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  • Frontiers Media SA  (9)
  • 1
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    Table1.DOCX
    Frontiers Media SA ; 2022
    In:  Frontiers in Cell and Developmental Biology Vol. 10 ( 2022-9-8)
    Details
    In: Frontiers in Cell and Developmental Biology, Frontiers Media SA, Vol. 10 ( 2022-9-8)
    Abstract: Aggregation of blastomeres is a promising method to improve the developmental competence of blastocysts and may be useful for the production of chimeric animals and the establishment of embryonic stem cell lines by increasing inner cell masses. Here, we determined the optimal conditions for blastomere aggregation using phytohemagglutinin-L (PHA-L) and examined PHA-L efficiency by comparing it with Well of the Well (WOW), a general blastomere aggregation method. As a result, we confirmed that treatment with 15 μg/ml PHA-L for 144 h was effective for blastomere aggregation and embryonic development of three zona-free 2-cell stage embryos (TZ2Es) after parthenogenetic activation (PA). The TZ2Es cultured with PHA-L showed a significantly ( p & lt; 0.05) higher blastomere aggregation rate than the WOW method (93.5 ± 1.9% vs. 78.0 ± 8.5%). In addition, our results demonstrated that TZ2Es aggregation through PHA-L improved the quality of PA-derived blastocysts and improved porcine embryonic stem-like cell (pESLCs) seeding efficiency and quality of colonies. It was also observed that PHA-L-derived pESLC could remain undifferentiated and exhibit typical embryonic stem cell pluripotency markers, embryoid body (EB)-forming ability, and differentiation into cell lineages of three germ layers. Pig blastomere aggregation technology is expected to improve embryo quality and the efficiency of embryonic stem cell establishment and embryoid-body formation. It can also be used in blastocyst complementation systems and in the production of chimeric animals.
    Type of Medium: Online Resource
    ISSN: 2296-634X
    URL: Article
    URL: Article
    DOI: 10.3389/fcell.2022.948778
    DOI: 10.3389/fcell.2022.948778.s001
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2022
    detail.hit.zdb_id: 2737824-X
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  • 2
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    Table1.docx
    Frontiers Media SA ; 2022
    In:  Frontiers in Cell and Developmental Biology Vol. 10 ( 2022-7-8)
    Details
    In: Frontiers in Cell and Developmental Biology, Frontiers Media SA, Vol. 10 ( 2022-7-8)
    Abstract: Neurotrophin-4 (NT-4), a granulosa cell-derived factor and a member of the neurotrophin family, is known to promote follicular development and oocyte maturation in mammals. However, the physiological and functional roles of NT-4 in porcine ovarian development are not yet known. The aim of this study was to investigate the physiological role of NT-4-related signaling in the in vitro maturation (IVM) of porcine cumulus–oocyte complexes (COCs). The NT-4 protein and its receptors were detected in matured porcine COCs via immunofluorescence analysis. NT-4 was shown to promote the maturation of COCs by upregulating NFKB1 transcription via the neurotrophin/p75 NTR signaling pathway. Notably, the mRNA expression levels of the oocyte-secreted factors GDF9 and BMP15 , sperm–oocyte interaction regulator CD9 , and DNA methylase DNMT3A were significantly upregulated in NT-4-treated than in untreated porcine oocytes. Concurrently, there were no significant differences in the levels of total and phosphorylated epidermal growth factor receptor and p38 mitogen-activated protein kinase between NT-4-treated and untreated cumulus cells (CCs); however, the level of phosphorylated ERK1/2 was significantly higher in NT-4-treated CCs. Both total and phosphorylated ERK1/2 levels were significantly higher in NT-4-treated than in untreated oocytes. In addition, NT-4 improved subsequent embryonic development after in vitro fertilization and somatic cell nuclear transfer. Therefore, the physiological and functional roles of NT-4 in porcine ovarian development include the promotion of oocyte maturation, CC expansion, and ERK1/2 phosphorylation in porcine COCs during IVM.
