Yeast cell wall capsules for delivery of oat biomarker avenanthramide-C.

Avenanthramide-C (AVN-C) is the biomarker for oat with a variety of physiological functions, whereas its application is constrained by low stability and bioavailability. Avenanthramide-C is the biomarker for oat with a variety of physiological functions, whereas its application is constrained by low stability and bioavailability. This study evaluated the potential of yeast cell (YC) and yeast cell wall (YCW) capsules as delivery systems for stabilizing AVN-C. It was observed that these yeast capsules possessed the ellipsoidal morphology and intact structure without visual pores. Additionally, the YCW capsules exhibited higher encapsulation and loading capacity due to the large internal space. The interaction of yeast capsules with AVN-C involved the hydrophobic interactions and hydrogen bonding. Moreover, the loading of AVN-C induced high hydrophobicity inside the yeast capsules, which helped to protect AVN-C against degradation and release AVN-C in a slow and sustained manner in the simulated gastrointestinal tract. The YCW capsules have potential as controlled delivery system for AVN-C, which could be further used as a nutraceutical and added to functional foods.

OCT4 expression transactivated by GATA protein is essential for non-rodent trophectoderm early development.

Oct4 is exclusively expressed in rodent inner cell mass (ICM) but silenced in its trophectoderm (TE). However, for many non-rodent animals, including pig, cattle, rabbit, goat, and human, OCT4 has a remarkable expression in early TE. This study, applying pig as the main research model, proves that OCT4 expression in TE is supported by a unique GATA motif in the OCT4 upstream conserved regulatory region, and GATA4 is responsible for its activation. Moreover, OCT4 acts as a specific regulator of a narrow range of genes (including BCL2A1 and HNRNP2AB1) that are essential for the first wave of rapid proliferation in early TE. This study describes the regulatory mechanism to direct the OCT4 expression and its significance in TE of porcine preimplantation embryo.

The Effects of Daxx Knockout on Pluripotency and Differentiation of Mouse Induced Pluripotent Stem Cells.

Induced pluripotent stem cell (iPSC) technology refers to the reprogramming of terminally differentiated somatic cells into pluripotent stem cells by introducing specific transcription factors that are known to regulate pluripotency, including Oct4, Sox2, Klf4, and c-Myc. In this study, we reprogrammed the primary fibroblasts isolated from the Daxxflox/flox mice, which carry the Oct4-green fluorescent protein reporter, and employed wild-type littermates as a control to induce iPSCs, then knocked out Daxx by infecting with Cre virus at the cellular level. The pluripotency and self-renewal capacity of iPSCs were determined. In addition, Daxx deletion altered the pluripotency marker (Nanog, Oct4) expression and displayed neural differentiation defects. Particularly, by performing transcriptome analysis, we observed that numerous ribosome biogenesis-related genes were altered, and quantitative polymerase chain reaction revealed that the expression of rDNA-related genes, 47S and 18S, was elevated after Daxx deletion. Finally, we illustrated that the expression of the neurodevelopment-related gene was upregulated both in iPSCs and differentiated neurospheres. Taken together, we demonstrated that Daxx knockout promotes the expression of rDNA, pluripotency, and neurodevelopment genes, which may improve the differentiation abilities of mouse iPSCs (miPSCs).

Ratio of the zygote cytoplasm to the paternal genome affects the reprogramming and developmental efficiency of androgenetic embryos.

Uniparental embryos have uniparental genomes and are very useful models for studying the specific gene expression of parents or for exploring the biological significance of genomic imprinting in mammals. However, the early developmental efficiency of androgenetic embryos is significantly lower than that of parthenogenetic embryos. In addition, oocytes are able to reprogram sperm nuclei after fertilization to guarantee embryonic development by maternally derived reprogramming factors, which accumulate during oogenesis. However, the importance of maternal material in the efficiency of reprogramming the pronucleus of androgenetic embryos is not known. In this study, androgenetic embryos were constructed artificially by pronucleus transfer (PT) or double sperm injection (DS). Compared with DS embryos, PT embryos that were derived from two zygotes contained more maternal material, like 10-11 translocation methylcytosine deoxygenase 3 (Tet3) and histone variant 3.3 (H3.3). Our experiments confirmed the better developmental potential of PT embryos, which had higher blastocyst rates, a stronger expression of pluripotent genes, a lower expression of apoptotic genes, and superior blastocyst quality. Our findings indicate that the aggregation of more maternal materials in the paternal pronucleus facilitate the reprogramming of the paternal genome, improving embryonic development in PT androgenesis.

