l-carnitine reduces the adverse effects of ROS and up-regulates the expression of implantation related genes in in vitro developed mouse embryos

Abstract

In vitro developed embryos are inevitably exposed to various reactive oxygen species (ROS) which may decrease the embryo's competence in assisted reproductive technology (ART) procedures. Optimization of embryo culture media using antioxidant agents could help to improve embryo quality and could overcome failures in current ART. The aim of this study was to evaluate the effects of l-carnitine (LC), an enhancer of mitochondrial activity and free radical scavenger, in culture media on early embryo competence and expression of ErbB1 and ErbB4 implantation related genes. Two-cell mouse embryos were cultured in the following four conditions: 1. LC group in media containing LC; 2.H ₂O₂ group exposed to H₂O₂ for 30 min and then transferred into a simple media; 3.H₂O₂+LC group exposed to H₂O₂ for 30 min and then transferred into a simple media containing LC; 4.the control group kept throughout in simple media. All groups were allowed to develop until the blastocyst stage. ErbB1 and ErbB4 expression were evaluated by Real-time PCR and immunocytochemistry. The expression of Sirt3 gene was also evaluated. Intracellular ROS levels were examined by DCFH-DA fluorescence intensity. In order to assess the morphological quality of the embryos, ICM and OCM number blastocyst cells were evaluated by using Hoechst and propidium iodide (PI) staining. ErbB1, ErbB4, ROS levels and cell number were compared across all in vitro groups. Our data reveal that LC significantly increases ErbB1 and ErbB4 gene and protein expression with intracellular ROS levels and Sirt3 gene expression significantly decreased after LC treatment. It is worth noting that an elevated cell number was observed in the LC-treated group compared with the other groups suggesting increased viability and/or proliferation. Our findings suggest that the use of LC could be helpful to improve preimplantation embryo culture media through its effects in decreasing ROS levels and the increase of implantation-related genes.

Keywords: Embryonic implantation genes; Gene expression; L-carnitine; ROS.