Increased Blastocyst Formation Rates Research Articles

Sequential treatment with resveratrol-trolox improves development of porcine embryos derived from parthenogenetic activation and somatic cell nuclear transfer

We investigated the effect of resveratrol supplementation during IVM and/or trolox during IVC on the development of porcine embryos derived from parthenogenetic activation (PA) and SCNT. In this study, we evaluated intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, gene expression in blastocysts, and embryonic development after PA and SCNT. To determine the combined effects of resveratrol during IVM and trolox during IVC on PA embryos, we selected optimal concentrations (2 μM of resveratrol and 200 μM of trolox) and designed four groups: (1) control, (2) resveratrol, (3) trolox, and (4) combined. All treatment groups showed significantly increased intracellular GSH levels and decreased ROS levels. Resveratrol supported significantly higher cleavage and blastocyst formation rates than the control (80.3% and 38.0% vs. 71.1% and 22.4%, respectively) by downregulating Bax/Bcl-2, Caspase-3, and Bak. Trolox showed significantly increased blastocyst formation rates (36.7%) compared with the control (22.4%) by downregulating only Caspase-3. The combined group had significantly higher cleavage and blastocyst formation rates and greater total cell numbers than the control (81.7%, 36.3%, and 67.1 vs. 71.1%, 22.4%, and 47.8, respectively) by downregulating Bax/Bcl-2, Caspase-3, and Bak. On the basis of these results, we applied sequential treatments with resveratrol and trolox to SCNT, and blastocyst formation rates and total cell numbers were significantly increased compared with the control (17.2% and 52.1 vs. 11.8% and 36.6, respectively), with increased GSH, decreased ROS levels, upregulated proliferating cell nuclear antigen, and downregulated Bax/Bcl-2 and Caspase-3. These results indicate that sequential treatment with resveratrol during IVM and trolox during IVC improved the development of PA and SCNT porcine embryos by regulating intracellular GSH, ROS levels, and gene expression.

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.

Biological transcomplementary activation as a novel and effective strategy applied to the generation of porcine somatic cell cloned embryos

A novel method termed the biological transcomplementary activation (B-TCA) has been recently utilized for the stimulation of porcine oocytes reconstituted by somatic cell nuclear transfer (SCNT). The use of cytosolic components originating from fertilized (FE) rabbit zygotes as the stimuli for the B-TCA of SCNT-derived pig oocytes appeared to be a highly efficient strategy applied to promote the in vitro development of cloned embryos, leading to a significant improvement in the blastocyst yield (43.6%) compared to the yields achieved using the standard protocol of simultaneous fusion and electrical activation (SF-EA; [31.3%]) or the protocol of delayed electrical activation (D-EA) independent of extracellular Ca2+ ions (0%). The FE rabbit zygote cytoplast-mediated B-TCA resulted in the increased blastocyst formation rate of porcine cloned embryos as compared to the B-TCA triggered by either cytoplasts isolated from pig parthenogenotes (PAs; [27.8%]) or rabbit PA-descended cytoplasts (0%). A considerably lower percentage of blastocysts containing apoptotic and/or necrotic (annexin V-eGFP-positive) cells were obtained from the SCNT-derived oocytes stimulated by the FE rabbit zygote cytoplast-based B-TCA (22.2%) compared to those stimulated using the SF-EA protocol (35.1%). In contrast to the B-TCA induced by FE rabbit zygote cytoplasts, apoptosis/necrosis incidence decreased totally among the cloned pig blastocysts that developed from reconstituted oocytes undergoing the porcine PA cytoplast-evoked B-TCA. In conclusion, the FE rabbit zygote cytoplast-mediated B-TCA turned out to be a relatively effective strategy for the in vitro production of porcine blastocyst clones of higher quality compared to those created using the standard SF-EA approach.

Cell Colony Formation Induced byXenopusEgg Extract as a Marker for Improvement of Cloned Blastocyst Formation in the Pig

Treatment with cytoplasmic extracts from Xenopus laevis eggs represents a potential tool for universal cellular reprogramming. However, the biochemical activity and quality of the extract vary from batch to batch. This study aimed to evaluate three different extract batches prepared by the same method based on the colony formation of cells after extract treatment, and subsequent in vitro cloning efficiency using treated cells as chromatin donors. Porcine fetal fibroblasts were treated with each batch of extract, and cultured in embryonic stem cell (ES) medium for 12 days. The number of forming colonies in treated cells was counted on Day 7 after extract treatment and significant variability was detected between different batches of extract. Similarly, when using cells from colonies at Days 7 to 8 after treatment for handmade cloning, increased blastocyst formation rates were observed after the cells were treated with a batch showing higher colony formation. In conclusion, assessment of cell colony formation may be used as selection marker for Xenopus egg extract used for pretreatment of donor cells prior to cloning.

