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Sample records for activated porcine embryos

  1. ROCK activity regulates functional tight junction assembly during blastocyst formation in porcine parthenogenetic embryos

    PubMed Central

    Kwon, Jeongwoo

    2016-01-01

    The Rho-associated coiled-coil-containing protein serine/threonine kinases 1 and 2 (ROCK1 and ROCK2) are Rho subfamily GTPase downstream effectors that regulate cell migration, intercellular adhesion, cell polarity, and cell proliferation by stimulating actin cytoskeleton reorganization. Inhibition of ROCK proteins affects specification of the trophectoderm (TE) and inner cell mass (ICM) lineages, compaction, and blastocyst cavitation. However, the molecules involved in blastocyst formation are not known. Here, we examined developmental competence and levels of adherens/tight junction (AJ/TJ) constituent proteins, such as CXADR, OCLN, TJP1, and CDH1, as well as expression of their respective mRNAs, after treating porcine parthenogenetic four-cell embryos with Y-27632, a specific inhibitor of ROCK, at concentrations of 0, 10, 20, 100 µM for 24 h. Following this treatment, the blastocyst development rates were 39.1, 20.7, 10.0, and 0% respectively. In embryos treated with 20 µM treatment, expression levels of CXADR, OCLN, TJP1, and CDH1 mRNA and protein molecules were significantly reduced (P < 0.05). FITC-dextran uptake assay revealed that the treatment caused an increase in TE TJ permeability. Interestingly, the majority of the four-cell and morula embryos treated with 20 µM Y-27643 for 24 h showed defective compaction and cavitation. Taken together, our results indicate that ROCK activity may differentially affect assembly of AJ/TJs as well as regulate expression of genes encoding junctional proteins. PMID:27077008

  2. RNA Profiles of Porcine Embryos during Genome Activation Reveal Complex Metabolic Switch Sensitive to In Vitro Conditions

    PubMed Central

    Østrup, Olga; Olbricht, Gayla; Østrup, Esben; Hyttel, Poul; Collas, Philippe; Cabot, Ryan

    2013-01-01

    Fertilization is followed by complex changes in cytoplasmic composition and extensive chromatin reprogramming which results in the abundant activation of totipotent embryonic genome at embryonic genome activation (EGA). While chromatin reprogramming has been widely studied in several species, only a handful of reports characterize changing transcriptome profiles and resulting metabolic changes in cleavage stage embryos. The aims of the current study were to investigate RNA profiles of in vivo developed (ivv) and in vitro produced (ivt) porcine embryos before (2-cell stage) and after (late 4-cell stage) EGA and determine major metabolic changes that regulate totipotency. The period before EGA was dominated by transcripts responsible for cell cycle regulation, mitosis, RNA translation and processing (including ribosomal machinery), protein catabolism, and chromatin remodelling. Following EGA an increase in the abundance of transcripts involved in transcription, translation, DNA metabolism, histone and chromatin modification, as well as protein catabolism was detected. The further analysis of members of overlapping GO terms revealed that despite that comparable cellular processes are taking place before and after EGA (RNA splicing, protein catabolism), different metabolic pathways are involved. This strongly suggests that a complex metabolic switch accompanies EGA. In vitro conditions significantly altered RNA profiles before EGA, and the character of these changes indicates that they originate from oocyte and are imposed either before oocyte aspiration or during in vitro maturation. IVT embryos have altered content of apoptotic factors, cell cycle regulation factors and spindle components, and transcription factors, which all may contribute to reduced developmental competence of embryos produced in vitro. Overall, our data are in good accordance with previously published, genome-wide profiling data in other species. Moreover, comparison with mouse and human embryos

  3. The effect of horse serum on in vitro development of porcine parthenogenetic embryos.

    PubMed

    Hwang, In-Sul; Kim, Sung Woo; Byun, Sung June; Kim, Kyung-Woon; Chung, Hee Kyoung; Park, Jong-Ju; Gobianand, Kuppannan; Park, Jin-Ki; Yoo, Jae Gyu

    2012-03-01

    The objective of this study was to examine the effect of different sera and serum-like substances on the preimplantation development of porcine parthenogenetic embryos. Chemically activated (calcium ionophore A23187+cytochalasin B) pig oocytes were pre-cultured for five days. On day 5, the parthenogenetic embryos were treated with porcine follicular fluid (PFF), fetal bovine serum (FBS), horse serum (HS) or porcine serum albumin (PSA), and were cultured two more days. Horse serum was found to be the most effective protein source in enhancing parthenote development judging by blastocyst formation and hatching. Next, three different concentrations of HS (10, 20 and 30%) were used to determine the optima HS concentration needed to improve the development of porcine parthenogenetic embryos. All HS concentrations increased the blastocyst cell number and decreased the incidence of blastocyst apoptotic cells with 20% being the most effective. In conclusion, horse serum enhanced parthenogenetic embryo development and the quality of porcine parthenogenetic embryos. PMID:22472938

  4. Cloning of Porcine Pituitary Tumor Transforming Gene 1 and Its Expression in Porcine Oocytes and Embryos.

    PubMed

    Xie, Bingkun; Qin, Zhaoxian; Liu, Shuai; Nong, Suqun; Ma, Qingyan; Chen, Baojian; Liu, Mingjun; Pan, Tianbiao; Liao, D Joshua

    2016-01-01

    The maternal-to-embryonic transition (MET) is a complex process that occurs during early mammalian embryogenesis and is characterized by activation of the zygotic genome, initiation of embryonic transcription, and replacement of maternal mRNA with embryonic mRNA. The objective of this study was to reveal the temporal expression and localization patterns of PTTG1 during early porcine embryonic development and to establish a relationship between PTTG1 and the MET. To achieve this goal, reverse transcription-polymerase chain reaction (RT-PCR) was performed to clone porcine PTTG1. Subsequently, germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, zygotes, 2-, 4-, and 8-cell-stage embryos, morulas, and blastocysts were produced in vitro and their gene expression was analyzed. The results revealed that the coding sequence of porcine PTTG1 is 609-bp in length and that it encodes a 202-aa polypeptide. Using qRT-PCR, PTTG1 mRNA expression was observed to be maintained at high levels in GV- and MII-stage oocytes. The transcript levels in oocytes were also significantly higher than those in embryos from the zygote to blastocyst stages. Immunohistochemical analyses revealed that porcine PTTG1 was primarily localized to the cytoplasm and partially localized to the nucleus. Furthermore, the PTTG1 protein levels in MII-stage oocytes and zygotes were significantly higher than those in embryos from the 2-cell to blastocyst stage. After fertilization, the level of this protein began to decrease gradually until the blastocyst stage. The results of our study suggest that porcine PTTG1 is a new candidate maternal effect gene (MEG) that may participate in the processes of oocyte maturation and zygotic genome activation during porcine embryogenesis. PMID:27058238

  5. Cloning of Porcine Pituitary Tumor Transforming Gene 1 and Its Expression in Porcine Oocytes and Embryos

    PubMed Central

    Liu, Shuai; Nong, Suqun; Ma, Qingyan; Chen, Baojian; Liu, Mingjun; Pan, Tianbiao; Liao, D. Joshua

    2016-01-01

    The maternal-to-embryonic transition (MET) is a complex process that occurs during early mammalian embryogenesis and is characterized by activation of the zygotic genome, initiation of embryonic transcription, and replacement of maternal mRNA with embryonic mRNA. The objective of this study was to reveal the temporal expression and localization patterns of PTTG1 during early porcine embryonic development and to establish a relationship between PTTG1 and the MET. To achieve this goal, reverse transcription-polymerase chain reaction (RT-PCR) was performed to clone porcine PTTG1. Subsequently, germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, zygotes, 2-, 4-, and 8-cell-stage embryos, morulas, and blastocysts were produced in vitro and their gene expression was analyzed. The results revealed that the coding sequence of porcine PTTG1 is 609-bp in length and that it encodes a 202-aa polypeptide. Using qRT-PCR, PTTG1 mRNA expression was observed to be maintained at high levels in GV- and MII-stage oocytes. The transcript levels in oocytes were also significantly higher than those in embryos from the zygote to blastocyst stages. Immunohistochemical analyses revealed that porcine PTTG1 was primarily localized to the cytoplasm and partially localized to the nucleus. Furthermore, the PTTG1 protein levels in MII-stage oocytes and zygotes were significantly higher than those in embryos from the 2-cell to blastocyst stage. After fertilization, the level of this protein began to decrease gradually until the blastocyst stage. The results of our study suggest that porcine PTTG1 is a new candidate maternal effect gene (MEG) that may participate in the processes of oocyte maturation and zygotic genome activation during porcine embryogenesis. PMID:27058238

  6. Generation and Developmental Characteristics of Porcine Tetraploid Embryos and Tetraploid/diploid Chimeric Embryos

    PubMed Central

    He, Wenteng; Kong, Qingran; Shi, Yongqian; Xie, Bingteng; Jiao, Mingxia; Huang, Tianqing; Guo, Shimeng; Hu, Kui; Liu, Zhonghua

    2013-01-01

    The aim of this study was to optimize electrofusion conditions for generating porcine tetraploid (4n) embryos and produce tetraploid/diploid (4n/2n) chimeric embryos. Different electric field intensities were tested and 2 direct current (DC) pulses of 0.9 kV/cm for 30 μs was selected as the optimum condition for electrofusion of 2-cell embryos to produce 4n embryos. The fusion rate of 2-cell embryos and the development rate to blastocyst of presumably 4n embryos, reached 85.4% and 28.5%, respectively. 68.18% of the fused embryos were found to be 4n as demonstrated by fluorescent in situ hybridization (FISH). Although the number of blastomeres in 4n blastocysts was significantly lower than in 2n blastocysts (P < 0.05), there was no significant difference in developmental rates of blastocysts between 2n and 4n embryos (P > 0.05), suggesting that the blastocyst forming capacity in 4n embryos is similar to those in 2n embryos. Moreover, 4n/2n chimeric embryos were obtained by aggregation of 4n and 2n embryos. We found that the developmental rate and cell number of blastocysts of 4-cell (4n)/4-cell (2n) chimeric embryos were significantly higher than those of 2-cell (4n)/4-cell (2n), 4-cell (4n)/8-cell (2n), 4-cell (4n)/2-cell (2n) chimeric embryos (P < 0.05). Consistent with mouse chimeras, the majority of 4n cells contribute to the trophectoderm (TE), while the 2n cells are mainly present in the inner cell mass (ICM) of porcine 4n/2n chimeric embryos. Our study established a feasible and efficient approach to produce porcine 4n embryos and 4n/2n chimeric embryos. PMID:24120753

  7. Cryotop vitrification of porcine parthenogenetic embryos at the early developmental stages.

    PubMed

    Wu, Guo-Quan; Quan, Guo-Bo; Shao, Qing-Yong; Lv, Chun-Rong; Jiang, Yan-Ting; Zhao, Zhi-Yong; Hong, Qiong-Hua

    2016-02-01

    The objective of this study was to evaluate the effects of early developmental stages at which Cryotop vitrification is performed on subsequent survival and in vitro development of porcine parthenogenetic activation embryos. The zygotes that were cultured for 4, 8, and 18 hours post electric activation (h.p.a.) and two- and four-cell embryos were vitrified, warmed, and continuously cultured for the remaining period. The zygotes vitrified at 4, 8, and 18 h.p.a. showed similar percentages of survival, cleavage, and blastocyst formation. No difference in viability was observed after vitrification of two- and four-cell embryos, but the embryos vitrified at the two-cell stage exhibited significantly higher blastocyst formation rate than those vitrified at the four-cell stage. However, vitrifying embryos resulted in significantly decreased survival and development rates, regardless of the developmental stage of the embryos. In addition, the final developmental stage, diameter, apoptotic index, and the number of inner cell mass, trophectoderm, and total cells of blastocysts derived from embryos vitrified at any stage of the early culture were similar to those of fresh blastocysts. In conclusion, our data indicate that the early-stage porcine parthenogenetically activated embryos including the zygote, two cells, and four cells have a high ability to survive cryopreservation; these viable embryos after vitrification can produce respectable development rates and good-quality blastocysts. PMID:26462660

  8. PXD101 significantly improves nuclear reprogramming and the in vitro developmental competence of porcine SCNT embryos

    SciTech Connect

    Jin, Jun-Xue; Kang, Jin-Dan; Li, Suo; Jin, Long; Zhu, Hai-Ying; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun

    2015-01-02

    Highlights: • First explored that the effects of PXD101 on the development of SCNT embryos in vitro. • 0.5 μM PXD101 treated for 24 h improved the development of porcine SCNT embryos. • Level of AcH3K9 was significantly higher than control group at early stages. - Abstract: In this study, we investigated the effects of the histone deacetylase inhibitor PXD101 (belinostat) on the preimplantation development of porcine somatic cell nuclear transfer (SCNT) embryos and their expression of the epigenetic markers histone H3 acetylated at lysine 9 (AcH3K9). We compared the in vitro developmental competence of SCNT embryos treated with various concentrations of PXD101 for 24 h. Treatment with 0.5 μM PXD101 significantly increased the proportion of SCNT embryos that reached the blastocyst stage, in comparison to the control group (23.3% vs. 11.5%, P < 0.05). We tested the in vitro developmental competence of SCNT embryos treated with 0.5 μM PXD101 for various amounts of times following activation. Treatment for 24 h significantly improved the development of porcine SCNT embryos, with a significantly higher proportion of embryos reaching the blastocyst stage in comparison to the control group (25.7% vs. 10.6%, P < 0.05). PXD101-treated SCNT embryos were transferred into two surrogate sows, one of whom became pregnant and four fetuses developed. PXD101 treatment significantly increased the fluorescence intensity of immunostaining for AcH3K9 in embryos at the pseudo-pronuclear and 2-cell stages. At these stages, the fluorescence intensities of immunostaining for AcH3K9 were significantly higher in PXD101-treated embryos than in control untreated embryos. In conclusion, this study demonstrates that PXD101 can significantly improve the in vitro and in vivo developmental competence of porcine SCNT embryos and can enhance their nuclear reprogramming.

  9. Effects of Histone Deacetylase Inhibitor Oxamflatin on In Vitro Porcine Somatic Cell Nuclear Transfer Embryos

    PubMed Central

    Hou, Liming; Ma, Fanhua; Yang, Jinzeng; Riaz, Hasan; Wang, Yongliang; Wu, Wangjun; Xia, Xiaoliang; Ma, Zhiyuan; Zhou, Ying; Zhang, Lin; Ying, Wenqin; Xu, Dequan; Zuo, Bo; Ren, Zhuqing

    2014-01-01

    Abstract Low cloning efficiency is considered to be caused by the incomplete or aberrant epigenetic reprogramming of differentiated donor cells in somatic cell nuclear transfer (SCNT) embryos. Oxamflatin, a novel class of histone deacetylase inhibitor (HDACi), has been found to improve the in vitro and full-term developmental potential of SCNT embryos. In the present study, we studied the effects of oxamflatin treatment on in vitro porcine SCNT embryos. Our results indicated that the rate of in vitro blastocyst formation of SCNT embryos treated with 1 μM oxamflatin for 15 h postactivation was significantly higher than all other treatments. Treatment of oxamflatin decreased the relative histone deacetylase (HDAC) activity in cloned embryos and resulted in hyperacetylation levels of histone H3 at lysine 9 (AcH3K9) and histone H4 at lysine 5 (AcH4K5) at pronuclear, two-cell, and four-cell stages partly through downregulating HDAC1. The suppression of HDAC6 through oxamflatin increased the nonhistone acetylation level of α-tubulin during the mitotic cell cycle of early SCNT embryos. In addition, we demonstrated that oxamflatin downregulated DNA methyltransferase 1 (DNMT1) expression and global DNA methylation level (5-methylcytosine) in two-cell-stage porcine SCNT embryos. The pluripotency-related gene POU5F1 was found to be upregulated in the oxamflatin-treated group with a decreased DNA methylation tendency in its promoter regions. Treatment of oxamflatin did not change the locus-specific DNA methylation levels of Sus scrofa heterochromatic satellite DNA sequences at the blastocyst stage. Meanwhile, our findings suggest that treatment with HDACi may contribute to maintaining the stable status of cytoskeleton-associated elements, such as acetylated α-tubulin, which may be the crucial determinants of donor nuclear reprogramming in early SCNT embryos. In summary, oxamflatin treatment improves the developmental potential of porcine SCNT embryos in vitro. PMID

  10. Oxamflatin Treatment Enhances Cloned Porcine Embryo Development and Nuclear Reprogramming*

    PubMed Central

    Mao, Jiude; Zhao, Ming-Tao; Whitworth, Kristin M.; Spate, Lee D.; Walters, Eric M.; O'Gorman, Chad; Lee, Kiho; Samuel, Melissa S.; Murphy, Clifton N.; Wells, Kevin; Rivera, Rocio M.

    2015-01-01

    Abstract Faulty epigenetic reprogramming of somatic nuclei is thought to be the main reason for low cloning efficiency by somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi), such as Scriptaid, improve developmental competence of SCNT embryos in several species. Another HDACi, Oxamflatin, is about 100 times more potent than Scriptaid in the ability to inhibit nuclear-specific HDACs. The present study determined the effects of Oxamflatin treatment on embryo development, DNA methylation, and gene expression. Oxamflatin treatment enhanced blastocyst formation of SCNT embryos in vitro. Embryo transfer produced more pigs born and fewer mummies from the Oxamflatin-treated group compared to the Scriptaid-treated positive control. Oxamflatin also decreased DNA methylation of POU5F1 regulatory elements and centromeric repeat elements in day-7 blastocysts. When compared to in vitro–fertilized (IVF) embryos, the methylation status of POU5F1, NANOG, and centromeric repeat was similar in the cloned embryos, indicating these genes were successfully reprogrammed. However, compared to the lack of methylation of XIST in day-7 IVF embryos, a higher methylation level in day-7 cloned embryos was observed, implying that X chromosomes were activated in day-7 IVF blastocysts, but were not fully activated in cloned embryos, i.e., reprogramming of XIST was delayed. A time-course analysis of XIST DNA methylation on day-13, -15, -17, and -19 in vivo embryos revealed that XIST methylation initiated at about day 13 and was not completed by day 19. The methylation of the XIST gene in day-19 control cloned embryos was delayed again when compared to in vivo embryos. However, methylation of XIST in Oxamflatin-treated embryos was comparable with in vivo embryos, which further demonstrated that Oxamflatin could accelerate the delayed reprogramming of XIST gene and thus might improve cloning efficiency. PMID:25548976

  11. Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure.

    PubMed

    Jeon, Yubyeol; Nam, Yeong-Hee; Cheong, Seung-A; Kwak, Seong-Sung; Lee, Eunsong; Hyun, Sang-Hwan

    2016-08-25

    Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation. PMID:27064112

  12. Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure

    PubMed Central

    JEON, Yubyeol; NAM, Yeong-Hee; CHEONG, Seung-A; KWAK, Seong-Sung; LEE, Eunsong; HYUN, Sang-Hwan

    2016-01-01

    Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation. PMID:27064112

  13. In vitro effects of relaxin on gene expression in porcine cumulus ooxyte complexes and developing embryos

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Relaxin hormone peptide is found in porcine follicular and utero-tubal fluids, but its possible actions during early embryo development are still undetermined. Here, we investigated the effects of porcine relaxin during oocyte maturation and embryo development, and gene expression in the pig. Immat...

  14. Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

    SciTech Connect

    Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue; Pang, Daxin; Ouyang, Hongsheng

    2011-07-29

    Highlights: {yields} Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. {yields} The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. {yields} A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 {mu}g/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

  15. Transcriptomic Analysis of the Porcine Endometrium during Embryo Implantation.

    PubMed

    Lin, Haichao; Wang, Huaizhong; Wang, Yanping; Liu, Chang; Wang, Cheng; Guo, Jianfeng

    2015-01-01

    In pigs, successful embryo implantation is an important guarantee for producing litter size, and early embryonic loss occurring on day 12-30 of gestation critically affects the potential litter size. The implantation process is regulated by the expression of numerous genes, so comprehensive analysis of the endometrium is necessary. In this study, RNA sequencing (RNA-Seq) technology is used to analyze endometrial tissues during early pregnancy. We investigated the changes of gene expression between three stages (day 12, 18, and 25) by multiple comparisons. There were 1557, 8951, and 2345 differentially expressed genes (DEGs) revealed between the different periods of implantation. We selected several genes for validation by the use of quantitative real-time RT-PCR. Bioinformatic analysis of differentially expressed genes in the endometrium revealed a number of biological processes and pathways potentially involved in embryo implantation in the pig, most noticeably cell proliferation, regulation of immune response, interaction of cytokine-cytokine receptors, and cell adhesion. These results showed that specific gene expression patterns reflect the different functions of the endometrium in three stages (maternal recognition, conceptus attachment, and embryo implantation). This study identified comprehensive transcriptomic profile in the porcine endometrium and thus could be a foundation for targeted studies of genes and pathways potentially involved in abnormal endometrial receptivity and embryo loss in early pregnancy. PMID:26703736

  16. Transcriptomic Analysis of the Porcine Endometrium during Embryo Implantation

    PubMed Central

    Lin, Haichao; Wang, Huaizhong; Wang, Yanping; Liu, Chang; Wang, Cheng; Guo, Jianfeng

    2015-01-01

    In pigs, successful embryo implantation is an important guarantee for producing litter size, and early embryonic loss occurring on day 12–30 of gestation critically affects the potential litter size. The implantation process is regulated by the expression of numerous genes, so comprehensive analysis of the endometrium is necessary. In this study, RNA sequencing (RNA-Seq) technology is used to analyze endometrial tissues during early pregnancy. We investigated the changes of gene expression between three stages (day 12, 18, and 25) by multiple comparisons. There were 1557, 8951, and 2345 differentially expressed genes (DEGs) revealed between the different periods of implantation. We selected several genes for validation by the use of quantitative real-time RT-PCR. Bioinformatic analysis of differentially expressed genes in the endometrium revealed a number of biological processes and pathways potentially involved in embryo implantation in the pig, most noticeably cell proliferation, regulation of immune response, interaction of cytokine-cytokine receptors, and cell adhesion. These results showed that specific gene expression patterns reflect the different functions of the endometrium in three stages (maternal recognition, conceptus attachment, and embryo implantation). This study identified comprehensive transcriptomic profile in the porcine endometrium and thus could be a foundation for targeted studies of genes and pathways potentially involved in abnormal endometrial receptivity and embryo loss in early pregnancy. PMID:26703736

  17. Treating Cloned Embryos, But Not Donor Cells, with 5-aza-2’-deoxycytidine Enhances the Developmental Competence of Porcine Cloned Embryos

    PubMed Central

    HUAN, Yan Jun; ZHU, Jiang; XIE, Bing Teng; WANG, Jian Yu; LIU, Shi Chao; ZHOU, Yang; KONG, Qing Ran; HE, Hong Bin; LIU, Zhong Hua

    2013-01-01

    The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies. However, the parameters of 5-aza-dC treatment among species are different, and whether 5-aza-dC could enhance the developmental competence of porcine cloned embryos has still not been well studied. Therefore, in this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with 5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine cloned embryos were investigated by assessing pseudo-pronucleus formation, developmental potential and pluripotent gene expression of these reconstructed embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation level in PFF (0 nM vs. 10 nM vs. 25 nM vs. 50 nM, 58.70% vs. 37.37% vs. 45.43% vs. 39.53%, P<0.05), but did not improve the blastocyst rate of cloned embryos derived from these cells. Treating cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst rate compared with that of the untreated group. Furthermore, treating cloned embryos, but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for control, respectively, P<0.05) and enhanced the expression levels of pluripotent genes (Oct4, Nanog and Sox2) up to those of in vitro fertilized embryos during embryo development. In conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus formation and improvement of pluripotent gene expression. PMID

  18. KIF20A regulates porcine oocyte maturation and early embryo development.

    PubMed

    Zhang, Yu; Liu, Jun; Peng, Xu; Zhu, Cheng-Cheng; Han, Jun; Luo, Jia; Rui, Rong

    2014-01-01

    KIF20A (Kinesin-like family member 20A), also called mitotic kinesin-like proteins 2 (MKLP2), is a mammalian mitotic kinesin-like motor protein of the Kinesin superfamily proteins (KIFs), which was originally involved in Golgi apparatus dynamics and thought to essential for cell cycle regulation during successful cytokinesis. In the present study, we investigated whether KIF20A has roles on porcine oocyte meiotic maturation and subsequent early embryo development. By immunofluorescence staining, KIF20A was found to exhibit a dynamic localization pattern during meiosis. KIF20A was restricted to centromeres after germinal vesicle breakdown (GVBD), transferred to the midbody at telophase I (TI), and again associated with centromeres at metaphase II (MII). Inhibition of endogenous KIF20A via a specific inhibitor, Paprotrain, resulted in failure of polar body extrusion. Further cell cycle analysis showed that the percentage of oocytes that arrested at early metaphase I (MI) stage increased after KIF20A activity inhibition; however, the proportion of oocytes at anaphase/telophase I (ATI) and MII stages decreased significantly. Our results also showed that KIF20A inhibition did not affect spindle morphology. In addition, KIF20A was localized at the nucleus of early embryos, and KIF20A inhibition resulted in failure of early parthenogenetic embryo development. These results demonstrated that KIF20A is critical for porcine oocyte meiotic maturation and subsequent early embryo development. PMID:25036038

  19. KIF20A Regulates Porcine Oocyte Maturation and Early Embryo Development

    PubMed Central

    Zhang, Yu; Liu, Jun; Peng, Xu; Zhu, Cheng-Cheng; Han, Jun; Luo, Jia; Rui, Rong

    2014-01-01

    KIF20A (Kinesin-like family member 20A), also called mitotic kinesin-like proteins 2 (MKLP2), is a mammalian mitotic kinesin-like motor protein of the Kinesin superfamily proteins (KIFs), which was originally involved in Golgi apparatus dynamics and thought to essential for cell cycle regulation during successful cytokinesis. In the present study, we investigated whether KIF20A has roles on porcine oocyte meiotic maturation and subsequent early embryo development. By immunofluorescence staining, KIF20A was found to exhibit a dynamic localization pattern during meiosis. KIF20A was restricted to centromeres after germinal vesicle breakdown (GVBD), transferred to the midbody at telophase I (TI), and again associated with centromeres at metaphase II (MII). Inhibition of endogenous KIF20A via a specific inhibitor, Paprotrain, resulted in failure of polar body extrusion. Further cell cycle analysis showed that the percentage of oocytes that arrested at early metaphase I (MI) stage increased after KIF20A activity inhibition; however, the proportion of oocytes at anaphase/telophase I (ATI) and MII stages decreased significantly. Our results also showed that KIF20A inhibition did not affect spindle morphology. In addition, KIF20A was localized at the nucleus of early embryos, and KIF20A inhibition resulted in failure of early parthenogenetic embryo development. These results demonstrated that KIF20A is critical for porcine oocyte meiotic maturation and subsequent early embryo development. PMID:25036038

  20. Nuclear transfer of porcine embryos using cryopreserved delipated blastomeres as donor nuclei.

    PubMed

    Nagashima, H; Ashman, R J; Nottle, M B

    1997-11-01

    Nuclear transfer protocol for the pig using cryopreserved delipated four- to eight-cell and morula stage embryos as nucleus donors was developed. Donor embryos, which had been delipated by micromanipulation following centrifugation for polarizing cytoplasmic lipid droplets, were cryopreserved with 1.5 M 1,2-propanediol and 0.1 M sucrose. Recipient cytoplasts were prepared from ovulated oocytes. Activation of oocytes could be induced more efficiently when electric stimulation was given 53 hr after the hCG injection or later (66-83%), compared with 52 hr or earlier (11-16%, P < 0.05), suggesting that aging after ovulation may be required for in vivo matured porcine oocytes to be activated by electric stimuli. Membrane fusion rates between donor blastomeres and enucleated oocytes were 88% (127/144) and 97% (56/58, P > 0.05) for the four- to eight-cell and morula stage embryos, respectively. In vitro developmental rates to the two-cell (53/100 vs. 35/65), four-cell (34/100 vs. 26/65), and morula stage (17/100 vs. 18/65) were the same between the nuclear transfer embryos with four- to eight-cell and morula nuclei. However, more embryos reconstituted with morula nuclei developed to blastocysts (15% vs. 6%, P < 0.05). These data demonstrated that blastomeres of cryopreserved, delipated porcine embryos can be used as donor nuclei for nuclear transfer. Frozen-thawed, delipated blastomeres can be efficiently isolated and fused, and therefore provide a useful source of donor nuclei. PMID:9322245

  1. Cryopreservation of In Vitro-Produced Early-Stage Porcine Embryos in a Closed System

    PubMed Central

    Men, Hongsheng; Spate, Lee D.; Murphy, Clifton N.; Prather, Randall S.

    2015-01-01

    Abstract Cryostorage of porcine embryos in a closed pathogen-free system is essential for the maintenance and safeguard of swine models. Previously, we reported a protocol for the successful cryopreservation of porcine embryos at the blastocyst stage in 0.25 mL ministraws. In this experiment, we aimed at developing a protocol to apply the same concept for the cryopreservation of early-stage porcine embryos. Porcine embryos from day 2 through day 4 were delipidated by using a modified two-step centrifugation method and were then cryopreserved in sealed 0.25 mL straws by using a slow cooling method. Control groups included open pulled straw (OPS) vitrified embryos after delipidation and noncryopreserved embryos without delipidation. There were no significant differences in cryosurvival between embryos frozen in 0.25 mL straws and OPS vitrified embryos across all the stages (two cell to morula) examined (p>0.05). Similarly, in all groups examined, the blastocyst rates were not different between the two cryopreserved groups. However, the blastocyst rates from the cryopreserved groups were significantly lower than the noncryopreserved controls (p<0.05). This experiment demonstrated that early-stage porcine embryos can survive cryopreservation in a closed system by using a slow cooling method at a comparable rate to those vitrified by using an ultrarapid cooling method (p>0.05). However, the developmental competence was significantly reduced after cryopreservation compared to noncryopreserved embryos. Further research is needed to optimize the protocol to improve the developmental potential of cryopreserved early-stage porcine embryos in sealed straws. PMID:26309801

  2. Derivation of Porcine Embryonic Stem-Like Cells from In Vitro-Produced Blastocyst-Stage Embryos.

    PubMed

    Hou, Dao-Rong; Jin, Yong; Nie, Xiao-Wei; Zhang, Man-Ling; Ta, Na; Zhao, Li-Hua; Yang, Ning; Chen, Yuan; Wu, Zhao-Qiang; Jiang, Hai-Bin; Li, Yan-Ru; Sun, Qing-Yuan; Dai, Yi-Fan; Li, Rong-Feng

    2016-01-01

    Efficient isolation of embryonic stem (ES) cells from pre-implantation porcine embryos has remained a challenge. Here, we describe the derivation of porcine embryonic stem-like cells (pESLCs) by seeding the isolated inner cell mass (ICM) from in vitro-produced porcine blastocyst into α-MEM with basic fibroblast growth factor (bFGF). The pESL cells kept the normal karyotype and displayed flatten clones, similar in phenotype to human embryonic stem cells (hES cells) and rodent epiblast stem cells. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers such as OCT4, NANOG, SOX2, SSEA-4, TRA-1-60, and TRA-1-81 as determined by both immunofluorescence and RT-PCR. Additionally, these cells formed embryoid body (EB), teratomas and also differentiated into 3 germ layers in vitro and in vivo. Microarray analysis showed the expression of the pluripotency markers, PODXL, REX1, SOX2, KLF5 and NR6A1, was significantly higher compared with porcine embryonic fibroblasts (PEF), but expression of OCT4, TBX3, REX1, LIN28A and DPPA5, was lower compared to the whole blastocysts or ICM of blastocyst. Our results showed that porcine embryonic stem-like cells can be established from in vitro-produced blastocyst-stage embryos, which promote porcine naive ES cells to be established. PMID:27173828

  3. Derivation of Porcine Embryonic Stem-Like Cells from In Vitro-Produced Blastocyst-Stage Embryos

    PubMed Central

    Hou, Dao-Rong; Jin, Yong; Nie, Xiao-Wei; Zhang, Man-Ling; Ta, Na; Zhao, Li-Hua; Yang, Ning; Chen, Yuan; Wu, Zhao-Qiang; Jiang, Hai-Bin; Li, Yan-Ru; Sun, Qing-Yuan; Dai, Yi-Fan; Li, Rong-Feng

    2016-01-01

    Efficient isolation of embryonic stem (ES) cells from pre-implantation porcine embryos has remained a challenge. Here, we describe the derivation of porcine embryonic stem-like cells (pESLCs) by seeding the isolated inner cell mass (ICM) from in vitro-produced porcine blastocyst into α-MEM with basic fibroblast growth factor (bFGF). The pESL cells kept the normal karyotype and displayed flatten clones, similar in phenotype to human embryonic stem cells (hES cells) and rodent epiblast stem cells. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers such as OCT4, NANOG, SOX2, SSEA-4, TRA-1-60, and TRA-1-81 as determined by both immunofluorescence and RT-PCR. Additionally, these cells formed embryoid body (EB), teratomas and also differentiated into 3 germ layers in vitro and in vivo. Microarray analysis showed the expression of the pluripotency markers, PODXL, REX1, SOX2, KLF5 and NR6A1, was significantly higher compared with porcine embryonic fibroblasts (PEF), but expression of OCT4, TBX3, REX1, LIN28A and DPPA5, was lower compared to the whole blastocysts or ICM of blastocyst. Our results showed that porcine embryonic stem-like cells can be established from in vitro-produced blastocyst-stage embryos, which promote porcine naive ES cells to be established. PMID:27173828

  4. Effects of histone acetylation status on the early development of in vitro porcine transgenic cloned embryos.

    PubMed

    Luo, Biping; Ju, Shiqiang; Muneri, Caroline W; Rui, Rong

    2015-02-01

    The purpose of this study was to investigate the effects of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) on transgene expression and development of porcine transgenic cloned embryos, specifically focusing on effects derived from TSA-treated donor cells or TSA-treated reconstructed embryos. The results showed that TSA treatment on reconstructed embryos modified the acetylation status, which significantly improved the development of porcine somatic cell nuclear transfer (SCNT) embryos in vitro, but not donor cells. Furthermore, the treatment of reconstructed embryos with TSA enhanced expression of the pluripotency-related gene POU5F1 and stimulated expression of the anti-apoptotic gene BCL-2. Enhanced green fluorescent protein (EGFP) mRNA expression of every group dropped drastically from donor cells to blastocysts. Interestingly, TSA is likely to prevent a decline in EGFP expression in nuclear reprogramming of porcine SCNT embryos. However DNA hypomethylation induced by modified histone acetylation of donor cells treated with TSA was significantly more effective in increasing EGFP expression in SCNT blastocysts. In conclusion, the acetylation status of both donor cells and reconstructed embryos modified by TSA treatment increased transgene expression and improved nuclear reprogramming and the developmental potential of porcine transgenic SCNT embryos. PMID:25393500

  5. Birth of piglets from in vitro-produced, zona-intact porcine embryos vitrified in a closed system.

    PubMed

    Men, H; Zhao, C; Si, W; Murphy, C N; Spate, L; Liu, Y; Walters, E M; Samuel, M S; Prather, R S; Critser, J K

    2011-07-15

    As the importance of swine models in biomedical research increases, it is essential to develop low-cost, high-throughput systems to cryopreserve swine germplasm for maintenance of these models. However, porcine embryos are exceedingly sensitive to low temperature and successful cryopreservation is generally limited to the use of vitrification in open systems that allow direct contact of the embryos with liquid nitrogen (LN(2)). This creates a high risk of pathogen transmission. Therefore, cryopreservation of porcine embryos in a "closed" system is of very high importance. In this study, in vitro-produced (IVP) porcine embryos were used to investigate cryosurvival and developmental potential of embryos cryopreserved in a closed system. Optimal centrifugal forces to completely disassociate intracellular lipids from blastomeres were investigated using Day-4 embryos. Cryosurvival of delipidated embryos was investigated by vitrifying the embryos immediately after centrifugation, or after development to blastocysts. In this study, centrifugation for 30 min at 13,000 g was adequate to completely delipidate the embryos; furthermore, these embryos were able to survive cryopreservation at a rate comparable to those centrifuged for only 12 min. When delipidated embryos were vitrified at the blastocyst stage, there was no difference in survival between embryos vitrified using OPS and 0.25 mL straws. Some embryos vitrified by each method developed to term. These experiments demonstrated that porcine embryos can be cryopreserved in a closed system after externalizing their intracellular lipids. This has important implications for banking swine models of human health and disease. PMID:21458047

  6. OCT-4 expression is essential for the segregation of trophectoderm lineages in porcine preimplantation embryos

    PubMed Central

    EMURA, Natsuko; SAKURAI, Nobuyuki; TAKAHASHI, Kazuki; HASHIZUME, Tsutomu; SAWAI, Ken

    2016-01-01

    Oct-4, a member of the POU family of transcription factors, is a key factor that regulates the segregation of the inner cell mass (ICM) and the trophectoderm (TE) during the transition from morula to blastocyst in mice. However, little is known about its role in porcine early embryogenesis. To determine the function of OCT-4 in the ICM and TE segregation of porcine embryos, we studied the developmental morphology of porcine embryos using RNA interference technology. Our experiments demonstrated that when 1-cell stage embryos were co-injected with the small interfering RNA (siRNA)for targeted knockdown of OCT-4 (OCT-4-siRNA) and tetramethylrhodamine isothiocyanate (TRITC)-dextran conjugate (Dx), they failed to form blastocysts. Therefore, in this study, we constructed chimeric embryos comprising blastomeres that either expressed OCT-4 normally or showed downregulated OCT-4 expression by co-injection of OCT-4-siRNA and Dx into one blastomere in 2- to 4-cell stage embryos. In control embryos, which were co-injected with control siRNA and Dx, Dx-positive cells contributed to the TE lineage in almost all the blastocysts examined. In contrast, Dx-positive cells derived from a blastomere co-injected with OCT-4-siRNA and Dx were degenerated in almost half the blastocysts. This was probably due to the inability of these cells to differentiate into the TE lineage. Real-time RT-PCR analysis revealed no difference in the levels of SOX2, TEAD4, FGF4 and FGFR1-IIIc, all of which are known to be regulated by OCT-4, between the OCT-4-siRNA-injected morulae and the control ones. However, the level of CDX2, a molecule specifically expressed in the TE lineage, was significantly higher in the former than in the latter. Our results indicate that continuous expression of OCT-4 in blastomeres is essential for TE formation of porcine embryos. PMID:27210587

  7. Oct4 overexpression facilitates proliferation of porcine fibroblasts and development of cloned embryos.

    PubMed

    Kim, Su Jin; Koo, Ok Jae; Park, Hee Jung; Moon, Joon Ho; da Torre, Bego Roibas; Javaregowda, Palaksha Kanive; Kang, Jung Taek; Park, Sol Ji; Saadeldin, Islam M; Choi, Ji Yei; Lee, Byeong-Chun; Jang, Goo

    2015-10-01

    Octamer-binding transcription factor 4 (Oct4) is a critical molecule for the self-renewal and pluripotency of embryonic stem cells. Recent reports have shown that Oct4 also controls cell-cycle progression and enhances the proliferation of various types of cells. As the high proliferation of donor fibroblasts is critical to the production of transgenic pigs, using the somatic cell nuclear transfer technique, we analysed the effect of Oct4 overexpression on the proliferation of porcine fibroblasts and embryos. Porcine endogenous Oct4 cDNA was cloned, sequenced and inserted into an expression vector. The vector was transfected into porcine fibroblasts, and a stable Oct4-overexpressed cell line was established by antibiotic selection. Oct4 expression was validated by the immunostaining of Oct4. Cell morphology was changed to sharp, and both proliferation and migration abilities were enhanced in Oct4-overexpressed cells. Real-time RT-PCR results showed that p16, Bcl2 and Myc were upregulated in Oct4-overexpressed cells. Somatic cell nuclear transfer was performed using Oct4-overexpressed cells, and the development of Oct4 embryos was compared with that of wild-type cloned embryos. The cleavage and blastocyst formation rates were improved in the Oct4 embryos. Interestingly, blastocyst formation of the Oct4 embryos was observed as early as day 5 in culture, while blastocysts were observed from day 6 in wild-type cloned embryos. In conclusion, the overexpression of Oct4 enhanced the proliferation of both porcine fibroblasts and embryos. PMID:25181424

  8. Glutathione and cysteine enhance porcine preimplantation embryo development in vitro after intracytoplasmic sperm injection.

    PubMed

    Li, Xiao Xia; Lee, Kyung-Bon; Lee, Ji Hye; Kim, Keun Jung; Kim, Eun Young; Han, Kil-Woo; Park, Kang-Sun; Yu, Jung; Kim, Min Kyu

    2014-01-15

    Because intracytoplasmic sperm injection (ICSI) had been introduced to animal science, not only reproductive biology of domestic animals, but also medicine to treat infertility has been developed. This assisted reproductive technology is beneficial for generating transgenic animals, especially pigs, because polyspermy is the greatest hurdle in porcine IVF when researchers make highly qualified preimplantation embryos. However, ICSI-derived embryos expressed high level of reactive oxygen species (ROS), which are known to cause serious dysfunction during preimplantation development. The objective of this study was to investigate the developmental competence, ROS level, and apoptosis index when glutathione (GSH) or cysteine was supplemented into the in vitro culture medium for ICSI-derived porcine embryos. First, we evaluated the effect of different concentrations of GSH or cysteine on developmental ability of porcine ICSI-derived embryos. The cleavage rate (79.6%) and the blastocyst formation rate (20.9%) were significantly improved in culture medium supplemented with 1 mmol/L GSH compared with other concentrations or no supplementation. Also, 1.71 mmol/L cysteine showed a significantly higher proportion of cleavage (80.7%) and blastocyst formation (22.5%) than other cysteine-supplemented groups. Next, we confirmed that intracellular ROS level was significantly reduced in the group of blastocysts cultured with GSH or cysteine after ICSI compared with the no supplementation group. Finally, we found that terminal uridine nick-end labeling index, fragmentation, and total apoptosis were significantly decreased and the total cell number was significantly increased in blastocysts when ICSI-derived embryos were cultured with supplementation of 1.71 mmol/L cysteine or 1 mmol/L GSH. Taken together, these results strongly indicate that GSH or cysteine can improve the developmental competence of porcine ICSI-derived embryos by reducing intracellular ROS level and the apoptosis

  9. Development of a porcine (Sus scofa) embryo-specific microarray: array annotation and validation

    PubMed Central

    2012-01-01

    Background The domestic pig is an important livestock species and there is strong interest in the factors that affect the development of viable embryos and offspring in this species. A limited understanding of the molecular mechanisms involved in early embryonic development has inhibited our ability to fully elucidate these factors. Next generation deep sequencing and microarray technologies are powerful tools for delineation of molecular pathways involved in the developing embryo. Results Here we present the development of a porcine-embryo-specific microarray platform created from a large expressed sequence tag (EST) analysis generated by Roche/454 next-generation sequencing of cDNAs constructed from critical stages of in vivo or in vitro porcine preimplantation embryos. Two cDNA libraries constructed from in vitro and in vivo produced preimplantation porcine embryos were normalized and sequenced using 454 Titanium pyrosequencing technology. Over one million high-quality EST sequences were obtained and used to develop the EMbryogene Porcine Version 1 (EMPV1) microarray composed of 43,795 probes. Based on an initial probe sequence annotation, the EMPV1 features 17,409 protein-coding, 473 pseudogenes, 46 retrotransposed, 2,359 non-coding RNA, 4,121 splice variants in 2,862 genes and a total of 12,324 Novel Transcript Regions (NTR). After re-annotation, the total unique genes increased from 11,961 to 16,281 and 1.9% of them belonged to a large olfactory receptor (OR) gene family. Quality control on the EMPV1 was performed and revealed an even distribution of ten clusters of spiked-in control spots and array to array (dye-swap) correlation was 0.97. Conclusions Using next-generation deep sequencing we have produced a large EST dataset to allow for the selection of probe sequences for the development of the EMPV1 microarray platform. The quality of this embryo-specific array was confirmed with a high-level of reproducibility using current Agilent microarray technology

  10. Putative Porcine Embryonic Stem Cell Lines Derived from Aggregated Four-Celled Cloned Embryos Produced by Oocyte Bisection Cloning

    PubMed Central

    Siriboon, Chawalit; Lin, Yu-Hsuan; Kere, Michel; Chen, Chun-Da; Chen, Lih-Ren; Chen, Chien-Hong; Tu, Ching-Fu; Lo, Neng-Wen; Ju, Jyh-Cherng

    2015-01-01

    We attempted to isolate ES cell lines using inner cell masses from high-quality cloned porcine blastocysts. After being seeded onto feeders, embryos had better (P < 0.05) attachment, outgrowth formation and primary colonization in both 2× and 3× aggregated cloned embryos (62.8, 42.6 and12.8% vs. 76.2, 55.2 and 26.2%, respectively) compared to the non-aggregated group (41.6, 23.4 and 3.9%). Effects of feeder types (STO vs. MEF) and serum sources (FBS vs. KSR) on extraction of cloned embryo-derived porcine ES cells were examined. More (17.1%) ntES cell lines over Passage 3 were generated in the MEF/KSR group. However, ntES cells cultured in KSR-supplemented medium had a low proliferation rate with defective morphology, and eventually underwent differentiation or apoptosis subsequently. Approximately 26.1, 22.7 and 35.7% of primary colonies were formed after plating embryos in DMEM, DMEM/F12 and α-MEM media, respectively. Survival rates of ntES cells cultured in α-MEM, DMEM and DMEM/F12 were 16.7, 4.3 and 6.8%, respectively (P > 0.05). We further examined the beneficial effect of TSA treatment of 3× aggregated cloned embryos on establishment of ntES cell lines. Primary colony numbers and survival rates of ntES cells beyond passage 3 were higher (P < 0.05) in those derived from TSA-treated 3× blastocysts (36.7 and 26.7%) than from the non-treated aggregated group (23.1 and 11.5%). These cells, remaining undifferentiated over 25 passages, had alkaline phosphatase activity and expressed ES specific markers Oct4, Nanog, Sox2, and Rex01. Moreover, these ntES cells successfully differentiated into embryoid bodies (EBs) that expressed specific genes of all three germ layers after being cultured in LIF-free medium. In conclusion, we have successfully derived putative porcine ntES cells with high efficiency from quality cloned embryos produced by embryo aggregation, and optimized the ES cell culture system suitable for establishing and maintaining ntES cell lines in

  11. Ginsenoside Rg1 Improves In vitro-produced Embryo Quality by Increasing Glucose Uptake in Porcine Blastocysts.

    PubMed

    Kim, Seung-Hun; Choi, Kwang-Hwan; Lee, Dong-Kyung; Oh, Jong-Nam; Hwang, Jae Yeon; Park, Chi-Hun; Lee, Chang-Kyu

    2016-08-01

    Ginsenoside Rg1 is a natural compound with various efficacies and functions. It has beneficial effects on aging, diabetes, and immunity, as well as antioxidant and proliferative functions. However, its effect on porcine embryo development remains unknown. We investigated the effect of ginsenoside Rg1 on the in vitro development of preimplantation porcine embryos after parthenogenetic activation in high-oxygen conditions. Ginsenoside treatment did not affect cleavage or blastocyst formation rates, but did increase the total cell number and reduced the rate of apoptosis. In addition, it had no effect on the expression of four apoptosis-related genes (Bcl-2 homologous antagonist/killer, B-cell lymphoma-extra large, Caspase 3, and tumor protein p53) or two metabolism-related genes (mechanistic target of rapamycin, carnitine palmitoyltransferase 1B), but increased the expression of Glucose transporter 1 (GLUT1), indicating that it may increase glucose uptake. In summary, treatment with the appropriate concentration of ginsenoside Rg1 (20 μg/mL) can increase glucose uptake, thereby improving the quality of embryos grown in high-oxygen conditions. PMID:26954154

  12. Birth of piglets from in vitro-produced, zona-intact porcine embryos vitrified in a closed system

    PubMed Central

    Men, Hongsheng; Zhao, Chongbei; Wei, Si; Murphy, Clifton N.; Spate, Lee; Liu, Yang; Walters, Eric M.; Samuel, Melissa S.; Prather, Randall S.; Critser, John K.

    2011-01-01

    As the importance of swine models in biomedical research increases, it is essential to develop low-cost, high-throughput systems to cryopreserve swine germplasm for maintenance of these models. However, porcine embryos are exceedingly sensitive to low temperature and successful cryopreservation is generally limited to the use of vitrification in open systems that allow direct contact of the embryos with liquid nitrogen (LN2). This creates a high risk of pathogen transmission. Therefore, cryopreservation of porcine embryos in a “closed” system is of very high importance. In this study, in vitro-produced (IVP) porcine embryos were used to investigate cryosurvival and developmental potential of embryos cryopreserved in a closed system. Optimal centrifugal forces to completely disassociate intracellular lipids from blastomeres were investigated using Day-4 embryos. Cryosurvival of delipidated embryos was investigated by vitrifying the embryos immediately after centrifugation, or after development to blastocysts. In this study, centrifugation for 30 min at 13,000 g was adequate to completely delipidate the embryos; furthermore, these embryos were able to survive cryopreservation at a rate comparable to those centrifuged for only 12 min. When delipidated embryos were vitrified at the blastocyst stage, there was no difference in survival between embryos vitrified using OPS and 0.25 mL straws. Some embryos vitrified by each method developed to term. These experiments demonstrated that porcine embryos can be cryopreserved in a closed system after externalizing their intracellular lipids. This has important implications for banking swine models of human health and disease. PMID:21458047

  13. Disruption of the Myostatin Gene in Porcine Primary Fibroblasts and Embryos Using Zinc-Finger Nucleases

    PubMed Central

    Huang, Xian-Ju; Zhang, Hong-Xiao; Wang, Huili; Xiong, Kai; Qin, Ling; Liu, Honglin

    2014-01-01

    Myostatin represses muscle growth by negatively regulating the number and size of muscle fibers. Myostatin loss-of-function can result in the double-muscling phenotype and increased muscle mass. Thus, knockout of myostatin gene could improve the quality of meat from mammals. In the present study, zinc finger nucleases, a useful tool for generating gene knockout animals, were designed to target exon 1 of the myostatin gene. The designed ZFNs were introduced into porcine primary fibroblasts and early implantation embryos via electroporation and microinjection, respectively. Mutations around the ZFNs target site were detected in both primary fibroblasts and blastocysts. The proportion of mutant fibroblast cells and blastocyst was 4.81% and 5.31%, respectively. Thus, ZFNs can be used to knockout myostatin in porcine primary fibroblasts and early implantation embryos. PMID:24802055

  14. Analysis of Imprinted Gene Expression in Normal Fertilized and Uniparental Preimplantation Porcine Embryos

    PubMed Central

    Park, Chi-Hun; Uh, Kyung-Jun; Mulligan, Brendan P.; Jeung, Eui-Bae; Hyun, Sang-Hwan; Shin, Taeyoung; Ka, Hakhyun; Lee, Chang-Kyu

    2011-01-01

    In the present study quantitative real-time PCR was used to determine the expression status of eight imprinted genes (GRB10, H19, IGF2R, XIST, IGF2, NNAT, PEG1 and PEG10) during preimplantation development, in normal fertilized and uniparental porcine embryos. The results demonstrated that, in all observed embryo samples, a non imprinted gene expression pattern up to the 16-cell stage of development was common for most genes. This was true for all classes of embryo, regardless of parental-origins and the direction of imprint. However, several differentially expressed genes (H19, IGF2, XIST and PEG10) were detected amongst the classes at the blastocyst stage of development. Most interestingly and despite the fact that maternally and paternally expressed genes should not be expressed in androgenones and parthenogenones, respectively, both uniparental embryos expressed these genes when tested for in this study. In order to account for this phenomenon, we compared the expression patterns of eight imprinted genes along with the methylation status of the IGF2/H19 DMR3 in haploid and diploid parthenogenetic embryos. Our findings revealed that IGF2, NNAT and PEG10 were silenced in haploid but not diploid parthenogenetic blastocysts and differential methylation of the IGF2/H19 DMR3 was consistently observed between haploid and diploid parthenogenetic blastocysts. These results appear to suggest that there exists a process to adjust the expression status of imprinted genes in diploid parthenogenetic embryos and that this phenomenon may be associated with altered methylation at an imprinting control region. In addition we believe that imprinted expression occurs in at least four genes, namely H19, IGF2, XIST and PEG10 in porcine blastocyst stage embryos. PMID:21804912

  15. ADAM10 Is Involved in Cell Junction Assembly in Early Porcine Embryo Development

    PubMed Central

    Kwon, Jeongwoo; Jeong, Sung-min; Choi, Inchul; Kim, Nam-Hyung

    2016-01-01

    ADAM10 (A Disintegrin and Metalloprotease domain-containing protein 10) is a cell surface protein with a unique structure possessing both potential adhesion and protease domains. However, the role of ADAM10 in preimplantation stage embryos is not clear. In this study, we examined the expression patterns and functional roles of ADAM10 in porcine parthenotes during preimplantation development. The transcription level of ADAM10 dramatically increased from the morula stage onward. Immunostaining revealed that ADAM10 was present in both the nucleus and cytoplasm in early cleavage stage embryos, and localized to the apical region of the outer cells in morula and blastocyst embryos. Knockdown (KD) of ADAM10 using double strand RNA did not alter preimplantation embryo development until morula stage, but resulted in significantly reduced development to blastocyst stage. Moreover, the KD blastocyst showed a decrease in gene expression of adherens and tight junction (AJ/TJ), and an increase in trophectoderm TJ permeability by disrupting TJ assembly. Treatment with an ADAM10 specific chemical inhibitor, GI254023X, at the morula stage also inhibited blastocyst development and led to disruption of TJ assembly. An in situ proximity ligation assay demonstrated direct interaction of ADAM10 with coxsackie virus and adenovirus receptor (CXADR), supporting the involvement of ADAM10 in TJ assembly. In conclusion, our findings strongly suggest that ADADM10 is important for blastocyst formation rather than compaction, particularly for TJ assembly and stabilization in preimplantation porcine parthenogenetic development. PMID:27043020

  16. ADAM10 Is Involved in Cell Junction Assembly in Early Porcine Embryo Development.

    PubMed

    Kwon, Jeongwoo; Jeong, Sung-min; Choi, Inchul; Kim, Nam-Hyung

    2016-01-01

    ADAM10 (A Disintegrin and Metalloprotease domain-containing protein 10) is a cell surface protein with a unique structure possessing both potential adhesion and protease domains. However, the role of ADAM10 in preimplantation stage embryos is not clear. In this study, we examined the expression patterns and functional roles of ADAM10 in porcine parthenotes during preimplantation development. The transcription level of ADAM10 dramatically increased from the morula stage onward. Immunostaining revealed that ADAM10 was present in both the nucleus and cytoplasm in early cleavage stage embryos, and localized to the apical region of the outer cells in morula and blastocyst embryos. Knockdown (KD) of ADAM10 using double strand RNA did not alter preimplantation embryo development until morula stage, but resulted in significantly reduced development to blastocyst stage. Moreover, the KD blastocyst showed a decrease in gene expression of adherens and tight junction (AJ/TJ), and an increase in trophectoderm TJ permeability by disrupting TJ assembly. Treatment with an ADAM10 specific chemical inhibitor, GI254023X, at the morula stage also inhibited blastocyst development and led to disruption of TJ assembly. An in situ proximity ligation assay demonstrated direct interaction of ADAM10 with coxsackie virus and adenovirus receptor (CXADR), supporting the involvement of ADAM10 in TJ assembly. In conclusion, our findings strongly suggest that ADADM10 is important for blastocyst formation rather than compaction, particularly for TJ assembly and stabilization in preimplantation porcine parthenogenetic development. PMID:27043020

  17. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo

    PubMed Central

    Xue, Binghua; Li, Yan; He, Yilong; Wei, Renyue; Sun, Ruizhen; Yin, Zhi; Bou, Gerelchimeg; Liu, Zhonghua

    2016-01-01

    Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs. PMID:26991423

  18. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo.

    PubMed

    Xue, Binghua; Li, Yan; He, Yilong; Wei, Renyue; Sun, Ruizhen; Yin, Zhi; Bou, Gerelchimeg; Liu, Zhonghua

    2016-01-01

    Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs. PMID:26991423

  19. Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System

    PubMed Central

    Koo, Ok Jae; Park, Sol Ji; Lee, Choongil; Kang, Jung Taek; Kim, Sujin; Moon, Joon Ho; Choi, Ji Yei; Kim, Hyojin; Jang, Goo; Kim, Jin-Soo; Kim, Seokjoong; Lee, Byeong-Chun

    2014-01-01

    To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells (RFP+/eGFP+) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system. PMID:25049958

  20. Scriptaid Treatment Decreases DNA Methyltransferase 1 Expression by Induction of MicroRNA-152 Expression in Porcine Somatic Cell Nuclear Transfer Embryos

    PubMed Central

    Liang, Shuang; Zhao, Ming-Hui; Choi, Jeong-woo; Kim, Nam-Hyung; Cui, Xiang-Shun

    2015-01-01

    Abnormal epigenetic reprogramming of donor nuclei after somatic cell nuclear transfer (SCNT) is thought to be the main cause of low cloning efficiencies. A growing body of evidence has demonstrated a positive role of Scriptaid, a histone deacetylase inhibitor (HDACi) that belongs to an existing class of hydroxamic acid-containing HDACis, on the development competence of cloned embryos in many species. The present study investigated the effects of Scriptaid on the development of porcine SCNT embryos in vitro and its mechanism. Treatment with 300 or 500 nM Scriptaid for 20 h after activation significantly increased the percentage of SCNT embryos that developed to the blastocyst stage and the total number of cells per blastocyst and significantly decreased the percentage of apoptotic cells in blastocysts. Scriptaid treatment significantly increased the level of histone H3 acetylated at K9 and the conversion of 5-methylcytosine into 5-hydroxymethylcytosine and significantly decreased the level of histone H3 trimethylated at K9 at the pronuclear stage. As a potential mechanism for the DNA methylation changes, our results showed that the expression of DNA methyltransferase 1 was frequently down-regulated in Scriptaid-treated embryos in comparison with untreated embryos and was inversely correlated to endogenous microRNA-152 (miR-152). Taken together, these findings illustrated a crucial functional crosstalk between miR-152 and DNMT1. Meanwhile, mRNA and protein levels of POU5F1 and CDX2 were increased in Scriptaid-treated embryos. mRNA levels of Caspase3, and Bax were significantly decreased and that of Bcl-xL was significantly increased in Scriptaid-treated embryos. In conclusion, these observations would contribute to uncover the nuclear reprogramming mechanisms underlying the effects of Scriptaid on the improvement of porcine SCNT embryos. PMID:26261994

  1. Cell Synchronization by Rapamycin Improves the Developmental Competence of Porcine SCNT Embryos.

    PubMed

    Hyun, Hyuk; Lee, Seung-Eun; Son, Yeo-Jin; Shin, Min-Young; Park, Yun-Gwi; Kim, Eun-Young; Park, Se-Pill

    2016-06-01

    The cell cycle stage of donor cells influences the success of somatic cell nuclear transfer (SCNT). This study investigated the effects of rapamycin treatment on synchronization of porcine fibroblasts in comparison with control and serum-starved cells, SCNT donor cell viability, and SCNT-derived embryo development. Porcine fibroblasts were treated with 0.1, 1, 10, and 100 μM rapamycin for 1 or 3 days. The proportion of cells in G0/G1 phase was significantly higher among cells treated with 1 μM rapamycin for 3 days (D3-1R) than among control and serum-starved cells (p < 0.05). In comparison with control cells, rapamycin-treated cells exhibited reduced proliferation, similar to serum-starved cells. The viability (as assessed by the MTT assay) of D3-1R-treated cells was good, similar to control cells, showing their quality was maintained. To confirm nutrient regulation by rapamycin treatment, we checked the transcript levels of nutrient transporter genes (SLC2A2, SLC2A4, SLC6A14, and SLC7A1). These levels were significantly lower in D3-1R-treated cells than in control cells (p < 0.01). We performed SCNT with D3-1R-treated cells (SCNT(D3-1R)) to confirm the effect of cell cycle synchronization by rapamycin treatment. Although SCNT(D3-1R) embryos did not have an increased fusion rate, their cleavage and blastocyst formation rates were significantly higher than those of control embryos (p < 0.05). Regarding embryo quality, the numbers of total and apoptotic cells per blastocyst were increased and decreased, respectively, in SCNT(D3-1R) blastocysts. The mRNA levels of developmental (CDX2 and CDH1) and proapoptotic (FAS and CASP3) genes were significantly higher and lower, respectively, in SCNT(D3-1R) blastocysts than in control blastocysts (p < 0.05). These results demonstrate that rapamycin treatment affects the cell cycle synchronization of donor cells and enhances the developmental potential of porcine SCNT embryos. PMID:27253629

  2. Development of porcine tetraploid somatic cell nuclear transfer embryos is influenced by oocyte nuclei.

    PubMed

    Fu, Bo; Liu, Di; Ma, Hong; Guo, Zhen-Hua; Wang, Liang; Li, Zhong-Qiu; Peng, Fu-Gang; Bai, Jing

    2016-02-01

    Cloning efficiency in mammalian systems remains low because reprogramming of donor cells is frequently incomplete. Nuclear factors in the oocyte are removed by enucleation, and this removal may adversely affect reprogramming efficiency. Here, we investigated the role of porcine oocyte nuclear factors during reprogramming. We introduced somatic cell nuclei into intact MII oocytes to establish tetraploid somatic cell nuclear transfer (SCNT) embryos containing both somatic nuclei and oocyte nuclei. We then examined the influence of the oocyte nucleus on tetraploid SCNT embryo development by assessing characteristics including pronucleus formation, cleavage rate, and blastocyst formation. Overall, tetraploid SCNT embryos have a higher developmental competence than do standard diploid SCNT embryos. Therefore, we have established an embryonic model in which a fetal fibroblast nucleus and an oocyte metaphase II plate coexist. Tetraploid SCNT represents a new research platform that is potentially useful for examining interactions between donor nuclei and oocyte nuclei. This platform should facilitate further understanding of the roles played by nuclear factors during reprogramming. PMID:26503330

  3. An Improved System for Generation of Diploid Cloned Porcine Embryos Using Induced Pluripotent Stem Cells Synchronized to Metaphase.

    PubMed

    Kim, Eunhye; Zheng, Zhong; Jeon, Yubyeol; Jin, Yong-Xun; Hwang, Seon-Ung; Cai, Lian; Lee, Chang-Kyu; Kim, Nam-Hyung; Hyun, Sang-Hwan

    2016-01-01

    Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently. PMID:27472781

  4. An Improved System for Generation of Diploid Cloned Porcine Embryos Using Induced Pluripotent Stem Cells Synchronized to Metaphase

    PubMed Central

    Jeon, Yubyeol; Jin, Yong-Xun; Hwang, Seon-Ung; Cai, Lian; Lee, Chang-Kyu; Kim, Nam-Hyung; Hyun, Sang-Hwan

    2016-01-01

    Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently. PMID:27472781

  5. MOLECULAR CLONING OF PORCINE ESTROGEN RECEPTOR-BETA COMPLEMENTARY DNAS AND DEVELOPMENTAL EXPRESSION I PERIIMPLANTATION EMBRYOS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the pig, estrogens transiently produced by embryos and progestins of maternal origin target the uterine endometrium, causing alterations in gene expression and secretory activity, both of which are important for the initiation of embryo attachment. The potential direct embryotrophic roles of estr...

  6. Analysis of selected transcript levels in porcine spermatozoa, oocytes, zygotes and two-cell stage embryos.

    PubMed

    Kempisty, Bartosz; Antosik, Paweł; Bukowska, Dorota; Jackowska, Marta; Lianeri, Margarita; Jaśkowski, Jedrzej M; Jagodziński, Paweł P

    2008-01-01

    It has been suggested that spermatozoa can deliver mRNAs to the oocyte during fertilisation. Using reverse transcription and real-time quantitative polymerase chain reaction analysis (RQ-PCR), we evaluated the presence of clusterin (CLU), protamine 2 (PRM2), calmegin (CLGN), cAMP-response element modulator protein (CREM), methyltransferase 1 (DNMT1), linker histone 1 (H1), protamine 1 (PRM1), TATA box-binding protein associated factor 1 (TAF1) and TATA box-binding protein (TBP) in porcine mature oocytes, zygotes and two-cell stage embryos. Spermatozoa isolated from semen samples of boars contained all transcripts investigated, whereas oocytes contained only CREM, H1, TAF1, and TBP mRNAs. The zygote and two-cell stage embryos contained CLU, CREM, H1, PRM1, PRM2, TAF1 and TBP transcripts. Our observations suggest that porcine spermatozoa may delivery CLU, PRM1 and PRM2 mRNAs to the oocyte, which may contribute to zygotic and early embryonic development. PMID:18462614

  7. Stem Cell–Derived Bioactive Materials Accelerate Development of Porcine In Vitro–Fertilized Embryos

    PubMed Central

    Lee, Seung-Eun; Moon, Jeremiah Ji-Man; Kim, Eun-Young

    2015-01-01

    Abstract Stem cells show the capability to proliferate in an undifferentiated state with long-term self-renewal, which gives the cells advantages for use as bioactive material (BM) for embryo culture in vitro. The objective of this experiment was to investigate the effect of two BMs—human adipose tissue–derived mesenchymal stem cell BM (hAT-MSC-BM) and human embryonic stem cell–derived BM (hESC-BM)—on porcine embryo development compared to commonly used bovine serum albumin (BSA) or serum treatment groups. In vitro–fertilized (IVF) embryos were cultured in PZM-5 with 4 mg/mL BSA until day 4 and equally divided into four groups. Starting from day 4 (until day 6), each group was treated with the following protein additives: 4 mg/mL BSA (control), 10% fetal bovine serum (FBS), 10% hAT-MSC-BM, or 10% hESC-BM. Our results show FBS- and two other BM-treated groups showed significant increases in blastocyst formation rate, hatching rate, and total cell number compared with the control group (p<0.05). The hAT-MSC-BM and hESC-BM treatment groups presented better-quality embryo development, especially from the middle expanding stage to hatching. In particular, the hAT-MSC-BM–treated group showed the highest developmental potential of all groups and formed the most expanding-stage blastocysts. The relative expression of reprogramming-related transcription factor (POU5F1, SOX2, DPPA5, and CDH1), antioxidant (PRDX5), and apoptosis (BCL2L1 and BIRC5) genes also increased in two types of BMs compared to the control. In addition, we investigated the protein synthesis of the tight junction– and gap junction–related genes, connexin 43 and zonula occludens-1 (ZO-1); these increased more than in the control. These results demonstrate that stem cell–derived BMs accelerate porcine preimplantation embryo development and that the BMs would be helpful in the development of preimplantation embryos. PMID:26053518

  8. In vitro development of preimplantation porcine embryos using alginate hydrogels as a three-dimensional extracellular matrix

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Between day 10 and 12 of gestation, porcine embryos undergo a dramatic morphological change, known as elongation, with a corresponding increase in estrogen production for maternal recognition of pregnancy. Elongation deficiencies contribute to ~20% of embryonic loss, but exact mechanisms of elongati...

  9. In vitro development of preimplantation porcine embryos using alginate hydrogels as a three-dimensional extracellular matrix.

    PubMed

    Sargus-Patino, Catherine N; Wright, Elane C; Plautz, Sarah A; Miles, Jeremy R; Vallet, Jeff L; Pannier, Angela K

    2014-08-01

    Between Days 10 and 12 of gestation, porcine embryos undergo a dramatic morphological change, known as elongation, with a corresponding increase in oestrogen production that triggers maternal recognition of pregnancy. Elongation deficiencies contribute to embryonic loss, but exact mechanisms of elongation are poorly understood due to the lack of an effective in vitro culture system. Our objective was to use alginate hydrogels as three-dimensional scaffolds that can mechanically support the in vitro development of preimplantation porcine embryos. White cross-bred gilts were bred at oestrus (Day 0) to Duroc boars and embryos were recovered on Days 9, 10 or 11 of gestation. Spherical embryos were randomly assigned to be encapsulated within double-layered 0.7% alginate beads or remain as non-encapsulated controls (ENC and CONT treatment groups, respectively) and were cultured for 96h. Every 24h, half the medium was replaced with fresh medium and an image of each embryo was recorded. At the termination of culture, embryo images were used to assess morphological changes and cell survival. 17β-Oestradiol levels were measured in the removed media by radioimmunoassay. Real-time polymerase chain reaction was used to analyse steroidogenic transcript expression at 96h in ENC and CONT embryos, as well as in vivo-developed control embryos (i.e. spherical, ovoid and tubular). Although no differences in cell survival were observed, 32% (P<0.001) of the surviving ENC embryos underwent morphological changes characterised by tubal formation with subsequent flattening, whereas none of the CONT embryos exhibited morphological changes. Expression of steroidogenic transcripts STAR, CYP11A1 and CYP19A1 was greater (P<0.07) in ENC embryos with morphological changes (ENC+) compared with CONT embryos and ENC embryos with no morphological changes (ENC-), and was more similar to expression of later-stage in vivo-developed controls. Furthermore, a time-dependent increase (P<0.001) in 17

  10. Porcine gonadogenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Five images submitted for teaching purposes related to porcine gonadogenesis (2), porcine fetal testicular development (2), and porcine fetal ovarian development. Key words include: Egg cell nests, Embryo, GATA4, Genital ridge, Gonad, Leydig cell, Mesonephros, MIS, Ovary, P450c17, Porcine, Sertoli ...

  11. Development of porcine embryos reconstituted with somatic cells and enucleated metaphase I and II oocytes matured in a protein-free medium

    PubMed Central

    Miyoshi, Kazuchika; Rzucidlo, S Jacek; Gibbons, John R; Arat, Sezen; Stice, Steven L

    2001-01-01

    Background Many cloned animals have been created by transfer of differentiated cells at G0/G1 or M phase of the cell cycle into enucleated M II oocytes having high maturation/meiosis/mitosis-promoting factor activity. Because maturation/meiosis/mitosis-promoting factor activity during oocyte maturation is maximal at both M I and M II, M I oocytes may reprogram differentiated cell nuclei as well. The present study was conducted to examine the developmental ability in vitro of porcine embryos reconstructed by transferring somatic cells (ear fibroblasts) into enucleated M I or M II oocytes. Results Analysis of the cell cycle stages revealed that 91.2 ± 0.2% of confluent cells were at the G0/G1 phase and 54.1 ± 4.4% of nocodazole-treated cells were at the G2/M phase, respectively. At 6 h after activation, nuclear swelling was observed in 50.0-88.9% and 34.4-39.5% of embryos reconstituted with confluent cells and nocodazole-treated cells regardless of the recipient oocytes, respectively. The incidence of both a swollen nucleus and polar body was low (6.3-10.5%) for all nocodazole-treated donor cell regardless of the recipient oocyte. When embryos reconstituted with confluent cells and M I oocytes were cultured, 2 (1.5%) blastocysts were obtained and this was significantly (P < 0.05) lower than that (7.6%) of embryos produced by transferring confluent cells into M II oocytes. No reconstructed embryos developed to the blastocyst stage when nocodazole-treated cells were used as donors. Conclusions Porcine M I oocytes have a potential to develop into blastocysts after nuclear transfer of somatic cells. PMID:11476669

  12. Porcine Induced Pluripotent Stem Cells Require LIF and Maintain Their Developmental Potential in Early Stage of Embryos

    PubMed Central

    Cheng, De; Guo, Yanjie; Li, Zhenzhen; Liu, Yajun; Gao, Xing; Gao, Yi; Cheng, Xiang; Hu, Junhe; Wang, Huayan

    2012-01-01

    Porcine induced pluripotent stem (piPS) cell lines have been generated recently by using a cocktail of defined transcription factors, however, the features of authentic piPS cells have not been agreed upon and most of published iPS clones did not meet the stringent requirements of pluripotency. Here, we report the generation of piPS cells from fibroblasts using retrovirus carrying four mouse transcription factors (mOct4, mSox2, mKlf4 and mc-Myc, 4F). Multiple LIF-dependent piPS cell lines were generated and these cells showed the morphology similar to mouse embryonic stem cells and other pluripotent stem cells. In addition to the routine characterization, piPS cells were injected into porcine pre-compacted embryos to generate chimera embryos and nuclear transfer (NT) embryos. The results showed that piPS cells retain the ability to integrate into inner and outer layers of the blastocysts, and support the NT embryos development to blastocysts. The generations of chimera embryos and NT embryos derived from piPS clones are a practical means to determine the quality of iPS cells ex vivo. PMID:23251622

  13. Porcine embryos produced after intracytoplasmic sperm injection using xenogeneic pig sperm from neonatal testis tissue grafted in mice.

    PubMed

    Honaramooz, Ali; Cui, Xiang-Shun; Kim, Nam-Hyung; Dobrinski, Ina

    2008-01-01

    Embryo development after homologous intracytoplasmic sperm injection (ICSI) with sperm from testis tissue xenografts from pigs or any other farm animal species has not been evaluated critically. Here, we report development of porcine embryos in vitro following ICSI with sperm retrieved from xenografted neonatal pig testis. Small pieces of testis tissue from newborn piglets were grafted under the back skin of castrated immunodeficient mice (n = 4) and the xenografts were collected 8 months after grafting. Spermatozoa were recovered by mincing of the grafted tissue. For comparison, testicular, epididymal and ejaculated spermatozoa were also collected from mature boars. Oocytes injected with xenogeneic spermatozoa were either fixed to determine fertilisation processes (n = 89 in five replicates) or allowed to develop in vitro (n = 143 in four replicates). Xenogeneic porcine spermatozoa were fertilisation competent (24% v. 58%, 68%, 62% or 0% for xenogeneic v. control testicular, epididymal and ejaculated spermatozoa or no spermatozoa, respectively) and embryos developed to the blastocyst stage (8% v. 22%, 27%, 25% or 0%, respectively). These results demonstrate that porcine spermatozoa derived from immature testis tissue xenografted into mice are fertilisation competent, albeit at a lower rate than testicular, epididymal or ejaculated spermatozoa from control boars, and support embryo development after ICSI. PMID:18842182

  14. Involvement of mouse and porcine PLCζ-induced calcium oscillations in preimplantation development of mouse embryos

    SciTech Connect

    Yoneda, Akihiro; Watanabe, Tomomasa

    2015-05-01

    In mammals, phospholipase Cζ (PLCζ) has the ability to trigger calcium (Ca{sup 2+}) oscillations in oocytes, leading to oocyte activation. Although there is a species-specific difference in the PLCζ-induced Ca{sup 2+} oscillatory pattern, whether PLCζ-induced Ca{sup 2+} oscillations affect preimplantation embryonic development remains unclear. Here, we show that Ca{sup 2+} oscillations in mouse PLCζ cRNA-injected oocytes stopped just before pronuclear formation, while that in porcine PLCζ cRNA-injected oocytes continued for several hours after pronuclei had been formed. This difference of Ca{sup 2+} oscillations in oocytes after pronuclear formation was dependent on the difference in the nuclear localization signal (NLS) sequence of PLCζ between the mouse and pig. However, mouse and porcine PLCζ cRNA-injected oocytes parthenogenetically developed to blastocysts regardless of the absence or presence of Ca{sup 2+} oscillations after pronuclear formation. Furthermore, the developmental rate of mouse or porcine PLCζ-activated oocytes injected with round spermatids to the blastocyst stage was not significantly different from that of strontium-activated oocytes injected with round spermatids. These results suggest that the PLCζ-induced Ca{sup 2+} oscillatory pattern in mouse oocytes is dependent on the NLS sequence of PLCζ and injection of PLCζ may be a useful method for activation of round spermatid-injected and somatic nuclear transferred oocytes. - Highlights: • Porcine PLCζ-induced Ca{sup 2+} oscillations continued after pronuclear formation. • The Ca{sup 2+} oscillatory pattern was dependent on the difference in the NLS sequence of PLCζ. • PLCζ-activated oocytes parthenogenetically developed to blastocysts. • PLCζ-activated oocytes injected with round spermatids developed to blastocysts.

  15. Alteration of the DNA methylation status of donor cells impairs the developmental competence of porcine cloned embryos

    PubMed Central

    HUAN, Yan Jun; WU, Zhan Feng; ZHANG, Ji Guang; ZHU, Jiang; XIE, Bing Teng; WANG, Jian Yu; LI, Jing Yu; XUE, Bing Hua; KONG, Qing Ran; LIU, Zhong Hua

    2015-01-01

    Nuclear reprogramming induced by somatic cell nuclear transfer is an inefficient process, and donor cell DNA methylation status is thought to be a major factor affecting cloning efficiency. Here, the role of donor cell DNA methylation status regulated by 5-aza-2'-deoxycytidine (5-aza-dC) or 5-methyl-2'-deoxycytidine-5'-triphosphate (5-methyl-dCTP) in the early development of porcine cloned embryos was investigated. Our results showed that 5-aza-dC or 5-methyl-dCTP significantly reduced or increased the global methylation levels and altered the methylation and expression levels of key genes in donor cells. However, the development of cloned embryos derived from these cells was reduced. Furthermore, disrupted pseudo-pronucleus formation and transcripts of early embryo development-related genes were observed in cloned embryos derived from these cells. In conclusion, our results demonstrated that alteration of the DNA methylation status of donor cells by 5-aza-dC or 5-methyl-dCTP disrupted nuclear reprogramming and impaired the developmental competence of porcine cloned embryos. PMID:26537205

  16. Alteration of the DNA methylation status of donor cells impairs the developmental competence of porcine cloned embryos.

