Blastocyst Total Cell Number Research Articles

Astaxanthin Normalizes Epigenetic Modifications of Bovine Somatic Cell Cloned Embryos and Decreases the Generation of Lipid Peroxidation.

Astaxanthin is an extremely common antioxidant scavenging reactive oxygen species (ROS) and blocking lipid peroxidation. This study was conducted to investigate the effects of astaxanthin supplementation on oocyte maturation, and development of bovine somatic cell nuclear transfer (SCNT) embryos. Cumulus-oocyte complexes were cultured in maturation medium with astaxanthin (0, 0.5, 1.0, or 1.5mg/l), respectively. We found that 0.5mg/l astaxanthin supplementation significantly increased the proportion of oocyte maturation. Oocytes cultured in 0.5mg/l astaxanthin supplementation were used to construct SCNT embryos and further cultured with 0, 0.5, 1.0 or 1.5mg/l astaxanthin. The results showed that the supplementation of 0.5mg/l astaxanthin significantly improved the proportions of cleavage and blastulation, as well as the total cell number in blastocysts compared with the control group, yet this influence was not concentration dependent. Chromosomal analyses revealed that more blastomeres showed a normal chromosomal complement in 0.5mg/l astaxanthin treatment group, which was similar to that in IVF embryos. The methylation levels located on the exon 1 of the imprinted gene H19 and IGF2, pluripotent gene OCT4 were normalized, and global DNA methylation, H3K9 and H4K12 acetylation were also improved significantly, which was comparable to that invitro fertilization (IVF) embryos. Moreover, we also found that astaxanthin supplementation significantly decreased the level of lipid peroxidation. Our findings showed that the supplementation of 0.5mg/l astaxanthin to oocyte maturation medium and embryo culture medium improved oocyte maturation, SCNT embryo development, increased chromosomal stability and normalized the epigenetic modifications, as well as inhibited overproduction of lipid peroxidation.

Effect of C-Type Natriuretic Peptide on Maturation and Developmental Competence of Goat Oocytes Matured In Vitro

The developmental competence of oocytes during in vitro maturation (IVM) is compromised due to asynchronous nuclear and cytoplasmic maturation. To improve IVM efficiency, a pre-maturation culture or two-step maturation strategy has been established, involving meiosis arrest induced by pharmacological agents to provide oocytes with sufficient time to synchronize the maturation of the nucleus and cytoplasm. C-type natriuretic peptide (CNP), which has been demonstrated to function as an oocyte maturation inhibitor (OMI) in many species, provides a new alternative to improve the developmental capacity of oocytes matured in vitro. However, the effect of CNP on meiosis arrest and the maturation of goat oocytes remains unclear. In the present study, CNP was shown to function as an OMI in goat oocytes. CNP could temporarily maintain the meiotic arrest of goat oocytes cultured in vitro for 4 hours. This transient effect was partly due to the reduction of natriuretic peptide receptor 2 (Npr2). Estradiol could delay the decrease in Npr2 expression and prolong the duration of meiosis arrest up to 6 hours. Based on the above results, a two-step method was established for goat oocyte maturation, in which the oocyte maturation rate was significantly increased. After parthenogenetic activation, the cleavage rate, blastocyst rate and total cell number of blastocysts were significantly improved. Our results suggested that CNP can be used to delay meiotic resumption and enhance the developmental competence of goat oocytes matured in vitro.

The effects of human recombinant granulocyte-colony stimulating factor treatment during in vitro maturation of porcine oocyte on subsequent embryonic development

Granulocyte colony-stimulating factor (G-CSF) is required for proliferation, differentiation, and survival of cells. It is also a biomarker of human oocyte developmental competence for embryo implantation. In humans, the G-CSF concentration peaks during the ovulatory phase of the ovarian cycle. In this study, the expressions of G-CSF and its receptor were analyzed by polymerase chain reaction in granulosa cells (GCs), CL, cumulus cells (CCs), and oocytes. Cumulus–oocyte complexes were aspirated from antral follicles of 1 to 3 mm (small follicles) and 4 to 6 mm (medium follicles). Cumulus–oocyte complexes from two kinds of follicles were matured in protein-free maturation medium supplemented with various concentrations of G-CSF (0, 10, and 100 ng/mL). By real-time polymerase chain reaction, the expressions of G-CSF and its receptor were detected in GCs, CL, CCs, and oocytes. Interestingly, the G-CSF transcript levels were significantly lower in oocytes than in the other cell types, whereas the G-CSF receptor transcript levels in oocytes were similar to those in GCs. After 44 hours of IVM, no differences in the rate of nuclear maturation were detected; however, the intracellular reactive oxygen species levels in oocytes from both groups of follicles matured with 10 ng/mL of human recombinant G-CSF (hrG-CSF) groups were significantly lower (P < 0.05). After parthenogenetic activation, the cleavage rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF–treated small (63.3%) follicles than in 0, 10 ng/mL hrG-CSF–treated small (38.6% and 49.0%, respectively) follicles and 0 ng/mL hrG-CSF–treated medium (52.1%) follicles, and the cleavage rates were significantly (P < 0.05) higher in 10 ng/mL hrG-CSF–treated medium (76.3%) follicles than in all other groups. The blastocyst formation rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF–treated small (31.2%) follicles than in 0 and 10 ng/mL hrG-CSF small (10.4% and 15.6%, respectively) follicles, and the 10 ng/mL hrG-CSF medium (45.7%) follicle was significantly (P < 0.05) higher than in all other groups. The total cell number in blastocysts from the 10 ng/mL hrG-CSF medium (106.5) follicles was significantly (P < 0.05) increased compared to 0, 10, 100 ng/mL hrG-CSF small (55.0, 73.7 and 59.5, respectively) follicles and 0, 100 ng/mL hrG-CSF–treated medium (82.5 and 93.5, respectively) follicles. After IVF, the blastocysts stage was significantly (P < 0.05) increased in 10 ng/mL hrG-CSF–treated medium (36.4%) follicles. Fertilization efficiency was significantly high in 100 ng/mL of small (29.1%) and 10 ng/mL of medium (44.0%) follicles. We also examined the Bcl2 and ERK2 transcript levels and found that they were significantly higher in the small and medium follicle treatment groups. In conclusion, these results indicate that hrG-CSF improve the viability of porcine embryos.

