DOT1L is critical for adult vessel maintenance and functions.

Objective: DOT1L is the only known histone H3K79 methyltransferase essential for the development of the embryonic cardiovascular system, including the heart, blood vessels, and lymphatic vessels, through transcriptional regulation. Our previous study demonstrated that Dot1l deletion results in aberrant lymphatic development and function. However, its precise function in the postnatal cardiovascular system remains unknown. Methods: Using conditional and inducible Dot1l knockout (KO) mice, along with a reporter strain carrying the Geo gene at the Dot1l locus, DOT1L expression and its function in the vascular system during postnatal life were investigated. To assess vessel morphology and vascular permeability, we administered Latex or Evans blue dye to KO mice. In addition, in vitro tube formation and cell migration assays were performed using DOT1L-depleted human umbilical vein endothelial cells (HUVECs). Changes in the expression of vascular genes in HUVECs were measured by quantitative polymerase chain reaction. Results: Our findings demonstrate that conditional Dot1l knockout in the Tg (Tie2-cre) strain results in abnormal blood vessel formation and lymphatic anomalies in the intestine. In a mouse model of Rosa26-creER-mediated inducible Dot1l knockout, we observed vascular phenotypes, including increased vascular permeability and brain hemorrhage, when DOT1L was deleted in adulthood. Additionally, DOT1L depletion in cultured HUVECs led to impaired cell migration and tube formation, likely due to altered gene transcription. These findings highlight the essential role of DOT1L in maintaining vascular integrity and function during embryonic development and postnatal life. Conclusion: Our study revealed that DOT1L is required for the maintenance of adult vascular function through the regulation of gene expression.

RNA helicase DEAD-box-5 is involved in R-loop dynamics of preimplantation embryos.

OBJECTIVE: R-loops are DNA:RNA triplex hybrids, and their metabolism is tightly regulated by transcriptional regulation, DNA damage response, and chromatin structure dynamics. R-loop homeostasis is dynamically regulated and closely associated with gene transcription in mouse zygotes. However, the factors responsible for regulating these dynamic changes in the R-loops of fertilized mouse eggs have not yet been investigated. This study examined the functions of candidate factors that interact with R-loops during zygotic gene activation. METHODS: In this study, we used publicly available next-generation sequencing datasets, including low-input ribosome profiling analysis and polymerase II chromatin immunoprecipitation-sequencing (ChIP-seq), to identify potential regulators of R-loop dynamics in zygotes. These datasets were downloaded, reanalyzed, and compared with mass spectrometry data to identify candidate factors involved in regulating R-loop dynamics. To validate the functions of these candidate factors, we treated mouse zygotes with chemical inhibitors using in vitro fertilization. Immunofluorescence with an anti-R-loop antibody was then performed to quantify changes in R-loop metabolism. RESULTS: We identified DEAD-box-5 (DDX5) and histone deacetylase-2 (HDAC2) as candidates that potentially regulate R-loop metabolism in oocytes, zygotes and two-cell embryos based on change of their gene translation. Our analysis revealed that the DDX5 inhibition of activity led to decreased R-loop accumulation in pronuclei, indicating its involvement in regulating R-loop dynamics. However, the inhibition of histone deacetylase-2 activity did not significantly affect R-loop levels in pronuclei. CONCLUSION: These findings suggest that dynamic changes in R-loops during mouse zygote development are likely regulated by RNA helicases, particularly DDX5, in conjunction with transcriptional processes. Our study provides compelling evidence for the involvement of these factors in regulating R-loop dynamics during early embryonic development.

INO80 function is required for mouse mammary gland development, but mutation alone may be insufficient for breast cancer.