    PubMed

    Huan, Yan Jun; Wu, Zhan Feng; Zhang, Ji Guang; Zhu, Jiang; Xie, Bing Teng; Wang, Jian Yu; Li, Jing Yu; Xue, Bing Hua; Kong, Qing Ran; Liu, Zhong Hua

    2016-01-01

    Nuclear reprogramming induced by somatic cell nuclear transfer is an inefficient process, and donor cell DNA methylation status is thought to be a major factor affecting cloning efficiency. Here, the role of donor cell DNA methylation status regulated by 5-aza-2'-deoxycytidine (5-aza-dC) or 5-methyl-2'-deoxycytidine-5'-triphosphate (5-methyl-dCTP) in the early development of porcine cloned embryos was investigated. Our results showed that 5-aza-dC or 5-methyl-dCTP significantly reduced or increased the global methylation levels and altered the methylation and expression levels of key genes in donor cells. However, the development of cloned embryos derived from these cells was reduced. Furthermore, disrupted pseudo-pronucleus formation and transcripts of early embryo development-related genes were observed in cloned embryos derived from these cells. In conclusion, our results demonstrated that alteration of the DNA methylation status of donor cells by 5-aza-dC or 5-methyl-dCTP disrupted nuclear reprogramming and impaired the developmental competence of porcine cloned embryos. PMID:26537205

  17. Nonsurgical deep uterine transfer of vitrified, in vivo-derived, porcine embryos is as effective as the default surgical approach

    PubMed Central

    Martinez, Emilio A.; Martinez, Cristina A; Nohalez, Alicia; Sanchez-Osorio, Jonatan; Vazquez, Juan M.; Roca, Jordi; Parrilla, Inmaculada; Gil, Maria A.; Cuello, Cristina

    2015-01-01

    Surgical procedures are prevalent in porcine embryo transfer (ET) programs, where the use of vitrified embryos is quasi non-existent. This study compared the effectiveness of surgical vs nonsurgical deep uterine (NsDU) ET using vitrified, in vivo-derived embryos (morulae and blastocysts) on the reproductive performance and welfare of the recipients. The recipient sows (n = 122) were randomly assigned to one of the following groups: surgical ET with 30 vitrified-warmed embryos (S-30 group, control); NsDU-ET with 30 vitrified-warmed embryos (NsDU-30 group) and NsDU-ET with 40 vitrified-warmed embryos (NsDU-40 group). Regardless of embryo stage, the NsDU-ET with 40 embryos presented similar rates of farrowing (72.7%) and litter size (9.9 ± 2.1 piglets) as the customary surgical procedure (75.0% and 9.6 ± 2.7 piglets). Numbers of ET-embryos appeared relevant, since the NsDU-ET with 30 embryos resulted in a decrease (P < 0.05) in farrowing rates (38.9%) and litter sizes (5.7 ± 2.4 piglets). In conclusion, we demonstrate for the first time that farrowing rate and litter size following a NsDU-ET procedure increase in function of a larger number of transferred vitrified embryos, with fertility equalizing that obtained with the invasive surgical approach. The results open new possibilities for the widespread use of non-invasive ET in pigs. PMID:26030839

  18. Embryo development and sex ratio of in vitro-produced porcine embryos are affected by the energy substrate and hyaluronic acid added to the culture medium.

    PubMed

    Torner, Eva; Bussalleu, Eva; Briz, M Dolors; Yeste, Marc; Bonet, Sergi

    2014-01-01

    In the present study, the effects of replacing glucose with pyruvate-lactate and supplementing these in vitro culture (IVC) media with hyaluronic acid (HA) on porcine embryo development and sex ratio were examined. The in vitro-produced (IVP) porcine embryos were cultured in NCSU-23 medium with 0.0, 0.5 or 1.0mgmL(-1) HA, and with either 5.55mM glucose (IVC-Glu) or pyruvate (0.17mM)-lactate (2.73mM) from 0 to 48h post insemination (h.p.i.) and then with glucose from 48 to 168h.p.i. (IVC-PL). Those embryos cultured with IVC-PL had significantly higher blastocyst rates (23.7±1.5%) than those cultured with IVC-Glu (14.27±2.75%). At 1.0mgmL(-1), HA tended to skew the sex ratio of blastocysts towards males in those embryos cultured in IVC-PL, and led to a significant decrease in the blastocyst rate compared with embryos cultured in the presence of 0.5 and 0.0mgmL(-1) HA and IVC-Glu (4.28±0.28% vs 11.01±1.42% and 10.14±2.77%, respectively) and IVC-PL (14.37±1.35% vs 20.96±2.85% and 22.99±1.39%, respectively). In contrast, there were no significant differences in the total cell number per blastocyst or in apoptosis rates. In conclusion, pyruvate and lactate were the preferred energy substrates in the early stages of IVP porcine embryos. Moreover, 1.0mgmL(-1) HA significantly decreased the percentage of blastocyst rates in both the IVC-Glu and IVC-PL groups, but only by a preferential loss of female embryos for those cultured in IVC-PL. PMID:23657201

  19. Pretreating porcine sperm with lipase enhances developmental competence of embryos produced by intracytoplasmic sperm injection.

    PubMed

    Wei, Yinghui; Fan, Junhua; Li, Lin; Liu, Zhiguo; Li, Kui

    2016-08-01

    Intracytoplasmic sperm injection (ICSI) has been widely applied in humans, mice, and some domestic animals to cure human infertility, or produce genetically superior or genetically engineered animals. However, the production efficiency of ICSI in pigs remains quite low. In this study, we developed a new sperm pretreatment method to improve production efficiency of ICSI in pigs. Experiment 1 revealed that pretreating porcine sperm with 2.5 mg/ml lipase before ICSI operation, not only can reduce the adhesion between sperm and the injection pipette without adding polyvinylpyrrolidone (PVP) in the operating medium, but also significantly improve male pronuclei (MPN) formation rate (55.56% vs. 40.00% (0 mg/ml), 42.59% (5.0 mg/ml), 40.00% (10.0 mg/ml), P < 0.05) and enhance developmental competence of ICSI embryos (26.03% vs. 10.87% (0 mg/ml), 10.00% (5.0 mg/ml), 10.13% (10.0 mg/ml), P < 0.05). Experiment 2 showed that this method has a higher MPN formation rate (50.47% vs. 30.78%, P < 0.05) and blastocyst rate (18.81% vs. 7.41%, P < 0.05) than the PVP method, and was better than the Triton X-100 treatment method (50.47% vs. 46.23%, 18.81% vs. 12.75%). Therefore, pretreating porcine sperm with 2.5 mg/ml lipase before ICSI operation is highly recommended, instead of adding PVP in the operating medium. PMID:26443110

  20. Metabolism of energy substrates of in vitro and in vivo derived embryos from ewes synchronized and super ovulated with norgestomet and porcine follicle stimulating hormone

    PubMed Central

    2012-01-01

    The synchronization and ovulatory responses of Sangsari cross bred ewes and metabolism of energy substrates in 8-cell stage embryos to hatched blastocysts stage produced in vitro or in vivo were investigated. Ewes were assigned randomly to receive 37.5 IU of porcine follicle stimulating hormone (FSH-P) daily for the 3 days preceding implant removal (Day 0). Synchronization of estrus was carried out using a 1.5 mg norgestomet (Crestar) ear implant for 12 days. Ewes in estrus were mated two to three times with rams of proven fertility. At the time of first mating each ewe was administered 1000 IU of human chorionic gonadotrophin (hCG) to induce ovulation. Surgical embryo recovery was performed on Days 4 and 6 after onset of estrus (Day 0) and recovered embryos were subjected to comparative metabolism studies with in vitro derived embryos at the same stage of development. The number of corpora lutea (CL), unovulated follicles and overall ovarian activity were recorded for each ewe during the breeding and non-breeding seasons. While the pattern of oxidation was similar among in vitro and in vivo derived embryos, a low pyruvate to lactate ratio was the preferred substrate of embryos derived in vitro. A high level of production of CO2 and lactate resulted from a stress response to the suboptimal culture environment. The first marked increase in the metabolism of glucose by ovine embryos was detected in compact morula stage, but there was no significant increase in the oxidation of glucose after the morula stage. Two different concentrations of glucose were compared, but this did not affect metabolism. However, the rate of incorporation and metabolism of glucose tended to be higher at the 0.56 mmol/L glucose dosage. PMID:23157819

  1. Effect of ATM and HDAC Inhibition on Etoposide-Induced DNA Damage in Porcine Early Preimplantation Embryos

    PubMed Central

    Wang, HaiYang; Luo, YiBo; Lin, ZiLi; Lee, In-Won; Kwon, Jeongwoo; Cui, Xiang-Shun; Kim, Nam-Hyung

    2015-01-01

    Oocyte maturation and embryonic development are sensitive to DNA damage. Compared with somatic cells or oocytes, little is known about the response to DNA damage in early preimplantation embryos. In this study, we examined DNA damage checkpoints and DNA repair mechanisms in parthenogenetic preimplantation porcine embryos. We found that most of the etoposide-treated embryos showed delay in cleavage and ceased development before the blastocyst stage. In DNA-damaged embryos, the earliest positive TUNEL signals were detected on Day 5 of in vitro culture. Caffeine, which is an ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related protein) kinase inhibitor, and KU55933, which is an ATM kinase inhibitor, were equally effective in rescuing the etoposide-induced cell-cycle blocks. This indicates that ATM plays a central role in the regulation of the checkpoint mechanisms. Treating the embryos with histone deacetylase inhibitors (HDACi) increased embryonic development and reduced etoposide-induced double-strand breaks (DSBs). The mRNA expression of genes involved in non-homologous end-joining (NHEJ) or homologous recombination (HR) pathways for DSB repair was reduced upon HDACi treatment in 5-day-old embryos. Furthermore, HDACi treatment increased the expression levels of pluripotency-related genes (OCT4, SOX2 and NANOG) and decreased the expression levels of apoptosis-related genes (CASP3 and BAX). These results indicate that early embryonic cleavage and development are disturbed by etoposide-induced DNA damage. ATMi (caffeine or KU55933) treatment bypasses the checkpoint while HDACi treatment improves the efficiency of DSB repair to increase the cleavage and blastocyst rate in porcine early preimplantation embryos. PMID:26556501

  2. Cdx2 represses Oct4 function via inducing its proteasome-dependent degradation in early porcine embryos.

    PubMed

    Bou, Gerelchimeg; Liu, Shichao; Guo, Jia; Zhao, Yueming; Sun, Mingju; Xue, Binghua; Wang, Jiaqiang; Wei, Yanchang; Kong, Qingran; Liu, Zhonghua

    2016-02-01

    Reciprocal repression of inner cell mass specific factor OCT4 and trophectoderm specific factor CDX2 promotes mouse first lineage segregation. Studies in mouse embryonic stem (ES) cells revealed that they bind to each other's regulatory regions to reciprocally suppress transcription, additionally they form protein complex for mutual antagonism. However, so far the molecular interaction of Oct4 and Cdx2 in other mammal's early embryo is not yet investigated. Here, over-expression of Cdx2 in early porcine embryo showed CDX2 represses Oct4 through neither the transcriptional repression nor forming repressive complex, but promoting OCT4 nuclear export and proteasomal degradation. The results showed novel molecular regulation of CDX2 on Oct4, and provided important clues for clarifying the mechanism of interaction between CDX2 and Oct4 in embryo of mammals other than mouse. PMID:26708097

  3. Transcriptional regulators TRIM28, SETDB1, and TP53 are aberrantly expressed in porcine embryos produced by in vitro fertilization in comparison to in vivo- and somatic-cell nuclear transfer-derived embryos.

    PubMed

    Hamm, Jennifer; Tessanne, Kim; Murphy, Clifton N; Prather, Randall S

    2014-06-01

    In vitro embryo production is important for research in animal reproduction, embryo transfer, transgenics, and cloning. Yet, in vitro-fertilized (IVF) embryos are generally developmentally delayed and are inferior to in vivo-derived (IVV) embryos; this discrepancy is likely a result of aberrant gene expression. Transcription of three genes implicated to be important in normal preimplantation embryo development, TRIM28, SETDB1, and TP53, was determined by quanitative PCR in IVF, somatic-cell nuclear transfer (SCNT), parthenogenetic, and IVV porcine oocytes and embryos. There was no difference in TRIM28 or SETDB1 abundance between oocytes matured in vitro versus in vivo (P > 0.05), whereas TP53 levels were higher in in vitro-matured oocytes. TRIM28 increased from metaphase-II oocytes to the 4-cell and blastocyst stages in IVF embryos, whereas IVV embryos showed a reduction in TRIM28 abundance from maturation throughout development. The relative abundance of TP53 increased by the blastocyst stage in all treatment groups, but was higher in IVF embryos compared to IVV and SCNT embryos. In contrast, SETDB1 transcript levels decreased from the 2-cell to blastocyst stage in all treatments. For each gene analyzed, SCNT embryos of both hard-to-clone and easy-to-clone cell lines were more comparable to IVV than IVF embryos. Knockdown of TRIM28 also had no effect on blastocyst development or expression of SETDB1 or TP53. Thus, TRIM28, SETDB1, and TP53 are dynamically expressed in porcine oocytes and embryos. Furthermore, TRIM28 and TP53 abundances in IVV and SCNT embryos are similar, but different from quantities in IVF embryos. PMID:24659575

  4. Transcriptional regulators TRIM28, SETDB1, and TP53 are aberrantly expressed in porcine embryos produced by in vitro fertilization in comparison to in vivo- and somatic-cell nuclear transfer-derived embryos

    PubMed Central

    Hamm, Jennifer; Tessanne, Kim; Murphy, Clifton N; Prather, Randall S

    2014-01-01

    In vitro embryo production is important for research in animal reproduction, embryo transfer, transgenics, and cloning. Yet, in vitro-fertilized (IVF) embryos are generally developmentally delayed and are inferior to in vivo-derived (IVV) embryos; this discrepancy is likely a result of aberrant gene expression. Transcription of three genes implicated to be important in normal preimplantation embryo development, TRIM28, SETDB1, and TP53, was determined by quanitative PCR in IVF, somatic-cell nuclear transfer (SCNT), parthenogenetic, and IVV porcine oocytes and embryos. There was no difference in TRIM28 or SETDB1 abundance between oocytes matured in vitro versus in vivo (P > 0.05), whereas TP53 levels were higher in in vitro-matured oocytes. TRIM28 increased from metaphase-II oocytes to the 4-cell and blastocyst stages in IVF embryos, whereas IVV embryos showed a reduction in TRIM28 abundance from maturation throughout development. The relative abundance of TP53 increased by the blastocyst stage in all treatment groups, but was higher in IVF embryos compared to IVV and SCNT embryos. In contrast, SETDB1 transcript levels decreased from the 2-cell to blastocyst stage in all treatments. For each gene analyzed, SCNT embryos of both hard-to-clone and easy-to-clone cell lines were more comparable to IVV than IVF embryos. Knockdown of TRIM28 also had no effect on blastocyst development or expression of SETDB1 or TP53. Thus, TRIM28, SETDB1, and TP53 are dynamically expressed in porcine oocytes and embryos. Furthermore, TRIM28 and TP53 abundances in IVV and SCNT embryos are similar, but different from quantities in IVF embryos. Mol. Reprod. Dev. 81: 552–556, 2014. © 2014 The Authors. Published by Wiley Periodicals, Inc. PMID:24659575

  5. How Active Are Porcine Endogenous Retroviruses (PERVs)?

    PubMed Central

    Denner, Joachim

    2016-01-01

    Porcine endogenous retroviruses (PERVs) represent a risk factor if porcine cells, tissues, or organs were to be transplanted into human recipients to alleviate the shortage of human transplants; a procedure called xenotransplantation. In contrast to human endogenous retroviruses (HERVs), which are mostly defective and not replication-competent, PERVs are released from normal pig cells and are infectious. PERV-A and PERV-B are polytropic viruses infecting cells of several species, among them humans; whereas PERV-C is an ecotropic virus infecting only pig cells. Virus infection was shown in co-culture experiments, but also in vivo, in the pig, leading to de novo integration of proviruses in certain organs. This was shown by measurement of the copy number per cell, finding different numbers in different organs. In addition, recombinations between PERV-A and PERV-C were observed and the recombinant PERV-A/C were found to be integrated in cells of different organs, but not in the germ line of the animals. Here, the evidence for such in vivo activities of PERVs, including expression as mRNA, protein and virus particles, de novo infection and recombination, will be summarised. These activities make screening of pigs for provirus number and PERV expression level difficult, especially when only blood or ear biopsies are available for analysis. Highly sensitive methods to measure the copy number and the expression level will be required when selecting pigs with low copy number and low expression of PERV as well as when inactivating PERVs using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (CRISPR/Cas) technology. PMID:27527207

  6. Application of hollow fiber vitrification for cryopreservation of bovine early cleavage stage embryos and porcine morula-blastomeres.

    PubMed

    Uchikura, Ayuko; Matsunari, Hitomi; Nakano, Kazuaki; Hatae, Shota; Nagashima, Hiroshi

    2016-04-22

    A novel hollow fiber vitrification (HFV) method was applied to materials that have previously been difficult to cryopreserve, thereby expanding the potential application of this method. The results showed that zona-free porcine morulae and their isolated blastomeres remained viable even after vitrification. The rate of development to blastocysts after vitrification was similar for zona-free and zona-intact morulae (21/23, 91.3% for both). Vitrified blastomeres had a developmental potential equal to that of non-vitrified blastomeres (blastocyst formation rate after reaggregation: 16/17, 94.1% for both). The HFV method was also effective for the cryopreservation of in vitro matured/fertilized bovine embryos at the 2- to 4-cell, 8- to 16-cell and morula stages. The blastocyst formation rates of vitrified embryos (66.1-82.5%) were similar to those of non-vitrified embryos (74.5-82.5%). These results indicate that this novel HFV method is an effective tool for embryo cryopreservation that can enhance current practices in reproductive biology. PMID:26875691

  7. Application of hollow fiber vitrification for cryopreservation of bovine early cleavage stage embryos and porcine morula-blastomeres

    PubMed Central

    UCHIKURA, Ayuko; MATSUNARI, Hitomi; NAKANO, Kazuaki; HATAE, Shota; NAGASHIMA, Hiroshi

    2016-01-01

    A novel hollow fiber vitrification (HFV) method was applied to materials that have previously been difficult to cryopreserve, thereby expanding the potential application of this method. The results showed that zona-free porcine morulae and their isolated blastomeres remained viable even after vitrification. The rate of development to blastocysts after vitrification was similar for zona-free and zona-intact morulae (21/23, 91.3% for both). Vitrified blastomeres had a developmental potential equal to that of non-vitrified blastomeres (blastocyst formation rate after reaggregation: 16/17, 94.1% for both). The HFV method was also effective for the cryopreservation of in vitro matured/fertilized bovine embryos at the 2- to 4-cell, 8- to 16-cell and morula stages. The blastocyst formation rates of vitrified embryos (66.1–82.5%) were similar to those of non-vitrified embryos (74.5–82.5%). These results indicate that this novel HFV method is an effective tool for embryo cryopreservation that can enhance current practices in reproductive biology. PMID:26875691

  8. In vitro manipulation techniques of porcine embryos: a meta-analysis related to transfers, pregnancies and piglets.

    PubMed

    Liu, Ying; Li, Juan; Løvendahl, Peter; Schmidt, Mette; Larsen, Knud; Callesen, Henrik

    2015-03-01

    During the last 17 years, considerable advancements have been achieved in the production of pigs, transgenic and non-transgenic, by methods of somatic cell nuclear transfer, in vitro fertilisation, intracytoplasmic sperm injection, microinjection and sperm-mediated gene transfer by artificial insemination. Therefore, a review of the overall efficiency for the developmental competence of embryos produced by these in vitro methods would be useful in order to obtain a more thorough overview of this growing area with respect to its development and present status. In this review a meta-analysis was used to analyse data collected from all published articles with a focus on zygotes and embryos for transfer, pregnancy, full-term development and piglets born. It was generally concluded that an increasing level of in vitro manipulation of porcine embryos decreased the overall efficiency for production of piglets. The techniques of nuclear transfer have been developed markedly through the increasing number of studies performed, and the results have become more stable. Prolonged in vitro culture period did not lead to any negative effect on nuclear transfer embryos after their transfer and it resulted in a similar or even higher litter size. More complete information is needed in future scientific articles about these in vitro manipulation techniques to establish a more solid basis for the evaluation of their status and to reveal and further investigate any eventual problems. PMID:25482653

  9. In vitro effects of relaxin on gene expression in porcine cumulus-oocyte complexes and developing embryos

    PubMed Central

    2011-01-01

    Background Relaxin hormone peptide is found in porcine follicular and utero-tubal fluids, but its possible actions during early embryo development are still undetermined. Here, we investigated the effects of porcine relaxin during oocyte maturation and embryo development, and gene expression in the pig. Methods Immature cumulus-oocyte complexes (COCs) were obtained from ovarian follicles of sows. In experiment 1, COCs were matured in the presence of 0, 20, or 40 ng relaxin/ml, or 10% (v/v) porcine follicular fluid. In experiment 2, COCs were in vitro matured, fertilized and resulting embryos were cultured in the presence of 0, 20, or 40 ng relaxin/ml. In experiment 3, COCs were matured in the presence of 40 ng relaxin/ml, fertilized and zygotes were cultured as indicated in experiment 2. We evaluated the proportions of matured oocytes in experiment 1, cleaved and blastocysts on Day 2 and Day 7 post insemination in all experiments. The total cell number of blastocysts was also evaluated. In parallel, transcription levels of both relaxin and its receptors (RXFP1 and RXFP2), as well as a pro- (Bax) and anti- (Bcl2-like 1) apoptotic-related genes were determined. All data were analyzed by ANOVA and significant differences were fixed for P < 0.05. Results In experiment 1, relaxin significantly increased the proportions of matured oocytes and cleaved embryos, as well as the expression level of RXFP2 mRNA compared to RXFP1 (P < 0.05). There was no effect on endogenous expression of relaxin and Bcl2-like1/Bax ratios. In all experiments, relaxin did not affect the proportions of blastocysts, but did significantly increase their total cell numbers (P < 0.05). Furthermore, no effect of relaxin was observed on Bcl2-like1/Bax expression ratios, which were similar between groups. Conclusions Exogenous relaxin influences its own receptors expression, improves oocyte nuclear maturation. Its beneficial effect on total cell number of blastocysts appears to be through a Bcl2-like1

  10. Impairment of Preimplantation Porcine Embryo Development by Histone Demethylase KDM5B Knockdown Through Disturbance of Bivalent H3K4me3-H3K27me3 Modifications1

    PubMed Central

    Huang, Jiaojiao; Zhang, Hongyong; Wang, Xianlong; Dobbs, Kyle B.; Yao, Jing; Qin, Guosong; Whitworth, Kristin; Walters, Eric M.; Prather, Randall S.; Zhao, Jianguo

    2015-01-01

    ABSTRACT KDM5B (JARID1B/PLU1) is a H3K4me2/3 histone demethylase that is implicated in cancer development and proliferation and is also indispensable for embryonic stem cell self-renewal, cell fate, and murine embryonic development. However, little is known about the role of KDM5B during preimplantation embryo development. Here we show that KDM5B is critical to porcine preimplantation development. KDM5B was found to be expressed in a stage-specific manner, consistent with demethylation of H3K4me3, with the highest expression being observed from the 4-cell to the blastocyst stages. Knockdown of KDM5B by morpholino antisense oligonucleotides injection impaired porcine embryo development to the blastocyst stage. The impairment of embryo development might be caused by increased expression of H3K4me3 at the 4-cell and blastocyst stages, which disturbs the balance of bivalent H3K4me3-H3K27me3 modifications at the blastocyst stage. Decreased abundance of H3K27me3 at blastocyst stage activates multiple members of homeobox genes (HOX), which need to be silenced for faithful embryo development. Additionally, the histone demethylase KDM6A was found to be upregulated by knockdown of KDM5B, which indicated it was responsible for the decreased abundance of H3K27me3 at the blastocyst stage. The transcriptional levels of Ten-Eleven Translocation gene family members (TET1, TET2, and TET3) are found to be increased by knockdown of KDM5B, which indicates cross talk between histone modifications and DNA methylation. The studies above indicate that KDM5B is required for porcine embryo development through regulating the balance of bivalent H3K4me3-H3K27me3 modifications. PMID:25609834

  11. A comparison of different vitrification devices and the effect of blastocoele collapse on the cryosurvival of in vitro produced porcine embryos

    PubMed Central

    BARTOLAC, Louise Katherine; LOWE, Jenna Louise; KOUSTAS, George; SJÖBLOM, Cecilia; GRUPEN, Christopher Gerald

    2015-01-01

    The aim of this study was to determine the optimum conditions for vitrifying in vitro produced day 7 porcine embryos using different vitrification devices and blastocoele collapse methods. Firstly embryos were collapsed by micro-pipetting, needle puncture and sucrose with and without conducting vitrification. In the next experiment, non-collapsed embryos were vitrified in an open device using either superfine open-pulled straws (SOPS) or the CryoLoopTM system, or vitrified in a closed device using either the CryoTipTM or Cryo BioTM’s high security vitrification system (HSV). The post-thaw survival of embryos vitrified in the open devices did not differ significantly (SOPS: 37.3%; CryoLoopTM: 37.3%) nor did the post-thaw survival of embryos vitrified in the closed devices (CryoTip™: 38.5%; HSV: 42.5%). The re-expansion rate of embryos that were collapsed via micro-pipetting (76.0%) did not differ from those that were punctured (75.0%) or collapsed via sucrose (79.6%) when vitrification was not performed. However, embryos collapsed via sucrose solutions (24.5%) and needle puncture (16.0%) prior to vitrification were significantly less likely to survive vitrification than the control (non-collapsed) embryos (53.6%, P < 0.05). The findings show that both open and closed vitrification devices were equally effective for the vitrification of porcine blastocysts. Collapsing blastocysts prior to vitrification did not improve survival, which is inconsistent with the findings of studies in other species. This may be due to the extremely sensitive nature of porcine embryos, and/or the invasiveness of the collapsing procedures. PMID:26211782

  12. Antioxidant activities of chick embryo egg hydrolysates

    PubMed Central

    Sun, Hao; Ye, Ting; Wang, Yuntao; Wang, Ling; Chen, Yijie; Li, Bin

    2014-01-01

    Chick embryo egg hydrolysates (CEEH) were obtained by enzymatic hydrolysis of chick embryo egg in vitro-simulated gastrointestinal digestion. The antioxidant activities of CEEH were investigated by employing three in vitro assays, including the 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate)/1,1-diphenyl-2-picrylhydrazyl (ABTS/DPPH)/hydroxyl radical-scavenging assays. The radical-scavenging effect of CEEH (1.0 mg/mL) was in a dose-dependent manner, with the highest trolox equivalent antioxidant capacity for ABTS, DPPH, and that of hydroxyl radicals found to be 569, 2097, and 259.6 μmol/L, respectively; whereas the trolox equivalent antioxidant capacity of unhatched egg for ABTS, DPPH, and that of hydroxyl radicals were found to be 199, 993, and 226.5 μmol/L, respectively. CEEH showed stronger scavenging activity than the hydrolysates of unhatched egg against free radicals such as ABTS, DPPH, and hydroxyl radicals. The antioxidant amino acid analysis indicated that the 14-day CEEH possess more antioxidant amino acids than that of the unhatched egg. In addition, essential amino acids analysis showed that the 14-day CEEH have the highest nutritional value. Combined with the results of the amino acid profiles, CEEH were believed to have higher nutritive value in addition to antioxidant activities than the unhatched egg. PMID:24804065

  13. Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR.

    PubMed

    Blanes, Milena S; Tsoi, Stephen C M; Dyck, Michael K

    2016-01-01

    Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, the ability to determine an embryo's sex is necessary in many experiments particularly regarding early development. The present protocol demonstrates an inexpensive, rapid and non-toxic preparation of pig genomic DNA for use with PCR. Day 30 embryos must be humanely collected according to the guidelines established by Institutional Animal Policy and Welfare Committees for the present protocol. The preparation of the whole embryo for this PCR based sexing technique simply involves grinding the frozen embryo to a fine powder using a pre-chilled mortar and pestle. PCR-quality DNA is released from a small amount of embryo powder by applying a hot incubation in an alkaline lysis reagent. Next, the DNA solution is mixed with neutralization buffer and used directly for PCR. Two primer pairs are generated to detect specific sex determining region of the Y- chromosome (SRY) and ZFX region of the X- chromosome with high accuracy and specificity. The same protocol can be applied to other elongated embryos (Day 10 to Day 14) earlier than Day 30. Also, this protocol can be carried with 96-welled plates when screening a large number of embryos, making it feasible for automation and high-throughput sex typing. PMID:26966900

  14. Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities

    PubMed Central

    Fu, Bo; Ren, Liang; Liu, Di; Ma, Jian-Zhang; An, Tie-Zhu; Yang, Xiu-Qin; Ma, Hong; Zhang, Dong-Jie; Guo, Zhen-Hua; Guo, Yun-Yun; Zhu, Meng; Bai, Jing

    2015-01-01

    The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (−) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes. PMID:26580437

  15. Isolation and characterization of trophoblast-derived stem-like cells from peri-implantation porcine embryos.

    PubMed

    Suasnavas, Edison A; Heywood, Sierra; Ward, Anika; Cox, Lindsay; O'Grady, Merecedes; Zhao, Yuanfeng; Gilbert, Lacey; Isom, S Clay

    2015-03-01

    In mammals, the trophoblast lineage of the embryo is specified before attachment/implantation to become the fetal portion of the placenta. Trophoblast-derived cells were isolated and cultured from day 10 and day 13 porcine embryos and were grown in vitro in a defined, serum-free culture medium for over 2 years without showing any signs of senescence. However, trophoblast-derived cells placed into serum-containing medium rapidly senesce and fail to proliferate. Semiquantitative and quantitative gene expression analyses of cells in culture from 0 to 30 days confirmed the presence (and relative abundance) of mRNA transcripts from genes involved in trophoblast function (CDX2, TEAD4, CYP17A1, HSD17B1, FGFR2, PLET, HAND1) as well as some genes known to mediate pluripotency (POU5F1, KLF4, CMYC). Protein immunolocalization demonstrated expression of both trophoblast and mesenchymal cell markers. DNA methylation patterns in promoters of three critical developmental genes (HAND1, KLF4, TEAD4) did not change appreciably over 4 months of culture in vitro. It was demonstrated that these trophoblast-derived cells are easily stably transfected with an exogenous transgene (eGFP) by a variety of methods, and show the ability to survive and to be passaged repeatedly after transfection. In summary, early embryonic porcine trophoblast-derived cells have demonstrated unique characteristics, which means they could be used as valuable tools for laboratory work. Anticipated applications include the study of trophoblast physiology as well as possible solutions for improving efficiency of transgenesis by somatic cell nuclear transfer and for pluripotency reprogramming of cells. PMID:25660622

  16. Factors Determining the Efficiency of Porcine Somatic Cell Nuclear Transfer: Data Analysis with Over 200,000 Reconstructed Embryos.

    PubMed

    Liu, Tianbin; Dou, Hongwei; Xiang, Xi; Li, Lin; Li, Yong; Lin, Lin; Pang, Xinzhi; Zhang, Yijie; Chen, Yu; Luan, Jing; Xu, Ying; Yang, Zhenzhen; Yang, Wenxian; Liu, Huan; Li, Feida; Wang, Hui; Yang, Huanming; Bolund, Lars; Vajta, Gabor; Du, Yutao

    2015-12-01

    Data analysis in somatic cell nuclear transfer (SCNT) research is usually limited to several hundreds or thousands of reconstructed embryos. Here, we report mass results obtained with an established and consistent porcine SCNT system (handmade cloning [HMC]). During the experimental period, 228,230 reconstructed embryos and 82,969 blastocysts were produced. After being transferred into 656 recipients, 1070 piglets were obtained. First, the effects of different types of donor cells, including fetal fibroblasts (FFs), adult fibroblasts (AFs), adult preadipocytes (APs), and adult blood mesenchymal (BM) cells, were investigated on the further in vitro and in vivo development. Compared to adult donor cells (AFs, APs, BM cells, respectively), FF cells resulted in a lower blastocyst/reconstructed embryo rate (30.38% vs. 37.94%, 34.65%, and 34.87%, respectively), but a higher overall efficiency on the number of piglets born alive per total blastocysts transferred (1.50% vs. 0.86%, 1.03%, and 0.91%, respectively) and a lower rate of developmental abnormalities (10.87% vs. 56.57%, 24.39%, and 51.85%, respectively). Second, recloning was performed with cloned adult fibroblasts (CAFs) and cloned fetal fibroblasts (CFFs). When CAFs were used as the nuclear donor, fewer developmental abnormalities and higher overall efficiency were observed compared to AFs (56.57% vs. 28.13% and 0.86% vs. 1.59%, respectively). However, CFFs had an opposite effect on these parameters when compared with CAFs (94.12% vs. 10.87% and 0.31% vs. 1.50%, respectively). Third, effects of genetic modification on the efficiency of SCNT were investigated with transgenic fetal fibroblasts (TFFs) and gene knockout fetal fibroblasts (KOFFs). Genetic modification of FFs increased developmental abnormalities (38.96% and 25.24% vs. 10.87% for KOFFs, TFFs, and FFs, respectively). KOFFs resulted in lower overall efficiency compared to TFFs and FFs (0.68% vs. 1.62% and 1.50%, respectively). In conclusion, this is the

  17. Effects of protein source, vitamin E and phenazine ethosulfate on developmental competence and quality of porcine embryos cultured in vitro.

    PubMed

    Gajda, Barbara; Bryła, Magdalena; Smorag, Zdzisław

    2008-01-01

    Effects of fetal calf serum (FCS) or bovine serum albumin (BSA), with or without vitamin E (vit. E) or phenazine ethosulfate (PES) supplementation on developmental competence and quality of cultured porcine embryos were examined. The experiment was done on zygotes and 2-cell embryos obtained from superovulated gilts. Morphologically normal zygotes were cultured in vitro in NCSU-23 medium supplemented with: experiment 1-0.004 g/ml BSA, 10% FCS, protein-free (control); experiment 2-0 (control), 25, 50 or 100 microM vit. E; experiment 3-0 (control), 0.025, 0.05 or 0.075 microM PES. Embryo quality criteria were developmental competence (cleavage, morula and blastocyst rates), total cell number per blastocyst and degree of apoptosis as assessed by TUNEL staining. Presence of BSA in culture medium increased significantly morula and blastocysts production as compared to FCS (P < 0.001) and protein-free group (P < 0.05 and P < 0.001, respectively). The blastocysts cultured in protein-free medium had higher average number of apoptotic nuclei and DNA fragmented nucleus index as compared to the BSA (P < 0.05 and P < 0.01, respectively) and FCS (P < 0.5) group. Supplementation in culture medium of 100 microM vit. E increased blastocyst production as compared to control and 50 microM vit-E (P < 0.05). Both the number of cells per and percentage of TUNEL positive nuclei per blastocyst were slightly lower in PES treated than control groups. PMID:19055026

  18. Antiviral Regulation in Porcine Monocytic Cells at Different Activation States

    PubMed Central

    Rowland, Raymond R. R.

    2014-01-01

    ABSTRACT Monocytic cells, including macrophages and dendritic cells, exist in different activation states that are critical to the regulation of antimicrobial immunity. Many pandemic viruses are monocytotropic, including porcine reproductive and respiratory syndrome virus (PRRSV), which directly infects subsets of monocytic cells and interferes with antiviral responses. To study antiviral responses in PRRSV-infected monocytic cells, we characterized inflammatory cytokine responses and genome-wide profiled signature genes to investigate response pathways in uninfected and PRRSV-infected monocytic cells at different activation states. Our findings showed suppressed interferon (IFN) production in macrophages in non-antiviral states and an arrest of lipid metabolic pathways in macrophages at antiviral states. Importantly, porcine monocytic cells at different activation states were susceptible to PRRSV and responded differently to viral infection. Based on Gene Ontology (GO) analysis, two approaches were used to potentiate antiviral activity: (i) pharmaceutical modulation of cellular lipid metabolism and (ii) in situ PRRSV replication-competent expression of interferon alpha (IFN-α). Both approaches significantly suppressed exogenous viral infection in monocytic cells. In particular, the engineered IFN-expressing PRRSV strain eliminated exogenous virus infection and sustained cell viability at 4 days postinfection in macrophages. These findings suggest an intricate interaction of viral infection with the activation status of porcine monocytic cells. An understanding and integration of antiviral infection with activation status of monocytic cells may provide a means of potentiating antiviral immunity. IMPORTANCE Activation statuses of monocytic cells, including monocytes, macrophages (Mϕs), and dendritic cells (DCs), are critically important for antiviral immunity. Unfortunately, the activation status of porcine monocytic cells or how cell activation status

  19. In Vitro Elongation of Porcine Embryos Using Alginate Hydrogels as a Three-Dimensional Extracellular Matrix

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the pig, the pre-implantation period of pregnancy is highly influential on sow productivity and therefore the profitability of swine production. Between Day 11 and 12 of gestation, the embryo undergoes a significant morphological change, during which it transforms from an ovoid structure of about...

  20. PROTEOMICS STUDY OF THE TROPHOBLAST ELONGATION PHASE OF THE PORCINE EMBRYO

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In pigs, about 20% embryonic loss is observed after artificial insemination or natural mating during peri-implantation and is coincident with rapid embryo elongation. The objective of the present study was to establish a comprehensive profile of abundant proteins and identify the expression pattern...

  1. Cell cycle synchronization of leukemia inhibitory factor (LIF)-dependent porcine-induced pluripotent stem cells and the generation of cloned embryos.

    PubMed

    Yuan, Ye; Lee, Kiho; Park, Kwang-Wook; Spate, Lee D; Prather, Randall S; Wells, Kevin D; Roberts, R Michael

    2014-01-01

    Nuclear transfer (NT) from porcine iPSC to create cloned piglets is unusually inefficient. Here we examined whether such failure might be related to the cell cycle stage of donor nuclei. Porcine iPSC, derived here from the inner cell mass of blastocysts, have a prolonged S phase and are highly sensitive to drugs normally used for synchronization. However, a double-blocking procedure with 0.3 μM aphidicolin for 10 h followed by 20 ng/ml nocodazole for 4 h arrested 94.3% of the cells at G2/M and, after release from the block, provided 70.1% cells in the subsequent G1 phase without causing any significant loss of cell viability or pluripotent phenotype. Nuclei from different cell cycle stages were used as donors for NT to in vitro-matured metaphase II oocytes. G2/M nuclei were more efficient than either G1 and S stage nuclei in undergoing first cleavage and in producing blastocysts, but all groups had a high incidence of chromosomal/nuclear abnormalities at 2 h and 6 h compared with non-synchronized NT controls from fetal fibroblasts. Many G2 embryos extruded a pseudo-second polar body soon after NT and, at blastocyst, tended to be either polyploid or diploid. By contrast, the few G1 blastocysts that developed were usually mosaic or aneuploid. The poor developmental potential of G1 nuclei may relate to lack of a G1/S check point, as the cells become active in DNA synthesis shortly after exit from mitosis. Together, these data provide at least a partial explanation for the almost complete failure to produce cloned piglets from piPSC. PMID:24621508

  2. TERATOGENICITY OF CYCLOPHOSPHAMIDE IN A COUPLED MICROSOMAL ACTIVATING/EMBRYO CULTURE SYSTEM

    EPA Science Inventory

    Using the coupled microsomal activating/embryo culture system, in vitro experiments were performed to establish the role of metabolism in the embryo toxicity and teratogenicity of cyclophosphamide. Cyclophosphamide in the coupled microsomal activating/embryo culture system produc...

  3. Vesicles Cytoplasmic Injection: An Efficient Technique to Produce Porcine Transgene-Expressing Embryos.

    PubMed

    Luchetti, C G; Bevacqua, R J; Lorenzo, M S; Tello, M F; Willis, M; Buemo, C P; Lombardo, D M; Salamone, D F

    2016-08-01

    The use of vesicles co-incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co-incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx-egfp was injected circular (CP) at 3, 30 and 300 ng/μl and linear (LP) at 30 ng/μl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/μl (N = 105), 30 ng/μl (N = 95) and 300 ng/μl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/μl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p < 0.05). The parthenogenic CP naked group showed lower cleavage respect to control (p < 0.05). The highest concentration of plasmids to allow development to blastocyst stage was 30 ng/μl. There were no differences in DNA fragmentation between groups. The parthenogenic LP naked group resulted in high GFP rates (46%) and also allowed the production of GFP blastocysts (33%). The cytoplasmic injection with LP vesicles into parthenogenic zygotes allowed 100% GFP blastocysts. Injected IVF showed higher cleavage rates than control (p < 0.05). In IVF zygotes, only the use of vesicles produced GFP blastocysts. The use of vesicles co-incubated with plasmids improves the transgene expression efficiency for cytoplasmic injection in porcine zygotes and constitutes a simple technique for easy delivery of plasmids. PMID:27260090

  4. In vitro antiviral activity of germacrone against porcine parvovirus.

    PubMed

    Chen, Ye; Dong, Yunxia; Jiao, Yiren; Hou, Lianjie; Shi, Yuzhen; Gu, Ting; Zhou, Pei; Shi, Zhongyuan; Xu, Lulu; Wang, Chong

    2015-06-01

    Porcine parvovirus (PPV) infections can lead to significant losses to the swine industry by causing reproductive failure in pigs. Germacrone has been reported to efficiently suppress the replication of influenza virus. In this report, the antiviral activity of germacrone on PPV in swine testis (ST) cells was investigated. Here, we show for the first time that germacrone protects cells from PPV infection and suppresses the synthesis of viral mRNA and protein. Furthermore, we show that germacrone inhibits PPV replication at an early stage in a dose-dependent manner. These findings suggest that germacrone is a potential candidate for anti-PPV therapy. PMID:25813663

  5. The effect of soluble uterine factors on porcine embryo development within a three-dimensional alginate matrix system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Between day 10 and 12 of gestation in the pig, the embryo undergoes a dramatic morphological change, known as elongation. During elongation the embryo produces and secretes estrogen, which serves as a key signal for maternal recognition of pregnancy. The uterine environment prepares for embryo elong...

  6. Development of pre-implantation porcine embryos cultured within a three-dimensional alginate hydrogel system either conjugated with Arg-Gly-Asp (RGD) peptide or supplemented with secreted phosphoprotein 1 (SPP1)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many uterine specific factors have been shown to be increased within the uterine milieu as the porcine embryo initiates elongation. Secreted phosphoprotein 1 (SPP1) is increased during this time and contains an Arg-Gly-Asp (RGD) peptide sequence that has been shown to bind to cell surface integrins ...

  7. Induction of photolyase activity in wood frog (Rana sylvatica) embryos.

    PubMed

    Smith, M A; Kapron, C M; Berrill, M

    2000-10-01

    Rising ultraviolet-B (UVB, 280-320 nm) radiation has been proposed as a factor which may explain nonnormal amphibian population declines. Accordingly research has been directed toward estimating the photolyase activity of several amphibian species in order to predict a species' resilience to UV damage. Unfortunately, in spite of published research which demonstrated that the activity of one of the principal photorepair enzymes, photolyase, can be induced, these estimates did not address the potential for in vivo induction by environmental factors present in situ. We show here that wood frog (Rana sylvatica) embryos exposed to periods of ambient solar radiation (1) displayed significantly different photolyase activities from embryos exposed to equivalent periods of dark; and (2) were positively correlated with the UVB fluence received in vivo. Such results suggest that previous conclusions regarding the relationship between photorepair and population decline must be reevaluated. Estimating amphibian photorepair is a complicated process, and caution must be exercised when interpreting such data. PMID:11045732

  8. Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

    PubMed

    Gambini, Andrés; De Stéfano, Adrián; Jarazo, Javier; Buemo, Carla; Karlanian, Florencia; Salamone, Daniel Felipe

    2016-09-01

    The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes

  9. Effect of Cell Cycle Interactions and Inhibition of Histone Deacetylases on Development of Porcine Embryos Produced by Nuclear Transfer.

    PubMed

    Rissi, Vitor B; Glanzner, Werner G; Mujica, Lady K S; Antoniazzi, Alfredo Q; Gonçalves, Paulo B D; Bordignon, Vilceu

    2016-02-01

    The aim of this study was to evaluate if the positive effects of inhibiting histone deacetylase enzymes on cell reprogramming and development of somatic cell nuclear transfer (SCNT) embryos is affected by the cell cycle stage of nuclear donor cells and host oocytes at the time of embryo reconstruction. SCNT embryos were produced with metaphase II (MII) or telophase II (TII) cytoplasts and nuclear donor cells that were either at the G1-0 or G2/M stages. Embryos reconstructed with the different cell cycle combinations were treated or not with the histone deacetylase inhibitor (HDACi) Scriptaid for 15 h and then cultured in vitro for 7 days. Embryos reconstructed with MII-G1-0 and TII-G2/M developed to the blastocyst stage with a higher frequency compared to the other groups, confirming the importance of cell cycle interactions on cell reprogramming and SCNT embryo development. Treatment with HDACi improved development of SCNT embryos produced with MII but not TII cytoplasts, independently of the cell cycle stage of nuclear donor cells. These findings provide evidence that the positive effect of HDACi treatment on development of SCNT embryos depends upon cell cycle interactions between the host cytoplast and the nuclear donor cells. PMID:27281695

  10. Porcine pancreatic lipase related protein 2 has high triglyceride lipase activity in the absence of colipase.

    PubMed

    Xiao, Xunjun; Ross, Leah E; Sevilla, Wednesday A; Wang, Yan; Lowe, Mark E

    2013-09-01

    Efficient dietary fat digestion is essential for newborns who consume more dietary fat per body weight than at any other time of life. In many mammalian newborns, pancreatic lipase related protein 2 (PLRP2) is the predominant duodenal lipase. Pigs may be an exception since PLRP2 expression has been documented in the intestine but not in the pancreas. Because of the differences in tissue-specific expression, we hypothesized that the kinetic properties of porcine PLRP2 would differ from those of other mammals. To characterize its properties, recombinant porcine PLRP2 was expressed in HEK293T cells and purified to homogeneity. Porcine PLRP2 had activity against tributyrin, trioctanoin and triolein. The activity was not inhibited by bile salts and colipase, which is required for the activity of pancreatic triglyceride lipase (PTL), minimally stimulated PLRP2 activity. Similar to PLRP2 from other species, PLRP2 from pigs had activity against galactolipids and phospholipids. Importantly, porcine PLRP2 hydrolyzed a variety of dietary substrates including pasteurized human mother's milk and infant formula and its activity was comparable to that of PTL. In conclusion, porcine PLRP2 has broad substrate specificity and has high triglyceride lipase activity even in the absence of colipase. The data suggest that porcine PLRP2 would be a suitable lipase for inclusion in recombinant preparations for pancreatic enzyme replacement therapy. PMID:23770034

  11. Progressive hypoxia decouples activity and aerobic performance of skate embryos

    PubMed Central

    Di Santo, Valentina; Tran, Anna H.; Svendsen, Jon C.

    2016-01-01

    Although fish population size is strongly affected by survival during embryonic stages, our understanding of physiological responses to environmental stressors is based primarily on studies of post-hatch fishes. Embryonic responses to acute exposure to changes in abiotic conditions, including increase in hypoxia, could be particularly important in species exhibiting long developmental time, as embryos are unable to select a different environment behaviourally. Given that oxygen is key to metabolic processes in fishes and aquatic hypoxia is becoming more severe and frequent worldwide, organisms are expected to reduce their aerobic performance. Here, we examined the metabolic and behavioural responses of embryos of a benthic elasmobranch fish, the little skate (Leucoraja erinacea), to acute progressive hypoxia, by measuring oxygen consumption and movement (tail-beat) rates inside the egg case. Oxygen consumption rates were not significantly affected by ambient oxygen levels until reaching 45% air saturation (critical oxygen saturation, Scrit). Below Scrit, oxygen consumption rates declined rapidly, revealing an oxygen conformity response. Surprisingly, we observed a decoupling of aerobic performance and activity, as tail-beat rates increased, rather than matching the declining metabolic rates, at air saturation levels of 55% and below. These results suggest a significantly divergent response at the physiological and behavioural levels. While skate embryos depressed their metabolic rates in response to progressive hypoxia, they increased water circulation inside the egg case, presumably to restore normoxic conditions, until activity ceased abruptly around 9.8% air saturation. PMID:27293746

  12. Matrine displayed antiviral activity in porcine alveolar macrophages co-infected by porcine reproductive and respiratory syndrome virus and porcine circovirus type 2.

    PubMed

    Sun, Na; Sun, Panpan; Lv, Haipeng; Sun, Yaogui; Guo, Jianhua; Wang, Zhirui; Luo, Tiantian; Wang, Shaoyu; Li, Hongquan

    2016-01-01

    The co-infection of porcine reproductive respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) is quite common in clinical settings and no effective treatment to the co-infection is available. In this study, we established the porcine alveolar macrophages (PAM) cells model co-infected with PRRSV/PCV2 with modification in vitro, and investigated the antiviral activity of Matrine on this cell model and further evaluated the effect of Matrine on virus-induced TLR3,4/NF-κB/TNF-α pathway. The results demonstrated PAM cells inoculated with PRRSV followed by PCV2 2 h later enhanced PRRSV and PCV2 replications. Matrine treatment suppressed both PRRSV and PCV2 infection at 12 h post infection. Furthermore, PRRSV/PCV2 co- infection induced IκBα degradation and phosphorylation as well as the translocation of NF-κB from the cytoplasm to the nucleus indicating that PRRSV/PCV2 co-infection induced NF-κB activation. Matrine treatment significantly down-regulated the expression of TLR3, TLR4 and TNF-α although it, to some extent, suppressed p-IκBα expression, suggesting that TLR3,4/NF-κB/TNF-α pathway play an important role of Matrine in combating PRRSV/PCV2 co-infection. It is concluded that Matrine possesses activity against PRRSV/PCV2 co-infection in vitro and suppression of the TLR3,4/NF-κB/TNF-α pathway as an important underlying molecular mechanism. These findings warrant Matrine to be further explored for its antiviral activity in clinical settings. PMID:27080155

  13. Matrine displayed antiviral activity in porcine alveolar macrophages co-infected by porcine reproductive and respiratory syndrome virus and porcine circovirus type 2

    PubMed Central

    Sun, Na; Sun, Panpan; Lv, Haipeng; Sun, Yaogui; Guo, Jianhua; Wang, Zhirui; Luo, Tiantian; Wang, Shaoyu; Li, Hongquan

    2016-01-01

    The co-infection of porcine reproductive respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) is quite common in clinical settings and no effective treatment to the co-infection is available. In this study, we established the porcine alveolar macrophages (PAM) cells model co-infected with PRRSV/PCV2 with modification in vitro, and investigated the antiviral activity of Matrine on this cell model and further evaluated the effect of Matrine on virus-induced TLR3,4/NF-κB/TNF-α pathway. The results demonstrated PAM cells inoculated with PRRSV followed by PCV2 2 h later enhanced PRRSV and PCV2 replications. Matrine treatment suppressed both PRRSV and PCV2 infection at 12 h post infection. Furthermore, PRRSV/PCV2 co- infection induced IκBα degradation and phosphorylation as well as the translocation of NF-κB from the cytoplasm to the nucleus indicating that PRRSV/PCV2 co-infection induced NF-κB activation. Matrine treatment significantly down-regulated the expression of TLR3, TLR4 and TNF-α although it, to some extent, suppressed p-IκBα expression, suggesting that TLR3,4/NF-κB/TNF-α pathway play an important role of Matrine in combating PRRSV/PCV2 co-infection. It is concluded that Matrine possesses activity against PRRSV/PCV2 co-infection in vitro and suppression of the TLR3,4/NF-κB/TNF-α pathway as an important underlying molecular mechanism. These findings warrant Matrine to be further explored for its antiviral activity in clinical settings. PMID:27080155

  14. Differential expression and activity of the porcine type I interferon family.

    PubMed

    Sang, Yongming; Rowland, Raymond R R; Hesse, Richard A; Blecha, Frank

    2010-07-01

    Type I interferons (IFNs) are central to innate and adaptive immunity, and many have unique developmental and physiological functions. However, in most species, only two subtypes, IFN-alpha and IFN-beta, have been well studied. Because of the increasing importance of zoonotic viral diseases and the use of pigs to address human research questions, it is important to know the complete repertoire and activity of porcine type I IFNs. Here we show that porcine type I IFNs comprise at least 39 functional genes distributed along draft genomic sequences of chromosomes 1 and 10. These functional IFN genes are classified into 17 IFN-alpha subtypes, 11 IFN-delta subtypes, 7 IFN-omega subtypes, and single-subtype subclasses of IFN-alphaomega, IFN-beta, IFN-epsilon, and IFN-kappa. We found that porcine type I IFNs have diverse expression profiles and antiviral activities against porcine reproductive and respiratory syndrome virus (PRRSV) and vesicular stomatitis virus (VSV), with activity ranging from 0 to >10(5) U.ng(-1).ml(-1). Whereas most IFN-alpha subtypes retained the greatest antiviral activity against both PRRSV and VSV in porcine and MARC-145 cells, some IFN-delta and IFN-omega subtypes, IFN-beta, and IFN-alphaomega differed in their antiviral activity based on target cells and viruses. Several IFNs, including IFN-alpha7/11, IFN-delta2/7, and IFN-omega4, exhibited minimal or no antiviral activity in the tested target cell-virus systems. Thus comparative studies showed that antiviral activity of porcine type I IFNs is virus- and cell-dependent, and IFN-alphas are positively correlated with induction of MxA, an IFN-stimulated gene. Collectively, these data provide fundamental genomic information for porcine type I IFNs, information that is necessary for understanding porcine physiological and antiviral responses. PMID:20406849

  15. BACE1 and cholinesterase inhibitory activities of Nelumbo nucifera embryos.

    PubMed

    Jung, Hyun Ah; Karki, Subash; Kim, Ji Hye; Choi, Jae Sue

    2015-06-01

    The aim of the present study was to evaluate the comparative anti-Alzheimer's disease (AD) activities of different parts of Nelumbo nucifera (leaves, de-embryo seeds, embryos, rhizomes, and stamens) in order to determine the selectivity and efficient use of its individual components. Anti-AD activities of different parts of N. nucifera were evaluated via inhibitory activities on acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) along with scavenging activity on peroxynitrite (ONOO(-)). Among the evaluated parts of N. nucifera, the embryo extract exhibited significant inhibitory potential against BACE1 and BChE as well as scavenging activity against ONOO(-). Thus, the embryo extract was selected for detailed investigation on anti-AD activity using BACE1- and ChEs-inhibitory assays. Among the different solvent-soluble fractions, the dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH) fractions showed promising ChEs and BACE1 inhibitory activities. Repeated column chromatography of the CH2Cl2, EtOAc and n-BuOH fractions yielded compounds 1-5, which were neferine (1), liensinine (2), vitexin (3), quercetin 3-O-glucoside (4) and northalifoline (5). Compound 2 exhibited potent inhibitory activities on BACE1, AChE, and BChE with respective IC50 values of 6.37 ± 0.13, 0.34 ± 0.02, and 9.96 ± 0.47 µM. Likewise, compound 1 showed potent inhibitory activities on BACE1, AChE, and BChE with IC50 values of 28.51 ± 4.04, 14.19 ± 1.46, and 37.18 ± 0.59 µM, respectively; the IC50 values of 3 were 19.25 ± 3.03, 16.62 ± 1.43, and 11.53 ± 2.21 µM, respectively. In conclusion, we identified potent ChEs- and BACE1-inhibitory activities of N. nucifera as well as its isolated constituents, which may be further explored to develop therapeutic and preventive agents for AD and oxidative stress related diseases. PMID:25300425

  16. Evaluation of a quail embryo model for the detection of botulinum toxin type A activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The quail embryo was evaluated for use as a bioassay to detect biologically active botulinum toxin serotype A (BoNT/A). Day 15 of incubation embryos were injected with decreasing dosages of BoNT/A from 250 to 0.5 ng of toxin. At 1 day post-injection, embryos receiving 20 ng of BoNT or higher had m...

  17. Evaluation of a quali embryo model for the detection of botulism toxin type A activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Japanese quail embryo (Coturnix japonica) was evaluated for use as a bioassay to detect biologically active botulinum toxin serotype A (BoNT/A). Day 15 of incubation embryos were injected with decreasing dosages of BoNT/A from 250 to 0.5 ng of toxin. At 1 day post-injection, embryos receiving ...

  18. Comparative analysis of signature genes in porcine reproductive and respiratory syndrome virus (PRRSV)-infected porcine monocyte-derived dendritic cells at differential activation statuses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Activation statuses of monocytic cells, e.g. monocytes, macrophages and dendritic cells (DCs), are critically important for antiviral immunity. In particular, some devastating viruses, including porcine reproductive and respiratory syndrome virus (PRRSV), are capable of directly infecting these cell...

  19. Early Aberrations in Chromatin Dynamics in Embryos Produced Under In Vitro Conditions

    PubMed Central

    Deshmukh, Rahul S.; Strejcek, Frantisek; Vejlsted, Morten; Lucas-Hahn, Andrea; Petersen, Bjorn; Li, Juan; Callesen, Henrik; Niemann, Heiner; Hyttel, Poul

    2012-01-01

    Abstract In vitro production of porcine embryos by means of in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) is limited by great inefficienciy. The present study investigated chromatin and nucleolar dynamics in porcine embryos developed in vivo (IV) and compared this physiological standard to that of embryos produced by IVF, parthenogenetic activation (PA), or SCNT. In contrast to IV embryos, chromatin spatial and temporal dynamics in PA, IVF, and SCNT embryos were altered; starting with aberrant chromatin–nuclear envelope interactions at the two-cell stage, delayed chromatin decondensation and nucleolar development at the four-cell stage, and ultimately culminating in failure of proper first lineage segregation at the blastocyst stage, demonstrated by poorly defined inner cell mass. Interestingly, in vitro produced (IVP) embryos also lacked a heterochromatin halo around nucleolar precursors, indicating imperfections in global chromatin remodeling after fertilization/activation. Porcine IV-produced zygotes and embryos display a well-synchronized pattern of chromatin dynamics compatible with genome activation and regular nucleolar formation at the four-cell stage. Production of porcine embryos under in vitro conditions by IVF, PA, or SCNT is associated with altered chromatin remodeling, delayed nucleolar formation, and poorly defined lineage segregation at the blastocyst stage, which in turn may impair their developmental capacity. PMID:22468997

  20. Antiviral activity of recombinant porcine surfactant protein A against porcine reproductive and respiratory syndrome virus in vitro.

    PubMed

    Li, Lan; Zheng, Qisheng; Zhang, Yuanpeng; Li, Pengcheng; Fu, Yanfeng; Hou, Jibo; Xiao, Xilong

    2016-07-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) has caused significant economic losses in the swine industry worldwide. However, there is not an ideal vaccine to provide complete protection against PRRSV. Thus, the need for new antiviral strategies to control PRRSV still remains. Surfactant protein A (SP-A) belongs to the family of C-type lectins, which can exert antiviral activities. In this present study, we assessed the antiviral properties of recombinant porcine SP-A (RpSP-A) on PRRSV infection in Marc 145 cells and revealed its antiviral mechanism using a plaque assay, real-time qPCR, western blotting analysis and an attachment and penetration assay. Our results showed that RpSP-A could inhibit the infectivity of PRRSV in Marc 145 cells and could reduce the total RNA and protein level. The attachment assay indicated that RpSP-A in the presence of Ca(2+) could largely inhibit Marc 145 cell attachment; however, in the penetration assay, it was relatively inactive. Furthermore, our study suggested that virus progeny released from infected Marc145 cells were blocked by RpSP-A from infecting other cells. We conclude that RpSP-A has antiviral activity against PRRSV, most probably by blocking viral attachment and the cell-to-cell transmission pathway, and therefore, RpSP-A holds promise as a novel antiviral agent against PRRSV. PMID:27101074

  1. Fluorescence-based visualization of autophagic activity predicts mouse embryo viability

    NASA Astrophysics Data System (ADS)

    Tsukamoto, Satoshi; Hara, Taichi; Yamamoto, Atsushi; Kito, Seiji; Minami, Naojiro; Kubota, Toshiro; Sato, Ken; Kokubo, Toshiaki

    2014-03-01

    Embryo quality is a critical parameter in assisted reproductive technologies. Although embryo quality can be evaluated morphologically, embryo morphology does not correlate perfectly with embryo viability. To improve this, it is important to understand which molecular mechanisms are involved in embryo quality control. Autophagy is an evolutionarily conserved catabolic process in which cytoplasmic materials sequestered by autophagosomes are degraded in lysosomes. We previously demonstrated that autophagy is highly activated after fertilization and is essential for further embryonic development. Here, we developed a simple fluorescence-based method for visualizing autophagic activity in live mouse embryos. Our method is based on imaging of the fluorescence intensity of GFP-LC3, a versatile marker for autophagy, which is microinjected into the embryos. Using this method, we show that embryonic autophagic activity declines with advancing maternal age, probably due to a decline in the activity of lysosomal hydrolases. We also demonstrate that embryonic autophagic activity is associated with the developmental viability of the embryo. Our results suggest that embryonic autophagic activity can be utilized as a novel indicator of embryo quality.

  2. Evaluation of quail and chicken embryos for the detection of botulism toxin serotypes A, B E and F activity.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparison of quail (Coturnix japonica) and chicken (Gallus domesticus) embryos for the detection of BoNT/A activity was conducted using equal dosages of toxin/g of embryo (quail at 7 g and chickens at 48 g). Quail embryos were injected at 0, 0.5 to 50 ng and chicken embryos at 0, 3.4 to 342 ng and...

  3. Evaluation of quail and chicken embryos for the detection of botulinum toxin serotypes A, B, E and F activity.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Comparison of quail (Coturnix japonica) and chicken (Gallus domesticus) embryos for the detection of BoNT/A activity was conducted using equal dosages of toxin/g of embryo (quail at 7 g and chickens at 48 g). Quail embryos were injected at 0, 0.5 to 50 ng adn chicken embryos at 0, 3.4 to 342 ng and...

  4. Supplementation with spermine during in vitro maturation of porcine oocytes improves early embryonic development after parthenogenetic activation and somatic cell nuclear transfer.

    PubMed

    Jin, J X; Lee, S; Khoirinaya, C; Oh, A; Kim, G A; Lee, B C

    2016-03-01

    Spermine plays an important role in protection from reactive oxygen species (ROS) in bacteria, yeast, and mammalian cells, but there are few studies on the effects of spermine on porcine oocyte maturation and subsequent embryo development. The aim of this study was to determine the effects of spermine on in vitro maturation (IVM) of porcine oocytes and their developmental competence after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). We evaluated nuclear maturation, intracellular glutathione (GSH), and ROS levels in oocytes, and their subsequent embryonic development, as well as gene expression in mature oocytes, cumulus cells, and PA blastocysts. After treatment with various concentrations of spermine in IVM culture medium, there was no significant difference in nuclear maturation rate. However, spermine treatment groups (10- 500 µM) showed significantly increased intracellular GSH levels and decreased ROS levels compared to the control ( < 0.05). Furthermore, 10 µM spermine supported significantly higher blastocyst formation rates after PA than the control group ( < 0.05). According to the optimal condition from the PA results, we investigated the effects of 10 µM spermine on SCNT, and it also significantly improved blastocyst formation rates compared with the control group ( < 0.05). In evaluating the effects of 10 µM spermine on gene expression, there was significantly lower expression of a proapoptotic gene () and higher expression of an antiapoptotic gene () in cumulus cells ( < 0.05). was increased in spermine-treated oocytes. Levels of transcription for and were significantly increased in PA blastocysts. In conclusion, 10 µM spermine supplementation during IVM improved the development of porcine PA and SCNT embryos by increasing intracellular GSH, scavenging ROS levels, and regulating gene expression. PMID:27065258

  5. Immortalization of porcine placental trophoblast cells through reconstitution of telomerase activity.

    PubMed

    Zhang, Hongling; Huang, Yong; Wang, Lili; Yu, Tingting; Wang, Zengguo; Chang, Lingling; Zhao, Xiaomin; Luo, Xiaomao; Zhang, Liang; Tong, Dewen

    2016-05-01

    Placental trophoblast cells (PTCs) play a critical role in histotrophic nutrient absorption, gaseous exchange, endocrine activities, and barrier function between the maternal and fetal systems. Establishment of immortalized porcine PTCs will help us to investigate the potential effects of different viruses on porcine trophoblast. In the present study, primary porcine PTCs were isolated from healthy gilts at Day 30 to Day 50 of gestation through collagenase digestion, percoll gradient centrifugation, and anti-CD9 immunomagnetic negative selection. To provide stable and long lifespan cells, primary PTCs were transfected with human telomerase reverse transcriptase (hTERT) gene. One porcine placental trophoblast cell line, named as hTERT-PTCs, was chosen for characterization. Human telomerase reverse transcriptase-PTCs achieved an extended replicative lifespan without exhibiting any neoplastic transformation signs in vivo or in vitro. The morphologic and key physiological characteristics of the immortalized PTCs were similar to primary PTCs. The immortalized PTCs retained original cell polarity and normal karyotype, expressed trophoblast-specific marker cytokeratin 7 and E-cadherin but did not express vimentin and major histocompatibility complex class I antigens as well as primary PTCs. Human telomerase reverse transcriptase-PTCs secreted low levels of chorionic gonadotrophin β-subunit and placental lactogen that were coincident with primary PTCs. Taken together, our results demonstrated that the porcine PTCs could be immortalized through reconstitution of telomerase activity. The immortalized PTCs maintained its original characteristics and can be used as a model cells line to study the pathologic changes of porcine placental trophoblast in viruses infectious diseases. PMID:26850465

  6. Detection of botulinum toxin types A, B, E, and F activity using the quail embryo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We recently demonstrated an effective new model for the detection of botulinum toxin type A using quail embryos in place of the mouse model. These experiments demonstrated that the Japanese quail embryo at 15 days of incubation was an effective vertebrate animal model to detect the activity of botu...

  7. Phosphorylated H2AX in parthenogenetically activated, in vitro fertilized and cloned bovine embryos.

    PubMed

    Pereira, A F; Melo, L M; Freitas, V J F; Salamone, D F

    2015-08-01

    In vitro embryo production methods induce DNA damage in the embryos. In response to these injuries, histone H2AX is phosphorylated (γH2AX) and forms foci at the sites of DNA breaks to recruit repair proteins. In this work, we quantified the DNA damage in bovine embryos undergoing parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) by measuring γH2AX accumulation at different developmental stages: 1-cell, 2-cell and blastocyst. At the 1-cell stage, IVF embryos exhibited a greater number of γH2AX foci (606.1 ± 103.2) and greater area of γH2AX staining (12923.6 ± 3214.1) than did PA and SCNT embryos. No differences at the 2-cell stage were observed among embryo types. Although PA, IVF and SCNT were associated with different blastocyst formation rates (31.1%, 19.7% and 8.3%, P < 0.05), no differences in the number of γH2AX foci or area were detected among the treatments. γH2AX is detected in bovine preimplantation embryos produced by PA, IVF and SCNT; the amount of DNA damage was comparable among those embryos developing to the blastocyst stage among different methods for in vitro embryo production. While IVF resulted in increased damage at the 1-cell embryo stage, no difference was observed between PA and SCNT embryos at any developmental stage. The decrease in the number of double-stranded breaks at the blastocyst stage seems to indicate that DNA repair mechanisms are functional during embryo development. PMID:24735637

  8. Genetic Association of the Porcine C9 Complement Component with Hemolytic Complement Activity.

    PubMed

    Khoa, D V A; Wimmers, K

    2015-09-01

    The complement system is a part of the natural immune regulation mechanism against invading pathogens. Complement activation from three different pathways (classical, lectin, and alternative) leads to the formation of C5-convertase, an enzyme for cleavage of C5 into C5a and C5b, followed by C6, C7, C8, and C9 in membrane attack complex. The C9 is the last complement component of the terminal lytic pathway, which plays an important role in lysis of the target cells depending on its self-polymerization to form transmembrane channels. To address the association of C9 with traits related to disease resistance, the complete porcine C9 cDNA was comparatively sequenced to detect single nucleotide polymorphisms (SNPs) in pigs of the breeds Hampshire (HS), Duroc (DU), Berlin miniature pig (BMP), German Landrace (LR), Pietrain (PIE), and Muong Khuong (Vietnamese potbelly pig). Genotyping was performed in 417 F2 animals of a resource population (DUMI: DU×BMP) that were vaccinated with Mycoplasma hyopneumoniae, Aujeszky diseases virus and porcine respiratory and reproductive syndrome virus at 6, 14 and 16 weeks of age, respectively. Two SNPs were detected within the third exon. One of them has an amino acid substitution. The European porcine breeds (LR and PIE) show higher allele frequency of these SNPs than Vietnamese porcine breed (MK). Association of the substitution SNP with hemolytic complement activity indicated statistically significant differences between genotypes in the classical pathway but not in the alternative pathway. The interactions between eight time points of measurement of complement activity before and after vaccinations and genotypes were significantly different. The difference in hemolytic complement activity in the both pathways depends on genotype, kind of vaccine, age and the interaction to the other complement components. These results promote the porcine C9 (pC9) as a candidate gene to improve general animal health in the future. PMID:26194222

  9. Genetic Association of the Porcine C9 Complement Component with Hemolytic Complement Activity

    PubMed Central

    Khoa, D. V. A.; Wimmers, K.

    2015-01-01

    The complement system is a part of the natural immune regulation mechanism against invading pathogens. Complement activation from three different pathways (classical, lectin, and alternative) leads to the formation of C5-convertase, an enzyme for cleavage of C5 into C5a and C5b, followed by C6, C7, C8, and C9 in membrane attack complex. The C9 is the last complement component of the terminal lytic pathway, which plays an important role in lysis of the target cells depending on its self-polymerization to form transmembrane channels. To address the association of C9 with traits related to disease resistance, the complete porcine C9 cDNA was comparatively sequenced to detect single nucleotide polymorphisms (SNPs) in pigs of the breeds Hampshire (HS), Duroc (DU), Berlin miniature pig (BMP), German Landrace (LR), Pietrain (PIE), and Muong Khuong (Vietnamese potbelly pig). Genotyping was performed in 417 F2 animals of a resource population (DUMI: DU×BMP) that were vaccinated with Mycoplasma hyopneumoniae, Aujeszky diseases virus and porcine respiratory and reproductive syndrome virus at 6, 14 and 16 weeks of age, respectively. Two SNPs were detected within the third exon. One of them has an amino acid substitution. The European porcine breeds (LR and PIE) show higher allele frequency of these SNPs than Vietnamese porcine breed (MK). Association of the substitution SNP with hemolytic complement activity indicated statistically significant differences between genotypes in the classical pathway but not in the alternative pathway. The interactions between eight time points of measurement of complement activity before and after vaccinations and genotypes were significantly different. The difference in hemolytic complement activity in the both pathways depends on genotype, kind of vaccine, age and the interaction to the other complement components. These results promote the porcine C9 (pC9) as a candidate gene to improve general animal health in the future. PMID:26194222

  10. Production of porcine parvovirus empty capsids with high immunogenic activity.

    PubMed

    Martínez, C; Dalsgaard, K; López de Turiso, J A; Cortés, E; Vela, C; Casal, J I

    1992-01-01

    The VP2 gene of porcine parvovirus was cloned in the baculovirus system and expressed in insect cells. The resulting product was present in high yield. It self-assembled into particles which were structurally and antigenically indistinguishable from regular PPV capsids. A high degree of purity of the recombinant capsids was obtained by ammonium sulphate precipitation of cell lysates. These virus-like particles were used as antigen in the immunization of two pigs. The pigs elicited an immune response which, when assayed by standard serological techniques, was identical to that of a commercial vaccine. The amount of recombinant antigen needed in a vaccine dose was only 3 micrograms in a primary dose and 1.5 micrograms in the booster. PMID:1523879

  11. Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions

    PubMed Central

    Karja, Ni Wayan Kurniani; Kikuchi, Kazuhiro; Fahrudin, Mokhamad; Ozawa, Manabu; Somfai, Tamás; Ohnuma, Katsuhiko; Noguchi, Junko; Kaneko, Hiroyuki; Nagai, Takashi

    2006-01-01

    Background Recent work has shown that glucose may induce cell injury through the action of free radicals generated by autooxidation or through hypoxanthine phosphoribosyltransferase inhibition. The effect of glucose during early in vitro culture (IVC) period of porcine embryos on their developmental competence, contents of reactive oxygen species (ROS) and glutathione (GSH), and the quality of the blastocysts yielded was examined. Methods In vitro matured and fertilized porcine oocytes were cultured for the first 2 days (Day 0 = day of fertilization) of IVC in NCSU-37 added with 1.5 to 20 mM glucose (Gluc-1.5 to -20 groups) or pyruvate and lactate (Pyr-Lac group). The embryos in all groups were cultured subsequently until Day 6 in NCSU-37 with 5.5 mM added glucose. The ROS and GSH level were measured at Day 1 and 2. DNA-fragmented nuclei and the total cell numbers in blastocyst were evaluated by TUNEL-staining at Day 6. Results Under 5% oxygen the blastocyst rates and total cell numbers in the blastocysts in all glucose groups were significantly lower than that in the Pyr-Lac group. Similar result in blastocyst rate was found under 20% oxygen (excluding the Gluc-10 group), but total cell numbers in the blastocysts was similar among the groups. At both oxygen tensions, the H2O2 levels of Day 1 embryos in all glucose groups were significantly higher than that in the Pyr-Lac group, while only the Gluc-1.5 group of Day 2 embryos showed a significantly higher H2O2 level than that in the Pyr-Lac group. The GSH contents of either Day 1 or Day 2 embryos developed under 5% oxygen were similar among the groups. Only the content of Day 2 embryos in 1.5 mM group was significantly lower than the embryos in the Pyr-Lac group under 20% oxygen. Total cell numbers in the blastocysts (except in the Gluc-20 group) were significantly lower in the embryos cultured under 20% oxygen than 5% oxygen. Only the Gluc-20 blastocysts developed under 5% oxygen showed significantly higher DNA

  12. Comparative analysis of signature genes in PRRSV-infected porcine monocyte-derived dendritic cells at differential activation statuses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Activation statuses of monocytic cells including monocytes, macrophages and dendritic cells (DCs) are critically important for antiviral immunity. In particular, some devastating viruses, including porcine reproductive and respiratory syndrome virus (PRRSV), are capable of directly infecting these c...

  13. Metabolism of Preimplantation Embryo Development: A Bystander or an Active Participant?

    PubMed

    Kaneko, K J

    2016-01-01

    Unicellular organisms are exquisitely sensitive to nutrient availability in the environment and have evolved elaborate mechanisms to sense the levels and types of nutrients, altering gene expression patterns accordingly to adjust the metabolic activities required to survive. Thus, environmental cues induce adaptive metabolic differentiation through transcriptional and posttranscriptional changes. Similarly, preimplantation embryos are exposed to various environmental cues within the maternal reproductive tract prior to implantation. Because only "simple" culture conditions are required, it is assumed that these embryos are genetically preprogrammed to develop with little influence from the environment, with the exception of few "necessities" provided by the environment. However, a wealth of literature now suggests that the developing embryos are greatly influenced by the maternal environment. Even though the developing embryos have the capacity and plasticity to deal with nutritional imbalance posed by an altered maternal environment, there is often a trade-off to the overall fitness of those embryos later in life. Despite these studies that underline the general importance of the reproductive environment during development, it is thought that the primary driver of mammalian development is strictly genetic and that metabolic adaptation by the preimplantation embryo is secondary to genetic control. In this review, I propose that not only does the maternal environment of developing preimplantation embryos influence developmental potential, pregnancy outcomes, and postnatal disease states, but that it has an active role in induction and potentiation of the first differentiation event, the production of trophectoderm and inner cell mass lineages. PMID:27475855

  14. Regulation of acyltransferase activity in immature maize embryos by abscisic acid and the osmotic environment.

    PubMed Central

    Pacheco-Moisés, F; Valencia-Turcotte, L; Altuzar-Martínez, M; Rodríguez-Sotres, R

    1997-01-01

    Maize (Zes mays L.) embryos, isolated from the developing seed and incubated in dilute buffer, show reduced triacylglycerol (TAG) synthesis, and accumulation stops after 24 h. Synthesis and accumulation can be maintained at high levels if the incubation medium contains abscisic acid (ABA) and/or a high osmotic concentration. Radiolabeled free fatty acids accumulate at higher levels in embryos that contain less TAG, and acetyl coenzyme A carboxylase activity remains essentially unchanged under all of the conditions tested. In contrast, the activities of the acyltransferases required for TAG synthesis remain high only in embryos incubated with ABA and/or a high osmotic concentration. Dose-response curves showed that 4 microM of ABA or mannitol at -1.0 MPa elicits a full response; both values are within the range considered to be physiological. The TAG synthesis capacity and discylglycerol acyltransferase activity lost by pretreatment of the embryos can be restored by re-exposure to ABA or high osmoticum. Germination is not involved because isolated scutellum halves showed the same changes in enzyme activity found in the whole embryo but did not germinate. Our results provide direct evidence for the regulation of TAG-synthesizing activities in maize embryos by ABA and the osmotic potential of the environment. PMID:9232885

  15. Developmental kinetics of pig embryos by parthenogenetic activation or by handmade cloning.

    PubMed

    Li, J; Li, R; Liu, Y; Villemoes, K; Purup, S; Callesen, H

    2013-10-01

    The developmental kinetics of pig embryos produced by parthenogenetic activation without (PAZF) or with (PAZI) zona pellucida or by handmade cloning (HMC) was compared by time-lapse videography. After cumulus cell removal, the matured oocytes were either left zona intact (PAZI) or were made zona free by pronase digestion (PAZF) before they were activated (PA). Other matured oocytes were used for HMC based on foetal fibroblast cells. On Day 0 (day of PA or reconstruction), the embryos were cultured for 7 days in vitro in our time-lapse system. Pictures were taken every 30 min, and afterwards, each cell cycle was identified for each embryo to be analysed. Results showed that the PA embryos (both PAZF and PAZI) had shorter first cell cycle compared with HMC (17.4. 17.8 vs 23.6 h), but had a longer time length from four cell to morula stages (57.9, 53.8 vs 44.9 h). However, at the second cell cycle, PAZF embryos needed shorter time, while PAZI embryos had similar time length as HMC embryos, and both were longer than PAZF (23.4, 24.8 vs 14.6 h). Both PAZF and PAZI embryos used similar time to reach the blastocyst stage, and this was later than HMC embryos. In addition, when all of these embryos were grouped into viable (developed to blastocysts) and non-viable (not developed to blastocysts), the only difference in the time length was observed on the first cell cycle (18.6 vs 24.5 h), but not on the later cell cycles. In conclusion, our results not only give detailed information regarding the time schedule of in vitro-handled pig embryos, but also indicate that the first cell cycle could be used as a selecting marker for embryo viability. However, to evaluate the effect of the produced techniques, the whole time schedule of the pre-implantation developmental kinetics should be observed. PMID:23617742

  16. The Effect of UV-B Radiation on Bufo arenarum Embryos Survival and Superoxide Dismutase Activity

    PubMed Central

    Herkovits, J.; D’Eramo, J. L.; Fridman, O.

