Thiamethoxam Effects the Quality of Blastocyst via Activation of Ros-induced DNA Damage Checkpoint in Pigs

Zheng-Wen Nie,Ying-Jie Niu,Wenjun Zhou,Kyung-Tae Shin,Yong-Han Kim,Xiang-Shun Cui 한국동물생명공학회(구 한국동물번식학회) 2018 발생공학 국제심포지엄 및 학술대회 Vol.2018 No.06

Thiamethoxam Induces Meiotic Arrest and Affects Embryo Developmental Potential of Bovine Oocytes

Zheng-Wen Nie,Ying-Jie Niu,Wenjun Zhou,Yong-Han Kim,Kyung-Tae Shin,Xiang-Shun Cui 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

Thiamethoxam (TMX) is a neonicotinoid insecticide. Residues of TMX have been detected in various crops. Although it has specific high toxicity to insects and is designed to exterminate them, the toxicity has also found in mammals recently. Differ from acetylcholine toxicity, TMX has peroxide toxicity in mammals. Matured oocytes have the capacity of fertilization, but oocytes own abundant mitochondria and its maturation is vulnerable to reactive oxygen species (ROS). Excessive production of reactive oxygen species (ROS) can override antioxidant defenses, produce oxidative stress and DNA damage that triggers apoptosis and necrosis in organisms. However little is known about the harm of ROS induced by TMX during oocytes maturation. Here, bovine germ-vesicle (GV) oocytes were cultured to metaphase of the second meiosis (MII) stage in vitro with or without TMX. During this process, oocytes were evaluated by various methods. Microscopic examination showed that 1.6 mM TMX significantly inhibited the maturation process in which oocytes were arrested before MI stage or between MI and MII stage. Correspond to this two periods, immunofluorescence staining and enzyme activity analysis showed that active CDC25 and CDC2 reduced in TMX group compared to control; time lapse and immunofluorescence staining gave results that Cyclin B could not be degraded, actin cap could not form, and Bub3 could not be removed from kinetochores. In addition, MII oocytes exposed to TMX showed disordered chromosomes and spindle. To study further, oocytes cultured for 24 h were analyzed. On the one hand, these oocytes in TMX group accumulated more ROS and produced significantly decreased mitochondrial membrane potential and increased apoptotic signal compared to control by methods of quantities for dichlorodihydrofluorescein diacetate (DCHFDA), 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide and Annexin-V, but the level of γH2AX protein in TMX group did not decline significantly compared with control. On the another hand, these oocytes were activated to be parthenogenetic embryos and cultured. Assessment for embryo development showed decreased rates of cleavage, morula and blastocyst in TMX group compared to control in vitro. In conclusion, these results suggest that ROS induced by TMX results in dysfunction of mitochondria and apoptosis, which block bovine oocytes to MI stage, trap them at AI/TI stage and trigger disordered chromosomes and spindle at MII stage. Additionally, MII oocytes with poor qualities result from TMX lose abilities to cleavage and develop to be morulae and blastocysts.

Function and Regulation Mechanism of Cyclin E1 during Development of Early Porcine Embryos

Zheng-Wen Nie,Jing Guo,Duk-Jung Kim,Nam-Hyung Kim,Xiang-Shun Cui 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10

Cyclin E1 (CCNE1) is a core component of cell cycle regulation that drives the transition into the S phase. CCNE1 plays critical roles in cell cycle, cell proliferation, and cellular functions. However, the function of CCNE1 in early embryonic development is limited. In the present study, the function and expression of Ccne1 in porcine early parthenotes were examined. Immunostaining experiments showed that CCNE1 localized in the nucleus, starting at the four-cell stage. Knockdown of Ccne1 by double-stranded RNA resulted in the failure of blastocyst formation and induced blastocyst apoptosis. Ccne1 depletion increased expression of the pro-apoptotic gene Bax, and decreased the expression of Oct4 and the rate of inner cell mass (ICM)/trophectoderm formation. The results indicated that CCNE1 affects blastocyst formation by inducing cell apoptosis and ICM formation during porcine embryonic development.

