Porcine Parthenogenetic Activation Research Articles

Chlorogenic acid improves the development of porcine parthenogenetic embryos by regulating oxidative stress and ameliorating mitochondrial function.

Chlorogenic acid (CGA) is an effective phenolic antioxidant that can scavenge hydroxyl radicals and superoxide anions. Herein, the protective effects and mechanisms leading to CGA-induced porcine parthenogenetic activation (PA) in early-stage embryos were investigated. Our results showed that 50 μM CGA treatment during the invitro culture (IVC) period significantly increased the cleavage and blastocyst formation rates and improved the blastocyst quality of porcine early-stage embryos derived from PAs. Then, genes related to zygotic genome activation (ZGA) were identified and investigated, revealing that CGA can promote ZGA in porcine PA early-stage embryos. Further analysis revealed that CGA treatment during the IVC period decreased the abundance of reactive oxygen species (ROS), increased the abundance of glutathione and enhanced the activity of catalase and superoxide dismutase in porcine PA early-stage embryos. Mitochondrial function analysis revealed that CGA increased mitochondrial membrane potential and ATP levels and upregulated the mitochondrial homeostasis-related gene NRF-1 in porcine PA early-stage embryos. In summary, our results suggest that CGA treatment during the IVC period helps porcine PA early-stage embryos by regulating oxidative stress and improving mitochondrial function.

RNA-Seq Reveals the Underlying Molecular Mechanism of First Cleavage Time Affecting Porcine Embryo Development.

The selection and evaluation of high-quality embryos are the key factors affecting in vitro embryo development and pregnancy outcome. The timing of first embryonic cleavage has been considered a positive indicator of the in vitro developmental potential of embryos, while the underlying molecular mechanism is still not fully understood. In this study, the embryos generated by parthenogenetic activation (PA) or in vitro fertilization (IVF) were monitored and recorded every 2 h and divided into two groups (early cleavage or late cleavage) based on the cleavage rate and blastocyst formation data. RNA sequencing was used to analyze the gene expression pattern of the embryos. We identified 667 and 71 different expression genes (DEGs) in early cleavage and late cleavage porcine PA and IVF embryos, respectively. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the DEGs are mainly enriched in pathways concerning the proteasome, DNA repair, cell cycle arrest, autophagy, and apoptosis, suggesting that severe endoplasmic reticulum stress (ERS) and DNA damage may be the key factors that led to the low development potential of late cleavage embryos. This study provides a theoretical basis for the following application and offers important information about the understanding of the timely manner of porcine embryo development.

Cd26 Knockdown Negatively Affects Porcine Parthenogenetic Preimplantation Embryo Development.

cd26 is ubiquitously distributed in the body, particularly in the endothelial and epithelial cells, with the highest expression in the kidney, liver, and small intestine. In humans, cd26 serves as a marker for the embryo implantation phase. However, little is known about the role of cd26 in porcine pre-implantation embryo development. Here, we aimed to examine siRNA-induced cd26 downregulation in the cytoplasm of MII oocytes, to determine whether cd26 is involved in the regulation of porcine pre-implantation embryonic development. The cd26 siRNA was micro-injected into the cytoplasm of MII oocytes, which were then parthenogenetically activated electrically in a medium containing 0.3M Mannitol. Inhibition of the cd26 expression did not affect cleavage but stopped development in the blastocyst stage. Additionally, the cd26 siRNA-treated blastocysts had significantly more apoptotic cells than the untreated blastocysts. Among the 579 transcripts evaluated with transcriptome resequencing, 38 genes were differentially expressed between the treatment and control blastocysts (p < 0.05). Twenty-four genes were upregulated in cd26 siRNA-injected blastocysts, whereas 14 were downregulated. These genes are involved in apoptosis, accumulation of reactive oxygen species, and aberrant expression of ribosomal protein genes. Our results indicate that cd26 is required for proper porcine parthenogenetic activation during embryonic development.