    2006-01-01

    The exposure of Bufo arenarum embryos to 300–310 nm UV-B at a dose of 4,104 Joule/m2 resulted in 100% lethality within 24 hr while 820 Joule/m2 was the NOEC value for short-term chronic (10 days) exposure. The dose response curves show that lethal effects are proportional with the dose and achieve its highest value within 48 hr post exposure. The superoxide dismutase (SOD) activity in amphibian embryos for sublethal UV-B exposures was evaluated by means of UV-B treatments with 273 (A), 820(B), 1368(C) and 1915(D) Joule/m2 at 2 and 5 hours post irradiation. The SOD activity in units/mg protein in A, B, C and D at 2 hr after treatments were 80.72 ± 14.29, 74.5 ± 13.19, 39.5 ± 6.99 and 10.7 ± 1.89 respectively while for control embryos it was 10.88 ± 1.31. At 5 hr after treatments the SOD values were similar to those found in control embryos. The results confirm the high susceptibility of amphibian embryos to UV-B and point out that the SOD activity is enhanced by low doses of UV-B irradiation achieving significantly higher values than in control embryos at 2 hr post exposure. PMID:16823076

  17. Effects of fluoridation of porcine hydroxyapatite on osteoblastic activity of human MG63 cells

    NASA Astrophysics Data System (ADS)

    Li, Zhipeng; Huang, Baoxin; Mai, Sui; Wu, Xiayi; Zhang, Hanqing; Qiao, Wei; Luo, Xin; Chen, Zhuofan

    2015-06-01

    Biological hydroxyapatite, derived from animal bones, is the most widely used bone substitute in orthopedic and dental treatments. Fluorine is the trace element involved in bone remodeling and has been confirmed to promote osteogenesis when administered at the appropriate dose. To take advantage of this knowledge, fluorinated porcine hydroxyapatite (FPHA) incorporating increasing levels of fluoride was derived from cancellous porcine bone through straightforward chemical and thermal treatments. Physiochemical characteristics, including crystalline phases, functional groups and dissolution behavior, were investigated on this novel FPHA. Human osteoblast-like MG63 cells were cultured on the FPHA to examine cell attachment, cytoskeleton, proliferation and osteoblastic differentiation for in vitro cellular evaluation. Results suggest that fluoride ions released from the FPHA play a significant role in stimulating osteoblastic activity in vitro, and appropriate level of fluoridation (1.5 to 3.1 atomic percents of fluorine) for the FPHA could be selected with high potential for use as a bone substitute.

  18. Activation of bovine somatic cell nuclear transfer embryos by PLCZ cRNA injection.

    PubMed

    Ross, Pablo J; Rodriguez, Ramon M; Iager, Amy E; Beyhan, Zeki; Wang, Kai; Ragina, Neli P; Yoon, Sook-Young; Fissore, Rafael A; Cibelli, Jose B

    2009-03-01

    The production of cloned animals by the transfer of a differentiated somatic cell into an enucleated oocyte circumvents fertilization. During fertilization, the sperm delivers a sperm-specific phospholipase C (PLCZ) that is responsible for triggering Ca(2)(+) oscillations and oocyte activation. During bovine somatic cell nuclear transfer (SCNT), oocyte activation is artificially achieved by combined chemical treatments that induce a monotonic rise in intracellular Ca(2)(+) and inhibit either phosphorylation or protein synthesis. In this study, we tested the hypothesis that activation of bovine nuclear transfer embryos by PLCZ improves nuclear reprogramming. Injection of PLCZ cRNA into bovine SCNT units induced Ca(2)(+) oscillations similar to those observed after fertilization and supported high rates of blastocyst development similar to that seen in embryos produced by IVF. Furthermore, gene expression analysis at the eight-cell and blastocyst stages revealed a similar expression pattern for a number of genes in both groups of embryos. Lastly, levels of trimethylated lysine 27 at histone H3 in blastocysts were higher in bovine nuclear transfer embryos activated using cycloheximide and 6-dimethylaminopurine (DMAP) than in those activated using PLCZ or derived from IVF. These results demonstrate that exogenous PLCZ can be used to activate bovine SCNT-derived embryos and support the hypothesis that a fertilization-like activation response can enhance some aspects of nuclear reprogramming. PMID:19074500

  19. Trichostatin A-Mediated Epigenetic Transformation of Adult Bone Marrow-Derived Mesenchymal Stem Cells Biases the In Vitro Developmental Capability, Quality, and Pluripotency Extent of Porcine Cloned Embryos

    PubMed Central

    Samiec, Marcin; Opiela, Jolanta; Lipiński, Daniel

    2015-01-01

    The current research was conducted to explore the in vitro developmental outcome and cytological/molecular quality of porcine nuclear-transferred (NT) embryos reconstituted with adult bone marrow-derived mesenchymal stem cells (ABM-MSCs) that were epigenetically transformed by treatment with nonspecific inhibitor of histone deacetylases, known as trichostatin A (TSA). The cytological quality of cloned blastocysts was assessed by estimation of the total cells number (TCN) and apoptotic index. Their molecular quality was evaluated by real-time PCR-mediated quantification of gene transcripts for pluripotency- and multipotent stemness-related markers (Oct4, Nanog, and Nestin). The morula and blastocyst formation rates of NT embryos derived from ABM-MSCs undergoing TSA treatment were significantly higher than in the TSA-unexposed group. Moreover, the NT blastocysts generated using TSA-treated ABM-MSCs exhibited significantly higher TCN and increased pluripotency extent measured with relative abundance of Oct4 and Nanog mRNAs as compared to the TSA-untreated group. Altogether, the improvements in morula/blastocyst yields and quality of cloned pig embryos seem to arise from enhanced abilities for promotion of correct epigenetic reprogramming of TSA-exposed ABM-MSC nuclei in a cytoplasm of reconstructed oocytes. To our knowledge, we are the first to report the successful production of mammalian high-quality NT blastocysts using TSA-dependent epigenomic modulation of ABM-MSCs. PMID:25866813

  20. Genome-wide analysis of antiviral signature genes in porcine macrophages at different activation statuses.

    PubMed

    Sang, Yongming; Brichalli, Wyatt; Rowland, Raymond R R; Blecha, Frank

    2014-01-01

    Macrophages (MФs) can be polarized to various activation statuses, including classical (M1), alternative (M2), and antiviral states. To study the antiviral activation status of porcine MФs during porcine reproductive and respiratory syndrome virus (PRRSV) infection, we used RNA Sequencing (RNA-Seq) for transcriptomic analysis of differentially expressed genes (DEGs). Sequencing assessment and quality evaluation showed that our RNA-Seq data met the criteria for genome-wide transcriptomic analysis. Comparisons of any two activation statuses revealed more than 20,000 DEGs that were normalized to filter out 153-5,303 significant DEGs [false discovery rate (FDR) ≤0.001, fold change ≥2] in each comparison. The highest 5,303 significant DEGs were found between lipopolysaccharide- (LPS) and interferon (IFN)γ-stimulated M1 cells, whereas only 153 significant DEGs were detected between interleukin (IL)-10-polarized M2 cells and control mock-activated cells. To identify signature genes for antiviral regulation pertaining to each activation status, we identified a set of DEGs that showed significant up-regulation in only one activation state. In addition, pathway analyses defined the top 20-50 significantly regulated pathways at each activation status, and we further analyzed DEGs pertinent to pathways mediated by AMP kinase (AMPK) and epigenetic mechanisms. For the first time in porcine macrophages, our transcriptomic analyses not only compared family-wide differential expression of most known immune genes at different activation statuses, but also revealed transcription evidence of multiple gene families. These findings show that using RNA-Seq transcriptomic analyses in virus-infected and status-synchronized macrophages effectively profiled signature genes and gene response pathways for antiviral regulation, which may provide a framework for optimizing antiviral immunity and immune homeostasis. PMID:24505295

  1. Genome-Wide Analysis of Antiviral Signature Genes in Porcine Macrophages at Different Activation Statuses

    PubMed Central

    Sang, Yongming; Brichalli, Wyatt; Rowland, Raymond R. R.; Blecha, Frank

    2014-01-01

    Macrophages (MФs) can be polarized to various activation statuses, including classical (M1), alternative (M2), and antiviral states. To study the antiviral activation status of porcine MФs during porcine reproductive and respiratory syndrome virus (PRRSV) infection, we used RNA Sequencing (RNA-Seq) for transcriptomic analysis of differentially expressed genes (DEGs). Sequencing assessment and quality evaluation showed that our RNA-Seq data met the criteria for genome-wide transcriptomic analysis. Comparisons of any two activation statuses revealed more than 20,000 DEGs that were normalized to filter out 153–5,303 significant DEGs [false discovery rate (FDR) ≤0.001, fold change ≥2] in each comparison. The highest 5,303 significant DEGs were found between lipopolysaccharide- (LPS) and interferon (IFN)γ-stimulated M1 cells, whereas only 153 significant DEGs were detected between interleukin (IL)-10-polarized M2 cells and control mock-activated cells. To identify signature genes for antiviral regulation pertaining to each activation status, we identified a set of DEGs that showed significant up-regulation in only one activation state. In addition, pathway analyses defined the top 20–50 significantly regulated pathways at each activation status, and we further analyzed DEGs pertinent to pathways mediated by AMP kinase (AMPK) and epigenetic mechanisms. For the first time in porcine macrophages, our transcriptomic analyses not only compared family-wide differential expression of most known immune genes at different activation statuses, but also revealed transcription evidence of multiple gene families. These findings show that using RNA-Seq transcriptomic analyses in virus-infected and status-synchronized macrophages effectively profiled signature genes and gene response pathways for antiviral regulation, which may provide a framework for optimizing antiviral immunity and immune homeostasis. PMID:24505295

  2. Developmental onset of mixed-function oxidase activity in preimplantation mouse embryos

    SciTech Connect

    Filler, R.; Lew, K.J.

    1981-11-01

    Two-cell embryos, obtained from the C57BL/6N and DBA/2N strains, were cultured in media that supported in vitro differentiation and that contained (/sup 3/H)benzo(a)pyrene. High-pressure liquid chromatography of the activated intermediates formed during in vitro early embryonic development indicated that the onset of polynuclear aromatic hydrocarbon activation coincided with blastocyst formation. Comparison of individual oxygenated intermediates metabolically formed from embryos genetically ''responsive'' or ''nonresponsive'' to aromatic hydrocarbons revealed significant quantitative differences in the production of dihydrodiol, quinone, and phenolic derivatives. In addition to exhibiting basal mixed-function oxidase activity, blastocysts were also responsive to enzymatic induction when exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. The presence of operative metabolite-detoxifying pathways was also assayed. Enzymatic treatment of water-soluble metabolites with ..beta..-glucuronidase or arylsulfatase revealed that neither glucuronic acid conjugates nor sulfate ester derivatives were present. These data, therefore, provide direct evidence that late preimplanation mouse embryos (day 3 1/2 of gestation) are similar to later developmental stages in having the enzymatic capability for xenobiotic activation and enzyme induction but are dissimilar with respect to their detoxification mechanisms(s). Moreover, the ability of preimplantation embryos to activate directly polynuclear aromatic hydrocarbon to bioreactive intermediates may be of importance in assessing the ontological susceptibility of the developing embryo to carcinogenic or teratogenic chemicals.

  3. Effects of two types of cobra venom factor on porcine complement activation and pulmonary artery pressure.

    PubMed Central

    Cheung, A K; Parker, C J; Wilcox, L

    1989-01-01

    Autologous porcine plasma that has been incubated with cuprophan haemodialysis membranes causes pulmonary hypertension and peripheral leucopenia following reinfusion into swine. These effects appear to be mediated by biologically active fragments of C3 and C5 that are generated as a consequence of ex vivo activation of complement. Putatively, C5a induces the leucopenia; however, the specific contributions of products of C3 and C5 activation to the pulmonary vasoconstriction have not been elucidated. In the present study, the effects of in vivo infusion of two different types of cobra venom factor (CVF) on peripheral leucocyte count and pulmonary artery pressure in the swine are reported. The CVF from Naja n. naja (CVF(TN)) was shown to activate both porcine C3 and C5, whereas the CVF from Naja h. haje (CVF(NH)) activated only C3. Both types of CVF produced pulmonary hypertension. Significant peripheral leucopenia, however, was observed only with CVF(TN). These results suggest that activation products of C3 contribute to the pulmonary hypertension but not to the peripheral leucopenia observed during haemodialysis using dialysis membranes that activate complement. PMID:12412765

  4. [Comparison of expression and antibacterial activities of recombinant porcine lactoferrin expressed in four Lactobacillus species].

    PubMed

    Yu, Hui; Jiang, Yanping; Cui, Wen; Wu, Xiao; He, Jia; Qiao, Xinyuan; Li, Yijing; Tang, Lijie

    2014-09-01

    The coding sequence for the mature peptide of porcine lactoferrin (Plf) was synthesized according to the codon usage of lactobacillus, to establish optimized porcine lactoferrin Lactobacillus expression system. The gene was ligated into the Xho I/BamH I site of Lactobacillus expression vector pPG612.1 and the recombinant plasmid pPG612.1-plf was transformed individually into Lactobacillus casei ATCC393, Lactobacillus pentosus KLDS1.0413, Lactobacillus plantarum KLDS1.0344 or Lactobacillus paracasei KLDS1.0652 by electroporation. After induction with xylose, expression of the recombinant proteins was detected by Western blotting and confocal laser scanning microscopy. Secretion of recombinant Plf proteins from four recombinant species was determined quantitatively by ELISA. The antibacterial activities of recombinant proteins were measured by agar diffusion method. The result shows that Plf was correctly expressed in four species of recombinant lactobacillus, with molecular weight of about 73 kDa. The expression levels in recombinant Lactobacillus casei, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paracasei were 9.6 μg/mL, 10.8 μg/mL, 12.5 μg/mL and 9.9 μg/mL, respectively. Antimicrobial activity experiment shows that the recombinant proteins were active against E. coli, Staphylococcus aureus, Salmonella typhimurium, Listeria, Pasteurella. The recombinant Plf expressed by recombinant Lactobacillus plantarum showed the best antibacterial activity among all recombinant lactobacillus species. These data represent a basis for the development and application of porcine lactoferrin from recombinant lactobacillus. PMID:25720152

  5. Actinobacillus pleuropneumoniae Possesses an Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus

    PubMed Central

    Labrie, Josée; Hernandez Reyes, Yenney; Burciaga Nava, Jorge A.; Gagnon, Carl A.; Jacques, Mario

    2014-01-01

    Pigs are often colonized by more than one bacterial and/or viral species during respiratory tract infections. This phenomenon is known as the porcine respiratory disease complex (PRDC). Actinobacillus pleuropneumoniae (App) and porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that are frequently involved in PRDC. The main objective of this project was to study the in vitro interactions between these two pathogens and the host cells in the context of mixed infections. To fulfill this objective, PRRSV permissive cell lines such as MARC-145, SJPL, and porcine alveolar macrophages (PAM) were used. A pre-infection with PRRSV was performed at 0.5 multiplicity of infection (MOI) followed by an infection with App at 10 MOI. Bacterial adherence and cell death were compared. Results showed that PRRSV pre-infection did not affect bacterial adherence to the cells. PRRSV and App co-infection produced an additive cytotoxicity effect. Interestingly, a pre-infection of SJPL and PAM cells with App blocked completely PRRSV infection. Incubation of SJPL and PAM cells with an App cell-free culture supernatant is also sufficient to significantly block PRRSV infection. This antiviral activity is not due to LPS but rather by small molecular weight, heat-resistant App metabolites (<1 kDa). The antiviral activity was also observed in SJPL cells infected with swine influenza virus but to a much lower extent compared to PRRSV. More importantly, the PRRSV antiviral activity of App was also seen with PAM, the cells targeted by the virus in vivo during infection in pigs. The antiviral activity might be due, at least in part, to the production of interferon γ. The use of in vitro experimental models to study viral and bacterial co-infections will lead to a better understanding of the interactions between pathogens and their host cells, and could allow the development of novel prophylactic and therapeutic tools. PMID:24878741

  6. Phospholipid transfer activities in toad oocytes and developing embryos. [Bufo arenarum

    SciTech Connect

    Rusinol, A.; Salomon, R.A.; Bloj, B.

    1987-01-01

    The role of lipid transfer proteins during plasma membrane biogenesis was explored. Developing amphibia embryos were used because during their growth an active plasma membrane biosynthesis occurs together with negligible mitochondrial and endoplasmic reticulum proliferation. Sonicated vesicles, containing /sup 14/C-labeled phospholipids and /sup 3/H-labeled triolein, as donor particles and cross-linked erythrocyte ghosts as acceptor particles were used to measure phospholipid transfer activities in unfertilized oocytes and in developing embryos of the toad Bufo arenarum. Phosphatidylcholine transfer activity in pH 5.1 supernatant of unfertilized oocytes was 8-fold higher than the activity found in female toad liver supernatant, but dropped steadily after fertilization. After 20 hr of development, at the stage of late blastula, the phosphatidylcholine transfer activity had dropped 4-fold. Unfertilized oocyte supernatant exhibited phosphatidylinositol and phosphatidylethanolamine transfer activity also, but at the late blastula stage the former had dropped 18-fold and the latter was no longer detectable under our assay conditions. Our results show that fertilization does not trigger a phospholipid transport process catalyzed by lipid transfer proteins. Moreover, they imply that 75% of the phosphatidylcholine transfer activity and more than 95% of the phosphatidylinositol and phosphatidylethanolamine transfer activities present in pH 5.1 supernatants of unfertilized oocytes may not be essential for toad embryo development. Our findings do not rule out, however, that a phosphatidylcholine-specific lipid transfer protein could be required for embryo early growth.

  7. Polymerization-dependent activation of porcine γδ T-cells by proanthocyanidins.

    PubMed

    Williams, Andrew R; Fryganas, Christos; Reichwald, Kirsten; Skov, Søren; Mueller-Harvey, Irene; Thamsborg, Stig M

    2016-04-01

    Plant-derived proanthocyanidins (PAC) have been promoted as a natural method of improving health and immune function in livestock. It has previously been shown that PAC are effective agonists for activating ruminant γδ T-cells in vitro, however effects on other livestock species are not yet clear. Moreover, the fine structural characteristics of the PAC which contribute to this stimulatory effect have not been elucidated. Here, we demonstrate activation of porcine γδ T-cells by PAC via up-regulation of CD25 (IL-2Rα) and show that 1) activation is dependent on degree of polymerization (DP), with PAC fractions containing polymers with mean DP >6 significantly more effective than fractions with mean DP <6, whilst flavan-3-ol monomers (the constituent monomeric units of PAC) did not induce CD25 expression and 2) both procyanidin and prodelphinidin-type PAC are effective agonists. Furthermore, we show that this effect of PAC is restricted to the γδ T-cell population within porcine peripheral mononuclear cells as significant CD25 up-regulation was not observed in non γδ T-cells, and no activation (via CD80/86 up-regulation) was evident in monocytes. Our results show that dietary PAC may contribute to enhancement of innate immunity in swine via activation of γδ T-cells. PMID:27033935

  8. Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

    PubMed

    Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

    2016-01-01

    In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming. PMID:26894831

  9. Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality

    PubMed Central

    Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

    2016-01-01

    In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming. PMID:26894831

  10. H(+)/K(+) ATPase activity is required for biomineralization in sea urchin embryos.

    PubMed

    Schatzberg, Daphne; Lawton, Matthew; Hadyniak, Sarah E; Ross, Erik J; Carney, Tamara; Beane, Wendy S; Levin, Michael; Bradham, Cynthia A

    2015-10-15

    The bioelectrical signatures associated with regeneration, wound healing, development, and cancer are changes in the polarization state of the cell that persist over long durations, and are mediated by ion channel activity. To identify physiologically relevant bioelectrical changes that occur during normal development of the sea urchin Lytechinus variegatus, we tested a range of ion channel inhibitors, and thereby identified SCH28080, a chemical inhibitor of the H(+)/K(+) ATPase (HKA), as an inhibitor of skeletogenesis. In sea urchin embryos, the primary mesodermal lineage, the PMCs, produce biomineral in response to signals from the ectoderm. However, in SCH28080-treated embryos, aside from randomization of the left-right axis, the ectoderm is normally specified and differentiated, indicating that the block to skeletogenesis observed in SCH28080-treated embryos is PMC-specific. HKA inhibition did not interfere with PMC specification, and was sufficient to block continuing biomineralization when embryos were treated with SCH28080 after the initiation of skeletogenesis, indicating that HKA activity is continuously required during biomineralization. Ion concentrations and voltage potential were abnormal in the PMCs in SCH28080-treated embryos, suggesting that these bioelectrical abnormalities prevent biomineralization. Our results indicate that this effect is due to the inhibition of amorphous calcium carbonate precipitation within PMC vesicles. PMID:26282894

  11. Differential type I interferon activation and susceptibility of dendritic cell populations to porcine arterivirus

    PubMed Central

    Loving, Crystal L; Brockmeier, Susan L; Sacco, Randy E

    2007-01-01

    Dendritic cells (DCs) play a role in anti-viral immunity by providing early innate protection against viral replication and by presenting antigen to T cells for initiation of the adaptive immune response. Studies show the adaptive response to porcine reproductive and respiratory syndrome virus (PRRSV) is ineffective for complete viral elimination. Other studies describe the kinetics of the adaptive response to PRRSV, but have not investigated the early response by DCs. We hypothesize that there is an aberrant activation of DCs early in PRRSV infection; consequently, the adaptive response is triggered inadequately. The current study characterized a subtype of porcine lung DCs (L-DCs) and investigated the ability of PRRSV to infect and replicate in L-DCs and monocyte-derived DCs (MDDCs). Furthermore, the type I interferon anti-viral response to PRRSV with and without the addition of recombinant porcine IFN-α (rpIFN-α), an important cytokine that signals for anti-viral mediator activation, was analysed. Results show that PRRSV replicated in MDDCs but not L-DCs, providing evidence that these cells have followed distinct differentiation pathways. Although both cell types responded to PRRSV with an induction of IFN-β mRNA, the magnitude and duration of the response differed between cell types. The addition of rpIFN-α was protective in MDDCs, and mRNA synthesis of Mx (myxovirus resistant) and PKR (double-stranded RNA dependent protein kinase) was observed in both cell types after rpIFN-α addition. Overall, PRRSV replicated in MDDCs but not L-DCs, and rpIFN-α was required for the transcription of protective anti-viral mediators. DC response to PRRSV was limited to IFN-β transcription, which may be inadequate in triggering the adaptive immune response. PMID:17116172

  12. Differential type I interferon activation and susceptibility of dendritic cell populations to porcine arterivirus.

    PubMed

    Loving, Crystal L; Brockmeier, Susan L; Sacco, Randy E

    2007-02-01

    Dendritic cells (DCs) play a role in anti-viral immunity by providing early innate protection against viral replication and by presenting antigen to T cells for initiation of the adaptive immune response. Studies show the adaptive response to porcine reproductive and respiratory syndrome virus (PRRSV) is ineffective for complete viral elimination. Other studies describe the kinetics of the adaptive response to PRRSV, but have not investigated the early response by DCs. We hypothesize that there is an aberrant activation of DCs early in PRRSV infection; consequently, the adaptive response is triggered inadequately. The current study characterized a subtype of porcine lung DCs (L-DCs) and investigated the ability of PRRSV to infect and replicate in L-DCs and monocyte-derived DCs (MDDCs). Furthermore, the type I interferon anti-viral response to PRRSV with and without the addition of recombinant porcine IFN-alpha (rpIFN-alpha), an important cytokine that signals for anti-viral mediator activation, was analysed. Results show that PRRSV replicated in MDDCs but not L-DCs, providing evidence that these cells have followed distinct differentiation pathways. Although both cell types responded to PRRSV with an induction of IFN-beta mRNA, the magnitude and duration of the response differed between cell types. The addition of rpIFN-alpha was protective in MDDCs, and mRNA synthesis of Mx (myxovirus resistant) and PKR (double-stranded RNA dependent protein kinase) was observed in both cell types after rpIFN-alpha addition. Overall, PRRSV replicated in MDDCs but not L-DCs, and rpIFN-alpha was required for the transcription of protective anti-viral mediators. DC response to PRRSV was limited to IFN-beta transcription, which may be inadequate in triggering the adaptive immune response. PMID:17116172

  13. A Method of Permeabilization of Drosophila Embryos for Assays of Small Molecule Activity

    PubMed Central

    Rand, Matthew D.

    2014-01-01

    The Drosophila embryo has long been a powerful laboratory model for elucidating molecular and genetic mechanisms that control development. The ease of genetic manipulations with this model has supplanted pharmacological approaches that are commonplace in other animal models and cell-based assays. Here we describe recent advances in a protocol that enables application of small molecules to the developing fruit fly embryo. The method details steps to overcome the impermeability of the eggshell while maintaining embryo viability. Eggshell permeabilization across a broad range of developmental stages is achieved by application of a previously described d-limonene embryo permeabilization solvent (EPS1) and by aging embryos at reduced temperature (18 °C) prior to treatments. In addition, use of a far-red dye (CY5) as a permeabilization indicator is described, which is compatible with downstream applications involving standard red and green fluorescent dyes in live and fixed preparations. This protocol is applicable to studies using bioactive compounds to probe developmental mechanisms as well as for studies aimed at evaluating teratogenic or pharmacologic activity of uncharacterized small molecules. PMID:25046169

  14. Utilization of quail and chicken embryos for the detection of botulinum toxin type A activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium botulinum is a ubiquitous microorganism which can produce botulinum toxins and the ability to assess toxin activity in a food sample is critical. As an alternative to the mouse assay incubating quail (Coturnix coturnix japonica) and chicken (Gallus gallus domestics) embryos were evaluat...

  15. Dimerization and activation of porcine pancreatic phospholipase A2 via substrate level acylation of lysine 56.

    PubMed

    Tomasselli, A G; Hui, J; Fisher, J; Zürcher-Neely, H; Reardon, I M; Oriaku, E; Kézdy, F J; Heinrikson, R L

    1989-06-15

    The porcine pancreatic phospholipase A2-catalyzed hydrolysis of the water-soluble chromogenic substrate 4-nitro-3-octanoyloxybenzoate shows an initial latency phase similar to the one observed in the hydrolysis of aggregated phospholipids by the same enzyme. We report here that during the latency phase the enzyme undergoes a slow, autocatalytic, substrate-level acylation whereby in a few of the catalytic events the scissile octanoyl group of the substrate, normally transferred to water, is transferred to the epsilon-amino group of lysine 56. The N epsilon 56-octanoylphospholipase shows a strong tendency to dimerize in solution and thus may be separated from the monomeric native enzyme by gel filtration. Octanoylation of Lys-56 activates the enzyme some 180-fold toward 4-nitro-3-octanoyloxybenzoate and more than 100-fold toward monolayers of 1,2-didecanoyl-sn-glycero-3-phosphocholine. Acylation also attends the enzymatic hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine with the incorporation of 1 eq of palmitate. Kinetic analysis of the early phase of reaction with 4-nitro-3-octanoyloxybenzoate shows that in this initial step the rate of activation is first order with respect to enzyme and substrate. A much more rapid, autocatalytic activation occurs in the later phases of the reaction where the activation of the enzyme is catalyzed by the activated enzyme itself. These findings with porcine pancreatic phospholipase A2, together with those relative to a snake venom enzyme monomer (Cho, W., Tomasselli, A. G., Heinrikson, R. L., and Kézdy, F. J. (1988) J. Biol. Chem. 263, 11237-11241), strongly support the proposal that interfacial activation of monomeric phospholipases is due to substrate-level autoacylation resulting in fully potentiated dimeric enzymes. PMID:2498336

  16. Complement receptor activity of recombinant porcine CR1-like protein expressed in a eukaryotic system.

    PubMed

    Yin, Wei; Wei, Xiaoming; Jiang, Junbing; Fan, Kuohai; Zhao, Junxing; Sun, Na; Wang, Zhiwei; Sun, Yaogui; Ma, Haili; Zhao, Xin; Li, Hongquan

    2016-08-01

    Primate complement receptor type 1 (CR1) protein, a single-chain transmembrane glycoprotein, plays an important role in immune adherence and clearing complement-opsonized immune complexes. Here, the mRNA of the porcine primate-like complement receptor (CR1-like) gene was analyzed, and two domain sequences with potential functions were cloned into the pwPICZalpha vector for expression in Pichia pastoris. The recombinant proteins were purified with both Protein Pure Ni-NTA resin and strong anion exchange resin. The activities of the purified recombinant proteins were evaluated by SDS-PAGE, western blotting, and complement receptor assays. The results indicated that two domains of the CR1-like protein, CCP36 and CCP811 with molecular weights of 29.8 kDa and 30 kDa, respectively, were successfully expressed in P. pastoris. These two recombinant proteins possess some of the functions of the primate CR1 protein. Using these two proteins coupled with an antibody blocking technique, we also showed that CR1-like is expressed on natural porcine erythrocytes. PMID:26903010

  17. Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

    PubMed Central

    Spetzler, Vinzent N.; Goldaracena, Nicolas; Knaak, Jan M.; Louis, Kristine S.; Selzner, Nazia; Selzner, Markus

    2015-01-01

    The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care. PMID:25992583

  18. Evaluation of biological activity of Uncaria tomentosa (Willd.) DC. using the chicken embryo model.

    PubMed

    Pilarski, Radosław; Bednarczyk, Marek; Gulewicz, Krzysztof

    2009-01-01

    The biological activity of Uncaria tomentosa (Willd.) DC. (cat's claw) was evaluated by application of the chicken embryo model. Among three groups of eggs (n = 360) with twelve-day old embryos, two were injected with different doses of cat's claw extracts (0.0492 and 0.492 mg/200 lambda). To the third control group 200 lambda of physiological salt was applied. All eggs were incubated in conventional forced-air apparatus until hatched. Hatchability, chicken weight and wholesomeness were analyzed. Selected parameters of blood including number of erythrocytes (RBC), number of leukocytes (WBC), mean red cell volume (MCV), hematocrit (HCT), hemoglobin concentration (HGB), mean amount of cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC) and embryo weight (MAS) were assayed and compared. Significant differences with ANOVA were observed for MCV (P = 0.002), MCHC (P = 0.00001) and MCH (P = 0.02). Applying the chicken embryo model brought new information about the biological activity of U. tomentosa showing an unfavourable effect on some morphological blood parameters. PMID:19777966

  19. Bioluminescence imaging to track real-time armadillo promoter activity in live Drosophila embryos.

    PubMed

    Akiyoshi, Ryutaro; Kaneuch, Taro; Aigaki, Toshiro; Suzuki, Hirobumi

    2014-09-01

    We established a method for bioluminescence imaging (BLI) to track real-time gene expression in live Drosophila embryos. We constructed a transgenesis vector containing multiple cloning sites and enhanced green-emitting luciferase (ELuc; Emerald Luc), a brighter and pH-insensitive luciferase for promoter analysis. To evaluate the utility of BLI using an ELuc reporter together with an optimized microscope system, we visualized the expression pattern of armadillo (arm), a member of the Wnt pathway in Drosophila, throughout embryogenesis. We generated transgenic flies carrying the arm:: ELuc fusion gene, and successfully performed BLI continuously for 22 h in the same embryos. Our study showed, for the first time, that arm::Eluc expression was dramatically increased in the anterior midgut rudiment, myoblasts of the dorsal/lateral musculature, and the posterior spiracle after stage 13, and the cephalic region at stage 17. To further demonstrate the application of our BLI system, we revealed that arm transcriptional activity in embryos was modulated inversely by treatment with ionomycin or 6-bromoindirubin-3-oxime (BIO), an inhibitor and activator of Wnt/β-catenin signaling, respectively. Therefore, our microscopic BLI system is useful for monitoring gene expression in live Drosophila embryos, and for investigating regulatory mechanisms by using chemicals and mutations that might affect expression. PMID:25023969

  20. A COUPLED MICROSOMAL-ACTIVATING/EMBRYO CULTURE SYSTEM: TOXICITY OF REDUCED BETA-NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (NADPH)

    EPA Science Inventory

    An NADPH-dependent microsomal-activating system has been coupled to a rat embryo culture in vitro. No embryonic morphological abnormalities or decrease in final yolk sac or embryo DNA and protein contents occurred when 0.2 mM NADPH was used in this coupled system. In contrast, 1....

  1. Porcine pituitary peptides with opiate-like activity: partial purification and effects in the rat after intraventricular injection.

    PubMed

    Teschemacher, H; Bläsig, J; Kromer, W

    1976-09-01

    A peptide material with opiate-like activity in the guinea-pig ileum was extracted from porcine pituitaries using a hot glacial acetic acid extraction method and was partially purified by gel filtration. When injected intraventricularly in rats, these purified peptides induced strong analgesia, catelepsy, respiratory depression and other opiate-like effects, which lasted for several hours. PMID:1034219

  2. Hyperpolarization-activated cation and T-type calcium ion channel expression in porcine and human renal pacemaker tissues.

    PubMed

    Hurtado, Romulo; Smith, Carl S

    2016-05-01

    Renal pacemaker activity triggers peristaltic upper urinary tract contractions that propel waste from the kidney to the bladder, a process prone to congenital defects that are the leading cause of pediatric kidney failure. Recently, studies have discovered that hyperpolarization-activated cation (HCN) and T-type calcium (TTC) channel conductances underlie murine renal pacemaker activity, setting the origin and frequency and coordinating upper urinary tract peristalsis. Here, we determined whether this ion channel expression is conserved in the porcine and human urinary tracts, which share a distinct multicalyceal anatomy with multiple pacemaker sites. Double chromagenic immunohistochemistry revealed that HCN isoform 3 is highly expressed at the porcine minor calyces, the renal pacemaker tissues, whereas the kidney and urinary tract smooth muscle lacked this HCN expression. Immunofluorescent staining demonstrated that HCN(+) cells are integrated within the porcine calyx smooth muscle, and that they co-express TTC channel isoform Cav3.2. In humans, the anatomic structure of the minor calyx pacemaker was assayed via hematoxylin and eosin analyses, and enabled the visualization of the calyx smooth muscle surrounding adjacent papillae. Strikingly, immunofluorescence revealed that HCN3(+) /Cav3.2(+) cells are also localized to the human minor calyx smooth muscle. Collectively, these data have elucidated a conserved molecular signature of HCN and TTC channel expression in porcine and human calyx pacemaker tissues. These findings provide evidence for the mechanisms that can drive renal pacemaker activity in the multi-calyceal urinary tract, and potential causes of obstructive uropathies. PMID:26805464

  3. Pharmacological characterization of the activity of endogenous inotropic factor from porcine left ventricle.

    PubMed

    Chen, Q M; Chau, T; Agbanyo, M; Navaratnam, S; Khatter, J C; Bose, D

    1993-01-01

    We report some of the unique pharmacological properties of a semipurified endogenous inotropic factor (EIF) present in the extract of the porcine left ventricle. EIF produced the following effects: (a) increase in isometric contractile force developed by electrically driven canine right ventricular trabecula, reaching a maximum with 60-100 microliters/ml concentration; (b) inhibition of Na-pump activity in canine portal vein; (c) no digitalis-like cardiac toxicity, e.g., increased diastolic tension or spontaneous diastolic mechanical oscillatory activity, despite inhibition of the sodium pump; (d) a small increase in sarcoplasmic reticular Ca release from the heart but a large increase in transsarcolemmal Ca influx as seen in biphasic contractions, an action similar to that produced by digitalis-like substances; and (e) prolongation of the action potential duration and refractory period of the canine isolated trabeculae. This latter action may confer a unique antiarrhythmic property to EIF. PMID:7508042

  4. Integrin beta 8 (ITGB8) regulates embryo implantation potentially via controlling the activity of TGF-B1 in mice.

    PubMed

    Kumar, Vijay; Maurya, Vineet Kumar; Joshi, Anubha; Meeran, Syed Musthapa; Jha, Rajesh Kumar

    2015-04-01

    Integrins (ITGs) are mediators of cell-cell and cell-matrix interactions, which are also associated with embryo implantation processes by controlling the interaction of blastocyst with endometrium. During early pregnancy, ITGbeta8 (ITGB8) has been shown to interact with latent transforming growth factor (TGF) beta 1 (TGFB1) at the fetomaternal interface. However, the precise role of ITGB8 in the uterus and its association with embryo implantation has not been elucidated. Therefore, we attempted to ascertain the role of ITGB8 during the window of embryo implantation process by inhibiting its function or protein expression. Uterine plasma membrane-anchored ITGB8 was augmented at peri-implantation and postimplantation stages. A similar pattern of mRNA expression was also found during the embryo implantation period. An immunolocalization study revealed the presence of ITGB8 on luminal epithelial cells along with mild expression on the stromal cells throughout the implantation period studied; however, an intense fluorescence was noted only during the peri- and postimplantation stages. Bioneutralization and mRNA silencing of the uterine Itgb8 at preimplantation stage reduced the rate/frequency of embryo implantation and subsequent pregnancy, suggesting its indispensable role during the embryo implantation period. ITGB8 can also regulate the liberation of active TGFB1 from its latent complex, which, in turn, acts on SMAD2/3 phosphorylation (activation) in the uterus during embryo implantation. This indicates involvement of ITGB8 in the embryo implantation process through regulation of activation of TGFB1. PMID:25788663

  5. Activation of estrogen receptor alpha disrupts differentiation of the reproductive organs in chicken embryos.

    PubMed

    Mattsson, Anna; Olsson, Jan A; Brunström, Björn

    2011-06-01

    Gonadal estrogen plays an important role in the differentiation of a female phenotype in birds. Exogenous compounds that interfere with estrogen signaling, for instance by binding to the estrogen receptors alpha and beta (ERα and ERβ), are therefore potential disruptors of sexual differentiation in birds. The ERα agonist propyl-pyrazole-triol (PPT), the ERα antagonist methyl piperidino pyrazole (MPP) and the ERβ agonist diarylproprionitrile (DPN) were used in the present study to explore the roles of the ERs in normal and disrupted sex differentiation in the chicken embryo. Activation of ERα by PPT caused disturbed differentiation of the reproductive organs in both sexes. In male embryos, PPT caused left-side ovotestis formation and retention of the Müllerian ducts. In female embryos, PPT caused retention of the right Müllerian duct (which normally regresses) and malformation of both Müllerian ducts. PPT also induced hepatic expression of mRNA for the estrogen-regulated egg yolk protein apoVLDL II. Notably, none of these effects were observed following treatment with DPN. ERα-inactivation by MPP counteracted the action of PPT but had little effect by its own. Our results indicate that ERα plays an important role in sex differentiation of the reproductive tract in female chicken embryos and show that ERα can mediate xenoestrogen-induced disturbances of sex differentiation. PMID:21420409

  6. Lactobacillus isolates from weaned piglets' mucosa with inhibitory activity against common porcine pathogens.

    PubMed

    Hacin, B; Rogelj, I; Matijasić, B B

    2008-01-01

    Twelve lactobacilli isolates from mucosa of 3-5-week-old weaned pigs were found to exert good antimicrobial activity against common porcine pathogens (S. aureus, B. cereus, E. coli, C. perfringens). Two of them produced in addition to lactic acid also considerable amounts of acetic acid, and 6 of them produced hydrogen peroxide and metabolites other than organic acids. Isolates 4/26 and 2/25 (identified as L. crispatus or L. amylovorus) were inhibitory against most strains of S. aureus, B. cereus and E. coli, and especially the strain 4/26 survived well in simulated gastric and intestinal juice. Diarrhea-causing E. coli O8K88H9 Ent(+) was successfully inhibited by the growing culture as well as by the catalase-treated and neutralized supernatant of L. reuteri 12/26. Mucin degradation and multiple resistance to antibiotics were not observed. PMID:19381487

  7. Metabolic Profiling of Chicken Embryos Exposed to Perfluorooctanoic Acid (PFOA) and Agonists to Peroxisome Proliferator-Activated Receptors.

    PubMed

    Mattsson, Anna; Kärrman, Anna; Pinto, Rui; Brunström, Björn

    2015-01-01

    Untargeted metabolic profiling of body fluids in experimental animals and humans exposed to chemicals may reveal early signs of toxicity and indicate toxicity pathways. Avian embryos develop separately from their mothers, which gives unique possibilities to study effects of chemicals during embryo development with minimal confounding factors from the mother. In this study we explored blood plasma and allantoic fluid from chicken embryos as matrices for revealing metabolic changes caused by exposure to chemicals during embryonic development. Embryos were exposed via egg injection on day 7 to the environmental pollutant perfluorooctanoic acid (PFOA), and effects on the metabolic profile on day 12 were compared with those caused by GW7647 and rosiglitazone, which are selective agonists to peroxisome-proliferator activated receptor α (PPARα) and PPARγ, respectively. Analysis of the metabolite concentrations from allantoic fluid by Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) showed clear separation between the embryos exposed to GW7647, rosiglitazone, and vehicle control, respectively. In blood plasma only GW7647 caused a significant effect on the metabolic profile. PFOA induced embryo mortality and increased relative liver weight at the highest dose. Sublethal doses of PFOA did not significantly affect the metabolic profile in either matrix, although single metabolites appeared to be altered. Neonatal mortality by PFOA in the mouse has been suggested to be mediated via activation of PPARα. However, we found no similarity in the metabolite profile of chicken embryos exposed to PFOA with those of embryos exposed to PPAR agonists. This indicates that PFOA does not activate PPAR pathways in our model at concentrations in eggs and embryos well above those found in wild birds. The present study suggests that allantoic fluid and plasma from chicken embryos are useful and complementary matrices for exploring effects on the metabolic profile resulting

  8. Metabolic Profiling of Chicken Embryos Exposed to Perfluorooctanoic Acid (PFOA) and Agonists to Peroxisome Proliferator-Activated Receptors

    PubMed Central

    Mattsson, Anna; Kärrman, Anna; Pinto, Rui; Brunström, Björn

    2015-01-01

    Untargeted metabolic profiling of body fluids in experimental animals and humans exposed to chemicals may reveal early signs of toxicity and indicate toxicity pathways. Avian embryos develop separately from their mothers, which gives unique possibilities to study effects of chemicals during embryo development with minimal confounding factors from the mother. In this study we explored blood plasma and allantoic fluid from chicken embryos as matrices for revealing metabolic changes caused by exposure to chemicals during embryonic development. Embryos were exposed via egg injection on day 7 to the environmental pollutant perfluorooctanoic acid (PFOA), and effects on the metabolic profile on day 12 were compared with those caused by GW7647 and rosiglitazone, which are selective agonists to peroxisome-proliferator activated receptor α (PPARα) and PPARγ, respectively. Analysis of the metabolite concentrations from allantoic fluid by Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) showed clear separation between the embryos exposed to GW7647, rosiglitazone, and vehicle control, respectively. In blood plasma only GW7647 caused a significant effect on the metabolic profile. PFOA induced embryo mortality and increased relative liver weight at the highest dose. Sublethal doses of PFOA did not significantly affect the metabolic profile in either matrix, although single metabolites appeared to be altered. Neonatal mortality by PFOA in the mouse has been suggested to be mediated via activation of PPARα. However, we found no similarity in the metabolite profile of chicken embryos exposed to PFOA with those of embryos exposed to PPAR agonists. This indicates that PFOA does not activate PPAR pathways in our model at concentrations in eggs and embryos well above those found in wild birds. The present study suggests that allantoic fluid and plasma from chicken embryos are useful and complementary matrices for exploring effects on the metabolic profile resulting

  9. The Use of Chick Embryos to Study Wnt Activity Gradients.

    PubMed

    Galli, Lisa M; Barnes, Tiffany; Burrus, Laura W

    2016-01-01

    The chick spinal cord provides a valuable model for assessing Wnt signaling activity. Loss or gain of function constructs that are transfected by electroporation can be directed to a single side of the spinal cord, thus leaving the contralateral side as an internal control. Here, we describe a method for measuring Wnt signaling via the use of BAT-Gal, a β-catenin dependent Wnt reporter. PMID:27590153

  10. High-content screening assay for identification of chemicals impacting spontaneous activity in zebrafish embryos.

    PubMed

    Raftery, Tara D; Isales, Gregory M; Yozzo, Krystle L; Volz, David C

    2014-01-01

    Although cell-based assays exist, rapid and cost-efficient high-content screening (HCS) assays within intact organisms are needed to support prioritization for developmental neurotoxicity testing in rodents. During zebrafish embryogenesis, spontaneous tail contractions occur from late-segmentation (∼19 h postfertilization, hpf) through early pharyngula (∼29 hpf) and represent the first sign of locomotion. Using transgenic zebrafish (fli1:egfp) that stably express eGFP beginning at ∼14 hpf, we have developed and optimized a 384-well-based HCS assay that quantifies spontaneous activity within single zebrafish embryos after exposure to test chemicals in a concentration-response format. Following static exposure of one embryo per well from 5 to 25 hpf, automated image acquisition procedures and custom analysis protocols were used to quantify total body area and spontaneous activity in live embryos. Survival and imaging success rates across control plates ranged from 87.5 to 100% and 93.3-100%, respectively. Using our optimized procedures, we screened 16 chemicals within the US EPA's ToxCast Phase-I library, and found that exposure to abamectin and emamectin benzoate-both potent avermectins-abolished spontaneous activity in the absence of gross malformations. Overall, compared to existing locomotion-based zebrafish assays conducted later in development, this method provides a simpler discovery platform for identifying potential developmental neurotoxicants. PMID:24328182

  11. The Role of Protegrins and Other Elastase-Activated Polypeptides in the Bactericidal Properties of Porcine Inflammatory Fluids

    PubMed Central

    Shi, Jishu; Ganz, Tomas

    1998-01-01

    The mammalian host response to infection includes the production and secretion of antimicrobial peptides from phagocytes and epithelial cells. Protegrins, a group of broadly microbicidal peptides isolated originally from porcine neutrophil lysates, were found to be stored as inactive proforms in porcine neutrophil granules but could be activated extracellularly by neutrophil elastase. We assessed the biological role of protegrins and other elastase-activated polypeptides in the microbicidal activity of neutrophil secretions and inflammatory fluids. When stimulated with phorbol myristate acetate (PMA), neutrophils generated stable microbicidal activity against both Escherichia coli and Listeria monocytogenes under normal-salt conditions and in the presence of 0 to 10% serum. The generation of these antimicrobial substances was dependent on neutrophil elastase, since it was inhibited by 1 mM N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone when it was present during activation, but not when this inhibitor was added afterwards. However, elastase-dependent activation of proprotegrins to protegrins in PMA-stimulated neutrophils was not inhibited by the presence of 1 to 2% serum. Porcine neutrophils also released antibacterial activity during phagocytosis of latex beads, and this too was dependent in large part on elastase-activated polypeptides, including protegrins. Moreover, protegrins were found at bactericidal concentrations in cell-free abscess fluid from naturally infected pigs. Taken together, these studies show that protegrins and other elastase-activated polypeptides are important stable antibacterial factors in porcine neutrophil secretions. The potential host defense role of elastase as an activating enzyme for the precursors of microbicidal peptides must be taken into account when therapeutic inhibitors of neutrophil elastase are evaluated for clinical use as anti-inflammatory agents. PMID:9673240

  12. Positive Correlation Between the Efficiency of Induced Pluripotent Stem Cells and the Development Rate of Nuclear Transfer Embryos When the Same Porcine Embryonic Fibroblast Lines Are Used As Donor Cells

    PubMed Central

    Xie, Bingteng; Wang, Jianyu; Liu, Shichao; Wang, Jiaqiang; Xue, Binghua; Li, Jingyu; Wei, Renyue; Zhao, Yanhua

    2014-01-01

    Abstract Induced pluripotent stem cells (iPSCs) and nuclear transfer (NT) are two of the primary routes to reprogram differentiated cells back to the pluripotent state. However, it is still unknown whether there is any correlation between the reprogramming efficiency of iPSCs and NT if the same donor cells are employed. In this study, six porcine embryonic fibroblast (PEF) lines from Landrace (L1, L6, L9) or Congjiang local pigs (C4, C5, C6) were used for iPSC induction and NT. Furthermore, the resultant iPSCs from four PEF lines (L1, L6, C4, and C5) were used for NT (iPSC-NT), and the expression of exogenous genes was detected in iPSC-NT embryos by real-time PCR. The results showed that the efficiency of iPSC lines established from different PEF lines were significantly different. When the same PEF lines were used as donor cells for NT, the blastocysts rates were also different among different PEF lines and positively related with iPSCs induction efficiency. When the iPSCs were used as donor cells for NT, compared with the source PEFs, the blastocysts rates were significantly decreased. Real-time PCR results indicated that exogenous genes (Oct4, c-Myc) continued to be expressed in iPSC-NT embryos. In summary, our results demonstrate that there was a positive correlation between iPSCs and NT reprogramming efficiency, although the mechanism of these two routes is different. This may provide a new method to select the appropriate donor cells for inducing iPSCs. PMID:24738969

  13. In Vitro Gender-Dependent Inhibition of Porcine Cytochrome P450 Activity by Selected Flavonoids and Phenolic Acids

    PubMed Central

    Ekstrand, Bo; Rasmussen, Martin Krøyer; Woll, Felicia; Zlabek, Vladimir; Zamaratskaia, Galia

    2015-01-01

    We investigated gender-related differences in the ability of selected flavonoids and phenolic compounds to modify porcine hepatic CYP450-dependent activity. Using pools of microsomes from male and female pigs, the inhibition of the CYP families 1A, 2A, 2E1, and 3A was determined. The specific CYP activities were measured in the presence of the following selected compounds: rutin, myricetin, quercetin, isorhamnetin, p-coumaric acid, gallic acid, and caffeic acid. We determined that myricetin and isorhamnetin competitively inhibited porcine CYP1A activity in the microsomes from both male and female pigs but did not affect the CYP2A and CYP2E1. Additionally, isorhamnetin competitively inhibited CYP3A in both genders. Noncompetitive inhibition of CYP3A activity by myricetin was observed only in the microsomes from male pigs, whereas CYP3A in female pigs was not affected. Quercetin competitively inhibited CYP2E1 and CYP1A activity in the microsomes from male pigs and irreversibly CY3A in female pigs. No effect of quercetin on CYP2E1 was observed in the microsomes from female pigs. Neither phenolic acids nor rutin affected CYP450 activities. Taken together, our results suggest that the flavonoids myricetin, isorhamnetin, and quercetin may affect the activities of porcine CYP1A, CYP3A, and CYP2E1 in a gender-dependent manner. PMID:25685784

  14. Autoimmune activation toward embryo implantation is rare in immune-privileged human endometrium.

    PubMed

    Haller-Kikkatalo, Kadri; Altmäe, Signe; Tagoma, Aili; Uibo, Raivo; Salumets, Andres

    2014-09-01

    Human embryo implantation represents embryo apposition, adhesion to the endometrial epithelium, and invasion into the stromal extracellular matrix within 1 to 2 days during days 6 to 9 after ovulation. The major molecular mechanisms mediating implantation include adhesion molecules, including mucins, selectins, integrins, and cadherins; extracellular matrix components, such as laminins and collagens and their degrading enzymes; phospholipids and immune regulatory molecules, including prostaglandins, cytokines; and immunosuppressive molecules expressed by invasive trophoblasts and endometrial cells. Many of these molecules are the targets for autoimmune reactions in autoimmune diseases and cancer; however, the relevance of those in immune-mediated implantation failure has not been defined. In this review, we will describe the molecules involved in 2-day event of human embryo implantation, which may also be involved in immune system activation and subsequently cause immune-mediated implantation failure. We speculate that the data in the literature are limited concerning antiendometrial antibodies because the endometrium might be taken as an immune-privileged site that avoids autoimmune activation that might harm the implantation process. Antibodies affecting human fertility in ways other than impairing implantation are outside the scope of the current article and will not be discussed. PMID:24959819

  15. Metabolite fingerprinting of pennycress (Thlaspi arvense L.) embryos to assess active pathways during oil synthesis

    PubMed Central

    Tsogtbaatar, Enkhtuul; Cocuron, Jean-Christophe; Sonera, Marcos Corchado; Alonso, Ana Paula

    2015-01-01

    Pennycress (Thlaspi arvense L.), a plant naturalized to North America, accumulates high levels of erucic acid in its seeds, which makes it a promising biodiesel and industrial crop. The main carbon sinks in pennycress embryos were found to be proteins, fatty acids, and cell wall, which respectively represented 38.5, 33.2, and 27.0% of the biomass at 21 days after pollination. Erucic acid reached a maximum of 36% of the total fatty acids. Together these results indicate that total oil and erucic acid contents could be increased to boost the economic competitiveness of this crop. Understanding the biochemical basis of oil synthesis in pennycress embryos is therefore timely and relevant to guide future breeding and/or metabolic engineering efforts. For this purpose, a combination of metabolomics approaches was conducted to assess the active biochemical pathways during oil synthesis. First, gas chromatography–mass spectrometry (GC-MS) profiling of intracellular metabolites highlighted three main families of compounds: organic acids, amino acids, and sugars/sugar alcohols. Secondly, these intermediates were quantified in developing pennycress embryos by liquid chromatography–tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Finally, partitional clustering analysis grouped the intracellular metabolites that shared a similar pattern of accumulation over time into eight clusters. This study underlined that: (i) sucrose might be stored rather than cleaved into hexoses; (ii) glucose and glutamine would be the main sources of carbon and nitrogen, respectively; and (iii) glycolysis, the oxidative pentose phosphate pathway, the tricarboxylic acid cycle, and the Calvin cycle were active in developing pennycress embryos. PMID:25711705

  16. Metabolite fingerprinting of pennycress (Thlaspi arvense L.) embryos to assess active pathways during oil synthesis.

    PubMed

    Tsogtbaatar, Enkhtuul; Cocuron, Jean-Christophe; Sonera, Marcos Corchado; Alonso, Ana Paula

    2015-07-01

    Pennycress (Thlaspi arvense L.), a plant naturalized to North America, accumulates high levels of erucic acid in its seeds, which makes it a promising biodiesel and industrial crop. The main carbon sinks in pennycress embryos were found to be proteins, fatty acids, and cell wall, which respectively represented 38.5, 33.2, and 27.0% of the biomass at 21 days after pollination. Erucic acid reached a maximum of 36% of the total fatty acids. Together these results indicate that total oil and erucic acid contents could be increased to boost the economic competitiveness of this crop. Understanding the biochemical basis of oil synthesis in pennycress embryos is therefore timely and relevant to guide future breeding and/or metabolic engineering efforts. For this purpose, a combination of metabolomics approaches was conducted to assess the active biochemical pathways during oil synthesis. First, gas chromatography-mass spectrometry (GC-MS) profiling of intracellular metabolites highlighted three main families of compounds: organic acids, amino acids, and sugars/sugar alcohols. Secondly, these intermediates were quantified in developing pennycress embryos by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Finally, partitional clustering analysis grouped the intracellular metabolites that shared a similar pattern of accumulation over time into eight clusters. This study underlined that: (i) sucrose might be stored rather than cleaved into hexoses; (ii) glucose and glutamine would be the main sources of carbon and nitrogen, respectively; and (iii) glycolysis, the oxidative pentose phosphate pathway, the tricarboxylic acid cycle, and the Calvin cycle were active in developing pennycress embryos. PMID:25711705

  17. Effect of knockout serum replacement supplementation to culture medium on porcine blastocyst development and piglet production.

    PubMed

    Sakurai, Masahiro; Suzuki, Chie; Yoshioka, Koji

    2015-03-01

    We have previously developed chemically defined media suitable for in vitro production (IVP) of porcine embryos and subsequently generated piglets by nonsurgical embryo transfer. In this study, to further improve the culture conditions for IVP of porcine embryos, we evaluated the effect of knockout serum replacement (KSR), a substitute for serum or albumin, on the viability and development of porcine blastocysts. The addition of 5% (v:v) KSR to porcine blastocyst medium (PBM) on Day 5 (Day 0 = IVF) significantly increased the survival and hatching rates of blastocysts and the total cell number of Day-7 blastocysts compared with those in cultures without KSR or addition of 10% fetal bovine serum. Furthermore, the number of cells in the trophectoderm of Day-6 blastocysts and the ATP content of Day-7 blastocysts cultured with 5% KSR were significantly higher than those of blastocysts cultured without KSR. The mRNA expression of a rate-limiting enzyme in β-oxidation, carnitine palmitoyltransferase 1, in Day-6 blastocysts, and a serine proteinase, urokinase-type plasminogen activator, in Day-7 blastocysts cultured in 5% KSR-PBM was significantly higher than that of blastocysts cultured in PBM alone. Four of eight recipients (50%), in which Day-5 blastocysts treated with 5% KSR were transferred nonsurgically, became pregnant. However, the efficiency of piglet production (percentage of piglets born based on the number of embryos transferred) was similar to recipients with transferred blastocysts treated without KSR. The present study demonstrated that the addition of KSR to PBM enhanced the in vitro viability of porcine blastocysts. In addition, our data suggest that KSR improved development to the hatching stage and blastocyst quality by increasing ATP content and hatching-related mRNA expression of blastocysts. PMID:25434774

  18. The activation of DNA damage detection and repair responses in cleavage-stage rat embryos by a damaged paternal genome.

    PubMed

    Grenier, Lisanne; Robaire, Bernard; Hales, Barbara F

    2012-06-01

    Male germ cell DNA damage, after exposure to radiation, exogenous chemicals, or chemotherapeutic agents, is a major cause of male infertility. DNA-damaged spermatozoa can fertilize oocytes; this is of concern because there is limited information on the capacity of early embryos to repair a damaged male genome or on the fate of these embryos if repair is inadequate. We hypothesized that the early activation of DNA damage response in the early embryo is a critical determinant of its fate. The objective of this study was to assess the DNA damage response and mitochondrial function as a measure of the energy supply for DNA repair and general health in cleavage-stage embryos sired by males chronically exposed to an anticancer alkylating agent, cyclophosphamide. Male rats were treated with saline or cyclophosphamide (6 mg/kg/day) for 4 weeks and mated to naturally cycling females. Pronuclear two- and eight-cell embryos were collected for immunofluorescence analysis of mitochondrial function and biomarkers of the DNA damage response: γH2AX foci, 53BP1 reactivity, and poly(ADP-ribose) polymer formation. Mitochondrial activities did not differ between embryos sired by control- and cyclophosphamide-exposed males. At the two-cell stage, there was no treatment-related increase in DNA double-strand breaks; by the eight-cell stage, a significant increase was noted, as indicated by increased medium and large γH2AX foci. This was accompanied by a dampened DNA repair response, detected as a decrease in the nuclear intensity of poly(ADP-ribose) polymers. The micronuclei formed in cyclophosphamide-sired embryos contained large γH2AX foci and enhanced poly(ADP-ribose) polymer and 53BP1 reactivity compared with their nuclear counterparts. Thus, paternal cyclophosphamide exposure activated a DNA damage response in cleavage-stage embryos. Furthermore, this damage response may be useful in assessing embryo quality and developmental competence. PMID:22454429

  19. Porcine parvovirus infection induces apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated pathway.

    PubMed

    Zhang, Hongling; Huang, Yong; Du, Qian; Luo, Xiaomao; Zhang, Liang; Zhao, Xiaomin; Tong, Dewen

    2015-01-01

    Porcine parvovirus (PPV) infection has been reported to induce the cytopathic effects (CPE) in some special host cells and contribute the occurrence of porcine parvovirus disease, but the molecular mechanisms underlying PPV-induced CPE are not clear. In this study, we investigated the morphological and molecular changes of porcine kidney cell line (PK-15 cells) infected with PPV. The results showed that PPV infection inhibited the viability of PK-15 cells in a time and concentration dependent manner. PPV infection induced typical apoptotic features including chromatin condensation, apoptotic body formation, nuclear fragmentation, and Annexin V-binding activity. Further studies showed that Bax was increased and translocated to mitochondria, whereas Bcl-2 was decreased in PPV-infected cells, which caused mitochondrial outer-membrane permeabilization, resulting in the release of mitochondrial cytochrome c, followed by caspase-9 and caspase-3 activation. However, the expression of Fas and Fas ligand (FasL) did not appear significant changes in the process of PPV-induced apoptosis. Moreover, PPV infection activated p53 signaling, which was involved in the activation of apoptotic signaling induced by PPV infection via regulation of Bax and Bcl-2. Taken together, our results demonstrated that PPV infection induced apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated apoptosis pathway. This study may contribute to shed light on the molecular pathogenesis of PPV infection. PMID:25499817

  20. Chick embryos have the same pattern of hypoxic lower-brain activation as fetal mammals.

    PubMed

    Landry, Jeremy P; Hawkins, Connor; Lee, Aaron; Coté, Alexandra; Balaban, Evan; Pompeiano, Maria

    2016-01-01

    cFos expression (indicating a particular kind of neuronal activation) was examined in embryonic day (E) 18 chick embryos after exposure to 4 h of either normoxia (21% O2), modest hypoxia (15% O2), or medium hypoxia (10% O2). Eight regions of the brainstem and hypothalamus were surveyed, including seven previously shown to respond to hypoxia in late-gestation mammalian fetuses (Breen et al., 1997; Nitsos and Walker, 1999b). Hypoxia-related changes in chick embryo brain activation mirrored those found in fetal mammals with the exception of the medullary Raphe, which showed decreased hypoxic activation, compared with no change in mammals. This difference may be explained by the greater anapyrexic responses of chick embryos relative to mammalian fetuses. Activation in the A1/C1 region was examined in more detail to ascertain whether an O2-sensitive subpopulation of these cells containing heme oxygenase 2 (HMOX2) may drive hypoxic brain responses before the maturation of peripheral O2-sensing. HMOX2-positive and -negative catecholaminergic cells and interdigitating noncatecholaminergic HMOX2-positive cells all showed significant changes in cFos expression to hypoxia, with larger population responses seen in the catecholaminergic cells. Hypoxia-induced activation of lower-brain regions studied here was significantly better correlated with activation of the nucleus of the solitary tract (NTS) than with that of HMOX2-containing A1/C1 neurons. Together, these observations suggest that (1) the functional circuitry controlling prenatal brain responses to hypoxia is strongly conserved between birds and mammals, and (2) NTS neurons are a more dominant driving force for prenatal hypoxic cFos brain responses than O2-sensing A1/C1 neurons. PMID:25964066

  1. Porcine arterivirus activates the NF-{kappa}B pathway through I{kappa}B degradation

    SciTech Connect

    Lee, Sang-Myeong; Kleiboeker, Steven B. . E-mail: KleiboekerS@Missouri.edu

    2005-11-10

    Nuclear factor-kappaB (NF-{kappa}B) is a critical regulator of innate and adaptive immune function as well as cell proliferation and survival. The present study demonstrated for the first time that a virus belonging to the Arteriviridae family activates NF-{kappa}B in MARC-145 cells and alveolar macrophages. In porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells, NF-{kappa}B activation was characterized by translocation of NF-{kappa}B from the cytoplasm to the nucleus, increased DNA binding activity, and NF-{kappa}B-regulated gene expression. NF-{kappa}B activation was increased as PRRSV infection progressed and in a viral dose-dependent manner. UV-inactivation of PRRSV significantly reduced the level of NF-{kappa}B activation. Degradation of I{kappa}B protein was detected late in PRRSV infection, and overexpression of the dominant negative form of I{kappa}B{alpha} (I{kappa}B{alpha}DN) significantly suppressed NF-{kappa}B activation induced by PRRSV. However, I{kappa}B{alpha}DN did not affect viral replication and viral cytopathic effect. PRRSV infection induced oxidative stress in cells by generating reactive oxygen species (ROS), and antioxidants inhibited NF-{kappa}B DNA binding activity in PRRSV-infected cells, suggesting ROS as a mechanism by which NF-{kappa}B was activated by PRRSV infection. Moreover, NF-{kappa}B-dependent expression of matrix metalloproteinase (MMP)-2 and MMP-9 was observed in PRRSV-infected cells, an observation which implies that NF-{kappa}B activation is a biologically significant aspect of PRRSV pathogenesis. The results presented here provide a basis for understanding molecular pathways of pathology and immune evasion associated with disease caused by PRRSV.

  2. Porcine arterivirus activates the NF-kappaB pathway through IkappaB degradation.

    PubMed

    Lee, Sang-Myeong; Kleiboeker, Steven B

    2005-11-10

    Nuclear factor-kappaB (NF-kappaB) is a critical regulator of innate and adaptive immune function as well as cell proliferation and survival. The present study demonstrated for the first time that a virus belonging to the Arteriviridae family activates NF-kappaB in MARC-145 cells and alveolar macrophages. In porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells, NF-kappaB activation was characterized by translocation of NF-kappaB from the cytoplasm to the nucleus, increased DNA binding activity, and NF-kappaB-regulated gene expression. NF-kappaB activation was increased as PRRSV infection progressed and in a viral dose-dependent manner. UV-inactivation of PRRSV significantly reduced the level of NF-kappaB activation. Degradation of IkappaB protein was detected late in PRRSV infection, and overexpression of the dominant negative form of IkappaBalpha (IkappaBalphaDN) significantly suppressed NF-kappaB activation induced by PRRSV. However, IkappaBalphaDN did not affect viral replication and viral cytopathic effect. PRRSV infection induced oxidative stress in cells by generating reactive oxygen species (ROS), and antioxidants inhibited NF-kappaB DNA binding activity in PRRSV-infected cells, suggesting ROS as a mechanism by which NF-kappaB was activated by PRRSV infection. Moreover, NF-kappaB-dependent expression of matrix metalloproteinase (MMP)-2 and MMP-9 was observed in PRRSV-infected cells, an observation which implies that NF-kappaB activation is a biologically significant aspect of PRRSV pathogenesis. The results presented here provide a basis for understanding molecular pathways of pathology and immune evasion associated with disease caused by PRRSV. PMID:16129468

  3. A system for the determination of planar force vectors from spontaneously active chicken embryos.

    PubMed

    Sharp, Andrew A; Cain, Blake W; Pakiraih, Joanna; Williams, James L

    2014-11-01

    Generally, a combination of kinematic, electromyographic (EMG), and force measurements are used to understand how an organism generates and controls movement. The chicken embryo has been a very useful model system for understanding the early stages of embryonic motility in vertebrates. Unfortunately, the size and delicate nature of embryos makes studies of motility during embryogenesis very challenging. Both kinematic and EMG recordings have been achieved in embryonic chickens, but two-dimensional force vector recordings have not. Here, we describe a dual-axis system for measuring force generated by the leg of embryonic chickens. The system employs two strain gauges to measure planar forces oriented with the plane of motion of the leg. This system responds to forces according to the principles of Pythagorean geometry, which allows a simple computational program to determine the force vector (magnitude and direction) generated during spontaneous motor activity. The system is able to determine force vectors for forces >0.5 mN accurately and allows for simultaneous kinematic and EMG recordings. This sensitivity is sufficient for force vector measurements encompassing most embryonic leg movements in midstage chicken embryos allowing for a more complete understanding of embryonic motility. Variations on this system are discussed to enable nonideal or alternative sensor arrangements and to allow for translation of this approach to other delicate model systems. PMID:25143544

  4. CDK2 Is Required for the DNA Damage Response During Porcine Early Embryonic Development.

    PubMed

    Wang, HaiYang; Kim, Nam-Hyung

    2016-08-01

    Cyclin-dependent kinase (CDK) 2 inhibition plays a central role in DNA damage-induced cell cycle arrest and DNA repair. However, whether CDK2 also influences early porcine embryo development is unknown. In this study, we examined whether CDK2 is involved in the regulation of oocyte meiosis and early embryonic development of porcine embryos. We found that disrupting CDK2 activity with RNAi or an inhibitor did not affect meiotic resumption or meiosis II arrest. However, CDK2 inhibitor-treated embryos showed delayed cleavage and ceased development before the blastocyst stage. Disrupting CDK2 activity is able to induce sustained DNA damage, as demonstrated by the formation of distinct gammaH2AX foci in nuclei of Day-3 and Day-5 embryos. Inhibiting CDK2 triggers a DNA damage checkpoint by activation of the ataxia telangiectasia mutated (ATM)-P53-P21 pathway. However, the mRNA expression of genes involved in nonhomologous end joining or homologous recombination pathways for double-strand break repair were reduced after administering CDK2 inhibitor to 5-day-old embryos. Furthermore, CDK2 inhibition caused apoptosis in Day-7 blastocysts. Thus, our results indicate that an ATM-P53-P21 DNA damage checkpoint is intact in the absence of CDK2; however, CDK2 is important for proper repair of the damaged DNA by either directly or indirectly influencing DNA repair-related gene expression. PMID:27307074

  5. Extracellular signal-regulated kinase (ERK) activation is required for porcine epidemic diarrhea virus replication.

    PubMed

    Kim, Youngnam; Lee, Changhee

    2015-10-01

    Porcine epidemic diarrhea virus (PEDV) is a highly enteropathogenic coronavirus of swine that causes acute enteritis with high mortality in nursery piglets. To date, the cellular factors involved in PEDV replication have not been well defined. The extracellular signal-regulated kinase (ERK) that serves as a critical component of cellular signal transduction pathways to modulate a variety of cellular functions has been shown to regulate several viral infections. In the present study, we found that PEDV activates ERK1/2 early in infection independently of viral replication. The PEDV-induced ERK1/2 activation resulted in the phosphorylation of its downstream substrate Elk-1 in infected cells. Treatment with ERK inhibitors or ERK1/2 knockdown significantly suppressed viral progeny production. Inhibition of ERK activation also diminished viral protein expression and genomic and subgenomic RNA transcription. These findings indicate that the ERK signaling pathway plays an important role in the PEDV life cycle and beneficially contributes to viral infection. PMID:26115165

  6. Measuring the electric activity of chick embryos heart through 16 bit audio card monitored by the Goldwavetm software

    NASA Astrophysics Data System (ADS)

    Silva, Dilson; Cortez, Celia Martins

    2015-12-01

    In the present work we used a high-resolution, low-cost apparatus capable of detecting waves fit inside the sound bandwidth, and the software package GoldwaveTM for graphical display, processing and monitoring the signals, to study aspects of the electric heart activity of early avian embryos, specifically at the 18th Hamburger & Hamilton stage of the embryo development. The species used was the domestic chick (Gallus gallus), and we carried out 23 experiments in which cardiographic spectra of QRS complex waves representing the propagation of depolarization waves through ventricles was recorded using microprobes and reference electrodes directly on the embryos. The results show that technique using 16 bit audio card monitored by the GoldwaveTM software was efficient to study signal aspects of heart electric activity of early avian embryos.

  7. Proteolytic Activation of the Porcine Epidemic Diarrhea Coronavirus Spike Fusion Protein by Trypsin in Cell Culture

    PubMed Central

    Wicht, Oliver; Li, Wentao; Willems, Lione; Meuleman, Tom J.; Wubbolts, Richard W.; van Kuppeveld, Frank J. M.; Rottier, Peter J. M.

    2014-01-01

    ABSTRACT Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. IMPORTANCE Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV. PMID:24807723

  8. Inhibition of Porcine Pancreatic Amylase Activity by Sulfamethoxazole: Structural and Functional Aspect.

    PubMed

    Maity, Sujan; Mukherjee, Koel; Banerjee, Amrita; Mukherjee, Suman; Dasgupta, Dipak; Gupta, Suvroma

    2016-06-01

    Combating Type-2 diabetes mellitus is a pivotal challenge in front of the present world. Several lines of therapy are in practice for resisting this deadly disease which often culminates with cardiovascular complexities, neuropathy and retinopathy. Among various therapies, administration of alpha glucosidase inhibitors is common and widely practiced. Sulfonylurea category of anti diabetic drug often suffers from cross reactivity with sulfamethoxazole (SMX), a common drug in use to treat a handful of microbial infections. However the specific cellular target generating postprandial hypoglycemia on SMX administration is till date unraveled. The present work has been initiated to elucidate the effects of a group of sulfonamide drugs inclusive of SMX for their amylase inhibitory role. SMX inhibits porcine pancreatic amylase (PPA) in a noncompetitive mode with an average IC50 value 0.94 mM respectively. Interaction of SMX with PPA is manifested with gradual quenching of tryptophan fluorescence with concomitant shift in lambda max value (λmax). Binding is governed by entropy driven factor (24.8 cal mol(-1) K(-1)) with unfavorable contribution from enthalpy change. SMX interferes with the activity of acarbose in a synergistic mode to reduce the effective dose of acarbose as evident from the in vitro PPA inhibition study. In summary, loss of PPA activity in presence of SMX is indicative of structural changes of PPA which is further augmented in the presence of acarbose as explained in the schematic model and docking study. PMID:27272220

  9. Proteolytic activation of the porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture.

    PubMed

    Wicht, Oliver; Li, Wentao; Willems, Lione; Meuleman, Tom J; Wubbolts, Richard W; van Kuppeveld, Frank J M; Rottier, Peter J M; Bosch, Berend Jan

    2014-07-01

    Isolation of porcine epidemic diarrhea coronavirus (PEDV) from clinical material in cell culture requires supplementation of trypsin. This may relate to the confinement of PEDV natural infection to the protease-rich small intestine of pigs. Our study focused on the role of protease activity on infection by investigating the spike protein of a PEDV isolate (wtPEDV) using a reverse genetics system based on the trypsin-independent cell culture-adapted strain DR13 (caPEDV). We demonstrate that trypsin acts on the wtPEDV spike protein after receptor binding. We mapped the genetic determinant for trypsin-dependent cell entry to the N-terminal region of the fusion subunit of this class I fusion protein, revealing a conserved arginine just upstream of the putative fusion peptide as the potential cleavage site. Whereas coronaviruses are typically processed by endogenous proteases of the producer or target cell, PEDV S protein activation strictly required supplementation of a protease, enabling us to study mechanistic details of proteolytic processing. Importance: Recurring PEDV epidemics constitute a serious animal health threat and an economic burden, particularly in Asia but, as of recently, also on the North-American subcontinent. Understanding the biology of PEDV is critical for combatting the infection. Here, we provide new insight into the protease-dependent cell entry of PEDV. PMID:24807723

  10. High Pressure Homogenization of Porcine Pepsin Protease: Effects on Enzyme Activity, Stability, Milk Coagulation Profile and Gel Development.

    PubMed

    Leite Júnior, Bruno Ricardo de Castro; Tribst, Alline Artigiani Lima; Cristianini, Marcelo

    2015-01-01

    This study investigated the effect of high pressure homogenization (HPH) (up to 190 MPa) on porcine pepsin (proteolytic and milk-clotting activities), and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure). Although the proteolytic activity (PA) was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained with the enzyme processed at 50, 100 and 150 MPa. HPH increased the milk-clotting activity (MCA) of the enzyme processed at 150 MPa, being 15% higher than the MCA of non-processed samples after 60 days of storage. The enzyme processed at 150 MPa produced faster aggregation and a more consistent milk gel (G' value 92% higher after 90 minutes) when compared with the non-processed enzyme. In addition, the gels produced with the enzyme processed at 150 MPa showed greater syneresis after 40 minutes of coagulation (forming a more compact protein network) and lower porosity (evidenced by confocal microscopy). These effects on the milk gel can be associated with the increment in MCA and reduction in PA caused by the effects of HPH on pepsin during storage. According to the results, HPH stands out as a process capable of changing the proteolytic characteristics of porcine pepsin, with improvements on the milk coagulation step and gel characteristics. Therefore, the porcine pepsin submitted to HPH process can be a suitable alternative for the production of cheese. PMID:25938823

  11. High Pressure Homogenization of Porcine Pepsin Protease: Effects on Enzyme Activity, Stability, Milk Coagulation Profile and Gel Development

    PubMed Central

    Leite Júnior, Bruno Ricardo de Castro; Tribst, Alline Artigiani Lima; Cristianini, Marcelo

    2015-01-01

    This study investigated the effect of high pressure homogenization (HPH) (up to 190 MPa) on porcine pepsin (proteolytic and milk-clotting activities), and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure). Although the proteolytic activity (PA) was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained with the enzyme processed at 50, 100 and 150 MPa. HPH increased the milk-clotting activity (MCA) of the enzyme processed at 150 MPa, being 15% higher than the MCA of non-processed samples after 60 days of storage. The enzyme processed at 150 MPa produced faster aggregation and a more consistent milk gel (G’ value 92% higher after 90 minutes) when compared with the non-processed enzyme. In addition, the gels produced with the enzyme processed at 150 MPa showed greater syneresis after 40 minutes of coagulation (forming a more compact protein network) and lower porosity (evidenced by confocal microscopy). These effects on the milk gel can be associated with the increment in MCA and reduction in PA caused by the effects of HPH on pepsin during storage. According to the results, HPH stands out as a process capable of changing the proteolytic characteristics of porcine pepsin, with improvements on the milk coagulation step and gel characteristics. Therefore, the porcine pepsin submitted to HPH process can be a suitable alternative for the production of cheese. PMID:25938823

  12. Improvement of embryo production by the replacement of the last two doses of porcine follicle-stimulating hormone with equine chorionic gonadotropin in Sindhi donors.

    PubMed

    Mattos, M C C; Bastos, M R; Guardieiro, M M; Carvalho, J O; Franco, M M; Mourão, G B; Barros, C M; Sartori, R

    2011-05-01

    The aim of this study was to evaluate the superovulatory (SOV) response of Sindhi (Bos indicus) donors submitted to an ovarian follicular superstimulatory protocol replacing the last two doses of pFSH by eCG. Forty-eight SOV treatments were performed in a crossover design in 19 nulliparous and primiparous females that were randomly divided into two groups: FSH (n=24), which consisted of eight pFSH injections, or FSH/eCG (n=24), which consisted of six pFSH injections followed by two eCG injections. Each female underwent two or three SOV treatments that consisted of an i.m. injection of 2mg estradiol benzoate and the insertion of an intravaginal progesterone-releasing device on Day 0. On Day 4, superstimulatory treatments were initiated and 100mg pFSH was divided into twice daily decreasing doses over a 4-day period. In the FSH/eCG group, the last two doses of pFSH were replaced by two doses of eCG (150 IU eCG each). At the time of the fifth and sixth injections of FSH, 0.150 mg PGF(2α) was injected i.m. The intravaginal progesterone-releasing device was removed at the time of the last FSH or eCG injection and ovulation was induced with 0.2 mg GnRH 18 h later. All females were artificially inseminated with frozen-thawed semen from the same bull 6 and 18 h after GnRH treatment. Seven days after GnRH treatment, embryos/ova were recovered and classified. Follicular superstimulatory (number of follicles ≥6mm at the time of the last FSH or eCG injection) and SOV (CL number) responses were determined by transrectal ultrasonography. Data were analyzed using generalized linear models and results were presented as least squares means±standard error. The FSH/eCG group had higher superstimulatory (33.8±3.9 compared to 23.8±2.6 follicles; P=0.03) and SOV (16.8±2.9 compared to 10.8±2.1 CL; P=0.10) responses. Although the number of total ova/embryos was not different between groups (8.2±1.8 compared to 5.9±1.4 for FSH/eCG and FSH groups, respectively; P=0.25), the number

  13. Monooxygenase activity and contaminant burdens of pipping heron embryos in Virginia, the Great Lakes and San Francisco Bay

    USGS Publications Warehouse

    Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.