The aberrant function of ATP-dependent chromatin remodeler INO80 has been implicated in multiple types of cancers by altering chromatin architecture and gene expression; however, the underlying mechanism of the functional involvement of INO80 mutation in cancer etiology, especially in breast cancer, remains unclear. In the present study, we have performed a weighted gene co-expression network analysis (WCGNA) to investigate links between INO80 expression and breast cancer sub-classification and progression. Our analysis revealed that INO80 repression is associated with differential responsiveness of estrogen receptors (ERs) depending upon breast cancer subtype, ER networks, and increased risk of breast carcinogenesis. To determine whether INO80 loss induces breast tumors, a conditional INO80-knockout (INO80 cKO) mouse model was generated using the Cre-loxP system. Phenotypic characterization revealed that INO80 cKO led to reduced branching and length of the mammary ducts at all stages. However, the INO80 cKO mouse model had unaltered lumen morphology and failed to spontaneously induce tumorigenesis in mammary gland tissue. Therefore, our study suggests that the aberrant function of INO80 is potentially associated with breast cancer by modulating gene expression. INO80 mutation alone is insufficient for breast tumorigenesis.

Altered Mitochondrial Function and Accelerated Aging Phenotype in Neural Stem Cells Derived from Dnm1l Knockout Embryonic Stem Cells.

Mitochondria are crucial for cellular energy metabolism and are involved in signaling, aging, and cell death. They undergo dynamic changes through fusion and fission to adapt to different cellular states. In this study, we investigated the effect of knocking out the dynamin 1-like protein (Dnm1l) gene, a key regulator of mitochondrial fission, in neural stem cells (NSCs) differentiated from Dnm1l knockout embryonic stem cells (Dnm1l-/- ESCs). Dnm1l-/- ESC-derived NSCs (Dnm1l-/- NSCs) exhibited similar morphology and NSC marker expression (Sox2, Nestin, and Pax6) to brain-derived NSCs, but lower Nestin and Pax6 expression than both wild-type ESC-derived NSCs (WT-NSCs) and brain-derived NSCs. In addition, compared with WT-NSCs, Dnm1l-/- NSCs exhibited distinct mitochondrial morphology and function, contained more elongated mitochondria, showed reduced mitochondrial respiratory capacity, and showed a metabolic shift toward glycolysis for ATP production. Notably, Dnm1l-/- NSCs exhibited impaired self-renewal ability and accelerated cellular aging during prolonged culture, resulting in decreased proliferation and cell death. Furthermore, Dnm1l-/- NSCs showed elevated levels of inflammation and cell stress markers, suggesting a connection between Dnm1l deficiency and premature aging in NSCs. Therefore, the compromised self-renewal ability and accelerated cellular aging of Dnm1l-/- NSCs may be attributed to mitochondrial fission defects.

Pre-implantation development of cattle embryos produced from fresh bull semen enriched for X- chromosome-bearing spermatozoa using a monoclonal antibody.

Immunological approaches are gaining attention as a convenient and economical method for sex-sorting mammalian spermatozoa. A monoclonal antibody (WholeMom™) has previously been reported to cause agglutination of Y-chromosome-bearing spermatozoa in frozen-thawed semen for gender preselection. However, its usefulness for gender preselection in fresh semen and subsequent in vitro fertilization (IVF) after freeze-thawing has not been reported. This study investigated the in vitro development of cattle embryos produced from fresh bull semen pre-treated with WholeMom™ monoclonal antibody. Results showed that antibody-treated, non-agglutinated spermatozoa (presumably X-chromosome-bearing spermatozoa) could fertilize cattle oocytes in vitro. However, embryos generated from non-agglutinated (enriched in X-chromosome-bearing spermatozoa) had a lower (p < 0.05) ability to cleave (66.4 ± 2.5% vs. 75.1 ± 3.3%) than those of non-treated control sperm. Nevertheless, the percentage of blastocysts developed from cleaved embryos did not differ (p > 0.05) between the groups (34.8 ± 3.7% vs. 35.8 ± 3.4%). Duplex PCR of blastocysts, using a bovine-specific universal primer pair and a Y-chromosome-specific primer pair, showed a sex ratio of 95.8% females from sex-sorted spermatozoa, which was higher than those of non-treated control spermatozoa (46.4%). In conclusion, the results of the present study suggest that monoclonal antibody-based enrichment of X- chromosome-bearing spermatozoa can be applied to fresh bull semen without compromising their post-fertilization early embryonic development to the blastocyst stage. Future studies should investigate the term development and sex ratio of calves from antibody-treated spermatozoa.