Establishment of mouse androgenetic embryonic stem cells by double sperm injection and differentiation into beating embryoid body.

Androgenetic embryonic stem (AgES) cells offer a possible tool for patient-specific pluripotent stem cells that will benefit genomic imprinting studies and clinic applications. However, the difficulty in producing androgenetic embryos and the unbalanced expression of imprinted genes make the therapeutic applicability of AgES cells uncertain. In this study, we produced androgenetic embryos by injecting two sperm into an enucleated metaphase II (MII) oocyte. By this method, 88.48% of oocytes survived after injection, and 20.24% of these developed to the blastocyst stage. We successfully generated AgES cell lines from the androgenetic embryos and assayed the expression of imprinted genes in the cell lines. We found that the morphological characteristics of AgES cells were similar to that of fertilized embryonic stem cells (fES), such as expression of key pluripotent markers, and generation of cell derivatives representing all three germ layers following in vivo and in vitro differentiation. Furthermore, activation of paternal imprinted genes was detected, H19, ASC12 and Tss3 in AgES cell activation levels were lower while other examined genes showed no significant difference to that of fES cells. Interestingly, among examined maternal imprinted genes, only Mest and Igf2 were significantly increased, while levels of other detected genes were no different to that of fES cells. These results demonstrated that activation of some paternal imprinted genes, as well as recovery of maternal imprinted genes, was present in AgES cells. We differentiated AgES cells into a beating embryoid body in vitro, and discovered that the AgES cells did not show significant higher efficiency in myocardial differentiation potential.

Inhibition of DNA repair protein RAD51 affects porcine preimplantation embryo development.

Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free DNA repair of double-stranded breaks. DNA repair protein RAD51 homolog 1 (RAD51) plays a central role in HR. However, the role of RAD51 during porcine early embryo development is unknown. In the present study, we examined whether RAD51 is involved in the regulation of early embryonic development of porcine parthenotes. We found that inhibition of RAD51 delayed cleavage and ceased development before the blastocyst stage. Disrupting RAD51 activity with RNAi or an inhibitor induces sustained DNA damage, as demonstrated by the formation of distinct γH2AX foci in nuclei of four-cell embryos. Inhibiting RAD51 triggers a DNA damage checkpoint by activating the ataxia telangiectasia mutated (ATM)-p53-p21 pathway. Furthermore, RAD51 inhibition caused apoptosis, reactive oxygen species accumulation, abnormal mitochondrial distribution and decreased pluripotent gene expression in blastocysts. Thus, our results indicate that RAD51 is required for proper porcine parthenogenetic activation (PA) embryo development.

Laminarin enhances the quality of aged pig oocytes by reducing oxidative stress.

Laminarin (LAM) is a ß-glucan oligomer known to possess biological activities such as anticancer and antioxidant effects. This study explored the influence of LAM supplementation on in vitro aged porcine oocytes and the underlying mechanisms behind this influence. We found that LAM delayed the aging process and improved the quality of aged oocytes. LAM supplementation enhanced the subsequent developmental competence of aged oocytes during the in vitro aging process. The blastocyst formation rate was significantly increased in aged oocytes treated with 20 µg/ml LAM compared to non-treated aged oocytes (45.3% vs. 28.7%, P < 0.01). The mRNA levels of apoptosis-related genes, B cell lymphoma-2-associated X protein (Bax) and Caspase-3, were significantly lower in blastocysts derived from the LAM-treated aged oocytes during the in vitro aging process. Furthermore, the level of intracellular reactive oxygen species was significantly decreased and that of glutathione was significantly increased in aged oocytes following LAM treatment. Mitochondrial membrane potential was increased, and the activities of caspase-3 and cathepsin B were significantly reduced in the LAM-treated aged oocytes compared with the non-treated aged oocytes. Taken together, these results suggest that LAM is beneficial for delaying the aging process in porcine oocytes.

Autophagy is required for proper meiosis of porcine oocytes maturing in vitro.

Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components; however, the contribution of autophagy during meiosis has not been studied in porcine oocytes maturing in vitro. In this study, we observed that the autophagy-related gene, LC3, was expressed in porcine oocytes during maturation for 44 h in vitro. Knockdown of the autophagy-related gene, BECN1, reduced both BECN1 and LC3 protein expression levels. Moreover, BECN1 knockdown and treatment with the autophagy inhibitor, LY294002, during maturation of porcine oocytes in vitro impaired polar body extrusion, disturbed mitochondrial function, triggered the DNA damage response, and induced early apoptosis in porcine oocytes. Autophagy inhibition during oocyte maturation also impaired the further developmental potential of porcine oocytes. These results indicate that autophagy is required for the in vitro maturation of porcine oocytes.

Inhibition of cathepsin B activity prevents deterioration in the quality of in vitro aged porcine oocytes.

The activity of cathepsin B, a member of the lysosomal protease family, directly correlates with oocyte quality and subsequent embryonic development. However, its biological function during the progression of in vitro aging of oocytes in pigs has not been demonstrated. Here, we showed that cathepsin B activity was dramatically increased during in vitro aged oocytes. The inhibition of cathepsin B activity prevented the decline of the quality of aged oocytes and improved their subsequent developmental competence. Moreover, the inhibition of cathepsin B activity reduced aging-induced mitochondrial dysfunction and attenuated oxidative stress. The inhibition of cathepsin B activity also markedly decreased early apoptosis levels and the frequency of spindle anomalies during in vitro aging of oocytes. These results demonstrate that in vitro aging of oocytes induces cathepsin B activity, which is associated with a decline in oocyte quality. The inhibition of cathepsin B activity has a beneficial effect on oocytes during the process of in vitro aging.

Laminarin improves developmental competence of porcine early stage embryos by inhibiting oxidative stress.

Laminarin (LMA), a ß-glucan mixture with good biocompatibility, improves the growth performance and immune response when used as food additives and nutraceuticals. The aim of the present research was to explore the effects of LMA on porcine early stage embryo development, as well as the underlying mechanisms. The results showed that the developmental competence of porcine early stage embryos was dramatically improved after LMA supplementation during the in vitro culture period. The presence of 20 µg/mL LMA during the in vitro culture period significantly improved cleavage rate, blastocyst formation rates, hatching rate, and total cell number in the blastocyst compared to that in the control group. Notably, LMA attenuated the intracellular reactive oxygen species generation induced by H2O2. Furthermore, LMA not only increased intracellular glutathione levels, but also ameliorated mitochondrial membrane potential. In addition, the expression of a zygotic genome activation related gene (YAP1), pluripotency-related genes (OCT4, NANOG, and SOX2), and hatching-related genes (COX2, GATA4, and ITGA5) were up-regulated following LMA supplementation during porcine early stage embryo development. These results demonstrate that LMA has beneficial effects on the development of porcine early stage embryos via regulation of oxidative stress. This evidence provides a novel method for embryo development improvement associated with exposure to LMA.

A novel long intergenic noncoding RNA indispensable for the cleavage of mouse two-cell embryos.

Endogenous retroviruses (ERVs) are transcriptionally active in cleavage stage embryos, yet their functions are unknown. ERV sequences are present in the majority of long intergenic noncoding RNAs (lincRNAs) in mouse and humans, playing key roles in many cellular processes and diseases. Here, we identify LincGET as a nuclear lincRNA that is GLN-, MERVL-, and ERVK-associated and essential for mouse embryonic development beyond the two-cell stage. LincGET is expressed in late two- to four-cell mouse embryos. Its depletion leads to developmental arrest at the late G2 phase of the two-cell stage and to MAPK signaling pathway inhibition. LincGET forms an RNA-protein complex with hnRNP U, FUBP1, and ILF2, promoting the cis-regulatory activity of long terminal repeats (LTRs) in GLN, MERVL, and ERVK (GLKLTRs), and inhibiting RNA alternative splicing, partially by downregulating hnRNP U, FUBP1, and ILF2 protein levels. Hnrnpu or Ilf2 mRNA injection at the pronuclear stage also decreases the preimplantation developmental rate, and Fubp1 mRNA injection at the pronuclear stage causes a block at the two-cell stage. Thus, as the first functional ERV-associated lincRNA, LincGET provides clues for ERV functions in cleavage stage embryonic development.

