Publications by authors named "Il-Keun Kong"

90 Publications

Selected Thiadiazine-Thione Derivatives Attenuate Neuroinflammation in Chronic Constriction Injury Induced Neuropathy.

Front Mol Neurosci 2021 16;14:728128. Epub 2021 Dec 16.

Department of Pharmacy, University of Peshawar, Peshawar, Pakistan.

Neuropathic pain refers to a lesion or disease of peripheral and/or central somatosensory neurons and is an important body response to actual or potential nerve damage. We investigated the therapeutic potential of two thiadiazine-thione [TDT] derivatives, 2-(5-propyl-6-thioxo-1, 3, 5-thiadiazinan-3-yl) acetic acid [TDT1] and 2-(5-propyl-2-thioxo-1, 3, 5-thiadiazinan-3-yl) acetic acid [TDT2] against CCI (chronic constriction injury)-induced neuroinflammation and neuropathic pain. Mice were used for assessment of acute toxicity of TDT derivatives and no major toxic/bizarre responses were observed. Anti-inflammatory activity was assessed using the carrageenan test, and both TDT1 and TDT2 significantly reduced carrageenan-induced inflammation. We also used rats for the induction of CCI and performed allodynia and hyperalgesia-related behavioral tests followed by biochemical and morphological analysis using RT-qPCR, immunoblotting, immunohistochemistry and immunofluorescence. Our findings revealed that CCI induced clear-cut allodynia and hyperalgesia which was reversed by TDT1 and TDT2. To determine the function of TDT1 and TDT2 in glia-mediated neuroinflammation, Iba1 mRNA and protein levels were measured in spinal cord tissue sections from various experimental groups. Interestingly, TDT1 and TDT2 substantially reduced the mRNA expression and protein level of Iba1, implying that TDT1 and TDT2 may mitigate CCI-induced astrogliosis. molecular docking studies predicted that both compounds had an effective binding affinity for TNF-α and COX-2. The compounds interactions with the proteins were dominated by both hydrogen bonding and van der Waals interactions. Overall, these results suggest that TDT1 and TDT2 exert their neuroprotective and analgesic potentials by ameliorating CCI-induced allodynia, hyperalgesia, neuroinflammation and neuronal degeneration in a dose-dependent manner.
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http://dx.doi.org/10.3389/fnmol.2021.728128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8716630PMC
December 2021

Effects of Donor Cell Types on the Development of Bovine Embryos Using Cytoplasm Injection Cloning Technology.

Int J Mol Sci 2021 May 29;22(11). Epub 2021 May 29.

Division of Applied Life Science (BK21 Four), Department of Animal Science, Gyeongsang National University, Jinju 52828, Korea.

Cytoplasm injection cloning technology (CICT) is an efficient technique for evaluating the developmental potential of cloned embryos. In this study, we investigated the effects of donor cell type on the developmental potential and quality of cloned bovine embryos. Adult fibroblasts (AFs) and embryonic cells (ECs) were used as donor cells to clone bovine embryos using CICT. We initially used AF cells to develop cloned embryos and then cultured the cloned day-8 blastocysts for 10 days to obtain ECs as donor cells for second embryo cloning. We found that the bovine blastocysts cloned using AF cells had significantly reduced developmental rates, embryo quality, and ratios of inner cell mass (ICM) to the total number of cells compared to those using ECs as donor cells. Furthermore, there were significant differences in the DNA methyltransferase-, histone deacetylation-, apoptosis-, and development-related genes at the blastocyst stage in embryos cloned from AFs compared to those in embryos cloned from ECs. Our results suggest that using ECs as donor cells for nuclear transfer enhances the quantity and quality of cloned embryos. However, further investigation is required in terms of determining pregnancy rates and developing cloned embryos from different donor cell types.
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http://dx.doi.org/10.3390/ijms22115841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8197982PMC
May 2021

Constitutive Expression of TERT Enhances β-Klotho Expression and Improves Age-Related Deterioration in Early Bovine Embryos.

Int J Mol Sci 2021 May 18;22(10). Epub 2021 May 18.

Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea.

Age-associated decline in oocyte quality is one of the dominant factors of low fertility. Aging alters several key processes, such as telomere lengthening, cell senescence, and cellular longevity of granulosa cells surrounding oocyte. To investigate the age-dependent molecular changes, we examined the expression, localization, and correlation of telomerase reverse transcriptase () and β-Klotho () in bovine granulosa cells, oocytes, and early embryos during the aging process. Herein, cumulus-oocyte complexes (COCs) obtained from aged cows (>120 months) via ovum pick-up (OPU) showed reduced expression of β-Klotho and its co-receptor fibroblast growth factor receptor 1 (FGFR1). plasmid injection into pronuclear zygotes not only markedly enhanced day-8 blastocysts' development competence (39.1 ± 0.8%) compared to the control (31.1 ± 0.5%) and D-galactose (17.9 ± 1.0%) treatment groups but also enhanced and expression. In addition, plasmid-injected zygotes displayed a considerable enhancement in blastocyst quality and implantation potential. Cycloastragenol (CAG), an extract of saponins, stimulates telomerase enzymes and enhances expression and alleviates age-related deterioration in cultured primary bovine granulosa cells. In conclusion, telomerase activation or constitutive expression will increase expression and activate the FGFR1/β-Klotho pathway in bovine granulosa cells and early embryos, inhibiting age-related malfunctioning.
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http://dx.doi.org/10.3390/ijms22105327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158768PMC
May 2021

Melatonin Alleviates the Toxicity of High Nicotinamide Concentrations in Oocytes: Potential Interaction with Nicotinamide Methylation Signaling.

Oxid Med Cell Longev 2021 8;2021:5573357. Epub 2021 Apr 8.

Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Republic of Korea.

Despite the numerous studies on melatonin and nicotinamide (NAM, the active form of vitamin B3), the linkage between these two biomolecules in the context of signaling pathways regulating preimplantation embryo development has not yet been investigated. In this study, we used bovine oocyte model to elucidate the effect of melatonin on the developmental competence of oocytes under the stress of high NAM concentrations. Results showed that NAM (20 mM) administration during in vitro maturation (IVM) significantly reduced oocyte maturation and actin distribution, while induced reactive oxygen species (ROS) accumulation and mitochondrial dysfunction, the multiple deleterious effects that were alleviated by melatonin (10 M). The RT-qPCR and/or immunofluorescence showed upregulation of the apoptosis (Caspase-3, Caspase-9, and BAX), autophagy (Beclin-1, LC3A, LC3B, ATG7, LAMP1, and LAMP2), cell cycle (P21, P27, and P53), and DNA damage (COX2 and 8-OxoG) specific markers in oocytes matured under NAM treatment, compared to NAM-melatonin dual-treated and the untreated ones. In addition, the total cleavage and blastocyst development rate, as well as the total number of cells and the inner cell mass (ICM) per blastocyst, were reduced, while DNA fragmentation was induced, in the group of NAM sole treatment than NAM-melatonin cotreatment and control. Inspecting the underlying mechanisms behind NAM-associated toxicity revealed an increase in transcription pattern of NAM methylation (NNMT and AHCY) genes in NAM-treated oocytes while the opposite profile was observed upon melatonin supplementation. In conclusion, to our knowledge, this is the first study reporting that melatonin can protect oocytes and embryos from NAM-induced injury through its ROS-scavenging activity together with potential interaction with NAM methylation signaling.
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http://dx.doi.org/10.1155/2021/5573357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049830PMC
May 2021

Wnt/β-catenin Pathway-Mediated PPARδ Expression during Embryonic Development Differentiation and Disease.