Supplementation with asiatic acid during in vitro maturation improves porcine oocyte developmental competence by regulating oxidative stress

Asiatic acid is a natural triterpene found in Centella asiatica that acts as an effective free radical scavenger. Our previous research showed that asiatic acid delayed porcine oocyte ageing in vitro and improved preimplantation embryo development competence in vitro; however, the protective effects of asiatic acid against oxidative stress in porcine oocyte maturation are still unclear. Here, we investigated the effects of asiatic acid on porcine oocyte in vitro maturation (IVM) and subsequent embryonic development competence after parthenogenetic activation (PA) and in vitro fertilization (IVF). The results of the present research showed that 10 μM asiatic acid supplementation did not affect the expansion of cumulus cells or polar body extrusion of porcine oocytes, while asiatic acid application significantly increased the subsequent blastocyst formation rate and quality of porcine PA and IVF embryos. Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) that induces oxidative stress in porcine oocytes. As expected, asiatic acid supplementation not only decreased intracellular ROS levels but also attenuated H2O2-induced intracellular ROS generation. Further analysis revealed that asiatic acid supplementation enhanced intracellular glutathione production, mitochondrial membrane potential, and ATP generation at the end of IVM. In summary, our results reveal that asiatic acid supplementation exerts beneficial effects on porcine oocytes by regulating oxidative stress during the IVM process and could act as a potential antioxidant in porcine oocytes matured in vitro production systems.

Iron Overload-Induced Ferroptosis Impairs Porcine Oocyte Maturation and Subsequent Embryonic Developmental Competence in vitro.

Accumulating evidence indicates that ferroptosis is an iron-dependent form of regulated cell death. This type of iron-dependent programmed cell death is different from traditional forms of regulated cell death, such as apoptosis and autophagy. However, the role of ferroptosis in porcine oocyte maturation and the associated mechanism remain unclear. In the present research, we investigated the effects of ferric ammonium citrate (FAC), a specific ferroptosis inducer, on porcine oocyte meiotic maturation and quality and subsequent embryonic developmental competence. FAC treatment caused obvious accumulation of intracellular ferrous ions in porcine oocytes. At the end of the in vitro maturation (IVM) period, there was a significant decrease in the polar body (PB) extrusion rate and an increase in the percentage of abnormal oocytes in the FAC treatment groups, indicating that iron overload-induced ferroptosis may suppress the meiotic process during porcine oocyte maturation. We also found that after FAC treatment, the subsequent two-cell rate, four-cell rate and blastocyst formation rate were significantly decreased in porcine parthenogenetic activation (PA) embryos, indicating that iron overload-induced ferroptosis decreased porcine oocyte quality. Further analysis revealed that FAC treatment not only enhanced intracellular reactive oxygen species (ROS) generation, decreased intracellular free thiol levels and induced mitochondrial dysfunction but also triggered autophagy in porcine oocytes. Taken together, these findings suggest that iron overload-induced ferroptosis impairs porcine oocyte meiosis and decreases porcine oocyte quality, possibly by increasing oxidative stress, inducing mitochondrial dysfunction and triggering autophagy.

Developmental and Degenerative Characterization of Porcine Parthenogenetic Fetuses during Early Pregnancy.

