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LIMK1/2 are required for actin filament and cell junction assembly in porcine embryos developing in vitro

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...

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Autores principales: Kwon, Jeongwoo, Seong, Min-Jung, Piao, Xuanjing, Jo, Yu-Jin, Kim, Nam-Hyung
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Asian-Australasian Association of Animal Production Societies (AAAP) and Korean Society of Animal Science and Technology (KSAST) 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463081/
https://www.ncbi.nlm.nih.gov/pubmed/32054159
http://dx.doi.org/10.5713/ajas.19.0744
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author Kwon, Jeongwoo
Seong, Min-Jung
Piao, Xuanjing
Jo, Yu-Jin
Kim, Nam-Hyung
author_facet Kwon, Jeongwoo
Seong, Min-Jung
Piao, Xuanjing
Jo, Yu-Jin
Kim, Nam-Hyung
author_sort Kwon, Jeongwoo
collection PubMed
description 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.
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spelling pubmed-74630812020-10-01 LIMK1/2 are required for actin filament and cell junction assembly in porcine embryos developing in vitro Kwon, Jeongwoo Seong, Min-Jung Piao, Xuanjing Jo, Yu-Jin Kim, Nam-Hyung Asian-Australas J Anim Sci Article 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. Asian-Australasian Association of Animal Production Societies (AAAP) and Korean Society of Animal Science and Technology (KSAST) 2020-10 2020-01-13 /pmc/articles/PMC7463081/ /pubmed/32054159 http://dx.doi.org/10.5713/ajas.19.0744 Text en Copyright © 2020 by Asian-Australasian Journal of Animal Sciences This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Article
Kwon, Jeongwoo
Seong, Min-Jung
Piao, Xuanjing
Jo, Yu-Jin
Kim, Nam-Hyung
LIMK1/2 are required for actin filament and cell junction assembly in porcine embryos developing in vitro
title LIMK1/2 are required for actin filament and cell junction assembly in porcine embryos developing in vitro
title_full LIMK1/2 are required for actin filament and cell junction assembly in porcine embryos developing in vitro
title_fullStr LIMK1/2 are required for actin filament and cell junction assembly in porcine embryos developing in vitro
title_full_unstemmed LIMK1/2 are required for actin filament and cell junction assembly in porcine embryos developing in vitro
title_short LIMK1/2 are required for actin filament and cell junction assembly in porcine embryos developing in vitro
title_sort limk1/2 are required for actin filament and cell junction assembly in porcine embryos developing in vitro
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463081/
https://www.ncbi.nlm.nih.gov/pubmed/32054159
http://dx.doi.org/10.5713/ajas.19.0744
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