in the E-64 treatment group also increased relative to the control. Moreover, the cryosurvival rates of IVF and SCNT blastocysts were increased in E-64 treatment groups when compared with the control. On the other hand, we found that IVF and SCNT blastocysts derived from E-64-treated groups had increased total cell numbers and decreased apoptotic nuclei. Furthermore, assessment of the expression of apoptosis-related genes (Bax and Bcl-xL) in bovine IVF and SCNT blastocysts treated with E-64 by real-time RT-PCR analysis revealed suppressed expression of the pro-apoptotic gene Bax and stimulated expression of the anti-apoptotic gene Bcl-xL. Taken together, these finding indicate that addition of E-64 to embryo culture medium may have important implications for improving developmental competence and preimplantation quality in bovine IVF and SCNT embryos.

Cathepsin B is abundantly expressed peptidase of the papain family in the lysosomes, and closely related to the cell degradation system such as apoptosis, necrosis and autophagy. Abnormal degradation of organelles often occurs due to release of cathepsin B into the cytoplasm. Many studies have been reported that relationship between cathepsin B and intracellular mechanisms in various cell types, but porcine embryos has not yet been reported. Therefore, this study evaluated the effect of cathepsin B inhibitor (E-64) on preimplantation developmental competence and quality of porcine embryos focusing on apoptosis and oxidative stress. The expression of cathepsin B mRNA in porcine em-bryos was gradually decreased in inverse proportion to E-64 concentration by using real-time RT-PCR. When putative zygotes were cultured with E-64 for 24 h, the rates of early cleavage and blastocyst development were decreased by increasing E-64 concentration. However, the rate of blastocyst development in 5 μM treated group was similar to the control. On the other hand, both the index of apoptotic and reactive oxygen species (ROS) of blastocysts were sig-nificantly decreased in the 5 μM E-64 treated group compared with control. We also examined the mRNA expression levels of apoptosis related genes in the blastocysts derived from 5 μM E-64 treated and non-treated groups. Expre-ssion of the pro-apoptotic Bax gene was shown to be decreased in the E-64 treated blastocyst group, whereas expre-ssion of the anti-apoptotic Bcl-xL gene was increased. Taken together, these results suggest that proper inhibition of cathepsin B at early development stage embryos improves the quality of blastocysts, which may be related to not only the apoptosis reduction but also the oxidative stress reduction in porcine embryos.

The corpus luteum (CL) is a transient endocrine organ. Development, maintenance, and regression of CL are effectively controlled by dynamic changes in gene expression. However, it is unknown what types of gene are affected during the CL life span of the estrous cycle in bovine. Here, we determined whether unfolded protein response (UPR) signaling via eIF2α/ATF4/GADD34, p90ATF6/p50ATF6, and IRE1/XBP1, which is a cellular stress response associated with the endoplasmic reticulum (ER), is involved in the bovine CL life span. Our results indicated that expression of Grp78/Bip, the master UPR regulator, was increased during the maintenance stage and rapidly decreased at the regression stage. Additionally, UPR signaling pathways genes were found to be involved in luteal phase progression during the estrous cycle. Our findings suggested that Grp78/Bip, ATF6, and XBP1 act as ER chaperones for initiating CL development and maintaining the CL. In addition, we investigated whether ER stress-mediated apoptosis is occurred through three UPR signaling pathways in CL regression stage. Interestingly, pIRE1 and CHOP were found to be involved in both the adaptive response and ER stress-mediated apoptosis. During the CL regression stage, increased expression of pJNK and CHOP, two components of ER stress-mediated apoptotic cascades, occurred before increased level of cleaved caspase 3 were observed. The present investigation was performed to identify a functional link between UPR signaling and CL life span during the bovine estrous cycle. Taken together, results from this study demonstrated that UPR protein/gene expression levels were different at various stages of the bovine CL life span. Variations in the expression of these protein/genes may play important roles in luteal stage progression during the estrous cycle.

Freezing of bovine blastocysts has been widely used to improve the feasibility of cattle production by the embryo transfer technique. However, the low survival of vitrified-warmed embryos and their further development are crucial problems. Particularly, the production of offspring in vitrified-warmed bovine hatching/hatched blastocysts derived from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) is very low. Thus, we examined the effects of forced blastocoel collapse (FBC) before vitrification of bovine IVF- and SCNT-derived hatching/hatched embryos on the survival rate and apoptosis index after warming. Under optimal conditions, the overall survival rates in vitrified-warmed bovine IVF- and SCNT-derived hatching/hatched blastocysts were higher in FBC groups than in non-FBC groups (p

