Pluripotency-related Genes OCT4 Research Articles

Methoxychlor induces oxidative stress and impairs early embryonic development in pigs.

Introduction: Methoxychlor (MXC) is an organochlorine pesticide (OCP) that was formerly used worldwide as an insecticide against pests and mosquitoes. However, MXC is not biodegradable and has lipophilic characteristics; thus, it accumulates in organisms and affects reproductive function. MXC, as an estrogenic compound, promotes oxidative stress, induces oxidative stress damage to ovarian follicles, and causes miscarriages and stillbirths in females. In this research endeavor, our primary objective was to explore the ramifications of MXC regarding the developmental processes occurring during the initial stages of embryogenesis in pigs. Methods: In this study, we counted the blastocyst rate of early embryos cultured in vitro. We also examined the reactive oxygen species level, glutathione level, mitochondrial membrane potential, mitochondrial copy number and ATP level in four-cell stage embryos. Finally, apoptosis and DNA damage in blastocyst cells, as well as pluripotency-related and apoptosis-related genes in blastocyst cells were detected. The above experiments were used to evaluate the changes of MXC damage on early parthenogenetic embryo development. Results and Discussion: The results showed that early embryos exposed to MXC had a significantly lower cleavage rate, blastocyst rate, hatching rate, and total cell count compared with the control group. It was also of note that MXC not only increased the levels of reactive oxygen species (ROS), but also decreased the mitochondrial membrane potential (ΔΨm) and mitochondrial copy number during the development of early embryos. In addition, after MXC treatment, blastocyst apoptosis and DNA damage were increased, decreased cell proliferation, and the expression of pluripotency-related genes SOX2, NANOG, and OCT4 was down-regulated, while the expression of apoptosis-related genes BAX/BCL-2 and Caspase9 was up-regulated. Our results clearly show that MXC can have deleterious effects on the developmental processes of early porcine embryos, establishing the toxicity of MXC to the reproductive system. In addition, the study of this toxic effect may lead to greater concern about pesticide residues in humans and the use of safer pesticides, thus potentially preventing physiological diseases caused by chemical exposure.

TET Family Members Are Integral to Porcine Oocyte Maturation and Parthenogenetic Pre-Implantation Embryogenesis.

The ten-eleven translocation (TET) enzyme family, which includes TET1/2/3, participates in active DNA demethylation in the eukaryotic genome; moreover, TET1/2/3 are functionally redundant in mice embryos. However, the combined effect of TET1/2/3 triple-gene knockdown or knockout on the porcine oocytes or embryos is still unclear. In this study, using Bobcat339, a specific small-molecule inhibitor of the TET family, we explored the effects of TET enzymes on oocyte maturation and early embryogenesis in pigs. Our results revealed that Bobcat339 treatment blocked porcine oocyte maturation and triggered early apoptosis. Furthermore, in the Bobcat339-treated oocytes, spindle architecture and chromosome alignment were disrupted, probably due to the huge loss of 5-hydroxymethylcytosine (5hmC)and concurrent increase in 5-methylcytosine (5mC). After Bobcat339 treatment, early parthenogenetic embryos exhibited abnormal 5mC and 5hmC levels, which resulted in compromised cleavage and blastocyst rate. The mRNA levels of EIF1A and DPPA2 (ZGA marker genes) were significantly decreased, which may explain why the embryos were arrested at the 4-cell stage after Bobcat339 treatment. In addition, the mRNA levels of pluripotency-related genes OCT4 and NANOG were declined after Bobcat339 treatment. RNA sequencing analysis revealed differentially expressed genes in Bobcat339-treated embryos at the 4-cell stage, which were significantly enriched in cell proliferation, cell component related to mitochondrion, and cell adhesion molecule binding. Our results indicated that TET proteins are essential for porcine oocyte maturation and early embryogenesis, and they act by mediating 5mC/5hmC levels and gene transcription.

Culture of leukocyte-derived cells from human peripheral blood: Increased expression of pluripotent genes OCT4, NANOG, SOX2, self-renewal gene TERT and plasticity.

