DISSECTING THE ROLE OF REACTIVE OXYGEN SPECIES IN EARLY MAMMALIAN EMBRYOGENESIS

Reactive oxygen species (ROS) comprise a broad spectrum of molecules that are known to induce oxidative stress (OS) in both gametes and embryos. OS arises when ROS production exceeds a critical threshold, or when intracellular antioxidant systems are unable to neutralize them effectively. OS is a key factor impairing embryo quality during the processes of oocyte maturation, fertilization, and early embryonic development in vitro. However, ROS also play essential roles as regulators of physiological processes, leading to the widely accepted hypothesis that excessive suppression of ROS could negatively affect cellular function. This is particularly relevant in cattle, where assisted reproductive technologies increasingly depend on in vitro production (IVP). A deeper understanding of the specific roles of different ROS is required to optimize culture systems and enhance reproductive efficiency. One major limitation in this research area is the absence of molecular tools to quantitatively monitor ROS levels over time. To address this issue, we utilized novel probe called HyPer7, an ultrasensitive fluorescent ratiometric sensor capable of tracking real-time H₂O₂ fluctuations within cellular sub-compartments, surpassing traditional methodologies. The HyPer7 sensor has been used in Xenopus models to demonstrate that fertilization-induced calcium signaling triggers mitochondrial H₂O₂ production, which subsequently activates the cell cycle (Han et al., 2018). In our study, we utilized HyPer7-MLS mRNA encoded to the mitochondrial matrix, which was microinjected into immature oocytes (GV) and in vitro matured oocytes (MII). These microinjected oocytes were then matured or fertilized in vitro. To assess the sensor’s ability to detect H₂O₂ with high spatial and temporal resolution, samples were imaged under control conditions and following exposure to a prooxidant challenge. Image acquisitions were conducted at the NOLIMITS UNITECH microscopy facility at the University of Milan, using a Nikon-Eclipse Ti2-E with Yokogawa W1- SoRa spinning disk confocal microscope equipped with a temperature-controlled CO₂ chamber and appropriate lasers and filters at 30-second intervals. Our image analysis confirmed that HyPer7 successfully detects H₂O₂ in both oocytes and zygotes. However, under control conditions, a significant increase in signal was observed after 20 minutes imaging, suggesting that phototoxicity may contribute to this rise during the image acquisition procedure. Nevertheless, both oocytes and zygotes exposed to a prooxidant (tBOOH) exhibited higher H₂O₂ levels than the control group, validating the sensor's capacity to quantify increased H₂O₂ concentrations. When comparing H₂O₂ content between oocytes and zygotes our results showed no significant difference under standard culture conditions. However, upon exposure to the prooxidant, oocytes demonstrated nearly doubled H₂O₂ levels compared to zygotes. This result raises important biological questions regarding the distinct regulation of redox balance, particularly whether antioxidant mechanisms are more robust in zygotes than in oocytes. We then explored the hypothesis that increasing concentrations of an antioxidant cocktail could serve as a model for suppressing mitochondrial H₂O₂, providing a tool to study the role of mt-H₂O₂ in zygotes. Fertilized zygotes were exposed for six hours to either standard culture conditions or an antioxidant mixture at concentrations 10, 100, and 1000 times higher than those previously shown to reduce oxidative stress (Truong et al., 2016; Truong & Gardner, 2017). The zygotes were then cultured for seven days. Our analysis revealed that higher concentrations of antioxidants impaired blastocyst development. However, Contrary to our hypothesis, increasing antioxidant levels did not reduce mt-H₂O₂ but instead increased its production, as assessed by means of the Hyper7 sensor. Therefore, the observed reduction in embryonic developmental competence is likely not due to mt-H₂O₂ suppression but rather their increased production due to the paradoxical effect of high antioxidant concentrations. In conclusion, we established a method to quantitatively measure mitochondrial H₂O₂ in bovine oocytes and zygotes using the HyPer7 sensor, offering a valuable tool for further elucidating its role of mt-H₂O₂ during oocyte maturation and early embryogenesis. However, additional efforts are necessary to optimize methods and reduce phototoxicity, enabling longer acquisition times with the HyPer7 sensor to monitor ROS fluctuations throughout the critical stages of oocyte maturation and early embryo development and use of specific inhibitors or the genetic engineering techniques to suppress the mt-H₂O₂ levels and elucidate their role in the early embryogenesis and the oocyte maturation.