Metschnikowia ahupensis f.a., sp. nov., a new yeast species isolated from the gut of the wood-feeding termite Nasutitermes sp.

The gut of xylophagous insects such as termites harbours various symbiotic micro-organisms, including many yeast species. In a taxonomic study of gut-associated yeasts, two strains (ATS2.16 and ATS2.18) were isolated from the gut of the wood-feeding termite Nasutitermes sp. in Maharashtra, India. Morphological and physiological characteristics and sequence analyses of the ITS and D1/D2 region of the large subunit rRNA gene revealed that these two strains represent a novel asexual ascomycetous yeast species in the genus Metschnikowia. The species differs from some of its close affiliates in the genus in its inability to utilize ethanol and succinate as the sole carbon source and growth in high sugar concentrations (up to 50 % glucose). In contrast to most members of Metschnikowia, the formation of ascospores was not observed on various sporulation media. Moreover, whole-genome sequencing was used to further confirm the novelty of this species. When compared with other large-spored Metschnikowia species, average nucleotide identity values of 79-80 % and digital DNA-DNA hybridization values of 16-17 % were obtained. The name Metschnikowia ahupensis f.a., sp. nov. is proposed to accommodate this novel yeast species, with ATS2.16 as the holotype and strains NFCCI 4949, MTCC 13085 and PYCC 9152 as isotypes. The MycoBank number is MB 844210.

The yeast community of Conotelus sp. (Coleoptera: Nitidulidae) in Brazilian passionfruit flowers (Passiflora edulis) and description of Metschnikowia amazonensis sp. nov., a large-spored clade yeast.

Species of the nitidulid beetle Conotelus found in flowers of Convolvulaceae and other plants across the New World and in Hawaii consistently harbour a yeast community dominated by one or more large-spored Metschnikowia species. We investigated the yeasts found in beetles and flowers of cultivated passionfruit in Rondônia state, in the Amazon biome of Brazil, where a Conotelus species damages the flowers and hinders fruit production. A sample of 46 beetles and 49 flowers yielded 86 and 83 yeast isolates, respectively. Whereas the flower community was dominated by Kodamaea ohmeri and Kurtzmaniella quercitrusa, the major yeasts recovered from beetles were Wickerhamiella occidentalis, which is commonly isolated from this community, and a novel species of large-spored Metschnikowia in the arizonensis subclade, which we describe here as Metschnikowia amazonensis sp. nov. Phylogenetic analyses based on barcode sequences (ITS-D1/D2) and a multigene alignment of 11,917 positions (genes ura2, msh6, and pmt2) agreed to place the new species as a sister to Metschnikowia arizonensis, a rare species known only from one locality in Arizona. The two form sterile asci when mated, which is typical of related members of the clade. The α pheromone of the new species is unique but typical of the subclade. The type of M. amazonensis sp. nov. is UFMG-CM-Y6309T (ex-type CBS 16156T , mating type a), and the designated allotype (mating type α) is UFMG-CM-Y6307A (CBS 16155A ). MycoBank MB 833560.

Delineating yeast species with genome average nucleotide identity: a calibration of ANI with haplontic, heterothallic Metschnikowia species.

We determined pairwise average nucleotide identity (ANI) values for the genomes of 71 strains assigned to 36 Metschnikowia species, 28 of which were represented by multiple isolates selected to represent the range of genetic diversity of the species, and most of which were defined on the basis of reproductive isolation. Similar to what has been proposed for prokaryote species delineation, an ANI value of 95% emerged as a good guideline for the delineation of yeast species, although some overlap exists, whereby members of a reproductive community could have slightly lower values (e.g., 94.3% for M. kamakouana), and representatives of distinct sister species could give slightly higher values (e.g., 95.2% for the sister species M. drakensbergensis and M. proteae). Unlike what is observed in prokaryotes, a sizeable gap between intraspecific and interspecific ANI values was not encountered. Given the ease with which yeast draft genomes can now be obtained, ANI values are poised to become the new standard upon which yeast species may be delineated on genetic distance. As borderline cases exist, however, the delineation of yeast species will continue to require careful evaluation of all available data. We also explore the often-neglected distinction between phylogenetic relatedness and sequence identity through the analysis of a tree constructed from ANI' (100 - ANI) values.

Catching speciation in the act-act 2: Metschnikowia lacustris sp. nov., a sister species to Metschnikowia dekortorum.

The isolation of a single yeast strain in the clade containing Metschnikowia dekortorum, in the Amazon biome of Brazil, incited us to re-examine the species boundaries within the clade. The strain (UFMG-CM-Y6306) was difficult to position relative to neighbouring species using standard barcode sequences (ITS-D1/D2 rRNA gene region). Mating took place freely with α strains of M. bowlesiae, M. dekortorum, and M. similis, but two-spored asci, indicative of a fertile meiotic progeny, were formed abundantly only with certain strains of M. dekortorum. Accordingly, we examined mating success among every phylotype in the clade and constructed a phylogeny based on a concatenation of 100 of the largest orthologous genes annotated in draft genomes. The analyses confirmed membership of the Amazonian isolate in M. dekortorum, but also indicated that the species should be subdivided into two. As a result, we retain three original members of M. dekortorum in the species, together with the new isolate, and reassign six isolates recovered from Mesoamerican lacustrine habitats to Metschnikowia lacustris sp. nov. The type is UWOPS 12-619.2T (isotype CBS 16250T). MycoBank: MB 833751.