    Type of Medium: Online Resource
    ISSN: 2296-634X
    URL: Article
    URL: Article
    URL: Article
    URL: Article
    DOI: 10.3389/fcell.2022.908992
    DOI: 10.3389/fcell.2022.908992.s001
    DOI: 10.3389/fcell.2022.908992.s002
    DOI: 10.3389/fcell.2022.908992.s003
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2022
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  • 3
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    Table_1.DOCX
    Frontiers Media SA ; 2022
    In:  Frontiers in Veterinary Science Vol. 9 ( 2022-12-8)
    Details
    In: Frontiers in Veterinary Science, Frontiers Media SA, Vol. 9 ( 2022-12-8)
    Abstract: Interleukin-7 (IL-7), a vital factor that affects cell development, proliferation, and survival, plays an important role in oocyte maturation. However, its role in embryonic development remains unknown. Therefore, in this study, we aimed to investigate the effects of IL-7 supplementation on in vitro culture (IVC) of porcine embryos after parthenogenetic activation (PA) based on characteristics such as cleavage, blastocyst formation rate, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels in cleaved embryos, total cell number, apoptosis rate, and cell lineage specification in blastocysts. Immunofluorescence revealed that IL-7 and its receptor, IL-7Rα (IL-7R) localized in the cytoplasm of porcine parthenote embryos. By supplementing the IVC medium (PZM5) with various concentrations of IL-7, an optimal concentration that enhanced embryonic development, promoted intracellular GSH, and decreased ROS levels in the cleavage stage during porcine embryo IVC was determined. Investigation of mRNA expression patterns via qRT-PCR suggested that IL-7 possibly regulated maternal mRNA clearance and zygotic genome activation. Furthermore, IL-7 supplementation reduced blastocyst apoptosis, enhanced the expression of the inner cell mass marker SOX2, and phosphorylated STAT5 levels in the blastocysts. Moreover, it altered the transcription patterns of genes that regulate apoptosis, IL-7 signaling, and development. Thus, we demonstrated the localization of IL-7 and IL-7R in porcine preimplantation embryos in vitro for the first time. Furthermore, we suggest that IL-7 supplementation can be employed to enhance embryonic development and blastocyst quality based on the activation of the transcripts of genes that are involved in developmental competence and IL-7 signaling during in vitro porcine embryo development following PA.
    Type of Medium: Online Resource
    ISSN: 2297-1769
    URL: Article
    URL: Article
    DOI: 10.3389/fvets.2022.1052856
    DOI: 10.3389/fvets.2022.1052856.s001
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2022
    detail.hit.zdb_id: 2834243-4
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  • 4
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    Copper deficiency affects the developmental competence of porcine oocytes matured in vitro
    Frontiers Media SA ; 2022
    In:  Frontiers in Cell and Developmental Biology Vol. 10 ( 2022-9-7)
    Details
    In: Frontiers in Cell and Developmental Biology, Frontiers Media SA, Vol. 10 ( 2022-9-7)
    Abstract: The trace element Cu is required for the activity of various enzymes essential for physiological processes. In this study, we elucidated the copper transport system in porcine follicular cells and investigated the effect of Cu chelation during in vitro maturation (IVM) of porcine oocytes and subsequent embryonic development after parthenogenetic activation (PA). Cu chelation was induced by adding tetraethylenepentamine (TEPA) to the maturation media (TCM199-PVA). First, we identified the localization and relative levels of the copper transporter CTR1 in follicular cells. The level of CTR1 protein was the highest in mature cumulus cells; moreover, CTR1 was mainly localized in the cytoplasmic vesicular compartment in oocytes, whereas it was evenly distributed in the cytoplasm in cumulus cells. A total of 42 h after IVM, the TEPA-treated group showed reduced maturation rates compared to those of the control ( p & lt; 0.05). This negative effect of TEPA disappeared when it was added to the media with Cu (Cu + TEPA group). The TEPA treatment during IVM significantly increased the mRNA levels of the Has2 gene, which is related to cumulus expansion ( p & lt; 0.05). Both Cu supplementation and chelation significantly increased the reactive oxygen species (ROS) levels in porcine oocytes ( p & lt; 0.05). When we analyzed the transcript levels of folliculogenesis-related genes in Cu chelation conditions, only the expression of MAPK3 in cumulus cells significantly increased compared to that of the control. We also evaluated the subsequent embryonic development of PA embryos. TEPA-treated oocytes showed significantly decreased blastocyst formation rates compared to those of the control. The TEPA-induced toxic effect was alleviated when Cu was added with TEPA. Our findings suggest that the Cu transport system plays an important role in the porcine follicular development process and that the Cu deficiency negatively affects porcine oocyte maturation, as well as their subsequent developmental competence.