Exogenous glutathione supplementation in culture medium improves the bovine embryo development after in vitro fertilization

To determine the beneficial effects and mechanisms of exogenous glutathione (GSH) during IVC on the embryonic development, bovine IVF zygotes were cultured in medium containing different concentrations of GSH, and the rate of cleavage and blastocyst development, total cell number of blastocysts, the inner cell mass:total cell number ratio, and intracellular GSH and reactive oxygen species concentrations were investigated. Gene expressions associated with embryonic development and GSH metabolism were measured using quantitative real-time polymerase chain reaction. At the concentrations of 1, 3, and 5 mM, GSH significantly increased the blastocyst rate and embryo quality. The highest blastocyst rate (51%) and the best embryo quality appeared in the 3-mM GSH treatment. Intracellular content of GSH of embryos at the two- to four-cell stage significantly increased, and the reactive oxygen species level decreased accordingly with 3-mM GSH treatment, but no significant differences were found in the four- to eight-cell stage and blastocysts. Gene expression analysis of the embryo regulator genes (OCT4, SOX2, and KLF4), GSH synthesis genes (GCLM, GCLC, and GSS), and GSH utilization genes (GSTP, GSTM, and GPX) showed that the GSH had no significant impact on these genes. In conclusion, exogenous GSH during IVC improved developmental potential and quality of bovine IVF embryos, which was probably caused by the ability of GSH to maintain the redox balance.

Anti-Apoptotic Effects of Catalpol on Preimplantaion Porcine Embryos

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<0.05). On the other hand, blastocyst development was significantly improved in the catalpol (100 μM) treated group when compared with the untreated catalpol group under H2O2 (200 μM) induced oxidative stress (P<0.05). Otherwise, the intracellular levels of ROS in catalpol (100 μM) treated group were significantly decreased in the untreated catalpol group under H2O2 (200 μM) induced oxidative stress (P<0.05). Furthermore, the total cell numbers of blastocysts were significantly increased (P<0.05) in the catalpol (100 μM) treated group under H2O2 (200 μM) induced oxidative stress, whereas numbers of apoptoic nuclei were significantly reduced (P<0.05). In conclusion, our results indicate that treatment of catalpol may have important implications for improving developmental competence and preimplantation quality of porcine embryos through its anti-oxidant and anti-apoptotic effect

Effect of donor cell type on developmental competence, quality, gene expression, and epigenetic status of interspecies cloned embryos produced using cells from wild buffalo and oocytes from domestic buffalo

This study compared the cloning efficiency of donor cells of fibroblast and epithelial origin isolated from ear skin of a wild buffalo (Bubalus arnee) and used with cytoplasts from domestic buffalo (Bubalus bubalis) in interspecies SCNT by hand-made cloning. The cleavage (93.0 ± 2.8% vs. 85.6 ± 2.4%) and blastocyst rates (50.6 ± 4.0% vs. 20.5 ± 2.6%) were higher (P < 0.05) for fibroblasts than those for epithelial cells, whereas the total cell number (490 ± 42 and 492 ± 95, respectively) and apoptotic index (2.3 ± 0.3 and 2.5 ± 0.6, respectively) of blastocysts were similar. The global level of H3K18ac and H3K27me3 was lower (P < 0.05) in fibroblasts than that in epithelial cells. The global level of H3K18ac was higher (P < 0.05) in fibroblast than that in epithelial cell–derived blastocysts, whereas that of H3K27me3 was similar between the two groups. The expression level of HDAC1, DNMT1, DNMT3a, and P53 was higher (P < 0.05) in fibroblasts than that in epithelial cells; that of CASPASE3 showed an opposite pattern (P < 0.001), whereas CASPASE7 expression level was similar in the two groups. In the embryos, the expression level of HDAC1, DNMT3a, and CDX2 was lower (P < 0.05) in fibroblast than that in epithelial cell–derived blastocysts; that of NANOG showed an opposite pattern (P < 0.05), whereas that of OCT4 was similar between the two groups. In conclusion, donor cells of fibroblast origin are easier to reprogram than those of epithelial origin in interspecies SCNT, and cloning efficiency, epigenetic status, and gene expression pattern vary among cells having different origin although they may be from the same tissue.

