Porcine Preimplantation Embryo Development Research Articles

MCT1-mediated transport of valeric acid promotes porcine preimplantation embryo development by improving mitochondrial function and inhibiting the autophagic AMPK-ULK1 pathway

Oocytes and embryos are highly sensitive to environmental stress in vivo and in vitro. During in vitro culture, many stressful conditions can affect embryo quality and viability, leading to adverse clinical outcomes such as abortion and congenital abnormalities. In this study, we found that valeric acid (VA) increased the mitochondrial membrane potential and ATP content, decreased the level of reactive oxygen species that the mitochondria generate, and thus improved mitochondrial function during early embryonic development in pigs. VA decreased expression of the autophagy-related factors LC3B and BECLIN1. Interestingly, VA inhibited expression of autophagy-associated phosphorylation-adenosine monophosphate-activated protein kinase (p-AMPK), phosphorylation-UNC-51-like autophagy-activated kinase 1 (p-ULK1, Ser555), and ATG13, which reduced apoptosis. Short-chain fatty acids (SCFAs) can signal through G-protein-coupled receptors on the cell membrane or enter the cell directly through transporters. We further show that the monocarboxylate transporter 1 (MCT1) was necessary for the effects of VA on embryo quality, which provides a new molecular perspective of the pathway by which SCFAs affect embryos. Importantly, VA significantly inhibited the AMPK-ULK1 autophagic signaling pathway through MCT1, decreased apoptosis, increased expression of embryonic pluripotency genes, and improved embryo quality.

KLF4 facilitates chromatin accessibility remodeling in porcine early embryos.

Chromatin accessibility remodeling driven by pioneer factors is critical for the development of early embryos. Current studies have illustrated several pioneer factors as being important for agricultural animals, but what are the pioneer factors and how the pioneer factors remodel the chromatin accessibility in porcine early embryos is not clear. By employing low-input DNase-seq (liDNase-seq), we profiled the landscapes of chromatin accessibility in porcine early embryos and uncovered a unique chromatin accessibility reprogramming pattern during porcine preimplantation development. Our data revealed that KLF4 played critical roles in remodeling chromatin accessibility in porcine early embryos. Knocking down of KLF4 led to the reduction of chromatin accessibility in early embryos, whereas KLF4 overexpression promoted the chromatin openness in porcine blastocysts. Furthermore, KLF4 deficiency resulted in mitochondrial dysfunction and developmental failure of porcine embryos. In addition, we found that overexpression of KLF4 in blastocysts promoted lipid droplet accumulation, whereas knockdown of KLF4 disrupted this process. Taken together, our study revealed the chromatin accessibility dynamics and identified KLF4 as a key regulator in chromatin accessibility and cellular metabolism during porcine preimplantation 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.

ROMO1 is required for mitochondrial metabolism during preimplantation embryo development in pigs

BackgroundReactive oxygen species (ROS) modulator 1 (ROMO1) is a mitochondrial membrane protein that is essential for the regulation of mitochondrial ROS production and redox sensing. ROMO1 regulates ROS generation within cells and is involved in cellular processes, such as cell proliferation, senescence, and death. Our purpose is to investigates the impact of ROMO1 on the mitochondria during porcine embryogenesis.ResultsWe found that high expression of ROMO1 was associated with porcine preimplantation embryo development, indicating that ROMO1 may contribute to the progression of embryogenesis. Knockdown of ROMO1 disrupted porcine embryo development and blastocyst quality, thereby inducing ROS production and decreasing mitochondrial membrane potential. Knockdown of ROMO1 induced mitochondrial dysfunction by disrupting the balance of OPA1 isoforms to release cytochrome c, reduce ATP, and induce apoptosis. Meanwhile, ROMO1 overexpression showed similar effects as ROMO1 KD on the embryos. Overexpression of ROMO1 rescued the ROMO1 KD-induced defects in embryo development, mitochondrial fragmentation, and apoptosis.ConclusionsROMO1 plays a critical role in embryo development by regulating mitochondrial morphology, function, and apoptosis in pigs.