There are few stem cells in human peripheral blood (PB). Increasing the population and plasticity of stem cells in PB and applying it to regenerative medicine require suitable culture methods. In this study, leukocyte populations 250 mL of PB were collected using a blood separator before that were cultured in optimal cell culture medium for 4 to 7 days. After culturing, stemness characteristics were analyzed, and red blood cells were removed from the cultured cells. In our results, stemness markers of the leukocyte populations Sca-1+ CD45+, CD117+ CD45+, and very small embryonic-like stem cells CD34+ Lin- CD45- and CXCR4+ Lin- CD45- were significantly increased. Furthermore, the expression of stem cell genes OCT4 (POU5F1), NANOG, SOX2, and the self-renewal gene TERT was analyzed by quantitative real-time polymerase chain reaction in these cells, and it showed a significant increase. These cells could be candidates for multi-potential cells and were further induced using trans-differentiation culture methods. These cells showed multiple differentiation potentials for osteocytes, nerve cells, cardiomyocytes, and hepatocytes. These results indicate that appropriate culture methods can be applied to increase expression of pluripotent genes and plasticity. Leukocytes of human PB can be induced to trans-differentiate into pluripotent potential cells, which will be an important breakthrough in regenerative medicine.

Acceleration of Mesenchymal-to-Epithelial Transition (MET) during Direct Reprogramming Using Natural Compounds.

Induced pluripotent stem cells (iPSCs) can be generated from somatic cells using Oct4, Sox2, Klf4, and c-Myc (OSKM). Small molecules can enhance reprogramming. Licochalcone D (LCD), a flavonoid compound present mainly in the roots of Glycyrrhiza inflata, acts on known signaling pathways involved in transcriptional activity and signal transduction, including the PGC1-α and MAPK families. In this study, we demonstrated that LCD improved reprogramming efficiency. LCD-treated iPSCs (LCD-iPSCs) expressed pluripotency-related genes Oct4, Sox2, Nanog, and Prdm14. Moreover, LCD-iPSCs differentiated into all three germ layers in vitro and formed chimeras. The mesenchymal-to-epithelial transition (MET) is critical for somatic cell reprogramming. We found that the expression levels of mesenchymal genes (Snail2 and Twist) decreased and those of epithelial genes (DSP, Cldn3, Crb3, and Ocln) dramatically increased in OR-MEF (OG2+/+/ROSA26+/+) cells treated with LCD for 3 days, indicating that MET effectively occurred in LCD-treated OR-MEF cells. Thus, LCD enhanced the generation of iPSCs from somatic cells by promoting MET at the early stages of reprogramming.

Effects of LPA on the development of sheep in vitro fertilized embryos and attempt to establish sheep embryonic stem cells

Lysophosphatidic acid (LPA) is a small molecule glycerophospholipid, which regulates multiple downstream signalling pathways through G-protein-coupled receptors to achieve numerous functions on oocyte maturation and embryo development. In this study, sheep in vitro fertilized embryos were applied to investigate the effects of LPA on early embryos development and embryonic stem cell establishment. At first, the maturation medium containing estrus female sheep serum and synthetic oviduct fluid (SOF) were optimized for sheep IVF, and then the effects of LPA were investigated. From 0.1 to 10 μmol L–1, LPA had no significant effect on the cleavage rate (P>0.05), but the maturation rate and blastocyst rate increased dependently with LPA concentration (P<0.05), and the blastocyst morphology was normal. When the LPA concentration was 15 μmol L–1, the maturation rate, cleavage rate and blastocyst rate decreased significantly (P<0.05), and the blastocyst exhibited abnormal morphology and could not develop into high-quality blastocyst. Besides, the exogenous LPA increases the expression of LPAR2, LPAR4, TE-related gene CDX-2 and pluripotency-related gene OCT-4 in sheep early IVF embryos with the raise of LPA concentration from 0.1 to 10 μmol L–1. The expression of LPAR2, LPAR4, CDX-2 and OCT-4 from the LPA-0.1 μmol L–1 to LPA-10 μmol L–1 groups in early embryos were extremely significant (P<0.05), while the expression of these genes significantly decreased in 15 μmol L–1 LPA-treated embryos compared with LPA-10 μmol L–1 group (P<0.05). The inner cell mass in 15 μmol L–1 LPA-treated embryos was also disturbed, and the blastocysts formation was abnormal. Secondly, the sheep IVF blastocysts were applied to establish embryonic stem cells. The results showed that LPA made the blastocyst inoculated cells grow towards TSC-like cells. They enhanced the fluorescence intensity and mRNA abundance of OCT-4 and CDX-2 as the concentration increased from 0 to 10 μmol L–1, while 15 μmol L–1 LPA decreased OCT-4 and CDX-2 expression in the derived cells. The expression of CDX-2 and OCT-4 in the blastocyst inoculated cells of LPA-1 μmol L–1 group and LPA-10 μmol L–1 group extremely significantly increased (P<0.05), but there was significant decrease in LPA-15 μmol L–1 group compared with LPA-10 μmol L–1 group (P<0.05). Meanwhile, the protein expression of LPAR2 and LPAR4 remarkably increased after treatment of LPA at 10 μmol L–1 concentration. This study references the IVF embryo production and embryonic stem cell research of domestic animals.

