Porcine In Vitro Fertilization Embryos Research Articles

Anethole improves the developmental competence of porcine embryos by reducing oxidative stress via the sonic hedgehog signaling pathway

BackgroundAnethole (AN) is an organic antioxidant compound with a benzene ring and is expected to have a positive impact on early embryogenesis in mammals. However, no study has examined the effect of AN on porcine embryonic development. Therefore, we investigated the effect of AN on the development of porcine embryos and the underlying mechanism.ResultsWe cultured porcine in vitro-fertilized embryos in medium with AN (0, 0.3, 0.5, and 1 mg/mL) for 6 d. AN at 0.5 mg/mL significantly increased the blastocyst formation rate, trophectoderm cell number, and cellular survival rate compared to the control. AN-supplemented embryos exhibited significantly lower reactive oxygen species levels and higher glutathione levels than the control. Moreover, AN significantly improved the quantity of mitochondria and mitochondrial membrane potential, and increased the lipid droplet, fatty acid, and ATP levels. Interestingly, the levels of proteins and genes related to the sonic hedgehog (SHH) signaling pathway were significantly increased by AN.ConclusionsThese results revealed that AN improved the developmental competence of porcine preimplantation embryos by activating SHH signaling against oxidative stress and could be used for large-scale production of high-quality porcine embryos.

Porcine embryo development and inactivation of microorganisms after ultraviolet-C irradiation at 228 nm

It is important to prevent contamination inside the incubator as a method of preventing microbial infections during the embryo culture. In the present study, we examined the effects of ultraviolet-C (UV–C) irradiation, used for microorganism inactivation, on embryo development and the growth of bacteria, including Escherichia coli and Staphylococcus aureus, and the fungus Cladosporium cladosporioides. In the embryo irradiation experiment, we examined the effects of the plastic lid of the culture dish, irradiation distances (10, 20, and 25 cm), and different irradiation wavelengths (228 and 260 nm) during embryo culture for 7 days on the development and quality of porcine in vitro-fertilized embryos. None of the embryos cultured in dishes without plastic lids developed into blastocysts after irradiation with 228 nm UV-C. When porcine embryos were cultured in a culture dish with lids, the 228 nm UV-C irradiation decreased blastocyst formation rates of the embryos but not their quality, irrespective of the UV-C irradiation distance. Moreover, irradiation with 260 nm UV-C, even with plastic lids, had more detrimental effects on embryo development than irradiation with 228 nm UV-C. Investigation of the inactivating effects of UV-C irradiation at 228 nm and 260 nm on the growth of the bacteria and fungus showed that 260 nm UV-C reduced the viability to a greater extent than 228 nm UV-C. Moreover, the disinfection efficacy for the bacteria increased when the irradiation duration increased and the distance decreased. In conclusion, porcine embryos can develop into blastocysts without loss of quality even after continuous long-duration irradiation (7 days) with 228 nm UV-C, which can inactivate the growth of bacteria and the tested fungus; however, the development rate of the embryo is reduced.

Coordination of zygotic genome activation entry and exit by H3K4me3 and H3K27me3 in porcine early embryos.

Histone modifications are critical epigenetic indicators of chromatin state associated with gene expression. Although the reprogramming patterns of H3K4me3 and H3K27me3 have been elucidated in mouse and human preimplantation embryos, the relationship between these marks and zygotic genome activation (ZGA) remains poorly understood. By ultra-low-input native chromatin immunoprecipitation and sequencing, we profiled global H3K4me3 and H3K27me3 in porcine oocytes and in vitro fertilized (IVF) embryos. We observed sharp H3K4me3 peaks in promoters of ZGA genes in oocytes, and these peaks became broader after fertilization and reshaped into sharp peaks again during ZGA. By simultaneous depletion of H3K4me3 demethylase KDM5B and KDM5C, we determined that broad H3K4me3 domain maintenance impaired ZGA gene expression, suggesting its function to prevent premature ZGA entry. In contrast, broad H3K27me3 domains underwent global removal upon fertilization, followed by a re-establishment for H3K4me3/H3K27me3 bivalency in morulae. We also found that bivalent marks were deposited at promoters of ZGA genes, and inhibiting this deposition was correlated with the activation of ZGA genes. It suggests that promoter bivalency contributes to ZGA exit in porcine embryos. Moreover, we demonstrated that aberrant reprogramming of H3K4me3 and H3K27me3 triggered ZGA dysregulation in somatic cell nuclear transfer (SCNT) embryos, whereas H3K27me3-mediated imprinting did not exist in porcine IVF and SCNT embryos. Our findings highlight two previously unknown epigenetic reprogramming modes coordinated with ZGA in porcine preimplantation embryos. Finally, the similarities observed between porcine and human histone modification dynamics suggest that the porcine embryo may also be a useful model for human embryo research.