Gangliosides are key lipid molecules required for the regulation of cellular processes such as proliferation, differentiation, and cell signaling, including signaling of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF) has long been considered a potential regulator of meiotic and cytoplasmic maturation in mammalian oocytes. However, there is no report on the direct effect of ganglioside GD1a in porcine oocyte maturation. In this study, we first investigated a functional link between GD1a and meiotic maturation during in vitro maturation (IVM) of porcine embryos. Moreover, we confirmed the effect of exogenous GD1a treatment on blastocyst development, quality, and fertilization rate in early embryonic development. First, we observed that the protein level of ST3GAL2, a GD1a synthesizing enzyme, significantly increased (P < 0.01) in cumulus-oocyte-complexes (COCs) during IVM progress. The proportion of arrested germinal vesicles (GV) increased in oocytes treated with EGF+GD1a (41.6 ± 1.5%) at the IVM I stage. Upon completion of meiotic maturation, the proportion of metaphase II (M II) was significantly higher (P < 0.05) in the EGF+GD1a (89.9 ± 3.6%) treated group. After IVF, the percentage of penetrated oocytes was significantly higher (P < 0.05) in the EGF+GD1a (89.1 ± 2.3%) treated group than in the control group. Furthermore, exogenous GD1a treatment improved the developmental competence and quality of blastocysts during preimplantation embryo development stage. These results suggest that ganglioside GD1a may play an important role in IVM mechanisms of porcine maturation capacity. Furthermore, our findings will be helpful for better promoting the embryo development and blastocyst quality in pigs.

Lipocalin-2 (LCN2) is a 25-kDa protein in the lipocalin family that shows antibacterial activity. The aim of this study was to clone human LCN2 into the mammalian-specific pIRES2-AcGFP1 vector and to determine the antibacterial activity of the recombinant pIRES2-AcGFP1-hLCN2 plasmid against major bacterial pathogens that cause bovine mastitis. The amplified hLCN2 was successfully cloned into the vector, and the plasmid was transfected into bovine mammary epithelial stem cells using nucleofection. Immunochemistry, reverse transcription polymerase chain reaction, and ELISA were used to monitor and quantify hLCN2 expression in the cells. After 1 day of incubation, the concentration of hLCN2 secreted by cells in the medium (6.85 ± 0.11 μg) was higher than that stored within the cells (22 ± 2.52 ng). After 4 days of culture, the amount of hLCN2 was 300-fold higher in the medium than within the cells. The recombinant clone showed potential antibacterial activity against mastitis-causing bacteria, with greater inhibitory activity against Escherichia coli than against Staphylococcus aureus. Antibacterial activity remained strong after 4-5 h of culture. Thus, pIRES2-AcGFP1 was identified as an efficient vector for the delivery of the target gene to the mammary cells, and recombinant hLCN2 showed strong antibacterial activity against major mastitis-causing bacteria.

Information obtained from peripheral blood could help us understand the underlying mechanisms in autoimmune diseases, cancer, pregnancy, and other conditions. In this paper, we present the protein map of porcine peripheral blood mononuclear cells (PBMC) to better understand the molecular expression changes that occur during pregnancy using proteomic analysis. We detected 94 differentially expressed proteins in pregnant vs. non-pregnant (NP) pigs, and a representative set of the proteins was subjected to LC–MS/MS analysis. Furthermore, the identified proteins were categorized according to their biological process and molecular function. By classifying the proteins according to their functions, a large number of differentially regulated proteins involved in anti-oxidant, detoxification and stress response pathways were found, including peroxiredoxin (PRX) 1, 2, and 6, glutathione-S-transferase (GST), annexin A2, and A6, and heat shock protein 27 (HSP 27) during pregnancy (pregnancy d of E40, embryonic day 40; E70, embryonic day 70; and E93, embryonic day 93) compared with non-pregnancy. In this study, a proteomic approach utilizing 2-DE and LC–MS/MS was applied to evaluate specific molecular expression changes during pregnancy compared with non-pregnancy. Together, these data offer new information about the proteome map and factors that are differentially regulated during maintenance of normal pregnancy.

Myeloid-derived suppressor cells (MDSCs) play an important role in controlling the immune response against cancer and in suppression of autoimmunity and allergic inflammation. However, the beneficial effects of MDSCs on the experimental mouse model of psoriasis have not been reported. Therefore, we investigated the anti-psoriatic effect of MDSCs on IMQ-induced skin inflammation in mice and explored the mechanisms involved. Our results showed that administration of MDSCs (1 × 106 or 2 × 106 cells) suppressed the development of IMQ-induced skin inflammation in mice as exemplified by a significant reduction in clinical severity scores and was associated with a reduction of histopathological changes, including inflammatory infiltration, epidermal hyperplasia and hyperkeratosis. The immunosuppressive effect of MDSCs (1 × 106 or 2 × 106 cells) corresponded to the production of Th1 cytokines (TNF-α, IFN-γ) and Th17 cytokines (IL-17A and IL-23) in the serum and dorsal skin. Administration of MDSCs (1 × 106 or 2 × 106 cells) also inhibited splenomegaly. Moreover, an increased percentage of CD4+ CD25+ FoxP3+ regulatory T (Treg) cells and decreased percentage of Th1 and Th17 cells were found in mice treated with MDSCs. Taken together, these results imply that MDSCs have immunomodulatory and immunosuppressive effects on disease progression in a murine model of psoriasis and that MDSCs could be used in preventive or therapeutic strategies for the management of autoimmune inflammatory skin disorders, such as psoriasis.