Cattle In Vitro Induced Pluripotent Stem Cells Generated and Maintained in 5 or 20% Oxygen and Different Supplementation.

The event of cellular reprogramming into pluripotency is influenced by several factors, such as in vitro culture conditions (e.g., culture medium and oxygen concentration). Herein, bovine iPSCs (biPSCs) were generated in different levels of oxygen tension (5% or 20% of oxygen) and supplementation (bFGF or bFGF + LIF + 2i—bFL2i) to evaluate the efficiency of pluripotency induction and maintenance in vitro. Initial reprogramming was observed in all groups and bFL2i supplementation initially resulted in a superior number of colonies. However, bFL2i supplementation in low oxygen led to a loss of self-renewal and pluripotency maintenance. All clonal lines were positive for alkaline phosphatase; they expressed endogenous pluripotency-related genes SOX2, OCT4 and STELLA. However, expression was decreased throughout the passages without the influence of oxygen tension. GLUT1 and GLUT3 were upregulated by low oxygen. The biPSCs were immunofluorescence-positive stained for OCT4 and SOX2 and they formed embryoid bodies which differentiated in ectoderm and mesoderm (all groups), as well as endoderm (one line from bFL2i in high oxygen). Our study is the first to compare high and low oxygen environments during and after induced reprogramming in cattle. In our conditions, a low oxygen environment did not favor the pluripotency maintenance of biPSCs.

Multiple Intravenous Injections of Valproic Acid-Induced Mesenchymal Stem Cell from Human-Induced Pluripotent Stem Cells Improved Cardiac Function in an Acute Myocardial Infarction Rat Model.

Mounting evidence indicates that the mesenchymal stem cell (MSC) injection is safe and efficacious for treating cardiomyopathy; however, there is limited information relating to multiple intravenous injections of human-induced pluripotent stem cell-derived mesenchymal stem cell (hiPSC-MSC) and long-term evaluation of the cardiac function. In the current study, MSC-like cells were derived from human-induced pluripotent stem cells through valproic acid (VPA) induction and continuous cell passages. The derived spindle-like cells expressed MSC-related markers, secreted angiogenic and immune-regulatory factors, and could be induced to experience chondrogenic and adipogenic differentiation. During the induction process, expression of epithelial-to-mesenchymal transition- (EMT-) related gene N-cadherin and vimentin was upregulated to a very high level, and the expression of pluripotency-related genes Sox2 and Oct4 was downregulated or remained unchanged, indicating that VPA initiated EMT by upregulating the expression of EMT promoting genes and downregulating that of pluripotency-related genes. Two and four intravenous hiPSC-MSC injections (106 cells/per injections) were provided, respectively, to model rats one week after acute myocardial infarction (AMI). Cardiac function parameters were dynamically monitored during a 12-week period. Two and four cell injections significantly the improved left ventricular ejection fraction and left ventricular fractional shortening; four-injection markedly stimulated angiogenesis reduced the scar size and cell apoptosis number in the scar area in comparison with that of the untreated control model rats. Although the difference was insignificant, the hiPSC-MSC administration delayed the increase of left ventricular end-diastolic dimension to different extents compared with that of the PBS-injection control. No perceptible immune reaction symptom or hiPSC-MSC-induced tumour formation was found over 12 weeks. Compared with the PBS-injection control, four injections produced better outcome than two injections; as a result, at least four rounds of MSC injections were suggested for AMI treatment.

In Vitro Induction of Pluripotency from Equine Fibroblasts in 20% or 5% Oxygen.

