High concentration of iron ions contributes to ferroptosis-mediated testis injury

In order to explore the effect of different concentrations of iron ions on ferroptosis in mouse testes, Kunming mice were randomly divided into control group (normal saline), low iron concentration group (25mg/kg), high iron concentration group (70mg/kg) and deferoxamine group (40mg/kg). The mice were injected continuously for 7 days and their body weight was measured. At the end of the experiment, the organ weight, sperm count, and malformation rate were measured. Testicular tissue, the pathological and ultrastructural changes in spermatogenic tubules were also observed by using hematoxylin eosin (HE) staining and transmission electron microscopy. The changes in transcription levels of related genes and serum biochemical indicators were measured in mouse testicular tissues. The results showed that higher iron concentration may inhibit the growth of mice, reduce the organ coe�cients of testis, heart, and liver, and increase the rate of sperm malformation and mortality. Supplementation of iron ion in high concentration can negatively affect the male reproductive system by reducing the sperm count and causing malformation and structural damage in seminiferous tubules and sperm cells. In addition, the iron concentration also affected the immune response and blood coagulation ability by in�uencing the red blood cells, white blood cells and platelets. The results showed that iron ions may affect mice testicular tissue and induce ferroptosis by altering the expression of ferroptosis related genes. Though, the degree of effect was different for the different concentrations of iron ions. The study also revealed the potential role of deferoxamine to inhibit the occurrence of ferroptosis. Though, the damages caused to the testis by deferoxamine supplementation suggests the need for further researches in this direction.


Introduction
Male infertility is a common disease, affecting around 70 million people worldwide.As per the World Health Organization, approximately 9% of couples worldwide have fertility problems and 50% of these couples are affected by male infertility.There are many reasons for male infertility ranging from gene mutation to lifestyle selection, to medical disease or drug treatment [1] .Its pathogenesis can be roughly divided into three categories: (1) secondary hypogonadism caused by hypothalamic-pituitary diseases; (2) obstruction of semen out ow (commonly referred to as obstructive azoospermia); (3) testicular dysfunction (possibly related to primary hypogonadism) [2] .
Testis is an important part of male reproductive system.Spermatogenic cells in testis are responsible for the production of spermatozoa and maintaining their normal physiological structure and function.
Research shows that electromagnetic radiation, high temperature, viruses, and environmental endocrine disruptors such as polycyclic aromatic hydrocarbon compound, heavy metals (lead) and metalloids (arsenic) can all cause damage to the male reproductive system and affect male fertility [3,4] .Similarly, iron ions can also affect the testicular tissue with dense vascular distribution by damage its structure and potential functions [5] .
Ferroptosis is a form of regulatory cell death characterized by iron-dependent lipid peroxidation which was rst proposed in 2012 [6] .Morphologically, cells undergoing ferroptosis have typical characteristics of necrosis with small and deformed mitochondria, reduced crista, concentrated membranes, ruptured outer membranes, and no apoptotic characteristics [7,8] .Different from the apoptosis in immune silence, ferroptosis has immunogenicity as the affected cells release damage-related molecular patterns and alarmins that may amplify cell death and promote a series of responses associated with in ammation [9] .Endoplasmic reticulum-related oxidative stress, Golgi stress-related lipid peroxidation, and lysosomal dysfunction all contribute to the induction of ferroptosis.In the presence of the antioxidant glutathione (GSH), glutathione peroxidase 4 (GPX4) inhibits lipid peroxidation and protects the cells from ferroptosis.Since this discovery, the complex interaction between iron, cysteine and lipid metabolism has become an important regulatory factor for ferroptosis [9] .Some studies have shown that in order to promote growth, cancer cells have an increased iron requirement as compared with normal non-cancer cells [10] .In addition to cancer, ferroptosis has been found to be associated with degenerative diseases, ischemiareperfusion injuries, and cardiovascular diseases [11][12][13][14] .Similarly, male reproductive diseases are also found to be induced by ferroptosis and have received extensive attention.It has been reported that oxygen-glucose deprivation and reoxygenation injury of sertoli cells in testis can cause ferroptosis [15] .In addition, arsenite can cause ferroptosis in testicular cells by inducing oxidative stress [16] .It is implied that busulfan-induced ferroptosis might be mediated via inhibition of Nrf2-GPX4 (FPN1) signaling pathway, and highlight that targeting ferroptosis serves as a potential strategy for prevention of busulfan-induced damage and male infertility [17] .Furthermore, iron overload has previously been demonstrated to induce oxidative damage in the testes in a number of animal and human studies [18][19][20][21] .Iron toxicity results in morphological changes in the seminiferous tubules, epididymes and sertoli cells [22] .
As an iron supplement, ferrous sulfate is often used to treat iron de ciency anemia.The effect of excessive iron supplementation on testis and ferroptosis has been rarely studied.Therefore, in this study, after intraperitoneal injection of different concentrations of ferrous sulfate into mice, the transcription levels of ferroptosis-related genes including GPX4, ferritin heavy chain polypeptide 1 (FTH1), Heme oxygenase 1 (HO-1) were detected by uorescent quantitative polymerase chain reaction (PCR) technology, pathological tissue damage, and ultrastructural changes were observed by hematoxylin eosin (HE) staining and transmission electron microscopy.Various biochemical indicators for the blood and sperm quality were detected at the same time to lay a theoretical foundation for in-depth research on ferroptosis.
2 Experimental materials and methods