Schisanhenol improves early porcine embryo development by regulating the phosphorylation level of MAPK

Schisanhenol (SAL), a biphenyl cyclooctene-type lignin compound which can be extracted and isolated from many plants of the Schisandra family, exhibits a variety of biological activities including anti chronic cough, night sweating, thirst, diabetes, and obesity. However, its effects on the female reproductive system are unclear. Previous studies showed that SAL had potential antioxidant activity in heart, liver, and brain. Therefore, we hypothesized that SAL could improve porcine early development by reducing oxidative stress. The purpose of this study was to investigate the effects of SAL on preimplantation porcine embryos and the potential mechanisms. In this study, we analyzed the effects of SAL on embryo quality, reactive oxygen species (ROS) accumulation, mitochondrial function, cell proliferation and apoptosis, and the activation of MAPK pathway. The results showed that 10 μM SAL significantly increased the blastocyst formation rate, proliferation ability, and mitochondrial activity while reducing ROS accumulation and apoptosis level. During this process, the phosphorylation levels of ERK1/2, JNK1/2/3, and p38 were decreased. In summary, 10 μM SAL improves porcine preimplantation embryo development by reducing ROS accumulation.

Intrauterine Infusion of TGF-β1 Prior to Insemination, Alike Seminal Plasma, Influences Endometrial Cytokine Responses but Does Not Impact the Timing of the Progression of Pre-Implantation Pig Embryo Development.

Simple SummaryAlthough endometrial immune regulation in pigs during the early preimplantation period is poorly documented, particularly under conditions of embryo transfer (ET), it is recognized that seminal plasma (SP) induces molecular changes in the reproductive tract, influencing numerous reproductive functions. A principal constituent of SP is the cytokine transforming growth factor β1 (TGF-β1), which has an important role in embryo development, pregnancy establishment, and progression. The present study evaluated different intrauterine infusion treatments at estrus (40 mL of SP, porcine TGF-β1 in an extender, or an extender alone (control)) by mimicking an ET scenario in so-called “donor” (inseminated) and “recipient” (uninseminated) sows. We investigated the effects of these treatments on day 6 embryo development (“donors”) and endometrial explants’ cytokine production (“donors” and “recipients”). SP infusion positively influenced embryo development compared with TGF-β1 or extender infusions. Infusion treatments differentially affected endometrial cytokine production, with the effects being stronger in “donors” than in “recipients.” Increased knowledge of the effects of SP or some of its active components on the female immune system may help to develop strategies for increasing the reproductive efficiency for the benefit of pig ET.Seminal plasma (SP) in the female genital tract induces changes that affect multiple reproductive processes. One of the active components in SP is the transforming growth factor β1 (TGF-β1), which has major roles in embryo development and pregnancy. Embryo transfer (ET) technology is welcomed by the pig industry provided that embryo quality at embryo collection as well as the fertility and prolificacy of the recipients after the ET is increased. This study evaluated different intrauterine infusion treatments at estrus (40 mL of SP, TGF-β1 cytokine in the extender, or the extender alone (control)) by mimicking an ET scenario in so-called “donor” (inseminated) and “recipient” (uninseminated) sows. On day 6 (day 0—onset of estrus), all “donors” were laparotomized to determine their pregnancy status (presence and developmental stage of the embryos). In addition, endometrial explants were collected from pregnant “donors” and cyclic “recipients,” incubated for 24 h, and analyzed for cytokine production. SP infusions (unlike TGF-β1 infusions) positively influenced the developmental stage of day 6 embryos. Infusion treatments differentially influenced the endometrial cytokine production, mainly in donors. We concluded that SP infusions prior to AI not only impacted the porcine preimplantation embryo development but also influenced the endometrial cytokine production six days after treatment, both in donors and recipients.

