NANOG expression in parthenogenetic porcine blastocysts is required for intact lineage specification and pluripotency.

OBJECTIVE: Nanog homeobox (NANOG) is a core transcription factor that contributes to pluripotency along with octamer binding transcription factor-4 (OCT4) and sex determining region-Y box-2 (SOX2). It is an epiblast lineage marker in mammalian pre-implantation embryos and exhibits a species-specific expression pattern. Therefore, it is important to understand the lineage of NANOG, the trophectoderm, and the primitive endoderm in the pig embryo. METHODS: A loss- and gain-of-function analysis was done to determine the role of NANOG in lineage specification in parthenogenetic porcine blastocysts. We analyzed the relationship between NANOG and pluripotent core transcription factors and other lineage makers. RESULTS: In NANOG-null late blastocysts, OCT4-, SOX2-, and SOX17-positive cells were decreased, whereas GATA binding protein 6 (GATA6)-positive cells were increased. Quantitative real-time polymerase chain reaction revealed that the expression of SOX2 was decreased in NANOG-null blastocysts, whereas that of primitive endoderm makers, except SOX17, was increased. In NANOG-overexpressing blastocysts, caudal type homeobox 2 (CDX2-), SOX17-, and GATA6-positive cells were decreased. The results indicated that the expression of primitive endoderm markers and trophectoderm-related genes was decreased. CONCLUSION: Taken together, the results demonstrate that NANOG is involved in the epiblast and primitive endoderm differentiation and is essential for maintaining pluripotency within the epiblast.

Comparison data of transcriptomes from blastocyst seeding samples and cultured cell lines from pigs.

Fertilized embryos develop and move freely in the reproductive tract until implantation. Subsequently, the embryos continue to develop after attachment to the uterus. Because of the absence of the uterus, in vitro culturing of embryos is limited to a period of approximately a week. Hatched blastocysts were seeded on feeder cells to extend the culture period. We cultured the colonies formed from the blastocysts for an additional 14 days. From the colonies, four types of cells were established, and each type was isolated to extract RNA. RNA sequencing was conducted using NovaSeq6000. Sequencing reads were aligned to genes and transcripts. Raw data from our previous study were used to compare these samples with the cultured cell lines. We analyzed differentially expressed genes and Gene Ontology terms between new samples and cultured cell lines. Our data can provide essential information for extending the period of embryo culture in vitro.

The number of primitive endoderm cells in the inner cell mass is regulated by platelet-derived growth factor signaling in porcine preimplantation embryos.

OBJECTIVE: Discovering the mechanism of cell specification is important to manipulate cellular lineages. To obtain lineage-specific cell lines, the target lineage needs to be promoted, and counterpart lineages should be suppressed. Embryos in the early blastocyst stage possess two different cell populations, the inner cell mass (ICM) and trophectoderm. Then, cells in the ICM segregate into epiblasts (Epi) and primitive endoderm (PrE). PrE cells in embryos show specific expression of platelet-derived growth factor (PDGF) and its receptor, PDGF receptor A (PDGFRA). In this study, we suppressed PDGF signaling using two methods (CRISPR/Cas9 injection and inhibitor treatment) to provide insight into the segregation of embryonic lineages. METHODS: CRISPR/Cas9 RNAs were injected into parthenogenetically activated and in vitro fertilized embryos. The PDGF receptor inhibitor AG1296 was treated at 0, 5, 10, and 20 µM concentration. The developmental competence of the embryos and the number of cells expressing marker proteins (SOX2 for ICM and SOX17 for PrE) were measured after the treatments. The expression levels of the marker genes with the inhibitor were examined during embryo development. RESULTS: Microinjection targeting the PDGF receptor (PDGFR) A reduced the number of SOX17-positive cell populations in a subset of day 7 blastocysts (n = 9/12). However, microinjection accompanied diminution of Epi cells in the blastocyst. The PDGF receptor inhibitor AG1296 (5 µM) suppressed SOX17-positive cells without reducing SOX2-positive cells in both parthenogenetic activated and in vitro fertilized embryos. Within the transcriptional target of PDGF signaling, the inhibitor significantly upregulated the Txnip gene in embryos. CONCLUSION: We identified that PDGF signaling is important to sustain the PrE population in porcine blastocysts. Additionally, treatment with inhibitors was a better method to suppress PrE cells than CRISPR/Cas9 microinjection of anti-PDGF receptor α gene, because microinjection suppressed number of Epi cells. The PDGF receptor might control the number of PrE cells by repressing the proapoptotic gene Txnip. Our results can help to isolate Epi-specific cell lines from blastocysts.