    1991-01-01

    Black-crowned night-heron (Nvcticorax nvcticorax) pipping embryos were studied from undisturbed (Chincoteague National Wildl ife Refuge, VA) and industrialized (Cat Island, Green Bay WI, and Bair and W. Marin Islands, San Francisco Bay, CA) locations. Hepatic aryl hydrocarbon hydroxylase (AHH) , ethoxyresorufin-O-dealkylase, (EROD), benzyloxyROD (BROD), pentoxyROD (PROD) and ethoxycoumarinOD (ECOD) activities and burdens of organochlorines (embryo + yolk sac - liver) were quantified. AHH, BROD, ECOD and EROD were induced up to 100-fold (P<.O5) in embryos from Cat Island compared to the other sites. Greatest burdens of total PCBs and p,p?DDE were detected in Cat Island embryos. Monooxygenase activities (AHH, BROD, ECOD and EROD) and PCB concentrations were significantly correlated (r=O.50 to 0.72). These and other data indicate that monooxygenases may be rapid and inexpensive biomarkers of exposure to some PCB congeners. Current efforts include determination of PCB congeners and other contaminants in these embryos, additional characterization of the induced P-450 isozymes, and expanding the study to include heron embryos and nestlings at other estuaries.

  14. The antiviral activity of arctigenin in traditional Chinese medicine on porcine circovirus type 2.

    PubMed

    Chen, Jie; Li, Wentao; Jin, Erguang; He, Qigai; Yan, Weidong; Yang, Hanchun; Gong, Shiyu; Guo, Yi; Fu, Shulin; Chen, Xiabing; Ye, Shengqiang; Qian, Yunguo

    2016-06-01

    Arctigenin (ACT) is a phenylpropanoid dibenzylbutyrolactone lignan extracted from the traditional herb Arctium lappa L. (Compositae) with anti-viral and anti-inflammatory effects. Here, we investigated the antiviral activity of ACT found in traditional Chinese medicine on porcine circovirus type 2 (PCV2) in vitro and in vivo. Results showed that dosing of 15.6-62.5μg/mL ACT could significantly inhibit the PCV2 proliferation in PK-15 cells (P<0.01). Dosing of 62.5μg/mL ACT 0, 4 or 8h after challenge inoculation significantly inhibited the proliferation of 1MOI and 10MOI in PK-15 cells (P<0.01), and the inhibitory effect of ACT dosing 4h or 8h post-inoculation was greater than 0h after dosing (P<0.01). In vivo test with mice challenge against PCV2 infection demonstrated that intraperitoneal injection of 200μg/kg ACT significantly inhibited PCV2 proliferation in the lungs, spleens and inguinal lymph nodes, with an effect similar to ribavirin, demonstrating the effectiveness of ACT as an antiviral agent against PCV2 in vitro and in vivo. This compound, therefore, may have the potential to serve as a drug for protection of pigs against the infection of PCV2. PMID:27234554

  15. Wnt-Signaling-Mediated Antiosteoporotic Activity of Porcine Placenta Hydrolysates in Ovariectomized Rats

    PubMed Central

    Ko, Byoung-Seob; Kim, Da Sol; Kang, Suna; Lee, Na Ra; Ryuk, Jin Ah; Park, Sunmin

    2012-01-01

    Anti-osteoporotic effects of two types of porcine placenta hydrolysates (PPH) were evaluated in ovariectomized (OVX) rats orally administered PPH without (WPPH) or with (NPPH) ovarian hormones (1 g/kg bw/day). PPH groups were compared with OVX rats with estrogen replacement (0.1 mg/kg bw conjugated estrogen; EST), or dextrose (placebo; OVX-control) All rats received high-fat/calcium-deficient diets for 12 weeks. NPPH contained less estrogen and progesterone, but more essential amino acids, whereas the opposite was true for WPPH. NPPH decreased body weight and peri-uterine fat pads, and maintained uterus weight. NPPH rats had higher femur and lumbar spine bone mass density compared to controls; but less than those of EST rats. Serum phosphorus and urinary calcium and phosphorus levels were reduced in NPPH rats compared to OVX-controls. Serum bone-specific alkaline phosphatase, osteocalcin, and bone turnover marker levels were reduced NPPH rats compared to OVX-controls. WPPH produced results similar to those of NPPH, but less significant. Both NPPH and estrogen upregulated low-density lipoprotein receptor-related protein 5 and β-catenin in OVX rats, while the expression of dickkopf-related protein 1 was suppressed. In conclusion, NPPH exerted anti-osteoporotic effects by activating osteogenesis and stimulating Wnt signaling, possibly mediated by the various amino acids and not ovarian hormones. PMID:23258987

  16. In-vivo tissue repair using light-activated surgical adhesive in a porcine model

    NASA Astrophysics Data System (ADS)

    McNally-Heintzelman, Karen M.; Riley, Jill N.; Dickson, Tonya J.; Hou, Dong Ming; Rogers, Pamela; March, Keith L.

    2001-05-01

    An in vivo study was conducted to investigate the feasibility, mechanical function, and chronic biocompatibility of a new light-activated surgical adhesive for achieving rapid hemostasis of the puncture site following diagnostic catheterization and interventional cardiac procedures. Porcine carotid arteries (nequals6) and femoral arteries (nequals6) were exposed, and an incision was made in the arterial walls using a 16G needle. The surgical adhesive, composed of a poly(L-lactic-co-glycolic acid) scaffold doped with the traditional protein solder mix of serum albumin and indocyanine green dye, was used to close the incisions in conjunction with an 805-nm diode laser. Blood flow was restored to the vessels immediately after the procedure and the incision sites were checked for patency. The strength and hemostatic abilities of the new surgical adhesive were evaluated in the context of arterial pressure, persistence of hemostatis and presence of any inflammatory reaction after 3 days. After this evaluation period, the surgical procedure was repeated on the carotid arteries (nequals6) and femoral arteries (nequals6) of three additional animals that had been heparinized prior to surgery to closer approximate the conditions seen in a typical vascular surgical setting.

  17. Multidrug efflux transporter activity in sea urchin embryos:Does localization provide a diffusive advantage?

    NASA Astrophysics Data System (ADS)

    Song, Xianfeng; Setayeshgar, Sima; Cole, Bryan; Hamdoun, Amro; Epel, David

    2008-03-01

    Experiments have shown upregulation of multidrug efflux transporter activity approximately 30 min after fertilization in the sea urchin embryo [1]. These ATP-hydrolyzing transporter proteins pump moderately hydrophobic molecules out of the cell and represent the cell's first line of defense againstexogenous toxins. It has also been shown that transporters are moved in vesicles along microfilaments and localized to tips of microvilli prior to activation. We have constructed a geometrically realistic model of the embryo, including microvilli, to explore the functional role of this localization in the efficient elimination of toxins from the standpoint of diffusion. We compute diffusion of toxins in extracellular, membrane and intracellular spaces coupled with transporter activity, using experimentally derived values for physical parameters. For transporters uniformly distributed along microvilli and tip-localized transporters we compare regions in parameter space where each distribution provides diffusive advantage, and comment on the physically expected conditions. [1] A. M. Hamdoun, G. N. Cherr, T. A. Roepke and D. Epel, Developmental Biology 276 452 (2004).

  18. Waves of Cdk1 Activity in S Phase Synchronize the Cell Cycle in Drosophila Embryos.

    PubMed

    Deneke, Victoria E; Melbinger, Anna; Vergassola, Massimo; Di Talia, Stefano

    2016-08-22

    Embryos of most metazoans undergo rapid and synchronous cell cycles following fertilization. While diffusion is too slow for synchronization of mitosis across large spatial scales, waves of Cdk1 activity represent a possible process of synchronization. However, the mechanisms regulating Cdk1 waves during embryonic development remain poorly understood. Using biosensors of Cdk1 and Chk1 activities, we dissect the regulation of Cdk1 waves in the Drosophila syncytial blastoderm. We show that Cdk1 waves are not controlled by the mitotic switch but by a double-negative feedback between Cdk1 and Chk1. Using mathematical modeling and surgical ligations, we demonstrate a fundamental distinction between S phase Cdk1 waves, which propagate as active trigger waves in an excitable medium, and mitotic Cdk1 waves, which propagate as passive phase waves. Our findings show that in Drosophila embryos, Cdk1 positive feedback serves primarily to ensure the rapid onset of mitosis, while wave propagation is regulated by S phase events. PMID:27554859

  19. Detecting cardiac contractile activity in the early mouse embryo using multiple modalities

    PubMed Central

    Chen, Chiann-Mun; Miranda, António M. A.; Bub, Gil; Srinivas, Shankar

    2015-01-01

    The heart is one of the first organs to develop during mammalian embryogenesis. In the mouse, it starts to form shortly after gastrulation, and is derived primarily from embryonic mesoderm. The embryonic heart is unique in having to perform a mechanical contractile function while undergoing complex morphogenetic remodeling. Approaches to imaging the morphogenesis and contractile activity of the developing heart are important in understanding not only how this remodeling is controlled but also the origin of congenital heart defects (CHDs). Here, we describe approaches for visualizing contractile activity in the developing mouse embryo, using brightfield time lapse microscopy and confocal microscopy of calcium transients. We describe an algorithm for enhancing this image data and quantifying contractile activity from it. Finally we describe how atomic force microscopy can be used to record contractile activity prior to it being microscopically visible. PMID:25610399

  20. Triphenylphosphonium Cations of the Diterpenoid Isosteviol: Synthesis and Antimitotic Activity in a Sea Urchin Embryo Model.

    PubMed

    Strobykina, Irina Yu; Belenok, Mayya G; Semenova, Marina N; Semenov, Victor V; Babaev, Vasiliy M; Rizvanov, Ildar Kh; Mironov, Vladimir F; Kataev, Vladimir E

    2015-06-26

    A series of novel triphenylphosphonium (TPP) cations of the diterpenoid isosteviol (1, 16-oxo-ent-beyeran-19-oic acid) have been synthesized and evaluated in an in vivo phenotypic sea urchin embryo assay for antimitotic activity. The TPP moiety was applied as a carrier to provide selective accumulation of a connected compound into mitochondria. When applied to fertilized eggs, the targeted isosteviol TPP conjugates induced mitotic arrest with the formation of aberrant multipolar mitotic spindles, whereas both isosteviol and the methyltriphenylphosphonium cation were inactive. The structure-activity relationship study revealed the essential role of the TPP group for the realization of the isosteviol effect, while the chemical structure and the length of the linker only slightly influenced the antimitotic potency. The results obtained using the sea urchin embryo model suggested that TPP conjugates of isosteviol induced mitotic spindle defects and mitotic arrest presumably by affecting mitochondrial DNA. Since targeting mitochondria is considered as an encouraging strategy for cancer therapy, TPP-isosteviol conjugates may represent promising candidates for further design as anticancer agents. PMID:26042548

  1. Nucleologenesis and embryonic genome activation are defective in interspecies cloned embryos between bovine ooplasm and rhesus monkey somatic cells

    PubMed Central

    Song, Bong-Seok; Lee, Sang-Hee; Kim, Sun-Uk; Kim, Ji-Su; Park, Jung Sun; Kim, Cheol-Hee; Chang, Kyu-Tae; Han, Yong-Mahn; Lee, Kyung-Kwang; Lee, Dong-Seok; Koo, Deog-Bon

    2009-01-01

    Background Interspecies somatic cell nuclear transfer (iSCNT) has been proposed as a tool to address basic developmental questions and to improve the feasibility of cell therapy. However, the low efficiency of iSCNT embryonic development is a crucial problem when compared to in vitro fertilization (IVF) and intraspecies SCNT. Thus, we examined the effect of donor cell species on the early development of SCNT embryos after reconstruction with bovine ooplasm. Results No apparent difference in cleavage rate was found among IVF, monkey-bovine (MB)-iSCNT, and bovine-bovine (BB)-SCNT embryos. However, MB-iSCNT embryos failed to develop beyond the 8- or 16-cell stages and lacked expression of the genes involved in embryonic genome activation (EGA) at the 8-cell stage. From ultrastructural observations made during the peri-EGA period using transmission electron microscopy (TEM), we found that the nucleoli of MB-iSCNT embryos were morphologically abnormal or arrested at the primary stage of nucleologenesis. Consistent with the TEM analysis, nucleolar component proteins, such as upstream binding transcription factor, fibrillarin, nucleolin, and nucleophosmin, showed decreased expression and were structurally disorganized in MB-iSCNT embryos compared to IVF and BB-SCNT embryos, as revealed by real-time PCR and immunofluorescence confocal laser scanning microscopy, respectively. Conclusion The down-regulation of housekeeping and imprinting genes, abnormal nucleolar morphology, and aberrant patterns of nucleolar proteins during EGA resulted in developmental failure in MB-iSCNT embryos. These results provide insight into the unresolved problems of early embryonic development in iSCNT embryos. PMID:19635167

  2. Development of goat embryos after in vitro fertilization and parthenogenetic activation by different methods.

    PubMed

    Ongeri, E M; Bormann, C L; Butler, R E; Melican, D; Gavin, W G; Echelard, Y; Krisher, R L; Behboodi, E

    2001-06-01

    Effective activation protocols that can be used during nuclear transfer investigations in goats need to be developed. We compared the development of IVF goat embryos with those of nonfertilized parthogenetically developing oocytes activated by treatment with either ionomycin or ethanol, both followed by immediate exposure to 6-diethylaminopurine (6-DMAP). Cumulus oocyte complexes (COCs) recovered from abattoir goat ovaries were either matured in a conventional laboratory incubator or placed in pre-equilibrated maturation medium and shipped overnight in a battery-operated dry incubator to another laboratory. Mature COCs were allocated randomly to one of three treatment groups. Group 1 oocytes (n=169 shipped, n=253 not shipped) were fertilized in vitro at 24 h postmaturation (hpm). The remaining COCs were activated at 28 hpm in either ionomycin (Group 2: n=362 shipped, n=202 not shipped), or ethanol (Group 3: n=263 shipped, n=249 not shipped). Activated oocytes were immediately incubated in 6-DMAP for 4 h. Blastocyst development was evaluated on Day 8 post-insemination/activation. Percent cleavage was comparable in shipped and nonshipped oocytes and in all treatment groups. In both shipped and nonshipped oocytes, parthenotes developing from ionomycin- and ethanol-activated oocytes had significantly greater blastocyst development (P<0.01) compared to IVF embryos (28.5 +/- 3.0, 27.4 +/- 2.8, 10.3 +/- 3.0, respectively for the nonshipped oocytes and 9.9 +/- 2.1, 10.3 +/- 2.4, 3.7 +/- 4.7 respectively for the shipped oocytes). Shipped oocytes had lower blastocyst development compared to nonshipped oocytes in the three treatment groups. The mean blastocyst cell number was not statistically different between shipped and nonshipped oocytes or among treatment groups, suggesting that all were equally viable. PMID:11414497

  3. Porcine parvovirus infection induces apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated pathway

    SciTech Connect

    Zhang, Hongling; Huang, Yong; Du, Qian; Luo, Xiaomao; Zhang, Liang; Zhao, Xiaomin; Tong, Dewen

    2015-01-09

    Highlights: • PPV reduces PK-15 cells viability by inducing apoptosis. • PPV infection induces apoptosis through mitochondria-mediated pathway. • PPV infection activates p53 to regulate the mitochondria apoptotic signaling. - Abstract: Porcine parvovirus (PPV) infection has been reported to induce the cytopathic effects (CPE) in some special host cells and contribute the occurrence of porcine parvovirus disease, but the molecular mechanisms underlying PPV-induced CPE are not clear. In this study, we investigated the morphological and molecular changes of porcine kidney cell line (PK-15 cells) infected with PPV. The results showed that PPV infection inhibited the viability of PK-15 cells in a time and concentration dependent manner. PPV infection induced typical apoptotic features including chromatin condensation, apoptotic body formation, nuclear fragmentation, and Annexin V-binding activity. Further studies showed that Bax was increased and translocated to mitochondria, whereas Bcl-2 was decreased in PPV-infected cells, which caused mitochondrial outer-membrane permeabilization, resulting in the release of mitochondrial cytochrome c, followed by caspase-9 and caspase-3 activation. However, the expression of Fas and Fas ligand (FasL) did not appear significant changes in the process of PPV-induced apoptosis. Moreover, PPV infection activated p53 signaling, which was involved in the activation of apoptotic signaling induced by PPV infection via regulation of Bax and Bcl-2. Taken together, our results demonstrated that PPV infection induced apoptosis in PK-15 cells through activation of p53 and mitochondria-mediated apoptosis pathway. This study may contribute to shed light on the molecular pathogenesis of PPV infection.

  4. Transcriptional Regulation During Zygotic Genome Activation in Zebrafish and Other Anamniote Embryos.

    PubMed

    Wragg, J; Müller, F

    2016-01-01

    Embryo development commences with the fusion of two terminally differentiated haploid gametes into the totipotent fertilized egg, which through a series of major cellular and molecular transitions generate a pluripotent cell mass. The activation of the zygotic genome occurs during the so-called maternal to zygotic transition and prepares the embryo for zygotic takeover from maternal factors, in the control of the development of cellular lineages during differentiation. Recent advances in next generation sequencing technologies have allowed the dissection of the genomic and epigenomic processes mediating this transition. These processes include reorganization of the chromatin structure to a transcriptionally permissive state, changes in composition and function of structural and regulatory DNA-binding proteins, and changeover of the transcriptome as it is overhauled from that deposited by the mother in the oocyte to a zygotically transcribed complement. Zygotic genome activation in zebrafish occurs 10 cell cycles after fertilization and provides an ideal experimental platform for elucidating the temporal sequence and dynamics of establishment of a transcriptionally active chromatin state and helps in identifying the determinants of transcription activation at polymerase II transcribed gene promoters. The relatively large number of pluripotent cells generated by the fast cell divisions before zygotic transcription provides sufficient biomass for next generation sequencing technology approaches to establish the temporal dynamics of events and suggest causative relationship between them. However, genomic and genetic technologies need to be improved further to capture the earliest events in development, where cell number is a limiting factor. These technologies need to be complemented with precise, inducible genetic interference studies using the latest genome editing tools to reveal the function of candidate determinants and to confirm the predictions made by classic

  5. Fine mapping of genome activation in bovine embryos by RNA sequencing

    PubMed Central

    Graf, Alexander; Krebs, Stefan; Zakhartchenko, Valeri; Schwalb, Björn; Blum, Helmut; Wolf, Eckhard

    2014-01-01

    During maternal-to-embryonic transition control of embryonic development gradually switches from maternal RNAs and proteins stored in the oocyte to gene products generated after embryonic genome activation (EGA). Detailed insight into the onset of embryonic transcription is obscured by the presence of maternal transcripts. Using the bovine model system, we established by RNA sequencing a comprehensive catalogue of transcripts in germinal vesicle and metaphase II oocytes, and in embryos at the four-cell, eight-cell, 16-cell, and blastocyst stages. These were produced by in vitro fertilization of Bos taurus taurus oocytes with sperm from a Bos taurus indicus bull to facilitate parent-specific transcriptome analysis. Transcripts from 12.4 to 13.7 × 103 different genes were detected in the various developmental stages. EGA was analyzed by (i) detection of embryonic transcripts, which are not present in oocytes; (ii) detection of transcripts from the paternal allele; and (iii) detection of primary transcripts with intronic sequences. These strategies revealed (i) 220, (ii) 937, and (iii) 6,848 genes to be activated from the four-cell to the blastocyst stage. The largest proportion of gene activation [i.e., (i) 59%, (ii) 42%, and (iii) 58%] was found in eight-cell embryos, indicating major EGA at this stage. Gene ontology analysis of genes activated at the four-cell stage identified categories related to RNA processing, translation, and transport, consistent with preparation for major EGA. Our study provides the largest transcriptome data set of bovine oocyte maturation and early embryonic development and detailed insight into the timing of embryonic activation of specific genes. PMID:24591639

  6. Regulation of thyroid peroxidase activity by thyrotropin, epidermal growth factor and phorbol ester in porcine thyroid follicles cultured in suspension

    SciTech Connect

    Kasai, Kikuo; Hiraiwa, Masaki; Emoto, Tatsushi; Hattori, Yoshiyuki; Shimoda, Shin-Ichi ); Ohmori, Takeshi; Koizumi, Narumi; Hosoya, Toichiro )

    1989-01-01

    The activity of thyroid peroxidase (TPO) in porcine follicles cultured for 96 h in suspension with five hormones (5H) still attained over 50% of that in the freshly isolated follicles. On the other hand, the activity in those cultured with 5H + TSH (6H) was several times higher than that cultured with 5H after 96 h, although an initial decrease of TPO activity during the first 24 h of culture was observed in both conditions. The ability of follicles to metabolize iodide when cultured with 6H for 96 h was also several times higher than that of those cultured with 5H. The half-maximal dose of TSH for stimulation of TPO activity and iodide metabolism was 0.03 - 0.04 mU/ml and the effect was mediated by cAMP. These results indicate that in porcine thyroid follicles in primary suspension culture, TPO activity as well as the ability of iodide metabolism is induced by chronic TSH stimulation. In addition, epidermal growth factor and phorbol 12-myristate 13-acetate completely inhibited TSH stimulation on both activities and also basal (5H) activity of iodide metabolism.

  7. Relationships between environmental organochlorine contaminant residues, plasma corticosterone concentrations, and intermediary metabolic enzyme activities in Great Lakes herring gull embryos.

    PubMed Central

    Lorenzen, A; Moon, T W; Kennedy, S W; Glen, G A

    1999-01-01

    Experiments were conducted to survey and detect differences in plasma corticosterone concentrations and intermediary metabolic enzyme activities in herring gull (Larus argentatus) embryos environmentally exposed to organochlorine contaminants in ovo. Unincubated fertile herring gull eggs were collected from an Atlantic coast control site and various Great Lakes sites in 1997 and artificially incubated in the laboratory. Liver and/or kidney tissues from approximately half of the late-stage embryos were analyzed for the activities of various intermediary metabolic enzymes known to be regulated, at least in part, by corticosteroids. Basal plasma corticosterone concentrations were determined for the remaining embryos. Yolk sacs were collected from each embryo and a subset was analyzed for organochlorine contaminants. Regression analysis of individual yolk sac organochlorine residue concentrations, or 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs), with individual basal plasma corticosterone concentrations indicated statistically significant inverse relationships for polychlorinated dibenzo-p-dioxins/polychlorinated dibenzofurans (PCDDs/PCDFs), total polychlorinated biphenyls (PCBs), non-ortho PCBs, and TEQs. Similarly, inverse relationships were observed for the activities of two intermediary metabolic enzymes (phosphoenolpyruvate carboxykinase and malic enzyme) when regressed against PCDDs/PCDFs. Overall, these data suggest that current levels of organochlorine contamination may be affecting the hypothalamo-pituitary-adrenal axis and associated intermediary metabolic pathways in environmentally exposed herring gull embryos in the Great Lakes. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:10064546

  8. Oil refinery effluents: evidence of cocarcinogenic activity in the trout embryo microinjection assay

    SciTech Connect

    Metcalfe, C.D.; Sonstegard, R.A.

    1985-12-01

    Extracts prepared from oil refinery effluents (soxhlet and XAD-2) were tested for carcinogenic potential by means of the rainbow trout (Salmo gairdneri) embryo-injection bioassay. No neoplasms were detected in fish given injections of refinery extracts alone (with and without exogenous rat S-9 activation). Refinery extracts coinjected with aflatoxin B1 induced elevated frequencies of hepatic neoplasms. This cocarcinogenic effect was most pronounced when aflatoxin B1 was preincubated with rat S-9 prior to injection. Effluent extracts coinjected with a direct-acting carcinogen (N-methyl-N'-nitro-N-nitrosoguanidine (CAS: 56-57-5)) did not increase the incidence of hepatic neoplasms (with and without exogenous S-9 activation).

  9. GAGA mediates the enhancer blocking activity of the eve promoter in the Drosophila embryo

    PubMed Central

    Ohtsuki, Sumio; Levine, Michael

    1998-01-01

    Insulator DNAs and promoter competition regulate enhancer–promoter interactions within complex genetic loci. A transgenic embryo assay was used to obtain evidence that the Drosophila eve promoter possesses an insulator activity that can be uncoupled from the core elements that mediate competition. The eve promoter contains an optimal TATA element and a GAGA sequence. The analysis of various chimeric promoters provides evidence that TATA is essential for promoter competition, whereas GAGA mediates enhancer blocking. The Trithorax-like (Trl) protein interacts with GAGA, and mutations in trl attenuate eve promoter insulator activity. We suggest that Trl–GAGA increases the stability of enhancer–promoter interactions by creating an open chromatin configuration at the core promoter. PMID:9808619

  10. Porcine reproductive and respiratory syndrome virus nonstructural protein 1beta modulates host innate immune response by antagonizing IRF3 activation.

    PubMed

    Beura, Lalit K; Sarkar, Saumendra N; Kwon, Byungjoon; Subramaniam, Sakthivel; Jones, Clinton; Pattnaik, Asit K; Osorio, Fernando A

    2010-02-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine leads to a serious disease characterized by a delayed and defective adaptive immune response. It is hypothesized that a suboptimal innate immune response is responsible for the disease pathogenesis. In the study presented here we tested this hypothesis and identified several nonstructural proteins (NSPs) with innate immune evasion properties encoded by the PRRS viral genome. Four of the total ten PRRSV NSPs tested were found to have strong to moderate inhibitory effects on beta interferon (IFN-beta) promoter activation. The strongest inhibitory effect was exhibited by NSP1 followed by, NSP2, NSP11, and NSP4. We focused on NSP1alpha and NSP1beta (self-cleavage products of NSP1 during virus infection) and NSP11, three NSPs with strong inhibitory activity. All of three proteins, when expressed stably in cell lines, strongly inhibited double-stranded RNA (dsRNA) signaling pathways. NSP1beta was found to inhibit both IFN regulatory factor 3 (IRF3)- and NF-kappaB-dependent gene induction by dsRNA and Sendai virus. Mechanistically, the dsRNA-induced phosphorylation and nuclear translocation of IRF3 were strongly inhibited by NSP1beta. Moreover, when tested in a porcine myelomonocytic cell line, NSP1beta inhibited Sendai virus-mediated activation of porcine IFN-beta promoter activity. We propose that this NSP1beta-mediated subversion of the host innate immune response plays an important role in PRRSV pathogenesis. PMID:19923190

  11. Fluctuation of the CaS -sequestering activity of permeabilized sea urchin embryos during the cell cycle

    SciTech Connect

    Suprynowicz, F.A.; Mazia, D.

    1985-04-01

    The authors have followed the sequestration of CaS by intracellular compartments in sea urchin embryos through the first cell cycles. To gain biochemical access to these compartments, the embryos were permeabilized by brief exposure to an intense electric field. Sequestration was determined as the retention of tracer, UVCa, after filtration of aliquots on Millipore filters. The permeabilized cells sequester CaS at a constant rate for at least 20 min. The CaS -sequestering activities of embryos that are permeabilized at successive stages of the first cell cycle (one-cell stage) progressively increase to 5 times the initial level. The rate of sequestration is maximal during telophase and, in some populations of zygotes, is nearly as great throughout prophase. Over the course of the second cell cycle (two-cell stage), the activity undergoes a 2-fold oscillation that bears the same temporal relationship to mitosis as the previous fluctuation.

  12. Rosiglitazone stimulates peroxisome proliferator-activated receptor gamma expression and directly affects in vitro steroidogenesis in porcine ovarian follicles.

    PubMed

    Rak-Mardyła, Agnieszka; Karpeta, Anna

    2014-07-01

    Rosiglitazone is a peroxisome proliferator-activated receptor gamma (PPARγ) synthetic activator from the group of thiazolidinediones often used in the treatment of chronic diseases such as type 2 diabetes and other forms of insulin resistance. The present in vitro study assessed the direct effects of rosiglitazone at 25 and 50 μM doses on PPARγ gene expression, steroid secretion (progesterone [P4], androstenedione [A4], testosterone [T], and estradiol), and protein expression of PPARγ, 3βHSD, CYP17, 17βHSD, CYP19 by porcine ovarian follicles from prepubertal and cycling animals. We analyzed also steroid enzymatic activity by conversion of pregnen-3β-ol-20-one to P4, P4 to A4, and A4 to T. Our results indicated that rosiglitazone increased significantly PPARγ expression, P4 secretion, 3βHSD activity, and protein expression. Rosiglitazone decreased A4 and T secretion by reducing the expression and activity of CYP17 and 17βHSD and did not change estradiol secretion and CYP19. Similarly results was observed both in prepubertal and cycling pigs. Our results indicate that these direct effects of rosiglitazone on ovarian steroidogenesis provide a framework for testing several potential new mechanisms of PPAR-γ actions on porcine ovarian function. PMID:24681211

  13. Karyotype Characterization of In Vivo- and In Vitro-Derived Porcine Parthenogenetic Cell Lines

    PubMed Central

    Hou, Dongxia; Han, Xuejie; Jin, Yong; Zhao, Lihua; Nie, Xiaowei; Zhou, Xin; Yun, Ting; Zhao, Yuhang; Huang, Xianghua; Hou, Daorong; Yang, Ning; Wu, Zhaoqiang; Li, Xueling; Li, Rongfeng

    2014-01-01

    Mammalian haploid cell lines provide useful tools for both genetic studies and transgenic animal production. To derive porcine haploid cells, three sets of experiments were conducted. First, genomes of blastomeres from 8-cell to 16-cell porcine parthenogenetically activated (PA) embryos were examined by chromosome spread analysis. An intact haploid genome was maintained by 48.15% of blastomeres. Based on this result, two major approaches for amplifying the haploid cell population were tested. First, embryonic stem-like (ES-like) cells were cultured from PA blastocyst stage embryos, and second, fetal fibroblasts from implanted day 30 PA fetuses were cultured. A total of six ES-like cell lines were derived from PA blastocysts. No chromosome spread with exactly 19 chromosomes (the normal haploid complement) was found. Four cell lines showed a tendency to develop to polyploidy (more than 38 chromosomes). The karyotypes of the fetal fibroblasts showed different abnormalities. Cells with 19–38 chromosomes were the predominant karyotype (59.48–60.91%). The diploid cells were the second most observed karyotype (16.17%–22.73%). Although a low percentage (3.45–8.33%) of cells with 19 chromosomes were detected in 18.52% of the fetus-derived cell lines, these cells were not authentic haploid cells since they exhibited random losses or gains of some chromosomes. The haploid fibroblasts were not efficiently enriched via flow cytometry sorting. On the contrary, the diploid cells were efficiently enriched. The enriched parthenogenetic diploid cells showed normal karyotypes and expressed paternally imprinted genes at extremely low levels. We concluded that only a limited number of authentic haploid cells could be obtained from porcine cleavage-stage parthenogenetic embryos. Unlike mouse, the karyotype of porcine PA embryo-derived haploid cells is not stable, long-term culture of parthenogenetic embryos, either in vivo or in vitro, resulted in abnormal karyotypes. The

  14. Karyotype characterization of in vivo- and in vitro-derived porcine parthenogenetic cell lines.

    PubMed

    Liu, Qiang; Zhang, Manling; Hou, Dongxia; Han, Xuejie; Jin, Yong; Zhao, Lihua; Nie, Xiaowei; Zhou, Xin; Yun, Ting; Zhao, Yuhang; Huang, Xianghua; Hou, Daorong; Yang, Ning; Wu, Zhaoqiang; Li, Xueling; Li, Rongfeng

    2014-01-01

    Mammalian haploid cell lines provide useful tools for both genetic studies and transgenic animal production. To derive porcine haploid cells, three sets of experiments were conducted. First, genomes of blastomeres from 8-cell to 16-cell porcine parthenogenetically activated (PA) embryos were examined by chromosome spread analysis. An intact haploid genome was maintained by 48.15% of blastomeres. Based on this result, two major approaches for amplifying the haploid cell population were tested. First, embryonic stem-like (ES-like) cells were cultured from PA blastocyst stage embryos, and second, fetal fibroblasts from implanted day 30 PA fetuses were cultured. A total of six ES-like cell lines were derived from PA blastocysts. No chromosome spread with exactly 19 chromosomes (the normal haploid complement) was found. Four cell lines showed a tendency to develop to polyploidy (more than 38 chromosomes). The karyotypes of the fetal fibroblasts showed different abnormalities. Cells with 19-38 chromosomes were the predominant karyotype (59.48-60.91%). The diploid cells were the second most observed karyotype (16.17%-22.73%). Although a low percentage (3.45-8.33%) of cells with 19 chromosomes were detected in 18.52% of the fetus-derived cell lines, these cells were not authentic haploid cells since they exhibited random losses or gains of some chromosomes. The haploid fibroblasts were not efficiently enriched via flow cytometry sorting. On the contrary, the diploid cells were efficiently enriched. The enriched parthenogenetic diploid cells showed normal karyotypes and expressed paternally imprinted genes at extremely low levels. We concluded that only a limited number of authentic haploid cells could be obtained from porcine cleavage-stage parthenogenetic embryos. Unlike mouse, the karyotype of porcine PA embryo-derived haploid cells is not stable, long-term culture of parthenogenetic embryos, either in vivo or in vitro, resulted in abnormal karyotypes. The porcine PA

  15. Coordinated activation of the secretory pathway during notochord formation in the Xenopus embryo.

    PubMed

    Tanegashima, Kosuke; Zhao, Hui; Rebbert, Martha L; Dawid, Igor B

    2009-11-01

    We compared the transcriptome in the developing notochord of Xenopus laevis embryos with that of other embryonic regions. A coordinated and intense activation of a large set of secretory pathway genes was observed in the notochord, but not in notochord precursors in the axial mesoderm at early gastrula stage. The genes encoding Xbp1 and Creb3l2 were also activated in the notochord. These two transcription factors are implicated in the activation of secretory pathway genes during the unfolded protein response, where cells react to the stress of a build-up of unfolded proteins in their endoplasmic reticulum. Xbp1 and Creb3l2 are differentially expressed but not differentially activated in the notochord. Reduction of expression of Xbp1 or Creb3l2 by injection of antisense morpholinos led to strong deficits in notochord but not somitic muscle development. In addition, the expression of some, but not all, genes encoding secretory proteins was inhibited by injection of xbp1 morpholinos. Furthermore, expression of activated forms of Xbp1 or Creb3l2 in animal explants could activate a similar subset of secretory pathway genes. We conclude that coordinated activation of a battery of secretory pathway genes mediated by Xbp1 and Creb/ATF factors is a characteristic and necessary feature of notochord formation. PMID:19793890

  16. Coordinated activation of the secretory pathway during notochord formation in the Xenopus embryo

    PubMed Central

    Tanegashima, Kosuke; Zhao, Hui; Rebbert, Martha L.; Dawid, Igor B.

    2009-01-01

    Summary We compared the transcriptome in the developing notochord of Xenopus laevis embryos with that of other embryonic regions. A coordinated and intense activation of a large set of secretory pathway genes was observed in the notochord, but not in notochord precursors in the axial mesoderm at early gastrula stage. The genes encoding Xbp1 and Creb3l2 were also activated in the notochord. These two transcription factors are implicated in the activation of secretory pathway genes during the unfolded protein response, where cells react to the stress of a build-up of unfolded proteins in their endoplasmic reticulum. Xbp1 and Creb3l2 are differentially expressed but not differentially activated in the notochord. Reduction of expression of Xbp1 or Creb3l2 by injection of antisense morpholinos led to strong deficits in notochord but not somitic muscle development. In addition, the expression of some, but not all, genes encoding secretory proteins was inhibited by injection of xbp1 morpholinos. Furthermore, expression of activated forms of Xbp1 or Creb3l2 in animal explants could activate a similar subset of secretory pathway genes. We conclude that coordinated activation of a battery of secretory pathway genes mediated by Xbp1 and Creb/ATF factors is a characteristic and necessary feature of notochord formation. PMID:19793890

  17. MAT2B promotes adipogenesis by modulating SAMe levels and activating AKT/ERK pathway during porcine intramuscular preadipocyte differentiation.

    PubMed

    Zhao, Cunzhen; Chen, Xiaochang; Wu, Wenjing; Wang, Wusu; Pang, Weijun; Yang, Gongshe

    2016-05-15

    Intramuscular fat (IMF) has been demonstrated as one of the crucial factors of livestock meat quality. The MAT2B protein with MAT2α catalyzes the formation of methyl donor S- adenosylmethionine (SAMe) to mediate cell metabolism including proliferation and apoptosis. However, the regulatory effect of MAT2B on IMF deposition is still unclear. In this study, the effect of MAT2B on adipogenesis and its potential mechanism during porcine intramuscular preadipocyte differentiation was studied. The results showed that overexpression of MAT2B promoted adipogenesis and significantly up-regulated the mRNA and protein levels of adipogenic marker genes including FASN, PPARγ and aP2, consistently, knockdown of MAT2B inhibited lipid accumulation and down-regulated the mRNA and protein levels of the above genes. Furthermore, flow cytometry and EdU-labeling assay indicated that MAT2B regulate adipogenesis was partly due to influence intracellular SAMe levels and further affect cell clonal expansion. Also, increased expression of MAT2B activated the phosphorylations of AKT and ERK1/2, whereas knockdown of MAT2B blocked AKT signaling and repressed the phosphorylation of ERK1/2. Moreover, the inhibitory effect of LY294002 (a specific PI3K inhibitor) on the activities of AKT and ERK1/2 was partially recovered by overexpression of MAT2B in porcine intramuscular adipocytes. Finally, Co-IP experiments showed that MAT2B can directly interact with AKT. Taken together, our findings suggested that MAT2B acted as a positive regulator through modifying SAMe levels as well as activating AKT/ERK signaling pathway to promote porcine intramuscular adipocyte differentiation. PMID:26940012

  18. ORF2 protein of porcine circovirus type 2 promotes phagocytic activity of porcine macrophages by inhibiting proteasomal degradation of complement component 1, q subcomponent binding protein (C1QBP) through physical interaction.

    PubMed

    Choi, Chang-Yong; Oh, Hae-Na; Lee, Suk Jun; Chun, Taehoon

    2015-11-01

    Defining how each ORF of porcine circovirus type 2 (PCV2) manipulates the host immune system may be helpful to understand the disease progression of post-weaning multisystemic wasting syndrome. In this study, we demonstrated a direct interaction between the PCV2 ORF2 and complement component 1, q subcomponent binding protein (C1QBP) within the cytoplasm of host macrophages. The physical interaction between PCV2 ORF2 and C1QBP inhibited ubiquitin-mediated proteasomal degradation of C1QBP in macrophages. Increased stability of C1QBP by the interaction with PCV2 ORF2 further enhanced the phagocytic activity of porcine macrophages through the phosphoinositol 3-kinase signalling pathway. This may explain the molecular basis of how PCV2 ORF2 enhances the phagocytic activity of host macrophages. PMID:26361775

  19. The anti-porcine parvovirus activity of nanometer propolis flavone and propolis flavone in vitro and in vivo.

    PubMed

    Ma, Xia; Guo, Zhenhuan; Shen, Zhiqiang; Liu, Yonglu; Wang, Jinliang; Fan, Yunpeng

    2015-01-01

    Objectives. The present study was conducted to evaluate the activity of nanometer propolis flavone (NPF) on inhibiting porcine parvovirus (PPV) in vitro and in vivo. Methods. In vitro, the effect of NPF on cellular infectivity of PPV was carried out before and after adding drug and simultaneous adding and PPV after being mixed. In vivo, the anti-PPV effect of NPF in guinea pigs was performed. Results. The results showed that NPF could significantly inhibit PPV infecting porcine kidney- (PK-) 15 cells compared with propolis flavone (PF), and the activity of NPF was the best in preadding drug pattern. NPF at high and medium doses was able to observably restrain PPV copying in lung, gonad, blood, and spleen, decrease the impact of PPV on weight of guinea pigs, and improve hemagglutination inhibition (HI) of PPV in serum. In addition, it could also increase the contents of IL-2 and IL-6 in serum after PPV challenge. Conclusion. These results indicated that NPF could significantly improve the anti-PPV activity of PF, and its high concentration possessed the best efficacy. Therefore, NPF would be expected to be exploited into a new-style antiviral drug. PMID:25815034

  20. The Anti-Porcine Parvovirus Activity of Nanometer Propolis Flavone and Propolis Flavone In Vitro and In Vivo

    PubMed Central

    Ma, Xia; Guo, Zhenhuan; Shen, Zhiqiang; Liu, Yonglu; Wang, Jinliang; Fan, Yunpeng

    2015-01-01

    Objectives. The present study was conducted to evaluate the activity of nanometer propolis flavone (NPF) on inhibiting porcine parvovirus (PPV) in vitro and in vivo. Methods. In vitro, the effect of NPF on cellular infectivity of PPV was carried out before and after adding drug and simultaneous adding and PPV after being mixed. In vivo, the anti-PPV effect of NPF in guinea pigs was performed. Results. The results showed that NPF could significantly inhibit PPV infecting porcine kidney- (PK-) 15 cells compared with propolis flavone (PF), and the activity of NPF was the best in preadding drug pattern. NPF at high and medium doses was able to observably restrain PPV copying in lung, gonad, blood, and spleen, decrease the impact of PPV on weight of guinea pigs, and improve hemagglutination inhibition (HI) of PPV in serum. In addition, it could also increase the contents of IL-2 and IL-6 in serum after PPV challenge. Conclusion. These results indicated that NPF could significantly improve the anti-PPV activity of PF, and its high concentration possessed the best efficacy. Therefore, NPF would be expected to be exploited into a new-style antiviral drug. PMID:25815034

  1. COMBINED USE OF A WATER-INSOLUBLE CHEMICAL DELIVERY SYSTEM AND A METABOLIC ACTIVATION SYSTEM IN WHOLE EMBRYO CULTURE

    EPA Science Inventory

    An integrated water insoluble chemical delivery/metabolic activation/rat embryo culture system is described. In initial studies corn oil was used as the solvent and diallate as the substrate. Increasing concentrations of diallate dissolved in corn oil caused embryonic growth reta...

  2. bFGF signaling-mediated reprogramming of porcine primordial germ cells.

    PubMed

    Zhang, Yu; Ma, Jing; Li, Hai; Lv, Jiawei; Wei, Renyue; Cong, Yimei; Liu, Zhonghua

    2016-05-01

    Primordial germ cells (PGCs) have the ability to be reprogrammed into embryonic germ cells (EGCs) in vitro and are an alternative source of embryonic stem cells. Other than for the mouse, the systematic characterization of mammalian PGCs is still lacking, especially the process by which PGCs convert to pluripotency. This hampers the understanding of germ cell development and the derivation of authenticated EGCs from other species. We observed the morphological development of the genital ridge from Bama miniature pigs and found primary sexual differentiation in the E28 porcine embryo, coinciding with Blimp1 nuclear exclusion in PGCs. To explore molecular events involved in porcine PGC reprogramming, transcriptome data of porcine EGCs and fetal fibroblasts (FFs) were assembled and 1169 differentially expressed genes were used for Gene Ontology analysis. These genes were significantly enriched in cell-surface receptor-linked signal transduction, in agreement with the activation of LIF/Stat3 signaling and FGF signaling during the derivation of porcine EG-like cells. Using a growth-factor-defined culture system, we explored the effects of bFGF on the process and found that bFGF not only functioned at the very beginning of PGC dedifferentiation by impeding Blimp1 nuclear expression via a PI3K/AKT-dependent pathway but also maintained the viability of cultured PGCs thereafter. These results provide further insights into the development of germ cells from livestock and the mechanism of porcine PGC reprogramming. PMID:26613602

  3. Context-dependent transcriptional interpretation of mitogen activated protein kinase signaling in the Drosophila embryo

    NASA Astrophysics Data System (ADS)

    Kim, Yoosik; Iagovitina, Antonina; Ishihara, Keisuke; Fitzgerald, Kate M.; Deplancke, Bart; Papatsenko, Dmitri; Shvartsman, Stanislav Y.

    2013-06-01

    Terminal regions of the Drosophila embryo are patterned by the localized activation of Mitogen Activated Protein Kinase (MAPK), which induces zygotic genes through relief of their repression by transcriptional repressor Capicua. The levels of MAPK activation at the anterior and posterior termini are close to each other, but the expression patterns of MAPK-target genes, such as zerknüllt (zen) and tailless (tll), display strong anterior-posterior (AP) asymmetry. This region-specific response to MAPK activation provides a clear example of context-dependent interpretation of inductive signaling, a common developmental effect that remains poorly understood. In the past, the AP asymmetry of zen expression was attributed to a mechanism that depends on MAPK substrate competition. We present data suggesting that the asymmetric expression of tll is generated by a different mechanism, based on feedforward control and multiple enhancers of the tll gene. A simple mathematical model of this mechanism correctly predicts how the wild-type expression pattern of tll changes in mutants affecting the anterior, dorsoventral, and terminal patterning systems and some of their direct targets.

  4. Mechanisms Underlying Adaptation of Respiratory Network Activity to Modulatory Stimuli in the Mouse Embryo

    PubMed Central

    Chevalier, Marc; De Sa, Rafaël; Cardoit, Laura; Thoby-Brisson, Muriel

    2016-01-01

    Breathing is a rhythmic behavior that requires organized contractions of respiratory effector muscles. This behavior must adapt to constantly changing conditions in order to ensure homeostasis, proper body oxygenation, and CO2/pH regulation. Respiratory rhythmogenesis is controlled by neural networks located in the brainstem. One area considered to be essential for generating the inspiratory phase of the respiratory rhythm is the preBötzinger complex (preBötC). Rhythmogenesis emerges from this network through the interplay between the activation of intrinsic cellular properties (pacemaker properties) and intercellular synaptic connections. Respiratory activity continuously changes under the impact of numerous modulatory substances depending on organismal needs and environmental conditions. The preBötC network has been shown to become active during the last third of gestation. But only little is known regarding the modulation of inspiratory rhythmicity at embryonic stages and even less on a possible role of pacemaker neurons in this functional flexibility during the prenatal period. By combining electrophysiology and calcium imaging performed on embryonic brainstem slice preparations, we provide evidence showing that embryonic inspiratory pacemaker neurons are already intrinsically sensitive to neuromodulation and external conditions (i.e., temperature) affecting respiratory network activity, suggesting a potential role of pacemaker neurons in mediating rhythm adaptation to modulatory stimuli in the embryo. PMID:27239348

  5. Regulation of promoter occupancy during activation of cryptobiotic embryos from the crustacean Artemia franciscana.

    PubMed

    Martinez-Lamparero, Ana; Casero, Marie-Carmen; Ortiz-Caro, Javier; Sastre, Leandro

    2003-05-01

    Artemia franciscana embryos can suspend their development and metabolism at the gastrula stage to enter a state of cryptobiosis, forming cysts. Embryonic development and metabolism can be resumed under favorable environmental conditions to give rise to free-swimming larvae or nauplii. The mechanisms that mediate these processes are not completely known. Here, we report our studies of the mechanisms that regulate transcriptional activation upon exiting cryptobiosis. Regulatory regions of several A. franciscana gene promoters were identified. Functional analyses in mammalian cells allowed the identification of transcriptional activator regions in the Actin302 promoter and in promoter 2 of the sarco/endoplasmic reticulum Ca(2+)-ATPase-encoding gene. These regions were shown to specifically bind protein factors from nuclear extracts of A. franciscana nauplii by means of electrophoretic mobility shift assays. Several protein-binding regions were also detected by DNase I protection analysis in the promoters of the genes encoding the alpha1 subunit of Na(+)/K(+)-ATPase, actin 302 and sarco/endoplasmic reticulum Ca(2+)-ATPase. Specific DNA-binding proteins in nauplius nuclear extracts were detected for all the promoter regions analyzed. These proteins were either not present in cyst nuclear extracts or were present in much smaller concentrations. Three of the five regions analyzed also bound proteins present in cyst nuclear extracts. These data indicate that transcriptional activation upon exiting cryptobiosis in A. franciscana involves the expression/activation of DNA-binding transcription factors that are not present in cyst nuclei PMID:12654895

  6. Effects of Teratogenic Drugs on CYP1A1 Activity in Differentiating Rat Embryo Cells.

    PubMed

    Tayeboon, Gh S; Ostad, S N; Nasri, S; Nili-Ahmadabadi, A; Tavakoli, F; Sabzevari, O

    2015-05-01

    CYP1A1, a P450 isoenzyme, is involved in the phase I xenobiotic metabolism including teratogen drugs. In the present study, the ability of teratogens to elevate the embryonic expression of CYP1A1 was examined. Micromass cell cultures prepared from day 13 rat embryo limb buds (LB). LB cells were cultivated and exposed for 5 days to retinoic acid (RA), hydrocortisone (HC), caffeine (CA) and quinine (QN). CYP1A1 protein expression and activity were measured using immunofluorescence staining and ethoxyresorufin O-deethylation (EROD) assay, respectively. The EROD activity increased significantly following LB cells exposure to RA and HC (p<0.05) but the expression of CYP1A1 protein was reduced by these drugs, whereas the expression of CYP1A1 protein and EROD activity decreased significantly following the addition of CA and QN (p<0.05, p<0.01). Our findings show that studied teratogens have potency to increase CYP1A1 activity. PMID:24668578

  7. Context-dependent transcriptional interpretation of mitogen activated protein kinase signaling in the Drosophila embryo

    PubMed Central

    Kim, Yoosik; Iagovitina, Antonina; Ishihara, Keisuke; Fitzgerald, Kate M.; Deplancke, Bart; Papatsenko, Dmitri; Shvartsman, Stanislav Y.

    2013-01-01

    Terminal regions of the Drosophila embryo are patterned by the localized activation of Mitogen Activated Protein Kinase (MAPK), which induces zygotic genes through relief of their repression by transcriptional repressor Capicua. The levels of MAPK activation at the anterior and posterior termini are close to each other, but the expression patterns of MAPK-target genes, such as zerknüllt (zen) and tailless (tll), display strong anterior-posterior (AP) asymmetry. This region-specific response to MAPK activation provides a clear example of context-dependent interpretation of inductive signaling, a common developmental effect that remains poorly understood. In the past, the AP asymmetry of zen expression was attributed to a mechanism that depends on MAPK substrate competition. We present data suggesting that the asymmetric expression of tll is generated by a different mechanism, based on feedforward control and multiple enhancers of the tll gene. A simple mathematical model of this mechanism correctly predicts how the wild-type expression pattern of tll changes in mutants affecting the anterior, dorsoventral, and terminal patterning systems and some of their direct targets. PMID:23822503

  8. Epimedium koreanum Nakai Water Extract Exhibits Antiviral Activity against Porcine Epidermic Diarrhea Virus In Vitro and In Vivo

    PubMed Central

    Cho, Won-Kyung; Kim, Hyunil; Choi, Yu Jeong; Yim, Nam-Hui; Yang, Hye Jin; Ma, Jin Yeul

    2012-01-01

    Porcine epidemic diarrhea virus (PEDV) causes diarrhea of pigs age-independently and death of young piglets, resulting in economic loss of porcine industry. We have screened 333 natural oriental herbal medicines to search for new antiviral candidates against PEDV. We found that two herbal extracts, KIOM 198 and KIOM 124, contain significant anti-PED viral effect. KIOM 198 and KIOM 124 were identified as Epimedium koreanum Nakai and Lonicera japonica Thunberg, respectively. The further plaque and CPE inhibition assay in vitro showed that KIOM 198 has much stronger antiviral activity than KIOM 124. Additionally, KIOM 198 exhibited a similar extent of antiviral effect against other subtypes of Corona virus such as sm98 and TGE viruses. Cytotoxicity results showed that KIOM 198 is nontoxic on the cells and suggest that it can be delivered safely for therapy. Furthermore, when we orally administered KIOM 198 to piglets and then infected them with PEDV, the piglets did not show any disease symptoms like diarrhea and biopsy results showed clean intestine, whereas control pigs without KIOM 198 treatment exhibited PED-related severe symptoms. These results imply that KIOM 198 contains strong antiviral activity and has a potential to be developed as an antiviral phytomedicine to treat PEDV-related diseases in pigs. PMID:23259003

  9. Calcium signaling in mammalian egg activation and embryo development: Influence of subcellular localization

    PubMed Central

    Miao, Yi-Liang; Williams, Carmen J.

    2012-01-01

    Calcium (Ca2+) signals drive the fundamental events surrounding fertilization and the activation of development in all species examined to date. Initial studies of Ca2+ signaling at fertilization in marine animals were tightly linked to new discoveries of bioluminescent proteins and their use as fluorescent Ca2+ sensors. Since that time, there has been rapid progress in our understanding of the key functions for Ca2+ in many cell types and the impact of cellular localization on Ca2+ signaling pathways. In this review, which focuses on mammalian egg activation, we consider how Ca2+ is regulated and stored at different stages of oocyte development and examine the functions of molecules that serve as both regulators of Ca2+ release and effectors of Ca2+ signals. We then summarize studies exploring how Ca2+ directs downstream effectors mediating both egg activation and later signaling events required for successful preimplantation embryo development. Throughout this review, we focus attention on how localization of Ca2+ signals influences downstream signaling events, and attempt to highlight gaps in our knowledge that are ripe areas for future research. PMID:22888043

  10. Embryo as an active granular fluid: stress-coordinated cellular constriction chains.

    PubMed

    Jason Gao, Guo-Jie; Holcomb, Michael C; Thomas, Jeffrey H; Blawzdziewicz, Jerzy

    2016-10-19

    Mechanical stress plays an intricate role in gene expression in individual cells and sculpting of developing tissues. However, systematic methods of studying how mechanical stress and feedback help to harmonize cellular activities within a tissue have yet to be developed. Motivated by our observation of the cellular constriction chains (CCCs) during the initial phase of ventral furrow formation in the Drosophila melanogaster embryo, we propose an active granular fluid (AGF) model that provides valuable insights into cellular coordination in the apical constriction process. In our model, cells are treated as circular particles connected by a predefined force network, and they undergo a random constriction process in which the particle constriction probability P is a function of the stress exerted on the particle by its neighbors. We find that when P favors tensile stress, constricted particles tend to form chain-like structures. In contrast, constricted particles tend to form compact clusters when P favors compression. A remarkable similarity of constricted-particle chains and CCCs observed in vivo provides indirect evidence that tensile-stress feedback coordinates the apical constriction activity. Our particle-based AGF model will be useful in analyzing mechanical feedback effects in a wide variety of morphogenesis and organogenesis phenomena. PMID:27545101

  11. COMPARATIVE TRANSCRIPTOME ANALYSIS OF IN VIVO AND IN VITRO PORCINE BLASTOCYSTS BY SMALL AMPLIFIED RNA SERIAL ANALYSIS OF GENE EXPRESSION (SAR-SAGE)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Production of embryos in vitro has enormous potential for research and commercial applications. Unfortunately, in vitro production of porcine embryos is extremely inefficient. Despite the characterization of distinct phenotypes, little is known about the molecular mechanisms and altered physiologi...

  12. Porcine E. coli: Virulence-Associated Genes, Resistance Genes and Adhesion and Probiotic Activity Tested by a New Screening Method

    PubMed Central

    Schierack, Peter; Rödiger, Stefan; Kuhl, Christoph; Hiemann, Rico; Roggenbuck, Dirk; Li, Ganwu; Weinreich, Jörg; Berger, Enrico; Nolan, Lisa K.; Nicholson, Bryon; Römer, Antje; Frömmel, Ulrike; Wieler, Lothar H.; Schröder, Christian

    2013-01-01

    We established an automated screening method to characterize adhesion of Escherichia coli to intestinal porcine epithelial cells (IPEC-J2) and their probiotic activity against infection by enteropathogenic E. coli (EPEC). 104 intestinal E. coli isolates from domestic pigs were tested by PCR for the occurrence of virulence-associated genes, genes coding for resistances to antimicrobial agents and metals, and for phylogenetic origin by PCR. Adhesion rates and probiotic activity were examined for correlation with the presence of these genes. Finally, data were compared with those from 93 E. coli isolates from wild boars. Isolates from domestic pigs carried a broad variety of all tested genes and showed great diversity in gene patterns. Adhesions varied with a maximum of 18.3 or 24.2 mean bacteria adherence per epithelial cell after 2 or 6 hours respectively. Most isolates from domestic pigs and wild boars showed low adherence, with no correlation between adhesion/probiotic activity and E. coli genes or gene clusters. The gene sfa/foc, encoding for a subunit of F1C fimbriae did show a positive correlative association with adherence and probiotic activity; however E. coli isolates from wild boars with the sfa/foc gene showed less adhesion and probiotic activity than E. coli with the sfa/foc gene isolated from domestic pigs after 6 hour incubation. In conclusion, screening porcine E. coli for virulence associated genes genes, adhesion to intestinal epithelial cells, and probiotic activity revealed a single important adhesion factor, several probiotic candidates, and showed important differences between E. coli of domestic pigs and wild boars. PMID:23658605

  13. [In vitro evaluation of mucolytic activities of some expectorants using porcine gastric mucin].

    PubMed

    Misawa, M; Imamura, N

    1988-10-01

    The measurement of viscoelasticity of airway secretions (sputum) has been very difficult, because the secretions, mainly consisting of high molecular weight glycoproteins, are heterogeneous and non-Newtonian viscous fluid. In the present study, a new in vitro method was devised for evaluating the effects of mucolytic expectorants, using porcine gastric mucin as a mucous fluid. Twenty percent porcine gastric mucin solution was prepared by dissolving it in tris-HCl buffer solution. The mucolytics tested were incubated with the mucin solution at pH 7.0 and 37 degrees C for 30 min. The viscoelasticity of mucous fluid was determined by the glass plate method and rheometer method. The two cysteine-mucolytics, acetylcysteine (10(-3)-10(-1) M) and ethylcysteine++ (10(-3)-10(-1) M) showed a marked viscoelasticity-lowering effect with either method. On the other hand, another cysteine-mucolytic, carbocysteine had no mucolytic effect at pH 7.0, but showed its effect at pH 6.0. A protease-mucolytic, alpha-chymotrypsin (0.1-10 mg/ml), remarkably lowered the viscoelasticity of mucin fluid with either method. Bromhexine (3 X 10(-4)-3 X 10(-3) M) had no mucolytic effect even at the range of pH 6-8. From the above findings, it is indicated that distinct evaluation of the mucolytic actions of expectorants is feasible using porcine gastric mucin. The glass plate method has many advantages over the rheometer method in terms of required sample volume, measurement time, inexpensive, and so on. PMID:2468589

  14. Toxicity of petroleum crude oils and their effect on xenobiotic metabolizing enzyme activities in the chicken embryo in ovo

    SciTech Connect

    Lee, Y.Z.; O'Brien, P.J.; Payne, J.F.; Rahimtula, A.D.

    1986-01-01

    Microliter quantities of a Prudhoe Bay crude oil (PBCO) applied to the shell of fertile chick eggs during various stages of development induced cytochrome P-450 levels and mixed-function oxidase activities within the liver of the embryo. PBCO (5 ..mu..l) applied on Day 11 of incubation was found to maximally induce within 24 hr embryo hepatic cytochrome P-450 levels (fourfold), naphthalene hydroxylase (sixfold), benzo(a)pyrene 3-hyroxylase (14-fold), and 7-ethoxyresorufin O-deethylase (24-fold). Glutathione S-transferase was not induced. Crude oils are known to be highly toxic to avian embryos, especially during the early stages of development. The LD/sub 50/ of PBCO and Hibernia crude oil applied to the egg shell on Day 8 of incubation was found to be 1.3 and 2.2 ..mu..l, respectively. Mixed-function oxidase-dependent metabolism of crude oil components may be required for toxicity since administration of 20 ..mu..g of disulfiram in dioxane 1 hr prior to application of 1.3 ..mu..l of PBCO reduced embryo mortality from 60 to 20%.

  15. Spontaneous locomotor activity in late-stage chicken embryos is modified by stretch of leg muscles.

    PubMed

    Bradley, Nina S; Ryu, Young U; Yeseta, Marie C

    2014-03-15

    Chicks initiate bilateral alternating steps several days before hatching and adaptively walk within hours of hatching, but emergence of precocious walking skills is not well understood. One of our aims was to determine whether interactions between environment and movement experience prior to hatching are instrumental in establishing precocious motor skills. However, physiological evidence of proprioceptor development in the chick has yet to be established; thus, one goal of this study was to determine when in embryogenesis proprioception circuits can code changes in muscle length. A second goal was to determine whether proprioception circuits can modulate leg muscle activity during repetitive limb movements for stepping (RLMs). We hypothesized that proprioception circuits code changes in muscle length and/or tension, and modulate locomotor circuits producing RLMs in anticipation of adaptive locomotion at hatching. To this end, leg muscle activity and kinematics were recorded in embryos during normal posture and after fitting one ankle with a restraint that supported the limb in an atypical posture. We tested the hypotheses by comparing leg muscle activity during spontaneous RLMs in control posture and ankle extension restraint. The results indicated that proprioceptors detect changes in muscle length and/or muscle tension 3 days before hatching. Ankle extension restraint produced autogenic excitation of the ankle flexor and reciprocal inhibition of the ankle extensor. Restraint also modified knee extensor activity during RLMs 1 day before hatching. We consider the strengths and limitations of these results and propose that proprioception contributes to precocious locomotor development during the final 3 days before hatching. PMID:24265423

  16. Changes in Activities of Enzymes of Nitrogen Metabolism in Seedcoats and Cotyledons during Embryo Development in Pea Seeds.

    PubMed

    Murray, D R

    1980-10-01

    In the seedcoats of developing pea seeds, the maximal activities of asparaginase (EC 3.5.1.1) and aspartate: alpha-ketoglutarate aminotransferase (EC 2.6.1.1) are attained early in development, before the embryo has expanded to fill the embryo sac. These two enzyme activities could account for the early absence of asparagine and aspartate from the fluid secreted by the seedcoats into the embryo sac.CHANGES IN THE ACTIVITIES OF ALANINE: alpha-ketoglutarate aminotransferase (EC 2.6.1.2), glutamate dehydrogenase (EC 1.4.1.3), glutamine synthetase (EC 6.3.1.2), and glutamate synthase (EC 1.4.1.13) have also been measured, in cotyledons as well as seedcoats. On a fresh weight basis, the highest activities of asparaginase and both aminotransferases developed in the seedcoats, whereas the highest activities of the remaining enzymes developed in the cotyledons.The data indicate that the amide groups of imported asparagine and glutamine are metabolized differently, largely by asparaginase and glutamate synthase, respectively. The NH(4) (+) released by the action of asparaginase is evidently reassimilated in cotyledon cells by the joint action of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. The data emphasize the central importance of alpha-ketoglutarate-glutamate cycling in the redistribution of amino groups associated with the net synthesis of amino acids and reserve proteins. PMID:16661521

  17. MyoD Is a Novel Activator of Porcine FIT1 Gene by Interacting with the Canonical E-Box Element during Myogenesis

    PubMed Central

    Yan, Chi; Xia, Xiaoliang; He, Junxian; Ren, Zhuqing; Xu, Dequan; Xiong, Yuanzhu; Zuo, Bo

    2015-01-01

    Fat-induced transcript 1 (FIT1/FITM1) gene is a member of the conserved gene family important for triglyceride-rich lipid droplet accumulation. FIT1 gene displays a similar muscle-specific expression across pigs, mice, and humans. Thus pigs can act as a useful model of many human diseases resulting from misexpression of FIT1 gene. Triglyceride content in skeletal muscle plays a key role in pork meat quality and flavors. An insertion/deletion mutation in porcine FIT1 coding region shows a high correlation with a series of fat traits. To gain better knowledge of the potential role of FIT1 gene in human diseases and the correlations with pork meat quality, our attention is given to the region upstream of the porcine FIT1 coding sequence. We cloned ~1 kb of the 5′-flanking region of porcine FIT1 gene to define the role of this sequence in modulating the myogenic expression. A canonical E-box element that activated porcine FIT1 promoter activity during myogenesis was identified. Further analysis demonstrated that promoter activity was induced by overexpression of MyoD1, which bound to this canonical E-box during C2C12 differentiation. This is the first evidence that FIT1 as the direct novel target of MyoD is involved in muscle development. PMID:26492245

  18. The optimal period of Ca-EDTA treatment for parthenogenetic activation of porcine oocytes during maturation culture

    PubMed Central

    MORITA, Yasuhiro; TANIGUCHI, Masayasu; TANIHARA, Fuminori; ITO, Aya; NAMULA, Zhao; DO, Lanh Thi Kim; TAKAGI, Mitsuhiro; TAKEMOTO, Tatsuya; OTOI, Takeshige

    2016-01-01

    The changes triggered by sperm-induced activation of oocytes, which are required for normal oocyte development, can be mediated by other agents, thereby inducing the parthenogenesis. In this study, we exposed porcine oocytes to 1 mM Ca-EDTA, a metal-ion chelator, at various intervals during 48 hr of in vitro maturation to determine the optimum period of Ca-EDTA treatment for parthenogenetic activation. When the oocytes were cultured with or without Ca-EDTA from 36 hr (post-12), 24 hr (post-24), 12 hr (post-36) and 0 hr (post-48) after the start of maturation culture, the blastocyst formation rates were significantly higher (P<0.05) in the post-24, post-36 and post-48 groups (3.3%, 4.0% and 2.6%, respectively) than those in the control group without treatment (0%). Furthermore, when the oocytes were cultured with Ca-EDTA for 0 hr (control), 12 hr (pre-12), 24 hr (pre-24), 36 hr (pre-36) and 48 hr (pre-48) from the start of maturation culture, the oocytes formed blastocysts only in the pre-36 and pre-48 groups (0.4% or 0.8%, respectively). Pronuclei (<66.7%) were observed only when the periods of Ca-EDTA treatment were more than 12 hr during maturation culture. In the control group, no pronuclei were detected. Our findings demonstrate that porcine immature oocytes can be parthenogenetically activated by Ca-EDTA treatment for at least 24 hr to 36 hr during maturation culture, leading to pronucleus formation followed by the formation of blastocysts. PMID:26947170

  19. Diatom-derived oxylipins induce cell death in sea urchin embryos activating caspase-8 and caspase 3/7.

    PubMed

    Ruocco, Nadia; Varrella, Stefano; Romano, Giovanna; Ianora, Adrianna; Bentley, Matt G; Somma, Domenico; Leonardi, Antonio; Mellone, Stefano; Zuppa, Antonio; Costantini, Maria

    2016-07-01

    Diatoms are an important class of unicellular algae that produce bioactive secondary metabolites with cytotoxic activity collectively termed oxylipins, including polyunsaturated aldehydes (PUAs), hydroxyacids (HEPEs), oxo-acids and epoxyalcohols. Previous results showed that at higher concentrations, the PUA decadienal induced apoptosis on copepods and sea urchin embryos via caspase-3 activation; at lower concentrations decadienal affected the expression levels of the caspase-8 gene in embryos of the sea urchin Paracentrotus lividus. In the present work, we studied the effects of other common oxylipins produced by diatoms: two PUAs (heptadienal and octadienal) and four hydroxyacids (5-, 9- 11- and 15-HEPE) on P. lividus cell death and caspase activities. Our results showed that (i) at higher concentrations PUAs and HEPEs induced apoptosis in sea urchin embryos, detected by microscopic observation and through the activation of caspase-3/7 and caspase-8 measured by luminescent assays; (ii) at low concentrations, PUAs and HEPEs affected the expression levels of caspase-8 and caspase-3/7 (isolated for the first time here in P. lividus) genes, detected by Real Time qPCR. These findings have interesting implications from the ecological point of view, given the importance of diatom blooms in nutrient-rich aquatic environments. PMID:27130972

  20. Porcine circovirus type 2 induces the activation of nuclear factor kappa B by I{kappa}B{alpha} degradation

    SciTech Connect

    Wei Li; Kwang, Jimmy; Wang Jin; Shi Lei; Yang Bing; Li Yongqing; Liu Jue

    2008-08-15

    The transcription factor NF-{kappa}B is commonly activated upon virus infection and a key player in the induction and regulation of the host immune response. The present study demonstrated for the first time that porcine circovirus type 2 (PCV2), which is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome, can activate NF-{kappa}B in PCV2-infected PK15 cells. In PCV2-infected cells, NF-{kappa}B was activated concomitantly with viral replication, which was characterized by increased DNA binding activity, translocation of NF-{kappa}B p65 from the cytoplasm to the nucleus, as well as degradation and phosphorylation of I{kappa}B{alpha} protein. We further demonstrated PCV2-induced activation of NF-{kappa}B and colocalization of p65 nuclear translocation with virus replication in cultured cells. Treatment of cells with CAPE, a selective inhibitor of NF-{kappa}B activation, reduced virus protein expression and progeny production followed by decreasing PCV2-induced apoptotic caspase activity, indicating the involvement of this transcription factor in induction of cell death. Taken together, these data suggest that NF-{kappa}B activation is important for PCV2 replication and contributes to virus-mediated changes in host cells. The results presented here provide a basis for understanding molecular mechanism of PCV2 infection.

  1. A Single Amino Acid Deletion in the Matrix Protein of Porcine Reproductive and Respiratory Syndrome Virus Confers Resistance to a Polyclonal Swine Antibody with Broadly Neutralizing Activity

    PubMed Central

    Popescu, Luca N.; Monday, Nicholas; Calvert, Jay G.; Rowland, Raymond R. R.

    2015-01-01

    Assessment of virus neutralization (VN) activity in 176 pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) identified one pig with broadly neutralizing activity. A Tyr-10 deletion in the matrix protein provided escape from broad neutralization without affecting homologous neutralizing activity. The role of the Tyr-10 deletion was confirmed through an infectious clone with a Tyr-10 deletion. The results demonstrate differences in the properties and specificities of VN responses elicited during PRRSV infection. PMID:25855739

  2. In Vitro Antiviral Activity of Germacrone Against Porcine Reproductive and Respiratory Syndrome Virus.

    PubMed

    Feng, Jiaping; Bai, Xiaolei; Cui, Tiantian; Zhou, Han; Chen, Yao; Xie, Jiexiong; Shi, Qingwei; Wang, Heng; Zhang, Guihong

    2016-09-01

    Porcine reproductive and respiratory syndrome (PRRS) is one of the most serious diseases affecting the swine industry worldwide; however, there is no efficient control strategies against PRRSV at present. Therefore, development of new antiviral treatment strategies is urgently needed. As reported, germacrone can efficiently impair influenza virus replication. In this study, we exploited whether germacrone has the potential to inhibit PRRSV infection. Our results showed that the germacrone significantly inhibited replication of PRRSV in vitro and repressed the synthesis of viral RNA and protein. However, it did not block PRRSV binding and entry. Further studies confirmed that germacrone impaired PRRSV replication at an early stage, and inhibited infection of both classic and highly pathogenic type II PRRSV strains. Collectively, our findings imply that the germacrone has the potential to be used as an anti-PRRSV drug. PMID:27178541

  3. Downregulation of protein kinase PKR activation by porcine reproductive and respiratory syndrome virus at its early stage infection.

    PubMed

    Xiao, Yueqiang; Ma, Zexu; Wang, Rong; Yang, Liping; Nan, Yuchen; Zhang, Yan-Jin

    2016-05-01

    The interferon-induced double-strand RNA activated protein kinase (PKR) plays an important role in antiviral response. The objective of this study was to assess the effect of porcine reproductive and respiratory syndrome virus (PRRSV) on PKR activation. Here we report that PRRSV inhibited PKR activation during its early stage infection of primary pulmonary alveolar macrophages (PAMs). PRRSV infection led to lower level of phosphorylated PKR in comparison with mock-infected cells. The PKR inhibition was sustained until 10h post infection in the presence of polyI:C, a synthetic analog of double-stranded RNA activating PKR. PKR-mediated phosphorylation of the eukaryotic translation initiation factor eIF2α was also lower in the PRRSV-infected PAMs during the early stage infection. Interestingly, inactivated PRRSV was capable to inhibit the PKR activation until 6h post infection. This suggests that structural components of PRRSV virions were responsible for the inhibition, although PRRSV replication was needed for longer inhibition. These results indicate that the downregulation of PKR activation during early infection stage should be essential for PRRSV to avoid the antiviral response to initiate replication. This finding contributes to our understanding on PRRSV interaction with host innate immune response and reveal a target for control of PRRSV infection. PMID:27066702

  4. Impacts of oxidative stress on acetylcholinesterase transcription, and activity in embryos of zebrafish (Danio rerio) following Chlorpyrifos exposure.

    PubMed

    Rodríguez-Fuentes, Gabriela; Rubio-Escalante, Fernando J; Noreña-Barroso, Elsa; Escalante-Herrera, Karla S; Schlenk, Daniel

    2015-01-01

    Organophosphate pesticides cause irreversible inhibition of AChE which leads to neuronal overstimulation and death. Thus, dogma indicates that the target of OP pesticides is AChE, but many authors postulate that these compounds also disturb cellular redox processes, and change the activities of antioxidant enzymes. Interestingly, it has also been reported that oxidative stress plays also a role in the regulation and activity of AChE. The aims of this study were to determine the effects of the antioxidant, vitamin C (VC), the oxidant, t-butyl hydroperoxide (tBOOH) and the organophosphate Chlorpyrifos (CPF), on AChE gene transcription and activity in zebrafish embryos after 72h exposure. In addition, oxidative stress was evaluated by measuring antioxidant enzymes activities and transcription, and quantification of total glutathione. Apical effects on the development of zebrafish embryos were also measured. With the exception of AChE inhibition and enhanced gene expression, limited effects of CPF on oxidative stress and apical endpoints were found at this developmental stage. Addition of VC had little effect on oxidative stress or AChE, but increased pericardial area and heartbeat rate through an unknown mechanism. TBOOH diminished AChE gene expression and activity, and caused oxidative stress when administered alone. However, in combination with CPF, only reductions in AChE activity were observed with no significant changes in oxidative stress suggesting the adverse apical endpoints in the embryos may have been due to AChE inhibition by CPF rather than oxidative stress. These results give additional evidence to support the role of prooxidants in AChE activity and expression. PMID:25937383

  5. Detection and Characterization of Porcine Endogenous Retrovirus in Porcine Plasma and Porcine Factor VIII

    PubMed Central

    Takefman, Daniel M.; Wong, Susan; Maudru, Thomas; Peden, Keith; Wilson, Carolyn A.