Int J Mol Sci 2021 Feb 12;22(4). Epub 2021 Feb 12.

Department of Animal Science, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Korea.

The Wnt/β-catenin signaling pathway plays a crucial role in early embryonic development. Wnt/β-catenin signaling is a major regulator of cell proliferation and keeps embryonic stem cells (ESCs) in the pluripotent state. Dysregulation of Wnt signaling in the early developmental stages causes several hereditary diseases that lead to embryonic abnormalities. Several other signaling molecules are directly or indirectly activated in response to Wnt/β-catenin stimulation. The crosstalk of these signaling factors either synergizes or opposes the transcriptional activation of β-catenin/Tcf4-mediated target gene expression. Recently, the crosstalk between the peroxisome proliferator-activated receptor delta (PPARδ), which belongs to the steroid superfamily, and Wnt/β-catenin signaling has been reported to take place during several aspects of embryonic development. However, numerous questions need to be answered regarding the function and regulation of PPARδ in coordination with the Wnt/β-catenin pathway. Here, we have summarized the functional activation of the PPARδ in co-ordination with the Wnt/β-catenin pathway during the regulation of several aspects of embryonic development, stem cell regulation and maintenance, as well as during the progression of several metabolic disorders.
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http://dx.doi.org/10.3390/ijms22041854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918746PMC
February 2021

SHP2 Nuclear/Cytoplasmic Trafficking in Granulosa Cells Is Essential for Oocyte Meiotic Resumption and Maturation.

Front Cell Dev Biol 2020 22;8:611503. Epub 2021 Jan 22.

Division of Applied Life Science (BK21 Four), Institute of Agriculture and Life Science (IALS), Gyeongsang National University, Jinju, South Korea.

Src-homology-2-containing phosphotyrosine phosphatase (SHP2), a classic cytoplasmic protein and a major regulator of receptor tyrosine kinases and G protein-coupled receptors, plays a significant role in preimplantation embryo development. In this study, we deciphered the role of SHP2 in the somatic compartment of oocytes during meiotic maturation. SHP2 showed nuclear/cytoplasmic localization in bovine cumulus and human granulosa (COV434) cells. Follicle-stimulating hormone (FSH) treatment significantly enhanced cytoplasmic SHP2 localization, in contrast to the E treatment, which augmented nuclear localization. Enhanced cytoplasmic SHP2 was found to negatively regulate the expression of the ERα-transcribed and mRNAs, which are vital for oocyte meiotic arrest. The co-immunoprecipitation results revealed the presence of the SHP2/ERα complex in the germinal vesicle-stage cumulus-oocyte complexes, and this complex significantly decreased with the progression of meiotic maturation. The complex formation between ERα and SHP2 was also confirmed by using a series of computational modeling methods. To verify the correlation between SHP2 and /, SHP2 was knocked down RNA interference, and and mRNAs were analyzed in the control, E, and FSH-stimulated COV434 cells. Furthermore, phenyl hydrazonopyrazolone sulfonate 1, a site-directed inhibitor of active SHP2, showed no significant effect on the ERα-transcribed and mRNAs. Taken together, these findings support a novel nuclear/cytoplasmic role of SHP2 in oocyte meiotic resumption and maturation.
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http://dx.doi.org/10.3389/fcell.2020.611503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862566PMC
January 2021

Induction of Oxidative Stress and Mitochondrial Dysfunction by Juglone Affects the Development of Bovine Oocytes.

Int J Mol Sci 2020 Dec 26;22(1). Epub 2020 Dec 26.

Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Korea.

Juglone, a major naphthalenedione component of walnut trees, has long been used in traditional medicine as an antimicrobial and antitumor agent. Nonetheless, its impact on oocyte and preimplantation embryo development has not been entirely clarified. Using the bovine model, we sought to elucidate the impact of juglone treatment during the in vitro maturation (IVM) of oocytes on their maturation and development of embryos. Results showed a severe reduction in oocyte nuclear maturation and cumulus expansion and a significant increase in mitochondrial dysfunction and reactive oxygen species (ROS) levels in cumulus-oocyte complexes (COCs) treated with juglone (12.5, 25.0, and 50.0 µM). In addition, RT-qPCR showed downregulation of the expansion-related (HAS2, TNFAIP6, PTX3, and PTGS2) and mitochondrial (ATPase6 and ATP5F1E) genes in juglone-treated COCs. Moreover, the development rates of day 4 total cleavage and 8-16 cell stage embryos, as well as day 8 blastocysts, were significantly reduced following exposure to juglone. Using immunofluorescence, the apoptotic marker caspase-9 was overexpressed in oocytes exposed to juglone (25.0 µM) compared to the untreated control. In conclusion, our study reports that exposing bovine oocytes to 12.5-50.0 µM of juglone can reduce their development through the direct induction of ROS accumulation, apoptosis, and mitochondrial dysfunction.
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http://dx.doi.org/10.3390/ijms22010168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794829PMC
December 2020

Fibronectin protected bovine preantral follicles from the deleterious effects of kisspeptin.

Theriogenology 2021 Feb 18;161:301-312. Epub 2020 Dec 18.

Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju, 52828, Gyeongnam Province, Republic of Korea; Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, 52828, Gyeongnam Province, Republic of Korea; Thekingkong Co. Ltd., Gyeongsang National University, Jinju, 52828, Gyeongnam Province, Republic of Korea. Electronic address:

Kisspeptin (Kp), a multifunctional neuropeptide critical for initiating puberty and regulating ovulation, was reported to be expressed in mammalian ovaries. Fibronectin (FN), a major secretory product of granulosa cells, provided the extracellular environment for the cumulus cells during maturation. In the current study, we aimed to investigate the potential interplay between FN and Kp in bovine preantral follicles in the context of follicular development and quality. The results showed that Kp significantly reduced the follicular diameters after 14 days in culture, and this was prevented by the addition of FN. Follicles treated with Kp in the presence of FN showed lower levels of apoptotic cells compared to the Kp-treated group. The immunofluorescence analysis showed high levels of cyclooxygenase-2 (COX2), nuclear factor kappa B (NF-κB), and caspase 3, and low levels of sirtuin 1 (Sirt1) and Poly ADP-Ribose Polymerase 1 (PARP1) in the Kp-treated group compared to the control and FN-Kp co-treated groups. The protein expression levels of phosphoinositide 3 kinase (PI3K) increased significantly in the FN and FN-Kp combination treatment groups. Finally, we examined the signal pathway affecting the follicular development after Kp treatment. We detected a significant decrease in the mRNA levels of B-cell lymphoma 2 (BCL2), Sirt1, and PI3K, but the mRNA levels of NF-κB, Caspase3, COX2, P21, and P53 were significantly higher than in the control. Taken together, our results showed the importance of FN for preantral follicle developmental, and, for the first time, we reported that FN could neutralize the deleterious consequences of Kp, suggesting a potential role in the regulation of PI3K/Sirt1 signaling in bovine preantral follicle development.
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http://dx.doi.org/10.1016/j.theriogenology.2020.12.017DOI Listing
February 2021

Genetic Spectrum of Syndromic and Non-Syndromic Hearing Loss in Pakistani Families.

