In this context, oxidation conditions were created with the objective of utilizing H2O2 as an exogenous source of reactive oxygen species (ROS) to investigate the factors that influence the delivery of DNA to sperm. The results showed an increase in membrane permeability, including enhanced permeability to DNA. The process involves internalizing DNA into the sperm cytoplasm and subsequent transfer of DNA to the nucleus. Sperm cells characterized by high levels of membrane unsaturated fatty acids and low cytoplasmic volume are more susceptible to oxidative attacks. Conversely, oxidative stress diminishes motility and antioxidant capacity while promoting lipid peroxidation, membrane fluidity, and permeability. Under high oxidative stress conditions, cytotoxicity is escalated due to the accumulation of toxic by-products and sperm chromatin fragments. Importantly, this increase in ROS levels can lead to mitochondrial dysfunction, and the combined effects of endogenous and exogenous oxidative stress exhibit a synergistic relationship 25.
Furthermore, the type and concentration of ROS producers also play a significant role. Oxygen free radicals, including H2O2, possess the ability to penetrate the membrane surrounding the nucleus, leading to its damage, as well as guanosine oxidation and subsequent DNA fragmentation 26. Given that both oxidative stress and reductive stress can elevate ROS levels, it is necessary to examine the impact of both factors on sperm function, chromatin structure, DNA uptake, and cellular apoptosis in embryos. Evidence suggests that reactive oxygen species (ROS) can activate specific cell signaling pathways and trigger DNA damage repair mechanisms, potentially leading to DNA uptake 27. While the indirect negative impacts of ROS on sperm quality and function are well-documented, the direct effects of ROS on sperm remain unclear.
The findings from this experiment indicate that the elevation of ROS levels and subsequent increase in membrane permeability caused by H2O2 did not initially affect sperm function, but disturbances emerged thereafter. Based on this hypothesis, differen concentrations of H2O2 were examined to determine their effect on membrane permeability and transfection efficiency. It was observed that transfection efficiency increased up to a concentration of 100 µM H2O2, but at higher concentrations, a decline in transfection efficiency and adverse ROS effects were observed. Furthermore, the process of increasing DNA uptake at doses exceeding 100 µM H2O2 was associated with DNA fragmentation. Hence, the addition of antioxidants was explored to mitigate these detrimental effects and reduce DNA fragmentation. In this regard, the effects of different doses of melatonin were compared, revealing a significant decrease in the apoptosis process at a concentration of 10− 9 M melatonin.
Another notable finding of this study was the highest blastocyst yield, 93%, was obtained at a concentration of 50 µM H202, followed by 80% at a concentration of 10− 9 M melatonin. In comparison, the blastocyst rate in the control group was 89%, while it dropped to 67% in the presence of 3% DMSO, which served as the known substance control in SMGT. When evaluating the embryos using a fluorescent microscope, 92% of the blastocysts showed positive GFP fluorescence at a concentration of 50 µM H202, and 91% exhibited fluorescence at a concentration of 10− 9 M melatonin. However, in the presence of 3% DMSO, the fluorescence percentage dropped to 43%.
The highest percentage of apoptosis (47%) was observed at a concentration of 200 µM H202, followed by 20% at a concentration of 10− 3 M melatonin. The rate of plasmid uptake by sperm was assessed using aqRT-PCR, which showed a direct correlation between increasing H2O2 concentration and decreasing melatonin concentration. Moreover, the DNA absorption in treatments of 200 µM, 100 µM, and 50 µM H2O2, as well as 10− 9 M melatonin, was higher than the 3% DMSO treatment .
In this study, simultaneous treatment of H2O2 and sperm capacitation was conducted. However, in 2010, Du Plessis et al. conducted an experiment where sperm were exposed to H2O2 one hour after incubation to evaluate the impact of exogenous H2O2 on motility, reactive oxygen species (ROS), and intracellular products.