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.

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.

Improved early development potence of in vitro fertilization embryos by treatment with tubacin increasing acetylated tubulin of matured porcine oocytes

To improve the developmental potential of in vitro embryos is a long-term concern field for human assisted reproduction and animal in vitro embryo production practice. In the current study, we examined the effects and mechanism of an HDAC6 inhibitor, tubacin, on the maturation of porcine oocytes and in vitro development of porcine IVF embryos. It has been demonstrated the effect of tubacin on the acetylation level of α-tubulin in porcine oocytes. As a result, the maturation rate of porcine oocytes was significantly improved (P < 0.05), and the following development potent of blastocysts forming rate was also significantly increased (P < 0.05). We found that the increased acetylation of α-tubulin significantly reduced the abnormal rate of microtubule, furthermore, the proportion of mitochondria in the vicinity of in vitro fertilization (IVF) nucleus was significantly enhanced in Metaphase I (MI) and Metaphase II (MII) stages. The expression levels of microtubule assembly genes (TUBA1A, αTAT1 and MAP2) significantly up-regulated in MI and MII stages. Together, these results suggest that treatment of porcine oocytes during maturation with tubacin could promote their IVF embryos developmental competence by altering spindle formation, mitochondrial concentration and genes expression patterns of matured porcine oocytes.

Fatty acid metabolism as an indicator for the maternal–to–zygotic transition in porcine IVF embryos revealed by RNA sequencing

A number of fatty acids have been found in porcine oocytes and early embryos. Recent studies have indicated the importance of fatty acids in the development of pre-implantation porcine embryos, whether derived from in vivo or somatic cell nuclear transfer. However, the effects of fatty acids on porcine embryos produced by in vitro fertilization (IVF) remain poorly defined. This study aimed to investigate the patterns of gene expression and functions of fatty acids in pre-implantation IVF porcine embryos at different stages using transcriptome sequencing. We found that, in IVF porcine embryos, genes related to fatty acid metabolism were positively expressed during early embryonic development. Additionally, the expression of genes related to lipid metabolism changed dramatically during the maternal–to–zygotic transition (MZT), and the genes associated with lipid metabolism were correlated with zygotic genome activation in porcine IVF embryos, suggesting that fatty acid metabolism plays an important role in MZT. In summary, fatty acid metabolism may be an indicator of MZT in porcine IVF embryos, which presents new considerations for exploring the regulatory mechanisms of this process.

Selection based on morphological features of porcine embryos produced by in vitro fertilization: Timing of early cleavages and the effect of polyspermy.

The aim of this study was to examine whether a morphological approach is efficient for selecting high‐quality porcine embryos produced by in vitro fertilization (IVF) under high polyspermy conditions. Frozen‐thawed Meishan epididymal spermatozoa showing moderate and high polyspermy were subjected to IVF (1 × 105 sperms/ml). Under conditions of moderate polyspermy, 4‐cell embryos selected at 48 hr after IVF (single selection) and 8‐cell embryos selected at 79 hr after IVF from the collected 4‐cell embryos (double selection) showed high developmental competence. Likewise, 4‐ and 8‐cell embryos produced by IVF under high polyspermy conditions also showed high competence for development to blastocysts. However, blastocysts derived from high polyspermy conditions had significantly fewer cells than those produced under moderate polyspermy conditions. Furthermore, the frequency of nuclear and chromosomal abnormalities in 4‐ and 8‐cell embryos produced under conditions of high polyspermy was significantly (p < .05) higher in comparison to moderate polyspermy conditions. These findings suggest that although high polyspermy affects the frequency of nuclear and chromosomal anomalies in porcine IVF embryos, subsequent selection based on morphological features of 4‐ and 8‐cell embryos even under high polyspermy conditions, could be an alternative option for selecting porcine IVF embryos with high development ability.

Histone 3 lysine 4, 9, and 27 demethylases expression profile in fertilized and cloned bovine and porcine embryos.