Expression of focal adhesion kinase in mouse cumulus-oocyte complexes, and effect of phosphorylation at Tyr397 on cumulus expansion.

We investigated the expression of focal adhesion kinase (FAK) in mouse cumulus-oocyte complexes (COCs), as well as the role of FAK phosphorylation at Tyr397 during oocyte maturation. The effect of inhibiting FAK phosphorylation at Tyr397 during in vitro maturation (IVM) on subsequent fertilization and preimplantation embryo development was also examined. Western blotting analyses revealed that total and Tyr397-phosphorylated FAK were expressed in vivo in both cumulus cells and oocytes. Immunocytochemical studies localized this kinase throughout the cytoplasm of cumulus cells and oocytes; in particular, Tyr397-phosphorylated FAK tended to accumulate in regions where cumulus cells contact each other. Interestingly, the in vivo level of Tyr397 phosphorylation in cumulus cells was significantly lower after compared to before cumulus expansion. Addition of FAK inhibitor 14, which specifically blocks phosphorylation at Tyr397, stimulated oocyte meiotic maturation and cumulus expansion during IVM in the absence of follicle-stimulating hormone (FSH). Reverse-transcriptase PCR showed that the mRNA expression of hyaluronan synthase 2 (Has2), a marker of cumulus expansion, was significantly induced in cumulus cells. Subsequent in vitro fertilization and culture showed that more oocytes developed to the blastocyst stage when they were treated with FAK inhibitor 14 during IVM, although the blastocyst total cell number was lower than in oocytes stimulated with FSH. These results indicate that FAK is involved in the maturation of COCs; specifically, phosphorylation at Tyr397 may regulate cumulus expansion via the expression of Has2 mRNA in cumulus cells, which could affect the developmental competence of oocytes.

Regulation of embryonic development and apoptotic-related gene expression by brain-derived neurotrophic factor in two different culture conditions in ovine

In the present study, we aimed to evaluate effects of brain-derived neurotrophic factor (BDNF) which is a member of neurotrophic factor family on developmental competence of oocytes in sheep. In vitro maturation was performed in presence of various concentrations (0, 10, and 100 ng/mL) of BDNF. Meiotic maturation, levels of intracellular glutathione, embryonic developmental potential after parthenogenetic activation, number of total and apoptotic cells in blastocysts, and expression of Bax and Bcl-2 genes in blastocyst cells were determined. Under unstressed condition, while at 100 ng/mL concentration, BDNF increased the IVM rate; an increase of glutathione level was observed at 10 ng/mL concentration. Moreover, when BDNF-treated oocytes were used for parthenogenetic activation, more blastocyst at both 10 and 100 ng/mL levels was obtained in comparison with the untreated group. Under heat stress (HS), the blastocyst rate was dramatically reduced in untreated oocytes compared to that obtained from 10 ng/mL BDNF groups. Total cell number in blastocysts was not affected by the treatment groups. The mean of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei in blastocysts was not influenced by addition of BDNF in medium and that presence or absence of thermal stress during IVM than the control group. Moreover, our data revealed that the expression of Bax and Bcl-2 genes in blastocysts was affected by both BDNF concentration and HS. Conclusively, supplementation of IVM medium with 10 ng/mL BDNF had a beneficial effect on sheep oocyte competence by increasing the rate of blastocyst especially when HS exists.

Characterization of porcine partially reprogrammed iPSCs from adipose-derived stem cells.

Partially reprogrammed induced pluripotent stem cells (PiPSCs) have great potential for investigating reprogramming mechanisms and represent an alternative potential material for making genetically modified animals and regenerative medicine. To date, PiPSCs have scarcely been reported in detail when compared with mice and humans. In this study, we obtained PiPSCs from porcine adipose-derived stem cells (pADSCs) by ectopic expression of human transcription factors (OCT4, SOX2, c-MYC, and KLF4) in feeder-free condition. The morphology and proliferation activity of porcine PiPSCs (pPiPSCs) were similar to those of porcine fully reprogrammed iPSCs (pFiPSCs); furthermore, pPiPSCs expressed higher levels of the typical surface molecules (CD29) found in pADSCs. However, pPiPSCs were negative for key proteins (NANOG) connected with stemness and possessed lower differentiation ability in vivo and in vitro. When differentiation-inhibiting factors were withdrawn, pPiPSCs-derived cells (pPiPSC-DCs) showed similar features to pADSCs in many aspects, including proliferation, differentiation, and immunosuppression. When both types of cells were used to produce cloned embryos, we found that the blastocyst formation rate of 19DC (one of the pPiPSC-DC cell lines)-derived cloned embryos was obviously higher than that of others. The total cell number of 19DC-derived blastocysts was significantly higher than the 30DC (one pFiPSC-DC cell line)-derived blastocysts. In all, through limited differentiation ability, the proliferation activity of pPiPSCs is similar to that of pFiPSCs, and pPiPSCs can retain several of the features of pADSCs, which are beneficial to cell therapy. Furthermore, the differentiation of pPiPSCs is more favorable for producing high-quality reconstructed embryos.