    2001-01-01

    The pig genome contains porcine endogenous retroviruses (PERVs) capable of infecting human cells. Detection of infectious retrovirus in porcine peripheral blood mononuclear cells and endothelial cells suggested to us that pig plasma is likely to contain PERV. Both PERV env sequences and viral reverse transcriptase (RT) activity were detected in all plasma samples isolated from four NIH minipigs. To detect infectious virus from plasma, we performed a culture assay using three cell lines of feline, swine, and human origin that had previously been shown to be permissive for PERV. Infectious virus was successfully cultured from all four NIH minipig plasmas on the swine cell line ST-IOWA. Using RT-PCR with env-specific primers, we could detect expression of PERV class C envelope in the supernatant of ST-IOWA cells that had been exposed to each pig plasma. We next examined a pig plasma derivative, Hyate:C (porcine factor VIII), and found evidence of PERV particles, since all six lots examined were positive for PERV RNA and RT activity. However, infectious virus could not be detected in clinical lots of Hyate:C, suggesting that the manufacturing process might reduce the load of infectious virus to levels below detectable limits of the assay. Detection of infectious virus in porcine plasma confirms and extends the previous findings that certain porcine cells express PERV when manipulated in vitro and clearly demonstrates that there are porcine cells that express infectious PERV constitutively in vivo. PMID:11312325

  6. Activation of NF-κB by nucleocapsid protein of the porcine reproductive and respiratory syndrome virus.

    PubMed

    Luo, Rui; Fang, Liurong; Jiang, Yunbo; Jin, Hui; Wang, Yanwei; Wang, Dang; Chen, Huanchun; Xiao, Shaobo

    2011-02-01

    Nuclear factor kappa B (NF-κB) is a critical transcription factor in innate and adaptive immune response as well as cell proliferation and survival. Previous studies have demonstrated that porcine reproductive and respiratory syndrome virus (PRRSV) infection activated NF-κB pathways through IκB degradation in MARC-145 cells and alveolar macrophages. To evaluate the mechanisms behind this, we investigated the role of PRRSV structural proteins in the regulation of NF-κB. In this study, we screened the structural proteins of PRRSV by NF-κB DNA-binding assay and luciferase activity assay and demonstrated that PRRSV nucleocapsid (N) protein could activate NF-κB in MARC-145 cells. Furthermore, we revealed that the region between aa 30 and 73 of N protein was essential for its function in the activation of NF-κB. These results presented here provide a basis for understanding molecular mechanism of PRRSV infection and inflammation response. PMID:21063763

  7. Synthesis and anti-staphylococcal activity of TiO2 nanoparticles and nanowires in ex vivo porcine skin model.

    PubMed

    Nataraj, Namrata; Anjusree, G S; Madhavan, Asha Anish; Priyanka, P; Sankar, Deepthi; Nisha, N; Lakshmi, S V; Jayakumar, R; Balakrishnan, Avinash; Biswas, Raja

    2014-05-01

    Staphylococcus aureus is one of the major causes of skin and soft tissue infections. In this study we compared the antimicrobial activity of two different TiO2 nanoformulations against Staphylococcus aureus. We synthesized TiO2 nanoparticles of approximately 80 nm diameter and TiO2 nanowires of approximately 100 nm diameter. Both nanoformulations possess anti-microbial activity; were non-hemolytic and cytocompatible. However, the anti-staphylococcal activity of TiO2 nanowires was better than the nanoparticles. In broth culture, growth of S. aureus was only partially inhibited by 2% and 4 wt% TiO2 nanoparticles and completely inhibited by TiO2 nanowires till 24 h. TiO2 nanowires treated S. aureus cells exhibits diminished membrane potential than nanoparticle treated cells. The anti-microbial properties of both TiO2 nanoformulations were validated using ex vivo porcine skin model which supplements the in vitro assays. Anti-bacterial activity of the TiO2 nanowires were also validated against multi drug resistant pathogenic strains of S. aureus, showing the clinical potency of the TiO2 nanowires compared to its nanoparticles. PMID:24734539

  8. Porcine Circovirus Type 2 Activates CaMMKβ to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium.

    PubMed

    Gu, Yuanxing; Qi, Baozhu; Zhou, Yingshan; Jiang, Xiaowu; Zhang, Xian; Li, Xiaoliang; Fang, Weihuan

    2016-01-01

    Porcine circovirus type 2 (PCV2) induces autophagy via the 5' adenosine monophosphate-activated protein kinase (AMPK)/extracellular signal-regulated kinase (ERK)/tuberous sclerosis complex 2 (TSC2)/mammalian target of rapamycin (mTOR) pathway in pig kidney PK-15 cells. However, the underlying mechanisms of AMPK activation in autophagy induction remain unknown. With specific inhibitors and RNA interference (RNAi), we show that PCV2 infection upregulated calcium/calmodulin-dependent protein kinase kinase-beta (CaMKKβ) by increasing cytosolic Ca(2+) via inositol 1,4,5-trisphosphate receptor (IP3R). Elevation of cytosolic calcium ion (Ca(2+)) did not seem to involve inositol 1,4,5-trisphosphate (IP3) release from phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide phospholipase C-gamma (PLC-γ). CaMKKβ then activated both AMPK and calcium/calmodulin-dependent protein kinase I (CaMKI). PCV2 employed CaMKI and Trp-Asp (WD) repeat domain phosphoinositide-interacting protein 1 (WIPI1) as another pathway additional to AMPK signaling in autophagy initiation. Our findings could help better understanding of the signaling pathways of autophagy induction as part of PCV2 pathogenesis. Further research is warranted to study if PCV2 interacts directly with IP3R or indirectly with the molecules that antagonize IP3R activity responsible for increased cytosolic Ca(2+) both in PK-15 cells and PCV2-targeted primary cells from pigs. PMID:27213427

  9. Porcine Circovirus Type 2 Activates CaMMKβ to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium

    PubMed Central

    Gu, Yuanxing; Qi, Baozhu; Zhou, Yingshan; Jiang, Xiaowu; Zhang, Xian; Li, Xiaoliang; Fang, Weihuan

    2016-01-01

    Porcine circovirus type 2 (PCV2) induces autophagy via the 5′ adenosine monophosphate-activated protein kinase (AMPK)/extracellular signal-regulated kinase (ERK)/tuberous sclerosis complex 2 (TSC2)/mammalian target of rapamycin (mTOR) pathway in pig kidney PK-15 cells. However, the underlying mechanisms of AMPK activation in autophagy induction remain unknown. With specific inhibitors and RNA interference (RNAi), we show that PCV2 infection upregulated calcium/calmodulin-dependent protein kinase kinase-beta (CaMKKβ) by increasing cytosolic Ca2+ via inositol 1,4,5-trisphosphate receptor (IP3R). Elevation of cytosolic calcium ion (Ca2+) did not seem to involve inositol 1,4,5-trisphosphate (IP3) release from phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide phospholipase C-gamma (PLC-γ). CaMKKβ then activated both AMPK and calcium/calmodulin-dependent protein kinase I (CaMKI). PCV2 employed CaMKI and Trp-Asp (WD) repeat domain phosphoinositide-interacting protein 1 (WIPI1) as another pathway additional to AMPK signaling in autophagy initiation. Our findings could help better understanding of the signaling pathways of autophagy induction as part of PCV2 pathogenesis. Further research is warranted to study if PCV2 interacts directly with IP3R or indirectly with the molecules that antagonize IP3R activity responsible for increased cytosolic Ca2+ both in PK-15 cells and PCV2-targeted primary cells from pigs. PMID:27213427

  10. Inspection of the activator binding site for 4-alpha-glucanotransferase in porcine liver glycogen debranching enzyme with fluorogenic dextrins.

    PubMed

    Yamamoto, Eriko; Watanabe, Yumiko; Makino, Yasushi; Omichi, Kaoru

    2009-05-01

    Recently, we found that alpha-, beta- and gamma-cyclodextrins accelerated the 4-alpha-glucanotransferase action of porcine liver glycogen debranching enzyme (GDE) on Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5/84), and proposed the presence of an activator binding site in the GDE molecule. In liver cells, the structures of alpha-glucans proximal to the site GDE acts are not cyclodextrins, but glycogen and its degradation products. To estimate the structural characteristics of intrinsic activators and to inspect the features of the activator binding site, we examined the effects of four fluorogenic dextrins, (Glcalpha1-6)(m)Glcalpha1-4(Glcalpha1-4)(n)GlcPA (B5/51, m = 1, n = 3; B6/61, m = 1, n = 4; B7/71, m = 1, n = 5; G6PA, m = 0, n = 4), on the debranching of B5/84 by porcine liver GDE. The GDE 4-alpha-glucanotransferase removed the maltotriosyl residue from the maltotetraosyl branch of B5/84, producing Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5/81). In the presence of G6PA, the removed maltotriosyl residue was transferred to G6PA to give Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (G9PA). In the absence of G6PA, the removed maltotriosyl residue was transferred to water. B7/71, B6/61 and B5/51 did not undergo any changes by the GDE, but they accelerated the action of the 4-alpha-glucanotransferase in removing the maltotriosyl residue. Of the four fluorogenic dextrins examined, B6/61 most strongly accelerated the 4-alpha-glucanotransferase action. The activator binding site is likely to be a space that accommodates the structure of Glcalpha1-6Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glc. PMID:19155269

  11. Effects of preincubation of eggs and activation medium on the percentage of eyed embryos in ide (Leuciscus idus), an externally fertilizing fish.

    PubMed

    Siddique, Mohammad Abdul Momin; Linhart, Otomar; Krejszeff, Sławomir; Żarski, Daniel; Król, Jarosław; Butts, Ian Anthony Ernest

    2016-03-15

    Standardization of fertilization protocols is crucial for improving reproductive techniques for externally fertilizing fish in captive breeding. Therefore, the objectives of this study were to determine the effects of preincubation of eggs and activation medium on the percentage of eyed embryos for ide (Leuciscus idus). Pooled eggs from five females were preincubated in three different activating media for 0, 30, 60, 90, and 120 seconds and then fertilized by pooled sperm from five males. At the eyed-egg stage, the percentage of viable embryos was later calculated. Results showed that preincubation time was significant for the freshwater activation medium (P < 0.001), such that the percentage of eyed embryos declined across the preincubation time gradient. Additionally, there was an effect on the percentage of eyed embryos when eggs were incubated with Woynarovich solution (P < 0.001), such that a decline was detected at 90 seconds, whereas no effect was detected for the saline water medium. Activating medium had a significant effect on the percentage of eyed embryos for each preincubation time (P < 0.05). More precisely, freshwater produced the lowest percentage of eyed embryos at all preincubation times (ranged from 1.9% at 120 seconds to 43.6% at 0 seconds), whereas saline water and Woynarovich solution produced the highest percentage of eyed embryos at 0 seconds and 30 seconds before incubation. Woynarovich solution produced the highest percentage of eyed embryos at 60 seconds (65.26%), whereas saline water produced the highest percentage at 90 seconds (68.37%). No difference was detected between saline water and Woynarovich solution at 120 seconds. Examination of sperm traits showed no impact of activating medium on computer assisted sperm analysis parameters. Together, these results suggest that saline water or Woynarovich solution improve fertilization rate in ide during IVF; thus, these media are useful for standardizing fertilization protocols and

  12. Long-Chain Acyl CoA Synthetase 4A regulates Smad activity and dorsoventral patterning in the zebrafish embryo

    PubMed Central

    Miyares, Rosa Linda; Stein, Cornelia; Renisch, Björn; Anderson, Jennifer Lynn; Hammerschmidt, Matthias; Farber, Steven Arthur

    2013-01-01

    Summary Long-chain polyunsaturated fatty acids (LC-PUFA) and their metabolites are critical players in cell biology and embryonic development. Here we show that long-chain acyl CoA synthetase 4a (Acsl4a), an LC-PUFA activating enzyme, is essential for proper patterning of the zebrafish dorsoventral axis. Loss of Acsl4a results in dorsalized embryos due to attenuated Bmp signaling. We demonstrate that Acsl4a modulates the activity of Smad transcription factors, the downstream mediators of Bmp signaling. Acsl4a promotes the inhibition of p38 MAPK and the Akt-mediated inhibition of glycogen synthase kinase 3 (GSK3), critical inhibitors of Smad activity. Consequently, introduction of a constitutively active Akt can rescue the dorsalized phenotype of Acsl4a deficient embryos. Our results reveal a critical role for Acsl4a in modulating Bmp-Smad activity and provide a potential avenue for LC-PUFAs to influence a variety of developmental processes. PMID:24332754

  13. Activation of matrix metalloproteinase-26 by HOXA10 promotes embryo adhesion in vitro.

    PubMed

    Jiang, Yue; Yan, Guijun; Zhang, Hui; Shan, Huizhi; Kong, Chengcai; Yan, Qiang; Xue, Bai; Diao, Zhenyu; Hu, Yali; Sun, Haixiang

    2014-03-14

    Successful embryonic implantation requires an effective maternal-embryonic molecular dialogue. However, the detailed mechanisms of epithelial-embryo adhesion remain poorly understood. Here, we report that matrix metalloproteinase-26 (MMP-26) is a novel downstream target gene of homeobox a 10 (HOXA10) in human endometrial cells. HOXA10 binds directly to a conserved TTAT unit (-442 to -439) located within the 5' regulatory region of the MMP-26 gene and regulates the expression and secretion of MMP-26 in a concentration-dependent manner. Moreover, the adenovirus-mediated overexpression of MMP-26 in Ishikawa cells markedly increased BeWo spheroid adhesion. An antibody blocking assay further demonstrated that the promotion of BeWo spheroid adhesion by HOXA10 and MMP-26 was significantly inhibited by pre-treatment with a specific antibody against MMP-26. These results demonstrate that the HOXA10-mediated expression of MMP-26 promotes embryo adhesion during the process of embryonic implantation. PMID:24565841

  14. JNK and p38 mitogen-activated protein kinase pathways contribute to porcine epidemic diarrhea virus infection.

    PubMed

    Lee, Changhee; Kim, Youngnam; Jeon, Ji Hyun

    2016-08-15

    The mitogen-activated protein kinase (MAPK) pathways, which are central building blocks in the intracellular signaling network, are often manipulated by viruses of diverse families to favor their replication. Among the MAPK family, the extracellular signal-regulated kinase (ERK) pathway is known to be modulated during the infection with porcine epidemic diarrhea virus (PEDV); however, involvement of stress-activated protein kinases (SAPKs) comprising p38 MAPK and c-Jun NH2-terminal kinase (JNK) remains to be determined. Therefore, in the present study, we investigated whether activation of p38 MAPK and JNK cascades is required for PEDV replication. Our results showed that PEDV activates p38 MAPK and JNK1/2 up to 24h post-infection, whereas, thereafter their phosphorylation levels recede to baseline levels or even fall below them. Notably, UV-irradiated inactivated PEDV, which can enter cells but cannot replicate inside them, failed to induce phosphorylation of p38 MAPK and JNK1/2 suggesting that viral biosynthesis is essential for activation of these kinases. Treatment of cells with selective p38 or JNK inhibitors markedly impaired PEDV replication in a dose-dependent manner and these antiviral effects were found to be maximal during the early times of the infection. Furthermore, direct pharmacological inhibition of p38 MAPK or JNK1/2 activation resulted in a significant reduction of viral RNA synthesis, viral protein expression, and progeny release. However, independent treatments with either SAPK inhibitor did not inhibit PEDV-induced apoptotic cell death mediated by activation of mitochondrial apoptosis-inducing factor (AIF) suggesting that SAPKs are irrelevant to the apoptosis pathway during PEDV infection. In summary, our data demonstrated critical roles of the p38 and JNK1/2 signaling pathways in facilitating successful viral infection during the post-entry steps of the PEDV life cycle. PMID:27215486

  15. The study of a light-activated albumin protein solder to bond layers of porcine small intestinal submucosa.

    PubMed

    Ware, Mark H; Buckley, Christine A

    2003-01-01

    This study investigated the feasibility of bonding layers of porcine small intestinal submucosa (SIS, Cook Biotech, Inc.) with a light-activated protein solder. SIS is an acellular, collagen-based extracellular matrix material that is approximately 100 microns thick. The solder consists of bovine serum albumin and indocyanine green dye (ICG) in deionized water. The solder is activated by an 808 nm diode laser, which denatures the albumin, causing the albumin to bond with the collagen of the tissue. The predictable absorption and thermal energy diffusion rates of ICG increase the chances of reproducible results. To determine the optimal condition for laser soldering SIS, the following parameters were varied: albumin concentration (from 30-45% (w/v) in increments of 5%), the concentration of ICG (from 0.5-2.0 mg/ml H2O) and the irradiance of the laser (10-64 W/cm2). While many of the solder compositions and laser irradiance combinations resulted in no bonding, a solder composition of 45% albumin, ICG concentration of 0.5 mg/ml H2O, and a laser irradiance of 21 W/cm2 did produce a bond between two pieces of SIS. The average shear strength of this bond was 29.5 +/- 17.1 kPa (n = 14). This compares favorably to our previous work using fibrin glue as an adhesive, in which the average shear strength was 27 +/- 15.8 kPa (n = 40). PMID:12724859

  16. Rolling-circle amplification for the detection of active porcine circovirus type 2 DNA replication in vitro.

    PubMed

    Navidad, Paolo Dominic; Li, Hao; Mankertz, Annette; Meehan, Brian

    2008-09-01

    Porcine circovirus type 2 (PCV2) infections in pigs have diverse clinical presentations and are considered economically important diseases worldwide. However, despite intensive research, the early pathogenesis of PCV2 and the primary target cells for PCV2 infections and replication are still unknown. Rolling-circle amplification (RCA) is an amplification technique for small, circular DNA templates that essentially mimics rolling-circle replication in vitro. In this study, the amplification of PCV2-specific DNAs using randomly primed RCA has been demonstrated. This novel approach has circumvented the normal requirement for conventional virus isolation procedures for the characterization of PCV2 DNAs from clinical samples. In addition, the potential utility of a strand-specific derivative of RCA was further investigated. Specifically, strand-specific RCA for the detection of active virus replication following the amplification of complementary sense PCV2 DNAs, which occur as double-stranded replicative intermediates that are present only during de novo viral DNA replication both in vitro and in vivo has been demonstrated. PMID:18606463

  17. In vitro antiviral activity of phlorotannins isolated from Ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination.

    PubMed

    Kwon, Hyung-Jun; Ryu, Young Bae; Kim, Young-Min; Song, Naaleum; Kim, Cha Young; Rho, Mun-Chual; Jeong, Jae-Ho; Cho, Kyoung-Oh; Lee, Woo Song; Park, Su-Jin

    2013-08-01

    Despite the prepdominat agent causing severe entero-pathogenic diarrhea in swine, there are no effective therapeutical treatment of porcine epidemic diarrhea virus (PEDV). In this study, we evaluated the antiviral activity of five phlorotannins isolated from Ecklonia cava (E. cava) against PEDV. In vitro antiviral activity was tested using two different assay strategies: (1) blockage of the binding of virus to cells (simultaneous-treatment assay) and (2) inhibition of viral replication (post-treatment assay). In simultaneous-treatment assay, compounds 2-5 except compound 1 exhibited antiviral activities of a 50% inhibitory concentration (IC₅₀) with the ranging from 10.8 ± 1.4 to 22.5 ± 2.2 μM against PEDV. Compounds 1-5 were completely blocked binding of viral spike protein to sialic acids at less than 36.6 μM concentrations by hemagglutination inhibition. Moreover, compounds 4 and 5 of five phlorotannins inhibited viral replication with IC₅₀ values of 12.2 ± 2.8 and 14.6 ± 1.3 μM in the post-treatment assay, respectively. During virus replication steps, compounds 4 and 5 exhibited stronger inhibition of viral RNA and viral protein synthesis in late stages (18 and 24 h) than in early stages (6 and 12 h). Interestingly, compounds 4 and 5 inhibited both viral entry by hemagglutination inhibition and viral replication by inhibition of viral RNA and viral protein synthesis, but not viral protease. These results suggest that compounds isolated from E. cava have strong antiviral activity against PEDV, inhibiting viral entry and/or viral replication, and may be developed into natural therapeutic drugs against coronavirus infection. PMID:23746631

  18. Site-Specific Expression of Gelatinolytic Activity during Morphogenesis of the Secondary Palate in the Mouse Embryo

    PubMed Central

    Gkantidis, Nikolaos; Blumer, Susan; Katsaros, Christos; Graf, Daniel; Chiquet, Matthias

    2012-01-01

    Morphogenesis of the secondary palate in mammalian embryos involves two major events: first, reorientation of the two vertically oriented palatal shelves into a horizontal position above the tongue, and second, fusion of the two shelves at the midline. Genetic evidence in humans and mice indicates the involvement of matrix metalloproteinases (MMPs). As MMP expression patterns might differ from sites of activity, we used a recently developed highly sensitive in situ zymography technique to map gelatinolytic MMP activity in the developing mouse palate. At embryonic day 14.5 (E14.5), we detected strong gelatinolytic activity around the lateral epithelial folds of the nasopharyngeal cavity, which is generated as a consequence of palatal shelf elevation. Activity was concentrated in the basement membrane of the epithelial fold but extended into the adjacent mesenchyme, and increased in intensity with lateral outgrowth of the cavity at E15.5. Gelatinolytic activity at this site was not the consequence of epithelial fold formation, as it was also observed in Bmp7-deficient embryos where shelf elevation is delayed. In this case, gelatinolytic activity appeared in vertical shelves at the exact position where the epithelial fold will form during elevation. Mmp2 and Mmp14 (MT1-MMP), but not Mmp9 and Mmp13, mRNAs were expressed in the mesenchyme around the epithelial folds of the elevated palatal shelves; this was confirmed by immunostaining for MMP-2 and MT1-MMP. Weak gelatinolytic activity was also found at the midline of E14.5 palatal shelves, which increased during fusion at E15.5. Whereas MMPs have been implicated in palatal fusion before, this is the first report showing that gelatinases might contribute to tissue remodeling during early stages of palatal shelf elevation and formation of the nasopharynx. PMID:23091646

  19. Site-specific expression of gelatinolytic activity during morphogenesis of the secondary palate in the mouse embryo.

    PubMed

    Gkantidis, Nikolaos; Blumer, Susan; Katsaros, Christos; Graf, Daniel; Chiquet, Matthias

    2012-01-01

    Morphogenesis of the secondary palate in mammalian embryos involves two major events: first, reorientation of the two vertically oriented palatal shelves into a horizontal position above the tongue, and second, fusion of the two shelves at the midline. Genetic evidence in humans and mice indicates the involvement of matrix metalloproteinases (MMPs). As MMP expression patterns might differ from sites of activity, we used a recently developed highly sensitive in situ zymography technique to map gelatinolytic MMP activity in the developing mouse palate. At embryonic day 14.5 (E14.5), we detected strong gelatinolytic activity around the lateral epithelial folds of the nasopharyngeal cavity, which is generated as a consequence of palatal shelf elevation. Activity was concentrated in the basement membrane of the epithelial fold but extended into the adjacent mesenchyme, and increased in intensity with lateral outgrowth of the cavity at E15.5. Gelatinolytic activity at this site was not the consequence of epithelial fold formation, as it was also observed in Bmp7-deficient embryos where shelf elevation is delayed. In this case, gelatinolytic activity appeared in vertical shelves at the exact position where the epithelial fold will form during elevation. Mmp2 and Mmp14 (MT1-MMP), but not Mmp9 and Mmp13, mRNAs were expressed in the mesenchyme around the epithelial folds of the elevated palatal shelves; this was confirmed by immunostaining for MMP-2 and MT1-MMP. Weak gelatinolytic activity was also found at the midline of E14.5 palatal shelves, which increased during fusion at E15.5. Whereas MMPs have been implicated in palatal fusion before, this is the first report showing that gelatinases might contribute to tissue remodeling during early stages of palatal shelf elevation and formation of the nasopharynx. PMID:23091646

  20. Neferine, an alkaloid from lotus seed embryo, inhibits human lung cancer cell growth by MAPK activation and cell cycle arrest.

    PubMed

    Poornima, Paramasivan; Weng, Ching Feng; Padma, Viswanadha Vijaya

    2014-01-01

    Neferine is the major bisbenzylisoquinoline alkaloid isolated from the seed embryo of a traditional medicinal plant Nelumbo nucifera (Lotus). Epidemiological studies have revealed the therapeutic potential of lotus seed embryo. Although several mechanisms have been proposed, a clear anticancer action mechanism of neferine on lung cancer cells is still not known. Lung cancer is the most common cause of cancer death in the world, and the patients with advanced stage of nonsmall lung cancer require adjunct chemotherapy after surgical resection for the eradication of cancer cells. In this study, the effects of neferine were evaluated and characterized in A549 cells. Neferine induced apoptosis in a dose-dependent manner with the hypergeneration of reactive oxygen species, activation of MAPKs, lipid peroxidation, depletion of cellular antioxidant pool, loss of mitochondrial membrane potential, and intracellular calcium accumulation. Furthermore, neferine treatment leads to the inhibition of nuclear factor kappaB and Bcl2, upregulation of Bax and Bad, release of cytochrome C, activation of caspase cascade, and DNA fragmentation. In addition, neferine could induce p53 and its effector protein p21 and downregulation of cell cycle regulatory protein cyclin D1 thereby inducing G1 cell cycle arrest. These results suggest a novel function of neferine as an apoptosis inducer in lung cancer cells. PMID:23983146

  1. Chimerism in piglets developed from aggregated cloned embryos.

    PubMed

    Huang, Yongye; Li, Zhanjun; Wang, Anfeng; Han, Xiaolei; Song, Yuning; Yuan, Lin; Li, Tianye; Wang, Bing; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

    2016-04-01

    Porcine chimeras are valuable in the study of pluripotency, embryogenesis and development. It would be meaningful to generate chimeric piglets from somatic cell nuclear transfer embryos. In this study, two cell lines expressing the fluorescent markers enhanced green fluorescent protein (EGFP) and tdTomato were used as donor cells to produce reconstructed embryos. Chimeric embryos were generated by aggregating two EGFP-cell derived embryos with two tdTomato-cell derived embryos at the 4-cell stage, and embryo transfer was performed when the aggregated embryos developed into blastocysts. Live porcine chimeras were successfully born and chimerism was observed by their skin color, gene integration, microsatellite loci composition and fluorescent protein expression. The chimeric piglets were largely composed of EGFP-expressing cells, and this phenomenon was possibly due to the hyper-methylation of the promoter of the tdTomato gene. In addition, the expression levels of tumorigenicity-related genes were altered after tdTomato transfection in bladder cancer cells. The results show that chimeric pigs can be produced by aggregating cloned embryos and that the developmental capability of the cloned embryo in the subsequent chimeric development could be affected by the growth characteristics of its donor cell. PMID:27239442

  2. Somatic proembryo production from excised, wounded zygotic carrot embryos on hormone-free medium: evaluation of the effects of pH, ethylene and activated charcoal

    NASA Technical Reports Server (NTRS)

    Smith, D. L.; Krikorian, A. D.

    1990-01-01

    Wounded zygotic embryos of cultivated carrot produce somatic proembryos on hormone-free nutrient medium containing 1 mM NH4+ as the sole nitrogen source. Continued maintenance of proembryos on this medium leads to a "pure" culture of preglobular stage proembryos (PGSPs). Ethylene had no effect on this process. Also, somatic embryo production was not affected by growing cultures on activated charcoal-impregnated filter papers. However, somatic proembyros initiated on activated charcoal papers were not maintainable as PGSPs and developed into later embryo stages. Normally, medium pH dropped from 5.7 to 4 during each subculture period, but when using activated charcoal papers the pH endpoint was around 6 - 7 due to a leachable substance(s) within the filter papers. When powdered, activated charcoal was used in the medium as an adsorbent of products potentially released after wounding, pH dropped at the normal rate and to the expected levels; proembryos did not mature into later embryo stages and were maintainable exclusively as PGSPs. Low pH (approximately 4) is detrimental to proembyro production, but is essential to maintaining PGSPs on hormone-free nutrient medium, whereas a sustained pH > or = 5.7 allows continued development of PGSPs into later embryo stages.

  3. Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: characterization and application for enzymatic inhibition assays.

    PubMed

    Zhu, Yuan-Ting; Ren, Xiao-Yun; Liu, Yi-Ming; Wei, Ying; Qing, Lin-Sen; Liao, Xun

    2014-05-01

    Using carboxyl functionalized silica-coated magnetic nanoparticles (MNPs) as carrier, a novel immobilized porcine pancreatic lipase (PPL) was prepared through the 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. Transmission electron microscopic images showed that the synthesized nanoparticles (Fe3O4-SiO2) possessed three dimensional core-shell structures with an average diameter of ~20 nm. The effective enzyme immobilization onto the nanocomposite was confirmed by atomic force microscopic (AFM) analysis. Results from Fourier-transform infrared spectroscopy (FT-IR), Bradford protein assay, and thermo-gravimetric analysis (TGA) indicated that PPL was covalently attached to the surface of magnetic nanoparticles with a PPL immobilization yield of 50mg enzyme/g MNPs. Vibrating sample magnetometer (VSM) analysis revealed that the MNPs-PPL nanocomposite had a high saturation magnetization of 42.25 emu·g(-1). The properties of the immobilized PPL were investigated in comparison with the free enzyme counterpart. Enzymatic activity, reusability, thermo-stability, and storage stability of the immobilized PPL were found significantly superior to those of the free one. The Km and the Vmax values (0.02 mM, 6.40 U·mg(-1) enzyme) indicated the enhanced activity of the immobilized PPL compared to those of the free enzyme (0.29 mM, 3.16 U·mg(-1) enzyme). Furthermore, at an elevated temperature of 70 °C, immobilized PPL retained 60% of its initial activity. The PPL-MNPs nanocomposite was applied in the enzyme inhibition assays using orlistat, and two natural products isolated from oolong tea (i.e., EGCG and EGC) as the test compounds. PMID:24656379

  4. Transforming growth factor beta (TGFB) signaling is activated during porcine implantation: proposed role for latency-associated peptide interactions with integrins at the conceptus-maternal interface.

    PubMed

    Massuto, Dana A; Kneese, Eric C; Johnson, Gregory A; Burghardt, Robert C; Hooper, R Neil; Ing, Nancy H; Jaeger, Laurie A

    2010-02-01

    The process of implantation is mediated by a complex network of signaling and adhesive factors. In the pig, latent and active transforming growth factor beta (TGFB), TGFB receptors (TGFBR), and integrins (ITGs) are present during the peri-implantation period. TGFB signals via TGFBR and activates downstream effector SMAD proteins 2 and 3 (p-SMAD2/3). Latency-associated peptide (LAP), part of the latent TGFB complex, is known to bind to ITG heterodimers and activate TGFB. We hypothesize that active TGFBs and TGFBRs along with LAP and ITGs functionally interact at the conceptus-maternal interface to mediate events essential for conceptus development and attachment in pigs. Uteri and conceptuses from days 10, 12, 16, 20, and 24 pregnant gilts were immunostained for TGFB, LAP, and ITG subunits (ITGAV, ITGB1, ITGB3, ITGB5, ITGB6, and ITGB8). Activation of TGFBRs was evaluated by the presence of phosphorylated downstream effector SMAD2/3. Binding of LAP to ITGs was also evaluated using porcine trophectoderm cells. Abundant active TGFB was detected at the apical surfaces of epithelia at the conceptus-maternal interface, and p-SMAD2/3 was detected at both conceptus attachment and nonattachment sites during implantation. Separate aggregates of LAP, ITGB1, ITGB5, and later ITGB3 were detected at the porcine conceptus-maternal interface, and binding of LAP to ITGs on apical surfaces was demonstrated. Results suggest that functional LAP-ITG adhesion complexes support conceptus attachment and promote TGFB activation leading to TGFB interaction with TGFBR supporting events of porcine implantation. PMID:19920116

  5. Mitogen-activated protein kinases in the porcine retinal arteries and neuroretina following retinal ischemia-reperfusion

    PubMed Central

    Gesslein, Bodil; Håkansson, Gisela; Carpio, Ronald; Gustafsson, Lotta; Perez, Maria-Thereza

    2010-01-01

    Purpose The aim of the present study was to examine changes in the expression of intracellular signal-transduction pathways, specifically mitogen-activated protein kinases, following retinal ischemia-reperfusion. Methods Retinal ischemia was induced by elevating the intraocular pressure in porcine eyes, followed by 5, 12, or 20 h of reperfusion. The results were compared to those of the sham- operated fellow eye. The retinal arteries and neuroretina were isolated separately and examined. Tissue morphology and DNA fragmentation were studied using histology. Extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38, c-junNH2-terminal kinases (JNK), and c-jun protein and mRNA expression were examined using immunofluorescence staining, western blot, and real-time PCR techniques. Results Pyknotic cell nuclei, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, and glial fibrillary acidic protein mRNA expression were increased in ischemia, suggesting injury. Phosphorylated ERK1/2 protein levels were increased in the neuroretina following ischemia, while mRNA levels were unaltered. p38 protein and mRNA levels were not affected by ischemia. Immunofluorescence staining for phosphorylated p38 was especially intense in the retinal blood vessels, while only weak in the neuroretina. Phosphorylated JNK protein and mRNA were slightly decreased in ischemia. Phosphorylated c-jun protein and mRNA levels were higher in the neuroretina after ischemia-reperfusion. Conclusions Retinal ischemia-reperfusion alters expression of mitogen-activated protein kinases, particularly ERK1/2, in the neuroretina and retinal arteries. The development of pharmacological treatment targeting these intracellular transduction pathways may prevent injury to the eye following retinal circulatory failure. PMID:20300568

  6. Dynamic changes of histone H3 lysine 27 acetylation in pre-implantational pig embryos derived from somatic cell nuclear transfer.

    PubMed

    Zhou, Naru; Cao, Zubing; Wu, Ronghua; Liu, Xing; Tao, Jia; Chen, Zhen; Song, Dandan; Han, Fei; Li, Yunsheng; Fang, Fugui; Zhang, Xiaorong; Zhang, Yunhai

    2014-08-01

    Histone H3 lysine 27 acetylation (H3K27ac) is an active epigenetic modification which has been revealed to be associated with active gene expression. It was hypothesized that H3K27ac might also participate in the porcine somatic reprogramming process during early development of SCNT-derived embryos. The spatial and temporal expression profiles of H3K27ac were investigated at different developmental stages in SCNT embryos compared with in vitro fertilization (IVF) and parthenogenetic activation (PA) counterparts. Specifically, results showed that amounts of H3K27ac gradually decreased from the earliest pronuclear stage to 8-cell stage, corresponding to the major embryonic genome activation (EGA), followed by re-acetylation of H3K27 from the morula stage onwards accompanying the first cell lineage specification in IVF embryos. Similar dynamic patterns of H3K27ac signal was observed at all developmental stages of porcine SCNT and PA embryos except for the hatched stage in which amounts of H3K27ac in SCNT and PA embryos was slightly less than that in IVF counterparts. Moreover, the gradual decrease of H3K27ac before EGA was demonstrated to be an active process independent of DNA replication, RNA and protein synthesis. The expression of HDAC1, HDAC2, MBD3 and CBP genes were well correlated with the dynamic changes of H3K27ac mark. Overall, these results indicate that H3K27ac is only defective in late SCNT blastocysts, and that the dynamic changes of this marker might also underlie the EGA and initial cell lineage specification during early embryo development. PMID:24972950

  7. Aerobic heat shock activates trehalose synthesis in embryos of Artemia franciscana.

    PubMed

    Clegg, J S; Jackson, S A

    1992-05-25

    Encysted embryos (cysts) of the brine shrimp, Artemia franciscana, contain large amounts of trehalose which they use as a major substrate for energy metabolism and biosynthesis for development under aerobic conditions at 25 degrees C. When cysts are placed at 42 degrees C (heat shock) these pathways stop, and the cysts re-synthesize the trehalose that was utilized during the previous incubation at 25 degrees C. Glycogen and glycerol, produced from trehalose at 25 degrees C, appear to be substrates for trehalose synthesis during heat shock. Anoxia prevents trehalose synthesis in cysts undergoing heat shock. These results are consistent with the view that trehalose may play a protective role in cells exposed to heat shock, and other environmental insults, in addition to being a storage form of energy and organic carbon for development. PMID:1592115

  8. Local activation of uterine Toll-like receptor 2 and 2/6 decreases embryo implantation and affects uterine receptivity in mice.

    PubMed

    Sanchez-Lopez, Javier Arturo; Caballero, Ignacio; Montazeri, Mehrnaz; Maslehat, Nasim; Elliott, Sarah; Fernandez-Gonzalez, Raul; Calle, Alexandra; Gutierrez-Adan, Alfonso; Fazeli, Alireza

    2014-04-01

    Embryo implantation is a complex interaction between maternal endometrium and embryonic structures. Failure to implant is highly recurrent and impossible to diagnose. Inflammation and infections in the female reproductive tract are common causes of infertility, embryo loss, and preterm labor. The current work describes how the activation of endometrial Toll-like receptor (TLR) 2 and 2/6 reduces embryo implantation chances. We developed a morphometric index to evaluate the effects of the TLR 2/6 activation along the uterine horn (UH). TLR 2/6 ligation reduced the endometrial myometrial and glandular indexes and increased the luminal index. Furthermore, TLR 2/6 activation increased the proinflammatory cytokines such as interleukin (IL)-1beta and monocyte chemotactic protein (MCP)-1 in UH lavages in the preimplantation day and IL-1 receptor antagonist in the implantation day. The engagement of TLR 2/6 with its ligand in the UH during embryo transfer severely affected the rate of embryonic implantation (45.00% ± 6.49% vs. 16.69% ± 5.01%, P < 0.05, control vs. test, respectively). Furthermore, this interference with the embryo implantation process was verified using an in vitro model of human embryo implantation where trophoblast spheroids failed to adhere to a monolayer of TLR 2- and TLR 2/6-activated endometrial cells. The inhibition of TLR receptors 2 and 6 in the presence of their specific ligands restored the ability of the spheroids to bind to the endometrial cells. In conclusion, the activation of the innate immune system in the uterus at the time of implantation interfered with the endometrial receptivity and reduced the chances of implantation success. PMID:24621922

  9. Efficient biallelic mutation in porcine parthenotes using a CRISPR-Cas9 system.

    PubMed

    Tao, Li; Yang, Mingyao; Wang, Xiaodong; Zhang, Zhenni; Wu, Zhonghong; Tian, Jianhui; An, Lei; Wang, Shumin

    2016-08-01

    The parthenotes represent ideal models mimicking the embryonic development and characterizing the function of maternal genomes as well as an alternative source of pluripotent cell lines. Besides, parthenogenetically activated (PA) embryos serve as a rapid assay system to maximize the efficiency of generating genetically modified pig CRISPR/Cas9 system, an efficient and multiplex gene editing tool, has been utilized to modify the genome of porcine parthenotes. However, lower biallelic mutation rate and high mosaicism frequency were observed. Here, we aimed to enhance the biallelic mutation rate with reduced mosaicism by optimization of the concentration and injection time of the Cas9/sgRNA mixture in porcine parthenotes. The results showed that the efficient biallelic mutation (93%) and low mosaicism (33%) could be achieved in porcine parthenotes by cytoplasmic injection of Cas9 mRNA/sgRNA (125/12.5 ng/μl) after 8 h of parthenogenetical activation. Thus, our study provides an effective strategy for increasing the biallelic mutation rate and population homogeneity of genetically modified parthenotes, which will strengthen the role of parthenotes in uncovering early embryonic development and assessing the mutation efficiency due to the simplicity and adaptability of CRISPR/Cas9. PMID:27221047

  10. Influence of early pH decline on calpain activity in porcine muscle.

    PubMed

    Pomponio, Luigi; Ertbjerg, Per; Karlsson, Anders H; Costa, Leonardo Nanni; Lametsch, René

    2010-05-01

    This study investigated the influence of post-mortem pH decline on calpain activity and myofibrillar degradation. From 80 pigs, 30 Longissimus dorsi (LD) muscles were selected on the basis of pH values at 3h post-mortem and classified into groups of 10 as fast, intermediate and slow pH decline. The rate of pH decline early post-mortem differed between the three groups, but the ultimate pH values were similar at 24h. Calpain activity and autolysis from 1 to 72h post-mortem were determined using casein zymography and studied in relation to myofibrillar fragmentation. Colour and drip loss were measured. A faster decrease in pH resulted in reduced level of mu-calpain activity and increased autolysis of the enzyme, and hence an earlier loss of activity due to activation of mu-calpain in muscles with a fast pH decline. Paralleling the mu-calpain activation in muscles with a fast pH decline a higher myofibril fragmentation at 24h post-mortem was observed, which was no longer evident in the later phase of the tenderization process. In conclusion, the rate of early pH decline influenced mu-calpain activity and the rate but not the extent of myofibrillar degradation, suggesting an early effect of proteolysis on myofibril fragmentation that is reduced during ageing due to an earlier exhaustion of mu-calpain activity. PMID:20374873

  11. Putative phage-display epitopes of the porcine epidemic diarrhea virus S1 protein and their anti-viral activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Porcine epidemic diarrhea virus (PEDV) is a pathogen of swine that causes severe diarrhea and dehydration resulting in substantial morbidity and mortality in newborn piglets. Phage display is a technique with wide application, in particular, the identification of key antigen epitopes for the develop...

  12. Enhanced Antiviral Activity against Foot-and-Mouth Disease Virus by a Combination of Type 1 and 2 Porcine Interfereons

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previously, we showed that type I interferon (IFN alpha/beta) can inhibit foot-and-mouth disease virus (FMDV) replication in cell culture and swine inoculated with 109 pfu human adenovirus type 5 expressing porcine IFN-alpha (Ad5-pIFN alpha) were protected when challenged one day later. In this stud...

  13. Porcine circovirus type 2 detection in in vitro produced porcine blastocysts after virus sperm exposure.

    PubMed

    Galeati, Giovanna; Zannoni, Augusta; Spinaci, Marcella; Bucci, Diego; Ostanello, Fabio; Panarese, Serena; Tamanini, Carlo; Sarli, Giuseppe

    2016-04-01

    This study was aimed at assessing the capability of semen experimentally infected with porcine circovirus type 2 (PCV2) to produce porcine blastocysts PCR positive for PCV2. Embryos were obtained from in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes or by parthenogenesis. Sperm suspension was exposed to PCV2b and utilized for IVF. PCV2 spiked semen did not reveal any reduction in sperm viability or motility but its ability to produce infected blastocysts was irrelevant as only one out of 15 blastocysts obtained by IVF were PCV2b; however two blastocysts were PCV2a positive. Furthermore, the presence of PCV2 was demonstrated also in embryos obtained by parthenogenesis (one out of 17 was PCV2b and one PCV2a positive). Even if PCV2 firmly attaches to the surface of spermatozoa, experimentally spiked sperm were not effective in infecting oocytes during IVF and in producing PCR positive embryos. The infected blastocysts we obtained derived most probably from infected oocytes recovered at the abattoir. PMID:26434667

  14. [Apoptosis during embryo development].

    PubMed

    Jezek, Davor; Kozina, Viviana

    2009-10-01

    The development of human embryo includes two essential processes, i.e., rapid mitotic activity of cells and gradual differentiation of tissues and organs. The latter process is very often characterized by extensive migration of cells from their site of origin to the site of definitive location, inductive action of the neighboring germ layers and programmed cell death (apoptosis). This paper describes examples of proliferative and apoptotic processes during the development of human embryo. The development of trilaminar germ disk, skin, gonads, central and peripheral nerve system as well as limbs provides instructive examples of how apoptosis regulates the development and differentiation of cells. PMID:19999545

  15. High glucose environment inhibits cranial neural crest survival by activating excessive autophagy in the chick embryo

    PubMed Central

    Wang, Xiao-Yu; Li, Shuai; Wang, Guang; Ma, Zheng-Lai; Chuai, Manli; Cao, Liu; Yang, Xuesong

    2015-01-01

    High glucose levels induced by maternal diabetes could lead to defects in neural crest development during embryogenesis, but the cellular mechanism is still not understood. In this study, we observed a defect in chick cranial skeleton, especially parietal bone development in the presence of high glucose levels, which is derived from cranial neural crest cells (CNCC). In early chick embryo, we found that inducing high glucose levels could inhibit the development of CNCC, however, cell proliferation was not significantly involved. Nevertheless, apoptotic CNCC increased in the presence of high levels of glucose. In addition, the expression of apoptosis and autophagy relevant genes were elevated by high glucose treatment. Next, the application of beads soaked in either an autophagy stimulator (Tunicamycin) or inhibitor (Hydroxychloroquine) functionally proved that autophagy was involved in regulating the production of CNCC in the presence of high glucose levels. Our observations suggest that the ERK pathway, rather than the mTOR pathway, most likely participates in mediating the autophagy induced by high glucose. Taken together, our observations indicated that exposure to high levels of glucose could inhibit the survival of CNCC by affecting cell apoptosis, which might result from the dysregulation of the autophagic process. PMID:26671447

  16. Accessory Cell Mediated Activation of Porcine NK Cells by TLR7 and TLR8 Agonists

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The induction of innate immune responses by toll-like receptor (TLR) agonists is the subject of intense investigation in many different species. In large part, this reflects the potential of such compounds to be effective vaccine adjuvants. For that reason, we analyzed the activation of innate cells...

  17. Porcine malignant hyperthermia susceptibility: increased calcium-sequestering activity of skeletal muscle sarcoplasmic reticulum.

    PubMed Central

    O'Brien, P J

    1986-01-01

    This study tested the hypothesis that calcium-sequestration by isolated sarcoplasmic reticulum was abnormal in skeletal muscle of malignant hyperthermia-susceptible swine. A heavy sarcoplasmic reticulum fraction was isolated from malignant hyperthermia and control muscle using differential and density-gradient centrifugation. Prior to onset of malignant hyperthermia, calcium-sequestering activity (Vmax at 37 degrees C, mumol calcium/mg/min) was twofold increased in malignant hyperthermia sarcoplasmic reticulum compared to control sarcoplasmic reticulum (1.96 +/- 0.50 versus 4.00 +/- 0.87, P less than 0.01), although thermodynamic and kinetic properties of this activity were otherwise indistinguishable between groups. This increased activity of the malignant hyperthermia sarcoplasmic reticulum fraction was associated with twofold increased concentration of Ca-ATPase and calsequestrin protein. When a malignant hyperthermia-reaction developed, calcium-uptake was depressed to less than 5% of control values. These data indicate that malignant hyperthermia is not initiated due to a defect in the calcium-sequestration mechanism, however, loss of calcium-uptake activity occurring after the onset of malignant hyperthermia might result in the propagation and irreversibility of the malignant hyperthermia reaction. Images Fig. 1. PMID:3742368

  18. Activation of rhodopsin gene transcription in cultured retinal precursors of chicken embryo: role of Ca(2+) signaling and hyperpolarization-activated cation channels.

    PubMed

    Bernard, Marianne; Dejos, Camille; Bergès, Thierry; Régnacq, Matthieu; Voisin, Pierre

    2014-04-01

    This study reports that the spontaneous 50-fold activation of rhodopsin gene transcription, observed in cultured retinal precursors from 13-day chicken embryo, relies on a Ca(2+)-dependent mechanism. Activation of a transiently transfected rhodopsin promoter (luciferase reporter) in these cells was inhibited (60%) by cotransfection of a dominant-negative form of the cAMP-responsive element-binding protein. Both rhodopsin promoter activity and rhodopsin mRNA accumulation were blocked by Ca(2+)/calmodulin-dependent kinase II inhibitors, but not by protein kinase A inhibitors, suggesting a role of Ca(2+) rather than cAMP. This was confirmed by the inhibitory effect of general and T-type selective Ca(2+) channel blockers. Oscillations in Ca(2+) fluorescence (Fluo8) could be observed in 1/10 cells that activated the rhodopsin promoter (DsRed reporter). A robust and reversible inhibition of rhodopsin gene transcription by ZD7288 indicated a role of hyperpolarization-activated channels (HCN). Cellular localization and developmental expression of HCN1 were compatible with a role in the onset of rhodopsin gene transcription. Together, the data suggest that the spontaneous activation of rhodopsin gene transcription in cultured retinal precursors results from a signaling cascade that involves the pacemaker activity of HCN channels, the opening of voltage-gated Ca(2+)-channels, activation of Ca(2+)/calmodulin-dependent kinase II and phosphorylation of cAMP-responsive element-binding protein. Rhodopsin gene expression in cultured retinal precursors from chicken embryo relies on a Ca2+-dependent mechanism whereby hyperpolarization-activated cyclic nucleotide-gated channels (HCN) activate T-type voltage-dependent Ca2+ channels (VDCC) through membrane depolarization, causing calmodulin-dependent kinase II (CaMKII) to phosphorylate the cAMP-responsive element-binding protein (CREB) and leading to activation of rhodopsin gene transcription. Photoreceptor localization and development

  19. The Kinase Activity-deficient Isoform of the Protein Araf Antagonizes Ras/Mitogen-activated Protein Kinase (Ras/MAPK) Signaling in the Zebrafish Embryo*

    PubMed Central

    Xiong, Cong; Liu, Xingfeng; Meng, Anming

    2015-01-01

    Raf kinases are important components of the Ras-Raf-Mek-Erk pathway and also cross-talk with other signaling pathways. Araf kinase has been demonstrated to inhibit TGF-β/Smad2 signaling by directly phosphorylating and accelerating degradation of activated Smad2. In this study, we show that the araf gene expresses in zebrafish embryos to produce a shorter transcript variant, araf-tv2, in addition to the full-length variant araf-tv1. araf-tv2 is predicted to encode a C-terminally truncated peptide without the kinase activity domain. Araf-tv2 can physically associate with Araf-tv1 but does not antagonize the inhibitory effect of Araf-tv1 on TGF-β/Smad2 signaling. Instead, Araf-tv2 interacts strongly with Kras and Nras, ultimately blocking MAPK activation by these Ras proteins. In zebrafish embryos, overexpression of araf-tv2 is sufficient to inhibit Fgf/Ras-promoted Erk activation, mesodermal induction, dorsal development, and neuroectodermal posteriorization. Therefore, different isoforms of Araf may participate in similar developmental processes but by regulating different signaling pathways. PMID:26306042

  20. Angiotensin I-Converting Enzyme (ACE) Inhibitory Activity and ACE Inhibitory Peptides of Salmon (Salmo salar) Protein Hydrolysates Obtained by Human and Porcine Gastrointestinal Enzymes

    PubMed Central

    Darewicz, Małgorzata; Borawska, Justyna; Vegarud, Gerd E.; Minkiewicz, Piotr; Iwaniak, Anna

    2014-01-01

    The objectives of the present study were two-fold: first, to detect whether salmon protein fractions possess angiotensin I-converting enzyme (ACE) inhibitory properties and whether salmon proteins can release ACE inhibitory peptides during a sequential in vitro hydrolysis (with commercial porcine enzymes) and ex vivo digestion (with human gastrointestinal enzymes). Secondly, to evaluate the ACE inhibitory activity of generated hydrolysates. A two-step ex vivo and in vitro model digestion was performed to simulate the human digestion process. Salmon proteins were degraded more efficiently by porcine enzymes than by human gastrointestinal juices and sarcoplasmic proteins were digested/hydrolyzed more easily than myofibrillar proteins. The ex vivo digested myofibrillar and sarcoplasmic duodenal samples showed IC50 values (concentration required to decrease the ACE activity by 50%) of 1.06 and 2.16 mg/mL, respectively. The in vitro hydrolyzed myofibrillar and sarcoplasmic samples showed IC50 values of 0.91 and 1.04 mg/mL, respectively. Based on the results of in silico studies, it was possible to identify 9 peptides of the ex vivo hydrolysates and 7 peptides of the in vitro hydrolysates of salmon proteins of 11 selected peptides. In both types of salmon hydrolysates, ACE-inhibitory peptides IW, IY, TVY and VW were identified. In the in vitro salmon protein hydrolysates an ACE-inhibitory peptides VPW and VY were also detected, while ACE-inhibitory peptides ALPHA, IVY and IWHHT were identified in the hydrolysates generated with ex vivo digestion. In our studies, we documented ACE inhibitory in vitro effects of salmon protein hydrolysates obtained by human and as well as porcine gastrointestinal enzymes. PMID:25123137

  1. The Endoribonuclease Activity Essential for the Nonstructural Protein 11 of Porcine Reproductive and Respiratory Syndrome Virus to Inhibit NLRP3 Inflammasome-Mediated IL-1β Induction.

    PubMed

    Wang, Chao; Shi, Xibao; Zhang, Xiaozhuan; Wang, Aiping; Wang, Li; Chen, Jing; Deng, Ruiguang; Zhang, Gaiping

    2015-12-01

    NLRP3 inflammasome, which is multiprotein complex that induces the maturity and secretion of proinflammatory interleukin-1β (IL-1β), takes a bridge between the innate and adaptive immune responses to the invading pathogens. It has been shown that porcine reproductive and respiratory syndrome virus (PRRSV) could activate the NLRP3 inflammasome but induce the host's immunosuppression. This study aims to explore whether PRRSV could encode the component to antagonize the NLRP3 inflammasome. The obtained results showed that PRRSV could induce the expression and secretion of IL-1β in early infection through the pathway of NLRP3 inflammasome in porcine alveolar macrophages (PAMs), but the levels of pro-IL-1β mRNA and IL-1β protein decreased to a degree that was similar to the level of the mock-infected group in later infection. This work also found that PRRSV nonstructural protein (nsp) 11 could inhibit the expression of pro-IL-1β mRNA induced by lipopolysaccharide (LPS) and the secretion of IL-1β induced by LPS plus nigericin in PAMs. Furthermore, the mutation studies showed that the endoribonuclease activity was essential for nsp11 to inhibit the secretion of IL-1β. Therefore, it could be indicated that PRRSV could induce the activation of NLRP3 inflammasome, but the virus encoded nsp11 to inhibit this action. PMID:26398903

  2. [Activity of porcine anti-Brucella abortus immunoglobulins in the acid plate agglutination test (APAT)].

    PubMed

    Stryszak, A; Błaszczyk, B; Królak, M

    1987-01-01

    Serological activity of swine IgM and IgG against Brucella abortus in RBPT was determined in relation to four other reactions used in Poland for diagnosing brucellosis standard agglutination test, complement fixation test, antiglobulin test, 2-mercaptoethanol test). Isolation of IgG was performed by the method of filtration on Sephadex gel G-200 of swine sera raised against Brucella abortus S19 by double immunization with suspension of killed bacteria. The presence of a certain Ig class in the fractions thus obtained was confirmed by immunoelectrophoresis and immunodiffusion tests. RBPT revealed the reaction of antibodies of IgM and IgG class which proves usability of this reaction diagnosis both early (IgM) and chronic (IgG) infection with brucellosis. Both classes of antibodies mentioned above were active also in SAT and CTT. Also the results obtained in AGT and MET were found interesting. In one of the sera, the absence of incomplete antibodies was observed, whereas positive reaction in antiglobulin test was found in its fractions containing IgG. This phenomenon was determined as concealment of incomplete agglutinins through higher level of complete antibodies in normal serum. In swine (the results were different from those obtained for cattle), apart from incomplete antibodies in IgG class, the presence of these agglutinins in IgM class was noted. On the other hand, the results obtained in MET proved that IgM antibodies of swine were not totally reduced when affected by 2-mercaptoethanol. PMID:3137534

  3. Inhibitory activity of chlorogenic acids in decaffeinated green coffee beans against porcine pancreas lipase and effect of a decaffeinated green coffee bean extract on an emulsion of olive oil.

    PubMed

    Narita, Yusaku; Iwai, Kazuya; Fukunaga, Taiji; Nakagiri, Osamu

    2012-01-01

    A decaffeinated green coffee bean extract (DGCBE) inhibited porcine pancreas lipase (PPL) activity with an IC50 value of 1.98 mg/mL. Six different chlorogenic acids in DGCBE contributed to this PPL inhibition, accounting for 91.8% of the inhibitory activity. DGCBE increased the droplet size and decreased the specific surface area of an olive oil emulsion. PMID:23221697

  4. Porcine MHC classical class I genes are coordinately expressed in superantigen-activated mononuclear cells.

    PubMed

    Kametani, Yoshie; Ohshima, Shino; Kita, Yuki F; Shimada, Shin; Kamiguchi, Hiroshi; Shiina, Takashi; Inoko, Hidetoshi; Kulski, Jerzy K; Ando, Asako

    2012-08-15

    The expression of the major histocompatibility complex (MHC) classical class I genes is important for the adaptive immune response to target virus-infected cells and cancer cells. The up-regulation of the MHC is achieved by hormonal/cytokine signals including IFN-γ-inducible elements. The swine leukocyte antigen (SLA), the MHC class I region of pigs, consists of the duplicated classical class I genes, SLA-1, SLA-2 and SLA-3, but the molecular mechanisms involved in their up-regulation after T cell stimulation have not been fully elucidated. In order to better understand some of the putative regulatory mechanisms of SLA class I gene expression in activated T cells, we examined the coordinated expression of the SLA classical class I, IFN-γ and interferon regulatory factor-1 (IRF-1) genes in the peripheral blood mononuclear cells (PBMCs) of SLA homozygous Clawn miniature swine stimulated for 72 h with either IFN-γ or an enterotoxin produced by Staphylococcus aureus. This enterotoxin, toxic shock syndrome-1 (TSST-1), is known to act as a superantigen (sAG) to activate the T cells in various vertebrate species. We showed by using mAbs and flow cytometry that the CD4(+)CD25(+) cell number of swine PBMCs was also increased by TSST-1 and to a lesser degree by IFN-γ. Time course analyses of the expression of the IFN-γ, IRF-1 and the three classical class I genes, SLA-1, SLA-2, and SLA-3, in PBMCs by quantitative real-time PCR revealed a transitory response to TSST-1 or IFN-γ stimulation. The IFN-γ mRNA levels in the PBMCs were continuously up-regulated over the first 48 h by TSST-1 or IFN-γ. In contrast, SLA class I expression moderately increased at 24h and then decreased to a baseline level or less at 72 h of IFN-γ or TSST-1 stimulation. The three classical SLA class I genes showed similar expression kinetics, although SLA-3 mRNA level was consistently lower than those of SLA-1 and -2. The expression of IRF-1, a modulator of SLA expression, showed similar

  5. ACOUSTICALLY ACTIVE INJECTION CATHETER GUIDED BY ULTRASOUND: NAVIGATION TESTS IN ACUTELY ISCHEMIC PORCINE HEARTS

    PubMed Central

    Belohlavek, Marek; Katayama, Minako; Zarbatany, David; Fortuin, F. David; Fatemi, Mostafa; Nenadic, Ivan Z.; McMahon, Eileen M.

    2014-01-01

    Catheters are increasingly used therapeutically and investigatively. With complex usage comes a need for more accurate intracardiac localization than traditional guidance can provide. An injection catheter navigated by ultrasound was designed and then tested in an open-chest model of acute ischemia in eight pigs. The catheter is made “acoustically active” by a piezoelectric crystal near its tip, electronically controlled, vibrating in the acoustic frequency range, and uniquely identifiable using pulsed-wave (PW) Doppler. Another “target” crystal was sutured to the epicardium within the ischemic region. Sonomicrometry was used to measure distances between the two crystals and then compared to measurements from 2D echocardiographic images. Complete data were obtained from 7 pigs, and the correlation between sonomicrometry and ultrasound measurements was excellent (p < 0.0001, ρ = 0.9820), as was the intraclass correlation coefficient (0.96) between 2 observers. These initial experimental results suggest high accuracy of ultrasound navigation of the acoustically active catheter prototype located inside the beating left ventricle. PMID:24785441

  6. Porcine prion protein amyloid

    PubMed Central

    Hammarström, Per; Nyström, Sofie

    2015-01-01

    ABSTRACT Mammalian prions are composed of misfolded aggregated prion protein (PrP) with amyloid-like features. Prions are zoonotic disease agents that infect a wide variety of mammalian species including humans. Mammals and by-products thereof which are frequently encountered in daily life are most important for human health. It is established that bovine prions (BSE) can infect humans while there is no such evidence for any other prion susceptible species in the human food chain (sheep, goat, elk, deer) and largely prion resistant species (pig) or susceptible and resistant pets (cat and dogs, respectively). PrPs from these species have been characterized using biochemistry, biophysics and neurobiology. Recently we studied PrPs from several mammals in vitro and found evidence for generic amyloidogenicity as well as cross-seeding fibril formation activity of all PrPs on the human PrP sequence regardless if the original species was resistant or susceptible to prion disease. Porcine PrP amyloidogenicity was among the studied. Experimentally inoculated pigs as well as transgenic mouse lines overexpressing porcine PrP have, in the past, been used to investigate the possibility of prion transmission in pigs. The pig is a species with extraordinarily wide use within human daily life with over a billion pigs harvested for human consumption each year. Here we discuss the possibility that the largely prion disease resistant pig can be a clinically silent carrier of replicating prions. PMID:26218890

  7. Piglets born after intrauterine laparoscopic embryo transfer.

    PubMed

    Wieczorek, J; Koseniuk, J; Mandryk, I; Poniedziałek-Kempny, K

    2015-01-01

    The aim of the study was the preliminary development of laparoscopic transfer of embryos to the uterus in the pig, which can become the alternative for more invasive surgical methods. We proposed the original method of embryo transfer. Donors (n = 40) and recipients (n = 15) of embryos were sows of age of 6-8 months. The estrus cycle of both recipients and donors was routinely synchronized. The experimental animals were divided into two groups. In the first group (10 donors and 3 recipients) embryos were transplanted according to the method described earlier and in the second group (30 donors and 12 recipients) embryos were transplanted according to our own proposed method. As the control group, we used 16 sows after insemination (AI). In animals from both experimental groups pregnancy was diagnosed between 28-31 day after transplantation and in the control group between 28-31 day after insemination. All animals were observed during pregnancy and weaning period in pig farm. Embryos at the development stage of 2-4 cell were obtained surgically and cultured in vitro for 4 days. Obtained blastocysts were transferred to donors. The original set of catheters for blastocysts transfer to pig uterus was constructed. Three trocars were placed in abdominal cavity for inserting endoscope and 2 grasps for uterus stabilization. After uterus stabilization, the slide was inserted into abdomen which was used for putting the needle to puncture uterus. Through this needle catheter with embryos was inserted into the uterus cavity. Embryos were placed by injection into lumen of the one uterine horn. From 12 recipients pregnancy was diagnosed in 6 recipients. From 6 litters, 57 piglets were born. We weaned 41 piglets (71.9%). In our study we obtained 50% efficacy, with the mean number of 9.5 alive piglets in litter and 6.8 weaned piglets. The efficacy of developed method of laparoscopic transfer of porcine embryos allows it to be used in routine practice. PMID:26172194

  8. Upregulation of Intermediate-Conductance Ca2+-Activated K+ Channels (KCNN4) in Porcine Coronary Smooth Muscle Requires NADPH Oxidase 5 (NOX5)

    PubMed Central

    Gole, Hope K. A.; Tharp, Darla L.; Bowles, Douglas K.

    2014-01-01

    Aims NADPH oxidase (NOX) is the primary source of reactive oxygen species (ROS) in vascular smooth muscle cells (SMC) and is proposed to play a key role in redox signaling involved in the pathogenesis of cardiovascular disease. Growth factors and cytokines stimulate coronary SMC (CSMC) phenotypic modulation, proliferation, and migration during atherosclerotic plaque development and restenosis. We previously demonstrated that increased expression and activity of intermediate-conductance Ca2+-activated K+ channels (KCNN4) is necessary for CSMC phenotypic modulation and progression of stenotic lesions. Therefore, the purpose of this study was to determine whether NOX is required for KCNN4 upregulation induced by mitogenic growth factors. Methods and Results Dihydroethidium micro-fluorography in porcine CSMCs demonstrated that basic fibroblast growth factor (bFGF) increased superoxide production, which was blocked by the NOX inhibitor apocynin (Apo). Apo also blocked bFGF-induced increases in KCNN4 mRNA levels in both right coronary artery sections and CSMCs. Similarly, immunohistochemistry and whole cell voltage clamp showed bFGF-induced increases in CSMC KCNN4 protein expression and channel activity were abolished by Apo. Treatment with Apo also inhibited bFGF-induced increases in activator protein-1 promoter activity, as measured by luciferase activity assay. qRT-PCR demonstrated porcine coronary smooth muscle expression of NOX1, NOX2, NOX4, and NOX5 isoforms. Knockdown of NOX5 alone prevented both bFGF-induced upregulation of KCNN4 mRNA and CSMC migration. Conclusions Our findings provide novel evidence that NOX5-derived ROS increase functional expression of KCNN4 through activator protein-1, providing another potential link between NOX, CSMC phenotypic modulation, and atherosclerosis. PMID:25144362

  9. Cyclic AMP-dependent activation of rhodopsin gene transcription in cultured retinal precursor cells of chicken embryo.

    PubMed

    Voisin, Pierre; Bernard, Marianne

    2009-07-01

    The present study describes a robust 50-fold increase in rhodopsin gene transcription by cAMP in cultured retinal precursor cells of chicken embryo. Retinal cells isolated at embryonic day 8 (E8) and cultured for 3 days in serum-supplemented medium differentiated mostly into red-sensitive cones and to a lesser degree into green-sensitive cones, as indicated by real-time RT-PCR quantification of each specific opsin mRNA. In contrast, both rhodopsin mRNA concentration and rhodopsin gene promoter activity required the presence of cAMP-increasing agents [forskolin and 3-isobutyl-1-methylxanthine (IBMX)] to reach significant levels. This response was rod-specific and was sufficient to activate rhodopsin gene transcription in serum-free medium. The increase in rhodopsin mRNA levels evoked by a series of cAMP analogs suggested the response was mediated by protein kinase A, not by EPAC. Membrane depolarization by high KCl concentration also increased rhodopsin mRNA levels and this response was strongly potentiated by IBMX. The rhodopsin gene response to cAMP-increasing agents was developmentally gated between E6 and E7. Rod-specific transducin alpha subunit mRNA levels also increased up to 50-fold in response to forskolin and IBMX, while rod-specific phosphodiesterase-VI and rod arrestin transcripts increased 3- to 10-fold. These results suggest a cAMP-mediated signaling pathway may play a role in rod differentiation. PMID:19457115

  10. The Porcine Chloride Channel Calcium-Activated Family Member pCLCA4a Mirrors Lung Expression of the Human hCLCA4

    PubMed Central

    Plog, Stephanie; Grötzsch, Tanja; Klymiuk, Nikolai; Kobalz, Ursula; Gruber, Achim D.

    2012-01-01

    Pig models of cystic fibrosis (CF) have recently been established that are expected to mimic the human disease closer than mouse models do. The human CLCA (originally named chloride channels, calcium-activated) member hCLCA4 is considered a potential modifier of disease severity in CF, but its murine ortholog, mCLCA6, is not expressed in the mouse lung. Here, we have characterized the genomic structure, protein processing, and tissue expression patterns of the porcine ortholog to hCLCA4, pCLCA4a. The genomic structure and cellular protein processing of pCLCA4a were found to closely mirror those of hCLCA4 and mCLCA6. Similar to human lung, pCLCA4a mRNA was strongly expressed in porcine lungs, and the pCLCA4a protein was immunohistochemically detected on the apical membranes of tracheal and bronchial epithelial cells. This stands in sharp contrast to mouse mCLCA6, which has been detected exclusively in intestinal epithelia but not the murine lung. The results may add to the understanding of species-specific differences in the CF phenotype and support the notion that the CF pig model may be more suitable than murine models to study the role of hCLCA4. PMID:22205680

  11. Production of monoclonal antibodies to porcine interleukin-18 and their use for immunoaffinity purification of recombinant porcine interleukin-18.

    PubMed

    Muneta, Y; Shimoji, Y; Yokomizo, Y; Mori, Y

    2000-03-01

    We have recently reported the cloning and expression of porcine interleukin-18 (IL-18). In this study, we describe the production of anti-porcine IL-18 monoclonal antibodies (mAb) and their use in the purification of a large amount of recombinant porcine IL-18 by immunoaffinity column chromatography. Five monoclonal antibodies (2-2-B, 2-5-B, 2-13-C, 3-1-C and 5-3-B) were established and characterized. Three (2-2-B, 3-1-C and 5-3-B) of them were of IgG1 subclass, and the other two were IgMs. Epitope analysis of the three IgG1 mAbs showed that they recognized the same epitope. All five mAbs demonstrated reactivity with baculovirus generated porcine IL-18 by immunoblot analysis. Biologically active porcine IL-18 was obtained by immunoaffinity chromatography using anti-porcine IL-18 mAb at more than 85% purity from culture supernatants of Trichoplusia ni (Tn5) derived cells infected with recombinant baculovirus containing the coding sequence of porcine mature IL-18. These results suggest that the anti-porcine IL-18 mAbs established in this study are useful for one-step purification of porcine mature IL-18 as well as the detection of porcine IL-18 by immunoblotting. PMID:10699583

  12. [Influence of the activator of transcription GAL4 on growth and development of embryos and embryonic cells in primary cultures of sand dollar].

    PubMed

    Odintsova, N A; Kiselev, K V; Bulgakov, V P; Kol'tsova, E A; Iakovlev, K V

    2003-01-01

    In order to solve many tasks of biotechnology, constant lines of the cells of marine invertebrates with a high growth potential are required, which are absent at present. We used the universal activator of transcription gal4 to change the degree of expression of genes of growth factors in embryonic sea urchin cells and, thereby, increase their proliferative activity. The fertilized sea urchin eggs and dissociated embryonic cells at the blastula stage were treated with plasmids containing both the functional gene gal4 and the gene devoid of the regions encoding the activator domain. The transfection of embryonic sea urchin eggs with the functional gene led to cell dedifferentiation and formation of tumor-like structures in the embryos or increased number of embryonic cells in culture. In the cells obtained from the transfected embryos, the pigments were found within two months of cultivation, whose absorption spectrum coincided with that of echinochrome. PMID:12942737

  13. Polyalkoxybenzenes from plants. 5. Parsley seed extract in synthesis of azapodophyllotoxins featuring strong tubulin destabilizing activity in the sea urchin embryo and cell culture assays.

    PubMed

    Semenova, Marina N; Kiselyov, Alex S; Tsyganov, Dmitry V; Konyushkin, Leonid D; Firgang, Sergei I; Semenov, Roman V; Malyshev, Oleg R; Raihstat, Mikhail M; Fuchs, Fabian; Stielow, Anne; Lantow, Margareta; Philchenkov, Alex A; Zavelevich, Michael P; Zefirov, Nikolay S; Kuznetsov, Sergei A; Semenov, Victor V

    2011-10-27

    A series of 4-azapodophyllotoxin derivatives with modified rings B and E have been synthesized using allylpolyalkoxybenzenes from parsley seed oil. The targeted molecules were evaluated in vivo in a phenotypic sea urchin embryo assay for antimitotic and tubulin destabilizing activity. The most active compounds identified by the in vivo sea urchin embryo assay featured myristicin-derived ring E. These molecules were determined to be more potent than podophyllotoxin. Cytotoxic effects of selected molecules were further confirmed and evaluated by conventional assays with A549 and Jurkat human leukemic T-cell lines including cell growth inhibition, cell cycle arrest, cellular microtubule disruption, and induction of apoptosis. The ring B modification yielded 6-OMe substituted molecule as the most active compound. Finally, in Jurkat cells, compound induced caspase-dependent apoptosis mediated by the apical caspases-2 and -9 and not caspase-8, implying the involvement of the intrinsic caspase-9-dependent apoptotic pathway. PMID:21916509

  14. Polarity-Dependent Asymmetric Distribution and MEX-5/6–Mediated Translational Activation of the Era-1 mRNA in C. elegans Embryos

    PubMed Central

    Spiró, Zoltán; Gönczy, Pierre

    2015-01-01

    The early C. elegans embryo is an attractive model system to investigate fundamental developmental processes. With the exception of mex-3 mRNA, maternally contributed mRNAs are thought to be distributed uniformly in the one-cell embryo. Here, we report and characterize the striking distribution of the mRNA encoding the novel protein ERA-1. We found that era-1 mRNA is enriched in the anterior of the one-cell embryo and present solely in anterior blastomeres thereafter. Although era-1 is not an essential gene, we uncovered that era-1 null mutant embryos are sensitive to slight impairment of embryonic polarity. We found that the asymmetric distribution of era-1 mRNA depends on anterior-posterior polarity cues and on the era-1 3’UTR. Similarly to the era-1 mRNA, the YFP-ERA-1 protein is enriched in anterior blastomeres. Interestingly, we found that the RNA-binding protein MEX-5 is required for era-1 mRNA asymmetry. Furthermore, we show that MEX-5, together with its partially redundant partner MEX-6, are needed to activate era-1 mRNA translation in anterior blastomeres. These findings lead us to propose that MEX-5/6–mediated regulation of era-1 mRNA contributes to robust embryonic development. PMID:25821955

  15. Benzotriazole UV-stabilizers and benzotriazole: Antiandrogenic activity in vitro and activation of aryl hydrocarbon receptor pathway in zebrafish eleuthero-embryos.

    PubMed

    Fent, Karl; Chew, Geraldine; Li, Jun; Gomez, Elena

    2014-06-01

    Benzotriazole UV-stabilizers (BUVs) are applied in materials for protection against UV-irradiation. They are widely used, bioaccumulate and share structural similarities to benzotriazole. Benzotriazole (1HBT) finds application as corrosion inhibitor in dishwashing detergents, antifreeze (vehicles) and aircraft de-icing agent. BUVs and 1HBT are persistent and ubiquitous in the aquatic environment, but there is little understanding of the ecotoxicological implications. Here, we comparatively analyze the hormonal activity in vitro and effects in zebrafish eleuthero-embryos in vivo. 2-(2-Hydroxy-5-methylphenyl)benzotriazole (UV-P), 2-(3-t-butyl-2-hydroxy-5-methylphenyl)-5-chlorobenzotriazole (UV-326), UV-327, UV-328, UV-329 and UV-320 showed no estrogenicity (YES assay) and androgenicity (YAS assay). However, UV-P and 1HBT showed significant antiandrogenic activity. We assessed the transcription profiles of up to 26 genes associated with different toxicological pathways in zebrafish eleuthero-embryos to elucidate potential modes of action of UV-P, UV-326 and 1HBT. Embryos were experimentally exposed for 144hpf to three measured concentrations of 15.8, 70.8, and 690μg/L UV-P, 7.5, 31.7, and 84.3μg/L UV-326 and 7.9, 97.3 and 1197.3μg/L 1HBT. Among the 26 transcripts, the induction of the aryl hydrocarbon receptor (AHR) pathway by UV-P and UV-326 was the most significant finding. UV-P led to dose-related induction of AHR1, ARNT2 and cyp1a1, as well as of phase II enzymes glutathione-S-transferase (gstp1) and ugt1a. UV-326 led to a significant induction of cyp1a1 and AHR2, but down-regulation of gstp1 at 84μg/L. Only little transcriptional alterations occurred in genes related to apoptosis, oxidative stress, hormone receptors, and steroidogenesis including aromatase. 1HBT led to only a few expressional changes at 1197μg/L. Our data lead to the conclusion that UV-P and UV-326 activate the AHR-pathway, whereas 1HBT shows only little transcriptional alterations. It

  16. An extraovarian protein accumulated in mosquito oocytes is a carboxypeptidase activated in embryos

    SciTech Connect

    Wenlong Cho; Deitsch, K.W.; Raikhel, A.S. )

    1991-12-01

    The authors report a phenomenon previously unknown for oviparous animals; in Aedes aegypti mosquitoes a serine carboxypeptidase is synthesized extraovarially and then internalized by oocytes. The cDNA encoding mosquito vitellogenic carboxypeptidase (VCP) was cloned and sequenced. The VCP cDNA hybridizes to a 1.5-kilobase mRNA present only in the fat body of vitellogenic females. The deduced amino acid sequence of VCP shares significant homology with members of the serine carboxypeptidase family. Binding assays using a serine protease inhibitor, ({sup 3}H)diisopropyl fluorophosphate, showed that VCP is activated in eggs at the onset of embryonic development. Activation of VCP is associated with the reduction in its size from 53 kDa (inactive proenzyme) to 48 kDa (active enzyme). The active, 48-kDa, form of VCP is maximally present at the middle of embryonic development and disappears by the end.

  17. Construction and Evaluation of a Maize Chimeric Promoter with Activity in Kernel Endosperm and Embryo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chimeric promoters contain DNA sequences from different promoters. Chimeric promoters are developed to increase the level of recombinant protein expression, precisely control transgene activity, or to escape homology-based gene silencing. Sets of chimeric promoters, each containing different lengt...

  18. Context-dependent regulation of Dicer activity and small RNA production: Implications to oocyte-to-embryo transition.

    PubMed

    Arur, Swathi

    2015-01-01

    Cellular and molecular mechanisms that suppress small RNAs in oocytes while maintaining them in zygotes remain unknown. Signal-mediated regulation of small RNA biogenesis pathway is emerging as a theme for regulating small RNA production. We recently reported that ERK-mediated phosphorylation of Dicer, a central player in small RNA biogenesis, induced Dicer to move from the cytoplasm to the nucleus. Dicer phosphorylation inhibited its function, e.g., the production of 26G endo-siRNAs in the female germline. Moreover, our findings showed that the inhibition of Dicer function was necessary for normal progression of meiosis I and oogenesis, and that Dicer function had to be restored before fertilization for normal progression of embryogenesis. Thus, extracellular signal-dependent inhibition and then reactivation of Dicer is essential for oocyte-to-embryo transition. Strikingly, signal-induced Dicer translocation from the cytoplasm to nucleus is evolutionarily conserved from worm, flies, mice to humans thereby suggesting the ERK-mediated control of Dicer activity may be a generalized mechanism for regulating small RNA biogenesis. PMID:27123367

  19. Involvement of Sp1 and SREBP-1a in transcriptional activation of the LDL receptor gene by insulin and LH in cultured porcine granulosa-luteal cells.

