Validating hyper7 sensors for quantitative hydrogen peroxide measurement in bovine oocytes

Reactive Oxygen Species (ROS), particularly hydrogen peroxide (H2O2), are recognized for their role in inducing oxidative damage in oocytes, impacting critical biomolecules and oocyte quality. However, the physiological roles of H2O2 and other ROS during mammalian oogenesis are less understood, hindering advancements in fertility treatments and underscoring the need for tools to study ROS dynamics. This study aims to bridge this gap by taking advantage of a novel sensor capable of monitoring H2O2 fluctuations in real-time and surpassing traditional detection methods. Specifically, a novel experimental set-up employing Hyper7, an ultrasensitive genetically encoded fluorescent ratiometric sensor, was developed to directly quantify mitochondrial and cytosolic H2O2 in bovine oocytes. This sensor consists of a circularly permuted Yellow Fluorescence Protein (cpYFP) integrated into the OxyR domain from Neisseria meningitidis, which is sensitive to basal H2O2 concentrations. cpYFP has two excitation peaks at 405 and 499 nm and emits at 516 nm. Changes in the sensor oxidation state result in ratiometric changes with decreases in the excitation at 405 and increases at 499 nm upon oxidation. Thus, sequential excitation at 405 and 488 nm allows analysis of the F488/F405 ratio that serves as an index of H2O2 content. In our experiments, validating these sensors involved exposing bovine oocytes to a prooxidant challenge with 100 mM tert-Butyl hydroperoxide (t-BOOH). mRNAs encoding for the mitochondrial and the cytoplasmic-targeted Hyper7 sensors (Hyper7-MLS and Hyper7-NES, respectively) were obtained by in vitro transcription of the respective plasmids (Addgene). A total of 172 and 189 cumulus-oocyte complexes (COCs) were collected from abattoir-derived ovaries, freed from cumulus cells, and microinjected with Hyper7-MLS or Hyper7-NES mRNA. Diverging from the conventional In Vitro Maturation (IVM) protocol applied to COCs, a tailored protocol was adopted to align with specific research goals. These adjustments included denuding oocytes to facilitate microinjection, followed by a pre-IVM phase with 3-isobutyl-1-methyl-xanthine (IBMX) to delay meiotic resumption, critical for mRNA translation. The microinjected denuded oocytes (DO) were cocultured with intact COCs to support maturation. Finally, DOs were separated from the co-cultured COCs and imaged for 60 minutes, either under control IVM medium or ROS inducing conditions (IVM medium enriched with t-BOOH). Imaging was conducted using a Nikon ECLIPSE Ti2-E microscope with a Yokogawa W1-SoRa spinning disk, 405 and 488 nm lasers, and a multi-band filter. A high-resolution camera and a temperature-controlled CO2 chamber enabled time-lapse imaging. Exposures were set at 3 and 2 seconds for 405 and 488 nm, respectively, capturing emissions at 516 nm. Image analysis was performed using NIS ELEMENTS AR software. Both Hyper7-MLS and Hyper7-NES sensors demonstrated their ability to detect H2O2 levels within oocytes, revealing distinct fluorescence patterns. A significant increase in ratio values was observed after 20 minutes for Hyper7-MLS and 25 minutes for Hyper7-NES (Friedman test). This suggests phototoxicity as an exogenous causal factor during prolonged imaging. Nevertheless, oocytes treated with tBOOH exhibited significantly higher ratio values than those in the control group (two-way ANOVA), affirming the sensors' capacity to quantify elevated H2O2 levels. This validation step marks the first application of Hyper7 probes in mammalian oocytes, promising a novel tool for investigating the role of H2O2-mediated mechanisms in oocyte biology while posing the critical need to manage phototoxicity in subsequent research. Funded by H2020 MSCA-ITN-ETN n.860960 (EUROVA) and SEED2019 UNIMI N.1250 (cROSs-Talk)