Overexpression of MicroRNA-29b Decreases Expression of DNA Methyltransferases and Improves Quality of the Blastocysts Derived from Somatic Cell Nuclear Transfer in Cattle

Liang, Shuang,Nie, Zheng-Wen,Guo, Jing,Niu, Ying-Jie,Shin, Kyung-Tae,Ock, Sun A.,Cui, Xiang-Shun Cambridge University Press 2018 Microscopy and Microanalysis Vol.24 No.1

<B>Abstract</B><P>MicroRNA (miR)-29b plays a crucial role during somatic cell reprogramming. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos, as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared with <I>in vitro</I> fertilized embryos. In addition, miR-29b regulates the expression of DNA methyltransferases (<I>Dnmt3a/3b</I> and <I>Dnmt1</I>) in bovine SCNT embryos. We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency and downregulation inhibits developmental potency. Nevertheless, the quality of bovine SCNT embryos at the blastocyst stage improved significantly. The expression of pluripotency factors and cellular proliferation were significantly higher in blastocysts from the miR-29b overexpression group than the control and downregulation groups. In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and downregulation groups. Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.</P>

Nuclear Transportation of Pyruvate Dehydrogenase Controls the Zygotic Genome Activation in Pig

Wenjun Zhou,Ying-Jie Niu,Zheng-Wen Nie,Kyung-Tae Shin,Yong-Han Kim,Xiang-Shun Cui 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

The porcine zygotic genome activation occurs along with global epigenetic remolding at the 4-cell stage. The histone acetylation, regulating DNA transcription, replication and so on, requires adequate acetyl-CoA. Acetyl-CoA produced by translocated pyruvate dehydrogenase in the nucleus of mammalian cells has been reported, which is commonly considered locating in the mitochondria. To find out whether the nuclear pyruvate dehydrogenase regulating the histone acetylation by controlling generation of acetyl-CoA, a multiple sgRNAs-CRISPR/Cas9 targeting strategy was employed to generate a pyruvate dehydrogenase E1 alpha1 (Pdha1) knockout (KO) parthenogenetic embryo model. Results showed that the targeting efficiency of Pdha1 reached more than 90%. Hence, this model was used in the subsequent experiments. Furthermore, a translocation of Pdha1 during zygotic genome activation was found by immunofluorescent staining and was significantly inhibited by Pdha1 KO. Meanwhile, the 8-cell stage embryo rate significantly decreased after 72 h (24.19% vs 12.53%, control vs Pdha1 KO), indicating a 4-cell arrest. In addition, the nuclear histone acetylation level significantly decreased when Pdha1 was KO. To determine whether the zygotic genome transcription was affected, the qPCR was performed and showed that the mRNA level of Eif1A, Acly, Sqle and Pdha1 all dropped significantly in the Pdha1 KO group compared to the control. In conclusion, the translocated Pdha1 generates acetyl-CoA for histone acetylation inside the nucleus of porcine embryos, which promotes the zygotic genome activation of porcine embryos.

Nitric Oxide-induced Protein S-nitrosylation Causes Mitochondrial Dysfunction and Accelerates Post-ovulatory Aging of Oocytes in Cattle

Niu, Ying-Jie,Zhou, Dongjie,Zhou, Wenjun,Nie, Zheng-Wen,Kim, Ju-Yeon,Oh, YoungJin,Lee, So-Rim,Cui, Xiang-Shun The Korean Society of Animal Reproduction and Biot 2020 한국동물생명공학회지 Vol.35 No.1

Nitric oxide (NO)-induced protein S-nitrosylation triggers mitochondrial dysfunction and was related to cell senescence. However, the exact mechanism of these damages is not clear. In the present study, to investigate the relationship between in vitro aging and NO-induced protein S-nitrosylation, oocytes were treated with sodium nitroprusside dihydrate (SNP), and the resultant S-nitrosylated proteins were detected through biotin-switch assay. The results showed that levels of protein S-nitroso thiols (SNO)s and expression of S-nitrosoglutathione reductase (GSNOR) increased, while activity and function of mitochondria were impaired during oocyte aging. Addition of SNP, a NO donor, to the oocyte culture led to accelerated oocyte aging, increased mitochondrial dysfunction and damage, apoptosis, ATP deficiency, and enhanced ROS production. These results suggested that the increased NO signal during oocyte aging in vitro, accelerated oocyte degradation due to increased protein S-nitrosylation, and ROS-related redox signaling.