Successful production of transgenic pigs requires oocytes with a high developmental competence. However, cumulus–oocyte complexes (COCs) obtained from antral follicles have a heterogeneous morphology. COCs can be classified into one of two classes: class I, with five or more layers of cumulus cells; and class II, with one or two layers of cumulus cells. Activator [e.g., epidermal growth factor (EGF)] or inhibitors (e.g., wortmannin and U0126) are added to modulate kinases in oocytes during meiosis. In the present study, we investigated the effects of kinase modulation on nuclear and cytoplasmic maturation in COCs. Class I COCs showed a significantly higher developmental competence than class II COCs. Moreover, the expression of two kinases, AKT and ERK, differed between class I and class II COCs during in vitro maturation (IVM). Initially, inhibition of the PI3K/AKT signaling pathway in class I COCs during early IVM (0–22 h) decreased developmental parameters, such as blastocyst formation rate, blastomere number, and cell survival. Conversely, EGF-mediated AKT activation in class II COCs enhanced developmental capacity. Regarding the MAPK signaling pathway, inhibition of ERK by U0126 in class II COCs during early IVM impaired developmental competence. However, transient treatment with U0126 in class II COCs increased oocyte maturation and AKT activity, improving embryonic development. Additionally, western blotting showed that inhibition of ERK activity negatively regulated the AKT signaling pathway, indicative of a relationship between AKT and MAPK signaling in the process underlying meiotic progression in pigs. These findings may help increase the developmental competence and utilization rate of pig COCs with regard to the production of transgenic pigs and improve our understanding of kinase-associated meiosis events.

Animal bioreactors have been regarded as alternative tools for the production of limited human therapeutic proteins. The mammary glands of cattle are optimal tissues to produce therapeutic proteins that cannot be produced in large amounts in traditional systems based on microorganisms and eukaryotic cells. In this study, two knock-in vectors, pBCTPOKI-6 and pBCTPOKI-10, which target the hTPO gene on the bovine beta-casein locus, were designed to develop cloned transgenic cattle. The pBCTPOKI-6 and pBCTPOKI-10 vectors expressed hTPO protein in culture medium at a concentration of 774 pg/ml and 1,867 pg/ml, respectively. Successfully, two targeted cell clones were obtained from the bovine fibroblasts transfected with the pBCTPOKI-6 vector. Cloned embryos reconstructed with the targeted nuclei showed a lower in vitro developmental competence than those with the wild-type nuclei. After transfer of the cloned embryos into recipients, 7 pregnancies were detected at 40 to 60 days of gestation, but failed to develop to term. The results are the first trial for targeting of a human gene on the bovine milk protein gene locus, providing the potential for a large-scale production of therapeutic proteins in the animal bioreactor system.

Plasminogen activators (PAs) play a pivotal role in a variety of uterine physiologies, such as endometrial function, trophoblast invasion, and implantation process, but its alteration in expression or activity during cryopreservation of primary uterine cells has received little attention. In this study, we investigated whether PA expression and activity were modulated in first passage primary porcine uterus endometrial epithelium cells (PUEECs) treated with or without a freezing-thawing procedure. Western blotting and zymographic analysis showed that uPA expression and activity increased significantly in frozen-thawed PUEECs in a passage-dependent manner as compared to freshly prepared control cells. Moreover, intracellular reactive oxygen species (ROS) were increased by freezing-thawing and longer culturing, and were more prominent in frozen-thawed PUEECs than in control cells. However, the increase in both uPA expression and activity was greatly reduced or alleviated by treatment with either ROS scavenger N-acetylcysteine or extracellular signal-regulated kinase (ERK) inhibitor PD98059. These results suggest that ROS/ERK-mediated uPA activation may be an important factor in cryo-damage of primary uterine cells.

The production of reactive oxygen species (ROS) from mitochondria contributes to redox signalling, mitochondrial functions, and apoptosis. However, the specific effects of mitochondria target superoxide (O2 •–) on porcine embryo development remain unclear. The objective of present study was to examine the differences of mitochondrial functions and dynamics in 2 subpopulations of porcine zygotes (G1 and G2), and to investigate the effects of Mito-TEMPO on porcine embryo development. Porcine embryos were visually classified in 2 groups [Grade (G)1: over 90%, and G2: below 90%] according to the lipid distribution at the zygote stage. The blastocyst development rate was greater in G1 than in G2 embryos (G1: 26.5 ± 5.9% v. G2: 16.2 ± 7.9%; P < 0.05). To evaluate blastocyst quality, we performed a 4′,6-diamidino-2-phenylindole (DAPI)-TUNEL assay. The proportion of TUNEL-positive cells was higher (P