The cellular reprogramming into pluripotency is influenced by external and internal cellular factors, such as in vitro culture conditions (e.g., environmental oxygen concentration), and the aging process. Herein, we aimed to generate and maintain equine iPSCs (eiPSCs) derived from fibroblasts of a horse older than 20 years and to evaluate the effect of different levels of oxygen tension (atmospheric 20% O2, 5% O2, or 20% to 5% O2) on these cells. Fibroblasts were reprogrammed, and putative eiPSCs were positive for positive alkaline phosphatase detection; they were positive for pluripotency-related genes OCT4, REX1, and NANOG; immunofluorescence-positive staining was presented for OCT4 and NANOG (all groups), SOX2 (groups 5% O2 and 20% to 5% O2), and TRA-1-60, TRA-1-81, and SSEA-1 (only in 20% O2); they formed embryoid bodies; and there is spontaneous differentiation in mesoderm, endoderm, and ectoderm embryonic germ layers. In addition to the differences in immunofluorescence analysis results, the eiPSC colonies generated at 20% O2 presented a more compact morphology with a well-defined border than cells cultured in 5% O2 and 20% to 5% O2. Significant differences were also observed in the expression of genes related to glucose metabolism, mitochondrial fission, and hypoxia (GAPDH, GLUT3, MFN1, HIF1α, and HIF2α), after reprogramming. Our results show that the derivation of eiPSCs was not impaired by aging. Additionally, this study is the first to compare high and low oxygen cultures of eiPSCs, showing the generation of pluripotent cells with different profiles. Under the tested conditions, the lower oxygen tension did not favor the pluripotency of eiPSCs. This study shows that the impact of oxygen atmosphere has to be considered when culturing eiPSCs, as this condition influences the pluripotency characteristics.

Effect of Conditioned Medium from Human Umbilical Cord-Derived Mesenchymal Stromal Cells on Rejuvenation of Nucleus Pulposus Derived Stem/Progenitor Cells from Degenerated Intervertebral Disc.

Background and ObjectivesMesenchymal stromal cells (MSCs)-based treatment for degeneration of intervertebral disc (IVD) has been proposed recently. We here addressed whether MSC secreted factors can rejuvenate nucleus pulposus-derived stem/progenitor cells from degenerated disc (D-NPSCs) in vitro.Methods and ResultsWe analyzed the expression of MSCs and NP cell specific surface markers, pluripotency related genes, multilineage potential and cell proliferative capacity of D-NPSCs upon incubation with the conditioned medium which was collected from the umbilical cord derived MSCs (UCMSCs). Our results indicated that the conditioned medium restore the stemness of D-NPSCs by up-regulating the expression level of CD29 and CD105, pluripotency related genes OCT4 and Nanog, and NP progenitor marker Tie2. The increased stemness was accompanied by promoted cell proliferative capacity and improved osteogenic and chondrogenic differentiation potential.ConclusionsOur findings suggested that the UCMSCs derived conditioned medium might be used to rejuvenate the degenerated NP stem/progenitor cells.

Melatonin Enhances the Development of Porcine Cloned Embryos by Improving DNA Methylation Reprogramming.

Incomplete DNA methylation reprogramming in cloned embryos leads to poor cloning efficiency. Melatonin has been proven to improve the development of cloned embryos, however, the role of melatonin during somatic cell nuclear transfer remains unclear. This work demonstrated that 10-7 M melatonin significantly enhanced the developmental progress, reduced the arrested rate before zygotic genome activation, and upregulated the blastocyst rate of cloned embryos. Melatonin also promoted the pseudo-pronucleus formation, increased blastocyst cell number, and reduced embryo apoptosis through upregulating the expression of antiapoptosis factors while downregulating the transcription of proapoptosis genes. Further study displayed that DNA methylation reprogramming related genes were greatly improved in cloned embryos when treated with melatonin; then, melatonin effectively promoted genomic DNA demethylation and DNA remethylation, DNA demethylation of pluripotency related gene Oct4, DNA methylation maintenance of imprinted gene H19/Igf2, and DNA remethylation of tissue-specific gene Thy1 in cloned embryos. Thus, zygotic genome activation related gene Eif1a, pluripotency related genes Oct4, Nanog, and Sox2, imprinted genes Igf2 and H19, and blastocyst quality related genes Cdx2 and ATP1b1 were remarkably upregulated, and tissue-specific genes Thy1 and Col5a2 were considerably silenced. In conclusion, melatonin enhanced the development of cloned embryos by ameliorating DNA methylation reprogramming. This work reveals that melatonin can regulate DNA methylation reprogramming and provides a novel insight to improve cloning efficiency.