Simple SummaryTo increase the early implantation rate, oocytes and zygotes have been subjected to various artificial stimulations before and/or after in vitro fertilization, nuclear transfer, or sperm (spermatid) injection, etc. However, the stimulation process may induce parthenogenetic development. It is difficult to identify whether the embryo or fetus is normally fertilized or parthenogenetically activated in early pregnancy. In the present study, the porcine parthenotes originated from electric stimulation implanted and developed normally during the first month, in a manner similar to artificially inseminated embryos and fetuses. There were no statistical differences in the formation of the major organs such as the brain, liver, kidney, or heart in both groups. However, the implanted parthenotes radically ceased their development and degenerated after one month. It can be postulated that the parthenotes are one of the reasons for the gap between early pregnancy and delivery rate in assisted reproduction techniques.The difference between early pregnancy and delivery rate is quite large in assisted reproduction techniques (ARTs), including animal cloning. However, it is not clear why the implanted fetuses aborted after the early pregnancy stage. In the present study, we tried to evaluate the developmental and morphological characteristics of porcine parthenogenetically activated (PA) embryos or fetuses by electric stimulation during the early pregnancy period. The implanted PA and artificially inseminated (AI) embryos and fetuses were collected at day 26 and 35 after embryo transfer, respectively. The developmental and morphological parameters in the PA embryos at day 26 were similar to the AI embryos. The size, weight, formation of major organs, and apoptotic cells were not statistically different in both embryos at day 26. However, the PA fetuses at day 35 showed ceased fetal development and degenerated with abnormal morphologies in their organs. The day 35 PA fetuses showed significantly higher apoptotic cells and lower methylation status in three differentially methylated regions of the H19 gene compared to their comparators. Therefore, the normal development of PA embryos and fetuses during early gestation could lead to these pregnancies being misinterpreted as normal and become one of the main reasons for the gap between early pregnancy and delivery rate.

Induction of autophagy promotes porcine parthenogenetic embryo development under low oxygen conditions.

Autophagy plays an important role in embryo development; however, only limited information is available on how autophagy specifically regulates embryo development, especially under low oxygen culture conditions. In this study we used parthenogenetic activation (PA) of porcine embryos to test the hypothesis that a low oxygen concentration (5%) could promote porcine embryo development by activating autophagy. Immunofluorescence staining revealed that low oxygen tension activated autophagy and alleviated oxidative stress in porcine PA embryos. Development was significantly affected when autophagy was blocked by 3-methyladenine, even under low oxygen culture conditions, with increased reactive oxygen species levels and malondialdehyde content. Furthermore, the decreased expression of pluripotency-associated genes induced by autophagy inhibition could be recovered by treatment with the antioxidant vitamin C. Together, these results demonstrate that low oxygen-induced autophagy regulates embryo development through antioxidant mechanisms in the pig.

Inhibition of DNA repair protein RAD51 affects porcine preimplantation embryo development.

Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free DNA repair of double-stranded breaks. DNA repair protein RAD51 homolog 1 (RAD51) plays a central role in HR. However, the role of RAD51 during porcine early embryo development is unknown. In the present study, we examined whether RAD51 is involved in the regulation of early embryonic development of porcine parthenotes. We found that inhibition of RAD51 delayed cleavage and ceased development before the blastocyst stage. Disrupting RAD51 activity with RNAi or an inhibitor induces sustained DNA damage, as demonstrated by the formation of distinct γH2AX foci in nuclei of four-cell embryos. Inhibiting RAD51 triggers a DNA damage checkpoint by activating the ataxia telangiectasia mutated (ATM)-p53-p21 pathway. Furthermore, RAD51 inhibition caused apoptosis, reactive oxygen species accumulation, abnormal mitochondrial distribution and decreased pluripotent gene expression in blastocysts. Thus, our results indicate that RAD51 is required for proper porcine parthenogenetic activation (PA) embryo development.

Effects of manganese on maturation of porcine oocytes in vitro and their subsequent embryo development after parthenogenetic activation and somatic cell nuclear transfer.

This study was carried out to examine the effects of manganese (Mn) on the developmental competence of porcine oocytes during in vitro maturation (IVM) after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). Upon treatment of porcine oocytes with different concentrations (0, 3, 6, and 12 ng/ml) of Mn during IVM, PA was performed to determine the optimum concentration. Following PA, the rate of blastocyst formation was higher significantly in treated porcine oocytes at 6 ng/ml of Mn than in other groups (P < 0.05). However, there was no substantial difference in the cleavage rate and total blastocyst cell numbers among all groups. SCNT was performed using the optimal concentration of Mn from PA, which showed an improved blastocyst formation rate in treated oocytes compared to that in control group (P < 0.05). However, the cleavage rate and total cell numbers per blastocyst were not different between the control and the Mn treated groups after SCNT. Additionally, oocyte nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels were assessed. There was no significant difference observed in nuclear maturation among all the groups. However, enhanced intracellular GSH levels while lower levels of ROS were seen in the Mn treated group compared to the control group (P < 0.05). Thus, these results indicate that Mn supplementation can improve the developmental competence of porcine PA and SCNT embryos by increasing GSH and decreasing ROS levels.