The mammalian oocyte undergoes dynamic changes in chromatin structure to reach complete maturation. However, little known is about behaviors of ATP-dependent chromatin remodeling factors (ACRFs) during meiosis. Here, we found that respective ACRFs may differently behave in the process of oocyte maturation in the bovine. All ACRFs interacted with oocytic chromatin at the germinal vesicle (GV) stage. Mi-2 and hSNF2H disappeared from GV-chromatin within 1 hr of in vitro culture whereas Brg-1 and BAF-170 were retained throughout germinal vesicle break down (GVBD). Brg-1 was localized on the condensed chromatin outside, whereas BAF-170 was entirely excluded from condensed chromatin. Thereafter, Brg-1 and BAF-170 interacted with metaphase I and metaphase II chromosomes. These results imply that Mi-2 and hSNF2H may initiate the meiotic resumption, and Brg-1 and BAF-170 may support chromatin condensation during meiosis. In addition, DNA methylation and methylation of histone H3 at lysine 9 (H3K9) seem to be constantly retained in the oocyte chromatin throughout in vitro maturation. Inhibition of ACRF activity by treatment with the inhibitor apyrase led to retarded chromatin remodeling in bovine oocytes, thereby resulting in poor development of fertilized embryos. Therefore, these results indicate that precise behaviors of ACRFs during meiosis are critical for nuclear maturation and subsequent embryonic development in the bovine.

Porcine pancreas development is not well studied at the molecular level despite being a therapeutic resource for diabetic patients. In this study, we investigated expression of lineage markers and performed proteomic analysis. Expression of the early lineage markers Pdx1 and Ptf1a was developmentally conserved between mice and pigs, whereas expression of the islet differentiation marker Pax4 was delayed in porcine compared with murine pancreas development. Proteomic analysis found that expression levels of chymotrypsinogen were down-regulated during porcine pancreas development while those of digestive enzymes like lipases, elastase and serine protease were up-regulated. In addition, specific isoforms of protein folding assistants such as protein disulfide isomerase and prefoldin were expressed at specific stages during the maturation of digestive enzymes. Taken together, these results show that development of the porcine pancreas is regulated by a concerted interplay of pancreas lineage marker proteins and other specified proteins, resulting in a functional endocrine and exocrine organ.

Neuropeptide Y (NPY) and NPY receptors are widely expressed in various organs and cell types and have been shown to have pleiotropic functions. However, their presence or role in human embryonic stem cells (hESCs) remains unknown. We now show that undifferentiated hESCs primarily express NPY and its Y1 and Y5 receptors. Inhibition of NPY signalling using either the selective NPY Y1 or Y5 receptor antagonist reduces the maintenance of self-renewal and proliferation of undifferentiated hESCs. We also provide compelling evidence that exogenous NPY supports the long-term growth of undifferentiated hESCs in the absence of feeder cell factors using only knockout serum replacement media. Further, NPY facilitates the use of chemically defined medium made up of N2/B27 supplement and basic fibroblast growth factor (bFGF) for hESC feeder-free culture. Our results indicate that both Y1 and Y5 receptors appear to be involved in the NPY-mediated activation of AKT/protein kinase B and extracellular signal-regulated kinase 1/2 (ERK1/2) in hESCs. Notably, only Y1 receptor, but not Y5 receptor, is responsible for the NPY-induced activation of cAMP-response element binding (CREB) in hESCs. These results provide the first evidence that NPY and its Y1 and Y5 receptors have potential role in maintaining hESC self-renewal and pluripotency. We demonstrate the underlying importance of NPY signalling and its usefulness in the development of a defined and xeno-free culture condition for the large-scale propagation of undifferentiated hESCs.

Early embryonic losses are common in cloned embryos in the current cloning system. However, the reasons for embryonic losses in early developmental stages of cloned embryos remain unclear. To elucidate the cause of early defective development in cloned embryos, two porcine clones including extraembryonic tissues were obtained at 26 days of gestation. The expression of various molecules in developmentally important signaling pathways, including Notch, hedgehog (Hh), receptor tyrosine kinase (RTK), Janus kinase/signal transducer and activator of transcription (JAK/STAT), wingless related (Wnt), and transforming growth factor-beta (TGF-beta), was then examined in the extraembryonic tissues. Western blot analysis showed that the expression of key molecules involved in the Notch, Hh, RTK, and JAK/STAT signaling pathways was downregulated, whereas most Wnt and TGF-beta signaling pathway molecules were upregulated in cloned extraembryonic tissues compared to normal extraembryonic tissues. These results indicate that unbalanced coordination of signaling pathways might impair the early development of cloned embryos postimplantation, thereby resulting in embryonic losses during the first trimester.

The potential medical applications of animal cloning include xenotransplantation, but the complex molecular cascades that control porcine organ development are not fully understood. Still, it has become apparent that organs derived from cloned pigs may be suitable for transplantation into humans. In this study, we examined the pancreas of an adult cloned pig developed through somatic cell nuclear transfer (SCNT) using two-dimensional electrophoresis (2-DE) and Western blotting. Proteomic analysis revealed 69 differentially regulated proteins, including such apoptosis-related species as annexins, lamins, and heat shock proteins, which were unanimously upregulated in the SCNT sample. Among the downregulated proteins in SCNT pancreas were peroxiredoxins and catalase. Western blot results indicate that several antioxidant enzymes and the anti-apoptotic protein were downregulated in SCNT pancreas, whereas several caspases were upregulated. Together, these data suggest that the accumulation of reactive oxygen species (ROS) in the pancreas of an adult cloned pig leads to apoptosis.