212 Increased expression of YAP/TAZ encourages outgrowth establishment and three-dimensional colony formation and boosts plasticity of parthenogenetic stem cells

Cell proliferation, apoptosis, and differentiation are essential processes from the early phases of embryogenesis to adult tissue formation and maintenance. These mechanisms also play a key role in embryonic stem cells (ESCs), which are able to proliferate maintaining pluripotency and, at the same time, give rise to all populations belonging to the three germ layers in response to specific stimuli. ESCs are, therefore, considered a well-established invitro model to study the complexity of these processes. We previously generated porcine and human diploid parthenogenetic embryonic stem cells (ParthESC) that showed many features and regulatory pathways common to biparental ESCs. However, we observed that monoparental cells demonstrated a greater ability to form outgrowths and generate stable three-dimensional (3D) spheroid colonies, which are distinctive signs of high plasticity (Brevini et al. 2010 STCR 6; https://doi.org/10.1007/s12015-010-9153-2). In the present study, we compared porcine and human biparental ESCs and ParthESC. Pluripotency gene expression was analysed and ELISA tests for global DNA methylation and total YAP levels were performed. Our results showed that ParthESCs showed significantly higher expression levels of YAP and TAZ than biparental ESCs. In contrast, the analysis of the upstream genes involved in the Hippo signalling pathway revealed no differences. We demonstrated that YAP remains in a dephosphorylated form, encouraging its nuclear compartmentalization and direct interaction with the nuclear transcription factors TEADs and SMADs, that are also upregulated and maintain pluripotency, repressing differentiation processes (Beyer et al. 2013 Cell Rep. 5, 1611-1624; https://doi.org/10.1016/j.celrep.2013.11.021). These complex regulatory interactions accompanied the overexpression of the pluripotency-related genes OCT4, NANOG, REX1, SOX2, UTF1, and TERT. In parallel, we observed a decrease of DNMT3 and DNMT4 activity that resulted in a global DNA hypomethylation and a chromatin high-permissive state. Altogether, our results demonstrate a significant upregulation of YAP/TAZ activity in monoparental cell lines. This may account for their greater ability to form outgrowths and generate 3D spheroid colonies and increased plasticity compared with biparental counterparts. It is interesting to note that YAP mRNA is supplied and expressed by the oocyte and is maternally inherited (Yu et al. 2016 Cell Res. 26, 275-287; https://doi.org/10.1038/cr.2016.20). Although further clarifications are needed, we hypothesise that its higher expression in ParthESC may be related to the strictly maternal origin of these cells. This study was supported by the Carraresi Foundation. Authors are members of the COST Actions CA16119.

Kaempferol alleviates the reduction of developmental competence during aging of porcine oocytes.

Kaempferol (KAE) is a natural flavonoid present in different plant species and exhibits anti-inflammatory, antioxidant, and anticancer therapeutic properties. In the present study, we investigated the influence and underlying mechanisms of KAE supplementation on porcine oocytes during in vitro aging. The results show that KAE treatment can alleviate the aging-related reduction of developmental competence. We observed that the blastocyst production rate in aged oocytes treated with 0.1μM KAE was significantly higher than in untreated aging oocytes (36.78±0.86% vs. 27.55±2.60%, respectively, p<.05). The KAE-treated aging oocytes had significantly reduced levels of reactive oxygen species (p<.05). Furthermore, the mRNA levels of the embryonic pluripotency-related genes Oct4, NANOG, and ITGA5 were significantly increased in blastocysts derived from KAE-treated oocytes (p<.05). During excessive oocyte culture, KAE treatment maintained the mitochondrial membrane potential and reduced apoptosis; however, this was not observed in untreated aging oocytes. In conclusion, our results suggest that KAE treatment can alleviate the aging of porcine oocytes by reducing oxidative stress and improving mitochondrial function.

Extracellular matrix structure in SCNT and natural bovine placenta conditions

Somatic cell nuclear transfer (SCNT), using transgenic donor cells, is a highly efficient method for producing transgenic embryos. We compared the developmental competence, quality and gene expression of transgenic embryos produced by Hand-made cloning from buffalo fetal fibroblasts (BFFs) containing human insulin gene, with non-transgenic embryos produced from BFFs (Controls). The expression vector (pAcISUBC), constructed by inserting human insulin gene between DNA fragments containing mammary gland-specific buffalo β-lactoglobulin (buBLG) promoter and terminator buBLG 3′UTR regions into pAcGFP-N1 vector, was used for obtaining the 11 kb insert for transfection of BFFs by nucleofection. Presence of the transgene in embryos was confirmed by examining GFP expression by RT-PCR and immunofluorescence. The blastocyst rate was lower (P < 0.05) for transgenic embryos than for controls (35.7 ± 1.8% vs 48.7 ± 2.4%). The apoptotic index was higher (P < 0.05) for transgenic than for control blastocysts which, in turn, was higher (P < 0.05) than for IVF counterparts (6.9 ± 0.9, 3.8 ± 0.5 and 1.8 ± 0.3, respectively). The total cell number was similar for transgenic and non-transgenic blastocysts (143.2 ± 17.0 and 137.2 ± 7.6, respectively). The expression level of pro-apoptotic genes BAX and BID but not that of CASP3 and CASP9, and cell cycle check point control-related gene P53 was higher (P < 0.05), and that of development- (IGF-1R and G6PD) and pluripotency-related gene NANOG was lower (P < 0.05) in transgenic than in control embryos. The expression level of epigenetic-related genes DNMT1, DNMT3a and HDAC1 and pluripotency-related gene OCT4 was similar in the two groups. The expression level of BAX, BID, CASP9, P53, DNMT1 and DNMT3a was higher (P < 0.05) and that of OCT4, NANOG IGF-1R and G6PD was lower (P < 0.05) in cloned transgenic than in IVF blastocysts whereas, that of CASP3 and HDAC1 was similar between the two groups. In conclusion, these results suggest that transgenic embryos produced by SCNT have lower developmental competence and quality, and altered gene expression compared to non-transgenic embryos.