Epigenetic modifications in the C-terminal domain of histones coordinate important events during early development including embryo genome activation (EGA) and cell differentiation. In this study, the mRNA expression profile of the main lysine demethylases (KDMs) acting on the lysine 4 (H3K4), 9 (H3K9), and 27 (H3K27) of the histone H3 was determined at pre-, during and post-EGA stages of bovine and porcine embryos produced by in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). In IVF embryos, mRNA abundance of most KDMs revealed a bell-shaped profile with peak expression around the EGA period, i.e. Day 3 for porcine (KDM2B, KDM5B, KDM5C, KDM4B, KDM4C, KDM6A, KDM6B, and KDM7A), and Day 4 for bovine (KDM1A, KDM5A, KDM5B, KDM5C, KDM3A, KDM4A, KDM4C, and KDM7A). The mRNA profile of KDM1A, KDM2B, KDM3A, KDM3B, KDM6A, and KDM6B differed between porcine and bovine IVF embryos. Several differences were also observed between SCNT and IVF, which includes a precocious peak in the mRNA expression of KDM1A, KDM3A, KDM4C, KDM5A, KDM5B, KDM5C, KDM6A, and KDM7A in bovine SCNT embryos; absence of mRNA peak for KDM4B, KDM4C, and KDM6A in porcine SCNT embryos; and early decreasing in KDM5B and KDM5C mRNA in porcine SCNT embryos. Based on the mRNA profile, this study has identified several KDMs that are likely involved in the regulation of the EGA transition, KDMs that may have a species-specific role in bovine and porcine embryos, and KDMs that are improperly expressed during cell reprogramming in SCNT embryos.

Dynamic changes of SETD2, a histone H3K36 methyltransferase, in porcine oocytes, IVF and SCNT embryos.

SETD2 (SET domain containing protein 2) acts as a histone H3 lysine 36 (H3K36)-specific methyltransferase and may play important roles in active gene transcription in human cells. However, its expression and role in porcine oocytes and preimplantation embryos are not well understood. Here, we used immunofluorescence and laser scanning confocal microscopy to examine SETD2 expression in porcine fetal fibroblasts, oocytes, and preimplantation embryos derived from in vitro fertilization (IVF), parthenogenetic activation (PA), and somatic cell nuclear transfer (SCNT). In porcine fetal fibroblasts, SETD2 expression was detected in interphase cells, but not in M (mitotic)-phase cells. The SETD2 signal was observed in non-surrounded nucleolus (NSN)-stage oocytes, but not in surrounded nucleolus (SN)-, metaphase I (MI)-, or metaphase II (MII)-stage oocytes. The SETD2 signal was detectable in sperm, and undetectable immediately after fertilization, detectable at the 2-cell stage, and peaked at the 4-cell stage of IVF embryos in which porcine embryonic genome is activated. Similar to the pattern found in IVF embryos, the SETD2 signal was absent from PA embryos at the 1-cell stage, but it was detected at the 2-cell stage and thereafter maintained to the blastocyst stage. Interestingly, unlike the IVF and PA embryos, the SETD2 signal was detected throughout the development of SCNT embryos, including at the 1-cell stage. These data suggest that SETD2 may be functional for embryonic gene transcription in porcine preimplantation embryos. It is further speculated that the aberrant expression of SETD2 at the 1-cell stage of porcine SCNT embryos may be a factor in the low efficiency of cloning in pig.