S-nitrosoglutathione reductase maintains mitochondrial homeostasis by promoting clearance of damaged mitochondria in porcine preimplantation embryos.

ObjectivesS‐nitrosoglutathione reductase (GSNOR), a protein denitrosylase, protects the mitochondria from mitochondrial nitrosative stress. Mammalian preimplantation embryos are mitochondria‐rich, but the effects of GSNOR on mitochondrial function in preimplantation embryos are not well‐studied. In the present study, we investigate whether GSNOR plays a role in mitochondrial regulation during porcine preimplantation embryo development.Materials and MethodsGSNOR dsRNA was employed to knock down the expression of GSNOR, and Nω‐Nitro‐L‐arginine methyl ester hydrochloride (L‐NAME), a pan‐NOS inhibitor, was used to prevent protein S‐nitrosylation. Mitochondrial amount and function in embryo development were assessed by performing immunofluorescence staining, Western blot, fluorescent probe and real‐time reverse transcription PCR.ResultsGSNOR knock‐down significantly impaired blastocyst formation and quality and markedly induced the increase in protein S‐nitrosylation. Notably, GSNOR knock‐down‐induced overproduction of S‐nitrosylation caused mitochondrial dysfunction, including mitochondrial membrane potential depolarization, mitochondria‐derived reactive oxygen species (ROS) increase and ATP deficiency. Interestingly, GSNOR knock‐down‐induced total mitochondrial amount increase, but the ratio of active mitochondria reduction, suggesting that the damaged mitochondria were accumulated and mitochondrial clearance was inhibited. In addition, damaged mitochondria produced more ROS, and caused DNA damage and apoptosis. Importantly, supplementation with L‐NAME reverses the increase in S‐nitrosylation, accumulation of damaged mitochondria, and oxidative stress‐induced cell death. Interestingly, autophagy was downregulated after GSNOR knock‐down, but reversed by L‐NAME treatment. Thus, GSNOR maintains mitochondrial homeostasis by promoting autophagy and the clearing of damaged mitochondria in porcine preimplantation embryos.

Removal of hypotaurine from porcine embryo culture medium does not impair development of in vitro-fertilized or somatic cell nuclear transfer-derived embryos at low oxygen tension.

Hypotaurine (HT) is a routine component of porcine embryo culture medium, functioning as an antioxidant, but its requirement may be diminished as most embryo culture systems now use 5% O2 instead of atmospheric (20%) O2. Our objective was to determine the effects of removing HT from the culture medium on porcine preimplantation embryo development. Embryos cultured in 20% O2 without HT had decreased blastocyst development compared to culture with HT or in 5% O2 with or without HT. Notably, differences in blastocyst development or total cell number were not detected between embryos cultured in 5% O2 with or without HT. After culture in 5% O2 without HT and embryo transfer, healthy fetuses were retrieved from two pregnancies on Day 42, confirming in vivo developmental competence. Transcript abundance of proapoptotic markers was decreased in embryos cultured without HT regardless of oxygen tension; however, assays for apoptosis did not demonstrate differences between groups. Additionally, no differences were observed in the development or apoptosis of somatic cell nuclear transfer‐derived embryos cultured in 5% O2 with or without HT. With decreased utility in 5% O2, removing HT from porcine embryo culture medium would also have economic advantages because it is undoubtedly the most expensive component.

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.

A novel chemically defined serum- and feeder-free medium for undifferentiated growth of porcine pluripotent stem cells.

Development and improvement of in vitro culture system supporting self-renewal and unlimited proliferation of porcine pluripotent stem cells (pPSCs) is an indispensable process for the naïve pPSCs establishment. In this study, we modified the previous culture system and attempted to develop a novel chemically defined medium (KOFL) for the establishment of pPSCs. It has been cultured >45 passages with flat colony morphology and normal karyotypes in in vitro environment. These cells exhibited alkaline phosphatase activity and expressed pluripotency markers such as OCT4, SOX2, and NANOG, and also possessed differentiation abilities both in vitro and in vivo, proving by the formation of embryonic bodies and teratomas into three germ layers. Then the cells transfected with a green fluorescent protein (GFP) and the GFP positive cells contribute to the porcine preimplantation embryo development. In addition, these cells maintained long duration under feeder-free condition. In conclusion, our results demonstrated that the pPSCs could be derived from preimplantation porcine embryos in serum-free medium and cultured under the feeder-free condition, providing an effective reference for further optimization of the pPSCs culture system.