Expression of pluripotency-related genes is highly dependent on trichostatin A-assisted epigenomic modulation of porcine mesenchymal stem cells analysed for apoptosis and subsequently used for generating cloned embryos.

The present study sought to examine whether trichostatin A (TSA)-assisted epigenetic transformation of porcine bone marrow (BM)-derived mesenchymal stem cells (BM-MSCs) affects the transcriptional activities of pluripotency-related genes (Oct4, Nanog, c-Myc, Sox2 and Rex1), multipotent stemness-related gene (Nestin) and anti-apoptotic/anti-senescence-related gene (Survivin). Epigenetically transformed or non-transformed BM-MSCs that had been transcriptionally profiled by qRT-PCR and had been analysed for different stages of apoptosis progression provided a source of nuclear donor cells for the in vitro production of cloned pig embryos. TSA-mediated epigenomic modulation has been found to enhance the multipotency extent, stemness and intracellular anti-ageing properties of porcine BM-MSCs. This has been confirmed by the relative abundances for Nanog, c-Myc Rex1, Sox2 and Survivin mRNAs in TSA-exposed BM-MSCs that turned out to be significantly higher than those of TSA-unexposed BM-MSCs. Additionally, TSA-assisted epigenomic modulation of BM-MSCs did not impact the caspase-8 activity, Bax protein expression and the incidence of TUNEL-positive cells. In conclusion, the considerably elevated quantitative profiles of Sox2, Rex1, c-Myc, Nanog and Survivin mRNA transcripts seem to trigger improved reprogrammability of TSA-treated BM-MSC nuclei in cloned pig embryos that thereby displayed remarkably increased blastocyst formation rates as compared to those noticed for embryos derived from TSA-untreated BM-MSCs.

Comparative analysis of buffalo (Bubalus bubalis) non-transgenic and transgenic embryos containing human insulin gene, produced by SCNT

Somatic cell nuclear transfer (SCNT), using transgenic donor cells, is a highly efficient method for producing transgenic embryos. We compared the developmental competence, quality and gene expression of transgenic embryos produced by Hand-made cloning from buffalo fetal fibroblasts (BFFs) containing human insulin gene, with non-transgenic embryos produced from BFFs (Controls). The expression vector (pAcISUBC), constructed by inserting human insulin gene between DNA fragments containing mammary gland-specific buffalo β-lactoglobulin (buBLG) promoter and terminator buBLG 3′UTR regions into pAcGFP-N1 vector, was used for obtaining the 11 kb insert for transfection of BFFs by nucleofection. Presence of the transgene in embryos was confirmed by examining GFP expression by RT-PCR and immunofluorescence. The blastocyst rate was lower (P < 0.05) for transgenic embryos than for controls (35.7 ± 1.8% vs 48.7 ± 2.4%). The apoptotic index was higher (P < 0.05) for transgenic than for control blastocysts which, in turn, was higher (P < 0.05) than for IVF counterparts (6.9 ± 0.9, 3.8 ± 0.5 and 1.8 ± 0.3, respectively). The total cell number was similar for transgenic and non-transgenic blastocysts (143.2 ± 17.0 and 137.2 ± 7.6, respectively). The expression level of pro-apoptotic genes BAX and BID but not that of CASP3 and CASP9, and cell cycle check point control-related gene P53 was higher (P < 0.05), and that of development- (IGF-1R and G6PD) and pluripotency-related gene NANOG was lower (P < 0.05) in transgenic than in control embryos. The expression level of epigenetic-related genes DNMT1, DNMT3a and HDAC1 and pluripotency-related gene OCT4 was similar in the two groups. The expression level of BAX, BID, CASP9, P53, DNMT1 and DNMT3a was higher (P < 0.05) and that of OCT4, NANOG IGF-1R and G6PD was lower (P < 0.05) in cloned transgenic than in IVF blastocysts whereas, that of CASP3 and HDAC1 was similar between the two groups. In conclusion, these results suggest that transgenic embryos produced by SCNT have lower developmental competence and quality, and altered gene expression compared to non-transgenic embryos.