Embryonic Stem Cells Lacking DNA Methyltransferases Differentiate into Neural Stem Cells that Are Defective in Self-Renewal.

Background and Objectives: DNA methyltransferases (Dnmts) play an important role in regulating DNA methylation during early developmental processes and cellular differentiation. In this study, we aimed to investigate the role of Dnmts in neural differentiation of embryonic stem cells (ESCs) and in maintenance of the resulting neural stem cells (NSCs). Methods and Results: We used three types of Dnmt knockout (KO) ESCs, including Dnmt1 KO, Dnmt3a/3b double KO (Dnmt3 DKO), and Dnmt1/3a/3b triple KO (Dnmt TKO), to investigate the role of Dnmts in neural differentiation of ESCs. All three types of Dnmt KO ESCs could form neural rosette and differentiate into NSCs in vitro. Interestingly, however, after passage three, Dnmt KO ESC-derived NSCs could not maintain their self-renewal and differentiated into neurons and glial cells. Conclusions: Taken together, the data suggested that, although deficiency of Dnmts had no effect on the differentiation of ESCs into NSCs, the latter had defective maintenance, thereby indicating that Dnmts are crucial for self-renewal of NSCs.

INO80 Is Required for the Cell Cycle Control, Survival, and Differentiation of Mouse ESCs by Transcriptional Regulation.

Precise regulation of the cell cycle of embryonic stem cells (ESCs) is critical for their self-maintenance and differentiation. The cell cycle of ESCs differs from that of somatic cells and is different depending on the cell culture conditions. However, the cell cycle regulation in ESCs via epigenetic mechanisms remains unclear. Here, we showed that the ATP-dependent chromatin remodeler Ino80 regulates the cell cycle genes in ESCs under primed conditions. Ino80 loss led to a significantly extended length of the G1-phase in ESCs grown under primed culture conditions. Ino80 directly bound to the transcription start site and regulated the expression of cell cycle-related genes. Furthermore, Ino80 loss induced cell apoptosis. However, the regulatory mechanism of Ino80 in differentiating ESC cycle slightly differed; an extended S-phase was detected in differentiating inducible Ino80 knockout ESCs. RNA-seq analysis of differentiating ESCs revealed that the expression of genes associated with organ development cell cycle is persistently altered in Ino80 knockout cells, suggesting that cell cycle regulation by Ino80 is not limited to undifferentiated ESCs. Therefore, our study establishes the function of Ino80 in ESC cycle via transcriptional regulation, at least partly. Moreover, this Ino80 function may be universal to other cell types.

Inhibition of neural stem cell aging through the transient induction of reprogramming factors.

Adult stem cells age during long-term in vitro culture, and neural stem cells (NSCs), which can self-renew and differentiate into neurons and glial cells, also display reduced differentiation potential after repeated passaging. However, the mechanistic details underlying this process remain unclear. In this study, we found that long-term in vitro culture of NSCs resulted in aging-related upregulation of inflammatory- and endoplasmic reticulum (ER) stress-related genes, including the proinflammatory cytokines interleukin (IL)1ß and IL6, the senescence-associated enzyme matrix metallopeptidase 13 (MMP13), and the ER stress-responsive transcription factor activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). However, the cyclic and transient induction of four reprogramming factors (POU domain, class 5, transcription factor 1, also known as octamer-binding transcription factor 4; SRY [sex determining region Y]-box 2; Kruppel-like factor 4; and myelocytomatosis oncogene; collectively referred to as OSKM) can inhibit NSC aging, as indicated by the decreased expression of the inflammatory and ER stress-related genes. We used ROSA-4F NSCs, which express OSKM from only one allele, to minimize the potential for full reprogramming or tumor formation during NSC rejuvenation. We expect that this novel rejuvenation method will enhance the potential of NSCs as a clinical approach to the treatment of neurological diseases.

