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Kwon Jeongwoo,Seong Min-Jung,Piao Xuanjing,Jo Yu-Jin,김남형 아세아·태평양축산학회 2020 Animal Bioscience Vol.33 No.10
Objective: This study was conducted to investigate the roles of LIM kinases (LIMK1 and LIMK2) during porcine early embryo development. We checked the mRNA expression patterns and localization of LIMK1/2 to evaluate their characterization. We further explored the function of LIMK1/2 in developmental competence and their relationship between actin assembly and cell junction integrity, specifically during the first cleavage and compaction. Methods: Pig ovaries were transferred from a local slaughterhouse within 1 h and cumulus oocyte complexes (COCs) were collected. COCs were matured in in vitro maturation medium in a CO2 incubator. Metaphase II oocytes were activated using an Electro Cell Manipulator 2001 and microinjected to insert LIMK1/2 dsRNA into the cytoplasm. To confirm the roles of LIMK1/2 during compaction and subsequent blastocyst formation, we employed a LIMK inhibitor (LIMKi3). Results: LIMK1/2 was localized in cytoplasm in embryos and co-localized with actin in cell-to-cell boundaries after the morula stage. LIMK1/2 knockdown using LIMK1/2 dsRNA significantly decreased the cleavage rate, compared to the control group. Protein levels of E-cadherin and β-catenin, present in adherens junctions, were reduced at the cell-to-cell boundaries in the LIMK1/2 knockdown embryos. Embryos treated with LIMKi3 at the morula stage failed to undergo compaction and could not develop into blastocysts. Actin intensity at the cortical region was considerably reduced in LIMKi3-treated embryos. LIMKi3-induced decrease in cortical actin levels was attributed to the disruption of adherens junction and tight junction assembly. Phosphorylation of cofilin was also reduced in LIMKi3-treated embryos. Conclusion: The above results suggest that LIMK1/2 is crucial for cleavage and compaction through regulation of actin organization and cell junction assembly.
Roles of hnRNPA2/B1 in m6A RNA Modification during Mammalian Embryonic Development
JeongWoo Kwon,Suk Namgoong,Xiang-Shun Cui,Nam-Hyung Kim 한국수정란이식학회 2016 한국수정란이식학회 학술대회 Vol.2016 No.10
Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) have a important role in influence of pre-messenger RNA (pre-mRNA) processing and mRNA metabolism and transportation in cells. Recently, hnRNP A2/B1 can recognize m6A modifications on pre-mRNA or pre-miRNA and affect alternative splicing and miRNA processing in HeLa Cells. However, roles of hnRNP A2/B1 in various cells and tissues, especially in elary embryo development, are unclear. Here, we investigated the temporal and spatial expression patterns of hnRNPA2B13 during mammalian early embryo development. In mouse, hnRNPA2B1 was localized at the nucleus after 1-cell stage, however, hnRNPA2B1 was expressed after 2-cell stage in pig. Then, knockdown of hnRNP A2/B1 induced by RNA interference (RNAi) was used to analyze the effect of hnRNP A2/B1 in preimplantation develop in pigs. Knockdown of hnRNP A2/B1 delayed embryo development. Interestingly, ICM marker OCT4 and Sox2 was significantly decreased in blastocyst stage. mRNA expression show that transcription factors which is Pou5f1, Sox2, Nanog, Cdx2 and AP2γwas decreased the transcription levels without the changing of junction protein, ZO-1, occludin, and CXADR. Outgrowth results indicated that knock-down of hnRNPA2B1 embryos cannot format the colony. Knock-down of Methyltransferase like 3(METTL3) embryos mislocalized the hnRNPA2/B1 at the nucleus. In summary, the expression patterns of hnRNPA2/B1 differ between mouse and porcine embryos, and these differences may reflect species-specific functions during preimplantation embryo development. Our results suggested that hnRNPA2/B1 is necessary for newly synthesis of mRNA related with transcription factor, and early embryo development by the RNA epigenetic modification.