Characterization of multitype colonies originating from porcine blastocysts produced in vitro.

Many types of embryonic stem cells have been induced from pre-implantation blastocysts to study the specification of early lineages. Various cell lines have been established using chemicals, including excessive inhibitory molecules. Previous studies have also aimed to purify cell populations representing a single embryonic lineage from a protocol. In this study, we used a novel culture condition to induce cells from blastocyst seeding and analyzed their characteristics. Next, signaling inhibitors were introduced during the cell culture period. Furthermore, we investigated the cell types using RNA sequencing. Each type of cell population showed a distinct morphology and reactivity with alkaline phosphatase. Marker proteins enabled each cell type to be distinguished by immunocytochemistry, and genes such as Sox17, Gata4, Gata6, T, and Cdx2 showed applicability for the discrimination of cell types. Signaling inhibitors suppressed the production of some cell types, and gene expression and marker protein patterns were collapsed. RNA-sequencing suggested cell-type-specific marker genes and the correlation among samples. In conclusion, four types of cells could be induced from porcine embryos using a single protocol, and they could be isolated manually. Our data will help promote the study of lineage segregation based on embryonic cells.

Changes in OCT4 expression play a crucial role in the lineage specification and proliferation of preimplantation porcine blastocysts.

OBJECTIVES: Curiosity about the role of OCT4, a core transcription factor that maintains inner cell mass (ICM) formation during preimplantation embryogenesis and the pluripotent state in embryonic development, has long been an issue. OCT4 has a species-specific expression pattern in mammalian preimplantation embryogenesis and is known to play an essential role in ICM formation. However, there is a need to study new roles for OCT4-related pluripotency networks and second-cell fate decisions. MATERIALS AND METHODS: To determine the functions of OCT4 in lineage specification and embryo proliferation, loss- and gain-of-function studies were performed on porcine parthenotes using microinjection. Then, we performed immunocytochemistry and quantitative real-time polymerase chain reaction (PCR) to examine the association of OCT4 with other lineage markers and its effect on downstream genes. RESULTS: In OCT4-targeted late blastocysts, SOX2, NANOG, and SOX17 positive cells were decreased, and the total cell number of blastocysts was also decreased. According to real-time PCR analysis, NANOG, SOX17, and CDK4 were decreased in OCT4-targeted blastocysts, but trophoblast-related genes were increased. In OCT4-overexpressing blastocysts, SOX2 and NANOG positive cells increased, while SOX17 positive cells decreased, and while total cell number of blastocysts increased. As a result of real-time PCR analysis, the expression of SOX2, NANOG, and CDK4 was increased, but the expression of SOX17 was decreased. CONCLUSION: Taken together, our results demonstrated that OCT4 leads pluripotency in porcine blastocysts and also plays an important role in ICM formation, secondary cell fate decision, and cell proliferation.

Species-Specific Enhancer Activity of OCT4 in Porcine Pluripotency: The Porcine OCT4 Reporter System Could Monitor Pluripotency in Porcine Embryo Development and Embryonic Stem Cells.

The present study examined the activity and function of the pig OCT4 enhancer in the porcine early embryonic development stage and porcine authentic embryonic stem cells. OCT4 is known as a pluripotent regulator, and its upstream regulatory region-based dual-fluorescence protein reporter system controlled by distal and proximal enhancers is broadly used in studies examining the states and mechanism of pluripotency. We analyzed how this reporter system functions during early embryo development and in stem cells using a previously established porcine-specific reporter system. We demonstrated that the porcine OCT4 distal enhancer and proximal enhancer were activated with different expression patterns simultaneously as the expression of pluripotent marker genes changed during the development of in vitro pathenotes and the establishment of porcine embryonic stem cells (ESCs). This work demonstrates the applicability of the porcine OCT4 upstream region-derived dual-fluorescence reporter system, which may be applied to investigations of species-specific pluripotency in porcine-origin cells. These reporter systems may be useful tools for studies of porcine-specific pluripotency, early embryo development, and embryonic stem cells.