Astaxanthin present in the maturation medium reduces negative effects of heat shock on the developmental competence of porcine oocytes

Astaxanthin, one of the most common carotenoids, elicits antioxidant effects on cellular viability and embryonic development. This study was conducted to investigate the effects of astaxanthin on maturation, fertilization and development of porcine oocytes matured in vitro under heat stress conditions, and then fertilized and cultured under standard conditions. Porcine oocytes were cultured in maturation medium supplemented with different concentrations of astaxanthin (0, 0.25, 0.5 or 1ppm) for 46h at either 38.5 or 41°C. In comparison to oocytes cultured at 38.5°C, the exposure of porcine oocytes to 41.0°C during in vitro maturation (IVM) significantly inhibited maturation and development of fertilized oocytes to the blastocyst stage. Supplementation of maturation medium with astaxanthin (0.5ppm) significantly improved oocyte maturation, fertilization and development to the blastocysts stage in both oocyte groups. However, the total cell number and apoptosis index of blastocysts did not differ among groups. Moreover, astaxanthin (0.5ppm) significantly increased the rate of oocytes that reached metaphase II and decreased proportion of apoptotic oocytes exposed to H2O2 (1.0mM) during IVM. In summary, we demonstrated that supplementation of maturation medium with astaxanthin (0.5ppm) exerted antioxidative effects and improved the ability of maturation, fertilization, and development of porcine oocytes exposed to heat stress.

Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming.

Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The ChariotTM reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.

Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos.

This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages.

123 EFFECTS OF TAURO URSODEOXYCHOLIC ACID ON DEVELOPMENT OF BOVINE EMBRYOS FROM IN VITRO FERTILIZATION

Endoplasmic reticulum stress (ERS) is a novel apoptotic pathway and plays an important role for embryonic development. Tauro ursodeoxycholic acid (TUDCA) is a specific chemical chaperone that can inhibit ERS. In this study, we investigated the effects of TUDCA on the development and mRNA expression of ERS-related genes in bovine embryos from IVF in order to improve the efficiency of embryo in vitro culture. Bovine oocytes collected from ovaries at slaughter were cultured in the maturation medium (TCM-199 + 26.2 mmol L–1 NaHCO3 + 5 mmol L–1 HEPES + 5% fetal bovine serum) for 24 h and fertilized in vitro with bovine sperm. After fertilization, the embryos were respectively placed into the medium (TCM-199 + 3% fetal bovine serum) containing different concentrations of TUDCA (0, 100, 250, 500, 1000 μmol L–1) and cultured in the 5% CO2 at 38.5°C. Blastocyst development was evaluated after 7 days of culture, and then the total cell number and apoptosis index of blastocysts were detected with TUNEL. In addition, X-box binding protein 1 (XBP-1) of embryos at 2-cell, 4-cell, morula, and blastocyst stages was detected with RT-PCR, and the change of the mRNA expression of ERS-related (Grp78, Ire1, Chop) and apoptosis-related (Bax, Bcl-2) genes in blastocyst collected at 7 days of culture were analysed by QRT-PCR. A total of 1336 oocytes were used in this study, and each experimental group comprised 6 replicates. The results revealed that the splicing of XBP-1 was present during the development of bovine embryos, and especially obvious at the 4-cell, morula, and blastocyst stages. When embryos were cultured in medium with different concentrations of TUDCA, compared with the control group (0 μmol L–1), more embryos developed to blastocyst stage with 500 μmol L–1 TUDCA (31.86 ± 7.32% v. 21.11 ± 8.05%; P &lt; 0.05), but the cleavage rate was not significantly different among groups (P &gt; 0.05). The result for TUNEL found that when adding 500 μmol L–1 TUDCA to culture, the bovine embryos significantly improved the total cell number of blastocysts (110. ± 15.21 v. 102.3 ± 8.62; P &lt; 0.05), and the apoptosis index of blastocysts was markedly decreased (3.71 ± 0.91 v. 5.36 ± 1.92; P &lt; 0.05) relative to the control group. Moreover, the result of QRT-PCR analysis showed that treating embryos with 500 μmol L–1 TUDCA significantly reduced the mRNA expression level of Ire1 and Chop genes (P &lt; 0.05) and up-regulated the expression of anti-apoptotic Bcl-2 gene (P &lt; 0.05), while down-regulated the expression of pro-apoptotic Bax gene (P &lt; 0.05). Furthermore, XBP-1 splicing in blastocysts also abated after embryos were treated with 500 μmol L–1 TUDCA. In conclusion, ERS occurs in bovine embryos during in vitro culture, but treating embryos with 500 μmol L–1 TUDCA may reduce ERS to facilitate embryonic development. This work was funded by the China High Technology Development Program (2011AA100607), China Natural Science Foundation (31072033), and Guangxi Science Foundation (2011GXNSFA018084, 2012GXNSFFA060004).

