In this study, after juvenile male rats were exposed to PM2.5 for 4 weeks, their reproductive functions during the period of sexual maturity were studied using six parameters, including the sperm count, fertility rate, histological morphology, mitochondrial morphology, iron ion concentration, and protein expression level. The results showed that compared with rats in the control group, juvenile male rats exposed to PM2.5 had a decrease in the number of sperms and the fertility rate during the period of sexual maturity. Additionally, obvious atrophy of the epithelium of the seminiferous tubules, a distinct decrease in the number of spermatogenic cell layers and sperms in the seminiferous tubules, the detachment of some spermatogenic cells into the lumen in the testes of male rats during the period of sexual maturity were observed. These findings confirm that the reproductive function of juvenile male rats during the period of sexual maturity decreased after PM2.5 exposure, which enriches our previous research(Liu, Shen, Zhao, Sun, Wang, Long, He, Lin, Wu, & Wei, 2020; Liu, Wu, Shen, Zhao, Wei, Tang, Long, Zhou, He, Lin, & Wei, 2019).
Our previous studies confirmed that oxidative stress is one of the important mechanisms by which PM2.5 induces reproductive toxicity in adult male rats (Liu, Shen, Zhao, Sun, Wang, Long, He, Lin, Wu, & Wei, 2020; Liu, Wu, Shen, Zhao, Wei, Tang, Long, Zhou, He, Lin, & Wei, 2019). The decrease in the number of sperms in adult male rats may be partly attributed to systemic and testicular localized inflammatory responses caused by PM2.5 exposure during adolescence (Zhou, Su, Li, Chu, Sun, Zhang, Han, Li, Zou, Niu, & Zhang, 2019). Such a decrease may also be ascribed to the decreased testosterone concentration and hindered testosterone biosynthesis, which further affect sperm concentration, morphology, and viability (Y. Yang, Yang, Liu, Cao, Zhao, Su, Liao, Teng, & Hua, 2019). Some scholars have found that PM2.5 may lead to reproductive impairment by inducing decreased sperm quality and testosterone levels in juvenile male rats via the inositol-requiring enzyme (IRE1)/c-Jun NH (2)-terminal kinase (JNK)/ autophagy signaling pathway (Y. Yang, Feng, Huang, Cui, & Li, 2021). Additionally, after mother rats are exposed to PM2.5, PM2.5 may affect spermatogenesis by activating the unfolded protein response (UPR)-mediated JNK apoptosis pathway in the testes of their offspring to increase apoptosis and reduce testosterone secretion (Ren, Jiang, Huang, Zang, Huang, Zhang, Wei, Lu, Wu, & Zhou, 2022). Based on previous studies, we found that the expression level of antioxidant transcription factor Nrf2 significantly decreased, while that of lipid peroxide MDA distinctly increased in the testes of juvenile male rats during the period of sexual maturity after PM2.5 exposure, which again verified that oxidative stress is the initiating agent for PM2.5 to induce reproductive damage in male rats and that the redox is imbalanced and lipid peroxides are accumulated in the testes of juvenile male rats during the period of sexual maturity after PM2.5 exposure.
As the centers of energy metabolism in cells, mitochondria play an important role in maintaining cell homeostasis (Y. Yang, Karakhanova, Hartwig, D'Haese, Philippov, Werner, & Bazhin, 2016). However, mitochondria may be damaged due to increased ROS (M. Zhao, Wang, Li, Liu, Wang, Yuan, Yang, Chen, Cheng, Lu, & Liu, 2021), leading to abnormal cell death. Our previous work confirmed that PM2.5 can cause mitochondrial dysfunction by damaging the UPRmt pathway of mitochondria in spermatogenic cells, which further leads to spermatogenic impairment in juvenile rats (C. Wang, Liu, Shu, Yin, Xiao, Ai, Zhao, Luo, & Liu, 2022). Therefore, we further investigated mitochondrial damage. COXIV is considered a key marker of mitochondrial function because of its rate-limiting effect on oxidation (Holper, Ben-Shachar, & Mann, 2019; Srinivasan & Avadhani, 2012), while HSP60 plays a key role in regulating mitochondrial protein homeostasis (Duan, Tang, Mitchell-Silbaugh, Fang, Han, & Ouyang, 2020). Therefore, through the detection of the expression levels of associated proteins in the testicular tissues of juvenile male rats during the period of sexual maturity after PM2.5 exposure, we discovered a significant decrease in the expression levels of COXIV and HSP60 and observed reduced volume, atrophied membrane, and cristae decrease in some mitochondria of testicular cells via electron microscopy, which indicated that mitochondria in the testicular tissues of juvenile male rats during the period of sexual maturity are seriously damaged after PM2.5 exposure. Studies have confirmed that mitochondria regulate cysteine metabolism through the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC) and that disruption of TCA metabolism, ETC activity, and mitochondrial membrane potential (MMP) hyperpolarization resulting in cysteine deficiency cause lipid ROS accumulation, which in turn leads to cellular ferroptosis following mitochondrial damage(Gao, Yi, Zhu, Minikes, Monian, Thompson, & Jiang, 2019).