This paper reports a new dielectrophoretic separation method of porcine oocytes for in vitro fertilization. Conventional manual selection of oocyte highly depends on the expert's experience and lacks universal standards for identifying the quality of oocyte. In this study, an electrode array chip with castellated shape was developed to evaluate dielectrophoretic velocities of oocytes, under applied bias conditions with an AC 3 V waveform at 1 MHz for 15 s. Based on different dielectrophoresis (DEP) response, the selected group of oocytes that moved showed a better developmental potential than the group of oocytes that stayed, representing a higher rate of blastocyst formation and a lower rate of polyspermic fertilization. In addition, the overall developmental potential of oocytes selected by the DEP device was comparable to that of oocytes selected by conventional manual method. These results demonstrate that the difference in dielectrophoretic velocity can be used to establish an objective criterion for the selection of oocytes. Consequently, this method will open the possibility to develop an automatic tool for oocyte selection, which would be helpful for assisted reproductive technologies such as transgenic and clonal animal production.

Gangliosides are implicated in neuronal development processes. The regulation of ganglioside levels is closely related to the induction of neuronal cell differentiation. In this study, the relationship between ganglioside expression and neuronal cell development was investigated using an in vitro model of neural differentiation from mouse embryonic stem (mES) cells. Daunorubicin (DNR) was applied to induce the expression of gangliosides in embryoid body (EB) (4+). We observed an increase in expression of gangliosides in all stages of EBs by treatment of DNR (2microM). High-performance thin-layer chromatography (HPTLC) showed that gangliosides GD3, GD1a, GT1b, and GQ1b increased in DNR-treated 7-day-old EB (4+) [EB (4+):7]. DNR treatment significantly increased the expression of gangliosides, especially GT1b and GQ1b in comparison to control cells. Interestingly, GQ1b co-localized with microtubule-associated protein 2 (MAP-2) expressing cells in DNR-treated EB (4+):7. The co-localization of GQ1b and MAP-2 was found in neurite-bearing cells in DNR-treated 15-day-old EB (4+) [EB (4+):15], whereas no significant expression of GQ1b and less neurite formation were observed in untreated control. Also, the expression of synaptophysin and NF200, both neuronal markers associated with neruites, was increased by DNR treatment. These results demonstrate that DNR increases expression of gangliosides, especially GQ1b, in differentiating neuronal cells. Further, neurite-bearing neuronal cell differentiation can be facilitated by DNR, possibly through the induction of gangliosides. Thus, the present data suggest that DNR is beneficial for facilitating neuronal differentiation from ES cells and among the gangliosides analyzed in the present study, GQ1b is mainly involved in neurite formation.

Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce first-generation (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second-generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third-generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age- and sex-matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the phenotypic abnormality in the G1 clones and that the abnormality was acquired by only a few of the G1 clone's cells during its gestational development.

DNA methylation reprogramming (DMR) during preimplantation development erases differentiation-associated, unessential epigenetic information accumulated during gametogenesis, and ultimately brings pluripotency to the resulting embryo. Two patterns of DMR of sperm-derived pronucleus have been reported in mammals. In the first, the male pronucleus is actively demethylated whereas in the second, the methylation state seems to be maintained. The maintenance-type DMR has been seen only through immunocytochemical observations, and waits to be proven by additional molecular-level evidence. We demonstrate that, in pig, paternally derived DNA methylation is preserved during pronucleus development, based on the following observations. First, immunostaining of pig zygotes at different time points showed the DNA methylation state to be balanced between parental pronuclei throughout pronucleus development. Second, bisulfite analysis of PRE-1 repetitive sequences found mono- and polyspermic eggs to have similar methylation states. Third, the methylation state of a human erythropoietin gene delivered by transgenic pig spermatozoa was maintained in the male pronucleus. Finally, 5-aza-2'-deoxycytidine treatment, which blocks re-methylation, did not show the male pronucleus to be stalled in a demethylated state. In pig zygotes, paternally derived cytosine methylation was preserved throughout pronucleus development. These findings from multilateral DMR analyses provide further support to the view that DMR occurs in a non-conserved manner during early mammalian development.