Combinatorial ethanol treatment increases the overall productivity of recombinant hG-CSF in E. coli: a comparative study.

Human granulocyte colony-stimulating factor (hG-CSF) is a cytokine that regulates the proliferation, maturation, and differentiation of precursor cells to neutrophils. In the present study, we report the feasibility of inducing recombinant hG-CSF expression (rhG-CSF) in a pET vector system by combinatorial induction using low-concentration ethanol, IPTG, and lactose and auto-induction media (AIM). The coding sequence of hG-CSF transcript variant 2 was expressed in pET14 vector, and the effect of combinatorial induction was analyzed on inclusion body (IB) formation, biomass, protein purification, and bioactivity. Results showed that there was an inverse relationship between the temperature and soluble expression of rhG-CSF. Three-step washing with Triton-X, 2 M, and 5 M urea resulted in the maximum recovery of IBs. Combinatorial single-spike induction with IPTG, ethanol, and lactose in a batch culture led to a 3-fold increase in the expression of rhG-CSF. It was also observed that low concentration of ethanol (1-3% v/v) could be used in lieu of IPTG for inducing the rhG-CSF protein expression without adversely affecting biomass production. A 2.4-fold increase in productivity was obtained in LB-AIM media with combinatorial ethanol induction, and the overall yield of 2.8 g/L rhG-CSF was found. The purified rhG-CSF was bioactive and increased the cellular proliferation of umbilical cord blood-derived mesenchymal stem cells (U-MSC) by 29%. In conclusion, our study shows that combined ethanol induction can enhance the expression of rhG-CSF with three-step washing for recovery of the proteins from IBs and a single-step purification of rhG-CSF by affinity chromatography. KEY POINTS: • Low concentration of ethanol (1-3%) could be used in lieu of IPTG for inducing rhG-CSF expression. • Combinatorial single-spike induction with IPTG, ethanol, and lactose improved rhG-CSF expression. • Purified rhG-CSF was bioactive and increased the proliferation of U-MSC.

Differential Regulation of TLE3 in Sertoli Cells of the Testes during Postnatal Development.

Spermatogenesis is a process by which haploid cells differentiate from germ cells in the seminiferous tubules of the testes. TLE3, a transcriptional co-regulator that interacts with DNA-binding factors, plays a role in the development of somatic cells. However, no studies have shown its role during germ cell development in the testes. Here, we examined TLE3 expression in the testes during spermatogenesis. TLE3 was highly expressed in mouse testes and was dynamically regulated in different cell types of the seminiferous tubules, spermatogonia, spermatids, and Sertoli cells, but not in the spermatocytes. Interestingly, TLE3 was not detected in Sertoli cells on postnatal day 7 (P7) but was expressed from P10 onward. The microarray analysis showed that the expression of numerous genes changed upon TLE3 knockdown in a Sertoli cell line TM4. These include 1597 up-regulated genes and 1452 down-regulated genes in TLE3-knockdown TM4 cells. Ingenuity Pathway Analysis (IPA) showed that three factors were up-regulated and two genes were down-regulated upon TLE3 knockdown in TM4 cells. The abnormal expression of the three factors is associated with cellular malfunctions such as abnormal differentiation and Sertoli cell formation. Thus, TLE3 is differentially expressed in Sertoli cells and plays a crucial role in regulating cell-specific genes involved in the differentiation and formation of Sertoli cells during testicular development.

5-Azacytidine improves the meiotic maturation and subsequent in vitro development of pig oocytes.