Treatment with a Histone Deacetylase Inhibitor after Nuclear Transfer Improves the Preimplantation Development of Cloned Bovine Embryos

We examined the effects of treatment with histone deacetylase inhibitors (HDACi), trichostatin A (TSA) and scriptaid (SCR), on the blastocyst formation rate in bovine somatic cell nuclear transferred (SCNT) embryos derived from fibroblast cells. Three fibroblast cell lines (L1, L2 and L3) were used as somatic cell donors to produce SCNT embryos (L1, L2 and L3 embryos, respectively). In Experiment 1, we compared the in vitro developmental competence of L1 embryos treated with various concentrations of TSA for different time periods following chemical activation. Embryos treated with 5 nM TSA for 20 h showed a significantly increased blastocyst formation rate compared with untreated controls. In Experiment 2, we examined the effect of TSA (5 nM) treatment of L1, L2 and L3 embryos as well as the effect of treatment of L1, L2 and L3 embryos with various concentrations of SCR on in vitro developmental competence. It was found that 5 nM TSA treatment significantly increased the blastocyst formation rate in L1 and L3 embryos but did not have an influence on the development of L2 embryos. On the other hand, 5 nM SCR treatment significantly increased the blastocyst formation rates of L1 and L2 embryos compared with controls. However, there was no significant increase in the blastocyst formation rate of L3 embryos when they were treated with SCR. In Experiment 3, acetylation of H4K12 was examined in donor cells and pronuclear-stage L1, L2 and L3 embryos treated with 5 nM TSA or 5 nM SCR by immunostaining. The level of H4K12 acetylation was different among donor cells. The staining intensities in the TSA-treated L1 and L3 embryos and SCR-treated L2 embryos were significantly higher than those of untreated embryos. These results suggest that HDACi treatment of bovine SCNT embryos improves the blastocyst formation rate; however, the optimal treatment conditions may differ among donor cell lines.

Effects of Melatonin on In Vitro Development of Mouse Two-Cell Embryos Cultured in HTF Medium

Melatonin is capable of improving the developmental capacity of ovine, porcine and bovine embryos in vitro. However, whether melatonin possesses similar benefits to the in vitro mouse embryonic development has yet to be determined. In this study, we assessed the effects of various concentrations of melatonin (10–13 to 10–3 M) on the in-vitro development of mouse embryos cultured in HTF medium for 96 hr; embryos cultured without melatonin were used as control. The in vitro development of mouse two-cell embryos significantly benefited from treatment with melatonin in a concentration-dependent manner. The effects of melatonin on the rates of blastocyst formation, hatching/hatched blastocysts and cell number per blastocyst were bi-phasic; all significantly increased by melatonin at 10–13 to 10–5 M and decreased by melatonin at 10–3 M. Maximal benefit of melatonin on in vitro mouse 2-cell embryo development was achieved at a concentration of 10–9 M. In comparison to control, 10–9 M melatonin increased blastocyst formation rate from 48.08 ± 5.25% to 82.08 ± 2.34% (p < 0.05), hatched blastocyst rate from 25.65 ± 11.79% to 66.47 ± 4.94% (p < 0.05), and cell number per blastocyst 62.71 ± 5.97 to 77.91 ± 10.63 (p < 0.05). Thus, our datas demonstrated firstly that melatonin has beneficial effects on the in vitro development of 2-cell mouse embryos cultured in HTF medium.

Embryotropic effect of glycosaminoglycans and receptors in development of porcine pre-implantation embryos

The present study investigated the expression of receptors for glycosaminoglycans (GAGs) and the effect of GAGs supplementation on development of porcine IVF embryos. Total RNA was prepared from oocytes, 2-, 4- and 8-cell embryos, morulae and blastocysts. The expression of hyaluronic acid receptor (CD44) and heparin (HP) interacting protein (HIP) was determined using RT-PCR and Southern blot analysis. The CD44 and HIP mRNA were detected from in vitro matured oocytes and all stages of pre-implantation embryos. The IVF embryos were cultured in modified NCSU-23 medium supplemented with various concentrations (0, 0.1, 0.5 or 1.0 mg/mL) of hyaluronic acid (HA) or heparin. Supplementing with 0.5 mg/mL HA significantly increased total cell number compared to other experimental groups, due to increase in trophectoderm cells. Supplementing with 1.0 mg/mL, HP significantly increased blastocyst formation rate compared to the control group. Supplementing media, in which IVF embryos were cultured with 0.5 mg/mL HA + 1.0 mg/mL HP, significantly increased blastocyst formation rate compared to the control group. In conclusion, the present study demonstrated the expression of HA and HP receptors and the embryotrophic effect of HA or HP on porcine IVF embryos.

Evaluation of mitomycin-treated vero cells as a co-culture system for IVM/IVF-derived bovine embryos

Support of the in vitro development of IVM/IVF-derived bovine embryos by Vero cells was evaluated by comparing the following treatment groups: 1) proliferating (Unt-Vero) vs nonproliferating (Mit-Vero) cells; 2) supplementation of medium with estrous cow serum (ECS) vs bovine serum albumin (BSA); 3) Mit-Vero cells vs bovine oviduct epithelial cells (BOECs); and 4) addition of leukemia inhibitory factor (LIF) to Mit-Vero cell co-cultures at Day 1 vs Day 4. Mit-Vero cells stimulated higher rates of blastocysts (Day 7, 40 vs 27%) and hatched blastocyst (Day 10, 38 vs 12%) formation than Unt-Vero cells. These rates were comparable to those obtained with BOECs; blastocyst hatching was slightly higher following co-culture with Mit-Vero cells (36%) than BOECs (29%). Blastocyst formation was similar in ECS- vs BSA-supplemented medium; however, hatching was greatest (37%) during co-culture in medium +10% ECS. While the addition of LIF throughout the co-culture period was ineffective, addition of the cytokine beginning at Day 4 slightly increased blastocyst formation rates. Evaluation of LIF secretion using ELISA revealed detectable levels of the cytokine in Mit-Vero-conditioned medium (50 pg/10 5 cells); this may explain the minimal influence of exogenous LIF during embryo co-culture. Mit-Vero cells provided comparable support of bovine embryo development when used even up to 2 wk after establishment as monolayers. In conclusion, Mit-Vero cells provide a readily-available, safe and easy-to-use co-culture method which is at least as supporting of bovine embryo development as BOECs. One contribution of these cells may be secretion of the cytokine LIF.