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 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 ß-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.

Kaempferol attenuates mitochondrial dysfunction and oxidative stress induced by H2O2 during porcine embryonic development.

Kaempferol (3,4',5,7-tetrahydroxyflavone, KAE) is one of the most commonly occurring dietary flavonols. The biological and pharmacological effects of kaempferol depend upon whether it acts as an antioxidant, anti-inflammatory, or anticancer agent. The present study explored the influence of KAE supplementation on in vitro damage to porcine oocytes and its underlying mechanisms. Different concentrations of KAE (0.05, 0.1, 0.5, 1 µM) were added to porcine zygote medium 5 during in vitro culture. The results showed that supplementation with 0.1 µM KAE significantly increased the blastocyst formation rate. Blastocyst formation and quality were significantly increased in the 200 µM H2O2 treatment group following addition of 0.1 µM KAE. KAE prevented the H2O2-induced compromise of mitochondrial membrane potential and reactive oxygen species generation. Furthermore, the extent of autophagy and DNA damage in the blastocysts was attenuated by supplementation with KAE in the H2O2 induced oxidative injury group compared to that observed in controls. These results suggest that KAE has beneficial effects on the development of porcine parthenotes by attenuating oxidative stress and increasing mitochondrial function.