27 EFFECT OF A SPECIFIC INHIBITOR OF DOT1L ON PREIMPLANTATION DEVELOPMENT OF PORCINE SOMATIC CLONED EMBRYOS

EPZ004777 (EPZ), a specific inhibitor of DOT1L (a methyltransferase of H3K79), can significantly improve the generation and quality of mouse induced pluripotent stem cells [Onder et al. 2012 Nature 483(7391), 598–602), suggesting that H3K79 dimethylation (H3K79me2) is involved in controlling cell pluripotency. To date, however, it is unclear whether H3K79me2 regulates development competency of animal cloned embryos. Thus, we aimed to examine the dynamic changes of H3K79me2 in pre-implantation cloned embryos of pigs, and to explore effect of EPZ treatment of embryos on in vitro development fate in order to lay the foundation for revealing the role of H3K79me2 and mechanisms in controlling cell pluripotency. Porcine cloned embryos were treated immediately when fusion and activation stimuli were conducted, in vitro with porcine zygote medium (PZM)-3, including 0.5, 5, or 50 nM EPZ (S7353, Selleck Chemicals, Houston, TX, USA) and 1‰ DMSO (vol/vol, control group) for 24 h, respectively. Then, they were transferred into fresh PZM-3 without EPZ. We found that there was no significant difference in cleavage rate among groups, whereas the blastocyst rate of 0.5 nM EPZ group was higher than that of control group [28.97 ± 2.65% (28/96) v. 17.13 ± 2.69% (17/96)]. No obvious difference was observed for the total cell number of blastocyst among groups. We further treated the SCNT embryos with 0.5 nM EPZ for 0 (control group), 12, 24, and 36 h, respectively. No significant differences were found for cleavage rate among groups, whereas the blastocyst rates of the 12- and 24-h groups were significantly higher than that of control and 36-h groups [28.56 ± 3.51% (27/97), 28.34 ± 3.00% (25/88) v. 16.32 ± 1.93% (16/97), 17.93 ± 0.64% (18/100)]. Except for the remarkable decrease in the 36-h treatment group, no obvious difference was observed for the total cell number of blastocyst among the other 3 groups. All the above experiments were repeated at least 3 times. These results suggested that treatment of porcine SCNT embryos with 0.5 nM EPZ for 12 to 24 h could improve their development during the early stage. Then, we tested whether the EPZ favoured the in vitro development of porcine SCNT embryos by regulating H3K79me2 reprogramming. Porcine SCNT embryos were treated with 0.5 nM EPZ from the onset of electric activation and fusion stimuli was performed, and then the H3K79me2 signal (by immune-fluorescent staining) and expression of DOT1L (by RT-qPCR) at different development stages was analysed. We found that the H3K79me2 signal in control group (without EPZ treatment) decreased slowly from the time of electric stimulation to 4 hpa, and it disappeared in 8 hpa stage. In the EPZ treatment group, H3K79me2 signal started decreasing from 2 hpa, and disappeared in 8 hpa stage. The mRNA level of DOT1L in EPZ treatment group was lower than that in control group, although the difference was not significant. Taken together, treatment with EPZ at the appropriate concentration and for an appropriate time can improve the early in vitro development of pig SCNT embryos, probably by inhibiting expression of DOT1L and facilitating reprogramming of H3K79me2.Research was supported by NSFC No. 31272442.

93 THE INVOLVEMENT OF E-CADHERIN IN THERMOPROTECTIVE FUNCTION OF INSULIN-LIKE GROWTH FACTOR-1 IN 4-CELL HAMSTER EMBRYOS

Studies have demonstrated that the early pre-implantation embryo is very sensitive to effects of heat stress in vitro. Heat stress reduces the total cell number in blastocysts and increases apoptosis in blastomeres. Insulin-like growth factor-1 (IGF-1) has been widely studied as a thermoprotective agent for its anti-apoptotic actions. Addition of IGF-1 to the culture medium decreases the effects of heat stress on blastocysts but has no effects on 2-cell embryos. Molecular mechanisms by which IGF-1 decreases apoptosis involve activation of the PI3K/Akt pathway. It is also known that adherens junctions contribute to PI3K/AKT activation mediated by the transmembrane glycoprotein E-cadherin, which is involved in Ca2+-dependent cell-cell adhesion. Within 2- to 8-cell embryos, E-cadherin is mainly inactive and has cytoplasmic localization. 6-Dimethylaminopurine (6-DMAP) induces premature cell flattening and E-cadherin redistribution to adhesion sites in 4-cell embryos. The aim of this study was to induce E-cadherin redistribution in 4-cell hamster embryos and evaluate the thermoprotective function of IGF-1 in these embryos. Four-cell embryos were incubated in the presence of 6-DMAP to induce E-cadherin redistribution to adhesion sites and cultured for 24 h under conditions of heat stress and compared with controls without 6-DMAP. Culture medium was supplemented with IGF-1. At the end of culture, developmental stage and rate of apoptosis were determined and analysed by ANOVA using the General Linear Model (GLM) of SAS (SAS Institute Inc., Cary, NC, USA) procedure with statistical significance at P &lt; 0.05. E-Cadherin redistribution induced by 6-DMAP increased development to the 6-cell stage after 24 h (63.57% v. 38.81%, respectively; P &lt; 0.05) and reduced apoptosis (25% v. 33%, respectively; P &lt; 0.05) under heat-stress conditions. In conclusion, we hypothesise a role for E-cadherin-mediated cell flattening in promoting IGF-1-mediated thermoprotection in pre-compact 4-cell hamster embryos. Further studies are required to confirm this link.