    PubMed

    Sekar, Natesampillai; Veldhuis, Johannes D

    2004-07-01

    Luteinizing hormone (LH) and insulin stimulate transcriptional activity of the porcine low-density lipoprotein (LDL) receptor (LDLR) promoter supra-additively in primary cultures of granulosa-luteal cells. The mechanistic basis of this bihormonal interaction is unknown. The pig LDLR gene promoter includes three putative Sp1/Sp3-binding sites and one sterol response element (SRE) site 5' upstream to the transcriptional start site. To assess the role of SRE-binding protein (SREBP) in LDLR gene regulation, swine granulosa-luteal cells were cotransfected with CMV/SREBP-1a or SREBP-2 and the pLDLR1076/luc promoter. SREBP-1a and SREBP-2 stimulated LDLR gene transcription eight- and fourfold, respectively. LH alone augmented stimulation by SREBP-1 twofold. Conversely, cotransfection of a dominant-negative mutant form of SREBP-1a repressed basal and hormonally stimulated LDLR promoter activity by >80% (P < 0.01). Mutation of the SRE -167 ATCACCCCATG -157 to -167 ATCACCgCATG -157 bp decreased basal expression by 50% and LH + insulin- and LH + IGF-I-stimulated transcriptional activity by 80% and >90%, respectively (both P < 0.01). Mutations within each of the three flanking putative Sp1/Sp3 sites at -216/-211, -201/-196, and -151/-146 bp in the LDLR gene promoter also reduced basal activity (by >85%) and hormonal responsiveness (>95%, P < 0.05). EMSA confirmed that presumptive SRE-1 and Sp1/Sp3 elements bind respective peptides. Mithramycin, an inhibitor of Sp1/Sp3 protein(s) binding, blocked hormonally induced LDLR promoter expression by 80%. Basal transcription and supra-additive stimulation of porcine LDLR gene transcription by LH and insulin in granulosa-luteal cells require SREBP-1a and Sp1/Sp3-binding elements. PMID:14998783

  20. The role of porcine cytochrome b5A and cytochrome b5B in the regulation of cytochrome P45017A1 activities.

    PubMed

    Billen, M J; Squires, E J

    2009-01-01

    Male pigs are routinely castrated to prevent the accumulation of testicular 16-androstene steroids, in particular 5alpha-androst-16-en-3-one (5alpha-androstenone), which contribute to an off-odour and off-flavour known as boar taint. Cytochrome P450C17 (CYP17A1) catalyses the key regulatory step in the formation of the 16-androstene steroids from pregnenolone by the andien-beta synthase reaction or the synthesis of the glucocorticoid and sex steroids via 17alpha-hydroxylase and C17,20 lyase pathways respectively. We have expressed CYP17A1, along with cytochrome P450 reductase (POR), cytochrome b5 reductase (CYB5R3) and cytochrome b5 (CYB5) in HEK-293FT cells to investigate the importance of the two forms of porcine CYB5, CYB5A and CYB5B, in both the andien-beta synthase as well as the 17alpha-hydroxylase and C17,20 lyase reactions. Increasing the ratio of CYB5A to CYP17A1 caused a decrease in 17alpha-hydroxylase (p<0.013), a transient increase in C17,20 lyase, and an increase in andien-beta synthase activity (p<0.0001). Increasing the ratio of CYB5B to CYP17A1 also decreased 17alpha-hydroxylase, but did not affect the andien-beta synthase activity; however, the C17,20 lyase, was significantly increased. These results demonstrate the differential effects of two forms of CYB5 on the three activities of porcine CYP17A1 and show that CYB5B does not stimulate the andien-beta synthase activity of CYP17A1. PMID:19101629

  1. Cellular Transformation of Mouse Embryo Fibroblasts in the Absence of Activator E2Fs

    PubMed Central

    Gupta, Tushar; Sáenz Robles, Maria Teresa

    2015-01-01

    ABSTRACT The E2F family of transcription factors, broadly divided into activator and repressor E2Fs, regulates cell cycle genes. Current models indicate that activator E2Fs are necessary for cell cycle progression and tumorigenesis and are also required to mediate transformation induced by DNA tumor viruses. E2Fs are negatively regulated by the retinoblastoma (RB) family of tumor suppressor proteins, and virus-encoded oncogenes disrupt the RB-E2F repressor complexes. This results in the release of activator E2Fs and induction of E2F-dependent genes. In agreement, expression of large tumor T antigens (TAg) encoded by polyomaviruses in mammalian cells results in increased transcriptional levels of E2F target genes. In addition, tumorigenesis induced by transgenic expression of simian virus 40 (SV40) TAg in choroid plexus or intestinal villi requires at least one activator E2F. In contrast, we show that SV40 TAg-induced transformation in mouse embryonic fibroblasts is independent of activator E2Fs. This work, coupled with recent studies showing that proliferation in stem and progenitor cells is independent of activator E2Fs, suggests the presence of parallel pathways governing cell proliferation and tumorigenesis. IMPORTANCE The RB-E2F pathway is altered in many cancers and is also targeted by DNA tumor viruses. Viral oncoprotein action on RBs results in the release of activator E2Fs and upregulation of E2F target genes; thus, activator E2Fs are considered essential for normal and tumorigenic cell proliferation. However, we have observed that SV40 large T antigen can induce cell proliferation and transformation in the absence of activator E2Fs. Our results also suggest that TAg action on pRBs regulates both E2F-dependent and -independent pathways that govern proliferation. Thus, specific cell proliferation pathways affected by RB alterations in cancer may be a factor in tumor behavior and response to therapy. PMID:25717106

  2. Abcb4 acts as multixenobiotic transporter and active barrier against chemical uptake in zebrafish (Danio rerio) embryos

    PubMed Central

    2013-01-01

    Background In mammals, ABCB1 constitutes a cellular “first line of defense” against a wide array of chemicals and drugs conferring cellular multidrug or multixenobiotic resistance (MDR/MXR). We tested the hypothesis that an ABCB1 ortholog serves as protection for the sensitive developmental processes in zebrafish embryos against adverse compounds dissolved in the water. Results Indication for ABCB1-type efflux counteracting the accumulation of chemicals in zebrafish embryos comes from experiments with fluorescent and toxic transporter substrates and inhibitors. With inhibitors present, levels of fluorescent dyes in embryo tissue and sensitivity of embryos to toxic substrates were generally elevated. We verified two predicted sequences from zebrafish, previously annotated as abcb1, by cloning; our synteny analyses, however, identified them as abcb4 and abcb5, respectively. The abcb1 gene is absent in the zebrafish genome and we explored whether instead Abcb4 and/or Abcb5 show toxicant defense properties. Quantitative real-time polymerase chain reaction (qPCR) analyses showed the presence of transcripts of both genes throughout the first 48 hours of zebrafish development. Similar to transporter inhibitors, morpholino knock-down of Abcb4 increased accumulation of fluorescent substrates in embryo tissue and sensitivity of embryos toward toxic compounds. In contrast, morpholino knock-down of Abcb5 did not exert this effect. ATPase assays with recombinant protein obtained with the baculovirus expression system confirmed that dye and toxic compounds act as substrates of zebrafish Abcb4 and inhibitors block its function. The compounds tested comprised model substrates of human ABCB1, namely the fluorescent dyes rhodamine B and calcein-am and the toxic compounds vinblastine, vincristine and doxorubicin; cyclosporin A, PSC833, MK571 and verapamil were applied as inhibitors. Additionally, tests were performed with ecotoxicologically relevant compounds: phenanthrene (a

  3. Non-targeted metabolomic evaluation of the uterine milieu during the transitional period of embryo elongation in the pig

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alterations in the signaling of critical molecular factors within the uterine milieu lead to deficiencies in embryo elongation. The objective of this study was to identify metabolites within the uterine environment that are present as porcine embryos transition between spherical, ovoid, and tubular ...

  4. Novel porcine repetitive elements

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An analysis of 220 fully sequenced porcine BACs generated by the Comparative Vertebrate Sequencing Initiative (http://www.nisc.nih.gov/) revealed 27 distinct, novel porcine repetitive elements ranging in length from 55 to 1059 nucleotides. This set of fully sequenced BACs covers approximately 1% of...

  5. Fear is the mother of invention: anuran embryos exposed to predator cues alter life-history traits, post-hatching behaviour and neuronal activity patterns.

    PubMed

    Gazzola, Andrea; Brandalise, Federico; Rubolini, Diego; Rossi, Paola; Galeotti, Paolo

    2015-12-01

    Neurophysiological modifications associated to phenotypic plasticity in response to predators are largely unexplored, and there is a gap of knowledge on how the information encoded in predator cues is processed by prey sensory systems. To explore these issues, we exposed Rana dalmatina embryos to dragonfly chemical cues (kairomones) up to hatching. At different times after hatching (up to 40 days), we recorded morphology and anti-predator behaviour of tadpoles from control and kairomone-treated embryo groups as well as their neural olfactory responses, by recording the activity of their mitral neurons before and after exposure to a kairomone solution. Treated embryos hatched later and hatchlings were smaller than control siblings. In addition, the tadpoles from the treated group showed a stronger anti-predator response than controls at 10 days (but not at 30 days) post-hatching, though the intensity of the contextual response to the kairomone stimulus did not differ between the two groups. Baseline neuronal activity at 30 days post-hatching, as assessed by the frequency of spontaneous excitatory postsynaptic events and by the firing rate of mitral cells, was higher among tadpoles from the treated versus the control embryo groups. At the same time, neuronal activity showed a stronger increase among tadpoles from the treated versus the control group after a local kairomone perfusion. Hence, a different contextual plasticity between treatments at the neuronal level was not mirrored by the anti-predator behavioural response. In conclusion, our experiments demonstrate ontogenetic plasticity in tadpole neuronal activity after embryonic exposure to predator cues, corroborating the evidence that early-life experience contributes to shaping the phenotype at later life stages. PMID:26567349

  6. Porcine reproductive and respiratory syndrome virus activates the transcription of interferon alpha/beta (IFN-α/β) in monocyte-derived dendritic cells (Mo-DC).

    PubMed

    Zhang, Hanmo; Guo, Xueshui; Nelson, Eric; Christopher-Hennings, Jane; Wang, Xiuqing

    2012-10-12

    Porcine reproductive and respiratory syndrome virus (PRRSV) is known to be a poor inducer of interferon alpha/beta (IFN-α/β), which may contribute to the delayed development of adaptive immunity and the resultant viral persistence. However, the exact mechanism by which PRRSV inhibits the induction of IFN-α/β during infection of its natural host cells remains less well defined. Here, we show that PRRSV efficiently activates the transcription of IFN-α/β in porcine monocyte-derived dendritic cells (Mo-DC) in a time-dependent and transient manner; and this effect is dependent on the activation of phosphatidylinositol 3-kinase (PI3K). Despite the abundant IFN-α transcripts detected in PRRSV-infected Mo-DC, little or no detectable IFN-α is found in the supernatants and cell lysates of PRRSV-infected Mo-DC, suggesting that PRRSV either blocks the translation of IFN-α or inhibits the RNA processing and transport. Furthermore, we observed that PRRSV infection significantly reduced the induction of IFN-α by Poly I:C treatment; and virus replication is essential to the effect since heat-inactivated PRRSV has no effect on IFN-α induction by Poly I:C. Overall, our data provide evidence for the possible role of PI3K in the activation of the transcription of IFN-α/β by PRRSV. We conclude that PRRSV inhibits the induction of IFN-α in Mo-DC by as yet undefined post-transcriptional mechanisms. PMID:22592217

  7. Quantitative perturbation-based analysis of gene expression predicts enhancer activity in early Drosophila embryo.

    PubMed

    Sayal, Rupinder; Dresch, Jacqueline M; Pushel, Irina; Taylor, Benjamin R; Arnosti, David N

    2016-01-01

    Enhancers constitute one of the major components of regulatory machinery of metazoans. Although several genome-wide studies have focused on finding and locating enhancers in the genomes, the fundamental principles governing their internal architecture and cis-regulatory grammar remain elusive. Here, we describe an extensive, quantitative perturbation analysis targeting the dorsal-ventral patterning gene regulatory network (GRN) controlled by Drosophila NF-κB homolog Dorsal. To understand transcription factor interactions on enhancers, we employed an ensemble of mathematical models, testing effects of cooperativity, repression, and factor potency. Models trained on the dataset correctly predict activity of evolutionarily divergent regulatory regions, providing insights into spatial relationships between repressor and activator binding sites. Importantly, the collective predictions of sets of models were effective at novel enhancer identification and characterization. Our study demonstrates how experimental dataset and modeling can be effectively combined to provide quantitative insights into cis-regulatory information on a genome-wide scale. PMID:27152947

  8. Cyclin B Translation Depends on mTOR Activity after Fertilization in Sea Urchin Embryos.

    PubMed

    Chassé, Héloïse; Mulner-Lorillon, Odile; Boulben, Sandrine; Glippa, Virginie; Morales, Julia; Cormier, Patrick

    2016-01-01

    The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism. PMID:26962866

  9. Cyclin B Translation Depends on mTOR Activity after Fertilization in Sea Urchin Embryos

    PubMed Central

    Boulben, Sandrine; Glippa, Virginie; Morales, Julia; Cormier, Patrick

    2016-01-01

    The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism. PMID:26962866

  10. A method for using direct injection of plasmid DNA to study cis-regulatory element activity in F0 Xenopus embryos and tadpoles.

    PubMed

    Wang, Chen; Szaro, Ben G

    2015-02-01

    The ability to express exogenous reporter genes in intact, externally developing embryos, such as Xenopus, is a powerful tool for characterizing the activity of cis-regulatory gene elements during development. Although methods exist for generating transgenic Xenopus lines, more simplified methods for use with F0 animals would significantly speed the characterization of these elements. We discovered that injecting 2-cell stage embryos with a plasmid bearing a ϕC31 integrase-targeted attB element and two dual β-globin HS4 insulators flanking a reporter transgene in opposite orientations relative to each other yielded persistent expression with sufficiently high penetrance for characterizing the activity of the promoter without having to coinject integrase RNA. Expression began appropriately during development and persisted into swimming tadpole stages without perturbing the expression of the cognate endogenous gene. Coinjected plasmids having the same elements but expressing different reporter proteins were reliably coexpressed within the same cells, providing a useful control for variations in injections between animals. To overcome the high propensity of these plasmids to undergo recombination, we developed a method for generating them using conventional cloning methods and DH5α cells for propagation. We conclude that this method offers a convenient and reliable way to evaluate the activity of cis-regulatory gene elements in the intact F0 embryo. PMID:25448690

  11. Quantitative perturbation-based analysis of gene expression predicts enhancer activity in early Drosophila embryo

    PubMed Central

    Sayal, Rupinder; Dresch, Jacqueline M; Pushel, Irina; Taylor, Benjamin R; Arnosti, David N

    2016-01-01

    Enhancers constitute one of the major components of regulatory machinery of metazoans. Although several genome-wide studies have focused on finding and locating enhancers in the genomes, the fundamental principles governing their internal architecture and cis-regulatory grammar remain elusive. Here, we describe an extensive, quantitative perturbation analysis targeting the dorsal-ventral patterning gene regulatory network (GRN) controlled by Drosophila NF-κB homolog Dorsal. To understand transcription factor interactions on enhancers, we employed an ensemble of mathematical models, testing effects of cooperativity, repression, and factor potency. Models trained on the dataset correctly predict activity of evolutionarily divergent regulatory regions, providing insights into spatial relationships between repressor and activator binding sites. Importantly, the collective predictions of sets of models were effective at novel enhancer identification and characterization. Our study demonstrates how experimental dataset and modeling can be effectively combined to provide quantitative insights into cis-regulatory information on a genome-wide scale. DOI: http://dx.doi.org/10.7554/eLife.08445.001 PMID:27152947

  12. Effects of malathion, diazinon, and parathion on mallard embryo development and cholinesterase activity

    USGS Publications Warehouse

    Hoffman, D.J.; Eastin, W.C., Jr.

    1981-01-01

    The effects of external exposure of mallard (Anas platyrhynchos) eggs to malathion, diazinon, and parathion were examined using formulations and concentrations similar to field applications. Treatment with aqueous emulsion simulated exposure at the rate of 100 gal per acre (153 liters/hectare) with three to six different doses per compound with treatment at 3 and 8 days of embryonic development. Treatment with a nontoxic oil vehicle simulated exposure at the rate of 11 gal per acre (16.8 liters/hectare) with three to six different doses per compound. The order of embryotoxicity on a pounds-per-acre basis was parathion > diazinon > malathion with either vehicle. However, the potential hazard under conditions of up to five times the maximum field level of application was greater for malathion because of the high permissible level of application for malathion on certain crops. Parathion, the most embryotoxic of the three, had the most pronounced effects when an oil vehicle was used, as reflected by an LC50 of about 2 lb of active ingredient per acre, stunted growth, and a high frequency of malformations involving distortion of the axial skeleton, particularly in the cervical region. All three compounds resulted in significant depression of plasma and brain cholinesterase activity, but parathion caused the most depression throughout development, which was still apparent in hatchlings. Treatment with either distilled water or oil vehicle alone did not result in any of these effects seen with organophosphorous insecticides.

  13. Site-directed mutagenesis of porcine pepsin: Possible role of Asp32, Thr33, Asp215 and Gly217 in maintaining the nuclease activity of pepsin.

    PubMed

    Zhang, Yanfang; Liu, Yu; Guo, Hui; Jiang, Wei; Dong, Ping; Liang, Xingguo

    2016-07-01

    Site-directed mutagenesis of porcine pepsin was performed to identify its active sites that regulate nucleic acid (NA) digestion activity and to analyze the mechanism pepsin-mediated NA digestion. The mutation sites were distributed at the catalytic center of the enzyme (T33A, G34A, Y75H, T77A, Y189H, V214A, G217A and S219A) and at its active site (D32A and D215A) for protein digestion. Mutation of the active site residues Asp32 and Asp215 led to the inactivation of pepsin (both the NA and protein digestion activity), which demonstrated that the active sites of the pepsin protease activity were also important for its nuclease activity. Analysis of the variants revealed that T33A and G217A mutants showed a complete loss of NA digestion activity. In conclusion, residues Asp32, Thr33, Asp215 and Gly217 were related to the pepsin active sites for NA digestion. Moreover, the Y189H and V214A variants showed a loss of digestion activity on double-strand DNA (dsDNA) but only a decrease in digestion activity on single-strand DNA (ssDNA). On the contrary, the G34A variant showed a loss of digestion activity on ssDNA but only a decrease in digestion activity on dsDNA. Our findings are the first to identify the active sites of pepsin nuclease activity and lay the framework for further study of the mechanism of pepsin nuclease activity. PMID:27233129

  14. Ion currents in embryo development.

    PubMed

    Tosti, Elisabetta; Boni, Raffaele; Gallo, Alessandra

    2016-03-01

    Ion channels are proteins expressed in the plasma membrane of electrogenic cells. In the zygote and blastomeres of the developing embryo, electrical modifications result from ion currents that flow through these channels. This phenomenon implies that ion current activity exerts a specific developmental function, and plays a crucial role in signal transduction and the control of embryogenesis, from the early cleavage stages and during growth and development of the embryo. This review describes the involvement of ion currents in early embryo development, from marine invertebrates to human, focusing on the occurrence, modulation, and dynamic role of ion fluxes taking place on the zygote and blastomere plasma membrane, and at the intercellular communication between embryo cell stages. Birth Defects Research (Part C) 108:6-18, 2016. © 2016 Wiley Periodicals, Inc. PMID:26989869

  15. Detection of gelatinolytic activity in developing basement membranes of the mouse embryo head by combining sensitive in situ zymography with immunolabeling.

    PubMed

    Gkantidis, Nikolaos; Katsaros, Christos; Chiquet, Matthias

    2012-10-01

    Genetic evidence indicates that the major gelatinases MMP-2 and MMP-9 are involved in mammalian craniofacial development. Since these matrix metalloproteinases are secreted as proenzymes that require activation, their tissue distribution does not necessarily reflect the sites of enzymatic activity. Information regarding the spatial and temporal expression of gelatinolytic activity in the head of the mammalian embryo is sparse. Sensitive in situ zymography with dye-quenched gelatin (DQ-gelatin) has been introduced recently; gelatinolytic activity results in a local increase in fluorescence. Using frontal sections of wild-type mouse embryo heads from embryonic day 14.5-15.5, we optimized and validated a simple double-labeling in situ technique for combining DQ-gelatin zymography with immunofluorescence staining. MMP inhibitors were tested to confirm the specificity of the reaction in situ, and results were compared to standard SDS-gel zymography of tissue extracts. Double-labeling was used to show the spatial relationship in situ between gelatinolytic activity and immunostaining for gelatinases MMP-2 and MMP-9, collagenase 3 (MMP-13) and MT1-MMP (MMP-14), a major activator of pro-gelatinases. Strong gelatinolytic activity, which partially overlapped with MMP proteins, was confirmed for Meckel's cartilage and developing mandibular bone. In addition, we combined in situ zymography with immunostaining for extracellular matrix proteins that are potential gelatinase substrates. Interestingly, gelatinolytic activity colocalized precisely with laminin-positive basement membranes at specific sites around growing epithelia in the developing mouse head, such as the ducts of salivary glands or the epithelial fold between tongue and lower jaw region. Thus, this sensitive method allows to associate, with high spatial resolution, gelatinolytic activity with epithelial morphogenesis in the embryo. PMID:22688677

  16. Restriction of Porcine Endogenous Retrovirus by Porcine APOBEC3 Cytidine Deaminases ▿

    PubMed Central

    Dörrschuck, Eva; Fischer, Nicole; Bravo, Ignacio G.; Hanschmann, Kay-Martin; Kuiper, Heidi; Spötter, Andreas; Möller, Ronny; Cichutek, Klaus; Münk, Carsten; Tönjes, Ralf R.

    2011-01-01

    Xenotransplantation of porcine cells, tissues, and organs shows promise to surmount the shortage of human donor materials. Among the barriers to pig-to-human xenotransplantation are porcine endogenous retroviruses (PERV) since functional representatives of the two polytropic classes, PERV-A and PERV-B, are able to infect human embryonic kidney cells in vitro, suggesting that a xenozoonosis in vivo could occur. To assess the capacity of human and porcine cells to counteract PERV infections, we analyzed human and porcine APOBEC3 (A3) proteins. This multigene family of cytidine deaminases contributes to the cellular intrinsic immunity and act as potent inhibitors of retroviruses and retrotransposons. Our data show that the porcine A3 gene locus on chromosome 5 consists of the two single-domain genes A3Z2 and A3Z3. The evolutionary relationships of the A3Z3 genes reflect the evolutionary history of mammals. The two A3 genes encode at least four different mRNAs: A3Z2, A3Z3, A3Z2-Z3, and A3Z2-Z3 splice variant A (SVA). Porcine and human A3s have been tested toward their antiretroviral activity against PERV and murine leukemia virus (MuLV) using novel single-round reporter viruses. The porcine A3Z2, A3Z3 and A3Z2-Z3 were packaged into PERV particles and inhibited PERV replication in a dose-dependent manner. The antiretroviral effect correlated with editing by the porcine A3s with a trinucleotide preference for 5′ TGC for A3Z2 and A3Z2-Z3 and 5′ CAC for A3Z3. These results strongly imply that human and porcine A3s could inhibit PERV replication in vivo, thereby reducing the risk of infection of human cells by PERV in the context of pig-to-human xenotransplantation. PMID:21307203

  17. Interneurones in the Xenopus embryo spinal cord: sensory excitation and activity during swimming.

    PubMed Central

    Clarke, J D; Roberts, A

    1984-01-01

    The dorsolateral spinal cord of embryonic Xenopus laevis has previously been shown to contain two anatomical classes of interneurones with dendrites in the dorsal tract where they could be contacted by the central axons of Rohon-Beard cells (Roberts & Clarke, 1982). The activity of these neurones within the dorsolateral spinal cord has been examined using intracellular micro-electrodes. Following electrical stimulation of Rohon-Beard neurites within the ipsilateral skin, dorsolateral neurones receive a short-latency, compound, excitatory post-synaptic potential (e.p.s.p.). The amplitude of the e.p.s.p. depends upon the number of Rohon-Beard cells stimulated. The e.p.s.p. consists of early and later components. The early components may result from monosynaptic connexions from Rohon-Beard cells, the later components from some unidentified interposed neurones. During episodes of fictive swimming the dorsolateral neurones are inhibited by rhythmic inhibitory post-synaptic potentials. Following Rohon-Beard neurite stimulation, neurones in the contralateral spinal cord receive e.p.s.p.s. These contralateral e.p.s.p.s are probably one of the post-synaptic effects of one of the dorsolateral neurone classes. The results suggest that the dorsolateral neurones are responsible for amplifying and distributing the primary afferent signals of Rohon-Beard cells, and may be involved in the initiation of swimming and reflex movements. PMID:6481637

  18. Fertilization stimulates 8-hydroxy-2'-deoxyguanosine repair and antioxidant activity to prevent mutagenesis in the embryo.

    PubMed

    Lord, Tessa; Aitken, R John

    2015-10-01

    Oxidative DNA damage harbored by both spermatozoa and oocytes at the time of fertilization must be repaired prior to S-phase of the first mitotic division to reduce the risk of transversion mutations occurring in the zygote and subverting the normal patterns of cell differentiation and development. Of the characterised oxidative DNA lesions, 8-hydroxy-2'-deoxyguanosine (8OHdG) is particularly mutagenic. The current study reveals for the first time a marked acceleration of 8OHdG repair in the mouse oocyte/zygote by the base excision repair (BER) pathway following fertilization. Specifically, fertilization initiates post-translational modification to BER enzymes such as OGG1 and XRCC1, causing nuclear localisation and accelerated 8OHdG excision. Additionally, both the nuclear and mitochondrial genomes appear to benefit from increased protection against further 8OHdG formation by a fertilization-associated increase in glutathione peroxidase activity. The major limitation of the characterised 8OHdG repair system is the relatively low level of OGG1 expression in the oocyte, in contrast to the male germ line where it is the only constituent of the BER pathway. The male and female germ lines therefore collaborate in the repair of oxidative DNA damage, and oocytes are vulnerable to high levels of 8OHdG being carried into the zygote by the fertilizing spermatozoon. PMID:26234752

  19. Data on the concentrations of etoposide, PSC833, BAPTA-AM, and cycloheximide that do not compromise the vitality of mature mouse oocytes, parthenogencially activated and fertilized embryos.

    PubMed

    Martin, Jacinta H; Bromfield, Elizabeth G; Aitken, R John; Lord, Tessa; Nixon, Brett

    2016-09-01

    These data document the vitality of mature mouse oocytes (Metaphase II (MII)) and early stage embryos (zygotes) following exposure to the genotoxic chemotherapeutic agent, etoposide, in combination with PSC833, a selective inhibitor of permeability glycoprotein. They also illustrate the vitality of parthenogencially activated and fertilized embryos following incubation with the calcium chelator BAPTA-AM (1,2-Bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester)), cycloheximide (an antibiotic that is capable of inhibiting protein synthesis), and hydrogen peroxide (a potent reactive oxygen species). Finally, they present evidence that permeability glycoprotein is not represented in the proteome of mouse spermatozoa. Our interpretation and discussion of these data feature in the article "Identification of a key role for permeability glycoprotein in enhancing the cellular defense mechanisms of fertilized oocytes" (Martin et al., in press) [1]. PMID:27547800

  20. Anti-mitotic activity towards sea urchin embryos in extracts from the marine haptophycean Phaeocystis pouchetii (Hariot) Lagerheim collected along the coast of northern Norway.

    PubMed

    Hansen, Espen; Eilertsen, Hans Chr; Ernstsen, Arild; Genevière, Anne-Marie

    2003-06-01

    The marine bloom-forming alga Phaeocystis pouchetii is suspected to produce some toxic compound responsible for reduced growth, fecundity and survival of other marine organisms. Sea urchin early development was used as a model to investigate the degree and nature of toxicity. Colonial cells of P. pouchetii were collected during its spring-bloom along the coast of northern Norway and maintained in culture for a short period of time in order to evaluate the concentration of toxic compounds present inside the cells or excreted to the surrounding seawater medium. Cells were harvested by filtration and toxins were extracted separately from the collected cells and the filtrate using organic solvents. We found that extracts from the filtered seawater at a concentration corresponding to 9.0 x 10(5) cells ml(-1) completely blocked cell divisions in embryos of the sea urchin Sphaerechinus granularis, whereas extracts from intact algal cells were only mildly cytotoxic. When the extracts from seawater culture medium were purified by RP-HPLC, cytotoxic activity towards S. granularis embryos was recovered in three consecutive fractions. Moreover, unfertilised eggs incubated in the active HPLC fractions became unproductive, whereas incubation of sperm gave a reduced fertilisation rate. This anti-proliferative effect was further characterized by immunofluorescence staining of sea urchin embryos. DNA labelling revealed that incubating sea urchin embryos in the purified algal extracts inhibited both pronuclei migration and fusion. Incorporation and detection of the DNA-base analogue 5-bromo-2-deoxyuridine showed that DNA-replication was blocked. Furthermore, staining of alpha-tubulin subunits demonstrated that embryonic tubulin organisation was altered. We conclude that P. pouchetii produce some anti-mitotic compound, and that senescent colonial cells to a great extent excrete this compound to their surroundings. PMID:12782080

  1. Acute toxicity and sublethal effects of the mixture glyphosate (Roundup Active) and Cosmo-Flux 411F to anuran embryos and tadpoles of four Colombian species.

    PubMed

    Henao Muñoz, Liliana Marcela; Montes Rojas, Claudia Marsela; Bernal Bautista, Manuel Hernando

    2015-03-01

    Glyphosate is the most widely used herbicide in the world with application in agriculture, forestry, industrial weed control, garden and aquatic environments. However, its use is highly controversial for the possible impact on not-target organisms, such as amphibians, which are vanishing at an alarming and rapid rate. Due to the high solubility in water and ionic nature, the glyphosate requires of surfactants to increase activity. In addition, for the control of coca (Erythroxylum coca) and agricultural weeds in Colombia, formulated glyphosate is mixed and sprayed with the adjuvant Cosmo-Flux 411F to increase the penetration and activity of the herbicide. This study evaluates the acute toxic and sublethal effects (embryonic development, tadpole body size, tadpole swimming performance) of the mixture of the formulated glyphosate Roundup Active and Cosmo-Flux 411F to anuran embryos and tadpoles of four Colombian species under 96h laboratory standard tests and microcosms, which are more similar to field conditions as they include soil, sand and macrophytes. In the laboratory, embryos and tadpoles of Engystomops pustulosus were the most tolerant (LC50 = 3904 microg a.e./L; LC50=2 799 pg a.e./L, respectively), while embryos and tadpoles of Hypsiboas crepitans (LC50=2 203 microg a.e./L; LC50=1424 microgg a.e./L, respectively) were the most sensitive. R. humboldti and R. marina presented an intermediate toxicity. Embryos were significantly more tolerant to the mixture than tadpoles, which could be likely attributed to the exclusion of chemicals by the embryonic membranes and the lack of organs, such as gills, which are sensitive to surfactants. Sublethal effects were observed for the tadpole body size, but not for the embryonic development and tadpole swimming performance. In microcosms, no toxicity (LC50 could not be estimated), or sublethal responses were observed at concentrations up to fourfold (14.76 kg glyphosate a.e./ha) the highest field application rate of 3

  2. Immunohistochemistry of porcine skin.

    PubMed

    Wollina, U; Berger, U; Mahrle, G

    1991-01-01

    The present paper reports immunohistological findings in porcine skin, which were obtained by use of mono- and polyclonal antihuman antibodies and either alkaline phosphatase anti-alkaline phosphatase (APAAP) or peroxidase (POX) technique. Epidermal staining was observed with antibodies to keratins (K 8.12, RSKE 60), filaggrin, and calmodulin (ACAM). Staining of connective tissue and vessels was achieved using antibodies to vimentin (V9(1)), collagen type IV, and fibronectin. In general, these antibodies gave a staining pattern similar to that of normal human skin. The similarities of immunoreactivity to poly- and monoclonal antihuman antibodies in porcine and human skin render porcine skin a reliable model in biomedical research. PMID:1710864

  3. SNW1 is a critical regulator of spatial BMP activity, neural plate border formation, and neural crest specification in vertebrate embryos.

    PubMed

    Wu, Mary Y; Ramel, Marie-Christine; Howell, Michael; Hill, Caroline S

    2011-01-01

    Bone morphogenetic protein (BMP) gradients provide positional information to direct cell fate specification, such as patterning of the vertebrate ectoderm into neural, neural crest, and epidermal tissues, with precise borders segregating these domains. However, little is known about how BMP activity is regulated spatially and temporally during vertebrate development to contribute to embryonic patterning, and more specifically to neural crest formation. Through a large-scale in vivo functional screen in Xenopus for neural crest fate, we identified an essential regulator of BMP activity, SNW1. SNW1 is a nuclear protein known to regulate gene expression. Using antisense morpholinos to deplete SNW1 protein in both Xenopus and zebrafish embryos, we demonstrate that dorsally expressed SNW1 is required for neural crest specification, and this is independent of mesoderm formation and gastrulation morphogenetic movements. By exploiting a combination of immunostaining for phosphorylated Smad1 in Xenopus embryos and a BMP-dependent reporter transgenic zebrafish line, we show that SNW1 regulates a specific domain of BMP activity in the dorsal ectoderm at the neural plate border at post-gastrula stages. We use double in situ hybridizations and immunofluorescence to show how this domain of BMP activity is spatially positioned relative to the neural crest domain and that of SNW1 expression. Further in vivo and in vitro assays using cell culture and tissue explants allow us to conclude that SNW1 acts upstream of the BMP receptors. Finally, we show that the requirement of SNW1 for neural crest specification is through its ability to regulate BMP activity, as we demonstrate that targeted overexpression of BMP to the neural plate border is sufficient to restore neural crest formation in Xenopus SNW1 morphants. We conclude that through its ability to regulate a specific domain of BMP activity in the vertebrate embryo, SNW1 is a critical regulator of neural plate border formation and

  4. The nonstructural protein 11 of porcine reproductive and respiratory syndrome virus inhibits NF-κB signaling by means of its deubiquitinating activity.

    PubMed

    Wang, Dang; Fan, Jinxiu; Fang, Liurong; Luo, Rui; Ouyang, Haiping; Ouyang, Chao; Zhang, Huan; Chen, Huanchun; Li, Kui; Xiao, Shaobo

    2015-12-01

    Since its emergence in the late 1980s, porcine reproductive and respiratory syndrome (PRRS) has been devastating the swine industry worldwide. The causative agent is an Arterivirus, referred to as PRRS virus (PRRSV). The pathogenic mechanisms of PRRS are poorly understood, but are believed to correlate with the ability of PRRSV to inhibit immune responses of the host. However, precisely how the virus is capable of doing so remains obscure. In this study, we showed that PRRSV infection led to reduced ubiquitination of cellular proteins. Screening all of the 12 nonstructural proteins (Nsps) encoded by PRRSV revealed that, apart from the Nsp2 which contains the deubiqintinating (DUB) ovarian tumor (OTU) domain, Nsp11, which encodes a unique and conserved endoribonuclease (NendoU) throughout the Nidovirus order, also possesses DUB activity. In vivo assay demonstrated that Nsp11 specifically removed lysine 48 (K48)-linked polyubiquitin chains and the conserved sites C112, H144, D173, K180, and Y219 were critical for its DUB activity. Remarkably, DUB activity was responsible for the capacity of Nsp11 to inhibit nuclear factor κB (NF-κB) activation. Mutations abrogating the DUB activity of Nsp11 toward K48-linked polyubiquitin chains of IκBα nullified the suppressive effect on NF-κB. Our data add Nsp11 to the list of DUBs encoded by PRRSV and uncover a novel mechanism by which PRRSV cripples host innate immune responses. PMID:26342881

  5. Oral administration recombinant porcine epidermal growth factor enhances the jejunal digestive enzyme genes expression and activity of early-weaned piglets.

    PubMed

    Lee, D N; Chuang, Y S; Chiou, H Y; Wu, F Y; Yen, H T; Weng, C F

    2008-08-01

    This study attempted to determine ingested porcine epidermal growth factor (pEGF) on the gastrointestinal tract development of early-weaned piglets. Thirty-two piglets (14-day weaned) were randomly allotted to supplemented with 0 (control), 0.5, 1.0, or 1.5 mg pEGF/kg diet. Each treatment consisted of four replicates with two pigs per pen for a 14 days experimental period. Piglets were sacrificed and gastrointestinal tract samples were collected to measure mucosa morphology, mRNA expression and activities of digestive enzymes in the gastrointestinal tract of piglets at the end of the experiment. Diets supplemented with pEGF failed to influence growth performance but tended to increase jejunal mucosa weight (p < 0.09) and protein content (p < 0.07). Piglets supplemental pEGF induced incrementally the gastric pepsin activity (p < 0.05) and stimulated jejunal alkaline phosphatase (ALP) and lactase activities accompanied with the increase of jejunal ALP and maltase mRNA expression. No effect of pEGF on the activities of all enzymes in ileum except the stimulation of ileal aminopeptide N mRNA expression. These results reveal that dietary pEGF supplementation might enhance gene expression and activities of digestive enzymes in the stomach and jejunum of piglets. PMID:18662356

  6. The FRK1 mitogen-activated protein kinase kinase kinase (MAPKKK) from Solanum chacoense is involved in embryo sac and pollen development

    PubMed Central

    Lafleur, Edith; Kapfer, Christelle; Joly, Valentin; Liu, Yang; Tebbji, Faiza; Daigle, Caroline; Gray-Mitsumune, Madoka; Cappadocia, Mario; Nantel, André; Matton, Daniel P.

    2015-01-01

    The fertilization-related kinase 1 (ScFRK1), a nuclear-localized mitogen-activated protein kinase kinase kinase (MAPKKK) from the wild potato species Solanum chacoense, belongs to a small group of pMEKKs that do not possess an extended N- or C-terminal regulatory domain. Initially selected based on its highly specific expression profile following fertilization, in situ expression analyses revealed that the ScFRK1 gene is also expressed early on during female gametophyte development in the integument and megaspore mother cell and, later, in the synergid and egg cells of the embryo sac. ScFRK1 mRNAs are also detected in pollen mother cells. Transgenic plants with lower or barely detectable levels of ScFRK1 mRNAs lead to the production of small fruits with severely reduced seed set, resulting from a concomitant decline in the number of normal embryo sacs produced. Megagametogenesis and microgametogenesis were affected, as megaspores did not progress beyond the functional megaspore (FG1) stage and the microspore collapsed around the first pollen mitosis. As for other mutants that affect embryo sac development, pollen tube guidance was severely affected in the ScFRK1 transgenic lines. Gametophyte to sporophyte communication was also affected, as observed from a marked change in the transcriptomic profiles of the sporophytic tissues of the ovule. The ScFRK1 MAPKKK is thus involved in a signalling cascade that regulates both male and female gamete development. PMID:25576576

  7. Morphological transformation and effect on gap junction intercellular communication in Syrian hamster embryo cells as screening tests for carcinogens devoid of mutagenic activity.

    PubMed

    Rivedal, E; Mikalsen, S O; Sanner, T

    2000-04-01

    A large fraction of chemicals observed to cause cancer in experimental animals is devoid of mutagenic activity. It is therefore of importance to develop methods that can be used to detect and study environmental carcinogenic agents that do not interact directly with DNA. Previous studies have indicated that induction of in vitro cell transformation and inhibition of gap junction intercellular communication are endpoints that could be useful for the detection of non-genotoxic carcinogens. In the present work, 13 compounds [chlordane, Arochlor 1260, di(2-ethylhexyl)phthalate, 1,1,1-trichloro-2, 2-bis(4-chlorophenyl)ethane, limonene, sodium fluoride, ethionine, o-anisidine, benzoyl peroxide, o-vanadate, phenobarbital, 12-O-tetradecanoylphorbol 13-acetate and clofibrate] have been tested for their ability to induce morphological transformation and affect intercellular communication in Syrian hamster embryo cells. The substances were selected on the basis of being proven or suspected non-genotoxic carcinogens, and thus difficult to detect in short-term tests. The data show that nine of the 13 compounds induced morphological transformation, and seven of the 13 inhibited intercellular communication in hamster embryo cells. Taken together, 12 of the 13 substances either induced transformation or caused inhibition of communication. The data suggest that the combined use of morphological transformation and gap junction intercellular communication in Syrian hamster embryo cells may be beneficial when screening for non-genotoxic carcinogens. PMID:10793297

  8. The FRK1 mitogen-activated protein kinase kinase kinase (MAPKKK) from Solanum chacoense is involved in embryo sac and pollen development.

    PubMed

    Lafleur, Edith; Kapfer, Christelle; Joly, Valentin; Liu, Yang; Tebbji, Faiza; Daigle, Caroline; Gray-Mitsumune, Madoka; Cappadocia, Mario; Nantel, André; Matton, Daniel P

    2015-04-01

    The fertilization-related kinase 1 (ScFRK1), a nuclear-localized mitogen-activated protein kinase kinase kinase (MAPKKK) from the wild potato species Solanum chacoense, belongs to a small group of pMEKKs that do not possess an extended N- or C-terminal regulatory domain. Initially selected based on its highly specific expression profile following fertilization, in situ expression analyses revealed that the ScFRK1 gene is also expressed early on during female gametophyte development in the integument and megaspore mother cell and, later, in the synergid and egg cells of the embryo sac. ScFRK1 mRNAs are also detected in pollen mother cells. Transgenic plants with lower or barely detectable levels of ScFRK1 mRNAs lead to the production of small fruits with severely reduced seed set, resulting from a concomitant decline in the number of normal embryo sacs produced. Megagametogenesis and microgametogenesis were affected, as megaspores did not progress beyond the functional megaspore (FG1) stage and the microspore collapsed around the first pollen mitosis. As for other mutants that affect embryo sac development, pollen tube guidance was severely affected in the ScFRK1 transgenic lines. Gametophyte to sporophyte communication was also affected, as observed from a marked change in the transcriptomic profiles of the sporophytic tissues of the ovule. The ScFRK1 MAPKKK is thus involved in a signalling cascade that regulates both male and female gamete development. PMID:25576576

  9. Influence of chitosan nanofiber scaffold on porcine endogenous retroviral expression and infectivity in pig hepatocytes

    PubMed Central

    Han, Bing; Shi, Xiao-Lei; Xiao, Jiang-Qiang; Zhang, Yue; Chu, Xue-Hui; Gu, Jin-Yang; Tan, Jia-Jun; Gu, Zhong-Ze; Ding, Yi-Tao

    2011-01-01

    AIM: To investigate the influence of chitosan nanofiber scaffold on the production and infectivity of porcine endogenous retrovirus (PERV) expressed by porcine hepatocytes. METHODS: Freshly isolated porcine hepatocytes were cultured with or without chitosan nanofiber scaffold (defined as Nano group and Hep group) for 7 d. The daily collection of culture medium was used to detect reverse transcriptase (RT) activity with RT activity assay kits and PERV RNA by reverse transcription-polymerase chain reaction (PCR) and real time PCR with the PERV specific primers. And Western blotting was performed with the lysates of daily retrieved cells to determine the PERV protein gag p30. Besides, the in-vitro infectivity of the supernatant was tested by incubating the human embryo kidney 293 (HEK293) cells. RESULTS: The similar changing trends between two groups were observed in real time PCR, RT activity assay and Western blotting. Two peaks of PERV expression at 10H and Day 2 were found and followed by a regular decline. No significant difference was found between two groups except the significantly high level of PERV RNA at Day 6 and PERV protein at Day 5 in Nano group than that in Hep group. And in the in-vitro infection experiment, no HEK293 cell was infected by the supernatant. CONCLUSION: Chitosan nanofiber scaffold might prolong the PERV secreting time in pig hepatocytes but would not obviously influence its productive amount and infectivity, so it could be applied in the bioartificial liver without the increased risk of the virus transmission. PMID:21734784

  10. In situ autoradiography and ligand-dependent tyrosine kinase activity reveal insulin receptors and insulin-like growth factor I receptors in prepancreatic chicken embryos.

    PubMed Central

    Girbau, M; Bassas, L; Alemany, J; de Pablo, F

    1989-01-01

    We previously reported specific cross-linking of 125I-labeled insulin and 125I-labeled insulin-like growth factor I (IGF-I) to the alpha subunit of their respective receptors in chicken embryos of 20 somites and older. To achieve adequate sensitivity and localize spatially the receptors in younger embryos, we adapted an autoradiographic technique using whole-mounted chicken blastoderms. Insulin receptors and IGF-I receptors were expressed and could be localized as early as gastrulation, before the first somite is formed. Relative density was analyzed by a computer-assisted image system, revealing overall slightly higher binding of IGF-I than of insulin. Structures rich in both types of receptors were predominantly of ectodermal origin: Hensen's node in gastrulating embryos and neural folds, neural tube and optic vesicles during neurulation. The signal transduction capability of the receptors in early organogenesis was assessed by their ability to phosphorylate the exogenous substrate poly(Glu80Tyr20). Ligand-dependent tyrosine phosphorylation was demonstrable with both insulin and IGF-I in glycoprotein-enriched preparations from embryos at days 2 through 6 of embryogenesis. There was a developmentally regulated change in ligand-dependent tyrosine kinase activity, with a sharp increase from day 2 to day 4, in contrast with a small increase in the ligand binding. Binding of 125I-labeled IGF-I was, with the solubilized receptors, severalfold higher than binding of 125I-labeled insulin. However, the insulin-dependent phosphorylation was as high as the IGF-I-dependent phosphorylation at each developmental stage. Images PMID:2548191

  11. DIDS inhibits Na-K-ATPase activity in porcine nonpigmented ciliary epithelial cells by a Src family kinase-dependent mechanism.

    PubMed

    Shahidullah, Mohammad; Wei, Guojun; Delamere, Nicholas A

    2013-09-01

    The anion transport inhibitor DIDS is known to reduce aqueous humor secretion but questions remain about anion dependence of the effect. In some tissues, DIDS is reported to cause Na-K-ATPase inhibition. Here, we report on the ability of DIDS to inhibit Na-K-ATPase activity in nonpigmented ciliary epithelium (NPE) and investigate the underlying mechanism. Porcine NPE cells were cultured to confluence on permeable supports, treated with drugs added to both sides of the membrane, and then used for (86)Rb uptake measurements or homogenized to measure Na-K-ATPase activity or to detect protein phosphorylation. DIDS inhibited ouabain-sensitive (86)Rb uptake, activated Src family kinase (SFK), and caused a reduction of Na-K-ATPase activity. PP2, an SFK inhibitor, prevented the DIDS responses. In BCECF-loaded NPE, DIDS was found to reduce cytoplasmic pH (pHi). PP2-sensitive Na-K-ATPase activity inhibition, (86)Rb uptake suppression, and SFK activation were observed when a similar reduction of pHi was imposed by low-pH medium or an ammonium chloride withdrawal maneuver. PP2 and the ERK inhibitor U0126 prevented robust ERK1/2 activation in cells exposed to DIDS or subjected to pHi reduction, but U0126 did not prevent SFK activation or the Na-K-ATPase activity response. The evidence points to an inhibitory influence of DIDS on NPE Na-K-ATPase activity by a mechanism that hinges on SFK activation associated with a reduction of cytoplasmic pH. PMID:23677800

  12. Maternal immune activation evoked by polyinosinic:polycytidylic acid does not evoke microglial cell activation in the embryo

    PubMed Central

    Smolders, Silke; Smolders, Sophie M. T.; Swinnen, Nina; Gärtner, Annette; Rigo, Jean-Michel; Legendre, Pascal; Brône, Bert

    2015-01-01

    Several studies have indicated that inflammation during pregnancy increases the risk for the development of neuropsychiatric disorders in the offspring. Morphological brain abnormalities combined with deviations in the inflammatory status of the brain can be observed in patients of both autism and schizophrenia. It was shown that acute infection can induce changes in maternal cytokine levels which in turn are suggested to affect fetal brain development and increase the risk on the development of neuropsychiatric disorders in the offspring. Animal models of maternal immune activation reproduce the etiology of neurodevelopmental disorders such as schizophrenia and autism. In this study the poly (I:C) model was used to mimic viral immune activation in pregnant mice in order to assess the activation status of fetal microglia in these developmental disorders. Because microglia are the resident immune cells of the brain they were expected to be activated due to the inflammatory stimulus. Microglial cell density and activation level in the fetal cortex and hippocampus were determined. Despite the presence of a systemic inflammation in the pregnant mice, there was no significant difference in fetal microglial cell density or immunohistochemically determined activation level between the control and inflammation group. These data indicate that activation of the fetal microglial cells is not likely to be responsible for the inflammation induced deficits in the offspring in this model. PMID:26300736

  13. Differential Regulation of Disheveled in a Novel Vegetal Cortical Domain in Sea Urchin Eggs and Embryos: Implications for the Localized Activation of Canonical Wnt Signaling

    PubMed Central

    Peng, ChiehFu Jeff; Wikramanayake, Athula H.

    2013-01-01

    Pattern formation along the animal-vegetal (AV) axis in sea urchin embryos is initiated when canonical Wnt (cWnt) signaling is activated in vegetal blastomeres. The mechanisms that restrict cWnt signaling to vegetal blastomeres are not well understood, but there is increasing evidence that the egg’s vegetal cortex plays a critical role in this process by mediating localized “activation” of Disheveled (Dsh). To investigate how Dsh activity is regulated along the AV axis, sea urchin-specific Dsh antibodies were used to examine expression, subcellular localization, and post-translational modification of Dsh during development. Dsh is broadly expressed during early sea urchin development, but immunolocalization studies revealed that this protein is enriched in a punctate pattern in a novel vegetal cortical domain (VCD) in the egg. Vegetal blastomeres inherit this VCD during embryogenesis, and at the 60-cell stage Dsh puncta are seen in all cells that display nuclear β-catenin. Analysis of Dsh post-translational modification using two-dimensional Western blot analysis revealed that compared to Dsh pools in the bulk cytoplasm, this protein is differentially modified in the VCD and in the 16-cell stage micromeres that partially inherit this domain. Dsh localization to the VCD is not directly affected by disruption of microfilaments and microtubules, but unexpectedly, microfilament disruption led to degradation of all the Dsh pools in unfertilized eggs over a period of incubation suggesting that microfilament integrity is required for maintaining Dsh stability. These results demonstrate that a pool of differentially modified Dsh in the VCD is selectively inherited by the vegetal blastomeres that activate cWnt signaling in early embryos, and suggests that this domain functions as a scaffold for localized Dsh activation. Localized cWnt activation regulates AV axis patterning in many metazoan embryos. Hence, it is possible that the VCD is an evolutionarily conserved

  14. Induction of Angiogenesis in Zebrafish Embryos and Proliferation of Endothelial Cells by an Active Fraction Isolated from the Root of Astragalus membranaceus using Bioassay-guided Fractionation

    PubMed Central

    Lai, Patrick Kwok-Kin; Chan, Judy Yuet-Wa; Kwok, Hin-Fai; Cheng, Ling; Yu, Hua; Lau, Ching-Po; Leung, Ping-Chung; Fung, Kwok-Pui; Lau, Clara Bik-San

    2014-01-01

    The objective of the study was to identify the active fraction(s) from AR aqueous extract responsible for promoting angiogenesis using bioassay-guided fractionation. The angiogenic activity was screened by monitoring the increase of sprout number in sub-intestinal vessel (SIV) of the transgenic zebrafish embryos after they were treated with 0.06-0.25 mg/ml of AR aqueous extract or its fraction(s) for 96 h. Furthermore, the angiogenic effect was evaluated in treated zebrafish embryos by measuring the gene expression of angiogenic markers (VEGFA, KDR, and Flt-1) using real-time polymerase chain reaction (RT-PCR), and in human microvascular endothelial cell (HMEC-1) by measuring cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 3H-thymidine uptake assay, and cell cycle analysis. A major active fraction (P1-1-1), which was identified as glycoproteins, was found to significantly stimulate sprout formation (2.03 ± 0.27) at 0.125 mg/ml (P < 0.001) and up-regulate the gene expression of VEGFA, KDR, and Flt-1 by 2.6-fold to 8.2-fold. Additionally, 0.031-0.125 mg/ml of P1-1-1 was demonstrated to significantly stimulate cell proliferation by increasing cell viability (from 180% to 205%), 3H-thymidine incorporation (from 126% to 133%) during DNA synthesis, and the shift of cell population to S phase of cell cycle. A major AR active fraction consisting of glycoproteins was identified, and shown to promote angiogenesis in zebrafish embryos and proliferation of endothelial cells in vitro. PMID:25379465

  15. Peroxidase and catalase activities are involved in direct adventitious shoot formation induced by thidiazuron in eastern white pine (Pinus strobus L.) zygotic embryos.

    PubMed

    Tang, Wei; Newton, Ronald J

    2005-08-01

    We reported establishment of an efficient plant regeneration procedure through direct adventitious shoot (DAS) formation from cotyledons and hypocotyls of eastern white pine (Pinus strobus L.) mature embryos in this investigation. Multiple DASs were initiated from cotyledons of embryos on PS medium containing N6-benzyladenine (BA), thidiazuron (TDZ), or kinetin (KIN). Among different concentrations of casein enzymatic hydrosylate (CH) and glutamine used in this study, 500 mg l(-1) CH or 600 mg l(-1) glutamine induced the highest frequency of DAS formation. Rooting of regenerated shoots was obtained on PS medium supplemented with 0.01-0.1 microM indole-3-acetic acid (IAA) with the highest frequency on medium containing 0.01 muM IAA. No DASs were obtained on medium without TDZ. Measurement of peroxidase (POD) and catalase (CAT) activity during direct shoot induction and differentiation demonstrated that the lowest POD activity appeared in the 5-6th week of culture and lowest CAT activity occurred in the 7-8th week of culture on medium with TDZ. No such a change in POD and CAT activities was observed on medium without TDZ. These results demonstrated that POD and CAT activities were involved in DAS formation induced by TDZ in eastern white pine. PMID:16129608

  16. The N-terminus of porcine circovirus type 2 replication protein is required for nuclear localization and ori binding activities

    SciTech Connect

    Lin, W.-L.; Chien, M.-S.; Du, Y.-W.; Wu, P.-C.; Huang Chienjin

    2009-02-20

    Porcine circovirus type 2 possesses a circular, single-stranded DNA genome that requires the replication protein (Rep) for virus replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the Rep protein, the defined coding regions of rep gene were cloned and expressed. All of the recombinant proteins except for the N-terminal 110 residues deletion mutant could bind to the double-stranded minimal binding site of replication origin (ori). In addition, the N-terminal deletion mutant lacking 110 residues exhibited mainly cytoplasmic staining in the transfected cells in contrast to the others, which localized dominantly in the nucleus, suggesting that this N-terminal domain is essential for nuclear localization. Furthermore, a series of green fluorescence proteins (GFP) containing potential nuclear localization signal (NLS) sequences were tested for their cellular distribution. The ability of the utmost 20 residues of the N-terminal region to target the GFP to the nucleus confirmed its role as a functional NLS.

  17. Evaluation of anticoccidial drugs in chicken embryos.

    PubMed

    Xie, M Q; Fukata, T; Gilbert, J M; McDougald, L R

    1991-01-01

    Infections of Eimeria tenella in chicken embryos were used to compare the anticoccidial activity of ten drugs. The minimal inhibitory concentration (MIC) and minimal toxic concentration (MTC) were affected by the time of inoculation into the embryos and by the chemical nature of the compounds. Some compounds (nicarbazin, amprolium) had no effect on the development of coccidia when they were injected into embryos after the day of infection. Drugs that act early in the life cycle of coccidia (robenidine, clopidol, decoquinate, diclazuril, halofuginone, monensin, salinomycin, and lasalocid) were active at 5-125 micrograms/embryo when they were injected on the day of infection. The ionophores and halofuginone were highly toxic to embryos; most synthetic compounds were nontoxic. The incubation of merozoites in drug suspensions prior to the infection of embryos did not result in embryo toxicity, but the resultant MICs were much higher than those obtained when drugs were injected directly into the embryos. Several products were essentially inactive. Neither nicarbazin nor amprolium prevented oocyst formation. The widely divergent endpoints for the MIC and MTC of anticoccidials in embryos seriously limits the application of this technique as a screen for anticoccidial drugs. PMID:1792230

  18. Investigation on CAST, CAPN1 and CAPN3 porcine gene polymorphisms and expression in relation to post-mortem calpain activity in muscle and meat quality.

    PubMed

    Gandolfi, G; Pomponio, L; Ertbjerg, P; Karlsson, A H; Nanni Costa, L; Lametsch, R; Russo, V; Davoli, R

    2011-08-01

    This study aimed to detect variability in CAST, CAPN1 and CAPN3 porcine genes and to investigate the effect of CAST and CAPN1 polymorphisms on the activity of native and autolyzed μ-calpain and m-calpain, measured from 1 to 72 h post-mortem in Longissimus dorsi (LD) muscle of 30 pigs. Effects of polymorphisms on meat quality parameter such as pH, color and drip loss were also evaluated. Samples carrying CAST EU137105:g.76,872AA genotype showed higher autolyzed μ-calpain activity 24 and 72 h post-mortem, as well as lower drip loss values. Expression of CAST, CAPN1 and CAPN3 was assessed in LD muscles divergent for shear force. Higher CAST and CAPN3 expression was found in LD with high shear force (P<0.2), confirming a direct role for calpastatin but not for calpain 3 in meat tenderization. In conclusion, CAST gene affected post-mortem activation time of calpain and drip loss. PMID:21450414

  19. The Adjuvant Activity of Epimedium Polysaccharide-Propolis Flavone Liposome on Enhancing Immune Responses to Inactivated Porcine Circovirus Vaccine in Mice.

    PubMed

    Fan, Yunpeng; Guo, Liwei; Hou, Weifeng; Guo, Chao; Zhang, Weimin; Ma, Xia; Ma, Lin; Song, Xiaoping

    2015-01-01

    Objectives. The adjuvant activity of Epimedium polysaccharide-propolis flavone liposome (EPL) was investigated in vitro and in vivo. Methods. In vitro, the effects of EPL at different concentrations on splenic lymphocytes proliferation and mRNA expression of IFN-γ and IL-6 were determined. In vivo, the adjuvant activities of EPL, EP, and mineral oil were compared in BALB/c mice through vaccination with inactivated porcine circovirus type 2 (PCV2) vaccine. Results. In vitro, EPL promoted lymphocytes proliferation and increased the mRNA expression of IFN-γ and IL-6, and the effect was significantly better than EP at all concentrations. In vivo, EPL significantly promoted the lymphocytes proliferation and the secretion of cytokines and improved the killing activity of NK cells, PCV2-specific antibody titers, and the proportion of T-cell subgroups. The effects of EPL were significantly better than EP and oil adjuvant at most time points. Conclusion. EPL could significantly improve both PCV2-specific cellular and humoral immune responses, and its medium dose had the best efficacy. Therefore, EPL would be exploited in an effective immune adjuvant for inactivated PCV2 vaccine. PMID:26612996

  20. The Adjuvant Activity of Epimedium Polysaccharide-Propolis Flavone Liposome on Enhancing Immune Responses to Inactivated Porcine Circovirus Vaccine in Mice

    PubMed Central

    Fan, Yunpeng; Guo, Liwei; Hou, Weifeng; Guo, Chao; Zhang, Weimin; Ma, Xia; Ma, Lin; Song, Xiaoping

    2015-01-01

    Objectives. The adjuvant activity of Epimedium polysaccharide-propolis flavone liposome (EPL) was investigated in vitro and in vivo. Methods. In vitro, the effects of EPL at different concentrations on splenic lymphocytes proliferation and mRNA expression of IFN-γ and IL-6 were determined. In vivo, the adjuvant activities of EPL, EP, and mineral oil were compared in BALB/c mice through vaccination with inactivated porcine circovirus type 2 (PCV2) vaccine. Results. In vitro, EPL promoted lymphocytes proliferation and increased the mRNA expression of IFN-γ and IL-6, and the effect was significantly better than EP at all concentrations. In vivo, EPL significantly promoted the lymphocytes proliferation and the secretion of cytokines and improved the killing activity of NK cells, PCV2-specific antibody titers, and the proportion of T-cell subgroups. The effects of EPL were significantly better than EP and oil adjuvant at most time points. Conclusion. EPL could significantly improve both PCV2-specific cellular and humoral immune responses, and its medium dose had the best efficacy. Therefore, EPL would be exploited in an effective immune adjuvant for inactivated PCV2 vaccine. PMID:26612996

  1. Highly sensitive sequencing reveals dynamic modifications and activities of small RNAs in mouse oocytes and early embryos.

    PubMed

    Yang, Qiyuan; Lin, Jimin; Liu, Miao; Li, Ronghong; Tian, Bin; Zhang, Xue; Xu, Beiying; Liu, Mofang; Zhang, Xuan; Li, Yiping; Shi, Huijuan; Wu, Ligang

    2016-06-01

    Small RNAs play important roles in early embryonic development. However, their expression dynamics and modifications are poorly understood because of the scarcity of RNA that is obtainable for sequencing analysis. Using an improved deep sequencing method that requires as little as 10 ng of total RNA or 50 oocytes, we profile small RNAs in mouse oocytes and early embryos. We find that microRNA (miRNA) expression starts soon after fertilization, and the mature miRNAs carried into the zygote by sperm during fertilization are relatively rare compared to the oocyte miRNAs. Intriguingly, the zygotic miRNAs display a marked increase in 3' mono- and oligoadenylation in one- to two-cell embryos, which may protect the miRNAs from the massive degradation taking place during that time. Moreover, bioinformatics analyses show that the function of miRNA is suppressed from the oocyte to the two-cell stage and appears to be reactivated after the two-cell stage to regulate genes important in embryonic development. Our study thus provides a highly sensitive profiling method and valuable data sets for further examination of small RNAs in early embryos. PMID:27500274

  2. Highly sensitive sequencing reveals dynamic modifications and activities of small RNAs in mouse oocytes and early embryos

    PubMed Central

    Yang, Qiyuan; Lin, Jimin; Liu, Miao; Li, Ronghong; Tian, Bin; Zhang, Xue; Xu, Beiying; Liu, Mofang; Zhang, Xuan; Li, Yiping; Shi, Huijuan; Wu, Ligang

    2016-01-01

    Small RNAs play important roles in early embryonic development. However, their expression dynamics and modifications are poorly understood because of the scarcity of RNA that is obtainable for sequencing analysis. Using an improved deep sequencing method that requires as little as 10 ng of total RNA or 50 oocytes, we profile small RNAs in mouse oocytes and early embryos. We find that microRNA (miRNA) expression starts soon after fertilization, and the mature miRNAs carried into the zygote by sperm during fertilization are relatively rare compared to the oocyte miRNAs. Intriguingly, the zygotic miRNAs display a marked increase in 3′ mono- and oligoadenylation in one- to two-cell embryos, which may protect the miRNAs from the massive degradation taking place during that time. Moreover, bioinformatics analyses show that the function of miRNA is suppressed from the oocyte to the two-cell stage and appears to be reactivated after the two-cell stage to regulate genes important in embryonic development. Our study thus provides a highly sensitive profiling method and valuable data sets for further examination of small RNAs in early embryos. PMID:27500274

  3. Benzo[a]pyrene effects on glycine N-methyltransferase mRNA expression and enzyme activity in Fundulus heteroclitus embryos

    PubMed Central

    Fang, Xiefan; Dong, Wu; Thornton, Cammi; Willett, Kristine L.

    2010-01-01

    Benzo[a]pyrene (BaP) is a ubiquitous environmental polycyclic aromatic hydrocarbon (PAH) contaminant that is both a carcinogen and a developmental toxicant. We hypothesize that some of BaP’s developmental toxicity may be mediated by effects on glycine N-methyltransferase (GNMT). GNMT is a mediator in the methionine and folate cycles, and the homotetrameric form enzymatically transfers a methyl group from S-adenosylmethionine (SAM) to glycine forming S-adenosylhomocysteine (SAH) and sarcosine. SAM homeostasis, as regulated by GNMT, is critically involved in regulation of DNA methylation, and altered GNMT expression is associated with liver pathologies. The homodimeric form of GNMT has been suggested as the 4S PAH-binding protein. To further study BaP-GNMT interactions, Fundulus heteroclitus embryos were exposed to waterborne BaP at 10 and 100 μg/L and both GNMT mRNA expression and enzyme activity were determined. Whole mount in situ hybridization showed GNMT mRNA expression was increased by BaP in the liver region of 7, 10 and 14 dpf F. heteroclitus embryos. In contrast to mRNA induction, in vivo BaP exposure decreased GNMT enzyme activity in 4, 10 and 14 dpf embryos. However, in vitro incubations of adult F. heteroclitus liver cytosol with BaP did not cause decreased enzyme activity. In conclusion, BaP exposure altered GNMT expression, which may represent a new target pathway for BaP-mediated embryonic toxicities and DNA methylation changes. PMID:20185185

  4. Relation among cytochrome P450, Ah-active PCB congeners and dioxin equivalents in pipping black- crowned night-heron embryos

    USGS Publications Warehouse

    Rattner, B.A.; Hatfield, J.S.; Melancon, M.J.; Custer, T.W.; Tillitt, D.E.

    1994-01-01

    Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from a relatively uncontaminated site (next to Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). Hepatic cytochrome P450-associated monooxygenases and cytochrome P450 proteins, induced up to 85- fold relative to the reference site, were associated with concentrations of total polychlorinated biphenyls (PCBs) and 11 PCB congeners that are presumed to express toxicity through the arylhydrocarbon (Ah) receptor. Multiple regression revealed that up to 86% of the variation of cytochrome P450 measurements was accounted for by variation in the concentration of these PCB congeners. Toxic equivalents (TEQs) of sample extracts, predicted mathematically (summed product of PCB congener concentrations and toxic equivalency factors), and dioxin equivalents (TCDD-EQs), derived by bioassay (ethoxyresorufin-O-dealkylase activity of treated H4IIE rat hepatoma cells), were greatest in Cat Island samples. Cytochrome P450-associated monooxygenases and cytochrome P450 proteins were related to TEQs and TCDD-EQs; adjusted r super(2) often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data extend previous observations in heron embryos of an association between P450 and total PCB burdens to include Ah- active PCB congeners, and presumably other compounds, which interact similarly with the Ah receptor. Benzyloxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase, and cytochrome P450 1A appear to be the most reliable measures of exposure to Ah-active PCB congeners in black-crowned night-heron embryos. These findings provide further evidence that cytochrome P450-associated parameters have considerable value as a biomarker for assessing environmental contamination of wetlands.

  5. Relation among cytochrome P450, AH-active PCB congeners and dioxin equivalents in pipping black-crowned night-heron embryos

    USGS Publications Warehouse

    Rattner, B.A.; Hatfield, J.S.; Melancon, M.J.; Custer, T.W.; Tillitt, D.E.

    1994-01-01

    Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from a relatively uncontaminated site (next to Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). Hepatic cytochrome P-450-associated monooxygenases and cytochrome P-450 proteins, induced up to 85-fold relative to the reference site, were associated with concentrations of total polychlorinated biphenyls (PCBs) and 11 PCB congeners that are presumed to express toxicity through the arylhydrocarbon (Ah) receptor. Multiple regression revealed that up to 86% of the variation of cytochrome P450 measurements was accounted for by variation in the concentration of these PCB congeners. Toxic equivalents (TEQs) of sample extracts, predicted mathematically (summed product of PCB congener concentrations and toxic equivalency factors), and dioxin equivalents (TCDD-EQs), derived by bioassay (ethoxyresorufin-O-dealkylase activity of treated H4IIE rat hepatoma cells), were greatest in Cat Island samples. Cytochrome P450-associated monooxygenases and cytochrome P450 proteins were related to TEQs and TCDD-EQs; adjusted r-2 often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data extend previous observations in heron embryos of an association between P450 and total PCB burdens to include Ah-active PCB congeners, and presumably other compounds, which interact similarly with the Ah receptor. Benzyloxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase, and cytochrome P450 1A appear to be the most reliable measures of exposure to Ah-active PCB congeners in black-crowned night-heron embryos. These findings provide further evidence that cytochrome P450-associated parameters have considerable value as a biomarker for assessing environmental contamination of wetlands.

  6. Embryos, microscopes, and society.

    PubMed

    Maienschein, Jane

    2016-06-01

    Embryos have different meanings for different people and in different contexts. Seen under the microscope, the biological embryo starts out as one cell and then becomes a bunch of cells. Gradually these divide and differentiate to make up the embryo, which in humans becomes a fetus at eight weeks, and then eventually a baby. At least, that happens in those cases that carry through normally and successfully. Yet a popular public perception imagines the embryo as already a little person in the very earliest stages of development, as if it were predictably to become an adult. In actuality, cells can combine, pull apart, and recombine in a variety of ways and still produce embryos, whereas most embryos never develop into adults at all. Biological embryos and popular imaginations of embryos diverge. This paper looks at some of the historical reasons for and social implications of that divergence. PMID:26996410

  7. The role of paf in embryo physiology.

    PubMed

    O'Neill, Chris

    2005-01-01

    Embryo-derived paf (1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is produced by de novo synthesis. This synthesis commences soon after fertilization and persists throughout the preimplantation phase. Paf is produced and released by the embryos of all mammalian species studied to date. Its release from the embryo involves binding to extracellular albumin in a manner that protects paf from enzymatic degradation. Released paf causes a range of alterations in maternal physiology, including platelet activation, changes in oviductal, endometrial and maternal immune function. Paf also acts in an autocrine fashion as a trophic/survival factor for the early embryo. In vitro, supplementation of culture media with paf improves embryo development. Embryo-derived paf's autocrine actions are transduced by 1-o-phosphatidylinositol-3-kinase, which induces characteristic calcium transients within the early embryo. The calcium transients require both the influx of external calcium and release of inositol trisphosphate-dependent internal calcium stores. Buffering these transients compromised embryo development in a manner that was reversed by exogenous paf. Assisted reproductive technologies compromise the production of paf by some embryos and retard the expression of the paf receptor. This deprivation of paf's action is one of the factors limiting the survivability of embryos produced by assisted reproductive technologies. Paf is one of several autocrine and paracrine trophic/survival factors that act on the early embryo. These factors probably act cooperatively and may, to some degree, be mutually redundant. As the earliest-released and the best-described embryotrophin, paf provides an important exemplar for understanding the role of ligand-mediated trophic support of the early embryo. PMID:15790601

  8. Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations.

    PubMed

    Sato, Masahiro; Koriyama, Miyu; Watanabe, Satoshi; Ohtsuka, Masato; Sakurai, Takayuki; Inada, Emi; Saitoh, Issei; Nakamura, Shingo; Miyoshi, Kazuchika

    2015-01-01

    Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B4 (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types. PMID:26247938

  9. Aronia melanocarpa juice, a rich source of polyphenols, induces endothelium-dependent relaxations in porcine coronary arteries via the redox-sensitive activation of endothelial nitric oxide synthase.

    PubMed

    Kim, Jong Hun; Auger, Cyril; Kurita, Ikuko; Anselm, Eric; Rivoarilala, Lalainasoa Odile; Lee, Hyong Joo; Lee, Ki Won; Schini-Kerth, Valérie B

    2013-11-30

    This study examined the ability of Aronia melanocarpa (chokeberry) juice, a rich source of polyphenols, to cause NO-mediated endothelium-dependent relaxations of isolated coronary arteries and, if so, to determine the underlying mechanism and the active polyphenols. A. melanocarpa juice caused potent endothelium-dependent relaxations in porcine coronary artery rings. Relaxations to A. melanocarpa juice were minimally affected by inhibition of the formation of vasoactive prostanoids and endothelium-derived hyperpolarizing factor-mediated responses, and markedly reduced by N(ω)-nitro-l-arginine (endothelial NO synthase (eNOS) inhibitor), membrane permeant analogs of superoxide dismutase and catalase, PP2 (Src kinase inhibitor), and wortmannin (PI3-kinase inhibitor). In cultured endothelial cells, A. melanocarpa juice increased the formation of NO as assessed by electron paramagnetic resonance spectroscopy using the spin trap iron(II)diethyldithiocarbamate, and reactive oxygen species using dihydroethidium. These responses were associated with the redox-sensitive phosphorylation of Src, Akt and eNOS. A. melanocarpa juice-derived fractions containing conjugated cyanidins and chlorogenic acids induced the phosphorylation of Akt and eNOS. The present findings indicate that A. melanocarpa juice is a potent stimulator of the endothelial formation of NO in coronary arteries; this effect involves the phosphorylation of eNOS via the redox-sensitive activation of the Src/PI3-kinase/Akt pathway mostly by conjugated cyanidins and chlorogenic acids. PMID:23973200

  10. Direct Injection of CRISPR/Cas9-Related mRNA into Cytoplasm of Parthenogenetically Activated Porcine Oocytes Causes Frequent Mosaicism for Indel Mutations

    PubMed Central

    Sato, Masahiro; Koriyama, Miyu; Watanabe, Satoshi; Ohtsuka, Masato; Sakurai, Takayuki; Inada, Emi; Saitoh, Issei; Nakamura, Shingo; Miyoshi, Kazuchika

    2015-01-01

    Some reports demonstrated successful genome editing in pigs by one-step zygote microinjection of mRNA of CRISPR/Cas9-related components. Given the relatively long gestation periods and the high cost of housing, the establishment of a single blastocyst-based assay for rapid optimization of the above system is required. As a proof-of-concept, we attempted to disrupt a gene (GGTA1) encoding the α-1,3-galactosyltransferase that synthesizes the α-Gal epitope using parthenogenetically activated porcine oocytes. The lack of α-Gal epitope expression can be monitored by staining with fluorescently labeled isolectin BS-I-B4 (IB4), which binds specifically to the α-Gal epitope. When oocytes were injected with guide RNA specific to GGTA1 together with enhanced green fluorescent protein (EGFP) and human Cas9 mRNAs, 65% (24/37) of the developing blastocysts exhibited green fluorescence, although almost all (96%, 23/24) showed a mosaic fluorescent pattern. Staining with IB4 revealed that the green fluorescent area often had a reduced binding activity to IB4. Of the 16 samples tested, six (five fluorescent and one non-fluorescent blastocysts) had indel mutations, suggesting a correlation between EGFP expression and mutation induction. Furthermore, it is suggested that zygote microinjection of mRNAs might lead to the production of piglets with cells harboring various mutation types. PMID:26247938

  11. BFCOD activity in fish cell lines and zebrafish embryos and its modulation by chemical ligands of human aryl hydrocarbon and nuclear receptors.

    PubMed

    Creusot, N; Brion, F; Piccini, B; Budzinski, H; Porcher, J M; Aït-Aïssa, S

    2015-11-01

    Assessment of exposure and effect of fish to pharmaceuticals that contaminate aquatic environment is a current major issue in ecotoxicology and there is a need to develop specific biological marker to achieve this goal. Benzyloxy-4-trifluoromethylcoumarin-O-debenzyloxylase (BFCOD) enzymatic activity has been commonly used to monitor CYP3A activity in fish. In this study, we assessed the capacity of a panel of toxicologically relevant chemicals to modulate BFCOD activity in fish, by using in vitro and in vivo bioassays based on fish liver cell lines (PLHC-1, ZFL, RTL-W1) and zebrafish embryos, respectively. Basal BFCOD activity was detectable in all biological models and was differently modulated by chemicals. Ligands of human androgens, glucocorticoids, or pregnanes X receptors (i.e., dexamethasone, RU486, rifampicin, SR12813, T0901317, clotrimazole, ketoconazole, testosterone, and dihydrotestosterone) moderately increased or inhibited BFCOD activity, with some variations between the models. No common feature could be drawn by regards to their capacity to bind to these receptors, which contrasts with their known effect on mammalian CYP3A. In contrast, dioxins and polycyclic aromatic hydrocarbons (PAHs) strongly induced BFCOD activity (up to 30-fold) in a time- and concentration-dependent manner, both in vitro in all cell lines and in vivo in zebrafish embryos. These effects were AhR dependent as indicated by suppression of induced BFCOD by the AhR pathway inhibitors 8-methoxypsoralen and α-naphthoflavone. Altogether our result further question the relevance of using liver BFCOD activity as a biomarker of fish exposure to CYP3A-active compounds such as pharmaceuticals. PMID:25471715

  12. DNA repair in mammalian embryos.

    PubMed

    Jaroudi, Souraya; SenGupta, Sioban

    2007-01-01

    Mammalian cells have developed complex mechanisms to identify DNA damage and activate the required response to maintain genome integrity. Those mechanisms include DNA damage detection, DNA repair, cell cycle arrest and apoptosis which operate together to protect the conceptus from DNA damage originating either in parental gametes or in the embryo's somatic cells. DNA repair in the newly fertilized preimplantation embryo is believed to rely entirely on the oocyte's machinery (mRNAs and proteins deposited and stored prior to ovulation). DNA repair genes have been shown to be expressed in the early stages of mammalian development. The survival of the embryo necessitates that the oocyte be sufficiently equipped with maternal stored products and that embryonic gene expression commences at the correct time. A Medline based literature search was performed using the keywords 'DNA repair' and 'embryo development' or 'gametogenesis' (publication dates between 1995 and 2006). Mammalian studies which investigated gene expression were selected. Further articles were acquired from the citations in the articles obtained from the preliminary Medline search. This paper reviews mammalian DNA repair from gametogenesis to preimplantation embryos to late gestational stages. PMID:17141556

  13. Nonstructural protein 1{alpha} subunit-based inhibition of NF-{kappa}B activation and suppression of interferon-{beta} production by porcine reproductive and respiratory syndrome virus

    SciTech Connect

    Song Cheng; Krell, Peter; Yoo, Dongwan

    2010-11-25

    Induction of type I interferon (IFN-{alpha}/{beta}) is an early antiviral response of the host, and porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to downregulate the IFN response during infection in cells and pigs. We report that the PRRSV nonstructural protein 1{alpha} (Nsp1{alpha}) subunit of Nsp1 is a nuclear-cytoplasmic protein distributed to the nucleus and contains a strong suppressive activity for IFN-{beta} production that is mediated through the retinoic acid-inducible gene I (RIG-I) signaling pathway. Nsp1{alpha} suppressed the activation of nuclear factor (NF)-{kappa}B when stimulated with dsRNA or tumor necrosis factor (TNF)-{alpha}, and NF-{kappa}B suppression was RIG-I-dependent. The suppression of NF-{kappa}B activation was associated with the poor production of IFN-{beta} during PRRSV infection. The C-terminal 14 amino acids of the Nsp1{alpha} subunit were critical in maintaining immunosuppressive activity of Nsp1{alpha} for both IFN-{beta} and NF-{kappa}B, suggesting that the newly identified zinc finger configuration comprising of Met180 may be crucial for inhibitory activities. Nsp1{alpha} inhibited I{kappa}B phosphorylation and as a consequence NF-{kappa}B translocation to the nucleus was blocked, leading to the inhibition of NF-{kappa}B stimulated gene expression. Our results suggest that PRRSV Nsp1{alpha} is a multifunctional nuclear protein participating in the modulation of the host IFN system.

  14. Cellular microRNA miR-26a suppresses replication of porcine reproductive and respiratory syndrome virus by activating innate antiviral immunity

    PubMed Central

    Jia, Xiaojuan; Bi, Yuhai; Li, Jing; Xie, Qing; Yang, Hanchun; Liu, Wenjun

    2015-01-01

    Porcine reproductive and respiratory syndrome (PRRS) has caused large economic losses in the swine industry in recent years. Current PRRS vaccines fail to effectively prevent and control this disease. Consequently, there is a need to develop new antiviral strategies. MicroRNAs play critical roles in intricate host-pathogen interaction networks, but the involvement of miRNAs during PRRS virus (PRRSV) infection is not well understood. In this study, pretreatment with miR-26a induced a significant inhibition of PRRSV replication and remission of the cytopathic effect in MARC-145 cells, and this antiviral effect was sustained for at least 120 h. Luciferase reporter analysis showed that the PRRSV genome was not the target of miRNA-26a. Instead, RNA-seq analysis demonstrated that miR-26a significantly up-regulated innate anti-viral responses, including activating the type I interferon (IFN) signaling pathway and promoting the production of IFN-stimulated genes. These findings suggest that delivery of miR-26a may provide a potential strategy for anti-PRRSV therapies. PMID:26013676

  15. Maneb-induced dopaminergic neuronal death is not affected by loss of mitochondrial complex I activity: Results from primary mesencephalic dopaminergic neurons cultured from individual Ndufs4+/+ and Ndufs4-/- mouse embryos

    PubMed Central

    Choi, Won-Seok; Xia, Zhengui

    2014-01-01

    Primary cultures from embryonic mouse ventral mesencephalon are widely used for investigating the mechanisms of dopaminergic neuronal death in Parkinson's disease models. Specifically, single mouse or embryo cultures from littermates can be very useful for comparative studies involving transgenic mice when the neuron cultures are to be prepared before genotyping. However, preparing single mouse embryo culture is technically challenging because of the small number of cells present in the mesencephalon of each embryo (150,000-300,000), of which only 0.5-5% are tyrosine hydroxylase (TH) -positive, dopaminergic neurons. In this study, we optimized the procedure for preparing primary mesencephalic neuron cultures from individual mouse embryos. Mesencephalic neurons that are dissociated delicately, plated on Aclar film coverslips, and incubated in DMEM supplemented with FBS for 5 days and then N2 supplement for 1 day resulted in the best survival of dopaminergic neurons from each embryo. Using this optimized method, we prepared mesencephalic neuron cultures from single Ndufs4+/+ or Ndufs4-/- embryos, and investigated the role of mitochondrial complex I in maneb-induced dopamine neuron death. Our results suggest that maneb toxicity to dopamine neurons is not affected by loss of mitochondrial complex I activity in Ndufs4-/- cultures. PMID:25275677

  16. Molecular characterization of porcine SARM1 and its role in regulating TLRs signaling during highly pathogenic porcine reproductive and respiratory syndrome virus infection in vivo.