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.

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.

Metschnikowia mating genomics.

Genes involved in mating type determination and recognition were examined in Metschnikowia and related species, to gather insights on factors affecting mating compatibility patterns among haplontic, heterothallic yeast species of the genus. We confirmed the universality of the special mating locus organisation found in Clavispora lusitaniae across and exclusive to the family Metschnikowiaceae (i.e., Metschnikowia and Clavispora). Timing of the divergence between idiomorphs was confirmed to coincide with the origin of the larger (CUG-ser) clade comprising the Debaryomycetaceae and the Metschnikowiaceae, exclusive of Cephaloascus fragrans. The sequence of the a mating pheromone is highly conserved within the large-spored Metschnikowia species, including Metschnikowia orientalis and Metschnikowia hawaiiana, but not Metschnikowia drosophilae or Metschnikowia torresii, which have a pattern of their own, as do other clades in the genus. In contrast, variation in α pheromones shows a more continuous, although imperfect correlation with phylogenetic distance as well as with in vivo mating compatibility.

Aggregation of cloned embryos in empty zona pellucida improves derivation efficiency of pig ES-like cells.

The development of embryonic stem cells (ESCs) from large animal species has become an important model for therapeutic cloning using ESCs derived by somatic cell nuclear transfer (SCNT). However, poor embryo quality and blastocyst formation have been major limitations for derivation of cloned ESCs (ntESCs). In this study, we have tried to overcome these problems by treating these cells with histone deacetylase inhibitors (HDACi) and aggregating porcine embryos. First, cloned embryos were treated with Scriptaid to confirm the effect of HDACi on cloned embryo quality. The Scriptaid-treated blastocysts showed significantly higher total cell numbers (29.50 ± 2.10) than non-treated blastocysts (22.29 ± 1.50, P < 0.05). Next, cloned embryo quality and blastocyst formation were analyzed in aggregates. Three zona-free, reconstructed, four-cell-stage SCNT embryos were injected into the empty zona of hatched parthenogenetic (PA) blastocysts. Blastocyst formation and total cell number of cloned blastocysts increased significantly for all aggregates (76.4% and 83.18 ± 8.33) compared with non-aggregates (25.5% and 27.11 ± 1.67, P < 0.05). Finally, aggregated blastocysts were cultured on a feeder layer to examine the efficiency of porcine ES-like cell derivation. Aggregated blastocysts showed a higher primary colony formation rate than non-aggregated cloned blastocysts (17.6 ± 12.3% vs. 2.2 ± 1.35%, respectively, P < 0.05). In addition, derived ES-like cells showed typical characters of ESCs. In conclusion, the aggregation of porcine SCNT embryos at the four-cell stage could be a useful technique for improving the development rate and quality of porcine-cloned blastocysts and the derivation efficiency of porcine ntESCs.

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.

Identification and differential expression patterns of porcine OCT4 variants.

OCT4 encoded by POU5F1 has a crucial role of maintaining pluripotency in embryonic stem cells during early embryonic development and several OCT4 variants have been identified in mouse and human studies. The objective of this study was to identify different variants of OCT4 and analyze their expression patterns in preimplantation porcine embryos and various tissues. In this study, we showed that POU5F1 transcribes its three variants, namely OCT4A, OCT4B, and OCT4B1. The OCT4B transcript consists of exons identical to the major form of the OCT4 variant, OCT4A, with a differential N-terminal domain-coding exon. The structure of OCT4B1 mRNA was the same as that of OCT4B mRNA, but harbored a cryptic exon. Based on these findings, the transcription levels were investigated and found that OCT4B and OCT4B1 made up ∼20% among the variants in the embryonic stage and this indicates that OCT4A mRNA is dominantly expressed during preimplantation embryo development. In addition, OCT4B mRNA was detected in all tissues examined, while OCT4A and OCT4B1 were detected only in testis but not in other tissues examined. OCT4B1 showed inversely correlated expression with SOX2 and NANOG expression. OCT4A protein was specifically localized to the nuclei, whereas OCT4B was mainly localized to the cytoplasm of the porcine embryos at the blastocyst stage. The findings of this study reveal that the porcine OCT4 gene can potentially encode three variants (OCT4A, OCT4B, and OCT4B1), and they are differentially expressed and would have roles dissimilar between each other in preimplantation embryos and various adult tissues.

Overexpression of OCT4A ortholog elevates endogenous XIST in porcine parthenogenic blastocysts.