Pelvic inflammatory disease (PID), which is one of the most problematic complications experienced by women with sexually transmitted diseases, frequently causes secondary infections after reproductive abnormalities in veterinary animals. Although the uterus is self-protective, it becomes fragile during periods or pregnancy. To investigate PID, bacteria or lipopolysaccharide (LPS) extracted from gram negative bacteria has been used to induce the disease in several animal models. However, when LPS is applied to the peritoneum, it often causes systemic sepsis leading to death and the PID was not consistently demonstrated. Hydrochloric acid (HCl) has been used to induce inflammation in the lungs and stomach but not tested for reproductive organs. In this study, we developed a PID model in mice by HCl and LPS sequential intracervical (i.c.) administration. The proinflammatory cytokines, interleukin (IL)-1β, IL-6 and tumor necrosis factor-α, were detected in the mouse uterus by western blot analysis and cytokine enzyme-linked immunosorbent assay after HCl (25 mg/kg) administration i.c. followed by four LPS (50 mg/kg) treatments. Moreover, mice exhibited increased infiltration of neutrophils in the endometrium and epithelial layer. These results suggest that ic co-administration of HCl and LPS induces PID in mice. This new model may provide a consistent and reproducible PID model for future research.

Human embryonic stem cells (hESCs) are able to differentiate into various cell types, including neuronal cells and glial cells. However, little information is available regarding astrocyte differentiation. This report describes the differentiation of hESCs into nestin- and GFAP-expressing astrocytes following treatment with cyclopamine, which is an inhibitor of Hedgehog (Hh) signaling, and culturing in human astrocyte medium (HAM). In hESCs, cyclopamine treatment suppressed the expression of Hh signaling molecules, the Hh signaling target gene, and ESC-specific markers. Clyclopamine also induced the differentiation of the cells at the edges of the hESC colonies, and these cells stained positively for the early neural marker nestin. Subsequent culturing in HAM promoted the expression of the astrocyte-specific marker GFAP, and these cells were also nestin-positive. These findings indicate that treatment with cyclopamine followed by culturing in HAM leads to the differentiation of hESCs into nestin- and GFAP-expressing astrocytic lineage.

A viable cloned animal indicates that epigenetic status of the differentiated cell nucleus is reprogrammed to an embryonic totipotent state. However, molecular events regarding epigenetic reprogramming of the somatic chromatin are poorly understood. Here we provide new insight that somatic chromatins are refractory to reprogramming of histone acetylation during early development. A low level of acetylated histone H4-lysine 5 (AcH4K5) of the somatic chromatin was sustained at the pronuclear stage. Unlike in vitro fertilized (IVF) embryos, the AcH4K5 level remarkably reduced at the 8-cell stage in cloned bovine embryos. The AcH4K5 status of somatic chromatins transmitted to cloned and even recloned embryos. Differences of AcH4K5 signal intensity were more distinguishable in the metaphase chromosomes between IVF and cloned embryos. Two imprinted genes, Ndn and Xist, were aberrantly expressed in cloned embryos as compared with IVF embryos, which is partly associated with the AcH4K5 signal intensity. Our findings suggest that abnormal epigenetic reprogramming in cloned embryos may be because of a memory mechanism, the epigenetic status itself of somatic chromatins.

This study was conducted to examine the activities of maturation-promoting factor (MPF) and mitogen-activated protein (MAP) kinase in the porcine oocytes after artificial activation. To determine optimal electrical activation condition, oocytes were exposed to single DC pulse in a variety of electric field strengths (120, 150, 180, and 210 V/mm) and pulse durations (15, 30, 45, and 60 µsec). After the artificial activation, 40–50 oocytes were cultured in a 50 µl drop of NCSU23 medium supplemented with 0.4% BSA at 39°C, 5% CO2 in air for 6 days. No difference was detected in the preimplantation development of pocine oocytes and the mean nuclei number of blastocysts between electric field strengths. Under the 180 V/mm electric field strength, short pulse durations (15 and 30 µsec) showed a higher preimplantation developmental rate of the oocytes and mean nuclei number of blastocysts than an extended electric pulse (60 µsec) (P

The present study was to investigate the effects of in vitro fertilization conditions on in vitro development and structural integrity of pig embryos. Porcine oocytes matured in vitro were co-incubated with four different spermatozoa concentrations (0.6 x 10(5), 1.2 x 10(5), 2.5 x 10(5) and 5 x 10(5) cells/ml) for 6 h, and at a spermatozoa concentration (1.2 x 10(5) cells/ml) for 2, 4 and 6 h, respectively. Spermatozoa penetration and blastocyst formation were observed at 10 and 144 h post insemination, respectively. The allocation of a blastocyst to inner cell mass (ICM) and trophectoderm (TE) cells was determined by using a differential staining method. Polyspermy frequency increased with increasing spermatozoa concentrations. The spermatozoa-oocyte co-incubation period of 2 h provided for decreased in vitro development rate than 4 and 6 h groups (P0.05), although no difference was detected in polyspermy frequency between spermatozoa-oocyte co-incubation periods. Interestingly, blastocysts derived from the groups with greater spermatozoa concentrations (2.5 x 10(5) and 5 x 10(5) cells/ml) had significantly fewer ICM cell nuclei as compared with those groups with lesser spermatozoa concentrations (0.6 x 10(5) and 1.2 x 10(5) cells/ml). There was no difference in the structural integrity of blastocysts among the co-incubation periods. Blastocysts derived from respective experiments were individually classified into three groups (I:20%; II: 20-40% and III:40%) based on the ratio of ICM to total cells. Proportion of blastocysts in Group II, with a presumptive normal range of structural integrity, was slightly decreased in the groups with greater spermatozoa concentrations (2.5 x 10(5) and 5 x 10(5) cells/ml). The results indicate that the spermatozoa concentration during in vitro fertilization may be important for developmental competence and quality of pig embryos.

