HyPer-7 sensor: a novel tool for unveiling the role of hydrogen peroxide in bovine zygotes

Despite being viewed as detrimental to cellular processes, hydrogen peroxide is an essential signaling molecule in biological processes, including early embryogenesis in Xenopus, where the interplay between calcium and mitochondrially produced H2O2 (mt-H2O2) mediates early cleavage. However, the dynamics of H2O2 fluctuation and its role in the mammalian zygote remain largely unexplored owing to the absence of imaging techniques to monitor H2O2 fluctuation in living embryos and the challenge of experimentally suppressing its production during culture. To address this issue, we first optimized a protocol to detect basal mt-H2O2 levels in bovine zygotes using a novel ratiometric sensor consisting of a mitochondrially targeted fluorescent protein called HyPer7 (Hyper7-MLS). This sensor utilizes sequential excitation to 405 and 488 nm wavelengths and emission peaks at 516 nm. As HyPer7 undergoes oxidation by H2O2, F405 intensity diminishes while F488 intensifies, resulting in an F488/F405 ratio that serves as an index of H2O2 content. The mRNA encoding Hyper7-MLS was produced by in vitro transcription of the pCS2+MLS-HyPer7 plasmid (Addgene). Cumulus oocyte complexes (COCs) were collected from abattoir-derived ovaries, cultured under meiotic arrest conditions for 3 hours to synchronize the onset of meiotic resumption, and then matured for 22 hours. In vitro matured oocytes were then freed of cumulus cells and microinjected with Hyper7-MLS mRNA. Oocytes were co-incubated with sperm for 10 hours in the presence of intact in vitro matured COCs. Zygotes were imaged for up to 1 hour using a Nikon-Eclipse Ti2-E with Yokogawa W1-SoRa spinning disk microscope equipped with a CO2 chamber using the appropriate Hyper7 settings. Subsequently, the ratio of fluorescent signal intensities was calculated after background subtraction using the NIS-Elements software (Nikon). The sensor's ability to detect increasing H2O2 levels was initially confirmed by comparing the Hyper7-MLS signal ratio in 154 zygotes, divided into control and tert-butyl hydroperoxide (t-BOOH)-treated groups. t-BOOH served as a pro-oxidant stimulus to induce mt-H2O2 production. Then, we tested the hypothesis that increasing concentrations of an antioxidant cocktail could serve as an experimental model to suppress mt-H2O2, thus providing a tool to assess the role of mt-H2O2 in zygotes. A total of 370 in vitro matured oocytes were fertilized, separated from the co-cultured COCs, and exposed for 6 hours to standard culture conditions or a mix of acetyl-L-carnitine, N-acetyl-L-cysteine, and α-lipoic acid at 10, 100 and 1000 times the concentration previously reported to reduce oxidative stress. The zygotes were then cultured for 7 days. Data analysis revealed that increasing antioxidant concentrations impaired blastocyst development with significant effects starting at 100X concentration (One-way ANOVA followed by Tukey’s test, p<0.05). To further assess whether the 100X antioxidant mix reduced mt-H2O2, 35 and 30 zygotes expressing Hyper7-MLS were cultured under control or 100X antioxidant condition, respectively, and imaged. Contrary to our hypothesis, increasing the concentration of antioxidants did not reduce mt-H2O2 but significantly increased its production (Two-way ANOVA followed by Šidák's test). Hence, the reduced embryonic developmental competence is not due to suppression of mt-H2O2, but rather to some paradoxical effect of antioxidants in high concentrations. In conclusion, we established a method to quantitatively measure mt-H2O2 in bovine zygotes that could serve as a tool to further elucidate its function during early embryogenesis. Additional efforts are needed to optimize methods to modulate its production. Funded by SEED2019 UNIMI No.1250 (cROSs-Talk) and RL PSR2014-2020 No.202102146691 (R-INNOVA).