85 CARBOXYETHYLGERMANIUM SESQUIOXIDE CONFERS ANTIOXIDATIVE PROTECTION FOR PORCINE IN VITRO-FERTILIZED EMBRYOS

Reactive oxygen species (ROS) are generated both from the environment as well as from within the embryos during normal embryo development. However, excessive production of ROS creates the defective effect on embryo development. The purpose of the present study was to determine the effect of antioxidant, carboxyethylgermanium sesquioxide (Ge-132), during in vitro culture (IVC) on embryonic development in porcine in vitro-fertilized (IVF) embryos. The different concentrations of Ge-132 (0, 100, 200, and 400 μg mL–1) were treated to the zygotes (n = 233, 234, 233, and 234, respectively). All of the experiments were repeated at least three times. To characterise putative porcine embryonic stem cell (ESC) derived from Ge-132 treated IVF blastocysts, several assays including an assessment of intensive alkaline phosphatase activity, embryoid body formation and expression analysis of pluripotency by immunofluorescence staining were performed. In the result of embryo developmental competence, the 100- and 200-μg mL–1 Ge-132 significantly (P &lt; 0.05) increased cleavage rates (67.4% and 67.1%, respectively; P &lt; 0.05) compared with the control group (61.6%). The 100 μg mL–1 Ge-132 had significantly (P &lt; 0.05) higher blastocyst formation rates (32.6%) than the control group (26.5%). The Ge-132 supplement during IVC significantly (P &lt; 0.05) increased GSH levels in embryos compared with those in the control, which led to the reduced ROS level. Two putative ESC lines were successfully derived from porcine IVF embryos cultured in 100 and 200 μg mL–1 Ge-132 treatment. The established putative porcine ESCs showed alkaline phosphatase activity, pluripotency-associated markers such as OCT4 and NANOG and the capacity for embryoid body formation, which confirm the differentiation potentials. Together, these results suggest that Ge-132 supplementations during IVC enhanced the developmental competence of porcine IVF embryos, most likely by increasing intracellular GSH synthesis, reducing ROS level, and it might help the derivation of putative porcine ESCs. This work was supported, in part, by a grant from the Cooperative Research Program for Agriculture Science &amp; Technology Development (Project No. PJ011077, PJ011288), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2013R1A2A2A04008751). These are the results of a study by the Leaders INdustry-University Cooperation Project 2015-B-0013–010118, supported by the Ministry of Education, Republic of Korea.

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.

Pro-apoptotic Effect of Pifithrin-α on Preimplantation Porcine In vitro Fertilized Embryo Development.

The aim of this study was to investigate the impact of a reported p53 inhibitor, pifithrin-α (PFT-α), on preimplantation porcine in vitro fertilized (IVF) embryo development in culture. Treatment of PFT-α was administered at both early (0 to 48 hpi), and later stages (48 to 168 hpi) of preimplantation development, and its impact upon the expression of five genes related to apoptosis (p53, bak, bcl-xL, p66Shc and caspase3), was assessed in resulting d 7 blastocysts, using real-time quantitative PCR. Total cell numbers, along with the number of apoptotic nuclei, as detected by the in situ cell death detection assay, were also calculated on d 7 in treated and non-treated control embryos. The results indicate that PFT-α, when administered at both early and later stages of porcine IVF embryo development, increases the incidence of apoptosis in resulting blastocysts. When administered at early cleavage stages, PFT-α treatment was shown to reduce the developmental competence of porcine IVF embryos, as well as reducing the quality of resulting blastocysts in terms of overall cell numbers. In contrast, at later stages, PFT-α administration resulted in marginally increased blastocyst development rates amongst treated embryos, but did not affect cell numbers. However, PFT-α treatment induced apoptosis and apoptotic related gene expression, in all treated embryos, irrespective of the timing of treatment. Our results indicate that PFT-α may severely compromise the developmental potential of porcine IVF embryos, and is a potent apoptotic agent when placed into porcine embryo culture media. Thus, caution should be exercised when using PFT-α as a specific inhibitor of p53 mediated apoptosis, in the context of porcine IVF embryo culture systems.

Effects of porcine granulocyte-macrophage colony-stimulating factor on porcine in vitro-fertilized embryos