    Type of Medium: Online Resource
    ISSN: 2296-634X
    URL: Article
    DOI: 10.3389/fcell.2022.993030
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2022
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  • 5
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    DataSheet1.PDF
    Frontiers Media SA ; 2022
    In:  Frontiers in Bioengineering and Biotechnology Vol. 9 ( 2022-1-28)
    Details
    In: Frontiers in Bioengineering and Biotechnology, Frontiers Media SA, Vol. 9 ( 2022-1-28)
    Abstract: The advent of tissue clearing methods, in conjunction with novel high-resolution imaging techniques, has enabled the visualization of three-dimensional structures with unprecedented depth and detail. Although a variety of clearing protocols have been developed, little has been done to quantify their efficacies in a systematic, reproducible fashion. Here, we present two simple assays, Punching-Assisted Clarity Analysis (PACA)-Light and PACA-Glow, which use easily accessible spectroscopy and gel documentation systems to quantify the transparency of multiple cleared tissues simultaneously. We demonstrate the use of PACA-Light and PACA-Glow to compare twenty-eight tissue clearing protocols on rodent brains. We also show that regional differences exist in tissue transparency in the rodent brain, with cerebellar tissue consistently achieving lower clearing levels compared to the prefrontal or cerebral cortex across all protocols. This represents the largest comparative study of tissue clearing protocols to date, made possible by the high-throughput nature of our PACA platforms.
    Type of Medium: Online Resource
    ISSN: 2296-4185
    URL: Article
    URL: Article
    DOI: 10.3389/fbioe.2021.784626
    DOI: 10.3389/fbioe.2021.784626.s001
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2022
    detail.hit.zdb_id: 2719493-0
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  • 6
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    Table1.docx
    Frontiers Media SA ; 2023
    In:  Frontiers in Cell and Developmental Biology Vol. 11 ( 2023-7-13)
    Details
    In: Frontiers in Cell and Developmental Biology, Frontiers Media SA, Vol. 11 ( 2023-7-13)
    Abstract: Neurotrophin-4 (NT-4), a neurotrophic factor, appears to affect early embryonic development because it is secreted not only by neurons but also by oviductal and uterine epithelial cells. However, no studies have characterized the effects of NT-4 on early embryonic development in pigs. In this study, we applied the experimental model of parthenogenetic-activation (PA)-derived embryos. Herein, we investigated the effect of NT-4 supplementation during the in vitro culture (IVC) of embryos, analyzed the transcription levels of specific genes, and outlined the first cell lineage specification for porcine PA-derived blastocysts. We confirmed that NT-4 and its receptor proteins were localized in both the inner cell mass (ICM) and trophectoderm (TE) in porcine blastocysts. Across different concentrations (0, 1, 10, and 100 ng/mL) of NT-4 supplementation, the optimal concentration of NT-4 to improve the developmental competence of porcine parthenotes was 10 ng/mL. NT-4 supplementation during porcine IVC significantly ( p & lt; 0.05) increased the proportion of TE cells by inducing the transcription of TE lineage markers ( CDX2 , PPAG3 , and GATA3 transcripts). NT-4 also reduced blastocyst apoptosis by regulating the transcription of apoptosis-related genes ( BAX and BCL2L1 transcripts) and improved blastocyst quality via the interaction of neurotrophin-, Hippo-yes-associated protein (Hippo-YAP) and mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway. Additionally, NT-4 supplementation during IVC significantly ( p & lt; 0.05) increased YAP1 transcript levels and significantly ( p & lt; 0.01) decreased LATS2 transcript levels, respectively, in the porcine PA-derived blastocysts. We also confirmed through fluorescence intensity that the YAP1 protein was significantly ( p & lt; 0.001) increased in the NT-4-treated blastocysts compared with that in the control. NT-4 also promoted differentiation into the TE lineage rather than into the ICM lineage during porcine early embryonic development. In conclusion, 10 ng/mL NT-4 supplementation enhanced blastocyst quality by regulating the apoptosis- and TE lineage specification-related genes and interacting with neurotrophin-, Hippo-YAP-, and MAPK/ERK signaling pathway during porcine in vitro embryo development.