The necessity of TEAD4 for early development and gene expression involved in differentiation in porcine embryos

TEA domain family transcription factor 4 (Tead4) is known to be important for the trophectoderm (TE) segregation in murine embryos. However, the role of TEAD4 in early development of porcine embryos is still unknown. We examined TEAD4 expression patterns and attempted to determine the functions of TEAD4 during porcine preimplantation development using RNA interference. TEAD4 mRNA was upregulated from the 2–4-cell to 8–16-cell stages and then decreased to the blastocyst stage. Nuclear localization of TEAD4 protein was detected at the 16-cell stage, as well as at subsequent developmental stages. In porcine embryos injected with TEAD4 siRNA, transformation from morula to blastocyst was inhibited. Although TEAD4 downregulation did not affect the expression levels of POU class 5 homeobox 1 (OCT-4), transcription of SRY-related HMG-box gene 2 (SOX2) was detected at high level in TEAD4-downregulated embryos. It is possible that TEAD4 contributes to blastocyst formation in porcine embryos through downregulation of SOX2 expression. Collectively, our results indicate that TEAD4 is an important factor for the preimplantation development of porcine embryos.

Effects of High Dose Lysophosphatidic Acid Supplement during IVC on Preimplantation Development of Porcine Embryos

Lysophosphatidic acid (LPA) is an important signaling molecule. Here, the effect and mechanism of LPA on the preimplantation development of porcine embryos during in vitro culture (IVC) was examined. Porcine embryos were cultured in porcine zygote medium (PZM-3) supplemented with 30 μM LPA during different days. There was a significantly higher cleavage rate in Day 1-7 and significantly higher total cell number of blastocysts in Day 1-3 and Day 4-7. It was also found that messenger RNA (mRNA) expression level of PCNA, BCL2 and BAX in blastocysts obtained from D1-7 group were significantly higher and BCL2/BAX mRNA ratio in D1-3 group was significantly lower than control group but Day 4-7 and Day 1-7 groups were comparable with control group. Treatment with 20 μM PLC inhibitor significantly decreased the embryo cleavage rate and blastocyst formation rate. Moreover, LPA as an activator of PLCs, enhanced the 30 μM LPA + 20 μM U73122 group embryo cleavage rate which similar with control group. In conclusion, the results suggest that treatment with LPA during IVC improves the porcine early embryo cleavage by activation of PLC signaling pathway and regulate the mRNA expression that contribute to total cell number of blastocysts during blastocyst formation.

