Iron is a crucial micronutrient for almost all living things. It can combine with different ligands and conduct electron transfer which is very important for the optimal functioning of living things. However, the excess iron can react with H2O2 in the form of ferrous ions in the redox cycle. This reaction that is called Fenton reaction generates hydroxyl radical (•OH) and increases malondialdehyde (MDA) content [23] causing harmful oxidative damage to DNA, protein and membrane lipid which in turn induce lipid peroxidation in the testicular tissue [24]. Iron ion intervention can lead to the vacuolization of mouse testicular tissue structure, the shedding of germ cells, the impairment of endocrine function, and the reduction of the number of mature sperm [25]. One study has shown that inhibition of ferroptosis by ferrostatin-1 or deferoxamine can partially alleviate mouse oligozoospermia induced by Busulfan [17]. However, the study on the effect of different iron concentration on mouse spermatogenesis and ferroptosis has not been reported. In this study, different concentrations of ferrous sulfate were injected intraperitoneally to study its effects on spermatogenesis and ferroptosis in mice which is not only of great significance for iron supplementation and its rational use as a drug. The study also lays a foundation for the mechanism research of iron ions affecting male reproductive health.
The results of this study show that high concentration of iron ion can inhibit weight growth of mice and reduce the viscera coefficient of testis, heart and liver. The presence of excess iron can increase the sperm deformity and death rate, and can also cause the malformation of seminiferous tubules and decrease the number of sperm cells. The structural characterization of sperm cells revealed that the high iron supply can cause the rupture or disappearance of mesenchymal cells, cell membrane rupture and flowing out of the cell contents. The results of ultrastructure observation showed that the high iron concentration and low iron concentration groups showed obvious ferroptosis due to the observation of cell membrane rupture and blebbing, mitochondria atrophy, mitochondrial crista reduction or even disappearance and increased membrane density in the cells of mice testicle tissue. These structural damages in cells were also observed for the group of mice injected with deferoxamine. These results show that too high or too low iron concentration is unfavorable for the growth and development of testicular tissue.
The content of iron ions in the body has significant effect on the level of blood oxygen transport, as well as the RBCs and HBG[26]. Different kinds of white blood cells participate in the body’s defense response in different ways[27–29]. The results showed that the HGB content in high-dose iron ion mice increased significantly, while the lymphocytes and white blood cells increased rather extremely along with the decrease in PLT volume. These findings indicate that the increase of iron ion content could increase the efficiency of blood oxygen transport. However, it can also cause a strong immune response in the testicular tissue of mice and can reduce the coagulation function. Domenico Girelli et al. had a research on iron metabolism in COVID-19 infections,which showed that iron metabolism also has implications on the functionality of cells of the immune system. Once primed by the contact with antigen presenting cells, lymphocytes need iron to sustain the metabolic burst required for mounting an effective cellular and humoral response[30]. These results suggest that the lymphocytes of mice in the state of high iron ions may secrete a large number of antibodies which can inhibit or promote the occurrence of various diseases. This result is similar to that of Xiaofei Gao's research[31]. From this perspective, it may be more effective to study the relationship between ferroptosis and the disease caused by increase of lymphocytes.
When cystine transport proteins are inhibited, intracellular GSH will be depleted, eventually leading to the inactivation of GSH peroxidase (GPX4), resulting in the accumulation of lipid peroxidation, which can induce ferroptosis to a certain extent. In this process, Fe2+ ions cause the increase in lipid peroxidation in testicular microsomes in a concentration dependent manner and the role of Fe2+ is far greater than that of Fe3+ in this process[32]. Studies showed that GPX4-deficient Treg cells can increase the production of mitochondrial superoxide and IL-1β and Gpx4 can prevent lipid peroxidation and ferroptosis of Treg cells in the regulation of immune homeostasis and anti-tumor immunity, which provides a potential therapeutic strategy for improving cancer treatment [33, 34]. In addition, FTH1, one of the antioxidants in cells, can inhibit the oxidation reaction of divalent iron ions, thus protecting cells from oxidative damage caused by excessive iron ions. HO-1 is a key regulator of ferroptosis in cells. Magnesium isoglycyrrhizinate can up-regulate the expression of HO-1 and further promote the overexpression of transferrin, transferrin receptor, and FTH1, and can cause the low expression of iron efflux pump - iron membrane transporter which may lead to intracellular iron deposition, lipid peroxides accumulation, and ferroptosis in hepatic stellate cells [35]. In the process of ferroptosis, the circulating iron binds to transferrin in the form of Fe3+ and then enters the cell through transferrin receptor 1 (TFR1). Fe3+ is deoxidized by iron oxide reductase six-transmembrane epithelial antigen of the prostate 3 (STEAP3) to Fe2+. Finally, Fe2+ is released into the unstable iron pool in the cytoplasm from DMT1 mediated endosome. In this study, the GSH content in the high iron group decreased with significant reduction in the mRNA expression of ferroptosis related genes GPX4 and FTH1. On the other hand, the transcription levels of DMT1 and HO-1 increased significantly which promoted the occurrence of testicular ferroptosis. The above results indicate that high concentration of iron ions can play a crucial role in the growth and development of testicular tissue and spermatogenesis by regulating the ferroptosis related genes and oxidative damage of mitochondria. Therefore, the amount of iron ions that people receive in their diet or other sources is crucial for male reproductive health and development. In this study, the ferroptosis related indicators and the expression level of ferroptosis regulatory genes in mouse testis were measured and analyzed to preliminarily investigate the possible involvement of iron ions in causing male infertility. At present, the research on ferroptosis is still in the initial stage and many inducing factors and mechanisms of ferroptosis need to be further studied. Although, ferroptosis can cause damage to normal cells, it can also eliminate the cells in a pathological state, thus maintaining the stability of the body. Further research in this direction may provide new ideas and research prospects for animal disease prevention and treatment.