Furthermore, a comparison between DMSO and H2O2 revealed that DMSO influenced acrosome reaction (AR), followed by a decrease in membrane degradation and fertilization. At controlled concentrations of 15 and 50, H2O2 demonstrated significant positive effects on transfection. However, DNA fragmentation increased at concentrations of 100 and higher, leading to diminished effects. The transfection rate of H2O2 at controlled concentrations of 100% was significantly higher than that of DMSO. This can be attributed to increased lipid peroxidation and enhanced membrane permeability, resulting in greater plasmid entry by H2O2 and, consequently, a higher transfection rate. These findings indicate that H2O2 can be an effective transfer agent at controlled concentrations of 100 M and an oxidizing agent at concentrations exceeding 100 M, similar to 28.
Prior to this study, there was limited focus on using H2O2 to enhance the rate of DNA entry into sperm in the SMGT method. Most previous studies had primarily explored the effects of H2O2, other oxidants, and antioxidants, as well as the adverse impacts of ROS levels. Some of these studies did not report significant differences in cleavage rates among the control group, the DMSO group, and the group treated with melatonin. Except for Tian's study [30], which observed an increase in the blastocyst formation rate in the group treated with 10− 12 M melatonin. Additionally, while that study showed the total number of blastocysts was higher at a melatonin concentration of 10− 9 M, the expression of the Hhmgb1 gene was higher at a melatonin concentration of 10− 12 M, indicating enhanced performance at this concentration 29. On the other hand, in a study involving sperm treatment with melatonin, the blastocyst rate at a melatonin concentration of 10− 12 M was found to be higher compared to embryos produced by in vitro maturation (IVM) 30. In Ishizuka's study 31 on embryos produced through somatic cell nuclear transfer (SCNT), a reduction in cell arrest was observed at a concentration of 10− 6 M melatonin, which aligns with the results obtained in our study.
In 2010, Du Plessis conducted a study using concentrations of 2.5, 7.5, and 15 µM H2O2 to evaluate their impact on sperm capacitation and motility parameters. It was observed that all concentrations resulted in an increase in reactive oxygen species (ROS). However, as the concentration of H2O2 increased, it led to a decrease in membrane fluidity and reduced movement parameters due to elevated lipid peroxidation of the sperm membrane 10. During another investigation on the effects of exogenous H2O2 on tyrosine phosphorylation, sperm function, and chromosomal exocytosis, an interesting finding was discovered. A significant increase in sperm-oocyte fusion was observed at a concentration of 50 µM H2O2, while the fusion rate decreased at 100 µM [33]. This increase in fusion was attributed to improved capacitation without a concurrent increase in the acrosomal reaction. These findings are consistent with the results obtained in this study, further contributing to our understanding of this phenomenon.
Furthermore, in agreement with [20], this study suggests it will be beneficial to enhance the efficiency of this method by introducing novel and diverse transfection reagents that promote gene transfer to sperm. In the majority of studies, DMSO was found to play a critical role in facilitating DNA entry. Initial research indicated that the optimal conditions for generating transgenic embryos using SMGT involved a 3% DMSO treatment, a sperm concentration of 107 ml, and a linear DNA concentration of 20 µg/ml. Studies have also revealed that an increase in DNA density results in enhanced DNA absorption in sperm, albeit at the cost of reduced sperm motility and fertility 32. Subsequent studies incorporating DMSO demonstrated a 42% transfection rate in embryos, accompanied by an accelerated progression toward the blastocyst stage and the production of GFP-positive blastocysts 33. The application of the SMGT method notably accelerated the progression to the blastocyst stage and resulted in an increased production of GFP-positive blastocysts 34. There is limited competition in the development of materials for enhancing transfection efficiency. In a few studies, X-tremeGENE HP, in conjunction with DMSO, was employed to effectively transfer a DNA concentration of 1 µg/ml to sperm; however, this transfection process did not proceed successfully to the embryo stage 35. In a dose-dependent study utilizing the NanoSMGT method, Colares et al. demonstrated that sperm can uptake exogenous DNA, with an optimal amount of 10 µg/106. They found the use of linear and circular plasmids did not impact sperm recruitment, and nanotransfection did not have any adverse effects on sperm motility or viability 36.