The cyclin-dependent kinase inhibitor, JNJ-7706621, improves in vitro developmental competence of porcine parthenogenetic activation and somatic cell nuclear transfer embryos.

In this study we examined the effects of JNJ-7706621, a cyclin-dependent kinase inhibitor, on the in vitro growth of pig embryos that had been produced either by parthenogenetic activation (PA) or somatic cell nuclear transfer (SCNT). A significantly higher percentage of PA embryos reached the blastocyst stage by Day 7 after exposure to 10µM JNJ-7706621 for 4h compared with embryos exposed to 5µgmL-1 cytochalasin B for 4h (P<0.05). Similarly, the rate of Tyr15 phosphorylation of the complex of cyclin and p34cdc2 (CDK1) was significantly elevated in the JNJ-7706621-treated embryos compared with embryos exposed to cytochalasin B or non-treated controls (P<0.05). In contrast, Thr161 phosphorylation of CDK1 was significantly lower in the JNJ-7706621-treated group compared with the cytochalasin B-treated as well as the non-treated group (P<0.05). Similarly, the level of M-phase-promoting factor (MPF) in embryos was significantly lower in the JNJ-7706621-treated group compared with the cytochalasin B-treated and non-treated groups (P<0.05). In addition, more SCNT embryos reached the blastocyst stage after treatment with JNJ-7706621 than following exposure to cytochalasin B (P<0.05). In conclusion, these results reveal that exposure to 10µM JNJ-7706621 for 4h improves early development of PA and SCNT porcine embryos by suppressing the activity of CDK1 and a concomitant reduction in the level of MPF.

74 Treatment with Melatonin During In Vitro Culture Enhances Porcine Parthenogenetically Activated Embryo Development

Melatonin and its metabolites are powerful antioxidants and free radical scavengers. Because porcine embryos are vulnerable to oxidative stress in vitro, the addition of various protective chemicals to the culture medium, including melatonin, has been explored. The aim of this study was to investigate the effect of melatonin on in vitro developmental competence of porcine parthenogenetically activated (PA) embryos. Immature cumulus–oocyte complexes (COC) were collected and cultured in medium comprising TCM-199 supplemented with 10 ng mL−1 epidermal growth factor, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 5 μL mL−1 insulin, transferrin selenium solution 100×, 10% porcine follicular fluid, 10 IU mL−1 eCG, and 10 IU mL−1 hCG for 44 h. Then, COC were denuded and PA with electrical stimulation, and PA embryos were cultured in porcine zygote medium 5 (PZM-5) supplemented with melatonin at increased concentrations (10−9, 10−7, 10−5 M) at 39°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 7 days. Subsequent embryo development, including cleavage rate, blastocyst rate, and blastocyst cell numbers, was compared between groups (mean no. of embryos; control, 27.14; 10−9 M, 28.86; 10−7 M, 27.71; 10−5 M, 26.43). The experiments were repeated 7 times for each treatment group. Statistical analyses of all data were performed using one-way ANOVA with Dunn’s multiple comparison test. Results are expressed as the mean ± SEM and all differences were considered significant at P &lt; 0.05. No apparent effect on cleavage rate of melatonin treatment of various concentrations was noted. Blastocyst cell number did not show any significant difference between groups. However, the potential of PA oocytes to develop into blastocysts was significantly higher in the group supplemented with 10−9 M melatonin compared with the control group (35.44 ± 3.84 v. 24.71 ± 1.59) and other melatonin treated groups (10−5 M, 21.35 ± 2.82; 10−7 M, 24.01 ± 2.31; P &lt; 0.05). These indicated that treatment with 10−9 M melatonin in embryo culture might reduce the oxidative stress properly compared with other concentrations, which results in improvement of blastocyst rate formation. In conclusion, treatment with 10−9 M melatonin positively promoted the blastocyst formation rate of porcine PA embryos with no beneficial effects on their blastocyst cell numbers or cleavage rate. This study was supported by the National Research Foundation (#2015R1C1A2A01054373; 2016M3A9B6903410), Research Institute for Veterinary Science and the BK21 PLUS Program.