The modification of mitochondrial energy metabolism and histone of goat somatic cells under small molecules compounds induction.

In recent years, induced pluripotent stem cells (iPSCs) technique is able to allow us to generate pluripotency from somatic cells in vitro through the over expression of several transcription factors. Normally, viral vectors and transcription factors are commonly used on iPSC technique, which could cause many barriers on further application. In this study, we attempt to process a new method to obtain pluripotency from goat somatic cells in vitro under fully chemically defined condition. The results showed that chemically induced pluripotent stem cells-like cells (CiPSC-like cells) colonies were generated from goat ear fibroblasts by fully small-molecule compounds. Those three dimensions colonies were similar with mouse iPSCs in morphology and had strong positive alkaline phosphatase (AP) activity and expressed pluripotency related genes OCT4, SOX2, NANOG, CDH1, TDGF, GDF3, DAX1, REX1, which determined by RT-PCR. Those colonies could also differentiate into different cell types derived from three germ layers proved by RT-PCR and immunofluorescence assays. The expression of glycolysis-related genes about PGAM1, KPYM2 and HXK2 in CiPSC-like colonies formation groups was significantly higher than their parental fibroblasts, but not in the non-CiPSC-like colonies formation group. The expression of histone acetylation and methylation-related genes, HAT1 and SMYD3, was not significantly up-regulated within different groups compared to their parental fibroblasts, respectively. Yet, the expression of histone methylation-related gene, KDM5B, was significantly up-regulated on the cells from non-colonies formation group compared to parental fibroblasts, but the expression of KDM5B of the cells from CiPSC-like cell colonies was not significantly difference compared to that of parental fibroblasts. In conclusion, this is the first report that CiPSC-like cells could be generated in vitro from goat rather than just mouse under fully chemically defined condition. The generation of CiPSC-like colonies may be depended on the correct modification of energy metabolism and histone epigenetic during the reprogramming, rather than just the over-expression of those pluripotency-related genes. This study will strongly support us to further establish the stable goat CiPSC lines without any integration of exogenous genes.

The effect of cell penetrating peptide-conjugated coactivator-associated arginine methyltransferase 1 (CPP-CARM1) on the cloned mouse embryonic development

Abnormalities in gene expression that negatively affect embryonic development are frequently observed in cloned embryos generated by somatic cell nuclear transfer (SCNT). In the present study, we successfully produced a cell-penetrating peptide (CPP)-conjugated with coactivator-associated arginine methyltransferase 1 (CARM1) protein from mammalian cells and confirmed introduction into donor somatic cells and cloned 8-cell embryos within 3 hours after addition to culture medium. In addition, H3R17 dimethylation and embryonic development up to the blastocyst stage were increased in the group treated with exogenous CPP-CARM1 protein compared with the untreated group (control). Interestingly, the number of total cells and trophectoderm in blastocysts as well as implantation rate were significantly increased in the CPP-CARM1 protein-treated group. However, the cell number of inner cell mass (ICM) was not changed compared with the control group; similarly, expression of pluripotency-related genes Oct4 and Nanog (ICM markers) was not significantly different between groups. On the other hand, expression of the implantation-related gene Cdx2 (trophectoderm marker) was transiently increased after treatment with CPP-CARM1 protein. On the basis of these results, we conclude that supplementation with exogenous CPP-CARM1 protein improves embryonic development of cloned embryos through regulation of histone methylation and gene expression. In addition, our results suggest that CPP-CARM1 protein may be a useful tool for strengthening implantation of mammalian embryos.

Effects of PRDM14 Silencing on Parthenogenetically Activated Porcine Embryos.