Linoleic acid reduces apoptosis via NF-κB during the in vitro development of induced parthenogenic porcine embryos.

Fatty acid has a various role in preimplantation embryo development. Especially, Linoleic acid, polyunsaturated fatty acid, has been reported to affect the apoptosis pathway via nuclear transcription factor-kappa B. But to date, the function of NF-κB has not been demonstrated in porcine preimplantation embryos. We demonstrated that linoleic acid had a positive effect on embryo development at a certain concentration(25 µM), but developmental failure was observed at higher concentration. Furthermore, the expression level of NF-κB increased, unlike that of IL-6, as the concentration of linoleic acid increased. Interestingly, the concentration of NF-κB was found to increase even at the concentration of linoleic acid at which embryo development decreased. We found that pro-apoptotic gene expression was downregulated in the linoleic acid-treated group. It was also found that MCL-1, an anti-apoptotic gene known to be unaffected by IL-6, was found to be increased at the mRNA level in the linoleic acid-treated group. As the concentration of NF-kB increased, the nuclear translocation of C-JUN gradually increased dependent on the linoleic acid concentration. It was confirmed that NF-κB is an important factor in porcine embryos by treated ammonium pyrrolidinedithiocarbamate (APDC 0.1 µM, an inhibitor of NF-κB) affected NF-κB protein expression, IL-6 expression, and blastocyst production. These data supported porcine embryos can use exogenous linoleic acid as a metabolic energy source via NF-κB.

Pig embryonic stem cell line with porcine-specific OCT4 upstream region based dual reporter system.

Here, we report pig embryonic stem cells (ESCs) originating from parthenogenetic blastocysts which were developed from 2-cell embryos micro-injected with porcine OCT4 reporter system cultured in previous reported chemically defined culture media. The ESCs with reporter system expressed pluripotency markers and fluorescent signals produced by OCT4 reporter system. Also, they were capable of forming teratomas following subcutaneous injection into nude mice. Since reporter system enables the non-destructive classification of the condition of live pluripotent stem cells, this reporter cell line could be a useful resource for research on species-specific pluripotency.

SOX2 plays a crucial role in cell proliferation and lineage segregation during porcine pre-implantation embryo development.

OBJECTIVES: Gene regulation in early embryos has been widely studied for a long time because lineage segregation gives rise to the formation of a pluripotent cell population, known as the inner cell mass (ICM), during pre-implantation embryo development. The extraordinarily longer pre-implantation embryo development in pigs leads to the distinct features of the pluripotency network compared with mice and humans. For these reasons, a comparative study using pre-implantation pig embryos would provide new insights into the mammalian pluripotency network and help to understand differences in the roles and networks of genes in pre-implantation embryos between species. MATERIALS AND METHODS: To analyse the functions of SOX2 in lineage segregation and cell proliferation, loss- and gain-of-function studies were conducted in pig embryos using an overexpression vector and the CRISPR/Cas9 system. Then, we analysed the morphological features and examined the effect on the expression of downstream genes through immunocytochemistry and quantitative real-time PCR. RESULTS: Our results showed that among the core pluripotent factors, only SOX2 was specifically expressed in the ICM. In SOX2-disrupted blastocysts, the expression of the ICM-related genes, but not OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real-time PCR analysis, pluripotency-related genes, excluding OCT4, and proliferation-related genes were decreased in SOX2-targeted blastocysts. In SOX2-overexpressing embryos, the total blastocyst cell number was greatly increased but the ICM/TE ratio decreased. CONCLUSIONS: Taken together, our results demonstrated that SOX2 is essential for ICM formation and cell proliferation in porcine early-stage embryogenesis.

Genetic Defects in DNAH2 Underlie Male Infertility With Multiple Morphological Abnormalities of the Sperm Flagella in Humans and Mice.