Iloprost supports early development of in vitro-produced porcine embryos through activation of the phosphatidylinositol 3-kinase/AKT signalling pathway

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 (10-1000nM), and the beneficial effects of iloprost on the early development of porcine IVF and PA embryos was completely ablated by treatment with 2.5μM 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.

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

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

Melatonin protects porcine oocyte invitro maturation from heat stress.

Melatonin is a pleiotropic molecule which plays an important role in animal reproductive activities. Because of the increased global warming, the impact of heat stress (HS) on stockbreeding has become an inevitable issue to be solved. To investigate the potential effects of melatonin on the invitro maturation of porcine oocyte under the HS, a HS model for porcine oocyte maturation has been used in this study and the different concentrations of melatonin (10(-6) -10(-9) m) were also tested for their protective effects on oocytes. The polar body rate, the index of the nuclear maturation of the oocytes, and the cleavage rate as well as the blastocyst rate were measured to evaluate the developmental competence of the oocytes after parthenogenetic activation (PA). The results showed that HS [in vitro maturation (IVM) 20-24hr, 42°C] significantly reduced the polar body rate of oocytes and the blastocyte rate of porcine PA embryos, while melatonin (10(-7) m) application not only improved polar body rate and blastocyte rate, but also preserved the normal levels of steroid hormone which is disrupted by HS. The presence of melatonin (10(-7) m) during the oocyte maturation under the HS reduced reactive oxygen species (ROS) formation, enhanced glutathione (GSH) production, inhibited cell apoptosis, and increased the gene expressions of SIRT1, AKT2, and Polg2. Importantly, the endogenously occurring melatonin of cumulus-oocyte complexes was significantly induced by HS. The results indicated that melatonin application effectively protected the oocytes from HS. These observations warranted the further studies invivo regarding to improve the reproductive activities of animals under the global warming environment.

Cytoplasmic membrane activities during first cleavage of zona-free porcine embryos: description and consequences.

Already at first embryo cleavage subsequent blastocyst formation can be predicted based on morphology but the finer morphological details can be difficult to determine due to the presence of the zona pellucida (ZP). Therefore, we monitored zona-free porcine parthenogenetically activated (PA) embryos in a time-lapse system to: (1) describe and characterise the morphological activity of the cytoplasmic membrane and the distribution to the two nuclei during first cleavage and (2) determine the relationship between specific morphological activities and subsequent embryonic development. After ZP removal the membrane surface activities were clearly visible, so all cleaved embryos could be divided into two groups depending on the surface activity during first cleavage: regular morphology (MN) or irregular morphology with 'bumps' (MB). The two nuclei were more unequal in MB embryos in both nucleus size and DNA quantity. After first cleavage, MB embryos could be further divided into three types of irregularities (MB1, MB2, MB3) based on their subsequent behaviour. Clear differences in developmental patterns were found between MN and MB embryos, such as delayed first cleavage, compromised blastocyst formation and total cell number. The predictive value of these new types of morphological events was comparable to the more traditionally used time of first cleavage. In conclusion, zona-free embryos allow visualisation of finer morphological details that can provide an early prediction of embryo developmental potential, but further studies are needed on other type of embryos.