This study investigated the effects of porcine granulocyte-macrophage colony-stimulating factor (pGM-CSF) on the developmental potential of porcine in vitro-fertilized (IVF) embryos in chemically and semidefined (with BSA) medium. In experiment 1, zygotes were treated with different concentrations of pGM-CSF (0, 2, 10, 100 ng/mL). The results indicated that 10 ng/mL pGM-CSF significantly (P < 0.05) increased blastocyst development and total cell number (15.1% and 53.5, respectively) compared with the control (6.1%, and 38.8, respectively). Comparing blastocyst formation, early and expanded blastocyst formation was significantly higher in the 10 ng/mL-pGM-CSF group than in the control on Days 6 and 7 of the culture period. However, there was no significant difference in cleavage rate. Experiment 2 demonstrated that pGM-CSF influenced the percentage of blastocyst formation and total cell number when pGM-CSF was added during Days 4 to 7 (14.6% and 53.9, respectively) or Days 0 to 7 (15.2% and 54.0, respectively) compared with the control (7.8% and 43.1, respectively) and compared with Days 0 to 3 (8.7% and 42.5, respectively). Similarly, early blastocyst formation rates were significantly higher at Days 4 to 7 than in the control, and expanded blastocyst formation was significantly higher at Days 4 to 7 or Days 0 to 7. No significant difference in cleavage rates appeared among the groups. In experiment 3, in the presence of BSA, pGM-CSF also increased the percentage of embryos that developed to the blastocyst stage and the total cell number (20.3% and 59.8, respectively) compared with the control (14.9% and 51.4, respectively), whereas there was no significant difference in cleavage rate. Experiment 4 found that the total cell number and the number of cells in the inner cell mass (ICM) were significantly increased compared with the control when zygotes were cultured in either porcine zygotic medium (PZM)-3 or PZM-4 supplemented with 10 ng/mL pGM-CSF. The number of trophectoderm (TE) cells was significantly higher in PZM-3 medium supplemented with pGM-CSF than in the control, and the number tended to increase (P = 0.058) in PZM-4 medium supplemented with pGM-CSF. The ratio of inner cell mass to trophectoderm cells was significantly higher in PZM-4 supplemented with 10 ng/mL pGM-CSF, but not in PZM-3. In experiment 5, it was found that the male pronuclear formation rate, monospermic penetration and sperm/oocyte were 95.4%, 37.2%, and 2.4, respectively. Together, these results suggest that pGM-CSF may have a physiological role in promoting the development of porcine preimplantation embryos and regulating cell viability and that addition of pGM-CSF to IVC medium at Days 4 to 7 or 0 to 7 improves the developmental potential of porcine IVF embryos.

Supplementation with vascular endothelial growth factor during in vitro maturation of porcine cumulus oocyte complexes and subsequent developmental competence after in vitro fertilization

The aim of the present study was to investigate whether the effects of vascular endothelial growth factor (VEGF) on porcine cumulus oocyte complexes (COCs) and subsequent blastocyst formation following in vitro fertilization are attributable to improved fertilization and cytoplasmic maturation. Porcine COCs were cultured for 42 h in TCM199 medium with 5 ng/mL human recombinant VEGF, and the resultant metaphase II oocytes were fertilized in vitro. COCs without VEGF supplementation served controls. Supplementation with VEGF during in vitro maturation (IVM) significantly (P < 0.05) improved the blastocyst formation rate and total cell number (46.7 ± 3.1% and 82.8 ± 6.7, respectively) compared with controls (32.5 ± 3.4% and 64.1 ± 5.6, respectively). On day 2, the percentage of four-cell stage embryos was significantly higher in the VEGF-matured group (49.1 ± 2.7%) than in the control (33.1 ± 5.8%), and the percentage of two-cell stage embryos was significantly higher in the control group (10.4 ± 1.4%) than in the VEGF-matured group (6.6 ± 0.9%). At 10 h after the onset of in vitro fertilization (IVF), oocytes with two pronuclei were considered as monospermically or normally fertilized, and oocytes with more than two pronuclei were considered as polyspermically fertilized. Monospermy was significantly higher in VEGF-matured oocytes (47.2 ± 4.3%) than in the control (20.0 ± 2.4%), and polyspermy and sperm penetration per oocyte were significantly higher in the control group (54.4 ± 3.8% and 2.3 ± 0.1, respectively) than in the VEGF-matured oocytes (43.9 ± 3.6% and 1.8 ± 0.1, respectively). Supplementation with VEGF during IVM significantly (P < 0.05) improved male pronuclear formation as compared with the control (91.1 ± 1.9 vs 74.4 ± 3.8%). Type III cortical granule distribution in oocytes was more common in VEGF-matured oocytes (78.0%) than in the control (52.1%). These results suggest that VEGF supplementation during IVM enhanced the developmental potential of porcine IVF embryos through higher male pronuclear formation and higher monospermic fertilization rates as a consequence of improved cytoplasmic maturation.

Amphiregulin promotes the proliferation of trophoblast cells during preimplantation development of porcine embryos

Our objective was to investigate the effects of in vitro culture (IVC) medium supplemented with amphiregulin (AREG) on the preimplantation embryonic development of porcine (Genus: Sus domestica, Species: Landrace) embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA). In vitro fertilization and PA embryos at the 1-cell stage were cultured in IVC medium supplemented with 0, 0.5, 5, or 50 ng/mL AREG for 7 d. There were significantly greater total numbers of cells in blastocysts of IVF and PA embryos cultured with 50 ng/mL AREG compared with that of controls. In vitro fertilization and PA embryos were then cultured in NCSU-23 medium supplemented with 50 ng/mL AREG on Days 1 through 7, Days 1 through 3 (early stage), or Days 4 through 7 (late stage), or without AREG. There were significantly greater numbers of trophoblast cells in the late-stage and full-term groups of IVF and PA embryos than in the early-stage and control groups. The presence of AREG protein in IVF-derived blastocysts was detected using a polyclonal AREG antibody and indirect immunofluorescence. Amphiregulin protein was localized in both the cytoplasm and nucleus. Using real-time polymerase chain reaction, we detected the expression of AREG mRNA in all developmental stages of IVF and PA embryos; however, the expression level varied according to stage. Thus, the incubation of porcine IVF and PA embryos in AREG-supplemented culture medium mainly at the late preimplantation stage increases the numbers of trophoblast cells.