Asthenozoospermia accounts for over 80% of primary male infertility cases. Reduced sperm motility in asthenozoospermic patients are often accompanied by teratozoospermia, or defective sperm morphology, with varying severity. Multiple morphological abnormalities of the flagella (MMAF) is one of the most severe forms of asthenoteratozoospermia, characterized by heterogeneous flagellar abnormalities. Among various genetic factors known to cause MMAF, multiple variants in the DNAH2 gene are reported to underlie MMAF in humans. However, the pathogenicity by DNAH2 mutations remains largely unknown. In this study, we identified a novel recessive variant (NM_020877:c.12720G > T;p.W4240C) in DNAH2 by whole-exome sequencing, which fully co-segregated with the infertile male members in a consanguineous Pakistani family diagnosed with asthenozoospermia. 80-90% of the sperm from the patients are morphologically abnormal, and in silico analysis models reveal that the non-synonymous variant substitutes a residue in dynein heavy chain domain and destabilizes DNAH2. To better understand the pathogenicity of various DNAH2 variants underlying MMAF in general, we functionally characterized Dnah2-mutant mice generated by CRISPR/Cas9 genome editing. Dnah2-null males, but not females, are infertile. Dnah2-null sperm cells display absent, short, bent, coiled, and/or irregular flagella consistent with the MMAF phenotype. We found misexpression of centriolar proteins and delocalization of annulus proteins in Dnah2-null spermatids and sperm, suggesting dysregulated flagella development in spermiogenesis. Scanning and transmission electron microscopy analyses revealed that flagella ultrastructure is severely disorganized in Dnah2-null sperm. Absence of DNAH2 compromises the expression of other axonemal components such as DNAH1 and RSPH3. Our results demonstrate that DNAH2 is essential for multiple steps in sperm flagella formation and provide insights into molecular and cellular mechanisms of MMAF pathogenesis.

Combination of cell signaling molecules can facilitate MYOD1-mediated myogenic transdifferentiation of pig fibroblasts.

BACKGROUND: Myogenic transdifferentiation can be accomplished through ectopic MYOD1 expression, which is facilitated by various signaling pathways associated with myogenesis. In this study, we attempted to transdifferentiate pig embryonic fibroblasts (PEFs) myogenically into skeletal muscle through overexpression of the pig MYOD1 gene and modulation of the FGF, TGF-ß, WNT, and cAMP signaling pathways. RESULTS: The MYOD1 overexpression vector was constructed based on comparative sequence analysis, demonstrating that pig MYOD1 has evolutionarily conserved domains across various species. Although forced MYOD1 expression through these vectors triggered the expression of endogenous muscle markers, transdifferentiated muscle cells from fibroblasts were not observed. Therefore, various signaling molecules, including FGF2, SB431542, CHIR99021, and forskolin, along with MYOD1 overexpression were applied to enhance the myogenic reprogramming. The modified conditions led to the derivation of myotubes and activation of muscle markers in PEFs, as determined by qPCR and immunostaining. Notably, a sarcomere-like structure was observed, indicating that terminally differentiated skeletal muscle could be obtained from transdifferentiated cells. CONCLUSIONS: In summary, we established a protocol for reprogramming MYOD1-overexpressing PEFs into the mature skeletal muscle using signaling molecules. Our myogenic reprogramming can be used as a cell source for muscle disease models in regenerative medicine and the production of cultured meat in cellular agriculture.

MicroRNA expression data of pluripotent and somatic cells and identification of cell type-specific MicroRNAs in pigs.

Typical models of pluripotency, humans and mice, have been used to analyse the characteristics of pluripotent stem cells. However, these species exhibit molecular differences in many aspects. With similar physiology and genomics as humans, pigs are promising model for the research of pluripotency. The data of porcine pluripotent cells would be helpful in understanding the molecular network of human pluripotency. Pluripotent cells of humans and mice exhibit specific MicroRNA (miRNA) expression patterns to maintain the pluripotent state. Information about miRNA expression in pig pluripotent cells is not sufficient, so we analysed miRNAs in pluripotent (blastocysts and ES-like) and somatic cell samples (PEB and PFF). We screened cell-type specific miRNAs and identified their target genes. Functional annotation of the target genes was also conducted. Our data may facilitate miRNA-based induction and maintenance of the pluripotent state of porcine cells and provide support to fill the gap between the pluripotency networks of humans and mice.

Pluripotent pig embryonic stem cell lines originating from in vitro-fertilized and parthenogenetic embryos.

Here, we report in vivo developmental competent pig embryonic stem cells (ESCs) originating from in vitro-fertilized and parthenogenetic embryos cultured in chemically defined culture media. The pig ESC lines expressed pluripotency markers and were capable of forming teratomas following subcutaneous injection into nude mice. These cell lines would be a useful resource for comparative developmental biology and agricultural biotechnology.