Treatment of donor cells and/or cloned embryos with cytidine analogues, having an Aza group at its 5th carbon (5-Aza), such as 5-Azacytidine (5-Aza-C) or 5-Aza-2'-deoxycytidine (5-Aza-dC) improves the in vitro development of cloned embryos produced by somatic cell nuclear transfer (SCNT). In vitro maturation (IVM) of immature pig oocytes treated with 5-Aza-C not only results in greater (P < 0.05) meiotic maturation to the MII stage but also enhances the capacity of 5-Aza-C treated oocytes for early embryonic development after parthenogenetic activation (PA), in vitro fertilization (IVF) or SCNT in a dose-dependent manner (0-10 µM). Cloned embryos generated from 5-Aza-C (0.01 µM) treated oocytes had an increased capacity to develop to the blastocyst stage (14.1 ±â€¯1.5% compared with 9.6 ±â€¯1.8%), greater probability of hatching (61.8 ±â€¯1.5% compared with 45.0 ±â€¯3.9%) and contained a greater number of cells per blastocyst (38.5 ±â€¯4.4 compared with 30.5 ±â€¯3.4) than those produced from non-treated control oocytes (P < 0.05). Data from the present study indicate that treatment of oocytes with 5-Aza-C may be an important approach to enhance the meiotic maturation and subsequent in vitro development of pig embryos. Future studies should be conducted to determine the underlying mechanism of improved early embryonic development of 5-Aza-C treated oocytes.

Evaluation of Graphene Oxide Induced Cellular Toxicity and Transcriptome Analysis in Human Embryonic Kidney Cells.

Graphene, a two-dimensional carbon sheet with single-atom thickness, shows immense promise in several nanoscientific and nanotechnological applications, including in sensors, catalysis, and biomedicine. Although several studies have shown the cytotoxicity of graphene oxide in different cell types, there are no comprehensive studies on human embryonic kidney (HEK293) cells that include transcriptomic analysis and an in vitro investigation into the mechanisms of cytotoxicity following exposure to graphene oxide. Therefore, we exposed HEK293 cells to different concentrations of graphene oxide for 24 h and performed several cellular assays. Cell viability and proliferation assays revealed a significant dose-dependent cytotoxic effect on HEK293 cells. Cytotoxicity assays showed increased lactate dehydrogenase (LDH) leakage and reactive oxygen species (ROS) generation, and decreased levels of reduced glutathione (GSH) and increased level of oxidized glutathione indicative of oxidative stress. This detailed mechanistic approach showed that graphene oxide exposure elicits significant decreases in mitochondrial membrane potential and ATP synthesis, as well as in DNA damage and caspase 3 activity. Furthermore, our RNA-Seq analysis revealed that HEK293 cells exposed to graphene oxide significantly altered the expression of genes involved in multiple apoptosis-related biological pathways. Moreover, graphene oxide exposure perturbed the expression of key transcription factors, promoting these apoptosis-related pathways by regulating their downstream genes. Our analysis provides mechanistic insights into how exposure to graphene oxide induces changes in cellular responses and massive cell death in HEK293 cells. To our knowledge, this is the first study describing a combination of cellular responses and transcriptome in HEK293 cells exposed to graphene oxide nanoparticles, providing a foundation for understanding the molecular mechanisms of graphene oxide-induced cytotoxicity and for the development of new therapeutic strategies.

Cytotoxicity and Transcriptomic Analyses of Biogenic Palladium Nanoparticles in Human Ovarian Cancer Cells (SKOV3).