    PubMed

    Zhou, Xiang; Jiang, Tengfei; Du, Xiaochuan; Zhou, Ping; Jiang, Zhihua; Michal, Jennifer J; Liu, Bang

    2013-01-01

    Toll-like receptors (TLRs) are important pattern-recognition receptors (PRRs) that trigger innate immune response and mediate acquired immunity. Evidence has shown that SARM1 (sterile-α and TIR motif containing protein 1) is one of five TIR domain-containing adaptor proteins involved in TLRs signaling transduction. In the present study, a full-length cDNA sequence was cloned for the porcine SARM1 gene, which contains nine exons. Using the radiation hybrid mapping approach, we assigned the porcine gene to SSC12 q13. Under the normal condition, porcine SARM1 was highly expressed in brain and spleen. Polyinosinic-polycytidylic acid (poly (I:C)) weakly induced the porcine SARM1 expression in the early stimulation. We found that porcine SARM1 protein is localized in mitochondria and attenuates NF-κB activation induced by stimulation and infection. The quantitative real-time PCR (Q-PCR) analysis showed that the expression of porcine SARM1 significantly decreased in several tissues of Tongcheng pigs infected with highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Gene-interaction network analysis for porcine SARM1 in porcine alveolar macrophages (PAMs) showed that down-regulation of SARM1 gene in infected Tongcheng pig may modulate TRIF-depend TLRs signaling and regulate the expression of disease-resistant genes and inflammatory genes. Our findings provide evidence that porcine SARM1 may play an important role in immune regulation with PRRSV infection. PMID:22366489

  17. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    SciTech Connect

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  18. Nuclear transfer procedures in the ovine can induce early embryo fragmentation and compromise cloned embryo development.

    PubMed

    Xue, Lian; Cheng, Lei; Su, Guanghua; Kang, Feng; Wu, Xia; Bai, Chunling; Zhang, Li; Li, Guang-Peng

    2011-07-01

    Cytoplasmic fragmentations are frequently observed in early mammalian embryos, and especially in the human. In our research we have observed subtle clues that the occurrence of fragmentation was most likely a result of somatic cell nuclear transfer (NT) protocols, and in particular, the in vitro culture system. In this study we examined various putative factors that might induce early embryo fragmentation in the ovine. The results indicate that nuclear transfer protocols such as the fusion parameter, activation treatment, and especially the choice of culture medium affected embryo cleavage rates and resulted in a higher incidence of fragmented embryos. Upon using the same fusion parameter, activation parameters that were based upon amino acids containing synthetic oviduct fluids (SOFaa) culture system resulted in significantly lower fragmentation rates than when utilizing a Charles Rosenkrans 1 (CR1aa) culture system. Fragmented embryos typically exhibited irregular numbers of blastomeres with the majority of blastomeres devoid of chromatin. Factors such as fusion DC pulse, activation treatment and culture system led to higher fragmentation and also affected in vitro and in vivo embryo development. The SOFaa based culture system produced a higher number of quality NT embryos resulting in higher pregnancy rates and the birth of live lambs as compared to the CR1aa based system (P<0.05). We conclude that early embryo fragmentation in the ovine is caused by suboptimal cloning protocols, and NT embryo development is especially affected by the culture system used. PMID:21700405

  19. Effects of Fluoxetine on Human Embryo Development.

    PubMed

    Kaihola, Helena; Yaldir, Fatma G; Hreinsson, Julius; Hörnaeus, Katarina; Bergquist, Jonas; Olivier, Jocelien D A; Åkerud, Helena; Sundström-Poromaa, Inger

    2016-01-01

    The use of antidepressant treatment during pregnancy is increasing, and selective serotonin reuptake inhibitors (SSRIs) are the most widely prescribed antidepressants in pregnant women. Serotonin plays a role in embryogenesis, and serotonin transporters are expressed in two-cell mouse embryos. Thus, the aim of the present study was to evaluate whether fluoxetine, one of the most prescribed SSRI antidepressant world-wide, exposure influences the timing of different embryo developmental stages, and furthermore, to analyze what protein, and protein networks, are affected by fluoxetine in the early embryo development. Human embryos (n = 48) were randomly assigned to treatment with 0.25 or 0.5 μM fluoxetine in culture medium. Embryo development was evaluated by time-lapse monitoring. The fluoxetine-induced human embryo proteome was analyzed by shotgun mass spectrometry. Protein secretion from fluoxetine-exposed human embryos was analyzed by use of high-multiplex immunoassay. The lower dose of fluoxetine had no influence on embryo development. A trend toward reduced time between thawing and start of cavitation was noted in embryos treated with 0.5 μM fluoxetine (p = 0.065). Protein analysis by shotgun mass spectrometry detected 45 proteins that were uniquely expressed in fluoxetine-treated embryos. These proteins are involved in cell growth, survival, proliferation, and inflammatory response. Culturing with 0.5 μM, but not 0.25 μM fluoxetine, caused a significant increase in urokinase-type plasminogen activator (uPA) in the culture medium. In conclusion, fluoxetine has marginal effects on the timing of developmental stages in embryos, but induces expression and secretion of several proteins in a manner that depends on dose. For these reasons, and in line with current guidelines, the lowest possible dose of SSRI should be used in pregnant women who need to continue treatment. PMID:27378857

  20. Effects of Fluoxetine on Human Embryo Development

    PubMed Central

    Kaihola, Helena; Yaldir, Fatma G.; Hreinsson, Julius; Hörnaeus, Katarina; Bergquist, Jonas; Olivier, Jocelien D. A.; Åkerud, Helena; Sundström-Poromaa, Inger

    2016-01-01

    The use of antidepressant treatment during pregnancy is increasing, and selective serotonin reuptake inhibitors (SSRIs) are the most widely prescribed antidepressants in pregnant women. Serotonin plays a role in embryogenesis, and serotonin transporters are expressed in two-cell mouse embryos. Thus, the aim of the present study was to evaluate whether fluoxetine, one of the most prescribed SSRI antidepressant world-wide, exposure influences the timing of different embryo developmental stages, and furthermore, to analyze what protein, and protein networks, are affected by fluoxetine in the early embryo development. Human embryos (n = 48) were randomly assigned to treatment with 0.25 or 0.5 μM fluoxetine in culture medium. Embryo development was evaluated by time-lapse monitoring. The fluoxetine-induced human embryo proteome was analyzed by shotgun mass spectrometry. Protein secretion from fluoxetine-exposed human embryos was analyzed by use of high-multiplex immunoassay. The lower dose of fluoxetine had no influence on embryo development. A trend toward reduced time between thawing and start of cavitation was noted in embryos treated with 0.5 μM fluoxetine (p = 0.065). Protein analysis by shotgun mass spectrometry detected 45 proteins that were uniquely expressed in fluoxetine-treated embryos. These proteins are involved in cell growth, survival, proliferation, and inflammatory response. Culturing with 0.5 μM, but not 0.25 μM fluoxetine, caused a significant increase in urokinase-type plasminogen activator (uPA) in the culture medium. In conclusion, fluoxetine has marginal effects on the timing of developmental stages in embryos, but induces expression and secretion of several proteins in a manner that depends on dose. For these reasons, and in line with current guidelines, the lowest possible dose of SSRI should be used in pregnant women who need to continue treatment. PMID:27378857

  1. Physiological and molecular determinants of embryo implantation

    PubMed Central

    Zhang, Shuang; Lin, Haiyan; Kong, Shuangbo; Wang, Shumin; Wang, Hongmei; Wang, Haibin; Armant, D. Randall

    2014-01-01

    Embryo implantation involves the intimate interaction between an implantation-competent blastocyst and a receptive uterus, which occurs in a limited time period known as the window of implantation. Emerging evidence shows that defects originating during embryo implantation induce ripple effects with adverse consequences on later gestation events, highlighting the significance of this event for pregnancy success. Although a multitude of cellular events and molecular pathways involved in embryo-uterine crosstalk during implantation have been identified through gene expression studies and genetically engineered mouse models, a comprehensive understanding of the nature of embryo implantation is still missing. This review focuses on recent progress with particular attention to physiological and molecular determinants of blastocyst activation, uterine receptivity, blastocyst attachment and uterine decidualization. A better understanding of underlying mechanisms governing embryo implantation should generate new strategies to rectify implantation failure and improve pregnancy rates in women. PMID:23290997

  2. Expression Pattern of Pluripotent Markers in Different Embryonic Developmental Stages of Buffalo (Bubalus bubalis) Embryos and Putative Embryonic Stem Cells Generated by Parthenogenetic Activation

    PubMed Central

    Singh, Karn P.; Kaushik, Ramakant; Garg, Veena; Sharma, Ruchi; George, Aman; Singh, Manoj K.; Manik, Radhey S.; Palta, Prabhat; Singla, Suresh K.

    2012-01-01

    Abstract In this study, we describe the production of buffalo parthenogenetic blastocysts and subsequent isolation of parthenogenetic embryonic stem cell (PGESC)-like cells. PGESC colonies exhibited dome-shaped morphology and were clearly distinguishable from the feeder layer cells. Different stages of development of parthenogenetic embryos and derived embryonic stem cell (ESC)-like cells expressed key ESC-specific markers, including OCT-4, NANOG, SOX-2, FOXD3, REX-1, STAT-3, TELOMERASE, NUCLEOSTEMIN, and cMYC. Immunofluorescence-based studies revealed that the PGESCs were positive for surface-based pluripotent markers, viz., SSEA-3, SSEA-4, TRA 1-80, TRA 1-60, CD-9, and CD-90 and exhibited high alkaline phosphatase (ALP) activity. PGEC cell-like cells formed embryoid body (EB)-like structures in hanging drop cultures and when cultured for extended period of time spontaneously differentiated into derivatives of three embryonic germ layers as confirmed by RT-PCR for ectodermal (CYTOKERATIN8, NF-68), mesodermal (MSX1, BMP-4, ASA), and endodermal markers (AFP, HNF-4, GATA-4). Differentiation of PGESCs toward the neuronal lineage was successfully directed by supplementation of serum-containing media with retinoic acid. Our results indicate that the isolated ESC-like cells from parthenogenetic blastocyst hold properties of ESCs and express markers of pluripotency. The pluripotency markers were also expressed by early cleavage-stage of buffalo embryos. PMID:23194456

  3. Relationship between colour flow Doppler sonographic assessment of corpus luteum activity and progesterone concentrations in mares after embryo transfer.

    PubMed

    Brogan, P T; Henning, H; Stout, T A E; de Ruijter-Villani, M

    2016-03-01

    Colour-flow Doppler sonography has been described as a means of assessing corpus luteum (CL) function rapidly, because area of luteal blood vessels correlates well with circulating progesterone (P4) concentrations [P4] in oestrous cycling mares. The aim of this study was to assess the relationships between CL size and vascularity, and circulating [P4] during early pregnancy in mares, and to determine whether luteal blood flow was a useful aid for selecting an embryo transfer recipient. Equine embryos (n=48) were recovered 8 days after ovulation and were transferred to available recipient mares as part of a commercial program with the degree of synchrony in timing of recipient ovulation ranging from 1 day before to 4 days after the donor. Immediately prior to embryo transfer (ET), maximum CL cross-section and blood vessel areas were assessed sonographically, and jugular blood was collected to measure plasma [P4]. Sonographic measurements and jugular blood collection were repeated at day 4 after ET for all mares, and again at days 11, 18 and 25 after ET in mares that were pregnant. The number of grey-scale and colour pixels within the CL was subsequently quantified using ImageJ software. The CL blood flow correlated significantly but weakly with plasma [P4] on the day of transfer and on day 4 after ET in all mares, and on days 11 and 25 after ET in pregnant mares (r=0.30-0.36). The CL area and plasma [P4] were also correlated on each day until day 11 after ET (r=0.49-0.60). The CL colour pixel area decreased significantly after day 18, whereas CL area was already decreasing by day 4 after ET. The CL area, area of blood flow, or [P4] was predictive of pregnancy. Findings in the present study suggest that both CL area and blood flow are correlated with circulating [P4] at the time of transfer and in early pregnancy. Evaluation of the CL using B-mode or CF sonography, although practical, provides no improvement in the selection of recipients or prediction of pregnancy

  4. Enhanced Proliferation of Porcine Bone Marrow Mesenchymal Stem Cells Induced by Extracellular Calcium is Associated with the Activation of the Calcium-Sensing Receptor and ERK Signaling Pathway

    PubMed Central

    Ye, Jingjing; Ai, Wei; Zhang, Fenglin; Zhu, Xiaotong; Shu, Gang; Wang, Lina; Gao, Ping; Xi, Qianyun; Zhang, YongLiang; Jiang, Qingyan; Wang, Songbo

    2016-01-01

    Porcine bone marrow mesenchymal stem cells (pBMSCs) have the potential for application in regenerative medicine. This study aims to investigate the effects of extracellular calcium ([Ca2+]o) on pBMSCs proliferation and to explore the possible underlying mechanisms. The results demonstrated that 4 mM [Ca2+]o significantly promoted pBMSCs proliferation by reducing the G0/G1 phase cell percentage and by increasing the S phase cell proportion and the proliferation index of pBMSCs. Accordingly, [Ca2+]o stimulated the expression levels of proliferative genes such as cyclin A2, cyclin D1/3, cyclin E2, and PCNA and inhibited the expression of p21. In addition, [Ca2+]o resulted in a significant elevation of intracellular calcium and an increased ratio of p-ERK/ERK. However, inhibition of calcium-sensing receptor (CaSR) by its antagonist NPS2143 abolished the aforementioned effects of [Ca2+]o. Moreover, [Ca2+]o-induced promotion of pBMSCs proliferation, the changes of proliferative genes expression levels, and the activation of ERK1/2 signaling pathway were effectively blocked by U0126, a selective ERK kinase inhibitor. In conclusion, our findings provided evidence that the enhanced pBMSCs proliferation in response to [Ca2+]o was associated with the activation of CaSR and ERK1/2 signaling pathway, which may be useful for the application of pBMSCs in future clinical studies aimed at tissue regeneration and repair. PMID:27123007

  5. Tissue Distribution of Porcine FTO and Its Effect on Porcine Intramuscular Preadipocytes Proliferation and Differentiation

    PubMed Central

    Chen, Xiaoling; Zhou, Bo; Luo, Yanliu; Huang, Zhiqing; Jia, Gang; Liu, Guangmang; Zhao, Hua

    2016-01-01

    The fat mass and obesity associated (FTO) gene plays an important role in adipogenesis. However, its function during porcine intramuscular preadipocyte proliferation and differentiation remains poorly understood. In this study, we prepared the antiserum against porcine FTO (pFTO), which was used to determine its subcellular localization and tissue distribution. Our data indicated that pFTO was localized predominantly in the nucleus. Real-time quantitative PCR and western blot analysis showed that pFTO was highly expressed in the lung and subcutaneous adipose tissue. Overexpression of pFTO in porcine intramuscular preadipocytes significantly promoted cell proliferation and lipid deposition. Furthermore, overexpression of pFTO in differentiating porcine intramuscular preadipocytes also significantly increased the mRNA levels of adipocyte differentiation transcription factors peroxisome proliferators-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), lipoprotein lipase (LPL) and fatty acid synthase (FAS). Our findings provide the first functional evidence to reveal a role of pFTO in porcine intramuscular preadipocyte proliferation and differentiation. PMID:26964098

  6. A porcine model of osteosarcoma

    PubMed Central

    Saalfrank, A; Janssen, K-P; Ravon, M; Flisikowski, K; Eser, S; Steiger, K; Flisikowska, T; Müller-Fliedner, P; Schulze, É; Brönner, C; Gnann, A; Kappe, E; Böhm, B; Schade, B; Certa, U; Saur, D; Esposito, I; Kind, A; Schnieke, A

    2016-01-01

    We previously produced pigs with a latent oncogenic TP53 mutation. Humans with TP53 germline mutations are predisposed to a wide spectrum of early-onset cancers, predominantly breast, brain, adrenal gland cancer, soft tissue sarcomas and osteosarcomas. Loss of p53 function has been observed in >50% of human cancers. Here we demonstrate that porcine mesenchymal stem cells (MSCs) convert to a transformed phenotype after activation of latent oncogenic TP53R167H and KRASG12D, and overexpression of MYC promotes tumorigenesis. The process mimics key molecular aspects of human sarcomagenesis. Transformed porcine MSCs exhibit genomic instability, with complex karyotypes, and develop into sarcomas on transplantation into immune-deficient mice. In pigs, heterozygous knockout of TP53 was sufficient for spontaneous osteosarcoma development in older animals, whereas homozygous TP53 knockout resulted in multiple large osteosarcomas in 7–8-month-old animals. This is the first report that engineered mutation of an endogenous tumour-suppressor gene leads to invasive cancer in pigs. Unlike in Trp53 mutant mice, osteosarcoma developed in the long bones and skull, closely recapitulating the human disease. These animals thus promise a model for juvenile osteosarcoma, a relatively uncommon but devastating disease. PMID:26974205

  7. Induction of autophagy improves embryo viability in cloned mouse embryos

    PubMed Central

    Shen, XingHui; Zhang, Na; Wang, ZhenDong; Bai, GuangYu; Zheng, Zhong; Gu, YanLi; Wu, YanShuang; Liu, Hui; Zhou, DongJie; Lei, Lei

    2015-01-01

    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. PMID:26643778

  8. African Swine Fever Virus Infection of Porcine Aortic Endothelial Cells Leads to Inhibition of Inflammatory Responses, Activation of the Thrombotic State, and Apoptosis

    PubMed Central

    Vallée, Isabelle; Tait, Stephen W. G.; Powell, Penelope P.

    2001-01-01

    African swine fever (ASF) is an asymptomatic infection of warthogs and bushpigs, which has become an emergent disease of domestic pigs, characterized by hemorrhage, lymphopenia, and disseminated intravascular coagulation. It is caused by a large icosohedral double-stranded DNA virus, African swine fever virus (ASFV), with infection of macrophages well characterized in vitro and in vivo. This study shows that virulent isolates of ASFV also infect primary cultures of porcine aortic endothelial cells and bushpig endothelial cells (BPECs) in vitro. Kinetics of early and late gene expression, viral factory formation, replication, and secretion were similar in endothelial cells and macrophages. However, ASFV-infected endothelial cells died by apoptosis, detected morphologically by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and nuclear condensation and biochemically by poly(ADP-ribose) polymerase (PARP) cleavage at 4 h postinfection (hpi). Immediate-early proinflammatory responses were inhibited, characterized by a lack of E-selectin surface expression and interleukin 6 (IL-6) and IL-8 mRNA synthesis. Moreover, ASFV actively downregulated interferon-induced major histocompatibility complex class I surface expression, a strategy by which viruses evade the immune system. Significantly, Western blot analysis showed that the 65-kDa subunit of the transcription factor NF-κB, a central regulator of the early response to viral infection, decreased by 8 hpi and disappeared by 18 hpi. Both disappearance of NF-κB p65 and cleavage of PARP were reversed by the caspase inhibitor z-VAD-fmk. Interestingly, surface expression and mRNA transcription of tissue factor, an important initiator of the coagulation cascade, increased 4 h after ASFV infection. These data suggest a central role for vascular endothelial cells in the hemorrhagic pathogenesis of the disease. Since BPECs infected with ASFV also undergo apoptosis, resistance of the natural host must involve

  9. Embryo adoption: Some further considerations.

    PubMed

    Patterson, Colin

    2015-02-01

    Recent discussions of embryo adoption have sought to make sense of the teaching of the Congregation for the Doctrine of the Faith (CDF) document Dignitas personae which appeared to provide a negative judgment on such a practice. This article aims to provide a personalist account of the process of fertilization and implantation that might serve as the basis for the negative judgment of the CDF document. In doing so, it relies upon the idea that a person, including an embryo, is not to be considered in isolation, but always in relation to God and to others. This approach extends the substantialist conceptualizations commonly employed in discussions of this issue. More generally, the article seeks to highlight the value of a personalist re-framing for an understanding of the moral questions surrounding the beginning of life. Lay summary: This article seeks to make sense of what appears to be a clear-cut rejection, set out in the Congregation for the Doctrine of the Faith (CDF) document Dignitas personae, of the proposal for women to "adopt" surplus frozen embryos. It draws upon more recently developed modes of philosophical/theological reasoning to argue that, in human procreation, both fertilization and implantation represent constitutive dimensions of divine creative activity and so must be protected from manipulative technological intervention. Since embryo adoption requires this kind of technology, it makes sense for the Church document not to approve it. PMID:25698841

  10. Embryo adoption: Some further considerations

    PubMed Central

    Patterson, Colin

    2015-01-01

    Recent discussions of embryo adoption have sought to make sense of the teaching of the Congregation for the Doctrine of the Faith (CDF) document Dignitas personae which appeared to provide a negative judgment on such a practice. This article aims to provide a personalist account of the process of fertilization and implantation that might serve as the basis for the negative judgment of the CDF document. In doing so, it relies upon the idea that a person, including an embryo, is not to be considered in isolation, but always in relation to God and to others. This approach extends the substantialist conceptualizations commonly employed in discussions of this issue. More generally, the article seeks to highlight the value of a personalist re-framing for an understanding of the moral questions surrounding the beginning of life. Lay summary: This article seeks to make sense of what appears to be a clear-cut rejection, set out in the Congregation for the Doctrine of the Faith (CDF) document Dignitas personae, of the proposal for women to “adopt” surplus frozen embryos. It draws upon more recently developed modes of philosophical/theological reasoning to argue that, in human procreation, both fertilization and implantation represent constitutive dimensions of divine creative activity and so must be protected from manipulative technological intervention. Since embryo adoption requires this kind of technology, it makes sense for the Church document not to approve it. PMID:25698841

  11. Morphology and metabolic activity of a porcine liver stem cell line (PICM-19) maintained in a multicompartment hollow fiber bioreactor for two weeks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A functional hepatocyte cell line that differentiates normally is needed to develop a bioartificial liver (BAL). A porcine hepatic stem cell line, PICM-19H, maintained in a 3D multicompartmental hollow-fiber bioreactor (Hepalife Technologies) was studied. The bioreactor was filled with 400 million...

  12. ANALYSIS OF PORCINE TRANSCRIPTIONAL RESPONSE TO SALMONELLA ENTERICA SEROVAR CHOLERAESUIS SUGGESTS NOVEL TARGETS OF NFKAPPAB ARE ACTIVATED IN THE MESENTERIC LYMPH NODE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Affymetrix GeneChip® porcine genome array was used to identify differentially expressed genes in pig mesenteric lymph nodes (MLN) responding to infection with Salmonella enterica serovar Choleraesuis (S. Choleraesuis) at acute (8 hours (h), 24h and 48h post-inoculation (pi)) and chronic stages (...

  13. Functionalized Fullerene Increases NF-κB Activity and Blocks Genotoxic Effect of Oxidative Stress in Serum-Starving Human Embryo Lung Diploid Fibroblasts

    PubMed Central

    Ershova, E. S.; Sergeeva, V. A.; Tabakov, V. J.; Kameneva, L. A.; Voronov, I. I.; Khakina, E. A.; Troshin, P. A.; Kutsev, S. I.; Veiko, N. N.; Kostyuk, S. V.

    2016-01-01

    The influence of a water-soluble [60] fullerene derivative containing five residues of 3-phenylpropionic acid and a chlorine addend appended to the carbon cage (F-828) on serum-starving human embryo lung diploid fibroblasts (HELFs) was studied. Serum deprivation evokes oxidative stress in HELFs. Cultivation of serum-starving HELFs in the presence of 0.1–1 µM F-828 significantly decreases the level of free radicals, inhibits autophagy, and represses expression of NOX4 and NRF2 proteins. The activity of NF-κB substantially grows up in contrast to the suppressed NRF2 activity. In the presence of 0.2–0.25 µM F-828, the DSB rate and apoptosis level dramatically decrease. The maximum increase of proliferative activity of the HELFs and maximum activity of NF-κB are observed at these concentration values. Conclusion. Under the conditions of oxidative stress evoked by serum deprivation the water-soluble fullerene derivative F-828 used in concentrations of 0.1 to 1 µM strongly stimulates the NF-κB activity and represses the NRF2 activity in HELFs.

  14. Virogenic BrdU and BrdU-sensitive DNA sequences are disproportionately concentrated in the template-active chromatin of rat embryo cells

    PubMed Central

    Schwartz, Stephen A.

    1979-01-01

    In order to characterize the molecular mechanism responsible for the BrdU-mediated activation of endogenous retrovirus from normal rat embryo cells, the previously reported selective distribution of bromouracil in DNA was correlated with the corresponding organization of the nucleo-protein complex in regard to nucleosome structure and template - active and -inactive chromatin. Following micrococcal nuclease digestion of chromatin labeled with either [3H]thymidine or [3H]BrdU, the amount and specific activities of the respective nucleosomal DNA were indistinguishable. Comparable findings were obtained following direct examination of the nuclease-sensitive, “spacer” DNA. However, when each chromatin type was fractionated into template-active and -inactive components, it was evident that [3H]bromouracil was nonrandomly more concentrated in the template-active portion in comparison to the random distribution of [3H]thymine moieties. Furthermore, it was apparent that the template-active chromatin fraction was substantially enriched in the nucleotide sequences of rat DNA known to be sensitive to the virogenic action of BrdU. PMID:424312

  15. GlutaMAX prolongs the shelf life of the culture medium for porcine parthenotes.

    PubMed

    Zhao, Ming-Hui; Kim, Nam-Hyung; Cui, Xiang-Shun

    2016-02-01

    In vitro porcine embryo production systems have been established and well characterized. However, the efficiency of embryo development during IVC is still very low. In the present study, we have investigated the development of parthenogenetic porcine embryos in the well-known PZM-5 medium for porcine embryos, which was modified by replacing glutamine with the GlutaMAX supplement. We revealed that blastocyst apoptosis was significantly lower in the presence of GlutaMAX, which reduced the release of mitochondrial cytochrome c. Furthermore, the expression of apoptosis genes was significantly lower during GlutaMAX treatment (P < 0.05). The modified medium was also examined for the eventual loss of its efficacy in the presence of GlutaMAX. Three, 6, and 12 months after medium preparation, blastocyst formation in the GlutaMAX-supplemented medium was significantly higher than the number of blastocysts in the medium containing glutamine. After a long period of storage, ammonia concentration was significantly increased in the glutamine medium, whereas it was not statistically different in the GlutaMAX medium. Elevated ammonia concentrations reduced the mitochondrial membrane potential and ATP content of blastocysts in the glutamine medium. These results demonstrate that GlutaMAX can reduce blastocyst apoptosis via inhibition of the cytochrome c pathway and significantly extend the shelf life of the culture medium to at least 1 year. PMID:26462658

  16. Direct recognition of SLA- and HLA-like class II antigens on porcine endothelium by human T cells results in T cell activation and release of interleukin-2.

    PubMed

    Bravery, C A; Batten, P; Yacoub, M H; Rose, M L

    1995-11-15

    To investigate whether human T cells can directly recognize pig xenoantigens, highly purified human CD4+ and CD8+ T cells were incubated with pig aortic endothelial cells (PAEC). The response was measured by [3H]thymidine uptake and release of bioactive interleukin-2. A detailed examination of MHC expression by cultured PAEC and tissue sections of porcine aorta and heart showed porcine endothelial cells (EC) to be constitutively positive for SLA class II and antigens that crossreact with HLA class II molecules. Low level expression of B7 receptors was detected by binding of both human and mouse CTLA-4-Ig to untreated PAEC, which was enhanced significantly by treatment with recombinant porcine interferon-gamma. Human T cells, purified by positive selection and residual DR+ cells removed by lymphocytolysis, were shown to be functionally free of monocytes. Untreated PAEC elicited strong proliferation by human CD4+ T cells: CD8+ T cells also proliferated, but more weakly. This response was inhibited by CTLA-4-Ig. Blocking studies were performed with mAbs that bind to PAEC and not human EC (MSA3, TH16B), an mAb that binds to human and porcine EC (DA6.231), and L243, which binds to human and not porcine EC. The proliferative response of CD4+ T cells to PAEC was inhibited significantly by mAbs against swine and human determinants. In contrast, the response of CD4+ T cells to human EC was inhibited only by mAbs against human determinants. Experiments that directly compared the CD4+ and CD8+ T cell responses to PAEC and the human EC line EAhy.926, both with and without prior treatment with species-specific interferon gamma, demonstrated greater proliferation and 5-10 times more interleukin-2 in response to pig EC than to human EC. PMID:7491676

  17. Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos

    SciTech Connect

    Zhong Zhisheng; Zhang Gang; Meng Xiaoqian; Zhang Yanling; Chen Dayuan; Schatten, Heide; Sun Qingyuan . E-mail: sunqy1@yahoo.com

    2005-05-15

    Centrosomes, the main microtubule-organizing centers (MTOCs) in most animal cells, are important for many cellular activities such as assembly of the mitotic spindle, establishment of cell polarity, and cell movement. In nuclear transfer (NT), MTOCs that are located at the poles of the meiotic spindle are removed from the recipient oocyte, while the centrosome of the donor cell is introduced. We used mouse MII oocytes as recipients, mouse fibroblasts, rat fibroblasts, or pig granulosa cells as donor cells to construct intraspecies and interspecies nuclear transfer embryos in order to observe centrosome dynamics and functions. Three antibodies against centrin, {gamma}-tubulin, and NuMA, respectively, were used to stain the centrosome. Centrin was not detected either at the poles of transient spindles or at the poles of first mitotic spindles. {gamma}-tubulin translocated into the two poles of the transient spindles, while no accumulated {gamma}-tubulin aggregates were detected in the area adjacent to the two pseudo-pronuclei. At first mitotic metaphase, {gamma}-tubulin was translocated to the spindle poles. The distribution of {gamma}-tubulin was similar in mouse intraspecies and rat-mouse interspecies embryos. The NuMA antibody that we used can recognize porcine but not murine NuMA protein, so it was used to trace the NuMA protein of donor cell in reconstructed embryos. In the pig-mouse interspecies reconstructed embryos, NuMA concentrated between the disarrayed chromosomes soon after activation and translocated to the transient spindle poles. NuMA then immigrated into pseudo-pronuclei. After pseudo-pronuclear envelope breakdown, NuMA was located between the chromosomes and then translocated to the spindle poles of first mitotic metaphase. {gamma}-tubulin antibody microinjection resulted in spindle disorganization and retardation of the first cell division. NuMA antibody microinjection also resulted in spindle disorganization. Our findings indicate that (1) the

  18. Loss of DDB1 Leads to Transcriptional p53 Pathway Activation in Proliferating Cells, Cell Cycle Deregulation, and Apoptosis in Zebrafish Embryos

    PubMed Central

    Hu, Zhilian; Holzschuh, Jochen; Driever, Wolfgang

    2015-01-01

    DNA damage-binding protein 1 (DDB1) is a large subunit of the heterodimeric DDB complex that recognizes DNA lesions and initiates the nucleotide excision repair process. DDB1 is also a component of the CUL4 E3 ligase complex involved in a broad spectrum of cellular processes by targeted ubiquitination of key regulators. Functions of DDB1 in development have been addressed in several model organisms, however, are not fully understood so far. Here we report an ENU induced mutant ddb1 allele (ddb1m863) identified in zebrafish (Danio rerio), and analyze its effects on development. Zebrafish ddb1 is expressed broadly, both maternally and zygotically, with enhanced expression in proliferation zones. The (ddb1m863 mutant allele affects the splice acceptor site of exon 20, causing a splicing defect that results in truncation of the 1140 amino acid protein after residue 800, lacking part of the β-propeller domain BPC and the C-terminal helical domain CTD. ddb1m863 zygotic mutant embryos have a pleiotropic phenotype, including smaller and abnormally shaped brain, head skeleton, eyes, jaw, and branchial arches, as well as reduced dopaminergic neuron groups. However, early forming tissues develop normally in zygotic ddb1m863 mutant embryos, which may be due to maternal rescue. In ddb1m863 mutant embryos, pcna-expressing proliferating cell populations were reduced, concurrent with increased apoptosis. We also observed a concomitant strong up-regulation of transcripts of the tumor suppressor p53 (tp53) and the cell cycle inhibitor cdkn1a (p21a/bCIP1/WAF1) in proliferating tissues. In addition, transcription of cyclin genes ccna2 and ccnd1 was deregulated in ddb1m863 mutants. Reduction of p53 activity by anti-sense morpholinos alleviated the apoptotic phenotype in ddb1m863 mutants. These results imply that Ddb1 may be involved in maintaining proper cell cycle progression and viability of dividing cells during development through transcriptional mechanisms regulating genes

  19. Gene Expression in Pre-MBT Embryos and Activation of Maternally-Inherited Program of Apoptosis to be Executed at around MBT as a Fail-Safe Mechanism in Xenopus Early Embryogenesis

    PubMed Central

    Shiokawa, Koichiro; Aso, Mai; Kondo, Takeshi; Uchiyama, Hiroaki; Kuroyanagi, Shinsaku; Takai, Jun-Ichi; Takahashi, Senji; Kajitani, Masayuki; Kaito, Chikara; Sekimizu, Kazuhisa; Takayama, Eiji; Igarashi, Kazuei; Hara, Hiroshi

    2008-01-01

    S-adenosylmethionine decarboxylase (SAMDC) is an enzyme which converts S-adenosylmethione (SAM), a methyl donor, to decarboxylated SAM (dcSAM), an aminopropyl donor for polyamine biosynthesis. In our studies on gene expression control in Xenopus early embryogenesis, we cloned the mRNA for Xenopus SAMDC, and overexpressed the enzyme by microinjecting its mRNA into Xenopus fertilized eggs. In the mRNA-injected embryos, the level of SAMDC was enormously increased, the SAM was exhausted, and protein synthesis was greatly inhibited, but cellular polyamine content did not change appreciably. SAMDC-overexpressed embryos cleaved and developed normally up to the early blastula stage, but at the midblastula stage, or the stage of midblastula transition (MBT), all the embryos were dissociated into cells, and destroyed due to execution of apoptosis. During cleavage SAMDC-overexpressed embryos transcribed caspase-8 gene, and this was followed by activation of caspase-9. When we overexpressed p53 mRNA in fertilized eggs, similar apoptosis took place at MBT, but in this case, transcription of caspase-8 did not occur, however activation of caspase-9 took place. Apoptosis induced by SAMDC-overexpression was completely suppressed by Bcl-2, whereas apoptosis induced by p53 overexpression or treatments with other toxic agents was only partially rescued. When we injected SAMDC mRNA into only one blastomere of 8- to 32-celled embryos, descendant cells of the mRNA-injected blastomere were segregated into the blastocoel and underwent apoptosis within the blastocoel, although such embryos continued to develop and became tadpoles with various extents of anomaly, reflecting the developmental fate of the eliminated cells. Thus, embryonic cells appear to check themselves at MBT and if physiologically severely-damaged cells occur, they are eliminated from the embryo by activation and execution of the maternally-inherited program of apoptosis. We assume that the apoptosis executed at MBT is a

  20. Glassfrog embryos hatch early after parental desertion.

    PubMed

    Delia, Jesse R J; Ramírez-Bautista, Aurelio; Summers, Kyle

    2014-06-22

    Both parental care and hatching plasticity can improve embryo survival. Research has found that parents can alter hatching time owing to a direct effect of care on embryogenesis or via forms of care that cue the hatching process. Because parental care alters conditions critical for offspring development, hatching plasticity could allow embryos to exploit variation in parental behaviour. However, this interaction of parental care and hatching plasticity remains largely unexplored. We tested the hypothesis that embryos hatch early to cope with paternal abandonment in the glassfrog Hyalinobatrachium fleischmanni (Centrolenidae). We conducted male-removal experiments in a wild population, and examined embryos' response to conditions with and without fathers. Embryos hatched early when abandoned, but extended development in the egg stage when fathers continued care. Paternal care had no effect on developmental rate. Rather, hatching plasticity was due to embryos actively hatching at different developmental stages, probably in response to deteriorating conditions without fathers. Our experimental results are supported by a significant correlation between the natural timing of abandonment and hatching in an unmanipulated population. This study demonstrates that embryos can respond to conditions resulting from parental abandonment, and provides insights into how variation in care can affect selection on egg-stage adaptations. PMID:24789892

  1. Glassfrog embryos hatch early after parental desertion

    PubMed Central

    Delia, Jesse R. J.; Ramírez-Bautista, Aurelio; Summers, Kyle

    2014-01-01

    Both parental care and hatching plasticity can improve embryo survival. Research has found that parents can alter hatching time owing to a direct effect of care on embryogenesis or via forms of care that cue the hatching process. Because parental care alters conditions critical for offspring development, hatching plasticity could allow embryos to exploit variation in parental behaviour. However, this interaction of parental care and hatching plasticity remains largely unexplored. We tested the hypothesis that embryos hatch early to cope with paternal abandonment in the glassfrog Hyalinobatrachium fleischmanni (Centrolenidae). We conducted male-removal experiments in a wild population, and examined embryos' response to conditions with and without fathers. Embryos hatched early when abandoned, but extended development in the egg stage when fathers continued care. Paternal care had no effect on developmental rate. Rather, hatching plasticity was due to embryos actively hatching at different developmental stages, probably in response to deteriorating conditions without fathers. Our experimental results are supported by a significant correlation between the natural timing of abandonment and hatching in an unmanipulated population. This study demonstrates that embryos can respond to conditions resulting from parental abandonment, and provides insights into how variation in care can affect selection on egg-stage adaptations. PMID:24789892

  2. Mouse Embryo Compaction.

    PubMed

    White, M D; Bissiere, S; Alvarez, Y D; Plachta, N

    2016-01-01

    Compaction is a critical first morphological event in the preimplantation development of the mammalian embryo. Characterized by the transformation of the embryo from a loose cluster of spherical cells into a tightly packed mass, compaction is a key step in the establishment of the first tissue-like structures of the embryo. Although early investigation of the mechanisms driving compaction implicated changes in cell-cell adhesion, recent work has identified essential roles for cortical tension and a compaction-specific class of filopodia. During the transition from 8 to 16 cells, as the embryo is compacting, it must also make fundamental decisions regarding cell position, polarity, and fate. Understanding how these and other processes are integrated with compaction requires further investigation. Emerging imaging-based techniques that enable quantitative analysis from the level of cell-cell interactions down to the level of individual regulatory molecules will provide a greater understanding of how compaction shapes the early mammalian embryo. PMID:27475854

  3. Nuclear AP/sub 4/A-binding activity of sea urchin embryos changes in relation to the initiation of S phase

    SciTech Connect

    Morioka, M.; Shimada, H.

    1986-01-01

    The AP/sub 4/A-binding activity of sea urchin embryos was studied using radioactively labelled diadenosine 5', 5'''-P/sup 1/,P/sup 4/-tetraphosphate (Ap/sub 4/A). Among various subcellular components that can bind (/sup 3/H)AP/sub 4/A, nuclei alone showed the highly specific Ap/sub 4/A-binding activity which was not influenced by the presence of AP/sub 4/A, AP/sub 5/A and GP/sub 4/G. The addition of an excess amount of ATP only slightly reduced the binding of (/sup 3/H)AP/sub 4/A to the nuclei. It was found that AP/sub 4/A binds to the residual proteinaceous structure of nuclei which was resistant to the extraction with 2 M NaCl. The nuclear AP/sub 4/A-binding activity fluctuated cyclically during each cell cycle, with at transient increase at the beginning of S phase followed by an abrupt-decrease within 10 min. When the initiation of S phase was blocked, the increase in the AP/sub 4/A-binding activity was also prevented. It seems that the binding of AP/sub 4/A to the nuclear structural protein is involved in the initiation of S phase.

  4. Features of complement activation-related pseudoallergy to liposomes with different surface charge and PEGylation: comparison of the porcine and rat responses.

    PubMed

    Dézsi, László; Fülöp, Tamás; Mészáros, Tamás; Szénási, Gábor; Urbanics, Rudolf; Vázsonyi, Csenge; Őrfi, Erik; Rosivall, László; Nemes, Réka; Kok, Robbert Jan; Metselaar, Josbert M; Storm, Gert; Szebeni, János

    2014-12-10

    Pigs are known to provide a sensitive model for studying complement (C) activation-related pseudoallergy (CARPA), a hypersensitivity reaction to liposomal and many other nanomedicines that limits their clinical use. The utility of rats as a CARPA model has, however, not been analyzed to date in detail. The present study compared the two models by inducing CARPA with i.v. bolus injections of two reactogenic liposomes that differed from each other in surface properties: one was AmBisome, a strong anionic, free-surface small unilamellar liposome (SUV), while the other was neutral, polyethylene glycol (PEG)-grafted SUV wherein the 2 kDa-PEG was anchored to the membrane via cholesterol (Chol-PEG). Both in pigs and rats AmBisome caused significant consumption of C3, indicating C activation, along with paralleling massive changes in blood pressure, white blood cell, platelet counts and in plasma thromboxane B2 levels, indicating CARPA. These processes were similar in the two species in terms of kinetics, but significantly differed in the doses that caused major hemodynamic changes (~0.01 and ~22 mg phospholipid (PL)/kg in pigs and rats, respectively). Pigs responded to AmBisome with pulmonary hypertension and systemic hypotension, and the reaction was not tachyphylactic. The major response of rats was systemic hypotension, leukopenia followed by leukocytosis, and thrombocytopenia. Chol-PEG liposomes caused severe reaction in pigs at 0.1 mg/kg, while the reaction they caused in rats was mild even at 300 mg PL/kg. Importantly, the reaction to Chol-PEG in pigs was partly tachyphylactic. These observations highlight fundamental differences in the immune mechanisms of porcine and rat CARPA, and also show a major impact of liposome surface characteristics, determining the presence or absence of tachyphylaxis. The data suggest that rats are 2-3 orders of magnitude less sensitive to liposomal CARPA than pigs; however, the causes of these differences, the PEG

  5. Establishment, differentiation, electroporation, viral transduction, and nuclear transfer of bovine and porcine mesenchymal stem cells.

    PubMed

    Colleoni, S; Donofrio, G; Lagutina, I; Duchi, R; Galli, C; Lazzari, G

    2005-01-01

    Mesenchymal stem cells (MSCs) reside in the bone marrow and have the potential for multilineage differentiation, into bone, cartilage, and fat, for example. In this study, bovine and porcine MSCs were isolated, cultured to determine their replication ability, and differentiated with osteogenic medium and 5-azacytine. Both bovine and porcine undifferentiated MSCs were electroporated and virally transduced to test the efficiency of genetic modification and the maintainance of differentiation ability thereafter. Nuclear transfer experiments were carried out with bovine and porcine MSCs, both at the undifferentiated state and following differentiation. Our results indicate that bovine and porcine MSCs have limited lifespans in vitro--approximately 50 population doublings. They can be efficiently differentiated and characterized along the osteogenic lineage by morphology, alkaline phosphatase, Von Kossa, oil red stainings, and RT-PCR. Electroporation and selection induce high levels of EGFP expression in porcine but not in bovine MSCs. Following genetic modification, MSCs retain their pluridifferentiation ability as parental cells. Cloned embryos derived from bovine and porcine undifferentiated MSCs and their derivatives along the osteogenic lineage give rise to consistently high preimplantation development comparable to adult fibroblasts. PMID:16176125

  6. Recent advances toward the practical application of embryo transfer in pigs.

    PubMed

    Martinez, Emilio A; Cuello, Cristina; Parrilla, Inmaculada; Martinez, Cristina A; Nohalez, Alicia; Vazquez, Jose L; Vazquez, Juan M; Roca, Jordi; Gil, Maria A

    2016-01-01

    Porcine embryo transfer (ET) technology has been in demand for decades because of its potential to provide considerable improvements in pig production with important sanitary, economic, and animal welfare benefits. Despite these advantages, the commercial use of ET is practically nonexistent. However, the two main obstacles hindering the commercial use of ET in pigs in the past several decades (i.e., surgical transfer and embryo preservation) have recently been overcome. A technique for nonsurgical deep-uterine (NsDU) ET of nonsedated gilts and sows, which was seemingly an impossible challenge just a few years ago, is a reality today. The improvements in embryo preservation that have been achieved in recent years and the excellent reproductive performance of the recipients after the NsDU-ET technique coupled with short-term and long-term-stored embryos represent essential progress for the international trade of porcine embryos and the practical use of ET by the pig industry. This review focuses, with an emphasis on our own findings, on the recent advances in embryo preservation and NsDU-ET technologies, which are starting to show potential for application under field conditions. PMID:26164803

  7. C/EBPβ (NF-M) Is Essential for Activation of the p20K Lipocalin Gene in Growth-Arrested Chicken Embryo Fibroblasts

    PubMed Central

    Kim, Selena; Mao, Pei-Lin; Gagliardi, Mark; Bédard, Pierre-André

    1999-01-01

    The p20K gene is induced in conditions of reversible growth arrest in chicken embryo fibroblasts (CEF). This expression is dependent on transcriptional activation and on a region of the promoter designated the quiescence-responsive unit (QRU). In this report, we describe the regulatory elements of the QRU responsible for activation in resting cells and characterize the trans-acting proteins interacting with these elements. We show that the QRU consists of functionally distinct domains including quiescence-specific and weak proliferation-responsive elements. The quiescence responsiveness of the QRU was mapped to two C/EBP binding sites, and the activity of the p20K promoter and its QRU was inhibited by the expression of a dominant negative mutant of C/EBPβ in nondividing cells. The activation of QRU in response to serum starvation and contact inhibition correlated with the presence of a growth arrest-specific complex in electrophoretic mobility shift assays. This complex was supershifted by antibody for C/EBPβ. C/EBPβ accumulated in conditions of contact inhibition as a result of transcriptional activation. Therefore, C/EBPβ was itself regulated as a growth arrest-specific gene in CEF. Finally, we show that the expression of p20K is regulated by linoleic acid, an essential fatty acid binding to p20K. The addition of linoleic acid to contact-inhibited CEF markedly repressed the synthesis of p20K without inducing mitogenesis. The activity of the QRU was inhibited by linoleic acid or the peroxisome proliferator-activated receptor PPARγ2 in transient expression assays. Therefore, we have identified C/EBPβ as a key activator of a growth arrest-specific gene in CEF and implicated an essential fatty acid, linoleic acid, in regulation of the QRU and the p20K lipocalin gene. PMID:10409760

  8. Novel derivatives of 1,3,4-oxadiazoles are potent mitostatic agents featuring strong microtubule depolymerizing activity in the sea urchin embryo and cell culture assays.

    PubMed

    Kiselyov, Alex S; Semenova, Marina N; Chernyshova, Natalya B; Leitao, Andrei; Samet, Alexandr V; Kislyi, Konstantine A; Raihstat, Mikhail M; Oprea, Tudor; Lemcke, Heiko; Lantow, Margaréta; Weiss, Dieter G; Ikizalp, Nazli N; Kuznetsov, Sergei A; Semenov, Victor V

    2010-05-01

    A series of novel 1,3,4-oxadiazole derivatives based on structural and electronic overlap with combretastatins have been designed and synthesized. Initially, we tested all new compounds in vivo using the phenotypic sea urchin embryo assay to yield a number of agents with anti-proliferative, anti-mitotic, and microtubule destabilizing activities. The experimental data led to identification of 1,3,4-oxadiazole derivatives with isothiazole (5-8) and phenyl (9-12) pharmacophores featuring activity profiles comparable to that of combretastatins, podophyllotoxin and nocodazole. Cytotoxic effects of the two lead molecules, namely 6 and 12, were further confirmed and evaluated by conventional assays with the A549 human cancer cell line including cell proliferation, cell cycle arrest at the G2/M phase, cellular microtubule distribution, and finally in vitro microtubule assembly with purified tubulin. The modeling results using 3D similarity (ROCS) and docking (FRED) correlated well with the observed activity of the molecules. Docking data suggested that the most potent molecules are likely to target the colchicine binding site. PMID:20110137

  9. In vivo studies on angiogenic activity of two designer self-assembling peptide scaffold hydrogels in the chicken embryo chorioallantoic membrane

    NASA Astrophysics Data System (ADS)

    Liu, Xi; Wang, Xiumei; Horii, Akihiro; Wang, Xiujuan; Qiao, Lin; Zhang, Shuguang; Cui, Fu-Zhai

    2012-03-01

    The rapid promotion of angiogenesis is critical for tissue engineering and regenerative medicine. The angiogenic activity of tissue-engineered scaffolds has already been the major criterion for choosing and designing ideal biological materials. We here report systematic in vivo studies on the angiogenic activity of two functionalized self-assembling peptides PRG (Ac-(RADA)4GPRGDSGYRGDS-CONH2) and KLT (Ac-(RADA)4G4KLTWQELYQLKYKGI-CONH2) using the chicken embryo chorioallantoic membrane (CAM) assay. 3D migration/sprouting bead assays showed that the two functional motifs PRGDSGYRGDS and KLTWQELYQLKYKGI improved the bioactivities of the self-assembling peptide RADA16-I (Ac-(RADA)4-CONH2) dramatically and provided ideal synthetic microenvironments for endothelial cell migration and cordlike structure sprout formation. A CAM assay was carried out to assess the efficiency of various peptide scaffolds in inducing capillary invasion in vivo. Among these three peptide scaffolds, the functionalized peptide scaffold RAD/KLT presented a significantly better angiogenic activity inducing CAM tissue invasion and new capillary vessel formation within the scaffolds in the absence of VEGF. With the addition of VEGF, more newly formed vessel lumen could be observed in all peptide scaffolds. Our results suggested that the functionalized peptide scaffolds had satisfactory angiogenic properties, and may also have wide potential applications in tissue regeneration.

  10. In vivo studies on angiogenic activity of two designer self-assembling peptide scaffold hydrogels in the chicken embryo chorioallantoic membrane.

    PubMed

    Liu, Xi; Wang, Xiumei; Horii, Akihiro; Wang, Xiujuan; Qiao, Lin; Zhang, Shuguang; Cui, Fu-Zhai

    2012-04-21

    The rapid promotion of angiogenesis is critical for tissue engineering and regenerative medicine. The angiogenic activity of tissue-engineered scaffolds has already been the major criterion for choosing and designing ideal biological materials. We here report systematic in vivo studies on the angiogenic activity of two functionalized self-assembling peptides PRG (Ac-(RADA)(4)GPRGDSGYRGDS-CONH(2)) and KLT (Ac-(RADA)(4)G(4)KLTWQELYQLKYKGI-CONH(2)) using the chicken embryo chorioallantoic membrane (CAM) assay. 3D migration/sprouting bead assays showed that the two functional motifs PRGDSGYRGDS and KLTWQELYQLKYKGI improved the bioactivities of the self-assembling peptide RADA16-I (Ac-(RADA)(4)-CONH(2)) dramatically and provided ideal synthetic microenvironments for endothelial cell migration and cordlike structure sprout formation. A CAM assay was carried out to assess the efficiency of various peptide scaffolds in inducing capillary invasion in vivo. Among these three peptide scaffolds, the functionalized peptide scaffold RAD/KLT presented a significantly better angiogenic activity inducing CAM tissue invasion and new capillary vessel formation within the scaffolds in the absence of VEGF. With the addition of VEGF, more newly formed vessel lumen could be observed in all peptide scaffolds. Our results suggested that the functionalized peptide scaffolds had satisfactory angiogenic properties, and may also have wide potential applications in tissue regeneration. PMID:22430460

  11. Monomeric adiponectin increases cell viability in porcine aortic endothelial cells cultured in normal and high glucose conditions: Data on kinases activation.

    PubMed

    Grossini, Elena; Farruggio, Serena; Qoqaiche, Fatima; Raina, Giulia; Camillo, Lara; Sigaudo, Lorenzo; Mary, David; Surico, Nicola; Surico, Daniela

    2016-09-01

    We found that monomeric adiponectin was able to increase cell viability in porcine aortic endothelial cells (PAE) cultured both in normal and high glucose condition. Moreover, in normal glucose condition monomeric adiponectin increased p38MAPK, Akt, ERK1/2 and eNOS phosphorylation in a dose- and time-dependent way. Also in high glucose condition monomeric adiponectin increased eNOS and above kinases phosphorylation with similar patterns but at lower extent. For interpretation of the data presented in this article, please see the research article "Monomeric adiponectin modulates nitric oxide release and calcium movements in porcine aortic endothelial cells in normal/high glucose conditions" (Grossini et al., in press) [1]. PMID:27583345

  12. Evaluation of a new range of light-activated surgical adhesives for tissue repair in a porcine model

    NASA Astrophysics Data System (ADS)

    Riley, Jill N.; Hodges, Diane E.; March, Keith L.; McNally-Heintzelman, Karen M.

    2001-05-01

    An in vitro study was conducted to determine the feasibility of using a new range of light-activated surgical adhesives for incision repair in a wide range of tissue types. Biodegradable polymer membranes of controlled porosity were fabricated with poly(L-lactic-co-glycolic acid) (PLGA) and salt particles using a solvent-casting and particulate- leaching technique. The porous membranes were doped with protein solder composed of 50%(w/v) bovine serum albumin solder and 0.5 mg/ml indocyanine green (ICG) dye mixed in deionized water. Tissue incisions were repaired using the surgical adhesive in conjunction with an 805-nm diode laser. Nine organs were tested ranging from skin to liver to the small intestine, as well as the coronary, pulmonary, carotid, femoral and splenetic arteries. Acute breaking strengths were measured and the data were analyzed by Student's T-test. Repairs formed on the small intestine were most successful followed by spleen, atrium, kidney, muscle and skin. The strongest vascular repairs were achieved in the carotid artery and femoral artery. The new surgical adhesive could possibly be used as a simple and effective method to stop bleeding and repair tissue quickly in an emergency situation, or as a substitute to mechanical staples or sutures in many clinical applications.

  13. Mitochondrial respiratory activity in porcine longissimus muscle fibers of different pig genetics in relation to their meat quality.

    PubMed

    Werner, C; Natter, R; Schellander, K; Wicke, M

    2010-05-01

    The pig genetics of Duroc, Pietrain (MHS homozygote negative, PiNN), Pietrain (MHS homozygote positive, PiPP) and a F2-Duroc-Pietrain cross-breed were analyzed. The animals had comparable (P>0.05) carcass weights, but the PiPP pigs had higher carcass yield and lean meat values (P<0.05). Considering the meat quality characteristics, the PiPP showed a faster pH drop and higher electrical conductivity, drip loss, shear force as well as lightness and redness values (P<0.05). The PiPP animals had less slow-twitch-oxidative (STO) and more fast-twitch-glycolytic (FTG) muscle fibers, whereas the results of the Duroc animals were converse (P<0.05). The STO and FTG fibers of the PiPP animals were larger than those of the other genetics (P<0.05). The analysis of the mitochondrial respiratory activity (MRA) using permeabilized longissimus muscle fibers resulted in no differences between the pig genetics before and immediately after slaughter. During chilling the MRA decreased in all pigs but to a higher extent in the PiPP pigs (P<0.05). PMID:20374876

  14. Zeatin reductase in Phaseolus embryos

    SciTech Connect

    Martin, R.C.; Mok, David, W.S.; Mok, M.C. )

    1989-04-01

    Zeatin was converted to O-xylosylzeatin in embryos of Phaseolus vulgaris . O-xylosyldihydrozeatin was also identified as a zeatin metabolite. Incubation of embryo extracts with {sup 14}C-zeatin and {sup 14}C-O-xylosylzeatin revealed that reduction preceeds the O-xylosylation of zeatin. An enzyme responsible for reducing the N{sup 6}-side chain was isolated and partially purified using ammonium sulfate fractionation and affinity, gel filtration and anion exchange chromatography. The NADPH dependent reductase was zeatin specific and did not recognize cis-zeatin, ribosylzeatin, i{sup 6}Ade or i{sup 6}Ado. Two forms of the reductase could be separated by either gel filtration or anion exchange HPLC. The HMW isozyme (Mr. 55,000) eluted from the anion exchange column later than the LMW isozyme (Mr. 25,000). Interspecific differences in zeatin reductase activity were also detected.

  15. Phase III interlaboratory study of FETAX, Part 2: interlaboratory validation of an exogenous metabolic activation system for frog embryo teratogenesis assay--Xenopus (FETAX).

    PubMed

    Fort, D J; Stover, E L; Bantle, J A; Rayburn, J R; Hull, M A; Finch, R A; Burton, D T; Turley, S D; Dawson, D A; Linder, G; Buchwalter, D; Dumont, J N; Kumsher-King, M; Gaudet-Hull, A M

    1998-02-01

    Interlaboratory validation of an exogenous metabolic activation system (MAS) developed for the alternative, short-term developmental toxicity bioassay, Frog Embryo Teratogenesis Assay-Xenopus (FETAX) was performed with cyclophosphamide and caffeine. Seven study groups within six separate laboratories participated in the study in which three definitive concentration-response experiments were performed with and without the MAS in a side-by-side format for each chemical. Since both chemicals had been previously tested in FETAX, the test concentrations were provided to each laboratory prior to testing. Interlaboratory coefficient of variation (CV) values for unactivated cyclophosphamide (no MAS) were 15%, 15%, 29%, and 25% for the 96-hr LC50, 96-hr EC50 (malformation), Minimum Concentration to Inhibit Growth (MCIG), and Teratogenic Index (TI) values, respectively. Addition of the MAS increased the CV values of each endpoint at least 3.9-fold. Interlaboratory CV values for unactivated caffeine were 31%, 18%, 31%, and 46% for the 96-hr LC50, 96-hr EC50 (malformation), MCIG, and TI values, respectively. Addition of the MAS decreased the CV values of each respective endpoint by at least 1.6-fold. Results indicated that bioactivated toxicants may be prone to greater variability in response amongst laboratories than compounds, which are detoxified. Even though more variability was noted with activated cyclophosphamide, results were within interlaboratory variation expected for other aquatic-based bioassays. Thus, results from these studies warrant the continued use and further refinement of FETAX for alternative developmental toxicity assessment. PMID:9530526

  16. Porcine circovirus type 2 activates PI3K/Akt and p38 MAPK pathways to promote interleukin-10 production in macrophages via Cap interaction of gC1qR.

    PubMed

    Du, Qian; Huang, Yong; Wang, Tongtong; Zhang, Xiujuan; Chen, Yu; Cui, Beibei; Li, Delong; Zhao, Xiaomin; Zhang, Wenlong; Chang, Lingling; Tong, Dewen

    2016-04-01

    Porcine circovirus type 2 (PCV2) infection caused PCV2-associated diseases (PCVAD) is one of the major emerging immunosuppression diseases in pig industry. In this study, we investigated how PCV2 inoculation increases interleukin (IL)-10 expression in porcine alveolar macrophages (PAMs). PCV2 inoculation significantly upregulated IL-10 expression compared with PCV1. Upon initial PCV2 inoculation, PI3K/Akt cooperated with NF-κB pathways to promote IL-10 transcription via p50, CREB and Ap1 transcription factors, whereas inhibition of PI3K/Akt activation blocked Ap1 and CREB binding to the il10 promoter, and decreased the binding level of NF-κB1 p50 with il10 promoter, leading to great reduction in early IL-10 transcription. In the later phase of inoculation, PCV2 further activated p38 MAPK and ERK pathways to enhance IL-10 production by promoting Sp1 binding to the il10 promoter. For PCV2-induced IL-10 production in macrophages, PCV2 capsid protein Cap, but not the replicase Rep or ORF3, was the critical component. Cap activated PI3K/Akt, p38 MAPK, and ERK signaling pathways to enhance IL-10 expression. In the whole process, gC1qR mediated PCV2-induced PI3K/Akt and p38 MAPK activation to enhance IL-10 induction by interaction with Cap. Depletion of gC1qR blocked PI3K/Akt and p38 MAPK activation, resulting in significant decrease in IL-10 production in PCV2-inoculated cells. Thus, gC1qR might be a critical functional receptor for PCV2-induced IL-10 production. Taken together, these data demonstrated that Cap protein binding with host gC1qR induction of PI3K/Akt and p38 MAPK signalings activation is a critical process in enhancing PCV2-induced IL-10 production in porcine alveolar macrophages. PMID:26883107

  17. Porcine circovirus type 2 activates PI3K/Akt and p38 MAPK pathways to promote interleukin-10 production in macrophages via Cap interaction of gC1qR

    PubMed Central

    Wang, Tongtong; Zhang, Xiujuan; Chen, Yu; Cui, Beibei; Li, Delong; Zhao, Xiaomin; Zhang, Wenlong; Chang, Lingling; Tong, Dewen

    2016-01-01

    Porcine circovirus type 2 (PCV2) infection caused PCV2-associated diseases (PCVAD) is one of the major emerging immunosuppression diseases in pig industry. In this study, we investigated how PCV2 inoculation increases interleukin (IL)-10 expression in porcine alveolar macrophages (PAMs). PCV2 inoculation significantly upregulated IL-10 expression compared with PCV1. Upon initial PCV2 inoculation, PI3K/Akt cooperated with NF-κB pathways to promote IL-10 transcription via p50, CREB and Ap1 transcription factors, whereas inhibition of PI3K/Akt activation blocked Ap1 and CREB binding to the il10 promoter, and decreased the binding level of NF-κB1 p50 with il10 promoter, leading to great reduction in early IL-10 transcription. In the later phase of inoculation, PCV2 further activated p38 MAPK and ERK pathways to enhance IL-10 production by promoting Sp1 binding to the il10 promoter. For PCV2-induced IL-10 production in macrophages, PCV2 capsid protein Cap, but not the replicase Rep or ORF3, was the critical component. Cap activated PI3K/Akt, p38 MAPK, and ERK signaling pathways to enhance IL-10 expression. In the whole process, gC1qR mediated PCV2-induced PI3K/Akt and p38 MAPK activation to enhance IL-10 induction by interaction with Cap. Depletion of gC1qR blocked PI3K/Akt and p38 MAPK activation, resulting in significant decrease in IL-10 production in PCV2-inoculated cells. Thus, gC1qR might be a critical functional receptor for PCV2-induced IL-10 production. Taken together, these data demonstrated that Cap protein binding with host gC1qR induction of PI3K/Akt and p38 MAPK signalings activation is a critical process in enhancing PCV2-induced IL-10 production in porcine alveolar macrophages. PMID:26883107

  18. Tribolium embryo morphogenesis

    PubMed Central

    Benton, Matthew A; Pavlopoulos, Anastasios

    2014-01-01

    Development of multicellular organisms depends on patterning and growth mechanisms encoded in the genome, but also on the physical properties and mechanical interactions of the constituent cells that interpret these genetic cues. This fundamental biological problem requires integrated studies at multiple levels of biological organization: from genes, to cell behaviors, to tissue morphogenesis. We have recently combined functional genetics with live imaging approaches in embryos of the insect Tribolium castaneum, in order to understand their remarkable transformation from a uniform single-layered blastoderm into a condensed multi-layered embryo covered by extensive extra-embryonic tissues. We first developed a quick and reliable methodology to fluorescently label various cell components in entire Tribolium embryos. Live imaging of labeled embryos at single cell resolution provided detailed descriptions of cell behaviors and tissue movements during normal embryogenesis. We then compared cell and tissue dynamics between wild-type and genetically perturbed embryos that exhibited altered relative proportions of constituent tissues. This systematic comparison led to a qualitative model of the molecular, cellular and tissue interactions that orchestrate the observed epithelial rearrangements. We expect this work to establish the Tribolium embryo as a powerful and attractive model system for biologists and biophysicists interested in the molecular, cellular and mechanical control of tissue morphogenesis. PMID:24451992

  19. Biphasic effect of arsenite on cell proliferation and apoptosis is associated with the activation of JNK and ERK1/2 in human embryo lung fibroblast cells

    SciTech Connect

    He Xiaoqing; Chen Rui; Yang Ping; Li Aiping; Zhou Jianwei; Liu Qizhan . E-mail: drqzliu@126.com

    2007-04-01

    Biphasic dose-response relationship induced by environmental agents is often characterized with the effect of low-dose stimulation and high-dose inhibition. Some studies showed that arsenite may induce cell proliferation and apoptosis via biphasic dose-response relationship in human cells; however, mechanisms underlying this phenomenon are not well understood. In the present study, we aimed at investigating the relationship between biphasic effect of arsenite on cell proliferation and apoptosis and activation of JNK and ERK1/2 in human embryo lung fibroblast (HELF) cells. Our results demonstrated that cell proliferation may be stimulated at lower concentrations (0.1 and 0.5 {mu}M) arsenite but inhibited at higher concentrations (5 and 10 {mu}M). When cell apoptosis was used as the endpoint, the concentration-response curves were changed to U-shapes. During stimulation phospho-JNK levels were significantly increased at 3, 6, and 12 h after 0.1 or 0.5 {mu}M arsenite exposure. Phospho-ERK1/2 levels were increased with different concentrations (0.1-10 {mu}M) of arsenite at 6, 12, and 24 h. Blocking of JNK pathway with 20 {mu}M SP600125 or ERK1/2 by 100 {mu}M PD98059 significantly inhibited biphasic effect of arsenite in cells. Data in the present study suggest that activation of JNK and ERK1/2 may be involved in biphasic effect of arsenite when measuring cell proliferation and apoptosis in HELF cells. JNK activation seems to play a more critical role than ERK1/2 activation in the biphasic process.

  20. Comparison of the metabolic activation of 7, 12-dimethylbenz(a)anthracene by a human hepatoma cell line (HepG2) and low passage hamster embryo cells

    SciTech Connect

    DiGiovanni, J.; Singer, J.M.; Diamond, L.

    1984-07-01

    Under similar conditions of cell-mediated mutagenesis, secondary hamster embryo (HE) cells were much more effective than were cells of the human hepatoma cell line, HepG2 , in activating 7, 12-dimethylbenz(a)anthracene (DMBA) to metabolites mutagenic for V79 Chinese hamster cells. At the same dose of DMBA (0.1 microgram/ml), mutation induction (6-thioguanine resistance) with HE cells as activators was about ten times greater than with HepG2 cells as activators. Both cell types rapidly metabolized DMBA. HepG2 cells converted DMBA primarily to water-soluble derivatives that were neither sulfates nor glucuronides, whereas HE cells converted DMBA to a variety of organic solvent-soluble and water-soluble metabolites. The major water-soluble metabolites produced by HE cells were phenol-glucuronides. In HepG2 cells, binding of DMBA to DNA reached a maximum value of 12.1 pmol/mg DNA at 12 hr, whereas in HE cells, binding reached a peak value of 180.7 pmol/mg DNA at 24 hr. Despite this difference in total binding between the two cell types, the pattern of DNA adducts formed was nearly identical. The results indicate that the marked difference in the ability of HepG2 and HE cells to activate DMBA in cell-mediated mutation assays is not due to a lower metabolizing capacity of HepG2 cells for DMBA. Rather, significant differences in the metabolic pathways used by the two cell types lead to a marked reduction in DNA-binding metabolites in one cell type (HepG2) compared to the other (HE).

  1. Evaluation of porcine mesenchymal stem cells for therapeutic use in human liver cancer.

    PubMed

    Groth, Ariane; Ottinger, Sabine; Kleist, Christian; Mohr, Elisabeth; Golriz, Mohammad; Schultze, Daniel; Bruns, Helge; Mehrabi, Arianeb; Schemmer, Peter; Büchler, Markus W; Herr, Ingrid

    2012-02-01

    Mesenchymal stem cell (MSC) transplantation is suggested for therapy of end-stage liver disease, due to e.g. liver cancer and metastasis. Liver transplantation is the only therapeutic option so far but donor organs are short. Also, the availability of allogeneic human MSCs for liver regeneration is limited. Therefore, we evaluated the suitability of porcine bone marrow MSCs from semi-adult pigs and found that morphology, surface expression pattern and multilineage differentiation are similar to those of human MSCs. Porcine MSCs differentiated to a hepatocyte-like phenotype and expressed porcine mRNA of typical liver proteins. However, hepatocyte-like MSCs failed to express the corresponding proteins and did not produce glycogen and urea as primary porcine hepatocytes do. Porcine MSCs were immunotolerated, since they did not activate resting human PBMCs, and were not attacked by human activated PBMCs. However, porcine MSCs led to enhanced proliferation of human pre-activated PBMCs suggesting that immunotoleration of porcine MSCs in the human system has limitations. Together, the potential of porcine MSCs for xenogenous use in human liver therapy is promising but needs further evaluation prior to clinical use. PMID:21964567

  2. In vitro development of canine somatic cell nuclear transfer embryos in different culture media.

    PubMed

    Kim, Dong-Hoon; No, Jin-Gu; Choi, Mi-Kyung; Yeom, Dong-Hyeon; Kim, Dong-Kyo; Yang, Byoung-Chul; Yoo, Jae Gyu; Kim, Min Kyu; Kim, Hong-Tea

    2015-01-01

    The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos. PMID:25549216

  3. In vitro development of canine somatic cell nuclear transfer embryos in different culture media

    PubMed Central

    No, Jin-Gu; Choi, Mi-Kyung; Yeom, Dong-Hyeon; Kim, Dong-Kyo; Yang, Byoung-Chul; Yoo, Jae Gyu; Kim, Min Kyu; Kim, Hong-Tea

    2015-01-01

    The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%) or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos. PMID:25549216

  4. Heterologous expression of newly identified galectin-8 from sea urchin embryos produces recombinant protein with lactose binding specificity and anti-adhesive activity

    PubMed Central

    Karakostis, Kostantinos; Costa, Caterina; Zito, Francesca; Matranga, Valeria

    2015-01-01

    Galectin family members specifically bind beta-galactoside derivatives and are involved in different cellular events, including cell communication, signalling, apoptosis, and immune responses. Here, we report a tandem-repeat type galectin from the Paracentrotus lividus sea urchin embryo, referred to as Pl-GAL-8. The 933nt sequence encodes a protein of 34.73 kDa, containing the conserved HFNPRF and WGxExR motifs in the two highly similar carbohydrate-recognition domains (CRD). The three-dimensional protein structure model of the N-CRD confirms the high evolutionary conservation of carbohydrate binding sites. The temporal gene expression is regulated during development and transcripts localize at the tip of the archenteron at gastrula stage, in a subset of the secondary mesenchyme cells that differentiate into blastocoelar (immune) cells. Functional studies using a recombinant Pl-GAL-8 expressed in bacteria demonstrate its hemo-agglutinating activity on human red blood cells through the binding to lactose, as well as its ability in inhibiting the adhesion of human Hep-G2 cells to the substrate. The recent implications in autoimmune diseases and inflammatory disorders make Gal-8 an attractive candidate for therapeutic purposes. Our results offer a solid basis for addressing the use of the new Pl-GAL-8 in functional and applicative studies, respectively in the developmental and biomedical fields. PMID:26640155

  5. Functional verification of a porcine myostatin propeptide mutant.

    PubMed

    Ma, Dezun; Jiang, Shengwang; Gao, Pengfei; Qian, Lili; Wang, Qingqing; Cai, Chunbo; Xiao, Gaojun; Yang, Jinzeng; Cui, Wentao

    2015-10-01

    Myostatin is a member of TGF-β superfamily that acts as a key negative regulator in development and growth of embryonic and postnatal muscles. In this study, the inhibitory activities of recombinant porcine myostatin propeptide and its mutated form (at the cleavage site of metalloproteinases of BMP-1/TLD family) against murine myostatin was evaluated in vivo by intraperitoneal injection into mice. Results showed that both wild type and mutated form of porcine propeptide significantly inhibited myostatin activity in vivo. The average body weight of mice receiving wild type propeptide or its mutated form increased by 12.5 % and 24.14%, respectively, compared to mice injected with PBS, implying that the in vivo efficacy of porcine propeptide mutant is greater than its wild type propeptide. Transgenic mice expressing porcine myostatin propeptide mutant were generated to further verify the results obtained from mice injected with recombinant porcine propeptide mutant. Compared with wild type (non-transgenic) mice, relative weight of gastrocnemius, rectusfemoris, and tibialis anterior increased by 22.14 %, 34.13 %, 25.37%, respectively, in transgenic male mice, and by 19.90 %, 42.47 %, 45.61%, respectively, in transgenic female mice. Our data also demonstrated that the mechanism by which muscle growth enhancement is achieved by these propeptides is due to an increase in fiber sizes, not by an increase in number of fiber cells. PMID:26174475

  6. High incubation temperatures enhance mitochondrial energy metabolism in reptile embryos

    PubMed Central

    Sun, Bao-Jun; Li, Teng; Gao, Jing; Ma, Liang; Du, Wei-Guo

    2015-01-01

    Developmental rate increases exponentially with increasing temperature in ectothermic animals, but the biochemical basis underlying this thermal dependence is largely unexplored. We measured mitochondrial respiration and metabolic enzyme activities of turtle embryos (Pelodiscus sinensis) incubated at different temperatures to identify the metabolic basis of the rapid development occurring at high temperatures in reptile embryos. Developmental rate increased with increasing incubation temperatures in the embryos of P. sinensis. Correspondingly, in addition to the thermal dependence of mitochondrial respiration and metabolic enzyme activities, high-temperature incubation further enhanced mitochondrial respiration and COX activities in the embryos. This suggests that embryos may adjust mitochondrial respiration and metabolic enzyme activities in response to developmental temperature to achieve high developmental rates at high temperatures. Our study highlights the importance of biochemical investigations in understanding the proximate mechanisms by which temperature affects embryonic development. PMID:25749301

  7. The effects of superovulation of donor sows on ovarian response and embryo development after nonsurgical deep-uterine embryo transfer.

    PubMed

    Angel, M A; Gil, M A; Cuello, C; Sanchez-Osorio, J; Gomis, J; Parrilla, I; Vila, J; Colina, I; Diaz, M; Reixach, J; Vazquez, J L; Vazquez, J M; Roca, J; Martinez, E A

    2014-04-01

    This study aimed to evaluate the effectiveness of superovulation protocols in improving the efficiency of embryo donors for porcine nonsurgical deep-uterine (NsDU) embryo transfer (ET) programs. After weaning (24 hours), purebred Duroc sows (2-6 parity) were treated with 1000 IU (n = 27) or 1500 IU (n = 27) of eCG. Only sows with clear signs of estrus 4 to 72 hours after eCG administration were treated with 750 IU hCG at the onset of estrus. Nonhormonally treated postweaning estrus sows (n = 36) were used as a control. Sows were inseminated and subjected to laparotomy on Days 5 to 6 (Day 0 = onset of estrus). Three sows (11.1%) treated with the highest dosage of eCG presented with polycystic ovaries without signs of ovulation. The remaining sows from nonsuperovulated and superovulated groups were all pregnant, with no differences in fertilization rates among groups. The number of CLs and viable embryos was higher (P < 0.05) in the superovulated groups compared with the controls and increased (P < 0.05) with increasing doses of eCG. There were no differences among groups in the number of oocytes and/or degenerated embryos. The number of transferable embryos (morulae and unhatched blastocysts) obtained in pregnant sows was higher (P < 0.05) in the superovulated groups than in the control group. In all groups, there was a significant correlation between the number of CLs and the number of viable and transferable embryos, but the number of CLs and the number of oocytes and/or degenerated embryos were not correlated. A total of 46 NsDU ETs were performed in nonhormonally treated recipient sows, with embryos (30 embryos per transfer) recovered from the 1000-IU eCG, 1500-IU eCG, and control groups. In total, pregnancy and farrowing rates were 75.1% and 73.2%, respectively, with a litter size of 9.4 ± 0.6 piglets born, of which 8.8 ± 0.5 were born alive. There were no differences for any of the reproductive parameters evaluated among groups. In conclusion, our results

  8. Implantation: mutual activity of sex steroid hormones and the immune system guarantee the maternal-embryo interaction.

    PubMed

    Gnainsky, Yulia; Dekel, Nava; Granot, Irit

    2014-09-01

    Implantation is strictly dependent on the mutual interaction between a receptive endometrium and the blastocyst. Hence, synchronization between blastocyst development and the acquisition of endometrial receptivity is a prerequisite for the success of this process. This review depicts the cellular and molecular events that coordinate these complex activities. Specifically, the involvement of the sex steroid hormones, estrogen and progesterone, as well as components of the immune system, such as cytokines and specific blood cells, is elaborated. PMID:24959815

  9. How couples who have undergone in vitro fertilization decide what to do with surplus frozen embryos.

    PubMed

    Nachtigall, Robert D; Mac Dougall, Kirstin; Harrington, Jennifer; Duff, Julia; Lee, Matthew; Becker, Gay

    2009-12-01

    In a qualitative interview study of 77 families with stored frozen embryos, we found that while embryo disposition decision making was influenced by individual life circumstances, embryo quantity/quality, personal values, embryo conceptualization, and clinic information, it was a stepwise process that could be represented as three sequential questions: (1) Will the embryos be used for additional attempts at conception? If not, (2) Will the embryos remain in storage? And if not, (3) Will the embryos be donated to other people or to science, or will they be destroyed? While almost two-thirds (63%) of participants kept their embryos in storage after 5 years, either passively through disagreement or indecision or actively to maintain embryo potential, avert feelings of loss, or as psychological or genetic "insurance," IVF clinic support and detailed information about options motivated families to make disposition decisions. PMID:19700150

  10. Acute toxic effects of the herbicide formulation and the active ingredient used in cycloxydim-tolerant maize cultivation on embryos and larvae of the African clawed frog, Xenopus laevis.