Cryotop vitrification of porcine parthenogenetic embryos at the early developmental stages

The objective of this study was to evaluate the effects of early developmental stages at which Cryotop vitrification is performed on subsequent survival and in vitro development of porcine parthenogenetic activation embryos. The zygotes that were cultured for 4, 8, and 18 hours post electric activation (h.p.a.) and two- and four-cell embryos were vitrified, warmed, and continuously cultured for the remaining period. The zygotes vitrified at 4, 8, and 18 h.p.a. showed similar percentages of survival, cleavage, and blastocyst formation. No difference in viability was observed after vitrification of two- and four-cell embryos, but the embryos vitrified at the two-cell stage exhibited significantly higher blastocyst formation rate than those vitrified at the four-cell stage. However, vitrifying embryos resulted in significantly decreased survival and development rates, regardless of the developmental stage of the embryos. In addition, the final developmental stage, diameter, apoptotic index, and the number of inner cell mass, trophectoderm, and total cells of blastocysts derived from embryos vitrified at any stage of the early culture were similar to those of fresh blastocysts. In conclusion, our data indicate that the early-stage porcine parthenogenetically activated embryos including the zygote, two cells, and four cells have a high ability to survive cryopreservation; these viable embryos after vitrification can produce respectable development rates and good-quality blastocysts.

Improvement in the blastocyst quality and efficiency of putative embryonic stem cell line derivation from porcine embryos produced in vitro using a novel culturing system.

Porcine embryonic stem cells (pESCs) have great potential for application in translational biomedical research, including xenotransplantation and disease models. Obtaining high-quality blastocysts is the most important factor in the isolation and colonization of primary ESCs and the establishment of ESC lines. In pigs, in vitro-derived blastocysts have a limited cell number compared to in vivo-derived blastocysts and show an indefinite inner cell mass, which may result in failure to establish pESC lines. In the present study, the effects of resveratrol (RES), granulocyte-macrophage colony stimulating factor (GM-CSF) and β-mercaptoethanol (β-ME) on the quality of blastocysts and the efficiency of colony derivation were investigated for the establishment of ESCs. A novel culturing system was developed in which 2 µM RES was added to the oocyte in vitro maturation (IVM) medium, and 10 ng/ml pGM-CSF and 10 µM β-ME were added to embryo in vitro culture (IVC) medium. This novel system showed significantly more parthenogenetic activation (PA) blastocysts (54.5 ± 1.8% vs. 43.4 ± 1.2%; P<0.05) and in vitro fertilization (IVF) blastocysts (36.9 ± 3.3% vs. 26.2 ± 2.9%; P<0.06) at day seven as compared with that in the control system. The PA and IVF blastocysts from the novel system showed a significantly greater hatching rate (P<0.05) and greater cell numbers (55.1 ± 2.0 vs. 45.6 ± 2.0; P<0.05 and 78.9 ± 6.8 vs. 58.5 ± 7.2; P<0.06, for PA and IVF, respectively) at day seven compared to that in the control system. After seeding on feeder cells, the PA blastocysts produced by the novel system showed a significantly increased rate of attachment (28.8 ± 3.9% vs. 17.2 ± 2.4%; P<0.062). Finally, two putative pESC lines from PA embryos produced by the novel system and one by the control system were established. In conclusion, the novel system improved blastocyst quality and increased the derivation efficiency of putative pESC lines from porcine PA and IVF embryos produced in vitro.

Distribution and content of lipid droplets and mitochondria in pig parthenogenetically activated embryos after delipation