Animals
Twelve healthy male Kunming mice weighing 25-30 g were purchased from Experimental Animal Center of Zhengzhou University and free to eat and drink.Mice were randomly divided into control Group (normal saline, C Group), high iron concentration group (75 mg/kg, H Group, n = 3), low iron concentration group (25 mg/kg, L Group, n = 3) and deferoxamine group (40 mg/kg, F Group, n = 3).Continuous intraperitoneal injections were performed, and testicular tissue of mice were collected 7 days later.
Animal experimental studies conducted in this manuscript were performed in accordance with the laid down ethical standards and approved by the Major science and technology special committee of Henan Province and Huanghuai University.

Sample collection
At the end of the experiment, the mice were anesthetized and blood samples were taken from the eyeballs before sacri cing them by cervical dislocation.Testicular tissue and epididymis were quickly removed by laparotomy and sperm were counted followed by motility test using a sperm analyzer.The left testis of each mouse was taken out and xed with 10% formalin and pathological sections were made to observe the histological changes.The other testis was rapidly frozen in liquid nitrogen and stored in an ultra-low temperature refrigerator at -80℃ for later use.

Sperm count and vitality test
The bilateral epididymis of mice was removed, weighed, and placed into a 1.5 ml centrifuge tube containing 1 ml warm phosphate-buffered saline.The epididymis was longitudinally cut and mixed with ophthalmic scissors to make sperm suspension.10 µl of the sperm suspension was collected by pipetting gun and examined on the cell counting plate using high-power microscope.Finally, the sperm were counted by a sperm counter and the sperm malformation rate was analyzed by taking photos.

Determination of serum reduced GSH content
Mouse blood was collected and stored in the refrigerator overnight at 4°C.Supernatant was taken out and centrifuged at 3000 rpm for 10 min at 4°C.After centrifugation, the supernatant was removed and transferred into a new eppendorf (EP) tube.According to the instructions of the GSH kit, the OD value of the sample was measured by microplate reader(Biotek, ELX800) at 405 nm and the GSH content in the sample was calculated.

Pathological examination of testicular tissue
Testicular tissue blocks were xed in formalin followed by dehydration with different concentrations of ethanol and subsequent treatment with xylene for transparency.The testicular tissue was moved into the melted para n and then taken out and put into the embedding frame lled with melted para n for embedding.Finally, it was cut into slices with a thickness of 5 µm.The tissue sections were baked in an oven at 60℃ for 60 min.Afterwards, the tissue sections were dewaxed and subjected to HE staining after a thorough rinse.The sections were stained with hematoxylin and eosin dye solution in turns followed by treatment with hydrochloric acid alcohol as a differentiating reagent.The stained tissue sections were then observed and photographed under the optical microscope.

Observation of ultrastructural changes of testicular tissue
After anesthesia, the mice were sacri ced by cervical dislocation.After collecting the testicular tissue, a small piece of tissue was taken by dissecting scissors and placed on a clean cardboard.A drop of cooled 2.5% glutaraldehyde solution was dropped on the tissue for xation.After xation, the tissue was cut into small pieces of around 1 mm wide and 2 ~ 3 mm long by using a new, oil-free sharp blade and then they were nally cut into smaller pieces of 1 mm 3 .These small pieces were put into glutaraldehyde xation solution at 4℃ one by one, and then sent to Wuhan Servicebio Technology Co., Ltd. for ultrastructural observation.

Detection of biochemical index
The obtained blood samples were stored in anticoagulant tube at 4℃ overnight and then sent to Wuhan Servicebio Technology Co., Ltd. for detection of blood related indicators such as lymph content, hemoglobin (HGB), platelets (PLT), red blood cells (RBC) etc.