    PubMed

    Wagner, Norman; Lötters, Stefan; Veith, Michael; Viertel, Bruno

    2015-04-01

    Most genetically engineered herbicide-tolerant crops are still awaiting approval in Europe. There is, however, a recent trend for the cultivation of cycloxydim-tolerant maize hybrids for use in maize production. We studied the acute toxic effects of the complementary herbicide Focus(®) Ultra and its active ingredient cycloxydim on embryos and early-stage larvae of the African clawed frog (Xenopus laevis). The results indicate that the herbicide formulation is significantly more toxic than the active ingredient alone. Therefore, it is suggested that the added substances either solely or in a synergistic action with the active ingredient are responsible for adverse effects. The formulation was found to be moderately toxic to embryos but highly toxic to early larvae. Based on calculated teratogenic indices, both cycloxydim and Focus(®) Ultra seem to be non-teratogenic and also the minimum Focus(®) Ultra concentration to inhibit growth in embryos and larvae was close to the LC50 values. The data suggest that tests with the rainbow trout are not in all cases appropriate to assess the risk in aquatically developing anurans. This is demonstrated by 96-h LC50 values, which are for rainbow trout more than 50- to 20-fold higher than for early X. laevis larvae. However, based on worst-case predicted environmental concentrations for surface waters, there is apparently a large safety margin in field use of Focus(®) Ultra if buffer strips between the farm land and the amphibian habitats are regarded. PMID:25634323

  11. Dimethyl Sulfoxide Perturbs Cell Cycle Progression and Spindle Organization in Porcine Meiotic Oocytes

    PubMed Central

    Li, Xuan; Wang, Yan-Kui; Song, Zhi-Qiang; Du, Zhi-Qiang; Yang, Cai-Xia

    2016-01-01

    Meiotic maturation of mammalian oocytes is a precisely orchestrated and complex process. Dimethyl sulfoxide (DMSO), a widely used solvent, drug, and cryoprotectant, is capable of disturbing asymmetric cytokinesis of oocyte meiosis in mice. However, in pigs, DMSO’s effect on oocyte meiosis still remains unknown. We aimed to evaluate if DMSO treatment will affect porcine oocyte meiosis and the underlying molecular changes as well. Interestingly, we did not observe the formation of the large first polar body and symmetric division for porcine oocytes treated with DMSO, contrary to findings reported in mice. 3% DMSO treatment could inhibit cumulus expansion, increase nuclear abnormality, disturb spindle organization, decrease reactive oxygen species level, and elevate mitochondrial membrane potential of porcine oocytes. There was no effect on germinal vesicle breakdown rate regardless of DMSO concentration. 3% DMSO treatment did not affect expression of genes involved in spindle organization (Bub1 and Mad2) and apoptosis (NF-κB, Pten, Bcl2, Caspase3 and Caspase9), however, it significantly decreased expression levels of pluripotency genes (Oct4, Sox2 and Lin28) in mature oocytes. Therefore, we demonstrated that disturbed cumulus expansion, chromosome alignment, spindle organization and pluripotency gene expression could be responsible for DMSO-induced porcine oocyte meiotic arrest and the lower capacity of subsequent embryo development. Our results provide new insights on DMSO’s effect on porcine oocyte meiosis and raise safety concerns over DMSO’s usage on female reproduction in both farm animals and humans. PMID:27348312

  12. Gene expression of porcine blastocysts from gilts fed organic or inorganic selenium and pyridoxine.

    PubMed

    Dalto, B D; Tsoi, S; Audet, I; Dyck, M K; Foxcroft, G R; Matte, J J

    2015-01-01

    In this study, we determined how maternal dietary supplementation with pyridoxine combined with different sources of selenium (Se) affected global gene expression of porcine expanded blastocysts (PEB) during pregnancy. Eighteen gilts were randomly assigned to one of the three experimental diets (n=6 per treatment): i) basal diet without supplemental Se or pyridoxine (CONT); ii) CONT+0.3 mg/kg of Na-selenite and 10 mg/kg of HCl-pyridoxine (MSeB610); and iii) CONT+0.3 mg/kg of Se-enriched yeast and 10 mg/kg of HCl-pyridoxine (OSeB610). All gilts were inseminated at their fifth post-pubertal estrus and killed 5 days later for embryo harvesting. A porcine embryo-specific microarray was used to detect differentially gene expression between MSeB610 vs CONT, OSeB610 vs CONT, and OSeB610 vs MSeB610. CONT gilts had lower whole blood Se and erythrocyte pyridoxal-5-P concentrations than supplemented gilts (P<0.05). No treatment effect was observed on blood plasma Se-glutathione peroxidase activity (P=0.57). There were 10, 247, and 96 differentially expressed genes for MSeB610 vs CONT, OSeB610 vs CONT, and OSeB610 vs MSeB610 respectively. No specific biological process was associated with MSeB610 vs CONT. However, for OSeB610 vs CONT, upregulated genes were related with global protein synthesis but not to selenoproteins. The stimulation of some genes related with monooxygenase and thioredoxin families was confirmed by quantitative real-time RT-PCR. In conclusion, OSeB610 affects PEB metabolism more markedly than MSeB610. Neither Se sources with pyridoxine influenced the Se-glutathione peroxidase metabolic pathway in the PEB, but OSeB610 selectively stimulated genes involved with antioxidant defense. PMID:25326430

  13. Impact of EmbryoGlue as the embryo transfer medium.

    PubMed

    Hazlett, William David; Meyer, Liza R; Nasta, Tricia E; Mangan, Patricia A; Karande, Vishvanath C

    2008-07-01

    Routine use of EmbryoGlue did not significantly improve pregnancy or implantation rates in nonselected patients receiving either a day 3 or day 5 embryo transfer compared with standard culture media. Future prospective randomized studies need to be performed to determine whether EmbryoGlue is beneficial in a selected patient population. PMID:17765233

  14. Detection of phosphorylated mitogen-activated protein kinase in the developing spinal cord of the mouse embryo

    SciTech Connect

    Teraishi, Toshiya; Miura, Kenji

    2011-09-16

    Highlights: {yields} We detected physiologically phosphorylated MAPKs in developing spinal cord. {yields} We detected physiologically phosphorylated MAPKs by an improved method. {yields} p-ERK1/2 and p-JNK1/2 were detected in the marginal layer and the dorsal horn. {yields} p-ERK1/2 and p-JNK1/2 might play critical roles in the developing spinal cord. {yields} Constructing phosphoprotein atlases will be possible if expanding this work. -- Abstract: Global understanding of the proteome is a major research topic. The comprehensive visualization of the distribution of proteins in vivo or the construction of in situ protein atlases may be a valuable strategy for proteomic researchers. Information about the distribution of various proteins under physiological and pathological conditions should be extremely valuable for the basic and clinical sciences. The mitogen-activated protein kinase (MAPK) cascade plays an essential role in intracellular signaling in organisms. This cascade also regulates biological processes involving development, differentiation, and proliferation. Phosphorylation and dephosphorylation are integral reactions in regulating the activity of MAPKs. Changes in the phosphorylation state of MAPKs are rapid and reversible; therefore, the localizations of physiologically phosphorylated MAPKs in vivo are difficult to accurately detect. Furthermore, phosphorylated MAPKs are likely to change phosphorylated states through commonly used experimental manipulations. In the present study, as a step toward the construction of in situ phosphoprotein atlases, we attempted to detect physiologically phosphorylated MAPKs in vivo in developing spinal cords of mice. We previously reported an improved immunohistochemical method for detecting unstable phosphorylated MAPKs. The distribution patterns of phosphorylated MAPKs in the spinal cords of embryonic mice from embryonic day 13 (E13) to E17 were observed with an improved immunohistochemical method. Phosphorylated

  15. Effect of the tripeptide glycyl-L-histidyl-L-lysine on the proliferation and synthetic activity of chick embryo chondrocytes.

    PubMed

    Pesáková, V; Novotná, J; Adam, M

    1995-08-01

    Under certain conditions chondrocytes form lattices with cartilage collagens, which may serve as cartilage implants. It is necessary to find the optimal conditions for culturing chondrocytes. Three different supports are compared: (a) plastic; (b) cartilage collagens; and (c) insoluble skin collagen solubilized under denaturing conditions (ISC-40). The effect of culture medium supplementation with the tripeptide (Gly-His-Lys)2.Cu.2H2O.2NaCl (GHK) on chondrocyte proliferation and synthetic activity is studied, with particular attention paid to collagen types I, II and III. The collagen supports stimulated chondrocyte proliferation, but on the ISC-40 support they started to dedifferentiate rather early. In the primary culture, chondrocytes on all three supports synthesized mainly collagen type II, and only small amounts of types I and III. In the first passage the synthesis of these two collagen types increased, relative to collagen type II, at least on the cartilage collagen support. Supplementation of culture medium with GHK stimulated chondrocyte proliferation in the primary structure mostly on the ISC-40 support. On the other two types of supports the stimulatory effect of GHK was expressed mostly in the first passages. The collagen synthetic rate was increased by GHK on both of the collagen supports; on the cartilage collagen support collagen type II was synthesized predominantly and on the ISC-40 support types I and III were mostly formed. It is suggested that supplementation of culture medium with GHK may be useful in the preparation of cartilage implants. PMID:8562779

  16. The First Human Cloned Embryo.

    ERIC Educational Resources Information Center

    Cibelli, Jose B.; Lanza, Robert P.; West, Michael D.; Ezzell, Carol

    2002-01-01

    Describes a process known as parthenogenesis which produces cloned, early-stage embryos and human embryos generated only from eggs. Speculates that this technology puts therapeutic cloning within reach. (DDR)

  17. Fructated apolipoprotein A-I exacerbates cellular senescence in human umbilical vein endothelial cells accompanied by impaired insulin secretion activity and embryo toxicity.

    PubMed

    Park, Ki-Hoon; Kim, Jae-Yong; Choi, Inho; Kim, Jae-Ryong; Won, Kyu Chang; Cho, Kyung-Hyun

    2016-08-01

    Glycation of apolipoproteins is a major feature of the production of dysfunctional high-density lipoprotein (HDL), which is associated with the incidence of several metabolic diseases such as coronary artery disease and diabetes. In this report, fructated apoA-I (fA-I) induced by fructose treatment showed a covalently multimerized band without cross-linking, and lysine residues were irreversibly modified to prevent crosslinking. Using pancreatic β-cells, insulin secretion was impaired by fA-I in the lipid-free and reconstituted HDL (rHDL) states, by up to 35%, and 40%, respectively, under hyperglycemic conditions (25 mmol/L glucose). Treatment of human umbilical vein endothelial cells (HUVECs) with fA-I and HDL from elderly patients caused a 1.8-fold and 1.5-fold increased cellular senescence, respectively, along with increased lysosomal enlargement. In the lipid-free and rHDL states, fA-I increased embryo death by 1.5-fold and 2.5-fold, respectively, along with the production of oxidized species. Furthermore, rHDL containing fA-I (fA-I-rHDL) showed a higher isoelectric point (pI, approximately 8.5), whereas rHDL containing nA-I (nA-I-rHDL) showed a narrow band range with lower pI (around 8.0) as well as a much smaller particle size than that of nA-I-rHDL. In conclusion, fructose-mediated apoA-I fructation resulted in the severe loss of several beneficial functions of apoA-I and HDL, including anti-senescence and insulin secretion activities, accompanied with increased susceptibility to protein degradation and structural modification. PMID:27487295

  18. [On hybrid embryo culture in vitro of Syringa L].

    PubMed

    Zhou, Li; Luo, Fengxia; Dai, Limin

    2003-03-01

    Syringa L. is the famous ornamental shrub in China, but its embryo always dies before seed mature during cross breeding, and hence, the breeding work is very difficult. The main object of this study is using embryo culture in vitro to get the seedling directly and to improve the succeed rate of cross breeding. The factors that influenced the embryo culture were researched in detail. The results showed that the optimal medium for embryo culture was Monnier, and the second was MS or LS, which meant that the embryo of Syringa needed abundant macroelements and microelements, especially Ca2+ and K+. The optimal sugar concentration was 50 g.L-1. At this level, the sugar could offer enough nutrition and high osmotic pressure for embryo. When the embryo age was 50-60 days, the culture was easy to be succeed. At this time, the cotyledon in ovule began to form, or organ began to differentiation, so, the embryo was very easy to germinate, and the seedling was very easy to form. Proper coconut milk, glutamic acid or glutamine, and activated charcoal could improve the germination and growth of the embryo. When the BA of low concentration (0.01 mg.L-1) was joined in the medium, the germination rate could be improved. The best NAA concentration was 0.01 mg.L-1. PMID:12836546

  19. Detection and Isolation of Swine Influenza A Virus in Spiked Oral Fluid and Samples from Individually Housed, Experimentally Infected Pigs: Potential Role of Porcine Oral Fluid in Active Influenza A Virus Surveillance in Swine

    PubMed Central

    Decorte, Inge; Steensels, Mieke; Lambrecht, Bénédicte

    2015-01-01

    Background The lack of seasonality of swine influenza A virus (swIAV) in combination with the capacity of swine to harbor a large number of co-circulating IAV lineages, resulting in the risk for the emergence of influenza viruses with pandemic potential, stress the importance of swIAV surveillance. To date, active surveillance of swIAV worldwide is barely done because of the short detection period in nasal swab samples. Therefore, more sensitive diagnostic methods to monitor circulating virus strains are requisite. Methods qRT-PCR and virus isolations were performed on oral fluid and nasal swabs collected from individually housed pigs that were infected sequentially with H1N1 and H3N2 swIAV strains. The same methods were also applied to oral fluid samples spiked with H1N1 to study the influence of conservation time and temperature on swIAV infectivity and detectability in porcine oral fluid. Results All swIAV infected animals were found qRT-PCR positive in both nasal swabs and oral fluid. However, swIAV could be detected for a longer period in oral fluid than in nasal swabs. Despite the high detectability of swIAV in oral fluid, virus isolation from oral fluid collected from infected pigs was rare. These results are supported by laboratory studies showing that the PCR detectability of swIAV remains unaltered during a 24 h incubation period in oral fluid, while swIAV infectivity drops dramatically immediately upon contact with oral fluid (3 log titer reduction) and gets lost after 24 h conservation in oral fluid at ambient temperature. Conclusions Our data indicate that porcine oral fluid has the potential to replace nasal swabs for molecular diagnostic purposes. The difficulty to isolate swIAV from oral fluid could pose a drawback for its use in active surveillance programs. PMID:26431039

  20. [Production of porcine blastocysts expressed EGFP by handmade cloning].

    PubMed

    Zhang, Peng; Yang, Zhen-Zhen; Dou, Hong-Wei; Li, Wei-Hang; Lv, Bo; Bolund, Lars; DU, Yu-Tao; Tan, Ping-Ping; Ma, Run-Lin

    2011-05-01

    Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been widely used worldwide. However, the application of SCNT is impeded by overall high costs and low efficiency. Here, we reported a modification of the existing technology in order to overcome some of the disadvantages associated with SCNT. Firstly, a marker gene, enhanced green fluorescent gene (EGFP), was transfected into pig fetal fibroblast cells, and was subsequently screened by fluorescent expression to ensure donor cells expressing EGFP. Porcine embryos expressing EGFP were then produced by a method called handmade cloning (HMC), a simplified method for micromanipulation. To demonstrate the concept, we collected a total of 378 fresh swine oocytes, from which 266 with the nucleus removed, obtained a total of 127 viable recombinant oocytes after fusion with EGFP-expressing cells. In vitro incubation of the 127 recombinant oocytes for approximately 144 hours resulted in successful generation of 65 viable embryos, with an average success rate of 52.1±8.3%. Compared with the traditional SCNT, the method of HMC is not only easy to operate, but also increases the rate of recombinant embryo significantly. Furthermore, the modified method no longer relies on expensive instrument like micromanipulator, facilitating the industrialization of transgenic animal production. PMID:21586400

  1. Cytological and physiological changes in orthodox maize embryos during cryopreservation.

    PubMed

    Wen, Bin; Wang, Ruling; Cheng, Hongyan; Song, Songquan

    2010-03-01

    Cytological and physiological changes during cryopreservation were studied in maize embryos at 35 days after pollination (DAP). Both dehydration and freezing caused cytological damage, such as plasmolysis, swelled mitochondria, increased heterochromatin, and nuclear shrinkage. Dehydration alone slightly impaired plasma membrane integrity while a drastic increase in electrolyte leakage was observed after freezing of embryos with moisture content above 23%. Damage to cellular ultrastructure and plasmalemma integrity was negatively related to moisture content in unfrozen embryos and positively related in frozen embryos. The pattern of changes in activity of antioxidant enzymes differed from one another during dehydration and/or freezing-thawing treatment. Dehydration increased activity of ascorbate peroxidase (APX) and glutathione reductase (GR) but decreased activity of superoxide dismutase (SOD) and dehydroascorbate reductase (DHAR). Freezing further decreased GR and SOD activity and resulted in extremely low DHAR activity. Embryos at intermediate moisture contents had low catalase (CAT) activity before freezing but highest CAT activity after freeze-thaw. Both dehydration and freezing promoted membrane lipid peroxidation which resulted in an approximately threefold increase at most in the malondialdehyde content in postthaw embryos. Changes in viability of postthaw embryos can be closely related to damage in cellular ultrastructure and plasmalemma integrity but directly related neither to antioxidants nor lipid peroxidation levels. PMID:19904484

  2. The Virtual Embryo Project

    EPA Science Inventory

    The v-Embryo™ is a far reaching new research program at the US EPA to develop a working computer model of a mammalian embryo that can be used to better understand the prenatal risks posed by environmental chemicals and to eventually predict a chemical’s potential developmental to...

  3. Culture of bovine embryos on a polydimethylsiloxane (PDMS) microwell plate.

    PubMed

    Akagi, Satoshi; Hosoe, Misa; Matsukawa, Kazutsugu; Ichikawa, Akihiko; Tanikawa, Tamio; Takahashi, Seiya

    2010-08-01

    We fabricated a polydimethylsiloxane (PDMS)-based microwell plate (PDMS-MP) containing 100 microwells with a rounded bottom and examined whether it can be used for culture of individual in vitro fertilized (IVF) embryos or parthenogenetically activated zona-free embryos in cattle. In Experiment 1, we examined the in vitro developmental ability of IVF embryos cultured individually on PDMS-MP. After IVF, 20 embryos were transferred into 100 microl drops on PDMS-MP and cultured individually in each well of PDMS-MP (PDMS group). After 7 days of culture, the embryos in the PDMS group developed to the blastocyst stage at the same rate of those in the control group cultured in a group of 20 embryos without PDMS-MP. There were no differences in total number of cells and the ratio of inner cell mass to total cells between the PDMS and control groups. In Experiment 2, we examined the in vitro developmental ability of parthenogenetically activated zona-free bovine embryos cultured individually on PDMS-MP. The zona-free embryos were cultured individually in each well of a PDMS-MP or in each well produced by pressing a darning needle onto the bottom of a culture dish (WOW group). After 7 days of culture, the blastocyst formation rate and cell number of blastocysts in the PDMS group did not differ from those of the zona-intact embryos in the control group. Also, there were no differences in the blastocyst formation rate and cell number of blastocysts between the WOW and PDMS groups. These results suggest that the culture system using PDMS-MP is useful for individual embryos or zona-free embryos in cattle. PMID:20484872

  4. 7 CFR 1230.611 - Porcine animal.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 10 2011-01-01 2011-01-01 false Porcine animal. 1230.611 Section 1230.611 Agriculture... CONSUMER INFORMATION Procedures for the Conduct of Referendum Definitions § 1230.611 Porcine animal. The term Porcine animal means a swine, that is raised: (a) As a feeder pig, that is, a young pig sold...

  5. 7 CFR 1230.611 - Porcine animal.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Porcine animal. 1230.611 Section 1230.611 Agriculture... CONSUMER INFORMATION Procedures for the Conduct of Referendum Definitions § 1230.611 Porcine animal. The term Porcine animal means a swine, that is raised: (a) As a feeder pig, that is, a young pig sold...

  6. 7 CFR 1230.611 - Porcine animal.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 10 2012-01-01 2012-01-01 false Porcine animal. 1230.611 Section 1230.611 Agriculture... CONSUMER INFORMATION Procedures for the Conduct of Referendum Definitions § 1230.611 Porcine animal. The term Porcine animal means a swine, that is raised: (a) As a feeder pig, that is, a young pig sold...

  7. 7 CFR 1230.611 - Porcine animal.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 10 2013-01-01 2013-01-01 false Porcine animal. 1230.611 Section 1230.611 Agriculture... CONSUMER INFORMATION Procedures for the Conduct of Referendum Definitions § 1230.611 Porcine animal. The term Porcine animal means a swine, that is raised: (a) As a feeder pig, that is, a young pig sold...

  8. 7 CFR 1230.611 - Porcine animal.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 10 2014-01-01 2014-01-01 false Porcine animal. 1230.611 Section 1230.611 Agriculture... CONSUMER INFORMATION Procedures for the Conduct of Referendum Definitions § 1230.611 Porcine animal. The term Porcine animal means a swine, that is raised: (a) As a feeder pig, that is, a young pig sold...

  9. Hydrogen Sulfide Donor Protects Porcine Oocytes against Aging and Improves the Developmental Potential of Aged Porcine Oocytes

    PubMed Central

    Krejcova, Tereza; Smelcova, Miroslava; Petr, Jaroslav; Bodart, Jean-Francois; Sedmikova, Marketa; Nevoral, Jan; Dvorakova, Marketa; Vyskocilova, Alena; Weingartova, Ivona; Kucerova-Chrpova, Veronika; Chmelikova, Eva; Tumova, Lenka; Jilek, Frantisek

    2015-01-01

    Porcine oocytes that have matured in in vitro conditions undergo the process of aging during prolonged cultivation, which is manifested by spontaneous parthenogenetic activation, lysis or fragmentation of aged oocytes. This study focused on the role of hydrogen sulfide (H2S) in the process of porcine oocyte aging. H2S is a gaseous signaling molecule and is produced endogenously by the enzymes cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST). We demonstrated that H2S-producing enzymes are active in porcine oocytes and that a statistically significant decline in endogenous H2S production occurs during the first day of aging. Inhibition of these enzymes accelerates signs of aging in oocytes and significantly increases the ratio of fragmented oocytes. The presence of exogenous H2S from a donor (Na2S.9H2O) significantly suppressed the manifestations of aging, reversed the effects of inhibitors and resulted in the complete suppression of oocyte fragmentation. Cultivation of aging oocytes in the presence of H2S donor positively affected their subsequent embryonic development following parthenogenetic activation. Although no unambiguous effects of exogenous H2S on MPF and MAPK activities were detected and the intracellular mechanism underlying H2S activity remains unclear, our study clearly demonstrates the role of H2S in the regulation of porcine oocyte aging. PMID:25615598

  10. Porcine Dentin Sialophosphoprotein

    PubMed Central

    Yamakoshi, Yasuo; Lu, Yuhe; Hu, Jan C.-C.; Kim, Jung-Wook; Iwata, Takanori; Kobayashi, Kazuyuki; Nagano, Takatoshi; Yamakoshi, Fumiko; Hu, Yuanyuan; Fukae, Makoto; Simmer, James P.

    2008-01-01

    Dentin sialophosphoprotein (DSPP) is critical for proper mineralization of tooth dentin, and mutations in DSPP cause inherited dentin defects. Dentin phosphoprotein (DPP) is the C-terminal cleavage product of DSPP that binds collagen and induces intrafibrillar mineralization. We isolated DPP from individual pigs and determined that its N-terminal and C-terminal domains are glycosylated and that DPP averages 155 phosphates per molecule. Porcine DPP is unstable at low pH and high temperatures, and complexing with collagen improves its stability. Surprisingly, we observed DPP size variations on SDS-PAGE for DPP isolated from individual pigs. These variations are not caused by differences in proteolytic processing or degrees of phosphorylation or glycosylation, but rather to allelic variations in Dspp. Characterization of the DPP coding region identified 4 allelic variants. Among the 4 alleles, 27 sequence variations were identified, including 16 length polymorphisms ranging from 3 to 63 nucleotides. None of the length variations shifted the reading frame, and all localized to the highly redundant region of the DPP code. The 4 alleles encode DPP domains having 551, 575, 589, or 594 amino acids and completely explain the DPP size variations. DPP length variations are polymorphic and are not associated with dentin defects. PMID:18359767

  11. Improved efficiency of the walnut somatic embryo gene transfer system.

    PubMed

    McGranahan, G H; Leslie, C A; Uratsu, S L; Dandekar, A M

    1990-01-01

    AnAgrobacterium-mediated gene transfer system which relies on repetitive embryogenesis to regenerate transgenic walnut plants has been made more efficient by using a more virulent strain ofAgrobacterium and vectors containing genes for both kanamycin resistance and beta-glucuronidase (GUS) activity to facilitate early screening and selection. Two plasmids (pCGN7001 and pCGN7314) introduced individually into the disarmedAgrobacterium host strain EHA101 were used as inoculum. Embryos maintained on medium containing 100 mg/l kanamycin after co-cultivation produced more transformed secondary embryos than embryos maintained on kanamycin-free medium. Of the 186 GUS-positive secondary embryo lines identified, 70% were regenerated from 3 out of 16 primary embryos inoculated with EHA101/pCGN7314 and grown on kanamycin- containing medium, 28% from 4 out of 17 primary embryos inoculated with EHA101/ pCGN7001 and grown on kanamycin medium, and 2% from one out of 13 primary embryos inoculated with EHA101/pCGN7001 but not exposed to kanamycin. Because kanamycin inhibits but does not completely block new embryo formation in controls, identification of transformants formerly required repetitive selection on kanamycin for several months. Introduction of the GUS marker gene allowed positive identification of transformant secondary embryos as early as 5-6 weeks after inoculation. DNA analysis of a representative subset of lines (n=13) derived from secondary embryos confirmed transformation and provided evidence for multiple insertion events in single inoculated primary embryos. PMID:24226275

  12. Extracellular acidification synergizes with PDGF to stimulate migration of mouse embryo fibroblasts through activation of p38MAPK with a PTX-sensitive manner

    SciTech Connect

    An, Caiyan; Sato, Koichi; Wu, Taoya; Bao, Muqiri; Bao, Liang; Tobo, Masayuki; Damirin, Alatangaole

    2015-05-01

    The elucidation of the functional mechanisms of extracellular acidification stimulating intracellular signaling pathway is of great importance for developing new targets of treatment for solid tumors, and inflammatory disorders characterized by extracellular acidification. In the present study, we focus on the regulation of extracellular acidification on intracellular signaling pathways in mouse embryo fibroblasts (MEFs). We found extracellular acidification was at least partly involved in stimulating p38MAPK pathway through PTX-sensitive behavior to enhance cell migration in the presence or absence of platelet-derived growth factor (PDGF). Statistical analysis showed that the actions of extracellular acidic pH and PDGF on inducing enhancement of cell migration were not an additive effect. However, we also found extracellular acidic pH did inhibit the viability and proliferation of MEFs, suggesting that extracellular acidification stimulates cell migration probably through proton-sensing mechanisms within MEFs. Using OGR1-, GPR4-, and TDAG8-gene knock out technology, and real-time qPCR, we found known proton-sensing G protein-coupled receptors (GPCRs), transient receptor potential vanilloid subtype 1 (TRPV1), and acid-sensing ion channels (ASICs) were unlikely to be involved in the regulation of acidification on cell migration. In conclusion, our present study validates that extracellular acidification stimulates chemotactic migration of MEFs through activation of p38MAPK with a PTX-sensitive mechanism either by itself, or synergistically with PDGF, which was not regulated by the known proton-sensing GPCRs, TRPV1, or ASICs. Our results suggested that others proton-sensing GPCRs or ion channels might exist in MEFs, which mediates cell migration induced by extracellular acidification in the presence or absence of PDGF. - Highlights: • Acidic pH and PDGF synergize to stimulate MEFs migration via Gi/p38MAPK pathway. • Extracellular acidification inhibits the

  13. NK-lysin, a novel effector peptide of cytotoxic T and NK cells. Structure and cDNA cloning of the porcine form, induction by interleukin 2, antibacterial and antitumour activity.

    PubMed Central

    Andersson, M; Gunne, H; Agerberth, B; Boman, A; Bergman, T; Sillard, R; Jörnvall, H; Mutt, V; Olsson, B; Wigzell, H

    1995-01-01

    A 78 residue antimicrobial, basic peptide, NK-lysin, with three intrachain disulfide bonds was purified from pig small intestine and characterized. A corresponding clone was isolated from a porcine bone marrow cDNA library. The 780 bp DNA sequence had a reading frame of 129 amino acids which corresponded to NK-lysin. The clone was used to show that stimulation with human interleukin-2 induced synthesis of NK-lysin-specific mRNA in a lymphocyte fraction enriched for T and NK cells. Lower levels of mRNA were detected in tissues known to contain T and NK cells, such as small intestine, spleen and colon. Interleukin-2 also induced both proliferation of the lymphocyte fraction and cytolytic function in these cells. Immunostaining showed that NK-lysin was present in cells positive for CD8, CD2 and CD4. NK-lysin showed high anti-bacterial activity against Escherichia coli and Bacillus megaterium and moderate activity against Acinetobacter calcoaceticus and Streptococcus pyogenes. The peptide showed a marked lytic activity against an NK-sensitive mouse tumour cell line, YAC-1, but it did not lyse red blood cells. The amino acid sequence of NK-lysin exhibits 33% identity with a putative human preproprotein, NKG5, of unknown function but derived from a cDNA clone of activated NK cells. We suggest that NK-lysin is a new effector molecule of cytotoxic T and NK cells. Images PMID:7737114

  14. Obesity does not aggravate vitrification injury in mouse embryos: a prospective study

    PubMed Central

    2012-01-01

    Background Obesity is associated with poor reproductive outcomes, but few reports have examined thawed embryo transfer in obese women. Many studies have shown that increased lipid accumulation aggravates vitrification injury in porcine and bovine embryos, but oocytes of these species have high lipid contents (63 ng and 161 ng, respectively). Almost nothing is known about lipids in human oocytes except that these cells are anecdotally known to be relatively lipid poor. In this regard, human oocytes are considered to be similar to those of the mouse, which contain approximately 4 ng total lipids/oocyte. To date, no available data show the impact of obesity on vitrification in mouse embryos. The aim of this study was to establish a murine model of maternal diet-induced obesity and to characterize the effect of obesity on vitrification by investigating the survival rate and embryo developmental competence after thawing. Methods Prospective comparisons were performed between six–eight-cell embryos from obese and normal-weight mice and between fresh and vitrified embryos. Female C57BL/6 mice were fed standard rodent chow (normal-weight group) or a high-fat diet (obese group) for 6 weeks. The mice were mated, zygotes were collected from oviducts and cultured for 3 days, and six–eight-cell embryos were then selected to assess lipid content in fresh embryos and to evaluate differences in apoptosis, survival, and development rates in response to vitrification. Results In fresh embryos from obese mice, the lipid content (0.044 vs 0.030, P<0.01) and apoptosis rate (15.1% vs.9.3%, P<0.05)were significantly higher, the survival rate (83.1% vs. 93.1%, P<0.01) on day 5 was significantly lower, and embryo development was notably delayed on days 3–5 compared with the normal-weight group. After vitrification, no significant difference was found between thawed embryos from obese and normal-weight mice in apoptosis, survival, and development rates on days 4 and 5. In both groups

  15. Ensoulment and IVF embryos.

    PubMed Central

    Shea, M C

    1987-01-01

    This paper examines the metaphysical question of 'ensoulment' in relation to the theory, put forward in an earlier paper, that human life begins when the newly formed body organs and systems of the embryo begin to function as an organised whole, at which stage there is evidence of a change of nature. Although Roman Catholic theology teaches that a human being is a union of physical body and spiritual soul, it is incorrect to interpret this in a dualistic sense. The meaning of 'soul' is considered and the conclusion reached that although both in the religious context and apart from it abortion is difficult to justify at any stage after conception, it does not follow that the use of 'spare' In Vitro Fertilisation (IVF) embryos should be rejected. If 'ensoulment' does not occur until the new organism functions as a whole then a decision not to make use of IVF embryos for medical purposes would be a heavy responsibility and not a 'safe' way out. PMID:3612702

  16. Early embryo development in Fucus distichus is auxin sensitive

    NASA Technical Reports Server (NTRS)

    Basu, Swati; Sun, Haiguo; Brian, Leigh; Quatrano, Ralph L.; Muday, Gloria K.

    2002-01-01

    Auxin and polar auxin transport have been implicated in controlling embryo development in land plants. The goal of these studies was to determine if auxin and auxin transport are also important during the earliest stages of development in embryos of the brown alga Fucus distichus. Indole-3-acetic acid (IAA) was identified in F. distichus embryos and mature tissues by gas chromatography-mass spectroscopy. F. distichus embryos accumulate [(3)H]IAA and an inhibitor of IAA efflux, naphthylphthalamic acid (NPA), elevates IAA accumulation, suggesting the presence of an auxin efflux protein complex similar to that found in land plants. F. distichus embryos normally develop with a single unbranched rhizoid, but growth on IAA leads to formation of multiple rhizoids and growth on NPA leads to formation of embryos with branched rhizoids, at concentrations that are active in auxin accumulation assays. The effects of IAA and NPA are complete before 6 h after fertilization (AF), which is before rhizoid germination and cell division. The maximal effects of IAA and NPA are between 3.5 and 5 h AF and 4 and 5.5 h AF, respectively. Although, the location of the planes of cell division was significantly altered in NPA- and IAA-treated embryos, these abnormal divisions occurred after abnormal rhizoid initiation and branching was observed. The results of this study suggest that auxin acts in the formation of apical basal patterns in F. distichus embryo development.

  17. Early Embryo Development in Fucus distichus Is Auxin Sensitive1

    PubMed Central

    Basu, Swati; Sun, Haiguo; Brian, Leigh; Quatrano, Ralph L.; Muday, Gloria K.

    2002-01-01

    Auxin and polar auxin transport have been implicated in controlling embryo development in land plants. The goal of these studies was to determine if auxin and auxin transport are also important during the earliest stages of development in embryos of the brown alga Fucus distichus. Indole-3-acetic acid (IAA) was identified in F. distichus embryos and mature tissues by gas chromatography-mass spectroscopy. F. distichus embryos accumulate [3H]IAA and an inhibitor of IAA efflux, naphthylphthalamic acid (NPA), elevates IAA accumulation, suggesting the presence of an auxin efflux protein complex similar to that found in land plants. F. distichus embryos normally develop with a single unbranched rhizoid, but growth on IAA leads to formation of multiple rhizoids and growth on NPA leads to formation of embryos with branched rhizoids, at concentrations that are active in auxin accumulation assays. The effects of IAA and NPA are complete before 6 h after fertilization (AF), which is before rhizoid germination and cell division. The maximal effects of IAA and NPA are between 3.5 and 5 h AF and 4 and 5.5 h AF, respectively. Although, the location of the planes of cell division was significantly altered in NPA- and IAA-treated embryos, these abnormal divisions occurred after abnormal rhizoid initiation and branching was observed. The results of this study suggest that auxin acts in the formation of apical basal patterns in F. distichus embryo development. PMID:12226509

  18. Abamectin induces rapid and reversible hypoactivity within early zebrafish embryos.

    PubMed

    Raftery, Tara D; Volz, David C

    2015-01-01

    During early zebrafish embryogenesis, spontaneous tail contractions represent the first sign of locomotion and result from innervation of primary motoneuron axons to target axial muscles. Based on a high-content screen, we previously demonstrated that exposure of zebrafish embryos to abamectin--an avermectin insecticide--from 5-25 hours post-fertilization (hpf) abolished spontaneous activity in the absence of effects on survival and gross morphology. Therefore, the objective of this study was to begin investigating the mechanism of abamectin-induced hypoactivity in zebrafish. Similar to 384-well plates, static exposure of embryos to abamectin from 5-25 hpf in glass beakers resulted in elimination of activity at low micromolar concentrations. However, abamectin did not affect neurite outgrowth from spinal motoneurons and, compared with exposure from 5-25 hpf, embryos were equally susceptible to abamectin-induced hypoactivity when exposures were initiated at 10 and 23 hpf. Moreover, immersion of abamectin-exposed embryos in clean water resulted in complete recovery of spontaneous activity relative to vehicle controls, suggesting that abamectin reversibly activated ligand-gated chloride channels and inhibited neurotransmission. To test this hypothesis, we pretreated embryos to vehicle or non-toxic concentrations of fipronil or endosulfan--two insecticides that antagonize the γ-aminobutyric acid (GABA) receptor--from 5-23 hpf, and then exposed embryos to vehicle or abamectin from 23-25 hpf. Interestingly, activity levels within abamectin-exposed embryos pretreated with either antagonist were similar to embryos exposed to vehicle alone. Using quantitative PCR and phylogenetic analyses, we then confirmed the presence of GABA receptor α1 and β2 subunits at 5, 10, and 23 hpf, and demonstrated that zebrafish GABA receptor subunits are homologous to mammalian GABA receptor subunits. Overall, our data collectively suggest that abamectin induces rapid and reversible

  19. VIP treatment prevents embryo resorption by modulating efferocytosis and activation profile of maternal macrophages in the CBAxDBA resorption prone model

    PubMed Central

    Gallino, Lucila; Calo, Guillermina; Hauk, Vanesa; Fraccaroli, Laura; Grasso, Esteban; Vermeulen, Mónica; Leirós, Claudia Pérez; Ramhorst, Rosanna

    2016-01-01

    Successful embryo implantation occurs followed by a local pro-inflammatory response subsequently shifted toward a tolerogenic one. VIP (vasoactive intestinal peptide) has embryotrofic, anti-inflammatory and tolerogenic effects. In this sense, we investigated whether the in vivo treatment with VIP contributes to an immunosuppressant local microenvironment associated with an improved pregnancy outcome in the CBA/J × DBA/2 resorption prone model. Pregnancy induced the expression of VIP, VPAC1 and VPAC2 in the uterus from CBA/J × DBA/2 mating females on day 8.5 of gestation compared with non-pregnant mice. VIP treatment (2 nmol/mouse i.p.) on day 6.5 significantly increased the number of viable implantation sites and improved the asymmetric distribution of implanted embryos. This effect was accompanied by a decrease in RORγt and an increase in TGF-β and PPARγ expression at the implantation sites. Moreover, VIP modulated the maternal peritoneal macrophages efferocytosis ability, tested using latex beads-FITC or apoptotic thymocytes, displaying an increased frequency of IL-10-producer F4/80 cells while did not modulate TNF-α and IL-12 secretion. The present data suggest that VIP treatment increases the number of viable embryos associated with an increase in the efferocytic ability of maternal macrophages which is related to an immunosuppressant microenvironment. PMID:26733206

  20. Dual Positive Regulation of Embryo Implantation by Endocrine and Immune Systems--Step-by-Step Maternal Recognition of the Developing Embryo.

    PubMed

    Fujiwara, Hiroshi; Araki, Yoshihiko; Imakawa, Kazuhiko; Saito, Shigeru; Daikoku, Takiko; Shigeta, Minoru; Kanzaki, Hideharu; Mori, Takahide

    2016-03-01

    In humans, HCG secreted from the implanting embryo stimulates progesterone production of the corpus luteum to maintain embryo implantation. Along with this endocrine system, current evidence suggests that the maternal immune system positively contributes to the embryo implantation. In mice, immune cells that have been sensitized with seminal fluid and then the developing embryo induce endometrial differentiation and promote embryo implantation. After hatching, HCG activates regulatory T and B cells through LH/HCG receptors and then stimulates uterine NK cells and monocytes through sugar chain receptors, to promote and maintain pregnancy. In accordance with the above, the intrauterine administration of HCG-treated PBMC was demonstrated to improve implantation rates in women with repeated implantation failures. These findings suggest that the maternal immune system undergoes functional changes by recognizing the developing embryos in a stepwise manner even from a pre-fertilization stage and facilitates embryo implantation in cooperation with the endocrine system. PMID:26755274

  1. Production of an enzymatically active and immunogenic form of ectodomain of Porcine rubulavirus hemagglutinin-neuraminidase in the yeast Pichia pastoris.

    PubMed

    Cerriteño-Sánchez, José Luis; Santos-López, Gerardo; Rosas-Murrieta, Nora Hilda; Reyes-Leyva, Julio; Cuevas-Romero, Sandra; Herrera-Camacho, Irma

    2016-04-10

    Blue-eye disease (BED) of swine is a viral disease endemic in Mexico. The etiological agent is a paramyxovirus classified as Porcine rubulavirus (PoRV-LPMV), which exhibits in its envelope the hemagglutinin-neuraminidase (HN) glycoprotein, the most immunogenic and a major target for vaccine development. We report in this study the obtaining of ectodomain of PoRV HN (eHN) through the Pichia pastoris expression system. The expression vector (pPICZαB-HN) was integrated by displacement into the yeast chromosome and resulted in a Mut(+) phenotype. Expressed eHN in the P. pastoris X33 strain was recovered from cell-free medium, featuring up to 67nmol/min/mg after 6 days of expression. eHN was recognized by the serum of infected pigs with strains currently circulating in the Mexican Bajio region. eHN induces antibodies in mice after 28 days of immunization with specific recognition in ELISA test. These antibodies were able to inhibit >80% replication by viral neutralization assays in cell culture. These studies show the obtaining of a protein with similar characteristics to the native HN and which may be a candidate to propose a vaccine or to use the antigen in a serologic diagnostic test. PMID:26940828

  2. INFECTIVITY AND TETRATOGENICITY OF BEAUVERIA BASSIANA IN MENDIA BERYLLINA EMBRYOS

    EPA Science Inventory

    Developing embryos of the inland silverside fish, Menidia eryllina, were exposed to conidiospores of the insect pathogenic fungus, Beauveria bassiana, that possessed activity against the migratory grasshopper, Melanoplus sanquinioes. arious adverse effects were observed in Menidi...

  3. GENE EXPRESSION IN PRE-IMPLANTATION MAMMALIAN EMBRYOS

    EPA Science Inventory

    The pre-implantation mammalian embryo is initially under the control of maternal informational macromolecules that are accumulated during oogenesis. ubsequently, the genetic program of development becomes dependent upon new transcription derived from activation of the embryonic g...

  4. Protein Phosphorylation during Coconut Zygotic Embryo Development1

    PubMed Central

    Islas-Flores, Ignacio; Oropeza, Carlos; Hernández-Sotomayor, S.M. Teresa

    1998-01-01

    Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L.) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [γ-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60src (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species. PMID:9733545

  5. Role of Phosphotyrosine Interaction Domain Containing 1 in Porcine Intramuscular Preadipocyte Proliferation and Differentiation.

    PubMed

    Chen, Xiaoling; Luo, Yanliu; Huang, Zhiqing; Jia, Gang; Liu, Guangmang; Zhao, Hua

    2016-10-01

    Phosphotyrosine interaction domain containing 1 (PID1), a recently identified gene involved in obesity-associated insulin resistance, plays an important role in fat deposition. However, its effect on porcine intramuscular preadipocyte proliferation and differentiation remains poorly understood. In this study, the plasmid pcDNA3.1(+)-pPID1 was transfected into porcine intramuscular preadipocytes with Lipofectamine 3000 reagent to over-express porcine PID1 (pPID1). Over-expression of pPID1 significantly promoted porcine intramuscular preadipocyte proliferation. Expression of pPID1 mRNA was significantly increased upon porcine intramuscular preadipocyte differentiation. Indirect fluorescent immunocytochemistry demonstrated that pPID1 protein was localized predominantly in the nucleus of porcine intramuscular preadipocyte. The mRNA levels of peroxisome proliferators-activated receptor γ, CCAAT/enhancer binding protein α and lipoprotein lipase were significantly increased by pPID1 over-expression. Over-expression of pPID1 also led to an increase in lipid accumulation which was detected by Oil Red O staining, and significantly increased the intramuscular triacylglycerol content. These results indicate that pPID1 may play a role in enhancing porcine intramuscular preadipocyte proliferation and differentiation. PMID:27565873

  6. Real protection for the embryo.

    PubMed

    Mazzoni, Cosimo Marco

    2005-01-01

    This article fundamentally analyses the current protection that the Italian law offers to the embryo. Likewise, the author, contrary to those who are of the opinion that the embryo is a person subject to law, exposes his polemic theory in which he places the embryo within the scope of things. Specifically, he argues that the embryo has a quasi-personal category. In order to justify this, he analyses the moral and legal history of the statute of the embryo, he makes a difference between the biological life and the legal life. The author establishes that the concept of the person has been and will continue to be a very controversial concept, concluding with a study on the Italian legislation in respect to the protection of the embryo. PMID:16385794

  7. Sterilising embryos for transgenic chimaeras.

    PubMed

    Aige-Gil, V; Simkiss, K

    1991-07-01

    1. Experiments were undertaken to attempt to sterilise fowl embryos with ultraviolet light. Such sterilised embryos would be useful as recipients of genetically manipulated germ cells. 2. The germinal crescents of embryos were exposed to a calibrated UV source at stages 4 and 8 to 10 of incubation for 30 s, 3 min and 10 min. Teratological and sterility effects were studied at periods up to 6 d of incubation. 3. Simply exposing embryos by opening the shell produced a number of abnormalities and mortalities. These decreased with the age of the embryo but increased with the dosage of irradiation. 4. Although there was abundant evidence for UV-induced cell damage, the sterility of the embryos was usually less than 75%. PMID:1893258

  8. Porcine Reproductive and Respiratory Syndrome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Porcine reproductive and respiratory syndrome (PRRS) first appeared in the late 1980s, though serologic evidence indicates that it had been circulating in swine for some time prior to being recognized. PRRS has since become a highly significant infectious disease affecting swine production worldwid...

  9. Gender determination of avian embryo

    DOEpatents

    Daum, Keith A.; Atkinson, David A.

    2002-01-01

    Disclosed is a method for gender determination of avian embryos. During the embryo incubation process, the outer hard shells of eggs are drilled and samples of allantoic fluid are removed. The allantoic fluids are directly introduced into an ion mobility spectrometer (IMS) for analysis. The resulting spectra contain the relevant marker peaks in the positive or negative mode which correlate with unique mobilities which are sex-specific. This way, the gender of the embryo can be determined.

  10. Contribution of in vitro systems to preservation and utilization of porcine genetic resources.

    PubMed

    Kikuchi, Kazuhiro; Kaneko, Hiroyuki; Nakai, Michiko; Somfai, Tamas; Kashiwazaki, Naomi; Nagai, Takashi

    2016-07-01

    Historically, the conservation or preservation of mammalian genetic resources, especially farm animals, has been conducted under in situ conditions by maintaining living individuals as "livestock." However, systems for laboratory in vitro embryo production using gametes such as spermatozoa and oocytes are now available, in addition to ex situ preservation methods for mammalian genetic resources. One of these methods is the cryopreservation of gametes, embryos, and gonadal tissues. In pigs, freezing of sperm is the most reliable and well-established method for this purpose. On the other hand, cryopreservation of female gametes (oocytes) and gonadal tissues-usually by vitrification-has been associated with very low efficacies. Recently, in our laboratory, some research themes related to this issue have been pursued. We have been focusing on advances in porcine in vitro embryo production systems, and here, we introduce recent data on the vitrification of porcine immature oocytes and gonadal tissues followed by their xenografting into host mice to produce gametes. PMID:27142488

  11. A three-dimensional culture system using alginate hydrogel prolongs hatched cattle embryo development in vitro.

    PubMed

    Zhao, Shuan; Liu, Zhen-Xing; Gao, Hui; Wu, Yi; Fang, Yuan; Wu, Shuai-Shuai; Li, Ming-Jie; Bai, Jia-Hua; Liu, Yan; Evans, Alexander; Zeng, Shen-Ming

    2015-07-15

    No successful method exists to maintain the three-dimensional architecture of hatched embryos in vitro. Alginate, a linear polysaccharide derived from brown algae, has characteristics that make it an ideal material as a three-dimensional (3D) extracellular matrix for in vitro cell, tissue, or embryo culture. In this study, alginate hydrogel was used for IVC of posthatched bovine embryos to observe their development under the 3D system. In vitro-fertilized and parthenogenetically activated posthatched bovine blastocysts were cultured in an alginate encapsulation culture system (AECS), an alginate overlay culture system (AOCS), or control culture system. After 18 days of culture, the survival rate of embryos cultured in AECS was higher than that in the control group (P < 0.05), and the embryos were expanded and elongated in AECS with the maximal length of 1.125 mm. When the AECS shrinking embryos were taken out of the alginate beads on Day 18 and cultured in the normal culture system, 9.09% of them attached to the bottoms of the plastic wells and grew rapidly, with the largest area of an attached embryo being 66.00 mm(2) on Day 32. The embryos cultured in AOCS developed monovesicular or multivesicular morphologies. Total cell number of the embryos cultured in AECS on Day 19 was significantly higher than that of embryos on Day 8. Additionally, AECS and AOCS supported differentiation of the embryonic cells. Binuclear cells were visible in Day-26 adherent embryos, and the messenger RNA expression patterns of Cdx2 and Oct4 in AOCS-cultured embryos were similar to those in vivo embryos, whereas IFNT and ISG15 messenger RNA were still expressed in Day-26 and Day-32 prolong-cultured embryos. In conclusion, AECS and AOCS did support cell proliferation, elongation, and differentiation of hatched bovine embryos during prolonged IVC. The culture system will be useful to further investigate the molecular mechanisms controlling ruminant embryo elongation and implantation. PMID

  12. Transcriptomic and proteomic analysis reveals mechanisms of embryo abortion during chrysanthemum cross breeding

    PubMed Central

    Zhang, Fengjiao; Wang, Zhiquan; Dong, Wen; Sun, Chunqing; Wang, Haibin; Song, Aiping; He, Lizhong; Fang, Weimin; Chen, Fadi; Teng, Nianjun

    2014-01-01

    Embryo abortion is the main cause of failure in chrysanthemum cross breeding, and the genes and proteins associated with embryo abortion are poorly understood. Here, we applied RNA sequencing and isobaric tags for relative and absolute quantitation (iTRAQ) to analyse transcriptomic and proteomic profiles of normal and abortive embryos. More than 68,000 annotated unigenes and 700 proteins were obtained from normal and abortive embryos. Functional analysis showed that 140 differentially expressed genes (DEGs) and 41 differentially expressed proteins (DEPs) were involved in embryo abortion. Most DEGs and DEPs associated with cell death, protein degradation, reactive oxygen species scavenging, and stress-response transcriptional factors were significantly up-regulated in abortive embryos relative to normal embryos. In contrast, most genes and proteins related to cell division and expansion, the cytoskeleton, protein synthesis and energy metabolism were significantly down-regulated in abortive embryos. Furthermore, abortive embryos had the highest activity of three executioner caspase-like enzymes. These results indicate that embryo abortion may be related to programmed cell death and the senescence- or death-associated genes or proteins contribute to embryo abortion. This adds to our understanding of embryo abortion and will aid in the cross breeding of chrysanthemum and other crops in the future. PMID:25288482

  13. Amino-Acid Sequence of Porcine Pepsin

    PubMed Central

    Tang, J.; Sepulveda, P.; Marciniszyn, J.; Chen, K. C. S.; Huang, W-Y.; Tao, N.; Liu, D.; Lanier, J. P.

    1973-01-01

    As the culmination of several years of experiments, we propose a complete amino-acid sequence for porcine pepsin, an enzyme containing 327 amino-acid residues in a single polypeptide chain. In the sequence determination, the enzyme was treated with cyanogen bromide. Five resulting fragments were purified. The amino-acid sequence of four of the fragments accounted for 290 residues. Because the structure of a 37-residue carboxyl-terminal fragment was already known, it was not studied. The alignment of these fragments was determined from the sequence of methionyl-peptides we had previously reported. We also discovered the locations of activesite aspartyl residues, as well as the pairing of the three disulfide bridges. A minor component of commercial crystalline pepsin was found to contain two extra amino-acid residues, Ala-Leu-, at the amino-terminus of the molecule. This minor component was apparently derived from a different site of cleavage during the activation of porcine pepsinogen. PMID:4587252

  14. Osteogenic and adipogenic potential of porcine adipose mesenchymal stem cells.

    PubMed

    Qu, Chang-qing; Zhang, Guo-hua; Zhang, Li-jie; Yang, Gong-she

    2007-02-01

    Human, rat, and mouse studies have demonstrated the existence of a population of adipose mesenchymal stem cells (AMSCs) that can undergo multilineage differentiation in vitro. Understanding the clinical potential of AMSCs may require their use in preclinical large-animal models such as pigs. Thus, the objectives of this study were to establish a protocol for the isolation of porcine AMSCs from adipose tissue and to examine their ex vivo differentiation potential to adipocytes and osteoblast. The porcine AMSCs from passage 4 were selected for differentiation analysis. The adipocytes were identified morphologically by staining with Oil Red O, and the adipogenic marker genes were examined by RT-PCR technique. Osteogenic lineage was documented by deposition of calcium stained with Alzarin Red S, visualization of alkaline phosphatase activity, and expression of marker gene. Our result indicates that porcine AMSCs have been successfully isolated and induced differentiation into adipocytes and osteoblasts. This study suggested that porcine AMSCs are also a valuable model system for the study on the mesenchymal lineages for basic research and tissue engineering. PMID:17570023

  15. Detection of Smad Signaling in Zebrafish Embryos.

    PubMed

    Liu, Xingfeng; Wang, Qiang; Meng, Anming

    2016-01-01

    Nodal and BMPs play critical roles in germ layer induction and patterning in early zebrafish embryos. Smad2/3 and Smad1/5/8 are intracellular effectors of Nodal and BMPs, respectively. These Smads regulate, in cooperation with other factors, transcription of hundreds of target genes in the nucleus. The activity and stability of Smads are regulated by phosphorylation modifications. To better understand the regulatory network of Smads-mediated signaling and its biological implications, it is necessary to monitor the signaling activity in an in vivo model system. In this chapter, we describe the methods used in zebrafish embryos for dissecting Smads signaling, including TGF-β/Nodal- and BMP-responsive luciferase reporter assays, Western blotting for Smads, co-immunoprecipitation for Smads and their interacting proteins, chromatin-immunoprecipitation for identification of Smad2-binding sites, and immunostaining for detection of active Smad1/5/8. PMID:26520131

  16. Functional activity of the porcine Gnrhr2 gene promoter in testis-derived cells is partially conferred by nuclear factor-κB, specificity protein 1 and 3 (SP1/3) and overlapping early growth response 1/SP1/3 binding sites.

    PubMed

    Brauer, Vanessa M; Wiarda-Bell, Jocelyn R; Desaulniers, Amy T; Cederberg, Rebecca A; White, Brett R

    2016-08-10

    Unlike the classical gonadotropin-releasing hormone (GnRH1), the second mammalian isoform (GnRH2) is ubiquitously expressed, suggesting a divergent function. Indeed, we demonstrated that GnRH2 governs LH-independent testosterone secretion in porcine testes via interaction with its receptor (GnRHR2) on Leydig cells. Transient transfections with luciferase reporter vectors containing 3009bp of 5' flanking sequence for the porcine Gnrhr2 gene (-3009pGL3) revealed promoter activity in all 15 cell lines examined, including swine testis-derived (ST) cells. Therefore, ST cells were utilized to explore the molecular mechanisms underlying transcriptional regulation of the porcine Gnrhr2 gene in the testis. Reporter plasmids containing progressive 5' deletions of the Gnrhr2 promoter indicated that the -708/-490 region contained elements critical to promoter activity. Electrophoretic mobility shift assays (EMSAs) with radiolabeled oligonucleotides spanning the -708/-490bp region and ST nuclear extracts, identified specific binding complexes for the -513/-490, -591/-571 and -606/-581bp segments of promoter. Antibody addition to EMSAs indicated that the p65 and p52 subunits of nuclear factor-κB (NF-κB) comprised the specific complex bound to the oligonucleotide probe for the -513/-490bp promoter region, specificity protein (SP) 1 and 3 bound the -591/-571bp probe and early growth response 1 (EGR1), SP1 and SP3 bound the -606/-581 radiolabeled oligonucleotide. Transient transfections with vectors containing mutations of the NF-κB (-499/-493), SP1/3 (-582/-575) or overlapping EGR1/SP1/3 (-597/-587) binding sites reduced luciferase activity by 26%, 61% and 56%, respectively (P<0.05). Thus, NF-κB, SP1/3 and overlapping EGR1/SP1/3 binding sites are critical to expression of the porcine Gnrhr2 gene in ST cells. PMID:27134031

  17. Cryopreservation of embryos: an overview.

    PubMed

    Engelmann, Florent

    2011-01-01

    Cryopreservation (liquid nitrogen, -196°C) is the only safe and cost-effective option for long-term -conservation of genetic resources of non-orthodox seed species. Cryopreservation protocols have been developed for various materials including seeds, dormant buds, cell suspensions, calli, apices, zygotic, and somatic embryos of numerous plant species. Zygotic embryos or embryonic axes of almost 100 different species and somatic embryos of almost 40 different species from both temperate and tropical climates, comprising crops, fruit, and forest trees as well as wild species, whose seeds displayed orthodox, intermediate, and recalcitrant storage characteristics, have been successfully cryopreserved. With zygotic embryos and embryonic axes, the desiccation technique has been used with the majority of the species tested, leading to highly variable survival and recovery after freezing, especially during earlier experiments. More recently, new cryopreservation techniques viz. encapsulation-dehydration and vitrification have been employed, leading to generally improved results. With somatic embryos, different cryopreservation methods have been used viz. desiccation, pre-growth-desiccation, encapsulation-dehydration, vitrification, encapsulation-vitrification, and droplet-vitrification. There are also a few examples of the utilisation of slow controlled freezing, which correspond to the earlier experiments performed with somatic embryos. The development and application of cryopreservation is significantly more advanced for somatic embryos, in comparison with zygotic embryos, mainly because of the different origin and characteristics of the species treated. In most cases, zygotic embryos originate from tropical, wild species, for which knowledge and techniques relevant to the development of cryopreservation protocols are limited, or even non-existent. By contrast, somatic embryos are generally produced from cultivated species, which have already been studied extensively

  18. Growth and metabolism of murine and bovine embryos in bovine uterine flushing-supplemented culture media.

    PubMed Central

    Rondeau, M; Guay, P; Goff, A K; Cooke, G M

    1996-01-01

    The aim of this study was to compare the development and metabolic activity of cultured murine and bovine embryos in 2 standard media (HAM F-10 and RPMI) in the presence or absence of bovine uterine flushings. Murine morulae (n = 653) and day 7 bovine embryos (n = 273) were cultured for 18 h or 36 h in either HAM F-10 or RPMI in the presence or absence of bovine uterine flushings. After culture, the development, quality, and metabolic activity (glucose utilization or methionine uptake and incorporation) of embryos was assessed. It was found that HAM F-10 (without uterine flushings) was a more suitable medium than RPMI for optimal development and metabolism of murine and bovine embryos. Poor quality and development, as well as decreased metabolism, were evident after culture of murine embryos in RPMI; in contrast, this medium had no adverse effects on bovine embryos in culture. Supplementation of HAM F-10 with bovine uterine flushings improved the growth of murine embryos and the protein synthesis (as measured by an increased methionine incorporation) for both murine and bovine embryos. However, supplementation with bovine uterine flushings could not overcome deficiencies of an inappropriate medium (RPMI) for murine embryos. Supplementation of a well-defined culture medium with uterine flushings increased metabolism of embryos in culture, and thus might help to increase pregnancy rates after transfer of such embryos to recipient cows. PMID:8825988

  19. Metabolomic assessment of embryo viability.

    PubMed

    Uyar, Asli; Seli, Emre

    2014-03-01

    Preimplantation embryo metabolism demonstrates distinctive characteristics associated with the developmental potential of embryos. On this basis, metabolite content of culture media was hypothesized to reflect the implantation potential of individual embryos. This hypothesis was tested in consecutive studies reporting a significant association between culture media metabolites and embryo development or clinical pregnancy. The need for a noninvasive, reliable, and rapid embryo assessment strategy promoted metabolomics studies in vitro fertilization (IVF) in an effort to increase success rates of single embryo transfers. With the advance of analytical techniques and bioinformatics, commercial instruments were developed to predict embryo viability using spectroscopic analysis of surplus culture media. However, despite the initial promising results from proof-of-principal studies, recent randomized controlled trials using commercial instruments failed to show a consistent benefit in improving pregnancy rates when metabolomics is used as an adjunct to morphology. At present, the application of metabolomics technology in clinical IVF laboratory requires the elimination of factors underlying inconsistent findings, when possible, and development of reliable predictive models accounting for all possible sources of bias throughout the embryo selection process. PMID:24515909

  20. Reprogrammed Transcriptome in Rhesus-Bovine Interspecies Somatic Cell Nuclear Transfer Embryos

    PubMed Central

    Wang, Kai; Otu, Hasan H.; Chen, Ying; Lee, Young; Latham, Keith; Cibelli, Jose B.

    2011-01-01

    Background Global activation of the embryonic genome (EGA), one of the most critical steps in early mammalian embryo development, is recognized as the time when interspecies somatic cell nuclear transfer (iSCNT) embryos fail to thrive. Methodology/Principal Findings In this study, we analyzed the EGA-related transcriptome of rhesus-bovine iSCNT 8- to 16-cell embryos and dissected the reprogramming process in terms of embryonic gene activation, somatic gene silencing, and maternal RNA degradation. Compared with fibroblast donor cells, two thousand and seven genes were activated in iSCNT embryos, one quarter of them reaching expression levels comparable to those found in in vitro fertilized (IVF) rhesus embryos. This suggested that EGA in iSCNT embryos had partially recapitulated rhesus embryonic development. Eight hundred and sixty somatic genes were not silenced properly and continued to be expressed in iSCNT embryos, which indicated incomplete nuclear reprogramming. We compared maternal RNA degradation in bovine oocytes between bovine-bovine SCNT and iSCNT embryos. While maternal RNA degradation occurred in both SCNT and iSCNT embryos, we saw more limited overall degradation of maternal RNA in iSCNT embryos than in SCNT embryos. Several important maternal RNAs, like GPF9, were not properly processed in SCNT embryos. Conclusions/Significance Our data suggested that iSCNT embryos are capable of triggering EGA, while a portion of somatic cell-associated genes maintain their expression. Maternal RNA degradation seems to be impaired in iSCNT embryos. Further understanding of the biological roles of these genes, networks, and pathways revealed by iSCNT may expand our knowledge about cell reprogramming, pluripotency, and differentiation. PMID:21799794

  1. Activation of the TLR/MyD88/NF-κB Signal Pathway Contributes to Changes in IL-4 and IL-12 Production in Piglet Lymphocytes Infected with Porcine Circovirus Type 2 In Vitro

    PubMed Central

    Duan, Dianning; Zhang, Shuxia; Li, Xiaolin; Guo, Hua; Chen, Mengmeng; Zhang, Yaqun; Han, Junyuan; Lv, Yingjun

    2014-01-01

    Porcine circovirus type 2 (PCV2) causes immunosuppression in pigs. One causative factor is an imbalance in cytokine levels in the blood and lymphoid tissues. Many studies have reported changes in cytokine production, but the regulatory mechanisms involved have not yet been elucidated. In this study, we investigated alteration and regulation of IL-4 and IL-12 production in lymphocytes following incubation with PCV2 in vitro. The levels of IL-4 decreased and levels of IL-12 increased in lymphocyte supernatants, and the DNA-binding activity of NF-κB and the expression of p65 in the nucleus and p-IκB in the cytoplasm of lymphocytes increased after incubation with PCV2. However, these effects were reversed when lymphocytes were coincubated with PCV2 and the NF-κB inhibitor BAY11-7082. In addition, the expression of MyD88 protein increased and the expression of mRNA for the toll-like receptors (TLRs) TLR2, TLR3, TLR4 and TLR9 was upregulated when lymphocytes were incubated with PCV2. However, no change was seen in TLR7 and TLR8 mRNA expression. In conclusion, this study showed that PCV2 induced a decrease in IL-4 and an increase in IL-12 production in lymphocytes, and these changes were regulated by the TLR-MyD88-NF-κB signal pathway. PMID:24841678

  2. Activation of the TLR/MyD88/NF-κB signal pathway contributes to changes in IL-4 and IL-12 production in piglet lymphocytes infected with porcine circovirus type 2 in vitro.

    PubMed

    Duan, Dianning; Zhang, Shuxia; Li, Xiaolin; Guo, Hua; Chen, Mengmeng; Zhang, Yaqun; Han, Junyuan; Lv, Yingjun

    2014-01-01

    Porcine circovirus type 2 (PCV2) causes immunosuppression in pigs. One causative factor is an imbalance in cytokine levels in the blood and lymphoid tissues. Many studies have reported changes in cytokine production, but the regulatory mechanisms involved have not yet been elucidated. In this study, we investigated alteration and regulation of IL-4 and IL-12 production in lymphocytes following incubation with PCV2 in vitro. The levels of IL-4 decreased and levels of IL-12 increased in lymphocyte supernatants, and the DNA-binding activity of NF-κB and the expression of p65 in the nucleus and p-IκB in the cytoplasm of lymphocytes increased after incubation with PCV2. However, these effects were reversed when lymphocytes were coincubated with PCV2 and the NF-κB inhibitor BAY11-7082. In addition, the expression of MyD88 protein increased and the expression of mRNA for the toll-like receptors (TLRs) TLR2, TLR3, TLR4 and TLR9 was upregulated when lymphocytes were incubated with PCV2. However, no change was seen in TLR7 and TLR8 mRNA expression. In conclusion, this study showed that PCV2 induced a decrease in IL-4 and an increase in IL-12 production in lymphocytes, and these changes were regulated by the TLR-MyD88-NF-κB signal pathway. PMID:24841678

  3. Ion/water channels for embryo implantation barrier.

    PubMed

    Liu, Xin-Mei; Zhang, Dan; Wang, Ting-Ting; Sheng, Jian-Zhong; Huang, He-Feng

    2014-05-01

    Successful implantation involves three distinct processes, namely the embryo apposition, attachment, and penetration through the luminal epithelium of the endometrium to establish a vascular link to the mother. After penetration, stromal cells underlying the epithelium differentiate and surround the embryo to form the embryo implantation barrier, which blocks the passage of harmful substances to the embryo. Many ion/water channel proteins were found to be involved in the process of embryo implantation. First, ion/water channel proteins play their classical role in establishing a resting membrane potential, shaping action potentials and other electrical signals by gating the flow of ions across the cell membrane. Second, most of ion/water channel proteins are regulated by steroid hormone (estrogen or progesterone), which may have important implications to the embryo implantation. Last but not least, these proteins do not limit themselves as pure channels but also function as an initiator of a series of consequences once activated by their ligand/stimulator. Herein, we discuss these new insights in recent years about the contribution of ion/water channels to the embryo implantation barrier construction during early pregnancy. PMID:24789983

  4. A Large Chondroitin Sulfate Proteoglycan, Versican, in Porcine Predentin.

    PubMed

    Okahata, Saori; Yamamoto, Ryuji; Yamakoshi, Yasuo; Fukae, Makoto

    2011-01-01

    Proteoglycans and their constituent glycosaminoglycan (GAG) have been proposed to be involved in the inhibition of mineralization in unmineralized tissue, predentin. Among the proteoglycans secreted by odontoblasts, we focused on the large chondroitin sulfate proteoglycan, versican, for its large binding capacity for calcium ions. The aims of this study were the determination of the full-length sequence and splicing variants of the porcine versican, and the detection of versican in the porcine predentin. The complete coding sequence of the porcine versican mRNA was cloned to be 11,775 nucleotides long and encode 3,924 amino acids, and four splicing variants, V0, V1, V2 and V3, were characterized in the isolated porcine cartilage cells. The number of potential GAG attachment sites was 15 in the V0 variant, 13 in the V1 variant, 2 in the V2 variant and 0 in the V3 variant. They were deposited in DDBJ. The V1 variant was determined by RT-PCR in the odontoblasts, dental papilla cells, dental follicle cells, periodontal ligament cells, dental pulp cells, and gingival cells of pigs, although a small amount of the V0 valiant was found in the dental papilla cells. The predentin was prepared from developing porcine permanent incisor tooth germs and its soluble proteins were extracted in order to be partially characterized by protein and proteinase profiles. The versican V1 cleavage products were detected in the predentin extract by Western blotting analysis. These results suggested that the versican splice variant V1 implicates both the control of the mineralization and the activities of the predentin metalloproteinases, because it has 13 GAG chains that bind a large amount of calcium. PMID:22200993

  5. Antiviral activity of Inonotus obliquus fungus extract towards infection caused by hepatitis C virus in cell cultures.

    PubMed

    Shibnev, V A; Mishin, D V; Garaev, T M; Finogenova, N P; Botikov, A G; Deryabin, P G

    2011-09-01

    Fractions of Inonotus obliquus fungus water extract exhibited a virucidal effect towards hepatitis C virus: it 100-fold reduced its infective properties within 10 min. The antiviral effects of fungus extracts manifested after preventive (24 h before infection) and therapeutic use (during infection of porcine embryo kidney cells). Moreover, the data indicate that the birch fungus extracts inhibit production of infective virus by porcine embryo kidney cells. PMID:22462058

  6. Developmental toxicity of cartap on zebrafish embryos.

    PubMed

    Zhou, Shengli; Dong, Qiaoxiang; Li, Shaonan; Guo, Jiangfeng; Wang, Xingxing; Zhu, Guonian

    2009-12-13

    Cartap is a widely used insecticide which belongs to a member of nereistoxin derivatives and acts on nicotinic acetylcholine receptor site. Its effects on aquatic species are of grave concern. To explore the potential developmental toxicity of cartap, zebrafish embryos were continually exposed, from 0.5 to 144h post-fertilization, to a range of concentrations of 25-1000microg/l. Results of the experiment indicated that cartap concentrations of 100microg/l and above negatively affected embryo survival and hatching success. Morphological analysis uncovered a large suite of abnormalities such as less melanin pigmentation, wavy notochord, crooked trunk, fuzzy somites, neurogenesis defects and vasculature defects. The most sensitive organ was proved to be the notochord which displayed defects at concentrations as low as 25microg/l. Both sensitivity towards exposure and localization of the defect were stage specific. To elucidate mechanisms concerning notochord, pigmentation, and hatching defects, enzyme assay, RT Q-PCR, and different exposure strategies were performed. For embryos with hatching failure, chorion was verified not to be digested, while removing cartap from exposure at early pre-hatching stage could significantly increase the hatching success. However, cartap was proved, via vitro assay, to have no effect on proteolytic activity of hatching enzyme. These findings implied that the secretion of hatching enzyme might be blocked. We also revealed that cartap inhibited the activity of melanogenic enzyme tyrosinase and matrix enzyme lysyl oxidase and induced expression of their genes. These suggested that cartap could impaired melanin pigmentation of zebrafish embryos through inhibiting tyrosinase activity, while inhibition of lysyl oxidase activity was responsible for notochord undulation, which subsequently caused somite defect, and at least partially responsible for defects in vasculature and neurogenesis. PMID:19923012

  7. Requirement of nuclear localization and transcriptional activity of p53 for its targeting to the yolk syncytial layer (YSL) nuclei in zebrafish embryo and its use for apoptosis assay

    SciTech Connect

    Chen, G.-D.; Chou, C.-M.; Hwang, S.-P.L.; Wang, F.-F.; Chen, Y.-C.; Hung, C.-C.; Chen, Jeou-Yuan . E-mail: bmchen@ibms.sinica.edu.tw; Huang, C.-J. . E-mail: cjibc@gate.sinica.edu.tw

    2006-05-26

    We expressed zebrafish p53 protein fused to GFP by a neuron-specific HuC promoter in zebrafish embryos. Instead of displaying neuronal expression patterns, p53-GFP was targeted to zebrafish YSL nuclei. This YSL targeting is p53 sequence-specific because GFP fusion proteins of p63 and p73 displayed neuronal-specific patterns. To dissect the underlying mechanisms, various constructs encoding a series of p53 mutant proteins under the control of different promoters were generated. Our results showed that expression of p53, in early zebrafish embryo, is preferentially targeted to the nuclei of YSL, which is mediated by importin. Similarly, this targeting is abrogated when p53 nuclear localization signal is disrupted. In addition, the transcriptional activity of p53 is required for this targeting. We further showed that fusion of pro-apoptotic BAD protein to p53-GFP led to apoptosis of YSL cells, and subsequent imperfect microtubule formation and abnormal blastomere movements.

  8. Protein deubiquitination during oocyte maturation influences sperm function during fertilisation, antipolyspermy defense and embryo development.

    PubMed

    Yi, Young-Joo; Sutovsky, Miriam; Song, Won-Hee; Sutovsky, Peter

    2015-11-01

    Ubiquitination is a covalent post-translational modification of proteins by the chaperone protein ubiquitin. Upon docking to the 26S proteasome, ubiquitin is released from the substrate protein by deubiquitinating enzymes (DUBs). We hypothesised that specific inhibitors of two closely related oocyte DUBs, namely inhibitors of the ubiquitin C-terminal hydrolases (UCH) UCHL1 (L1 inhibitor) and UCHL3 (L3 inhibitor), would alter porcine oocyte maturation and influence sperm function and embryo development. Aberrant cortical granule (CG) migration and meiotic spindle defects were observed in oocytes matured with the L1 or L3 inhibitor. Embryo development was delayed or blocked in oocytes matured with the general DUB inhibitor PR-619. Aggresomes, the cellular stress-inducible aggregates of ubiquitinated proteins, formed in oocytes matured with L1 inhibitor or PR-619, a likely consequence of impaired protein turnover. Proteomic analysis identified the major vault protein (MVP) as the most prominent protein accumulated in oocytes matured with PR-619, suggesting that the inhibition of deubiquitination altered the turnover of MVP. The mitophagy/autophagy of sperm-contributed mitochondria inside the fertilised oocytes was hindered by DUB inhibitors. It is concluded that DUB inhibitors alter porcine oocyte maturation, fertilisation and preimplantation embryo development. By regulating the turnover of oocyte proteins and mono-ubiquitin regeneration, the DUBs may promote the acquisition of developmental competence during oocyte maturation. PMID:24848520

  9. Mechanical characterization of porcine corneas.

    PubMed

    Boschetti, F; Triacca, V; Spinelli, L; Pandolfi, A

    2012-03-01

    An experimental program has been carried out in order to investigate the mechanical behavior of porcine corneas. We report the results of inflation tests on the whole cornea and uniaxial tests on excised corneal strips, performed on 51 fresh porcine eyes. Uniaxial tests have been performed on specimens cut from previously inflated corneas. The cornea behavior is characterized by means of elastic stiffness, measured on both average pressure-apex displacement and average uniaxial stress-strain curves; and by means of transversal contraction coefficient, peak stress, and failure stress measured on uniaxial stress-strain curves. Uniaxial tests performed on excised strips allowed to measure the anisotropy in the corneal stiffness and to compare the stiffness of the cornea with the one of the sclera. Viscous properties of the cornea have been obtained through uniaxial relaxation curves on excised corneal strips. The relevant geometrical parameters have been measured and, with the aid of the elastic thin shell theory, a stress-strain curve has been derived from the average inflation test data and compared with similar data available in the literature. The experimental system has been developed in view of future applications to the mechanical testing of both porcine and human corneas. PMID:22482683

  10. Identification and characterization of promoters specifically and strongly expressed in maize embryos.

    PubMed

    Liu, Xiaoqing; Tian, Jian; Zhou, Xiaojin; Chen, Rumei; Wang, Lei; Zhang, Chunyi; Zhao, Jun; Fan, Yunliu

    2014-12-01

    The use of maize seeds as bioreactors has several advantages for the production of recombinant proteins in plant biotechnology, but available embryo-specific and strong promoters are limited. Here, we describe a genome-scale microarray-based approach to identify embryo-specifically and strongly expressed genes and their promoters in maize. We identified 28 embryo-preferred and abundantly expressed genes based on our microarray data. These embryo-preferred genes were further analysed using the UniGene database and by quantitative reverse transcriptase-PCR leading to the identification of seven genes (Zm.2098, Zm.13387, Zm.66589, Zm.85502, Zm.68129, Zm.3896 and Zm.2941) as embryo-specific genes with higher expression levels relative to maize globulin-1. The putative promoters of five embryo-specific genes (Zm.13387, Zm.66589, Zm.85502, Zm.3896 and Zm.2941) were isolated and all exhibited strong promoter activities when transiently expressed in maize embryos of 20 DAP. The embryo specificity and expression levels of the promoters of four genes (Zm.13387, Zm.85502, Zm.3896 and Zm.2941) were further examined in transgenic maize plants, revealing that they are strong promoters in embryos of all four developmental stages tested compared with reference globulin-1 promoter. Moreover, Zm.2941 and Zm.3896 promoters are stringently embryo-specific promoters, while Zm.85502 promoter is basically embryo specific yet wounding inducible in non-seed tissues, and Zm.13387 promoter is developmentally expressed in both embryo and aleurone with wounding-induced activity in non-seed tissues. Our study provides novel embryo-specific and strong promoters that are suitable for production of high-level recombinant proteins in maize embryos. PMID:25052028

  11. Effect of recombinant porcine IGF-binding protein-3 on proliferation of embryonic porcine myogenic cell cultures in the presence and absence of IGF-I.

    PubMed

    Pampusch, M S; Kamanga-Sollo, E; White, M E; Hathaway, M R; Dayton, W R

    2003-02-01

    IGF-binding protein (IGFBP)-3 is produced by cultured porcine embryonic myogenic cell (PEMC) cultures and is secreted into the medium. Levels of secreted IGFBP-3 and IGFBP-3 mRNA are significantly reduced during differentiation and increase after differentiation is complete, suggesting that IGFBP-3 may play some role in myogenesis and/or in changes in myogenic cell proliferation that accompany differentiation. IGFBP-3 reportedly may either suppress or stimulate proliferation of cultured cells depending on cell type. Additionally, IGFBP-3 has been shown to affect proliferation via both IGF-dependent and IGF-independent mechanisms in some cell types but not all. Currently, the effect, if any, of IGFBP-3 on myogenic cell proliferation is not known. Consequently, the goal of this study was to assess the IGF-I-dependent and IGF-I-independent actions of recombinant porcine IGFBP-3 on proliferation of cultured porcine myogenic cells. To facilitate these investigations, we have expressed porcine IGFBP-3 in the baculovirus system, purified and characterized the expressed recombinant porcine IGFBP-3 (rpIGFBP-3), and produced and characterized an anti-porcine IGFBP-3 antibody that neutralizes the biological activity of porcine IGFBP-3. rpIGFBP-3 suppressed IGF-I-stimulated proliferation of PEMCs in a concentration-dependent manner with equimolar concentrations of IGF-I and rpIGFBP-3, resulting in complete suppression of IGF-I-stimulated proliferation. rpIGFBP-3 also suppressed Long-R3-IGF-I-stimulated proliferation of PEMC, indicating that rpIGFBP-3 possesses IGF-independent activity in this cell system. These data have established that IGFBP-3 has the potential to affect proliferation of PEMCs during critical periods of muscle development that may impact ultimate muscle mass achievable postnatally. PMID:12553871

  12. The Meganuclease I-SceI Containing Nuclear Localization Signal (NLS-I-SceI) Efficiently Mediated Mammalian Germline Transgenesis via Embryo Cytoplasmic Microinjection

    PubMed Central

    Xiang, Peng-Ying; Wang, Lu-Lu; Tang, Huan; Xie, Fei; Li, Liang; Wei, Hong

    2014-01-01

    The meganuclease I-SceI has been effectively used to facilitate transgenesis in fish eggs for nearly a decade. I-SceI-mediated transgenesis is simply via embryo cytoplasmic microinjection and only involves plasmid vectors containing I-SceI recognition sequences, therefore regarding the transgenesis process and application of resulted transgenic organisms, I-SceI-mediated transgenesis is of minimal bio-safety concerns. However, currently no transgenic mammals derived from I-SceI-mediated transgenesis have been reported. In this work, we found that the n