Genes (Basel) 2020 11 11;11(11). Epub 2020 Nov 11.

Institute of Human Genetics, Julius Maximilians University Würzburg, 97074 Würzburg, Germany.

The current molecular genetic diagnostic rates for hereditary hearing loss (HL) vary considerably according to the population background. Pakistan and other countries with high rates of consanguineous marriages have served as a unique resource for studying rare and novel forms of recessive HL. A combined exome sequencing, bioinformatics analysis, and gene mapping approach for 21 consanguineous Pakistani families revealed 13 pathogenic or likely pathogenic variants in the genes , , , , , , and , with an overall resolve rate of 61.9%. and were the most frequently involved genes in this cohort. All the identified variants were either homozygous or compound heterozygous, with two of them not previously described in the literature (15.4%). Overall, seven missense variants (53.8%), three nonsense variants (23.1%), two frameshift variants (15.4%), and one splice-site variant (7.7%) were observed. Syndromic HL was identified in five (23.8%) of the 21 families studied. This study reflects the extreme genetic heterogeneity observed in HL and expands the spectrum of variants in deafness-associated genes.
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http://dx.doi.org/10.3390/genes11111329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709052PMC
November 2020

Bovine Oviduct Epithelial Cell-Derived Culture Media and Exosomes Improve Mitochondrial Health by Restoring Metabolic Flux during Pre-Implantation Development.

Int J Mol Sci 2020 Oct 14;21(20). Epub 2020 Oct 14.

Department of Animal Science, Division of Applied Life Science (BK21 Four), Gyeongsang National University, Jinju 52828, Korea.

Oviduct flushing is enriched by a wide variety of nutrients that guide the 3-4 days journey of pre-implantation embryo through the oviduct as it develops into a competent blastocyst (BL). However, little is known about the specific requirement and role of these nutrients that orchestrate the early stages of embryonic development. In this study, we aimed to characterize the effect of in vitro-derived bovine oviduct epithelial cell (BOECs) secretion that mimics the in vivo oviduct micro-fluid like environment, which allows successful embryonic development. In this study, the addition of an in vitro derived BOECs-condition media (CM) and its isolated exosomes (Exo) significantly enhances the quality and development of BL, while the hatching ability of BLs was found to be high (48.8%) in the BOECs-Exo supplemented group. Surprisingly, BOECs-Exo have a dynamic effect on modulating the embryonic metabolism by restoring the pyruvate flux into TCA-cycle. Our analysis reveals that Exo treatment significantly upregulates the pyruvate dehydrogenase (PDH) and glutamate dehydrogenase (GLUD1) expression, required for metabolic fine-tuning of the TCA-cycle in the developing embryos. Exo treatment increases the influx into TCA-cycle by strongly suppressing the PDH and GLUD1 upstream inhibitors, i.e., PDK4 and SIRT4. Improvement of TCA-cycle function was further accompanied by higher metabolic activity of mitochondria in BOECs-CM and Exo in vitro embryos. Our study uncovered, for the first time, the possible mechanism of BOECs-derived secretion in re-establishing the TCA-cycle flux by the utilization of available nutrients and highlighted the importance of pyruvate in supporting bovine in vitro embryonic development.
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http://dx.doi.org/10.3390/ijms21207589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593913PMC
October 2020

Effect of nicotinamide supplementation in in vitro fertilization medium on bovine embryo development.

Mol Reprod Dev 2020 10 4;87(10):1070-1081. Epub 2020 Sep 4.

Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, Gyeongnam, Republic of Korea.

Increased oxidative stress is one of the main causes of poorly developed embryos in assisted reproductive technologies. Nicotinamide (NAM) has been shown to suppress reactive oxygen species (ROS) production through its potent antioxidative and anti-senescent effects. In the present study, we explored the effects of short-term NAM-treatment (3 and 5 h) during in vitro fertilization (IVF) on the development of bovine embryos. Treatment with 10 mM NAM for 3 h significantly increased the blastocyst formation but extending the treatment to 5 h did not enhance the benefits any further. Immunofluorescence analysis demonstrated that treatment with 10 mM NAM for 3 h decreased the expression of intracellular ROS, 8-oxo-7,8-dihydroguanine, caspase-3, and increased the expression of Sirt1, and incorporation of bromodeoxyuridine in one-cell stage embryos. Similarly, the level of H3K56ac significantly increased in the NAM-treated (3 and 5 h) one-cell stage embryos. Contrastingly, the treatment with 10 mM NAM for 5 h increased the caspase-9 level in blastocysts. Collectively, these findings suggest that NAM possesses antioxidant activity and supplementation of IVF medium with 10 mM NAM for 3 h improves the in vitro developmental competence of bovine embryos.
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http://dx.doi.org/10.1002/mrd.23417DOI Listing
October 2020

Effect of Additional Cytoplasm of Cloned Embryo on Developmental Competence and Reprogramming Efficiency in Mice.

Cell Reprogram 2020 10 24;22(5):236-243. Epub 2020 Aug 24.

Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, Republic of Korea.

Somatic cell nuclear transfer (SCNT) is an important technique for biological science research. Cytoplasm injection cloning technology (CICT) was developed to improve the reprogramming efficiency as well as to overcome the limitations of SCNT. CICT uses an additional cytoplasm fused with an enucleated oocyte to restore the cytoplasmic volume of the cloned embryo, and this method could improve the reprogramming efficiency of the cloned embryo. In this study, we show that CICT can be adapted to mouse species to overcome the inefficiency of the SCNT method. In this study, results indicate that the two-cell embryo and blastocyst rates of cloned embryos with the use of the CICT method were significantly higher ( < 0.05) than that of the SCNT method (96.6% ± 1.1% vs. 86.7% ± 6.0%, 29.5% ± 2.6% vs. 22.1% ± 3.0%, respectively). Furthermore, the apoptotic cell number per blastocyst was significantly lower in the CICT group than that in the SCNT group (1.7 ± 0.2 vs. 2.9 ± 0.3,  < 0.05). Moreover, the acH3K9/K14 expression level in the CICT group was greater than that of the SCNT group ( < 0.05), and the relative acH3K56 level in the CICT group was significantly ( < 0.05) higher than that in the SCNT group. These results indicate that CICT helps improve the developmental competence and quality of cloned embryos.
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http://dx.doi.org/10.1089/cell.2020.0022DOI Listing
October 2020

Growth Factors, and Cytokines; Understanding the Role of Tyrosine Phosphatase SHP2 in Gametogenesis and Early Embryo Development.