2i System Enhances Porcine Blastocyst Quality by Regulating of Epigenetic Modification
Jeongwoo Kwon,XingHui Shen,Jiang Hao,Yao Xuerui,Nam-Hyung Kim 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10
2i system is useful for maintenance of navie state of embryonic stem cells in various species. However, the role of 2i system in porcine preimplantation stage embryos unclear. In this study, we investigated the quality of blastocyst and pluripotent-related factor by 2i system. And, we also investigated about DNA and RNA epigenetic modification status by 2i system during porcine early embryo development. After treatment of MEK1/2 inhibitor PD0325901(4 μM), GSK-3 inhibitor CHIR99021(0.3 μM) and 2i (PD0325901+CHIR99021) during pre-implantation development, blastocyst formation rate had no significantly difference. However, blastocyst size increased in 2i treatment embryos. Blastocyst quality also increased after treatment of CHIR and 2i using TUNEL assay and Brdu assay. Interestingly, main transcription factor OCT4 and SOX2 positive cells in blastocyst was significantly increased in 2i groups. Especially, histone modification- relation proteins like h3k9me3 and h3k9ac changed after treatment of CHIR and 2i. Global gene expression patterns related with pluripotency and epigenetic also changed in inhibitor treatment groups. Furthermore, bi-sulfite sequencing results showed that 2i treatment groups demethylated in satellite I region. In conclusion, our findings strongly suggest that 2i system can be useful method for increasing of blastocyst quality by affecting of ICM/TE formation by regulating epigenetic modification during early embryo development.
M6A reader hnRNPA2/B1 is essential for porcine embryo development via gene expression regulation
Jeongwoo Kwon,Yu-Jin Jo,Seung-Bin Yoon,Hyeong-ju You,Changsic Youn,Yejin Kim,Jiin Lee,Nam-Hyung Kim,Ji-Su Kim 한국동물생명공학회(구 한국동물번식학회) 2022 Journal of Animal Reproduction and Biotechnology Vol.37 No.2
Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) is an N6-methyladenosine (m6A) RNA modification regulator and a key determinant of premRNA processing, mRNA metabolism and transportation in cells. Currently, m6A reader proteins such as hnRNPA2/B1 and YTHDF2 has functional roles in mice embryo. However, the role of hnRNPA2/B1 in porcine embryogenic development are unclear. Here, we investigated the developmental competence and mRNA expression levels in porcine parthenogenetic embryos after hnRNPA2/B1 knock-down. HhnRNPA2/B1 was localized in the nucleus during subsequent embryonic development since zygote stage. After hnRNPA2/B1 knock-down using double stranded RNA injection, blastocyst formation rate decreased than that in the control group. Moreover, hnRNPA2/B1 knock-down embryos show developmental delay after compaction. In blastocyste stage, total cell number was decreased. Interestingly, gene expression patterns revealed that transcription of Pou5f1, Sox2, TRFP2C, Cdx2 and PARD6B decreased without changing the junction protein, ZO1, OCLN, and CDH1. Thus, hnRNPA2/B1 is necessary for porcine early embryo development by regulating gene expression through epigenetic RNA modification.