PRDM14, a member of the PRDM family protein, plays an important role in the regulation of epigenetic reprogramming. Knockdown of Prdm14 in germ cells can lead to female and male subfertility, and the function of PRDM14 appears to be conserved across mammalian species. Thus, we analyzed the expression of Prdm14 in parthenogenetic embryos at all stages of preimplantation and then evaluated the effect of Prdm14 downregulation on porcine parthenogenetic embryonic development. We found that Prdm14 transcripts are highly expressed in the metaphase II (MII) oocyte, and their level gradually increased from the 2-cell to 8-cell stage and slightly declined at the blastocyst stage during the development of parthenogenetic porcine embryos. Prdm14 was knocked down in oocytes at the MII stage by the injection of siRNA to assess the role of this protein in epigenetic remodeling and embryo development. Prdm14 knockdown significantly decreased the cleavage and blastocyst rates without changing the total cell number of the blastocysts. In addition, the expression levels of the antiapoptotic gene BCL-2 and the pluripotency-related genes OCT4 and SOX2 were also significantly decreased. These results showed that PRDM14 could affect embryonic development through regulating the expression levels of the pluripotency and antiapoptotic genes during the development of parthenogenetically activated porcine embryos.

M-carboxycinnamic acid bishydroxamide improves developmental competence, reduces apoptosis and alters epigenetic status and gene expression pattern in cloned buffalo (Bubalus bubalis) embryos.

Incomplete or aberrant reprogramming of nuclear genome is one of the major problems in somatic cell nuclear transfer. In this study, we studied the effect of histone deacetylase inhibitor m-carboxycinnamic acid bishydroxamide (CBHA) on in vitro development of buffalo embryos produced by Hand-made cloning. Cloned embryos were treated with CBHA (0, 5, 10, 20 or 50μM) for 10hr from the start of reconstruction till activation. At 10μM, but not at other concentrations examined, CBHA increased (p<.05) the blastocyst rate (63.77±3.97% vs 48.63±3.55%) and reduced (p<.05) the apoptotic index of the cloned blastocysts (8.91±1.94 vs 4.36±1.08) compared to untreated controls, to levels similar to those in IVF blastocysts (4.78±0.74). CBHA treatment, at all the concentrations examined, increased (p<.05) the global level of H3K9ac in cloned blastocysts than in untreated controls to that observed in IVF blastocysts. Treatment with CBHA (10μM) decreased (p<.05) the global level of H3K27me3 in cloned blastocysts than in untreated controls but it was still higher (p<.05) than in IVF blastocysts. CBHA (10μM) treatment increased (p<.05) the relative expression level of pluripotency-related genes OCT-4 and NANOG, and anti-apoptotic gene BCL-XL, and decreased (p<.05) that of pro-apoptotic gene BAX than in untreated controls but did not affect the relative expression level of apoptosis-related genes p53 and CASPASE3 and epigenetics-related genes DNMT1, DNMT3a and HDAC1. These results suggest that treatment of cloned embryos with 10μM CBHA improves the blastocyst rate, reduces the level of apoptosis and alters the epigenetic status and gene expression pattern.

Temporal Gene Expression and DNA Methylation during Embryonic Stem Cell Derivation.

Objective Dual inhibition of mitogen-activated protein kinase (MAPK) kinase (also known as MEK) and transforming growth factor β (TGFβ) type I receptors by PD0325901 and SB431542, known as R2i has been introduced as a highly efficient approach to the generation of mouse embryonic stem cells (ESC). In the present study, we investigated the molecular mechanisms underlying ESC derivation in the R2i condition. Materials and Methods In this experimental study, zona-free whole E3.5 blastocysts were seeded on mouse embryonic fibroblast (MEF) feeder cells in both R2i and serum conventional media. The isolated inner cell mass (ICM), ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture for quantitative real time- polymerase chain reaction (qRT-PCR) analysis as well as to assess the DNA methylation status at the time points during the transition from ICM to ESC. Results qRT-PCR revealed a significantly higher expression of the pluripotency-related genes (Oct4, Nanog, Sox2, Rex1, Dppa3, Tcf3, Utf1, Nodal, Dax1, Sall4 and β-Catenin) and lower expression of early differentiation genes (Gata6, Lefty2 and Cdx2) in R2i condition compared to the serum condition. Moreover, the upstream region of Oct4 and Nanog showed a progressive increase in methylation levels in the upstream regions of the genes following in R2i or serum conditions, followed by a decrease of DNA methylation in ESCs obtained under R2i. However, the methylation level of ICM outgrowths in the serum condition was much higher than R2i, at levels that could have a repressive effect and therefore explain the absence of expression of these two genes in the serum condition. ConclusionOur investigation revealed that generation of ESCs in the ground-state of pluripotency could be achieved by inhibiting the MEK and TGF-β signaling pathways in the first 5 days of ESC derivation.