Cells 2020 07 29;9(8). Epub 2020 Jul 29.

Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju 52828, Korea.

Growth factors and cytokines have vital roles in germ cell development, gamete maturation, and early embryo development. Cell surface receptors are present for growth factors and cytokines to integrate with and trigger protein signaling in the germ and embryo intracellular milieu. Src-homology-2-containing phosphotyrosine phosphatase (SHP2) is a ubiquitously expressed, multifunctional protein that plays a central role in the signaling pathways involved in growth factor receptors, cytokine receptors, integrins, and G protein-coupled receptors. Over recent decades, researchers have recapitulated the protein signaling networks that influence gamete progenitor specification as well as gamete differentiation and maturation. SHP2 plays an indispensable role in cellular growth, survival, proliferation, differentiation, and migration, as well as the basic events in gametogenesis and early embryo development. SHP2, a classic cytosolic protein and a key regulator of signal transduction, displays unconventional nuclear expression in the genital organs. Several observations provided shreds of evidence that this behavior is essential for fertility. The growth factor and cytokine-dependent roles of SHP2 and its nuclear/cytoplasmic presence during gamete maturation, early embryonic development and embryo implantation are fascinating and complex subjects. This review is intended to summarize the previous and recent knowledge about the SHP2 functions in gametogenesis and early embryo development.
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http://dx.doi.org/10.3390/cells9081798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465981PMC
July 2020

Nicotinamide Supplementation during the In Vitro Maturation of Oocytes Improves the Developmental Competence of Preimplantation Embryos: Potential Link to SIRT1/AKT Signaling.

Cells 2020 06 25;9(6). Epub 2020 Jun 25.

Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju 52828, Korea.

Nicotinamide (NAM), the amide form of vitamin B3, plays pivotal roles in regulating various cellular processes including energy production and maintenance of genomic stability. The current study aimed at deciphering the effect of NAM, when administered during in vitro maturation (IVM), on the developmental competence of bovine preimplantation embryos. Our results showed that low NAM concentrations reduced the oxidative stress and improved mitochondrial profile, total cleavage and 8-16 cell stage embryo development whereas the opposite profile was observed upon exposure to high NAM concentrations (10 mM onward). Remarkably, the hatching rates of day-7 and day-8 blastocysts were significantly improved under 0.1 mM NAM treatment. Using RT-qPCR and immunofluorescence, the autophagy-related (Beclin-1 (BECN1), LC3B, and ATG5) and the apoptotic (Caspases; CASP3 and 9) markers were upregulated in oocytes exposed to high NAM concentration (40 mM), whereas only CASP3 was affected, downregulated, following 0.1 mM treatment. Additionally, the number of cells per blastocyst and the levels of SIRT1, PI3K, AKT, and mTOR were higher, while the inner cell mass-specific transcription factors GATA6, SOX2, and OCT4 were more abundant, in day-8 embryos of NAM-treated group. Taken together, to our knowledge, this is the first study reporting that administration of low NAM concentrations during IVM can ameliorate the developmental competence of embryos through the potential regulation of oxidative stress, apoptosis, and SIRT1/AKT signaling.
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http://dx.doi.org/10.3390/cells9061550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348965PMC
June 2020

Supplementation of insulin-transferrin-sodium selenite in culture medium improves the hypothermic storage of bovine embryos produced in vitro.

Theriogenology 2020 Aug 29;152:147-155. Epub 2020 Apr 29.

Department of Animal Science, Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, 52828, Gyeongnam Province, Republic of Korea; Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, 52828, Gyeongnam Province, Republic of Korea; The King Kong Corp. Ltd., Gyeongsang National University, Jinju, 52828, Gyeongnam Province, Republic of Korea. Electronic address:

Hypothermic storage of gametes and embryos at 4 °C can be used as an alternative to cryopreservation, but hypothermic preservation can maintain embryo viability for a short duration only. This study investigated the effect of insulin-transferrin-sodium selenite (ITS) in embryo culture medium on hypothermic storage of bovine embryos at 4 °C. Day 7 bovine embryos were subjected to hypothermic storage in tissue culture medium 199 supplemented with 50% fetal bovine serum and 25 mM HEPES for different time durations. After recovery, the embryos were assessed for survival and hatching rate and gene and protein expression levels. Supplementation of embryo culture medium with ITS significantly increased (P < 0.05) the survival and hatching ability of blastocysts stored at 4 °C for 72 h compared to the control group (100% and 76.3% vs 68.5% and 40.5%, respectively). Furthermore, the beneficial effects of ITS on embryos were associated with greater (P < 0.05) total cell number per blastocyst and lesser apoptotic cells number. Moreover, embryos cultured in ITS had lower intracellular lipid content. The protein expression of sirt1 was greater (P < 0.05) in the ITS group, however, caspase3 protein expression was significantly lesser (P < 0.05) in the ITS group. Quantitative reverse transcription PCR indicated that the mRNA levels of SIRT1 and HSP70 were (P < 0.05) increased upon culture with ITS; however, the mRNA levels of the pro-apoptotic genes BAX and CASP3 were reduced (P < 0.05). Taken together, these data suggest that supplementation of embryo culture medium with ITS improves in vitro bovine embryo quality and survival following hypothermic storage.
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http://dx.doi.org/10.1016/j.theriogenology.2020.04.036DOI Listing
August 2020

Matrix metalloproteinases improves trophoblast invasion and pregnancy potential in mice.

Theriogenology 2020 Jul 5;151:144-150. Epub 2020 Feb 5.

Department of Animal Science, Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, 52828, Gyeongnam Province, Republic of Korea; Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, 52828, Gyeongnam Province, Republic of Korea. Electronic address:

Successful implantation is closely linked to the expression of MMP-2 and MMP-9, which greatly influence the ability of an embryo to degrade the basement membrane of the uterine epithelium, mainly composed of type IV collagen, and invade the uterine stroma. The objective of this study was to determine the effect of MMP-2 and MMP-9 co-transfer with embryos on reproductive performance in mice. Using invasion assay, we tested the effect of MMP-2 and MMP-9 for their ability to support trophoblastic invasion in vitro. We performed co-transfer of MMP-2 and MMP-9 with mouse embryos to 2.5 days post-coitum (dpc) pseudo-pregnant uteri using nonsurgical embryo transfer (NSET) technique and evaluated the pregnancy outcomes. Uterine tissue samples were collected to determine collagen content by Masson's trichrome staining. Our results showed that in vitro treatment of MMP-2 and MMP-9 significantly promoted both spreading and invasion of mouse trophoblastic cells compared to the non-treated blastocysts. Moreover, embryo transfer results showed that MMP-9 co-transfer enhanced pregnancy outcome inform of live pup rate by degrading the extracellular matrix, collagen, and facilitate embryo implantation. Taken together our findings imply that MMP-9 can regulate trophoblastic cell invasion during preimplantation, which may have important consequences on embryo implantation, and shed the light on new strategies to avoid miscarriage and provides a platform for successful human embryo transfer technologies.
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http://dx.doi.org/10.1016/j.theriogenology.2020.02.002DOI Listing
July 2020

Role of Wnt Signaling During In-Vitro Bovine Blastocyst Development and Maturation in Synergism with PPARδ Signaling.