The Function of CX 43 in Porcine Embryos

Kyung-Tae Shin,Ying-Jie Niu,Zheng-Wen Nie,Wenjun Zhou,Yong-Han Kim,Xiang-Shun Cui 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

Connexin 43 (Cx43) is one of the gap junction proteins which are compounds of transmembrane proteins and transports the small-molecular-weight chemicals up to 2 kDa. Lacking of Cx43 influences the junctional protein, induces autophagy and apoptosis in somatic cells. However, the function of Cx43 in porcine early embryos is still unknown. Aim to find out the molecular mechanism of Cx43 on the developmental competence in early porcine embryos, Cx43 dsRNA (1 ㎍/㎕) was microinjected into the parthenogenetically activated porcine zygotes. Blastocyst rate (treatment, 8.8±1.6% vs. control, 38.6±4.3%) and total cell numbers in the blastocyst (treatment, 20.7±3.5 vs. control, 39.8±4.1) were significantly reduced following Cx43 knocking down. Results from FITC-dextran and Western blot assay show that knock down (KD) of Cx43 significantly increased membrane permeability through down regulation of genes which are component of both adherence and tight junction in the porcine blastocyst. Reactive oxygen species (ROS) was significantly increased in the Cx43 KD group compared to control. In addition, KD of Cx43 activated Caspase 3 and significantly increased ATG8 expression, induced autophagy and apoptosis. Results suggest that KD of Cx43 influences preimplantation porcine embryo development via increasing membrane permeability and ROS generation, and inducing autophagy and apoptosis.

Melatonin enhances mitochondrial biogenesis and function in porcine preimplantation embryos

Ying-Jie Niu,Wenjun Zhou,Zheng-Wen Nie,Kyung-Tae Shin,Yong-Han Kim,Xiang-Shun Cui 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

Melatonin (N-aceyl-5-methoxytryptamine) is the major hormone of the pineal gland. Melatonin and its metabolic derivatives possess extensive free-radical scavenging abilities and played critical roles in antioxidative stress, resisting apoptotic cell death. Melatonin also could enhance mitochondrial biogenesis in rats with carbon tetrachloride-induced liver fibrosis. In addition, melatonin attenuates myocardial ischemia/reperfusion injury by reducing oxidative stress damage via activation of SIRT1 signaling in a melatonin receptor 2-dependent manner. Activation or overexpression of SIRT1 could enhance mitochondrial biogenesis and function by inducing PGC-1α expression and deacetylation. The aim of this study was to investigate if melatonin enhances mitochondrial biogenesis and function via activation of melatonin receptor 2/SIRT1/PGC1-α Pathway. The results showed that Melatonin rescued rotenone-induced impairment of porcine embryo development. Treatment with rotenone could increase oxidative stress and apoptosis. Rotenone impaired mitochondrial functions by disrupting mitochondrial membrane potential, reducing mitochondrial DNA copy number and ATP production. Melatonin could improve SIRT1 and PGC-1α expression, inducing mitochondrial biogenesis. Rotenone-induced mitochondrial dysfunction and ATP deficiency was rescued by melatonin treatment, the oxidative stress and apoptosis was significantly decreased. Inhibition of melatonin receptor 2 or Knockdown of SIRT1 abolished the protective effects of melatonin on rotenone-induced impairments. Therefore, melatonin enhanced mitochondrial biogenesis and function, protected against rotenone-induced impairments.

Nitric Oxide-induced Protein S-nitrosylation Causes Mitochondrial Dysfunction and Accelerates Post-ovulatory Aging of Oocytes in Cattle

Ying-Jie Niu,Dongjie Zhou,Wenjun Zhou,Zheng-Wen Nie,Ju-Yeon Kim,YoungJin Oh,So-Rim Lee,Xiang-Shun Cui 한국동물생명공학회(구 한국동물번식학회) 2020 Journal of Animal Reproduction and Biotechnology Vol.35 No.1

Nitric oxide (NO)-induced protein S-nitrosylation triggers mitochondrial dysfunction and was related to cell senescence. However, the exact mechanism of these damages is not clear. In the present study, to investigate the relationship between in vitro aging and NO-induced protein S-nitrosylation, oocytes were treated with sodium nitroprusside dihydrate (SNP), and the resultant S-nitrosylated proteins were detected through biotin-switch assay. The results showed that levels of protein S-nitroso thiols (SNO)s and expression of S-nitrosoglutathione reductase (GSNOR) increased, while activity and function of mitochondria were impaired during oocyte aging. Addition of SNP, a NO donor, to the oocyte culture led to accelerated oocyte aging, increased mitochondrial dysfunction and damage, apoptosis, ATP deficiency, and enhanced ROS production. These results suggested that the increased NO signal during oocyte aging in vitro, accelerated oocyte degradation due to increased protein S-nitrosylation, and ROS-related redox signaling.

Fipronil Induces Apoptosis during Oocyte Meiotic Maturation in Pigs

Wenjun Zhou,Ying-Jie Niu,Zheng-Wen Nie,,Yong-Han Kim,Kyung-Tae Shin,Xiang-Shun Cui 한국동물생명공학회(구 한국동물번식학회) 2018 발생공학 국제심포지엄 및 학술대회 Vol.2018 No.06