Background

Fertilization triggers embryonic genome activation, whereby most maternal transcripts and proteins are degraded, followed by the generation of a new transcriptome and resulting proteome (Gao et al., 2017; Svoboda, 2018). These processes are required for the transition from maternal to embryonic control of development and create a cellular environment conducive of the totipotent state of the early embryo. There has been extensive analysis and development of tools for monitoring the changes to transcription at this time, but changes in translation have received less attention. New protein synthesis during the process of maternal to embryonic transition is tightly regulated and can be quantitatively evaluated with several methods, such as ³⁵S-Methionine (Crosby et al., 1988), two-dimensional gel electrophoresis (Latham et al., 1991), and mass spectrometry (Gao et al., 2017). Here we report the development and use of a rapid alternative method for the analysis of global changes in the level of protein synthesis in the early embryo. The Click-iT^® Plus OPP Protein Synthesis Assay is a non-toxic and non-radioactive method for the detection of nascent protein synthesis utilizing fluorescence microscopy and high-throughput imaging. O-propargyl-puromycin (OPP) is a membrane-permeable alkyne puromycin analog that forms covalent bonds with nascent polypeptide chains with cells. The addition of the Alexa Fluor^® picolyl azide and the Click reaction reagents leads to a chemoselective ligation or “click” reaction between the picolyl azide dye and the alkyne OPP, and the modified proteins are detected by imaged-based analysis. The assay has been demonstrated to preserve cell morphology and has been used in NIH3T3, HeLa, C2C12 cells, BPAE, U-2 OS, CHO-M1, and A549 (Mateu-Regue et al., 2019; Enam et al., 2020; Liu et al., 2012), and the embryonic cells of preimplantation development (Li et al., 2021).

This protocol is designed to semi-quantitatively assess the relative changes in protein synthesis during the period of mouse maternal to embryonic transition. The first stage is to test and optimize the concentration of the OPP, then confirm the specificity of the assay by use of the known protein synthesis inhibitor Cycloheximide and inhibitor of translation elongation, 4EGI-1 (eIF4E inhibitor) (Li et al., 2021).