X-chromosome inactivation (XCI) is an epigenetic process that equalizes expression of X-borne genes between male and female eutherians. This process is observed in early eutherian embryo development in a species-specific manner. Until recently, various pluripotent factors have been suggested to regulate the process of XCI by repressing XIST expression, which is the master inducer for XCI. Recent insights into the process and its regulation have been restricted in mouse species despite the evolutionary diversity of the process and molecular mechanism among the species. OCT4A is one of the represented pluripotent factors, the gate-keeper for maintaining pluripotency, and an XIST repressor. Therefore, in here, we examined the relation between OCT4A and X-linked genes in porcine preimplantation embryos. Three X-linked genes, XIST, LOC102165544, and RLIM, were selected in present study because their orthologues have been known to regulate XCI in mice. Expression levels of OCT4A were positively correlated with XIST and LOC102165544 in female blastocysts. Furthermore, overexpression of exogenous human OCT4A in cleaved parthenotes generated blastocysts with increased XIST expression levels. However, increased XIST expression was not observed when exogenous OCT4A was obtained from early blastocysts. These results suggest the possibility that OCT4A would be directly or indirectly involved in XIST expression in earlier stage porcine embryos rather than blastocysts.

Inhibition of BMP-mediated SMAD pathway supports the pluripotency of pig embryonic stem cells in the absence of feeder cells.

Here, we examined the effects of the BMP signaling pathway inhibitor LDN-193189 on the pluripotency of porcine embryonic stem cells (ESCs) in the absence of feeder cells using molecular and transcriptomic techniques. Additionally, the effects of some extracellular matrix components on porcine ESC pluripotency were evaluated to develop an optimized and sustainable feeder-free culture system for porcine ESCs. Feeder cells were found to play an important role in supporting the pluripotency of porcine ESCs by blocking trophoblast and mesodermal differentiation through the inhibition of the BMP pathway. Additionally, treatment with LDN-193189, an inhibitor of the BMP pathway, maintained the pluripotency and homogeneity of porcine ESCs for an extended period in the absence of feeder cells by stimulating the secretion of chemokines and suppressing differentiation, based on transcriptome analysis. Conclusively, these results suggest that LDN-193189 could be a suitable replacement for feeder cells in the maintenance of porcine ESC pluripotency during culture. Additionally, these findings contribute to the understanding of pluripotency gene networks and comparative embryogenesis.

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.

Development of Reproducible and Scalable Culture Conditions for In Vitro Maintenance of Pig Embryonic Stem Cells Using the Sandoz Inbred Swiss Mouse Thioguanine-Resistant Ouabain-Resistant Cell Line as a Feeder Layer.

Feeder cells play a crucial role in maintaining the pluripotency of embryonic stem cells (ESCs) by secreting various extrinsic regulators, such as extracellular matrix (ECM) proteins and growth factors. Although primary mouse embryonic fibroblasts (MEFs) are the most widely used feeder cell type for the culture of ESCs, they have inevitable disadvantages such as batch-to-batch variation and labor-intensive isolation processes. Here, we revealed that the Sandoz inbred Swiss Mouse (SIM) thioguanine-resistant ouabain-resistant (STO) cell line, an immortalized cell line established from mouse SIM embryonic fibroblasts, can be used as a feeder layer for in vitro culture of authentic pig ESCs instead of primary MEFs. First, the expression of genes encoding ECM proteins and growth factors was analyzed to compare their secretory functions as feeder cells. Quantitative real-time polymerase chain reaction (qPCR) showed that the gene expression of these pluripotency-associated factors was downregulated in STO cells compared to primary MEFs of similar density. Therefore, subsequent optimization of the culture conditions was attempted using higher STO cell densities. Notably, pig ESCs cultured on STO cell density of 3 × (187,500 cells/cm2) exhibited the most similar pluripotent state to pig ESCs cultured on primary MEF density of 1 × (62,500 cells/cm2), as determined by alkaline phosphatase staining, qPCR, and immunocytochemistry. In addition, pig ESCs cultured on STO cell density of 3 × formed complex teratoma containing multiple types of tissues derived from all three germ layers. Our culture conditions using optimal STO cell density can be applied to fields requiring reproducible and scalable production of pig ESCs, such as preclinical research and cellular agriculture.

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.

Unlocking the potential of stem cells: Their crucial role in the production of cultivated meat.

Cellular agriculture is an emerging research field of agribiotechnology that aims to produce agricultural products using stem cells, without sacrificing animals or cultivating crops. Cultivated meat, as a representative cellular product of cellular agriculture, is being actively researched due to global food insecurity, environmental, and ethical concerns. This review focuses on the application of stem cells, which are the seeds of cellular agriculture, for the production of cultivated meat, with emphasis on deriving and culturing muscle and adipose stem cells for imitating fresh meat. Establishing standards and safety regulations for culturing stem cells is crucial for the market entry of cultured muscle tissue-based biomaterials. Understanding stem cells is a prerequisite for creating reliable cultivated meat and other cellular agricultural biomaterials. The techniques and regulations from the cultivated meat industry could pave the way for new cellular agriculture industries in the future.

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.

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.