Methyl-CpG-Binding Protein 2 Improves the Development of Mouse Somatic Cell Nuclear Transfer Embryos.

Methyl-CpG-binding domain proteins (MBPs) connect DNA methylation and histone modification, which are the key changes of somatic cell reprogramming. Methyl-CpG-binding protein 2 (MeCP2) was the first discovered MBP that has been extensively studied in the neurodevelopmental disorder Rett syndrome. However, a role for MeCP2 during cellular reprogramming associated with somatic cell nuclear transfer (SCNT) has not been examined. In this study, we discovered that MeCP2 expression was significantly lower in embryos generated by SCNT compared with those generated by intracytoplasmic sperm injection (ICSI). We genetically modified mouse embryonic fibroblasts (MEFs) to overexpress MeCP2 and serve as donor cells for nuclear transfer (NT) to investigate the effects of MeCP2 on preimplantation development of SCNT embryos. The blastocyst rate (35.71%) of MeCP2 overexpressed embryos (NT(+)) was significantly greater than in nontransgenic embryos (NT(-), 24.29%). Furthermore, immunofluorescence experiments revealed that 5-methylcytosine (5mC) was transferred to 5-hydroxymethylcytosine (5hmC) to a greater extent in NT(+) embryos than in NT(-) embryos. Real-time PCR evaluation of gene expression also showed that embryonic development-associated genes, such as Oct4 and Nanog, were significantly higher in the NT(+) group compared to the NT(-) group. Collectively, these results suggested that MeCP2 facilitated Tet3 activity, enhanced expression of pluripotency-related genes, and eventually improved the development of NT embryos. Finally, we performed chromatin immunoprecipitation to identify direct targets of MeCP2 and constructed a protein interaction network to elucidate several putative MeCP2 targets.

Induction of autophagy improves embryo viability in cloned mouse embryos.

Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components. Moreover, autophagy is essential for preimplantation development in mammals. Here we show that autophagy is also important for reprogramming in somatic cell nuclear transfer (SCNT). Our data indicate that unlike fertilized oocytes, autophagy is not triggered in SCNT embryos during 6 hours of activation. Mechanistically, the inhibited autophagic induction during SCNT activation is due to the cytochalasin B (CB) caused depolymerization of actin filaments. In this study, we induced autophagy during SCNT activation by rapamycin and pp242, which could restore the expected level of autophagy and significantly enhance the development of SCNT embryos to the blastocyst stage when compared with the control (68.5% and 68.7% vs. 41.5%, P < 0.05). Furthermore, the treatment of rapamycin and pp242 accelerates active DNA demethylation indicated by the conversion of 5 mC to 5 hmC, and treatment of rapamycin improves degradation of maternal mRNA as well. Thus, our findings reveal that autophagy is important for development of SCNT embryos and inhibited autophagic induction during SCNT activation might be one of the serious causes of low efficiency of SCNT.

Morphological changes and germ layer formation in the porcine embryos from days 7-13 of development.

Morphogenesis and identification of embryonic differentiation in porcine embryos are crucial issues for developmental biology and laboratory animal science. The current paper presents a study on the asynchronous development of hatched porcine embryos from days 7 to 13 post-insemination. Examination of semi-thin sections of the hypoblast showed that it had characteristics similar to those of the mouse anterior visceral endoderm during embryonic disc formation. Also, a cavity appeared in the epiblast, which was similar to a mouse proamniotic cavity. With the gradual disappearance of Rauber's layer, the cavity opened and contacted the external environment directly, all of which formed the embryonic disc. To confirm the differentiation characteristics, we performed immunohistochemical analyses and showed that GATA6 was detected clearly in parietal endoderm cells during embryonic disc establishment. OCT4 was expressed in the inner cell mass (ICM) and trophoblast of hatched blastocysts and in the epiblast during formation of the embryonic disc. However, OCT4 showed comparatively decreased expression in the posterior embryonic disc, primitive streak and migrating cells. SOX2 was present in the ICM and epiblast. Therefore, both SOX2 and OCT4 can be used as markers of pluripotent cells in the porcine embryonic disc. At the start of gastrulation, staining revealed VIMENTIN in the posterior of the embryonic disc, primitive streak and in migrating cells that underlay the embryonic disc and was also expressed in epiblast cells located in the anterior primitive streak. Together with serial sections of embryos stained by whole mount immunohistochemistry, the mesoderm differentiation pattern was shown as an ingression movement that took place at the posterior of the embryonic disc and with bilateral migration along the embryonic disc borders.