Genes
Primer Sequence(5'-3') 2.9 Statistical analysis SPSS 26.0 software was used for statistical analysis and Least Signi cant Difference (LSD) method was used for one-way analysis of variance.The data were expressed as mean ± standard error.GraphPad Prism 6.0 software was used for plotting, and the signi cant difference was expressed as extremely different (P < 0.01) and signi cantly different (P < 0.05).
3 Results and Analysis

Effect of different concentrations of iron ions on body weight of mice
During the experiment, mice were weighed once a day and the change in weight was recorded as shown in Fig. 1.The body weight of mice in the control group gradually increased with time and the body weight of mice in the low iron concentration group was slightly higher than that in the control group after 2 days.The weight growth rate of the high iron concentration group was observed to be higher than that of the other groups from day 1 to 4. However, it began to decrease signi cantly from 5th day onwards.The weight growth rate of the deferoxamine group was found to be the slowest and signi cantly lower than that of the other three groups.The results indicated that the low concentration of iron ions may increase the blood content and weight of other tissues and organs, while excessive supplementation of iron ions and iron de ciency have obvious inhibitory effects on the body weight of mice.

Weight changes of testis and other organs in mice
The mouse organs were weighed and the organ coe cients were calculated during dissection.As compared to the control group, the testis weight of the three treatment groups were observed to be lower.The low concentration of iron ions had a signi cant effect on the testis of mice which was signi cantly different as compared to the control group (P < 0.01), to the high iron ion group(P < 0.05) and the deferoxamine group (P < 0.05) (Fig. 2).In addition, the growth and development of heart and liver in the low iron concentration group were signi cantly inhibited (P < 0.05), while no signi cant effect were observed for kidney, spleen, and lung.

Analysis of sperm count in mice
After the epididymis was fully cut, the sperms of control group, low iron concentration group, high iron concentration group, and deferoxamine group were observed by sperm analyzer (Mailang, China).According to the results (Fig. 3), the malformation rate and death rate of mouse sperm in the experimental group were signi cantly higher than those in the control group.The malformation rate and death rate of the high iron concentration group were extremely signi cantly higher than those in the low iron concentration group(P < 0.01) and the deferoxamine group (P < 0.01).These results showed that the excessive concentration of iron ions could signi cantly affect the spermatogenesis process and the male reproductive health.

HE staining of testis
HE staining was used to observe the effect of different concentrations of iron ions on the tissue injury of mice testis (Fig. 4).In the control group, the spermatogenic epithelium of the testis was observed to be thick, the seminiferous tubules were round or oval with complete and full structure, and the supporting cells and cells at all levels in the tubules were arranged regularly (Fig. 4A).In comparison to the control group, the seminiferous tubules of the deferoxamine group were arranged loosely, with a few malformations including fragmented or diminished interstitial cells, enlarged seminiferous tubule space with some seminiferous epithelial cells shed into the lumen (Fig. 4B).On the other hand, the spermatogenic epithelium in the low iron concentration group was thinner and the seminiferous tubules were deformed and arranged irregularly with the pathological damage similar to that in the deferoxamine group (Fig. 4C).In the high iron concentration group, the spermatogenic epithelium was observed even thinner than the other groups and a large amount of spermatogenic epithelium fell off into the lumen with deformed seminiferous tubules.The rate and degree of deformity for the seminiferous tubules were signi cantly higher and the arrangement was irregular in the high iron concentration group, while the number of sperm cells in the lumen was signi cantly less than that in the control group.There were almost no formed sperm cells and the interstitial cells were completely fragmented or disappeared.Most of the cell membranes were ruptured and the cell contents were discharged, resulting in serious pathological damage (Fig. 4D).

Ultrastructural observation
Transmission electron microscopy was used to observe the pathology of mice testis after different treatments.The results showed that there was no obvious damage to the cell structure in the control group and the structure of mitochondria (as shown by the red arrow) and nucleus (as shown by the asterisk) were normal (Figs.5A and 5a).However, in the high iron concentration and low iron concentration groups, there was obvious ferroptosis, cell membrane rupture and blebbing, mitochondrial atrophy, mitochondrial crista reduction or even disappearance (as shown by the red arrow), increased membrane density, and normal nuclear morphology (as shown by the asterisk) (Fig. 5B, Fig. 5b, Fig. 5C, Fig. 5c).In the deferoxamine group, intracellular mitochondrial deformation higher electron density (as shown by the red arrow), rupture and disappearance of nuclear and cell membrane (as shown by the blue and yellow arrows), and swelling and dilation of endoplasmic reticulum (as shown by the white arrows) were observed through electron microscopy (Figs.5D and 5d).The lesions in the high iron concentration group were signi cantly higher than those in the other three groups, indicating that the iron concentration played a decisive role in the occurrence of ferroptosis and the degree of cell damage.The higher the concentration, the more serious the damage.