56 Sirtinol Treatment Influences Preimplatation Development of Porcine Embryos via Regulation of Autophagy and Apoptosis

Sirtuin (Sirt), nicotinamide adenine dinucleotide dependent class III histone deacetylase, plays an important role in cellular processes including DNA repair, apoptosis, cell cycle, aging, and determining lifespan. In previous studies, levels of Sirt1 to Sirt3 mRNA were detected in porcine embryos for the first time and levels are lower in blastocysts relative to matured oocytes. In addition, mitochondrial dysfunction and hyperglycemia increases LC3 protein levels and apoptosis in porcine parthenotic embryos and modulation of autophagy also influences apoptosis, mitochondrial contents, abnormal autophagosome formation, and maternal mRNA degradation. However, Sirt-mediated mechanisms have not been examined in in vitro-produced embryos of pig. Therefore, we investigated the relationship between Sirt inhibition and autophagy/mitophagy in porcine pre-implantation embryos. After IVF, embryos were cultured in NCSU-23 media in the presence and absence of 100 μM sirtinol (Sirt inhibitor) until the expended blastocyst stage. As a result, there were no significant differences between the rate of cleavage in control (69.22 ± 1.29) and treated groups (72.66 ± 1.08). However, embryos treated with sirtinol had significantly decreased developmental rates to the morula as well as blastocyst stages. Especially, expanded blastocysts (9.90 ± 1.56 v. 2.92 ± 0.94%) were barely observed in sirtinol-treated group. In the levels of Sirt transcripts, Sirt2 mRNA was significantly lower in sirtinol-treated blastocysts compared with controls (P &lt; 0.05), but the levels of Sirt1 and Sirt3 mRNA were similar in both groups. In addition, we found that sirtinol treatment induced autophagy by increasing the expressions of LC3, Beclin1, and ATG5 in blastocysts. Furthermore, we observed that the abundance of mitochondria stained with mitotracker was lower in sirtinol-treated blastocysts than that of control. Finally, we found that sirtinol treatment resulted in a higher total apoptotic index (6.88 ± 0.84) compared with the control (12.84 ± 0.99) in blastocysts. In summary, our findings in this study demonstrated that Sirt inhibition by sirtinol led to lower levels of Sirt2 transcript in blastocysts, reduced developmental capability and embryo quality with regulation of ATGs, LC3 proteins, apoptosis-related genes, and mitochondrial abundance. Therefore, these results suggest that Sirt2 may play an important role in the pre-implantation development of porcine embryos and their quality through the regulation of autophagy/mitophagy and apoptosis pathways. This research was supported by a Grant from the Bio &amp; Medical Technology Development Program (2015M3A9C7030091) of the National Research Foundation (NRF) funded by the Korean government.

78 Mito-TEMPO, a Scavenger for Mitochondria-Derived Reactive Oxygen Species, Enhances Porcine Pre-Implantation Embryo Development

The production of reactive oxygen species (ROS) from mitochondria contributes to redox signalling, mitochondrial functions, and apoptosis. However, the specific effects of mitochondria target superoxide (O2 •–) on porcine embryo development remain unclear. The objective of present study was to examine the differences of mitochondrial functions and dynamics in 2 subpopulations of porcine zygotes (G1 and G2), and to investigate the effects of Mito-TEMPO on porcine embryo development. Porcine embryos were visually classified in 2 groups [Grade (G)1: over 90%, and G2: below 90%] according to the lipid distribution at the zygote stage. The blastocyst development rate was greater in G1 than in G2 embryos (G1: 26.5 ± 5.9% v. G2: 16.2 ± 7.9%; P &lt; 0.05). To evaluate blastocyst quality, we performed a 4′,6-diamidino-2-phenylindole (DAPI)-TUNEL assay. The proportion of TUNEL-positive cells was higher (P &lt; 0.05) in G2 than G1 embryos. We measured superoxide production by MitoSOX staining as mitochondrial superoxide specific fluorescence dye by iRiSTM Digital Cell Image System (Logos Biosystems Inc., Gyeonggi-do, South Korea). Red fluorescence intensity of superoxide in G2 embryos significantly increased (P &lt; 0.05) compared with that in G1. We investigated changes in mitochondrial functions using a Mitotracker JC-1 mitochondrial membrane potential assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and ATP determination kit, respectively. Mitochondria membrane potential and ATP production were lower (P &lt; 0.05) in G2 embryos than in G1 embryos. To confirm the protein levels of mitochondria fission protein DRP1, we performed Western blot analysis (per 40 embryos). Phosphorylation DRP1-Ser616 was increased (P &lt; 0.05) in G1 embryos at cleavage stage compared with that in zygote, but not significantly different in G2 embryos. Thus, G2 embryos showed low development rate until blastocyst via mitochondrial dysfunction, increase in fission protein expression and mitochondrial aggregation according to the elevation of mito-ROS. Subsequently, the effect of the adding superoxide scavenger Mito-TEMPO was investigated in G2 embryos. Blastocyst formation (G2+MitoTempo: 28.8 ± 4.0% v. G2: 19.1 ± 5.1%; P &lt; 0.05) and mitochondrial aggregation were recovered by mito-ROS reduction mediated by Mito-TEMPO. Our observations demonstrated that regulation of superoxide in mitochondria is important in pre-implantation development of porcine embryos. This work was supported by grants from the Next-Generation BioGreen 21 Program (PJ01117604) and the Bio-industry Technology Development Program (316037-04-2-HD020) through the Rural Development Administration and the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.