    Type of Medium: Online Resource
    ISSN: 2296-634X
    URL: Article
    URL: Article
    URL: Article
    DOI: 10.3389/fcell.2023.1194596
    DOI: 10.3389/fcell.2023.1194596.s001
    DOI: 10.3389/fcell.2023.1194596.s002
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2023
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  • 7
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    Table_1.DOCX
    Frontiers Media SA ; 2021
    In:  Frontiers in Veterinary Science Vol. 8 ( 2021-11-12)
    Details
    In: Frontiers in Veterinary Science, Frontiers Media SA, Vol. 8 ( 2021-11-12)
    Abstract: Neurotrophin-4 (NT-4) is a neurotrophic factor that plays an important role in follicular development and oocyte maturation. However, it is not yet known whether NT-4 is related to oocyte maturation and follicular development in pigs. This study aims to investigate the effects of NT-4 supplementation during in vitro maturation (IVM) of porcine oocytes and subsequent embryonic development after parthenogenetic activation (PA). First, NT-4 and its receptors (TrkB and p75 NTR ) were identified through fluorescent immunohistochemistry in porcine ovaries. NT-4 was mainly expressed in theca and granulosa cells; phospho-TrkB and total TrkB were expressed in theca cells, granulosa cells, and oocytes; p75 NTR was expressed in all follicular cells. During IVM, the defined maturation medium was supplemented with various concentrations of NT-4 (0, 1, 10, and 100 ng/mL). After IVM, the nuclear maturation rate was significantly higher in the 10 and 100 ng/mL NT-4 treated groups than in the control. There was no significant difference in the intracellular reactive oxygen species levels in any group after IVM, but the 1 and 10 ng/mL NT-4 treatment groups showed a significant increase in the intracellular glutathione levels compared to the control. In matured cumulus cells, the 10 ng/mL NT-4 treatment group showed significantly increased cumulus expansion-related genes and epidermal growth factor (EGF) signaling pathway-related genes. In matured oocytes, the 10 ng/mL treatment group showed significantly increased expression of cell proliferation-related genes, antioxidant-related genes, and EGF signaling pathway-related genes. We also investigated the subsequent embryonic developmental competence of PA embryos. After PA, the cleavage rates significantly increased in the 10 and 100 ng/mL NT-4 treatment groups. Although there was no significant difference in the total cell number of blastocysts, only the 10 ng/mL NT-4 treatment group showed a higher blastocyst formation rate than the control group. Our findings suggest that supplementation with the 10 ng/mL NT-4 can enhance porcine oocyte maturation by interacting with the EGF receptor signaling pathway. In addition, we demonstrated for the first time that NT-4 is not only required for porcine follicular development, but also has beneficial effects on oocyte maturation and developmental competence of PA embryos.