52 EFFECT OF CARBOXYLATED POLY-L-LYSINE ON THE POST-THAW VIABILITY OF IN VITRO-PRODUCED BOVINE BLASTOCYST

Recently, there has been development of an antifreeze polyamino acid (carboxylated poly-l-lysine: PLL) as new cryoprotectants (CPA). This compound counts as amphoteric macromolecular cationic and anionic substituents (polyampholyte) by chemically modifying poly-lysine. In addition, PLL is highly safe and frequently used as a food additive in substitution for other CPA. Other common CPA have high toxicity and caused physiological damage. In vitro-produced blastocysts were randomly assigned into 3 groups: (1) vitrified embryos with PLL vitrification solution [PLL-vit-1: 15% PLL + 15% ethylene glycol (EG); PLL-vit-2: 30% PLL + 30% EG + 0.5 M sucrose], (2) vitrified embryos with Vajta et al. solution (Conv-vit-1: 7.5% dimethyl sulfoxide + 7.5% EG; Conv-vit-2: 16.5% dimethyl sulfoxide + 16.5% EG + 0.5 M sucrose), and (3) nonvitrified blastocysts (control). All embryos were frozen by droplet vitrification method. First, the PLL-vitrified embryos were exposed to 5, 10, and 15 min in the PLL-vit-1 and then putted for 30 to about 60 s in the PLL-vit-2. Then, we compared with each group regarding exposure time of Vit-1. Post-thaw survival rate of each exposure time did not significantly differ among the 3 groups (100 v. 100 v. 100%). However, hatching rate of the 10-min exposure group was higher than that of 5- and 15-min groups (75.0 v. 25.0 v. 66.7; P &lt; 0.05). Therefore, we confirmed that exposure time of Vit-1 was exposed for a minimum of 10 min. The post-thawed survival rate of each vitrification method was not significantly different between PLL-vit and Conv-vit groups (97.7 v. 86.4%). The total cell numbers of blastocyst did not significantly differ among groups. However, the apoptotic cell numbers of blastocyst was significantly different between the control and Conv-vit groups (0.4 ± 0.6 v. 4.4 ± 3.9; P &lt; 0.05) but was not different in control v. PLL-vit (0.4 ± 0.6 v. 2.1 ± 2.4) and Conv-vit v. PLL-vit (4.4 ± 3.9 v. 2.1 ± 2.4). In conclusion, PLL-vit for bovine blastocyst could reduce toxicity and osmotic shock and showed high efficiency on the quality of post-thawed bovine blastocysts compared with that of Conv-vit.This work was partly supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ009587022014), IPET (Grant No. 112020-3), and a scholarship from the BK21 plus program. A-Na Ha, Kyeong-Lim Lee, and Md. Fakruzzaman were supported by BK21 plus fellowships at Gyeongsang National University, Republic of Korea.

303 HUMAN RECOMBINATION GRANULOCYTE-COLONY STIMULATING FACTOR (HRG-CSF) HAVE BENEFICIAL EFFECTS ON PORCINE OOCYTES QUALITY DURING IN VITRO MATURATION AND SUBSEQUENT VIABILITY OF EMBRYONIC DEVELOPMENT