Whole blood analysis of mice
After collection from the eyeball, the blood was transferred to the anticoagulant tube and stored at 4℃.Subsequently, the blood was sent to Wuhan Servicebio Technology Co., Ltd. to detect the changes in the biochemical indicators of blood.The results showed that in comparison to the other three groups, the number of white blood cells (Fig. 6M), lymphocytes (Fig. 6A), monocytes (Fig. 6B) and neutrophils (Fig. 6C) were extremely higher in the high iron concentration group (P < 0.01).The percentage of monocytes and neutrophils in the three experimental groups were higher than those in the control group, while the percentage of lymphocytes was observed to be lower.The number of RBCs, HBG, average RBC, HGB content in RBC and average HGB concentration in RBC for the high iron concentration group were signi cantly (P < 0.05) or extremely (P < 0.01) higher than those in the control group.In addition, the PLT count and mean PLT volume for high iron concentration group were signi cantly lower than those of control group (P < 0.01).

Effect of different concentrations of iron ions on serum reduced GSH content in mice
After the standard curve for GSH determination was constructed according to the instructions, samples were analyzed and the results were calculated according to the standard curve.The results showed that the reduced GSH content for high iron concentration group and low iron concentration group were extremely (P < 0.01) and signi cantly (P < 0.05) lower than the control group.The reduced GSH content in the deferoxamine group was also higher than the control group, but the difference was not signi cant (P > 0.05).

Transcription level of ferroptosis related genes
As shown in Fig. 8, the mRNA transcription level of GPX4 gene in the high iron concentration group was signi cantly lower than the control group (P < 0.01), while it was extremely higher in the deferoxamine group (P < 0.01).The transcription level of GPX4 gene in deferoxamine group was also extremely higher than the high iron concentration group (P < 0.01).As compared to the control group, the mRNA transcription level of divalent metal transporter 1 (DMT1) gene in the high iron concentration group was signi cantly higher (P < 0.01).After the mice were treated with deferoxamine mesylate, the expression of DMT1 gene was found to be decreased in the deferoxamine group and it was extremely lower than that in the high iron concentration group (P < 0.01).The transcription level of DMT1 in the high iron concentration group signi cantly increased (P < 0.01) in comparison to the low iron concentration group (P < 0.01).Similarly, the transcription level of HO-1 gene was observed to be increased in both the low and high iron concentration treatment groups and the difference was extremely signi cant as compared to the control group (P < 0.01).The expression level of HO-1 gene in high iron concentration group was signi cantly higher than the low iron concentration group (P < 0.05).After the mice were treated with deferoxamine, the expression level of HO-1 gene was still observed to be increased, but not signi cantly.
Compared with the control group, the expression level of FTH1 in the deferoxamine group signi cantly increased (P < 0.01) and the transcription level of FTH1 in the groups treated with different concentrations of iron ions was signi cantly higher (P < 0.01).The transcription of FTH1 in the high iron concentration group was signi cantly lower than the low iron concentration group.These results showed that the high concentration of iron ions can signi cantly promote ferroptosis.

Discussion
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 H 2 O 2 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 signi cance 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 coe cient 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 owing 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 signi cant 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][28][29] .The results showed that the HGB content in high-dose iron ion mice increased signi cantly, while the lymphocytes and white blood cells increased rather extremely along with the decrease in PLT volume.These ndings indicate that the increase of iron ion content could increase the e ciency 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, Fe 2+ ions cause the increase in lipid peroxidation in testicular microsomes in a concentration dependent manner and the role of Fe 2+ is far greater than that of Fe 3+ in this process [32] .Studies showed that GPX4-de cient 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 e ux 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 Fe 3+ and then enters the cell through transferrin receptor 1 (TFR1).Fe 3+ is deoxidized by iron oxide reductase six-transmembrane epithelial antigen of the prostate 3 (STEAP3) to Fe 2+ .Finally, Fe 2+ 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 signi cant 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 signi cantly 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.

Conclusion
Different concentrations of iron ions have different degrees of in uence on testicular tissue.High concentrations of iron ions obviously promote ferroptosis which in turn cause more damaging effects on the tissue structure of testicular tissue.Although deferoxamine can inhibit the occurrence of ferroptosis, it can still cause pathological changes in testicles.

Declarations Con icts of interest
The authors declare no con icts of interest.

Figures Figure 1 Figure 2 Testicular organ coe cient of mice Figure 3
Figures

Table 2
Organ coe cients of mice in different groups.* refers to a signi cant difference compared with the control group (P < 0.05); ** refers to an extremely signi cant difference compared with the control group (P < 0.01).