Abnormal dynamic changes in ß-tubulin in somatic nuclear transfer cloned mouse embryos.

The efficiency of somatic cell nuclear transfer (SCNT) cloning remains low, thus limiting the applications of this technique. In this study, we used immunochemistry and confocal microscopy to detect the microtubule component, ß-tubulin, in SCNT, parthenogenetic (PA), and intracytoplasmic sperm injection (ICSI) embryos before the first mitotic division. ß-Tubulin is the component subunit of microtubule, which plays critical roles in regulating localization of cellular organelles, and the growth, maturation and fertilization of oocytes. Our results demonstrated similar changes of spindle patterns in PA and ICSI embryos. The second meiotic division resumed 1 h post-treatment, and the cytoplasmic asters (CAs) disappeared. After about 4-6 h of treatment, pronuclei formed with the midbodies connecting each other. Meanwhile, the CAs reappeared and a microtubule network developed in the cytoplasm. However, SCNT embryos showed abnormal multipolar spindles, and the pseudopronuclei that contained many nucleoli existed after 6 h of SrCl2 activation. Enucleated oocytes alone did not form spindle-like structures when they were artificially activated for 6 h, indicating that somatic cell chromosomes might be necessary for spindle formation in SCNT embryos. These results demonstrated abnormal changes of ß-tubulin in mouse SCNT embryos, compared with PA and ICSI embryos.

Selection and expression profiles of reference genes in mouse preimplantation embryos of different ploidies at various developmental stages.

Real-time reverse transcription quantitative polymerase chain reaction (qPCR) has become the most frequently used system for studies of gene expression. Many studies have provided reliable evidence that the transcription levels of reference genes are not constant at different developmental stages and in different experimental conditions. However, suitable reference genes which are stably expressed in polyploid preimplantation embryos of different developmental stages have not yet been identified. Therefore, it is critical to verify candidate reference genes to analyze gene expression accurately in both diploid and polyploid embryos. We examined the expression levels of 12 candidate reference genes in preimplantation embryos of four different ploidies at six developmental stages. Stability analysis of the reference genes was performed by four independent software programs, and the stability of three genes was evaluated by comparison with the Oct4 expression level during preimplantation development in diploid embryos. The expression levels of most genes in the polyploid embryos were higher than that in the diploid embryos, but the increasing degree were disproportionate with the ploidies. There were no significant difference in reference gene expressions among embryos of different ploidies when they reached the morula stage, and the expression level remained flat until the blastocyst stage. Ubc, Ppia, and Pgk1 were the three most stable reference genes in diploid and polyploid embryos.

Effects of dimethyl sulfoxide on asymmetric division and cytokinesis in mouse oocytes.

BACKGROUND: Dimethyl sulfoxide (DMSO) is used extensively as a permeable cryoprotectant and is a common solvent utilized for several water-insoluble substances. DMSO has various biological and pharmacological activities; however, the effect of DMSO on mouse oocyte meiotic maturation remains unknown. RESULTS: In DMSO-treated oocytes, we observed abnormal MII oocytes that contained large polar bodies, including 2-cell-like MII oocytes, during in vitro maturation. Oocyte polarization did not occur, due to the absence of actin cap formation and spindle migration. These features are among the primary causes of abnormal symmetric division; however, analysis of the mRNA expression levels of genes related to asymmetric division revealed no significant difference in the expression of these factors between the 3% DMSO-treated group and the control group. After each "blastomere" of the 2-cell-like MII stage oocytes was injected by one sperm head respectively, the oocytes still possessed the ability to extrude the second polar body from each "blastomere" and to begin cleavage. However, MII oocytes with large polar bodies developed to the blastocyst stage after intracytoplasmic sperm injection (ICSI). Furthermore, other permeable cryoprotectants, such as ethylene glycol and glycerol, also caused asymmetric division failure. CONCLUSION: Permeable cryoprotectants, such as DMSO, ethylene glycol, and glycerol, affect asymmetric division. DMSO disrupts cytokinesis completion by inhibiting cortical reorganization and polarization. Oocytes that undergo symmetric division maintain the ability to begin cleavage after ICSI.