Granulocyte colony-stimulating factor (G-CSF), also known as colony-stimulating factor 3 (CSF3), is required for the proliferation, differentiation, and survival of cells. In humans, G-CSF is a biomarker of human oocyte developmental competence for embryo implantation. Furthermore, G-CSF concentration increases during the menstrual cycle and levels were significantly higher during ovulatory phase than the other phases. In this study, we examined G-CSF and its receptor gene expression in the porcine granulosa cells, corpus luteum, cumulus cells, and oocytes. The cumulus-oocyte complexes (COC) were aspirated from antral follicles 1 to 3 mm (small follicle) and 3 to 6 mm (medium follicle). The COC from 2 kinds of follicles were matured in protein-free maturation medium supplemented with various concentrations of hrG-CSF (0, 10, and 100 ng mL–1, respectively). Statistical analyses were done by one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests. After real time-PCR was performed, the CSF3 and its receptor (CSF3R) were observed all of granulosa cells, corpus luteum, cumulus cells, and oocytes. Interestingly, the CSF3 transcript levels were significantly lower in oocytes compared with other cell types, but the CSF3R transcript levels in oocytes were almost similar with granulosa cells. After 44 h of IVM, the rates of nuclear maturation had no difference, and the intracellular ROS levels of oocytes from both kind of follicle groups matured with 10 ng mL–1 were significantly decreased compared to other groups (P &lt; 0.05). After PA, the cleavage and blastocyst formation rates were significantly (P &lt; 0.05) increased for the 100 ng mL–1 of small follicle (SF; 63.29 and 31.18%) group compared to control and 10 ng mL–1 of SF (38.64, 10.4, and 49.0, 15.6%, respectively) group, and significantly (P &lt; 0.05) increased in the 10 ng mL–1 of medium follicle (MF; 76.32 and 45.61%) group compared with control and 100 ng mL–1 of MF (52.1, 32.8 and 61.3, 33.9%, respectively). The total cell numbers of blastocyst from SF and MF groups were significantly increased in the 10 ng mL–1 (73.67 and 106.52) groups. At IVF, the blastocysts formation rates were significantly increased in the 10 ng mL–1 of MF group compared to control, 100 ng mL–1 of SF, and control of MF (21.1, 22.8, and 27.8%, respectively). We also examined the Bcl2 and ERK2 transcript levels, which were significantly increased at 100 ng mL–1 of SF and 10 ng mL–1 of MF. These results suggest that hrG-CSF improved the quality of porcine oocyte and embryonic viability.This was supported, in part, by a grant from the National Research Foundation of Korea Grant Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

134 SERIAL TREATMENT OF RESVERATROL-TROLOX IMPROVED EMBRYONIC DEVELOPMENT OF PORCINE PARTHENOTES

Antioxidants are widely used for in vitro production of embryos due to their activity as reactive oxygen species scavengers. Among various antioxidants, resveratrol supplementation in in vitro-maturation (IVM) media and trolox supplementation in in vitro-culture (IVC) media improves oocyte maturation and embryonic development in other species, such as cattle and sheep. Limited information is available, however, on the effect of resveratrol and/or trolox on porcine embryos produced in vitro. In this study, we evaluated the effect of resveratrol supplemented to the media of IVM and trolox treatment during IVC on porcine parthenotes. We used TCM-199 as IVM media and porcine zygote medium (PZM)-5 as IVC media. For activation, matured oocytes after 44 h of IVM were electrically activated with 280 mM mannitol and cultured in IVC medium (PZM-5). Statistical analyses of all data were carried out using SPSS 17.0 (one-way ANOVA, followed by Duncan's multiple range test). In the experiment 1, a total of 618 oocytes were used in 4 independent replicates to evaluate the effect of 4 different concentrations (0, 1, 2, or 4 μM) of resveratrol during IVM on parthenotes. Oocytes treated with 2 μM resveratrol during IVM had significantly higher cleavage rates and blastocyst formation rates (73.0 and 34.4% v. 64.0 and 18.3%, respectively) than the control group. Experiment 2 involved supplementation with trolox (0 μM, 100 μM, 200 μM, 400 μM) to 957 parthenotes during IVC for 7 days (4 replicates). Cleavage rates significantly increased in the 100 μM group (75.6 v. 69.1%), and blastocyst formation rates in the 200 μM group were significantly higher compared to the control group (33.7 v. 23.8%). To determine the combined effects of resveratrol treatment during IVM and trolox treatment during IVC, in the experiment 3 we selected an optimized concentration (2 μM of resveratrol and 200 μM of trolox) from each experiment and evaluated the combined effects (3 times replicated). We designed 4 groups: (1) control, (2) resveratrol only (R), (3) trolox only (T), and (4) resveratrol-trolox (R-T). The R group and R-T group showed significantly higher cleavage rates than the control group (81.8 and 83.1% v. 72.3%). All treatment groups showed significantly increased blastocyst formation rates compared with the control group (39.2, 37.8, and 38.4% v. 23.7%). There is no significant difference in total cell numbers of blastocyst among the control, R, and T groups (47.8 v. 54.2 v. 54.7). However, the R-T group had significantly more cells than the control group (67.1 v. 47.8). Our results suggest that 2 μM resveratrol treatment during IVM, followed by 200 μM trolox treatment during IVC, improves developmental potential of the parthenotes. For a further study, we will apply this condition to somatic cell nuclear transfer, and we also will verify quantitative PCR analysis of apoptosis-related mRNA expression of PA and somatic cell nuclear transfer embryos. This study was supported by the MOTIE (#10033839), IPET (#311011-05-3-SB010), Research Institute for Veterinary Science, TS Corporation, and the BK21 plus program.