Ovarian cancer incidence continues to increase at an alarming rate. Although various therapeutic approaches exist for ovarian cancer, they have limitations, including undesired side effects. Therefore, nanoparticle (NP)-mediated therapy may be a viable, biocompatible, and suitable alternative. To the best of our knowledge, no comprehensive analysis has been undertaken on the cytotoxicity and cellular pathways involved in ovarian cancer cells, particularly SKOV3 cells. Here, we investigated the effect of palladium NPs (PdNPs) and the molecular mechanisms and cellular pathways involved in ovarian cancer. We assayed cell viability, proliferation, cytotoxicity, oxidative stress, DNA damage, and apoptosis and performed an RNA-Seq analysis. The results showed that PdNPs elicited concentration-dependent decreases in cell viability and proliferation and induced increasing cytotoxicity at increasing concentrations, as determined by leakage of lactate dehydrogenase, increased levels of reactive oxygen species and malondialdehyde, and decreased levels of antioxidants like glutathione and superoxide dismutase. Furthermore, our study revealed that PdNPs induce mitochondrial dysfunction by altering mitochondrial membrane potential, reducing adenosine triphosphate levels, inducing DNA damage, and activating caspase 3, all of which significantly induced apoptosis in SKOV3 cells following PdNPs treatment. Gene ontology (GO) term analysis of PdNPs-exposed SKOV3 cells showed various dysregulated pathways, particularly nucleosome assembly, telomere organization, and rDNA chromatin silencing. When genes downregulated by PdNPs were applied to GO term enrichment analysis, nucleosome assembly was the top-ranked biological pathway. We also provide evidence for an association between PdNPs exposure and multiple layers of epigenetic transcriptional control and establish a molecular basis for NP-mediated apoptosis. These findings provide a foundation, potential targets, and novel insights into the mechanism underlying toxicity and pathways in SKOV3 cells, and open new avenues to identify novel targets for ovarian cancer treatment.

Intermediate Reprogramming of Mouse Skin Fibroblasts into Stem-Like Cells by Bone Morphogenetic Protein 4.

Specific transcription factors are sufficient to reprogram fully induced pluripotent stem cells or other types of cells. These findings raise the question of whether chemical molecules or proteins can replace transcription factors to alter the defined cell fate. In this study, we treated mouse skin fibroblasts (MSFs) with bone morphogenetic protein 4 (BMP4) and examined intermediate reprogramming of MSFs into stem-like cells. Putative epidermal stem cells isolated from the ventral skin epidermis of an adult mouse were used to confirm the reprogramming activity of BMP4, which increased the proliferation of these cells. After these cells formed spheroids, they were treated with BMP4 and cultured for 5 days. Following BMP4 treatment, the characteristics of these cells changed, and they expressed Oct-4 and its target transcripts Nanog, Sox2, and alkaline phosphatase. To confirm the stem cell potency of these cells, we induced their differentiation into cardiomyocytes. Stem-like cell-derived cardiomyocytes exhibited mRNA expression of cardiac mesoderm markers such as Nk2 transcription factor-related locus 5 and connexin 40, and the cardiomyocyte marker troponin T. These differentiated cells exhibited contracting masses. These results suggest that BMP4-mediated somatic stem cell reprogramming may become an alternative approach for cell therapy.

Changes in Parthenogenetic Imprinting Patterns during Reprogramming by Cell Fusion.

Differentiated somatic cells can be reprogrammed into the pluripotent state by cell-cell fusion. In the pluripotent state, reprogrammed cells may then self-renew and differentiate into all three germ layers. Fusion-induced reprogramming also epigenetically modifies the somatic cell genome through DNA demethylation, X chromosome reactivation, and histone modification. In this study, we investigated whether fusion with embryonic stem cells (ESCs) also reprograms genomic imprinting patterns in somatic cells. In particular, we examined imprinting changes in parthenogenetic neural stem cells fused with biparental ESCs, as well as in biparental neural stem cells fused with parthenogenetic ESCs. The resulting hybrid cells expressed the pluripotency markers Oct4 and Nanog. In addition, methylation of several imprinted genes except Peg3 was comparable between hybrid cells and ESCs. This finding indicates that reprogramming by cell fusion does not necessarily reverse the status of all imprinted genes to the state of pluripotent fusion partner.

Pig Spermatozoa Defect in Acrosome Formation Caused Poor Motion Parameters and Fertilization Failure through Artificial Insemination and In vitro Fertilization.

