Effect of high hydrostatic pressure on mitochondrial activity, reactive oxygen species level and developmental competence of cultured pig embryos

Abstract

High hydrostatic pressure (HHP) has been previously used to increase mammalian oocyte and embryo tolerance on subsequent stresses related with different assisted reproductive technologies. Nevertheless, the mechanisms for HHP-induced stress responses in early embryos have not been yet well understood. Previous studies focused mainly on HHP-modified gene expression while possible changes in cellular functions, including modification of energy metabolism and oxidative stress were neglected. Therefore, we aimed to analyze the effect of HHP treatment on the efficiency of subsequent in vitro pig embryos culture in NCSU-23 medium, on mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) level during their pre-implantation development. Porcine embryos were exposed to the hydrostatic pressure of 20 MPa and their quick response to such stress was analyzed 1 h later. In comparison with control embryos, we detected lower ΔΨm by ∼13% only in expanded blastocysts as well as decreased ROS level by ∼30% and ∼42% at the morula and expanded blastocyst stages, respectively. After HHP-treatment at transcriptionally inactive zygote stage and subsequent embryo culture, long-time responses were found: (1) at expanded blastocyst stage manifesting by ΔΨm decrease by ∼16%, (2) at the morula and expanded blastocyst stages in the form of ROS level reduction by ∼38% and ∼33% respectively. Following HHP stress applied at the transcriptionally active morula stage the long-time response in the expanded blastocysts as a decrease of ΔΨm by ∼19% and ROS level by ∼37% was observed. The percentage of obtained expanded blastocysts was higher after culture of HHP-treated zygotes in comparison to the control. Moreover, expanded blastocysts developed in vitro from both HHP-treated zygotes or morulae, exhibited higher total number of cells per blastocyst, higher number of cells in the inner cell mass as well as lower number of TUNEL-positive nuclei per blastocyst and lower TUNEL index, when compared to untreated embryos. Therefore, the HHP stress applied at the zygote stage, enhances developmental potential and quality of in vitro obtained porcine blastocysts due to the both decreased ΔΨm and ROS level. Our findings may contribute to better understanding of the mechanism of HHP-mediated modifications of energy metabolism and oxidative stress during in vitro development of pig embryos.