Cells 2020 04 9;9(4). Epub 2020 Apr 9.

Department of Animal Science, Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea.

Wnt/β-catenin signaling plays vital role in the regulation of cellular proliferation, migration, stem cells cell renewal and genetic stability. This pathway is crucial during the early developmental process; however, the distinct role of Wnt/β-catenin signaling during pre-implantation period of bovine embryonic development is obscure. Here, we evaluated the critical role of Wnt/β-catenin pathway in the regulation of bovine blastocyst (BL) development and hatching. 6 bromoindurbin-3'oxime (6-Bio) was used to stimulate the Wnt signaling. Treatment with 6-Bio induced the expression of peroxisome proliferator-activated receptor-delta (PPARδ). Interestingly, the PPARδ co-localized with β-catenin and form a complex with TCF/LEF transcription factor. This complex potentiated the expression of several Wnt directed genes, which regulate early embryonic development. Inhibition of PPARδ with selective inhibitor 4-chloro-N-(2-{[5-trifluoromethyl]-2-pyridyl]sulfonyl}ethyl)benzamide (Gsk3787) severely perturbed the BL formation and hatching. The addition of Wnt agonist successfully rescued the BL formation and hatching ability. Importantly, the activation of PPARδ expression by Wnt stimulation enhanced cell proliferation and fatty acid oxidation (FAO) metabolism to improve BL development and hatching. In conclusion, our study provides the evidence that Wnt induced PPARδ expression co-localizes with β-catenin and is a likely candidate of canonical Wnt pathway for the regulation of bovine embryonic development.
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http://dx.doi.org/10.3390/cells9040923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226827PMC
April 2020

Comparative Genomic Mapping Implicates LRRK2 for Intellectual Disability and Autism at 12q12, and HDHD1, as Well as PNPLA4, for X-Linked Intellectual Disability at Xp22.31.

J Clin Med 2020 Jan 19;9(1). Epub 2020 Jan 19.

Section of Reproductive Endocrinology, Infertility & Genetics, Department of Obstetrics & Gynecology, Augusta University, Augusta, GA 30912, USA.

We report a genomic and phenotypic delineation for two chromosome regions with candidate genes for syndromic intellectual disability at 12q12 and Xp22.31, segregating independently in one family with four affected members. Fine mapping of three affected members, along with six unreported small informative CNVs, narrowed down the candidate chromosomal interval to one gene at 12q12. Expression studies revealed high levels of transcripts in the whole human brain, cerebral cortex and hippocampus. RT-qPCR assays revealed that transcripts were dramatically reduced in our microdeletion patient DGDP289A compared to his healthy grandfather with no deletion. The decreased expression of LRRK2 may affect protein-protein interactions between LRRK2 and its binding partners, of which eight have previously been linked to intellectual disability. These findings corroborate with a role for LRRK2 in cognitive development, and, thus, we propose that intellectual disability and autism, displayed in the 12q12 microdeletions, are likely caused by . Using another affected member, DGDP289B, with a microdeletion at Xp22.31, in this family, we performed the genomic and clinical delineation with six published and nine unreported cases. We propose and for X-linked intellectual disability in this region, since their high transcript levels in the human brain substantiate their role in intellectual functioning.
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http://dx.doi.org/10.3390/jcm9010274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019335PMC
January 2020

Novel Loss-of-Function Variants in are Associated with Recessive Sensorineural Hearing Loss in Iranian and Pakistani Patients.

Int J Mol Sci 2020 Jan 2;21(1). Epub 2020 Jan 2.

Institute of Human Genetics, Julius Maximilians University, 97074 Würzburg, Germany.

encodes the Cell Division Cycle 14A protein and has been associated with autosomal recessive non-syndromic hearing loss (DFNB32), as well as hearing impairment and infertile male syndrome (HIIMS) since 2016. To date, only nine variants have been associated in patients whose initial symptoms included moderate-to-profound hearing impairment. Exome analysis of Iranian and Pakistani probands who both showed bilateral, sensorineural hearing loss revealed a novel splice site variant (c.1421+2T>C, p.?) that disrupts the splice donor site and a novel frameshift variant (c.1041dup, p.Ser348Glnfs*2) in the gene , respectively. To evaluate the pathogenicity of both loss-of-function variants, we analyzed the effects of both variants on the RNA-level. The splice variant was characterized using a minigene assay. Altered expression levels due to the c.1041dup variant were assessed using RT-qPCR. In summary, cDNA analysis confirmed that the c.1421+2T>C variant activates a cryptic splice site, resulting in a truncated transcript (c.1414_1421del, p.Val472Leufs*20) and the c.1041dup variant results in a defective transcript that is likely degraded by nonsense-mediated mRNA decay. The present study functionally characterizes two variants and provides further confirmatory evidence that is associated with a rare form of hereditary hearing loss.
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http://dx.doi.org/10.3390/ijms21010311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6982189PMC
January 2020

Difference in Developmental Kinetics of Y-Specific Monoclonal Antibody Sorted Male and Female In Vitro Produced Bovine Embryos.

Int J Mol Sci 2019 Dec 30;21(1). Epub 2019 Dec 30.

Department of Animal Science, Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju 52828, Gyeongnam, Korea.

Sex-related growth differences between male and female embryos remain an attractive subject for reproductive biologists. This study aimed to investigate the endogenous factors that play a crucial role in the pace of early development between male and female bovine embryos. Using sex pre-selected semen by Y-specific monoclonal antibodies for the production of bovine embryos, we characterized the critical endogenous factors that are responsible for creating the development differences, especially during the pre-implantation period between male and female embryos. Our results showed that at day seven, (57.8%) Y-sperm sorted in vitro cultured embryos reached the expanded blastocyst (BL) stage, whereas the X-sperm sorted group were only 25%. Y-BLs showed higher mRNA abundance of pluripotency and developmental competency regulators, such as and . Interestingly, Y-sperm sorted BLs had a homogeneous mitochondrial distribution pattern, higher mitochondrial membrane potential (), efficient OXPHOS (oxidative phosphorylation) system and well-encountered production of ROS (reactive oxygen species) level. Moreover, Y-blastocysts (BLs) showed less utilization of glucose metabolism relative to the X-BLs group. Importantly, both sexes showed differences in the timing of epigenetic events. All these factors directly or indirectly orchestrate the whole embryonic progression and may help in the faster and better quality yield of BL in the Y-sperm sorted group compared to the X counterpart group.
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http://dx.doi.org/10.3390/ijms21010244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981608PMC
December 2019

The PPARδ Agonist GW501516 Improves Lipolytic/Lipogenic Balance through CPT1 and PEPCK during the Development of Pre-Implantation Bovine Embryos.