115 APOPTOSIS EVENT OF PREIMPLANTATION DEVELOPMENT STAGES IN PORCINE IN VITRO-FERTILIZED EMBRYOS

Little is known about apoptosis events in porcine preimplantation embryos. In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro-fertilized (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22 h postinsemination were treated at different concentrations of actinomycin D (0, 5, 50 and 500 ng mL–1 in NCSU medium). Four groups were incubated at 37°C in 5% CO2, 5%O2 for 8 h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2 days and BL development rate at 7 days after in vitro culture (IVC). A significantly less rate of cleavage was found in the 500 ng mL–1 group compared with others (500 ng mL–1 v. 0, 5, 50 ng mL–1; 15.4% v. 48.6%, 40%, 32%). In the results of BL formation rate, there was a significantly less BL formation rate in 500 ng mL–1 compared with others (500 ng mL–1 v. 0, 5, 50 ng mL–1; 0% v. 10%, 8.8%, 9%). In experiment 2, to evaluate apoptotic cells at different stage (2-cell, 4-cell, 8-cell and BL stage) of all groups, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng mL–1 actinomycin D). A high number of the BL derived control group contained at least one apoptotic cell. Actinomycin D treated BL responded to the presence of apoptotic inductor by a significant decrease in the average number of blastomeres and a significant increase in the incidence of apoptotic cell death. In the 500 ng mL–1 group, the incidence of apoptosis increased at the 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is a useful tool for the apoptosis study of porcine preimplantation embryos. This work was supported by a grant (#20070301034040) from BioGreen 21 program, Rural Development Administration, Republic of Korea.

Osmolarity at early culture stage affects development and expression of apoptosis related genes (bax‐α and bcl‐xl) in pre‐implantation porcine NT embryos

This study examined whether high osmolarity of culture medium at the early culture stage affects development and expression of apoptosis related genes (Bax-alpha and Bcl-xl) of porcine nuclear transfer (NT) and in vitro fertilization (IVF) embryos. NT and IVF embryos were divided into three groups and the basic medium was PZM-3 (260-270 mOsmol, control group). The control group of embryos was cultured in PZM-3 for whole culture period. Other two groups of embryos were cultured in a modified PZM-3 with 0.05 M sucrose (300-320 mOsmol, sucrose group) or increased NaCl to 138 mM (300-320 mOsmol, NaCl group) for the first 2 days, and then cultured in PZM-3 for 4 days. NT embryos cultured in NaCl group showed a significantly higher developmental rate to the blastocyst stage with a decreased apoptosis rate compared to the control (P < 0.05). There was no difference in blastocyst formation and apoptosis incidence among the three culture treatments for IVF-derived embryos. Bax-alpha mRNA expression was significantly higher in the control than sucrose or NaCl group for both NT and IVF embryos (P < 0.05). Moreover, the relative abundance of Bax-alpha/Bcl-xl was higher in the control than the treatment groups. These results indicate that the higher osmolarity at the early embryonic stage of porcine NT and IVF embryos can improve the in vitro development with reduced apoptosis through regulating the Bax-alpha/Bcl-xl gene expression.