Global reduction of DNA methylation, a part of genome reprogramming processes, occurs in a gradual manner until before implantation and is recognized as a conserved process in mammals. Here, we reported that in bovine, satellite regions exhibited varied patterns of methylation changes when one-cell egg advanced to the blastocyst; a maintenance methylation was observed in satellite I sequences, a decrease in alpha satellites, and an increase in satellite II regions. Cloned embryos exhibited similar changes for DNA methylation in the satellite I and alpha. We also observed that the satellite I and alpha sequences were methylated more in inner cell mass region of the blastocyst whereas the satellite II showed selective demethylation in this region. Together, these findings point that individual satellite sequences carry their own methylation patterns under the pressure of global demethylation, suggesting that local methylation control system acts on the satellite regions in early bovine embryos.

The structural integrity of blastocyst stage embryos, consisting of the inner cell mass (ICM) and trophectoderm (TE) cells, is a prerequisite for normal development after implantation in mammals. In this study, allocation of nuclear transfer (NT)-derived porcine blastocysts to the ICM and to the TE cells was examined and compared with IVF- and in vivo-derived embryos. NT-derived embryos had a lower developmental competence to the blastocyst stage than IVF-derived embryos (P0.05). Total cell number of NT-derived blastocysts was inferior to that of IVF-derived embryos (P0.05), although no difference was detected between the two groups in the ratio of ICM to total cells. However, in vivo-derived blastocysts had a higher proportion of ICM to total cells compared with in vitro-produced embryos (P0.01). To investigate what proportions of in vitro-produced porcine embryos represent normal structural integrity, differentially-stained blastocysts were individually classified into three presumptive groups (I:20%; II: 20-40%; III:40%) according to the ratio of ICM to total cells. Low proportions of NT- (12.5%, 7/56) and IVF-derived blastocysts (15.8%, 9/57) were assigned to Group II, presumptively having a normal range of structural integrity, whereas, almost all in vivo-derived embryos (97.5%, 39/40) were allocated to Group II. In conclusion, limited structural integrity may lead to the poor survival to term of NT- or IVF-derived porcine embryos produced in vitro.

Abortions of nuclear transfer (NT) embryos are mainly due to insufficient placentation. We hypothesized that the primary cause might be the aberrant allocations of two different cell lineages of the blastocyst stage embryos, the inner cell mass (ICM) and the trophectoderm (TE) cells. The potential for development of NT embryos to blastocysts was similar to that for in vitro fertilized (IVF) embryos. No difference in the total cell number was detected between NT and IVF blastocysts, but both types of embryos had fewer total cells than did in vivo-derived embryos (P 60%) according to the ratio of ICM:total cells. Most NT blastocysts were placed in groups III and IV, whereas most IVF and in vivo-derived blastocysts were distributed in group II. Our findings suggest that placental abnormalities or early fetal losses in the present cloning system may be due to aberrant allocations of NT embryos to the ICM and TE cells during early development.

Cloning by nuclear transfer (NT) has been riddled with difficulties: most clones die before birth and survivors frequently display growth abnormalities. The cross-species similarity in abnormalities observed in cloned fetuses/animals leads us to suspect the fidelity of epigenetic reprogramming of the donor genome. Here, we found that single-copy sequences, unlike satellite sequences, are demethylated in pre-implantation NT embryos. The differential demethylation pattern between genomic sequences was confirmed by analyzing single blastocysts. It suggests selective demethylation of other developmentally important genes in NT embryos. We also observed a reverse relationship between methylation levels and inner cell mass versus trophectoderm (ICM/TE) ratios, which was found to be a result of another type of differential demethylation occurring in NT blastocysts where unequal methylation was maintained between ICM and TE regions. TE-localized methylation aberrancy suggests a widespread gene dysregulation in an extra-embryonic region, thereby resulting in placental dysfunction familiar to cloned fetuses/animals. These differential demethylations among genomic sequences and between differently allocated cells produce varied overall, but specified, methylation patterns, demonstrating that epigenetic reprogramming occurs in a limited fashion in NT embryos.