Identification of the Lineage Markers and Inhibition of DAB2 in In Vitro Fertilized Porcine Embryos.

Specification of embryonic lineages is an important question in the field of early development. Numerous studies analyzed the expression patterns of the candidate transcripts and proteins in humans and mice and clearly determined the markers of each lineage. To overcome the limitations of human and mouse embryos, the expression of the marker transcripts in each cell has been investigated using in vivo embryos in pigs. In vitro produced embryos are more accessible, can be rapidly processed with low cost. Therefore, we analyzed the characteristics of lineage markers and the effects of the DAB2 gene (trophectoderm marker) in in vitro fertilized porcine embryos. We investigated the expression levels of the marker genes during embryonic stages and distribution of the marker proteins was assayed in day 7 blastocysts. Then, the shRNA vectors were injected into the fertilized embryos and the differences in the marker transcripts were analyzed. Marker transcripts showed diverse patterns of expression, and each embryonic lineage could be identified with localization of marker proteins. In DAB2-shRNA vectors injected embryos, HNF4A and PDGFRA were upregulated. DAB2 protein level was lower in shRNA-injected embryos without significant differences. Our results will contribute to understanding of the mechanisms of embryonic lineage specification in pigs.

Transcriptome profiling of pluripotent pig embryonic stem cells originating from uni- and biparental embryos.

OBJECTIVES: Pig pluripotent stem cells have tremendous potential because the pig is a valuable animal as both an agricultural resource and as a preclinical model of human therapy. To date, a lack of understanding of pig pluripotency has inhibited the derivation of embryonic stem cells (ESCs) and transgene-free induced pluripotent stem cells. Therefore, there has been no accessible or reliable transcriptome data for researching the genuine pig pluripotency network. Our previous study isolated authentic pig ESCs, which had teratoma-forming and direct differentiation ability, that were derived by activating the FGF2, ACTIVIN A, and WNT pathways. Here, we aimed to provide detailed information on transcriptome data of the newly derived pig ESCs and perform a comparative analysis with pig preimplantation embryo transcriptomes in a public database. DATA DESCRIPTION: The transcriptome data of ESCs derived from in vitro fertilized and parthenogenetic embryos were generated by HiSeq 2500. Then, differentially expressed genes (DEGs) from each sample were compared with fibroblasts, and gene expression profiling was carried out for comparative analysis. Our data, as the first transcriptome dataset for genuine pig pluripotent cells, could be a general reference for explaining the molecular mechanism of species-specific pluripotency and improving understanding of the embryo development of domestic animals.

Stearoyl-coenzyme A desaturase 1 is required for lipid droplet formation in pig embryo.

Lipid droplets (LD) provide a source of energy, and their importance during embryogenesis has been increasingly recognized. In particular, pig embryos have larger amounts of intercellular lipid bilayers than other mammalian species, suggesting that porcine embryos are more dependent on lipid metabolic pathways. The objective of the present study was to detect the effect of stearoyl-coenzyme A desaturase 1 (SCD1) on LD formation and to associate these effects with the mRNA abundance of LD formation-related genes (SREBP, ARF1, COPG2, PLD1 and ERK2) in in vitro-produced porcine embryos. To determine the effect of SCD1 on LD formation and related genes, we examined the effects of SCD1 inhibition using CAY10566 (an SCD1 inhibitor, 50 µM) on parthenogenetic embryos. SCD1 inhibition downregulated the mRNA levels of LD formation-related genes and embryo development. Our results revealed that SCD1 functions in the regulation of LD formation via phospholipid formation and embryo development. In addition, we treated parthenogenetic embryos with oleic acid (100 µM), which led to a significant increase in the blastocyst formation rate, LD size and number compared to controls. Remarkably, the adverse effects of the SCD1 inhibitor could be counteracted by oleic acid. These data suggest that porcine embryos can use exogenous oleic acid as a metabolic energy source.

Attempting to Convert Primed Porcine Embryonic Stem Cells into a Naive State Through the Overexpression of Reprogramming Factors.