295 EFFECT OF TREHALOSE DURING IN VITRO MATURATION OF PIG OOCYTES ON OOCYTE MATURATION AND EMBRYONIC DEVELOPMENT AFTER PARTHENOGENESIS

Autophagy is a critical process for the maintenance of cellular homeostasis and mammalian early embryogenesis. Autophagy can be regulated by various chemical inducers. However, there are few reports about effect of autophagy inducer in vitro maturation (IVM) of porcine oocyte. The present study investigated the effects of supplementary trehalose, a novel mTOR-independent autophagy enhancer, on oocyte maturation and embryonic development after parthenogenetic activation (PA). Immature oocytes were treated with various concentrations (0, 25, 50, and 100 mM) of trehalose in M-199 (Invitrogen, Carlsbad, CA) supplemented with 10 ng mL–1 of epidermal growth factor (EGF; Sigma-Aldrich Corp.), 1 ug mL–1 of insulin (Sigma-Aldrich Corp.), 4 IU mL–1 of pregnant mare serum gonadotropin (PMSG; Intervet, Boxmeer, Holland), 4 IU mL–1 of human chorionic gonadotropin (hCG; Intervet), and 10% (vol/vol) porcine follicular fluid (pFF) for 10 h, and transferred to another IVM medium without trehalose. Osmolality of each groups (0, 25, 50, and 100 mM trehalose) was in the 290 to 295, 310 to 315, 330 to 335, and 375 to 380 osmol range, respectively. After 44 h of IVM, trehalose treatment during IVM did not improve nuclear maturation rates of oocytes in any group (90.7, 92.1, 92.7, and 90.1%, respectively). The developmental competence of oocytes matured with different trehalose concentrations was evaluated after PA. There were no significant differences in cleavage rates. However, blastocyst (BL) formation was different. Oocytes treated with 25 mM of trehalose during IVM had a significantly higher (P &lt; 0.05) BL formation rate (64.2%) after PA compared with the control (52.0%). The BL quality was also improved in the 25 mM trehalose-treated group. Early BL rate significantly reduced in the 25 mM trehalose-treated group as compared to control (19.6 v. 29.9%, P &lt; 0.05). By contrast, expanded BL rate significantly increased in the 25 mM trehalose-treated group than of control (27.7 v. 11.0%, P &lt; 0.05). Total cell numbers of BL were significantly higher (P &lt; 0.05) in the 25 mM trehalose-treated group compared to those in the control group (52.2 v. 36.8). However, BL rate and quality of oocytes treated with 50 and 100 mM trehalose were similar with control group. In conclusion, these results indicate that 25 mM trehalose during IVM improved the developmental potential of porcine embryos. Trehalose will be useful for large-scale production of BL with good quality in porcine in vitro production.This work was supported by a grant from the Next-Generation Bio Green 21 Program (No. PJ009563032014), Rural Development Administration, Republic of Korea.

127 VERO CELLS CONDITIONED MEDIUM IMPROVES EMBRYONIC DEVELOPMENT DURING IN VITRO CULTURE OF BOVINE EMBRYOS

Beneficial effects on embryonic development of co-culture with Vero cells during in vitro culture have been previously reported and related to Vero cells growth factors/cytokines secretion into the medium (1998 Human Reprod. 1600–1605). However, co-culture increases the probability of microorganism contamination during culture. In this study, we aimed to overcome this problem by culturing bovine embryos in conditioned medium derived from Vero cells culture (CM). Vero cells (GenBiotech, Antibes, France) were cultured for 24 h, and then the medium was removed, filtered, and frozen until its use. A total of 701 slaughterhouse bovine cumulus oocytes complexes were matured and fertilized in vitro under standard conditions. After fertilization, zygotes were allocated to 4 different treatments [0 (control), 5, 10, and 20% of CM v/v] for in vitro culture in BBH7 medium (BoviPro®, MOFA Global, Verona, WI, USA) at 38.5°C in 5% O2, 5% CO2, 90% N2 atmosphere for 7 days. A total of 8 replicates were done. Cleavage rates were assessed at 2.5 days, blastocyst rates and embryonic stage at 7 days, and blastocyst total cell number at 7.5 days after fertilization. Data were analysed by ANOVA using GLM; the percentages were transformed using arcsin square root. If the ANOVA was significant (P &lt; 0.05), means were separated by Tukey's procedure using Statistix 10 software (Tallahassee, FL, USA). No differences in cleavage rates were found between treatments (P &gt; 0.1; Table 1). However, the addition of conditioned medium at all levels increased the blastocyst production at Day 7 of culture (between 11 to 19 percentage points) compared with the control group (P &lt; 0.01; Table 1); likewise, CM addition was superior in total embryo production (Day 7 morulae plus all blastocysts; P &lt; 0.01; Table 1), between 12.5 to 15 percentage points higher than the control group. No differences were found between treatments for blastocyst total cell number at 7.5 days of culture (P &gt; 0.1; Table 1) and overall embryonic developmental stage (P &gt; 0.1; Table 1). In conclusion, we found a superior embryonic development when Vero cells conditioned medium was added at Day 0 of in vitro culture; the positive effect on total embryo production was observed even at 5% of conditioned medium. Table 1.Means (± s.e.m.) of embryonic development of bovine embryos