    Type of Medium: Online Resource
    ISSN: 2297-1769
    URL: Article
    URL: Article
    URL: Article
    DOI: 10.3389/fvets.2021.779298
    DOI: 10.3389/fvets.2021.779298.s001
    DOI: 10.3389/fvets.2021.779298.s002
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2021
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  • 8
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    Table_1.docx
    Frontiers Media SA ; 2023
    In:  Frontiers in Veterinary Science Vol. 10 ( 2023-3-13)
    Details
    In: Frontiers in Veterinary Science, Frontiers Media SA, Vol. 10 ( 2023-3-13)
    Abstract: Porcine embryos are used for a variety of applications. However, the maturation rate in vitro remains low, and novel in vitro maturation (IVM) techniques that facilitate the collection of mature oocytes are necessary. C-C motif chemokine ligand 2 (CCL2) is a key periovulatory chemokine present in cumulus-oocyte complexes (COCs). We aimed to examine the effects of CCL2 supplementation during IVM on oocyte maturation and embryonic development. The CCL2 concentration was significantly higher in porcine follicular fluid (pFF) derived from follicles & gt;8 mm in size than in pFF derived from smaller follicles. There was a significant increase in CCL2 mRNA levels in all follicular cells after IVM compared with that before IVM. We analyzed the localization of CCL2 and its receptor, the CCL2 receptor, in follicular cells. During IVM, different concentrations of CCL2 were added to COCs cultured in a maturation medium. After IVM, the group treated with 100 ng/mL CCL2 showed significantly higher metaphase II rates than the control group. All CCL2-treatment groups showed a significant increase in intracellular glutathione levels and a significant decrease in reactive oxygen species levels, compared to the control. In CCs treated with 100 ng/mL CCL2, the mRNA levels of BAX, CASP3 , and NPR2 were significantly decreased. Furthermore, the mRNA levels of SOD1, SOD2 , and CD44 were significantly increased. In oocytes treated with 10 ng/mL CCL2, mRNA levels of BAX and CASP3 were significantly decreased, whereas, NRF2 and NPM2 were significantly increased. ERK1 exhibited significantly increased mRNA expression in both CCs and oocytes treated with 10 ng/mL CCL2. The protein expression ratio of phosphorylated ERK1/2 to total ERK1/2 was significantly increased in CCs treated with 10 ng/mL CCL2. After parthenogenetic activation, cleavage rates were significantly improved in the 100 ng/mL CCL2 treatment group, and blastocyst formation rates were significantly enhanced in the 10 ng/mL CCL2 treatment group. Overall, our results suggest that IVM medium along with CCL2 improves porcine oocyte maturation and the development of parthenogenetically-activated embryos.
    Type of Medium: Online Resource
    ISSN: 2297-1769
    URL: Article
    URL: Article
    DOI: 10.3389/fvets.2023.1136705
    DOI: 10.3389/fvets.2023.1136705.s001
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2023
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  • 9
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    Myo-inositol improves the viability of boar sperm during liquid storage
    Frontiers Media SA ; 2023
    In:  Frontiers in Veterinary Science Vol. 10 ( 2023-7-26)
    Details
    In: Frontiers in Veterinary Science, Frontiers Media SA, Vol. 10 ( 2023-7-26)
    Abstract: Liquid preservation of boar semen is a highly preferred method for semen preservation in pig production. However, oxidative stress is the main challenge during the liquid preservation of boar semen in a time dependent manner. Therefore, supplementation of sperm with antioxidants during storage to protect them from oxidative stress has been the focus of recent research. Myo-inositol (Myo-Ins), the most active form of inositol, which belongs to the vitamin (Vit.) (B1 group has been shown to improve semen quality) (1). This study aimed to investigate whether Myo-Ins supplementation protects boar sperm in liquid preservation against oxidative stress and determine the appropriate concentration of Myo-Ins to be used in this regard. Methods Boar sperm was diluted with a semen extender with different concentrations of Myo-Ins (2, 4, 6, and 8 mg/mL) depending on the previous studies (1, 24). Sperm motility and viability, plasma membrane and acrosome integrity, mitochondrial membrane potential (MMP), semen time survival, and gene expression were measured and analyzed on days 0, 1, 3, 5, and 7 for the different samples. Results Different concentrations of Myo-Ins exerted different protective effects on the boar sperm quality. The addition of 2 mg/mL Myo-Ins resulted in higher sperm motility and viability, plasma membrane and acrosome integrity, MMP, and effective survival time. Investigation of mRNA expression patterns via qRT-PCR suggested that the 2 mg/mL Myo-Ins sample had increased expression of antioxidative genes. Conclusion The addition of Myo-Ins to semen extender improved the boar semen quality by decreasing the effects of oxidative stress during liquid preservation at 17°C. Additionally, 2 mg/mL is the optimum inclusion concentration of Myo-Ins for semen preservation.
    Type of Medium: Online Resource
    ISSN: 2297-1769
    URL: Article
    DOI: 10.3389/fvets.2023.1150984
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2023
    detail.hit.zdb_id: 2834243-4
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