MLL2 is essential for porcine embryo development in vitro.

Several germ cell-specific transcription factors essential for ovarian formation and folliculogenesis have been identified and studied. However, their function during early embryo development has been poorly explored. In this study, we investigated the role of mixed-lineage leukemia protein 2 (MLL2) in the development of porcine preimplantation embryos. To explore the function of MLL2 in porcine embryo development, expression and localization of MLL2 were assessed by qRT-PCR and immunofluorescence assays. Results showed that expression of MLL2 was significantly reduced after the four-cell stage. However, immunofluorescence results showed that MLL2 only localized in the nucleus of blastocysts, revealing a potential role of MLL2 in regulating the gene expression in the blastocyst stage. Knockdown of Mll2 by double-stranded RNA (dsRNA) caused a reduction in MLL2 signal in porcine blastocyst cells and in blastocyst formation. No significant differences in the cleavage and morula stages were observed. The mechanism of MLL2 regulation in blastocysts was assessed by assaying the trimethylation of histone 3 at lysine 4 (H3K4m3). MLL2 knockdown significantly reduced H3K4m3 in the nucleus and further reduced expression of Sox2 and Magoh genes. In conclusion, MLL2 is essential for porcine embryo development by the regulation of methylation of H3K4 in vitro.

Antioxidant Effect of Edaravone on the Development of Preimplantation Porcine Embryos against Hydrogen Peroxide-Induced Oxidative Stress

Antioxidant Effect of Edaravone on the Development of Preimplantation Porcine Embryos against Hydrogen Peroxide-Induced Oxidative Stress

Poly(ADP-ribosyl)ation is involved in pro-survival autophagy in porcine blastocysts.

Poly(ADP-ribosyl)ation (PARylation) prevents apoptosis through its involvement in pro-survival autophagy in cultured cells; whether or not the same is true for pre-implantation embryos has not yet been documented. In this study, we investigated the participation of PARylation and autophagy in in vitro porcine pre-implantation embryo development. The transcript levels of autophagy-related genes and poly(ADP-ribose) polymerase 1 (PARP1), an enzyme required for PARylation, were transiently up-regulated by fertilization, decreased at the late 1-cell stage, and maintained until the blastocyst stage. LC3, a marker of autophagosomes, and poly(ADP-ribose) (PAR) polymer were present in all stages of pre-implantation development. Exposure of embryos to 3-methyladenine, an autophagy inhibitor, or 3-aminobenzamide, a PARP inhibitor, suppressed the development of blastocysts. Pharmacological inhibition of PARylation further suppressed pro-survival autophagy by decreasing the expression of autophagy-related genes (ATG5, BECLIN1, and LC3) and decreasing LC3 protein abundance while increasing the rate of apoptosis in blastocysts. Deficiency in autophagy also induced abnormal accumulation of SQSTM1/p62 aggregates in porcine blastocysts. Collectively, these data suggest that PARylation is involved in selective autophagic degradation of ubiquitinated proteins, functioning in a pro-survival role, in porcine in vitro-produced embryos. These pro-survival regulatory mechanisms may be important for the control of embryo quality.

Ginsenoside Rg1 Improves In vitro-produced Embryo Quality by Increasing Glucose Uptake in Porcine Blastocysts.