Int J Mol Sci 2019 Dec 2;20(23). Epub 2019 Dec 2.

Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea.

The PPARs (peroxisome proliferator-activated receptors) play critical roles in the regulation of lipid and glucose metabolism. PPARδ, a member of the PPARs family, is associated with decreased susceptibility to ectopic lipid deposition and is implicated in the regulation of mitochondrial processes. The current study aimed to determine the role of PPARδ in fatty acid β-oxidation and its influence on PEPCK for the lipogenic/lipolytic balance during in vitro bovine oocyte maturation and embryo development. Activation of PPARδ by GW501516, but not 2-BP, was indicated by intact embryonic PEPCK (cytosolic) and CPT1 expression and the balance between free fatty acids and mitochondrial β-oxidation that reduced ROS and inhibited p-NF-κB nuclear localization. Genes involved in lipolysis, fatty acid oxidation, and apoptosis showed significant differences after the GW501516 treatment relative to the control- and 2-BP-treated embryos. GSK3787 reversed the PPARδ-induced effects by reducing PEPCK and CPT1 expression and the mitochondrial membrane potential, revealing the importance of PPARδ/PEPCK and PPARδ/CPT1 for controlling lipolysis during embryo development. In conclusion, GW501516-activated PPARδ maintained the correlation between lipolysis and lipogenesis by enhancing PEPCK and CPT1 to improve bovine embryo quality.
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http://dx.doi.org/10.3390/ijms20236066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928732PMC
December 2019

Disruption of PHF21A causes syndromic intellectual disability with craniofacial anomalies, epilepsy, hypotonia, and neurobehavioral problems including autism.

Mol Autism 2019 22;10:35. Epub 2019 Oct 22.

21Department of Biology, Chungnam National University, Daejeon, Korea.

Background: has been associated with intellectual disability and craniofacial anomalies based on its deletion in the Potocki-Shaffer syndrome region at 11p11.2 and its disruption in three patients with balanced translocations. In addition, three patients with de novo truncating mutations in were reported recently. Here, we analyze genomic data from seven unrelated individuals with mutations in and provide detailed clinical descriptions, further expanding the phenotype associated with PHF21A haploinsufficiency.

Methods: Diagnostic trio whole exome sequencing, Sanger sequencing, use of GeneMatcher, targeted gene panel sequencing, and MiSeq sequencing techniques were used to identify and confirm variants. RT-qPCR was used to measure the normal expression pattern of in multiple human tissues including 13 different brain tissues. Protein-DNA modeling was performed to substantiate the pathogenicity of the missense mutation.

Results: We have identified seven heterozygous coding mutations, among which six are de novo (not maternal in one). Mutations include four frameshifts, one nonsense mutation in two patients, and one heterozygous missense mutation in the AT Hook domain, predicted to be deleterious and likely to cause loss of PHF21A function. We also found a new C-terminal domain composed of an intrinsically disordered region. This domain is truncated in six patients and thus likely to play an important role in the function of PHF21A, suggesting that haploinsufficiency is the likely underlying mechanism in the phenotype of seven patients. Our results extend the phenotypic spectrum of mutations by adding autism spectrum disorder, epilepsy, hypotonia, and neurobehavioral problems. Furthermore, is highly expressed in the human fetal brain, which is consistent with the neurodevelopmental phenotype.

Conclusion: Deleterious nonsense, frameshift, and missense mutations disrupting the AT Hook domain and/or an intrinsically disordered region in PHF21A were found to be associated with autism spectrum disorder, epilepsy, hypotonia, neurobehavioral problems, tapering fingers, clinodactyly, and syndactyly, in addition to intellectual disability and craniofacial anomalies. This suggests that is involved in autism spectrum disorder and intellectual disability, and its haploinsufficiency causes a diverse neurological phenotype.
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http://dx.doi.org/10.1186/s13229-019-0286-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805429PMC
June 2020

PTPN11 (SHP2) Is Indispensable for Growth Factors and Cytokine Signal Transduction During Bovine Oocyte Maturation and Blastocyst Development.

Cells 2019 10 18;8(10). Epub 2019 Oct 18.

Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju 52828, Gyeongnam Province, Korea.

This study was aimed to investigate the role of SHP2 (Src-homology-2-containing phosphotyrosine phosphatase) in intricate signaling networks invoked by bovine oocyte to achieve maturation and blastocyst development. (Protein Tyrosine Phosphatase, non-receptor type 11) encoding protein SHP2, a positive transducer of RTKs (Receptor Tyrosine Kinases) and cytokine receptors, can play a significant role in bovine oocyte maturation and embryo development, but this phenomenon has not yet been explored. Here, we used different growth factors, cytokines, selective activator, and a specific inhibitor of SHP2 to ascertain its role in bovine oocyte developmental stages in vitro. We found that SHP2 became activated by growth factors and cytokines treatment and was highly involved in the activation of oocyte maturation and embryo development pathways. Activation of SHP2 triggered MAPK (mitogen-activated protein kinases) and PI3K/AKT (Phosphoinositide 3-kinase/Protein kinase B) signaling cascades, which is not only important for GVBD (germinal vesical breakdown) induction but also for maternal mRNA translation. Inhibition of phosphatase activity of SHP2 with PHPS1 (Phenylhydrazonopyrazolone sulfonate 1) reduced oocytes maturation as well as bovine blastocyst ICM (inner cell mass) volume. Supplementation of LIF (Leukemia Inhibitory Factor) to embryos showed an unconventional direct relation between p-SHP2 and p-STAT3 (Signal transducer and activator of transcription 3) for blastocyst ICM development. Other than growth factors and cytokines, cisplatin was used to activate SHP2. Cisplatin activated SHP2 modulate growth factors effect and combine treatment significantly enhanced quality and rate of developed blastocysts.
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http://dx.doi.org/10.3390/cells8101272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6830097PMC
October 2019

Production of cloned cats using additional complimentary cytoplasm.

Anim Reprod Sci 2019 Sep 15;208:106125. Epub 2019 Jul 15.

Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, 52828, Gyeongnam Province, Republic of Korea; Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, 52828, Gyeongnam Province, Republic of Korea; The King Kong Corp. Ltd., Gyeongsang National University, Jinju, 52828, Gyeongnam Province, Republic of Korea. Electronic address:

Somatic cell nuclear transfer (SCNT) is an important technique for producing cloned animals. It, however, is inefficient when there is use of SCNT for cloned animal production. Cytoplasm injection cloning technology (CICT) was developed to overcome the inefficiencies of SCNT use of this purpose. The use of CICT involves additional cytoplasm fusing with enucleated oocytes to restore the cytoplasmic volume, thus improving the in vitro developmental competence and quality of cloned embryos. In this study, there was application of CICT in cats to improve the in vitro developmental competence of cloned embryos, as well as the production of the offspring. The results of this study were that fusion rate of the cloned embryos with use of the CICT method was greater than that with SCNT (80.0 ± 4.8% compared with 67.8 ± 11.3%, respectively), and more blastocysts developed with use of CICT than SCNT (20.0 ± 2.0% compared with 13.5 ± 5.0%, respectively). The 62 cloned embryos that were produced with use of CICT were transferred into five estrous synchronized recipients, and 151 cloned embryos produced using SCNT were transferred to 13 estrous-synchronized recipients. After the embryo transfer, there was birth from surrogate mothers of one live-born kitten that resulted using SCNT compared with three live-born kittens using CICT. The number of CICT-cloned embryos born was greater than that of SCNT-cloned embryos (4.8 ± 2.3% compared with 0.7 ± 1.3%, P <  0.05). These results indicate that the CICT technique can be used to produce cloned kittens, including endangered feline species.
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http://dx.doi.org/10.1016/j.anireprosci.2019.106125DOI Listing
September 2019

Melatonin Abrogates the Anti-Developmental Effect of the AKT Inhibitor SH6 in Bovine Oocytes and Embryos.

Int J Mol Sci 2019 Jun 17;20(12). Epub 2019 Jun 17.

Department of Animal Science, Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju 52828, Korea.

Melatonin, a nighttime-secreted antioxidant hormone produced by the pineal gland, and AKT, a serine/threonine-specific protein kinase, have been identified as regulators for several cellular processes essential for reproduction. The current study aimed to investigate the potential interplay between melatonin and AKT in bovine oocytes in the context of embryo development. Results showed that the inclusion of SH6, a specific AKT inhibitor, during in vitro maturation (IVM) significantly reduced oocyte maturation, cumulus cell expansion, cleavage, and blastocyst development that were rescued upon addition of melatonin. Oocytes treated with SH6 in the presence of melatonin showed lower levels of reactive oxygen species (ROS) and blastocysts developed exhibited low apoptosis while the mitochondrial profile was significantly improved compared to the SH6-treated group. The RT-qPCR results showed up-regulation of the mRNA of maturation-, mitochondrial-, and cumulus expansion-related genes including GDF-9, BMP-15, MARF1, ATPase, ATP5F1E, POLG2, HAS2, TNFAIP6, and PTGS2 and down-regulation of Bcl-2 associated X apoptosis regulator (BAX), caspase 3, and p21 involved in apoptosis and cell cycle arrest in melatonin-SH6 co-treated group compared to SH6 sole treatment. The immunofluorescence showed high levels of caspase 3 and caspase 9, and low AKT phosphorylation in the SH6-treated group compared to the control and melatonin-SH6 co-treatment. Taken together, our results showed the importance of both melatonin and AKT for overall embryonic developmental processes and, for the first time, we report that melatonin could neutralize the deleterious consequences of AKT inhibition, suggesting a potential role in regulation of AKT signaling in bovine oocytes.
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http://dx.doi.org/10.3390/ijms20122956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627520PMC
June 2019

Polydatin and I-CBP112 protects early bovine embryo against nicotinamide-induced mitochondrial dysfunction.

Theriogenology 2019 Aug 9;134:1-10. Epub 2019 May 9.

Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, 52828, Gyeongnam Province, Republic of Korea; Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, 52828, Gyeongnam Province, Republic of Korea. Electronic address:

The mammalian Sirtuin family of seven enzymes, members of the NAD-dependent histone deacetylase family that modify histones via direct deacetylation, is involved in the regulation of many antioxidant and oxidative stresses. In the present study, we explored the effects of nicotinamide (NAM)-induced oxidative stress on the in vitro development of bovine embryos, on the acetylation of histone H3 lysine 56 (H3K56ac) and on expression of apoptosis-related genes. Treatment with NAM (10, 20 or 40 mM for 24, 48 or 196 h) during IVC resulted in significantly decreased blastocyst formation (24 h: 38.8 vs. 33.1, 27.3 and 10.2%, with P > 0.05, P < 0.05 and P < 0.01, respectively; 48 h: 37.5 vs. 28.2, 13.4 and 0%, with P < 0.05 and P < 0.01, respectively; 196 h: 35.8 vs. 23.4, 0 and 0%, with P < 0.05, respectively). Treatment with NAM (20 and 40 mM for 24 h) resulted in increased intracellular reactive oxygen species (ROS) levels in 2-cell and blastocysts, and apoptotic cell numbers in blastocysts and decreased mitochondrial membrane potential (ΔΨ) in 2-cell embryos (P < 0.05). Polydatin (PD) and I-CBP112 rescued the 20 mM NAM-induced embryo developmental defects and reduced ROS levels and apoptotic cell numbers in blastocysts (P < 0.05). The gene expression of NF-κB, COX2 and p53 was significantly increased in the NAM-treated group. Immunofluorescence analysis confirmed that the protein levels of nuclear factor-kappa B (NF-κB) decreased significantly after PD and I-CBP112 treatment compared with the control (P < 0.05). High level of H3K56ac induced by NAM was decreased after PD and I-CBP112 treatment (P < 0.05). These findings suggest that NAM treatment induces high levels of H3K56 acetylation that may be involved in oxidative stress-induced bovine developmental defects, which can be tolerated by PD and I-CBP112 treatment.
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http://dx.doi.org/10.1016/j.theriogenology.2019.05.007DOI Listing
August 2019

Effect of Predator Stress on the Reproductive Performance of Female Mice After Nonsurgical Embryo Transfer.

J Am Assoc Lab Anim Sci 2019 05 10;58(3):304-310. Epub 2019 Apr 10.

Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Gyeongnam, Korea;, Email:

Predator Stress Can Exert Detrimental Effects on Female Mammals, Leading to Disrupted Reproduction. Although Many Studies Have Addressed the Effects of Predator Stress on Reproductive Output in Rodents, Few Studies Have Focused on the Effect of Visual or Auditory Stress on Pregnant Females. in This Study, We Investigated the Possible Effect of Predator Stress, Either Visual Only or Combined Visual and Auditory (visual+auditory), on the Reproductive Performance of Female Mice After Nonsurgical Embryo Transfer. Reproductive Performance Was Assessed As Pregnancy Rate, Implantation Rate, Gestation Length, Live Pup Rate, and Neonatal Birth Weight. Moreover, Serum Cortisol and Progesterone Levels in Dams Were Measured by Using Electrochemiluminescence Immunoassay. Exposure to Predator (cat) Stress Did Not Lead to a Significant Change in Pregnancy Rates in the Tested Mice. However, the Stressed Mice Showed Significantly Decreased Implantation Rates Compared with the Control Group. Similarly, the Live Pup Rate and Neonatal Birth Weight Were Significantly Lower in the Group Exposed to Preda- Tor Stress Than in the Control Group. Furthermore, Mice Exposed to Visual+auditory Stress Showed a Significant Reduction in Gestation Length Compared with the Control Mice. Our Data Showed That Predator Visual+auditory Stress As Combined Stimuli Significantly Increased Serum Cortisol Level. in Contrast, Progesterone Levels Did Not Significantly Vary Among the Experimental Groups. Taken Together, Our Findings Imply That Predator Stress Adversely Affects the Reproductive Efficiency of Pregnant Mice By Decreasing the Implantation Rate, Live Birth Rate, and Neonatal Birth Weight and by Prolonging Gestation Length.
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http://dx.doi.org/10.30802/AALAS-JAALAS-18-000085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6526485PMC
May 2019

Improves the In Vitro Developmental Competence and Reprogramming Efficiency of Cloned Bovine Embryos by Additional Complimentary Cytoplasm.

Cell Reprogram 2019 02;21(1):51-60

1 Division of Applied Life Science (BK21 Plus), Gyeongsang National University, Jinju, Republic of Korea.

Somatic cell nuclear transfer (SCNT) is a useful technology; however, its efficiency is low. In this study, we investigated the effects of cytoplasmic transfer into enucleated oocytes on the developmental competence and quality of cloned preimplantation bovine embryos via terminal deoxynucleotidyl transferase dUTP nick-end labeling, quantitative reverse transcription PCR, and immunocytochemistry. We used cytoplasm injection cloning technology (CICT), a new technique via which the cytoplasmic volume of an enucleated oocyte could be restored by injecting ∼30% of the cytoplasm of a donor oocyte. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly higher (p < 0.05) in the CICT group than in the SCNT group (28.9 ± 0.8% vs. 20.2 ± 1.3%, respectively). Furthermore, the total cell number per day 8 blastocyst was significantly higher in the CICT group than in the SCNT group (176.2 ± 6.5 vs. 119.3 ± 7.7, p < 0.05). Moreover, CICT increased mitochondrial activity, as detected using MitoTracker Green. The mRNA levels of DNA methyltransferase 1 and DNA methyltransferase 3a were significantly lower (p < 0.05) in the CICT group than in the SCNT group. The mRNA level of DNA methyltransferase 3b was lower in the CICT group than in the SCNT group; however, this difference was not significant (p > 0.05). Taken together, these data suggest that CICT improves the in vitro developmental competence and quality of cloned bovine embryos.
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http://dx.doi.org/10.1089/cell.2018.0050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383574PMC
February 2019

Silver Nanoparticles Potentiates Cytotoxicity and Apoptotic Potential of Camptothecin in Human Cervical Cancer Cells.

Oxid Med Cell Longev 2018 12;2018:6121328. Epub 2018 Dec 12.

Division of Applied Life Science (BK21 Plus), Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Gyeongnam Province, Republic of Korea.

Silver nanoparticles (AgNPs) are widely used metal nanoparticles in health care industries, particularly due to its unique physical, chemical, optical, and biological properties. It is used as an antibacterial, antiviral, antifungal, and anticancer agent. Camptothecin (CPT) and its derivatives function as inhibitors of topoisomerase and as potent anticancer agents against a variety of cancers. Nevertheless, the combined actions of CPT and AgNPs in apoptosis in human cervical cancer cells (HeLa) have not been elucidated. Hence, we investigated the synergistic combinatorial effect of CPT and AgNPs in human cervical cancer cells. We synthesized AgNPs using sinigrin as a reducing and stabilizing agent. The synthesized AgNPs were characterized using various analytical techniques. The anticancer effects of a combined treatment with CPT and AgNPs were evaluated using a series of cellular and biochemical assays. The expression of pro- and antiapoptotic genes was measured using real-time reverse transcription polymerase chain reaction. The findings from this study revealed that the combination of CPT and AgNPs treatment significantly inhibited cell viability and proliferation of HeLa cells. Moreover, the combination effect significantly increases the levels of oxidative stress markers and decreases antioxidative stress markers compared to single treatment. Further, the combined treatment upregulate various proapoptotic gene expression and downregulate antiapoptotic gene expression. Interestingly, the combined treatment modulates various cellular signaling molecules involved in cell survival, cytotoxicity, and apoptosis. Overall, these results suggest that CPT and AgNPs cause cell death by inducing the mitochondrial membrane permeability change and activation of caspase 9, 6, and 3. The synergistic cytotoxicity and apoptosis effect seems to be associated with increased ROS formation and depletion of antioxidant. Certainly, a combination of CPT and AgNPs could provide a beneficial effect in the treatment of cervical cancer compared with monotherapy.
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http://dx.doi.org/10.1155/2018/6121328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311846PMC
February 2019

In vitro production of sex preselected cattle embryos using a monoclonal antibody raised against bull sperm epitopes.

Anim Reprod Sci 2019 Jun 16;205:156-164. Epub 2018 Nov 16.

Department of Animal Science, Division of Applied Life Science (BK21 Plus), Republic of Korea; Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 52828, Gyeongsangnam-do Province, Republic of Korea. Electronic address:

Sex preselection has always generated great interest among livestock producers. Among the prevalent sperm sorting methods, there is much evidence that sex sorting has a negative effect on sperm quality with an altered pattern of sperm motility, ultimately reducing the period of cell viability. In this study, we have established a new approach for the preselected embryo production by using WholeMom®; a monoclonal antibody developed against bull sperm epitopes for simple and easy separation of X- and Y-sperm. There were no significant differences (P >  0.05) in the percentage of presumptive zygotes between the control and the X-sperm sorted group, but there was a difference in early cleaving embryos with there being 81.2 ± 1.4%, 78.3 ± 1.0%, and 66.7 ± 1.1% for the control, X-sperm sorted, and Y-sperm sorted groups, respectively. Similarly, the percentage of embryos that developed to the blastocyst stage (Day 7) were also greater (P <  0.05) in the control and X-sperm sorted group compared with the Y-sperm sorted group being 34.8 ± 1.0%, 32.1 ± 0.8%, and 23.7 ± 1.0% in the control, X-sperm sorted, and Y-sperm sorted groups, respectively. Furthermore, B-SRY F2 and B-SRY R2 gene expression data indicated there was a detection accuracy of 81.0% for the female embryos and 72.5% for the male embryos produced in vitro. In conclusion, in cattle in vitro derived embryo production using pre-selected sexed semen and subsequent embryo transfer can facilitate the mass production of individuals that are genetically superior.
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http://dx.doi.org/10.1016/j.anireprosci.2018.11.006DOI Listing
June 2019
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