Kwon, Jeongwoo,Park, Shuha,Seong, Min-Jung,Choi, Inchul,Kim, Nam-Hyung CSIRO Publishing 2019 Reproduction, fertility, and development Vol.31 No.2
<P> Cytoplasmic polyadenylation element binding protein (CPEB) is an RNA-binding protein that promotes elongation of poly(A) tails and regulates mRNA translation. CPEB depletion in mammary epithelium is known to disrupt tight-junction (TJ) assembly via mislocalisation of tight junction protein 1 (TJP1), but the role of CPEB in the biological functions associated with TJs has not yet been studied. The objective of this study was to investigate the roles of CPEB2 during porcine parthenote development. CPEB2 was detected in both the nuclei and apical cytoplasm at the 4- and 8-cell stages and was localised to cell-cell contact after the initiation of the morula stage. Its depletion led to retarded blastocyst formation caused by impaired TJ assembly. Moreover, transcription of TJ-associated genes, including TJP1, Coxsackie virus and adenovirus receptor (CXADR) and occludin (OCLN), was not affected, but the corresponding proteins were not properly localised at the apical cell membrane in morulae, suggesting that CPEB2 confers mRNA stability or determines subcellular localisation for translation. Remarkably reduced relative levels of TJP1 transcripts bearing the 3′-untranslated region were noted, indicating that CPEB2 mediates TJP1 mRNA stability. In conclusion, our findings demonstrate that because of its regulation of TJP1, CPEB2 is required for TJ assembly during porcine blastocyst development. </P>
Inhibition of MEK1/2 and GSK-3 Enhance Quality of Porcine Blastocyst Culturing In Vitro
Jeongwoo Kwon,Yujin Jo,XingHui Shen,Nam-Hyung Kim 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2
Inhibition of MEK1/2 and GSK-3(2i system) is useful for maintenance of navie state of embryonic stem cells in various species. However, the role of 2i system in porcine preimplantation stage embryos is not clear. In this study, we investigated the quality of blastocyst and pluripotent-related factors by 2i system and epigenetic modification status by 2i system during porcine early embryo development. Treatment of MEK1/2 inhibitor PD0325901 (4 uM), GSK-3 inhibitor CHIR99021 (0.3 uM) and 2i (PD0325901+ CHIR99021) did not enhanced the rate of blastocyst formation. However, the size of blastocyst was increased in 2i treated embryos. TUNEL assay showed that CHIR- 99021 and 2i treatment enhanced blastocyst quality. Furthermore, 2i treated blastocyst increased OCT4 and SOX2 positive cells and decreased GATA4 positive cells. Methylation of histone H3 lysin trime3 (h3k9me3) were reduced, and histone H3 deacetylaton (h3k9ac) were increased in 2i treated embryos. Furthermore, bi-sulfite sequencing results showed that 2i treatment groups demethylated in satellite I region. And, RNA methylatransferase protein METTL3 was increased while demethylase FTO was increased after treatment of 2i. In conclusion, our findings strongly suggest that 2i system can be useful method for increasing of blastocyst quality by affecting of ICM/TE formation by regulating epigenetic modification during early embryo development.
최정우(Jeongwoo Choi),권준화(Junhwa Kwon),한태욱(Tae Uk Han),김영란(Younglan Kim),강준구(Jun- Gu Kang),전태완(Tae-wan Jeon) 한국환경에너지공학회 2022 한국열환경공학회 학술대회지 Vol.2022 No.2
우리나라는 급격한 산업화와 코로나19 등으로 인하여 일회용 플라스틱 사용이 급속하게 증가하고 있다. 이에 따라 폐플라스틱의 발생량 또한 급격하게 증가하였으며, 환경 친화적인 재활용 및 처분방안이 요구되고 있다. 폐폴라스틱을 재활용하는 방법에는 물질 재활용과 화학적 재활용이 있다. 이 중 물질 재활용은 파쇄 및 세 척 등이 해당하며, 오염된 플라스틱의 경우 재활용하기에 어려움이 있다. 반면 화학적 재활용의 경우 플라스틱의 화학결합을 분해하는 기술로 폐폴라스틱 처리와 동시 에 생산부산물을 연료 또는 원료로 재활용할 수 있다는 장점 이 있다. 특히 여러 화학적 재활용 중 열분해 에 대한 연구가 활발하게 이루어 지고 있다. 열분해란 고분자 폐 기 물을 무산소 또는 회박산소 조건에서 간접 열을 가해 고분자 내에 존재하는 화학결합을 끊어 열분해 오일, 가스, 촤를 생산하는 기술이다. 현재 우리나라 열분해 공정은 대부분 회분식 설비이며, 업체마다 운전 조건과 공정 이 다양하다. 향후 열분해 공정 에 대한 기술력과 열분해유 품질을 단계적으로 높이기 위해선 공정별로 생산된 열분해유에 대한 기초분석 이 필요하다. 따라서 본 연구에서는 열분해 시설별 열분해유의 인화점, 잔류탄소 및 공업분석을 수행하여 열분해유의 기본 특성을 파악하고자 한다.