The selection of morphologically normal spermatozoa is critical to obtain high breeding performances in boar breeding farms and artificial insemination (AI) centers. Parameters for the selection of semen mainly include total sperm motility, concentration, and morphology. However, these primary parameters are often not reliable for discriminating between normal and abnormal, non-fertilizable spermatozoa. The present study was designed to compare the motion characteristics, fertilization ability using in vitro fertilization (IVF), and acrosome formation of the semen from boars having low (boar number 2012) and normal (boar number 2004 and 2023) breeding performances. The ultimate goal was to identify additional simple and easy criteria for the selection of normal sperm. There was no significant difference between boar 2004 and boar 2023 sperm total motility in computer assisted sperm analysis. However, boar number 2012 semen presented a significantly reduced population of rapid moving spermatozoa and an increased population of slow moving spermatozoa compared to boar numbers 2004 and 2023. Analysis of detailed motion characteristics revealed that sperm from boar number 2012 had significantly reduced motility in progressiveness, average path velocity, straight-line velocity (VSL), curvilinear velocity (VCL), straightness, and linearity. The assessment of the fertilizing ability by IVF also showed that sperm from boar number 2012 showed a fertility rate of 3.4%, whereas sperm from boar number 2023 had a fertility rate of 75.45%. Interestingly, most of the sperm nuclei were found on the peripheral area of the oocytes, suggesting that the sperm from boar number 2012 lacked penetration ability into the oocyte zonapellucida. The acrosome formation analysis using Pisum sativum agglutinin staining demonstrated that the sperm from boar number 2012 had a defect in acrosome formation. Consequently, primary parameters for selecting semen before AI such as motility are not sufficient to select normal and fertilizable spermatozoa. In conclusion, the present study suggests that the acrosome staining and detailed motion characteristics such as progressiveness, VCL, and VSL should be included in determining semen quality together with primary parameters for successful AI and high breeding performance in the swine industry.

Differential expression of ETS family transcription factors in NCCIT human embryonic carcinoma cells upon retinoic acid-induced differentiation.

E26 transformation-specific (ETS) transcription factors play important roles in normal and tumorigenic processes during development, differentiation, homeostasis, proliferation, and apoptosis. To identify critical ETS factor(s) in germ cell-derived cancer cells, we examined the expression patterns of the 27 ETS transcription factors in naive and differentiated NCCIT human embryonic carcinoma cells, which exhibit both pluripotent and tumorigenic characteristics. Overall, expression of ETS factors was relatively low in NCCIT cells. Among the 27 ETS factors, polyomavirus enhancer activator 3 (PEA3) and epithelium-specific ETS transcription factor-1 (ESE-1) exhibited the most significant changes in their expression levels. Western blot analysis confirmed these patterns, revealing reduced levels of PEA3 protein and elevated levels of ESE-1 protein in differentiated cells. PEA3 increased the proportion of cells in S-phase and promoted cell growth, whereas ESE-1 reduced proliferation potential. These data suggest that PEA3 and ESE-1 may play important roles in pluripotent and tumorigenic embryonic carcinoma cells. These findings contribute to our understanding of the functions of oncogenic ETS factors in germ cell-derived stem cells during processes related to tumorigenesis and pluripotency.

Supplementation of insulin-transferrin-selenium to embryo culture medium improves the in vitro development of pig embryos.