Under endoplasmic reticulum (ER)‐stress conditions, the unfolded protein response (UPR) generates a defense mechanism in mammalian cells. The regulation of UPR signaling is important in oocyte maturation, embryo development, and female reproduction of pigs. Recent studies have shown that melatonin plays an important role as an antioxidant to improve pig oocyte maturation. However, there is no report on the role of melatonin in the regulation of UPR signaling and ER‐stress during in vitro maturation (IVM) of porcine oocytes. Therefore, the objective of this study was to investigate the antioxidative effects of melatonin on porcine oocyte maturation through the regulation of ER‐stress and UPR signaling. We investigated the changes in the mRNA/protein expression levels of three UPR signal genes (Bip/Grp78, ATF4, P90/50ATF6, sXbp1, and CHOP) on oocytes, cumulus cells, and cumulus‐oocyte complexes (COCs) during IVM (metaphase I; 22 hours and metaphase II; 44 hours) by Western blot and reverse transcription‐polymerase chain reaction analysis. Treatment with the ER‐stress inducer, tunicamycin (Tm), significantly increased expression of UPR markers. Additionally, cumulus cell expansion and meiotic maturation of oocytes were reduced in COCs of Tm‐treated groups (1, 5, and 10 μg/mL). We confirmed the reducing effects of melatonin (0.1 μmol/L) on ER‐stress after pretreatment with Tm (5 μg/mL; 22 hours) in maturing COCs. Addition of melatonin (0.1 μmol/L) to Tm‐pretreated COCs recovered meiotic maturation rates and expression of most UPR markers. In conclusion, we confirmed a role for melatonin in the modulation of UPR signal pathways and reducing ER‐stress during IVM of porcine oocytes.

Bisphenol A (BPA) is well known as oestrogen-like chemical and it is widely used in plastic products. Many studies have reported that BPA exposure has a well-known toxicity effect on reproduction function, such as reducing the number of ovulated oocytes, oocyte quality, and maturation rate. Recently, BPA induced mitochondrial-derived reactive oxygen species (mito-ROS) and disrupted mitochondrial homeostasis by increasing of superoxide anions production. In this study, we investigated how the regulation of mito-ROS production may play a critical role in meiotic maturation and expansion of cumulus cells during the in vitro maturation progression of porcine oocytes. Furthermore, we investigated the toxicity effect of BPA exposure on mitochondrial functions and mito-ROS production during porcine oocyte maturation in vitro. All results were analysed using a 1-way ANOVA followed by Bonferroni’s and Tukey’s Multiple Comparison Test and t-tests. First, porcine oocytes were matured in NCSU-23 medium supplemented with BPA (50, 75, and 100 µM) for 44 h. Our results indicated that the rates of matured oocytes were significantly decreased by BPA exposure in a dose-dependent manner (69.4 ± 5.1, 50.9 ± 6.3, and 29.9 ± 5.8% for BPA treatments of 50, 75, and 100 μM) compared with control group (70.2 ± 7.8%; P

Unfolding protein response (UPR) is a defence mechanism during endoplasmic reticulum (ER) stress in mammalian cells. Especially, UPR genes and regulation of reactive oxygen species is involved in ER stress response on porcine oocyte maturation in vitro. Some studies have shown that melatonin treatment results in reducing oxidative stress, a protective function of free radical damage in oocyte maturation and embryo development. Also, melatonin has an important role in reducing reactive oxygen species and ER stress. However, it is unknown how the changes of UPR genes expression levels are affected the porcine oocyte maturation. In addition, there are no reports about ER stress recovery mechanism by melatonin during porcine oocyte maturation. Here, we investigated the UPR signal genes (Bip/Grp78, Atf4, p90/p50Atf6, and Xbp1) and ER-stress mediated apoptosis factors (Chop and Cleaved caspase 3) in porcine oocyte maturation in vitro. Expression of Chop and Cleaved caspase 3 mRNA levels were significantly increased (P

Despite evidence of the presence of prostaglandin (PG) I2 in mammalian oviducts, its role in early development of in vitro-produced (IVP) embryos is largely unknown. Thus, in the present study we examined the effects of iloprost, a PGI2 analogue, on the in vitro developmental competence of early porcine embryos and the underlying mechanism(s). To examine the effects of iloprost on the development rate of IVF embryos, iloprost was added to the in vitro culture (IVC) medium and cultured for 6 days. Supplementation of the IVC medium with iloprost significantly improved developmental parameters, such as blastocyst formation rate, the trophectoderm : inner cell mass ratio and cell survival in IVF and parthenogenetically activated (PA) embryos. In addition, post-blastulation development into the expanded blastocyst stage was improved in iloprost-treated groups compared with controls. Interestingly, the phosphatidylinositol 3-kinase (PI3K)/AKT signalling pathway was significantly activated by iloprost supplementation in a concentration-dependent manner (10–1000 nM), and the beneficial effects of iloprost on the early development of porcine IVF and PA embryos was completely ablated by treatment with 2.5 μM wortmannin, a PI3K/AKT signalling inhibitor. Importantly, expression of the PI3K/AKT signalling pathway was significantly reduced in somatic cell nuclear transfer (SCNT) compared with IVF embryos, and iloprost supported the early development of SCNT embryos, as was the case for IVF and PA embryos, suggesting a consistent effect of iloprost on the IVC of IVP porcine embryos. Together, these results indicate that iloprost can be a useful IVC supplement for production of IVP early porcine embryos with high developmental competence.