Comparison of reprogramming genes in induced pluripotent stem cells and nuclear transfer cloned embryos.

The most effective reprogramming methods, somatic cell nuclear transfer (SCNT) and induced pluripotent stem cells (iPSCs), are widely used in biological research and regenerative medicine, yet the mechanism that reprograms somatic cells to totipotency remains unclear and thus reprogramming efficiency is still low. Microarray technology has been employed in analyzing the transcriptomes changes during iPS reprogramming. Unfortunately, it is difficult to obtain enough DNA from SCNT reconstructed embryos to take advantage of this technology. In this study, we aimed to identify critical genes from the transcriptional profile for iPS reprogramming and compared expression levels of these genes in SCNT reprogramming. By integrating gene expression information from microarray databases and published studies comparing somatic cells with either miPSCs or mouse embryonic stem cells (ESCs), we obtained two lists of co-upregulated genes. The gene ontology (GO) enriched analysis of these two lists demonstrated that the reprogramming process is associated with numerous biological processes. Specifically, we selected 32 genes related to heterochromatin, embryonic development, and cell cycle from our co-upregulated gene datasets and examined the gene expression level in iPSCs and SCNT embryos by qPCR. The results revealed that some reprogramming related genes in iPSCs were also expressed in SCNT reprogramming. We established the network of gene interactions that occur with genes differentially expressed in iPS and SCNT reprogramming and then performed GO analysis on the genes in the network. The network genes function in chromatin organization, heterochromatin, transcriptional regulation, and cell cycle. Further researches to improve reprogramming efficiency, especially in SCNT, will focus on functional studies of these selected genes.

Continuous passages accelerate the reprogramming of mouse induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are usually generated by reprogramming somatic cells through transduction with a transcription factor cocktail. However, the low efficiency of this procedure has kept iPSCs away from the study of the clinical application of stem cell biology. Our research shows that continuous passage increases the efficiency of reprogramming. Compared with conventional method of establishment of iPSCs, more embryonic stem cell (ESC)-like clones are generated by continuous passage during early reprogramming. These inchoate clones, indistinguishable from genuine ESC clones, are closer to fully reprogrammed cells compared with those derived from classical iPSC induction, which increased the expression of pluripotent gene markers and the levels of demethylation of Oct4 and Nanog. These results suggested that full reprogramming is a gradual process that does not merely end at the point of the activation of endogenous pluripotency-associated genes. Continuous passage could increase the pluripotency of induced cells and accelerate the process of reprogramming by epigenetic modification. In brief, we have provided an advanced strategy to accelerate the reprogramming and generate more nearly fully reprogrammed iPSCs efficiently and rapidly.

Generation of neural progenitors from induced Bama miniature pig pluripotent cells.

Pig pluripotent cells may represent an advantageous experimental tool for developing therapeutic application in the human biomedical field. However, it has previously been proven to be difficult to establish from the early embryo and its pluripotency has not been distinctly documented. In recent years, induced pluripotent stem (iPS) cell technology provides a new method of reprogramming somatic cells to pluripotent state. The generation of iPS cells together with or without certain small molecules has become a routine technique. However, the generation of iPS cells from pig embryonic tissues using viral infections together with small molecules has not been reported. Here, we reported the generation of induced pig pluripotent cells (iPPCs) using the iPS technology in combination with valproic acid (VPA). VPA treatment significantly increased the expression of pluripotent genes and played an important role in early reprogramming. We showed that iPPCs resembled pig epiblast cells in their morphology and pluripotent markers, such as OCT4, NANOG, and SSEA1. It had a normal karyotype and could form embryoid bodies, which express three germ layer markers in vitro. In addition, the iPPCs might directly differentiate into neural progenitors after being induced with the retinoic acid and extracellular matrix. Our study established a reasonable method to generate pig pluripotent cells, which might be a new donor cell source for human neural disease therapy.