The present study examines the effect of delipation on developmental competence and the distribution pattern of lipid droplets (LDs) and mitochondria in parthenogenetically activated (PA) pig embryos. Mature oocytes were delipated by centrifugation after partial digestion of the zonae pellucidae, subjected to parthenogenetic activation after total removal of zonae pellucidae by pronase, and then cultured in vitro up to the blastocyst stage. The contents and distributions of LDs and mitochondria in the oocytes and/or embryos were observed by staining with Oil Red O and MitoTracker Red CMXRos, respectively. The LD and mitochondrial contents were significantly reduced by the delipation process, and only smaller LDs remained in the delipated oocytes and/or embryos. Their content remained constant from the metaphase II oocyte to the blastocyst stage, but they became gradually smaller as the oocytes and/or embryos developed. The distribution pattern of the LDs in the delipated embryos changed over time and in a manner different to that seen in the controls. In the early developmental stages (1- to 4-cell stages), they were distributed peripherally and formed a ring around the nucleus. However, by the blastocyst stage, a homogeneous distribution of LDs was observed in both the inner cell mass and trophectoderm. The distribution pattern of mitochondria also changed with the development of the delipated PA embryos and again, in ways different to those seen in the controls. In the early 1- to 4-cell stages, a peripheral distribution of mitochondrial foci was observed in each blastomere. However, in blastocysts, the mitochondria were homogeneously distributed throughout the inner cell mass and trophectoderm. Although the cleavage rate at the 2- and 4-cell stages of the PA embryos was not affected by delipation (95.83 ± 2.25% vs. 97.44 ± 0.67%; 79.17 ± 4.47% vs. 84.62 ± 1.19%), it was reduced significantly in the blastocyst compared with the controls (21.67 ± 3.78% vs. 49.36 ± 1.77%). The distribution pattern of the LDs in oocytes and/or embryos at different developmental stages, and that of the mitochondria in metaphase II oocytes, was affected by delipation. The developmental competence of porcine PA embryos would appear to be affected by delipation.

Dynamic changes of histone H3 lysine 27 acetylation in pre-implantational pig embryos derived from somatic cell nuclear transfer

Histone H3 lysine 27 acetylation (H3K27ac) is an active epigenetic modification which has been revealed to be associated with active gene expression. It was hypothesized that H3K27ac might also participate in the porcine somatic reprogramming process during early development of SCNT-derived embryos. The spatial and temporal expression profiles of H3K27ac were investigated at different developmental stages in SCNT embryos compared with in vitro fertilization (IVF) and parthenogenetic activation (PA) counterparts. Specifically, results showed that amounts of H3K27ac gradually decreased from the earliest pronuclear stage to 8-cell stage, corresponding to the major embryonic genome activation (EGA), followed by re-acetylation of H3K27 from the morula stage onwards accompanying the first cell lineage specification in IVF embryos. Similar dynamic patterns of H3K27ac signal was observed at all developmental stages of porcine SCNT and PA embryos except for the hatched stage in which amounts of H3K27ac in SCNT and PA embryos was slightly less than that in IVF counterparts. Moreover, the gradual decrease of H3K27ac before EGA was demonstrated to be an active process independent of DNA replication, RNA and protein synthesis. The expression of HDAC1, HDAC2, MBD3 and CBP genes were well correlated with the dynamic changes of H3K27ac mark. Overall, these results indicate that H3K27ac is only defective in late SCNT blastocysts, and that the dynamic changes of this marker might also underlie the EGA and initial cell lineage specification during early embryo development.

Effect of red light on the development and quality of mammalian embryos.

To assess irradiance and total energy dose from different microscopes during the in-vitro embryonic developmental cycle in mouse and pig and to evaluate its effect on embryonic development and quality in pig. Spectral scalar irradiance (380-1050 nm) was measured by a fiber-optic microsensor in the focal plane of a dissection microscope, an inverted microscope and a time-lapse incubation system. Furthermore, the effect of three different red light levels was tested in the time-lapse system on mouse zygotes for 5 days, and on porcine zona-intact and zona-free parthenogenetically activated (PA) embryos for 6 days. The time-lapse system used red light centered at 625 nm and with a lower irradiance level as compared to the white light irradiance levels on the dissection and inverted microscopes, which included more energetic radiation <550 nm. Even after 1000 times higher total energy dose of red light exposure in the time-lapse system, no significant difference was found neither in blastocyst development of mouse zygotes nor in blastocyst rates and total cell number of blastocysts of porcine PA embryos. Our results indicate that red light (625 nm, 0.34 W/m(2)) used in the time-lapse incubation system does not decrease the development and quality of blastocysts in both mouse zygotes and porcine PA embryos (both zona-intact and zona-free).