Insulin, transferrin and selenium (ITS) supplementation to oocyte maturation medium improves the post-fertilization embryonic development in pigs. ITS is also commonly used as a supplement for the in vitro culture (IVC) of embryos and stem cells in several mammalian species. However, its use during IVC of pig embryos has not been explored. This study investigated the effect of ITS supplementation to IVC medium on the in vitro development ability of pig embryos produced by parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). We observed that ITS had no significant effect on the rate of first cleavage (P > 0.05). However, the rate of blastocyst formation in ITS-treated PA (45.3 ± 1.9 versus 27.1 ± 2.3%), IVF (31.6 ± 0.6 versus 23.5 ± 0.6%) and SCNT (17.6 ± 2.3 versus 10.7 ± 1.4%) embryos was significantly higher (P < 0.05) than those of non-treated controls. Culture of PA embryos in the presence of ITS also enhanced the expansion and hatching ability (29.1 ± 3.0 versus 18.2 ± 3.8%; P < 0.05) of blastocysts and increased the total number of cells per blastocyst (53 ± 2.5 versus 40.9 ± 2.6; P < 0.05). Furthermore, the beneficial effect of ITS on PA embryos was associated with significantly reduced level of intracellular reactive oxygen species (ROS) (20.0 ± 2.6 versus 46.9 ± 3.0). However, in contrast to PA embryos, ITS had no significant effect on the blastocyst quality of IVF and SCNT embryos (P > 0.05). Taken together, these data suggest that supplementation of ITS to the IVC medium exerts a beneficial but differential effect on pig embryos that varies with the method of embryo production in vitro.

Clinical outcomes of vitrified-thawed embryo transfer using a pull and cut straw method.

OBJECTIVE: To compare the clinical outcomes of patients with vitrified-thawed embryos transferred using either the 0.25 mL straw method and the pull and cut straw (PNC) method. To evaluate the clinical outcomes of patients with transferred embryos that underwent assisted hatching at the cleaved embryo (day 3) or the blastocyst (day 5) stage. METHODS: The study population consisted of women who underwent vitrified-warmed embryo transfer between May 2000 and December 2011 and assisted hatching was performed after warming of embryos. Cycles of thawing between assisted hatching treated and non treated groups were compared for survival and pregnancy rates. RESULTS: The PNC vitrification method improved survival and pregnancy rates in partial lysed embryos. While assisted hatching did not affect the developmental and clinical pregnancy rates of the vitrified-warmed blastocyst group, it did increase the pregnancy rate of poor quality vitrified-warmed cleaved embryos. CONCLUSION: These results suggest that PNC may increase the number of clinical pregnancies via the vitrification of both cleaved embryos and blastocysts. In addition, selective assisted hatching treatment of embryos that show a poor prognosis after warming may increase the rate of clinical pregnancy.

Transgenic chicken, mice, cattle, and pig embryos by somatic cell nuclear transfer into pig oocytes.

This study explored the possibility of producing transgenic cloned embryos by interspecies somatic cell nuclear transfer (iSCNT) of cattle, mice, and chicken donor cells into enucleated pig oocytes. Enhanced green florescent protein (EGFP)-expressing donor cells were used for the nuclear transfer. Results showed that the occurrence of first cleavage did not differ significantly when pig, cattle, mice, or chicken cells were used as donor nuclei (p>0.05). However, the rate of blastocyst formation was significantly higher in pig (14.9±2.1%; p<0.05) SCNT embryos than in cattle (6.3±2.5%), mice (4.2±1.4%), or chicken (5.1±2.4%) iSCNT embryos. The iSCNT embryos also contained a significantly less number of cells per blastocyst than those of SCNT pig embryos (p<0.05). All (100%) iSCNT embryos expressed the EGFP gene, as evidenced by the green florescence under ultraviolet (UV) illumination. Microinjection of purified mitochondria from cattle somatic cells into pig oocytes did not have any adverse effect on their postfertilization in vitro development and embryo quality (p>0.05). Moreover, NCSU23 medium, which was designed for in vitro culture of pig embryos, was able to support the in vitro development of cattle, mice, and chicken iSCNT embryos up to the blastocyst stage. Taken together, these data suggest that enucleated pig oocytes may be used as a universal cytoplast for production of transgenic cattle, mice, and chicken embryos by iSCNT. Furthermore, xenogenic transfer of mitochondria to the recipient cytoplast may not be the cause for poor embryonic development of cattle-pig iSCNT embryos.