Establishing pig embryonic stem cells (pESCs) remains a challenge due to differences in the genetic backgrounds of mouse, human, and pig. Therefore, pig-specific pluripotency markers and cellular signaling must be identified. In this study, doxycycline (DOX)-inducible vectors carrying Oct4, sex-determining region Y-box 2 (Sox2), Nanog, Kruppel-like family 4 (Klf4), or Myc, which are known reprogramming factors, were transduced into pESCs. And pluripotency genes were analyzed in one or two reprogramming factor-expressed pESCs. When cultured without DOX, pESCs were stably maintained in basic fibroblast growth factor-supplemented media. However, when treated with DOX, the cells lost their alkaline phosphatase (AP) activity and differentiated within 2 weeks. Subsequently, we investigated the expression of genes related to pluripotency in DOX-treated pESCs using quantitative reverse transcription-polymerase chain reaction (PCR). Expression levels of Oct4, E-cadherin, and Fut4 were significantly increased by Oct4 overexpression, and Oct4 and Fut4 were upregulated in the Sox2-infected group. When a combination of two reprogramming factors, including Oct4 or Sox2, was introduced, weak AP activity remained. In addition, several of the two reprogramming factor transduction groups could be maintained after subculturing with transgene activation. Although long-term culture failed, pESCs transduced with Oct4 and Nanog, Oct4 and Klf4, or Sox2 and Nanog combinations could be subcultured even under transgene activation conditions. Analysis of the cause of long-term culture failure by quantitative PCR confirmed that the expression of intermediate reprogramming markers was not maintained. Given these results, additional methods are needed to support the completion of each reprogramming phase to succeed in the conversion of the pluripotent state of pESCs. This study improves our understanding of pluripotent networks and can be used to aid in the establishment of bona fide pig pluripotent stem cells.

FGF2 Signaling Plays an Important Role in Maintaining Pluripotent State of Pig Embryonic Germ Cells.

Germ cells are alternative sources for deriving pluripotent stem cells. Because embryonic germ cells (EGCs) possess physiological and developmental features similar to those of embryonic stem cells, pig EGCs are considered a potential tool for generating transgenic animals for agricultural usage. Therefore, in this study, we attempted to establish and characterize pig EGCs from fetal gonads. EGC lines were derived from the genital ridges of porcine fetuses in media containing leukemia inhibitory factor (LIF), fibroblast growth factor 2 (FGF2), and stem cell factor. After establishment, these cells were cultured and stabilized in LIF- or FGF2-containing media. The cell lines were maintained under both conditions over an extended time period and spontaneously differentiated into the three germ layers in vitro. Interestingly, expression of pluripotency markers showed different patterns between cell lines cultured in LIF or FGF2. SSEA4 was only expressed in FGF2-treated pig EGCs (FGF2-pEGCs), not LIF-treated pig EGCs (LIF-pEGCs). Pluripotency genes were upregulated in FGF2-pEGCs, and germline markers were highly expressed, indicating that FGF2 supplements are more efficient in supporting the pluripotency of pEGCs. In conclusion, we verified that FGF2 signaling plays an important role in reprogramming and maintaining pEGCs from fetal gonads.

Multi-resistance strategy for viral diseases and in vitro short hairpin RNA verification method in pigs.

OBJECTIVE: Foot and mouth disease (FMD) and porcine reproductive and respiratory syndrome (PRRS) are major diseases that interrupt porcine production. Because they are viral diseases, vaccinations are of only limited effectiveness in preventing outbreaks. To establish an alternative multi-resistant strategy against FMD virus (FMDV) and PRRS virus (PRRSV), the present study introduced two genetic modification techniques to porcine cells. METHODS: First, cluster of differentiation 163 (CD163), the PRRSV viral receptor, was edited with the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 technique. The CD163 gene sequences of edited cells and control cells differed. Second, short hairpin RNA (shRNAs) were integrated into the cells. The shRNAs, targeting the 3D gene of FMDV and the open reading frame 7 (ORF7) gene of PRRSV, were transferred into fibroblasts. We also developed an in vitro shRNA verification method with a target gene expression vector. RESULTS: shRNA activity was confirmed in vitro with vectors that expressed the 3D and ORF7 genes in the cells. Cells containing shRNAs showed lower transcript levels than cells with only the expression vectors. The shRNAs were integrated into CD163-edited cells to combine the two techniques, and the viral genes were suppressed in these cells. CONCLUSION: We established a multi-resistant strategy against viral diseases and an in vitro shRNA verification method.

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.