Ginsenoside Rg1 is a natural compound with various efficacies and functions. It has beneficial effects on aging, diabetes, and immunity, as well as antioxidant and proliferative functions. However, its effect on porcine embryo development remains unknown. We investigated the effect of ginsenoside Rg1 on the in vitro development of preimplantation porcine embryos after parthenogenetic activation in high-oxygen conditions. Ginsenoside treatment did not affect cleavage or blastocyst formation rates, but did increase the total cell number and reduced the rate of apoptosis. In addition, it had no effect on the expression of four apoptosis-related genes (Bcl-2 homologous antagonist/killer, B-cell lymphoma-extra large, Caspase 3, and tumor protein p53) or two metabolism-related genes (mechanistic target of rapamycin, carnitine palmitoyltransferase 1B), but increased the expression of Glucose transporter 1 (GLUT1), indicating that it may increase glucose uptake. In summary, treatment with the appropriate concentration of ginsenoside Rg1 (20 μg/mL) can increase glucose uptake, thereby improving the quality of embryos grown in high-oxygen conditions.

Stem Cell–Derived Bioactive Materials Accelerate Development of PorcineIn Vitro–Fertilized Embryos

Stem cells show the capability to proliferate in an undifferentiated state with long-term self-renewal, which gives the cells advantages for use as bioactive material (BM) for embryo culture in vitro. The objective of this experiment was to investigate the effect of two BMs-human adipose tissue-derived mesenchymal stem cell BM (hAT-MSC-BM) and human embryonic stem cell-derived BM (hESC-BM)-on porcine embryo development compared to commonly used bovine serum albumin (BSA) or serum treatment groups. In vitro-fertilized (IVF) embryos were cultured in PZM-5 with 4 mg/mL BSA until day 4 and equally divided into four groups. Starting from day 4 (until day 6), each group was treated with the following protein additives: 4 mg/mL BSA (control), 10% fetal bovine serum (FBS), 10% hAT-MSC-BM, or 10% hESC-BM. Our results show FBS- and two other BM-treated groups showed significant increases in blastocyst formation rate, hatching rate, and total cell number compared with the control group (p<0.05). The hAT-MSC-BM and hESC-BM treatment groups presented better-quality embryo development, especially from the middle expanding stage to hatching. In particular, the hAT-MSC-BM-treated group showed the highest developmental potential of all groups and formed the most expanding-stage blastocysts. The relative expression of reprogramming-related transcription factor (POU5F1, SOX2, DPPA5, and CDH1), antioxidant (PRDX5), and apoptosis (BCL2L1 and BIRC5) genes also increased in two types of BMs compared to the control. In addition, we investigated the protein synthesis of the tight junction- and gap junction-related genes, connexin 43 and zonula occludens-1 (ZO-1); these increased more than in the control. These results demonstrate that stem cell-derived BMs accelerate porcine preimplantation embryo development and that the BMs would be helpful in the development of preimplantation embryos.

Effects of WT1 down-regulation on oocyte maturation and preimplantation embryo development in pigs.

The Wilms' tumour 1 (WT1) gene originally identified as a tumour suppressor associated with WTs encodes a zinc finger-containing transcription factor that is expressed in multiple tissues and is an important regulator of cellular and organ growth, proliferation, development, migration and survival. However, there is a deficiency of data regarding the expression and function of WT1 during oocyte maturation and preimplantation embryonic development. Herein, we sought to define the expression characteristics and functions of WT1 during oocyte maturation and preimplantation embryonic development in pigs. We show that WT1 is expressed in porcine oocytes and at all preimplantation stages in embryos generated by ICSI. We then evaluated the effects of down-regulating WT1 expression at germinal vesicle and early ICSI stages using a recombinant plasmid (pGLV3-WT1-shRNA). Down-regulation of WT1 did not affect oocyte maturation but significantly decreased preimplantation embryonic development and increased apoptosis in blastocysts. These results indicate that WT1 plays important roles in the development of porcine preimplantation embryos.