Ferroptosis is a novel mode of cell death depending on iron ions, triggered by the excessive accumulation of the toxicity of lipid peroxides on the cell membrane (Dixon, Lemberg, Lamprecht, Skouta, Zaitsev, Gleason, Patel, Bauer, Cantley, Yang, Morrison, & Stockwell, 2012; Lei, Zhuang, & Gan, 2022; Stockwell, Friedmann Angeli, Bayir, Bush, Conrad, Dixon, Fulda, Gascon, Hatzios, Kagan, Noel, Jiang, Linkermann, Murphy, Overholtzer, Oyagi, Pagnussat, Park, Ran, Rosenfeld, Salnikow, Tang, Torti, Torti, Toyokuni, Woerpel, & Zhang, 2017; Wu, Minikes, Gao, Bian, Li, Stockwell, Chen, & Jiang, 2019). Some scholars have confirmed that PM2.5 leads to pulmonary fibrosis by inducing ferroptosis through the transforming growth factor beta (TGF-β) signaling pathway (Guo, Bai, Ding, Zhao, Xu, & Wang, 2022) and causes decreased reproductive function in adolescent male rats by inducing DNA damage and ferroptosis through oxidative stress (Shi, Zhang, Cui, Wang, Wang, Tang, Yang, Zou, Ling, Han, Liu, Chen, Liu, Cao, & Ao, 2022). When ferroptosis occurs in cells, mitochondrial damage is manifested as volume reduction, membrane atrophy, and cristae decrease or disappearance (Dixon, Lemberg, Lamprecht, Skouta, Zaitsev, Gleason, Patel, Bauer, Cantley, Yang, Morrison, & Stockwell, 2012; Xie, Hou, Song, Yu, Huang, Sun, Kang, & Tang, 2016). We observed these manifestations in the mitochondria of testicular cells in juvenile male rats during the period of sexual maturity after PM2.5 exposure, suggesting that ferroptosis also occurs in this experimental model.
To confirm the presence of ferroptosis, we further examined the iron ion content in testicular tissue, the results showed that male rats in the PM2.5 group had higher concentrations of total iron ions and ferrous ions and a metabolic disorder in the testicular tissues during the period of sexual maturity compared with rats in the normal group, further confirming that ferroptosis occurs in this experimental model.
Furthermore, ferroptosis is caused by the accumulation of cellular ROS exceeding the contents of glutathione (GSH) and phospholipid hydroperoxide peroxidase with GSH as the substrate in redox reactions (Gao, Yi, Zhu, Minikes, Monian, Thompson, & Jiang, 2019) and can be triggered by inhibiting the activity of System Xc-, leading to the depletion of cellular cysteine and GSH and redox homeostasis imbalance in cells. Some researchers found that the expression level of GPX4 significantly declined in infertile men diagnosed with azoospermia(Imai, Hakkaku, Iwamoto, Suzuki, Suzuki, Tajima, Konishi, Minami, Ichinose, Ishizaka, Shioda, Arata, Nishimura, Naito, & Nakagawa, 2009; W. S. Yang, SriRamaratnam, Welsch, Shimada, Skouta, Viswanathan, Cheah, Clemons, Shamji, Clish, Brown, Girotti, Cornish, Schreiber, & Stockwell, 2014). On this basis, we evaluated the expression levels of phospholipid hydroperoxide GPX4 and system Xc-associated protein SLC7A11 in the testicular tissues during the period of sexual maturity. The results showed that the expression levels of GPX4 and SLC7A11 in the testicular tissues decreased, indicating reduced ROS capacity, decreased activity of system Xc-, and redox imbalance in the testicular tissues of juvenile male rats during the period of sexual maturity after PM2.5 exposure, which were consistent with the manifestations of ferroptosis.
FPN1, the only transmembrane protein found to be related to the release of iron ions in mammals so far, is responsible for exporting ferrous ions from cells to the plasma (Tian, Xiong, Zhang, Leng, Tao, Li, Qiu, & Xia, 2021). The ferrous ion transfer protein DMT1 can regulate iron ion levels and is crucial for maintaining iron homeostasis (Song, Peng, Sun, Heng, & Zhu, 2021). Therefore, FPN1 and DMT1 are vital for iron ion homeostasis. After confirming the metabolic disorder of iron ions in this model, we further studied changes in the expression levels of proteins related to ferroptosis using WB. Compared with the normal group, the PM2.5 group displayed a significantly decreased level of FPN1 and a distinctly increased DMT1 level in the testicular tissues, proving that ferroptosis in the testicular tissues of juvenile male rats during the period of sexual maturity after PM2.5 exposure rises significantly, leading to impaired spermatogenic function.
Oxidative stress is known to be an “initiating agent” for PM2.5 to induce reproductive impairment in adult male rats, and the combined use of vitamins E and C can reduce oxidative stress by inhibiting the p38 MAPK signaling pathway and blocking DEHP-induced spermatogenic dysfunction and the destruction of the blood-testis barrier(Shen, Tang, Wei, Long, Tan, Wu, Sun, Zhou, Cao, & Wei, 2018). Thus, we added the vitamin intervention group to the model to investigate whether the early enhancement of the antioxidant capacity of juvenile male rats can reduce the reproductive toxicity of PM2.5. The results revealed an increase in spermatogenesis and a reduction in oxidative stress, mitochondrial damage, and ferroptosis in the testes of male rats during the period of sexual maturity after vitamin intervention compared with the PM2.5 group. In addition, the fertility rate of the rats rose significantly after they were subjected to the mating test.
Therefore, this study confirmed that the reproductive toxicity of PM2.5 in juvenile male rats was closely related to oxidative stress, mitochondrial damage, and ferroptosis. However, further research is needed to determine the specific underlying mechanism and target of action of oxidative stress leading to ferroptosis. Moreover, further studies are required regarding whether reproductive function can recover spontaneously and the recovery degree after rats are removed from the environment with PM2.5.