198 BENEFICIAL EFFECT OF GHRELIN ON IN VITRO DEVELOPMENT OF PORCINE IN VITRO-FERTILIZED AND PARTHENOGENETIC EMBRYOS

Accumulating evidence suggests that ghrelin plays an important role in female reproduction. The objective of the present study was to investigate the effect of ghrelin on pre-implantation development of porcine in vitro-fertilized (IVF) and parthenogenetic embryos. Cumulus–oocyte complexes were matured for 44 h in BSA-free NCSU23 supplemented with 10 ng mL-1 epidermal growth factor, 10 ng mL-1 leptin, 0.57 mM cysteine, 10 IU mL-1 pregnant mare serum gonadotropin, and 10 IU mL-1 hCG. After removal of the cumulus cells, some oocytes were fertilized with fresh boar semen (1 � 105 sperm mL-1) in modified Tween medium B with milk powder (Abeydeera and Day 1997 Theriogenology 48, 537–544) and some oocytes were activated by a single, 100-�s, direct current pulse of 1.4 kV cm-1. Presumptive zygotes (Experiment 1) and parthenogenetic oocytes (Experiment 2) were subsequently cultured in PZM3 (Yoshioka et al. 2002 Biol. Reprod. 66, 112–119) supplemented with ghrelin at 0 (control), 0.5, 5, 50, and 500 ng mL-1 (ghrelin 0.5, 5, 50, 500 groups, respectively) under 5% CO2, 5% O2, 90% N2 and 100% humidity at 39.0�C. Cleavage and blastocyst rates were assessed on Days 2 and 6 (Day 0: the day IVF and activation were conducted). The total cell number in blastocysts was determined by Hoechst 33342 staining on Day 6. All data were analyzed by using SPSS (13.0) with one-way ANOVA. All experiments were done at least 4 times. In Experiment 1, the rate of blasotcyst formation in IVF embryos was significantly (P &amp;lt; 0.05) increased in the ghrelin 500 group compared with that in the control group (26.1 � 1.8 vs. 12.4 � 6.0%, mean � SEM). Furthermore, increased total cell numbers (P &amp;lt; 0.05) were observed in the ghrelin 50 and 500 groups compared with that in the control group (63 � 6.6 and 64 � 5.5 vs. 42 � 6.6). In Experiment 2, we found that the blastocyst rate of parthenogenetic embryos was significantly (P &amp;lt; 0.05) higher in the ghrelin 5 and 500 groups than in the others (24.6 � 4.7 and 25.0 � 3.3 vs. 13.3 � 2.7, 14.9 � 2.4, 18.1 � 2.3% in the control and ghrelin 0.5 and 50 groups, respectively; P &amp;lt; 0.05). The total cell number per blastocyst was significantly increased in the ghrelin 50 group compared with that of the control group (85 � 10.2 vs. 56 � 8.0, P &amp;lt; 0.05). The maximum total cell number in the ghrelin treatment groups of parthenogenetic embryos was higher than in the control group (82, 93, 102, 100 in the ghrelin 0.5, 5, 50, 500 groups, respectively, vs. 69; P &amp;lt; 0.05). We also found that more embryos were developed to the morula stage and fewer embryos died early at the 2- to 4-cell stage in the ghrelin treatment groups than in the control group (data not shown) in both Experiments 1 and 2. The results suggest that supplementation with ghrelin in the embryo culture medium could enhance the pre-implantation development of porcine IVF and parthenogenetic embryos. This study was funded by the Natural Scientific Foundation of Beijing (5030001).

Embryotropic effect of insulin-like growth factor (IGF)-I and its receptor on development of porcine preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer

Insulin-like growth factor (IGF)-I is a receptor-mediated autocrine/paracrine growth/survival factor for mammalian embryo development. The present study investigated the temporal expression and regulation of porcine IGF-I receptor (IGF-IR) mRNA and the role of IGF-I on development of porcine in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. As assessed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), the level of IGF-IR mRNA expression was high in unfertilized oocytes, 2-cell and 4-cell embryos and gradually decreased in 8-cell embryos, morulae, and blastocysts in both IVF and SCNT series. The IVF or SCNT embryos were cultured with 0, 1, 10, 50, or 100 ng/ml IGF-I for 168 hr. Supplementing with 50 ng/ml IGF-I increased blastocyst formation and the number of cells in inner cell masses (ICMs) in both IVF and SCNT embryos. In a second experiment, more blastocysts were obtained when IVF or SCNT embryos were cultured for the first 48 hr or for the entire 168 hr with 50 ng/ml IGF-I compared to culturing without IGF-I for 48 hr or with IGF-I for the last 120 hr or without IGF-I for the entire 168 hr. Treating IVF or SCNT embryos with 50 ng/ml IGF-I significantly up-regulated IGF-IR mRNA compared to untreated control embryos. In conclusion, the present study demonstrated that IGF-IR mRNA is expressed in porcine IVF and SCNT embryos, and that IGF-I improved the developmental competence of IVF and SCNT embryos through its specific receptors.