Reproductive Toxicity of Chronic Exposure To Polystyrene Microplastics And The Molecular Mechanism of Decrease In Testosterone Levels In Male Mice

Background: Microplastics (MPs), which are smaller in size and dicult to degrade, can be easily ingested by marine life and enter mammals including human through the food chain. Previous study has demonstrated that acute exposure to MPs induced a reduction of testosterone levels and nally resulted in male reproductive toxicity. Therefore, assessing the harm that MPs dose to the male reproductive system under long-term exposure of environmental concentrations and comprehending the mechanism from cell and molecular levels are necessary. Results: We armed that male reproductive toxicity of chronic exposure to polystyrene MPs (PS-MPs) in environmental pollution concentrations and explored the underlying mechanism of MPs-induced decrease of testosterone in mice. Polystyrene MPs (PS-MPs) treatment resulted in the destruction of testicular histology, abnormal spermatogenesis, and interference of hormone secretion in serum. PS-MPs induced a reduction of testosterone levels through downregulation of LH-mediated LHR/cAMP/PKA/StAR pathway. Moreover, we discovered that PS-MPs attached to and became internalized by Leydig cells. Conclusions: In summary, our study showed that PS-MPs existed toxicity to male reproduction under long-term exposure and environmental pollution concentrations, and these potential hazards may ring alarm bells of public health. As well, the discussing of molecular mechanisms may be instructive to search novel therapeutic targets.

demonstrated that acute exposure to MPs induced a reduction of testosterone levels and nally resulted in male reproductive toxicity. Therefore, assessing the harm that MPs dose to the male reproductive system under long-term exposure of environmental concentrations and comprehending the mechanism from cell and molecular levels are necessary.
Results: We a rmed that male reproductive toxicity of chronic exposure to polystyrene MPs (PS-MPs) in environmental pollution concentrations and explored the underlying mechanism of MPs-induced decrease of testosterone in mice. Polystyrene MPs (PS-MPs) treatment resulted in the destruction of testicular histology, abnormal spermatogenesis, and interference of hormone secretion in serum. PS-MPs induced a reduction of testosterone levels through downregulation of LH-mediated LHR/cAMP/PKA/StAR pathway. Moreover, we discovered that PS-MPs attached to and became internalized by Leydig cells.
Conclusions: In summary, our study showed that PS-MPs existed toxicity to male reproduction under long-term exposure and environmental pollution concentrations, and these potential hazards may ring alarm bells of public health. As well, the discussing of molecular mechanisms may be instructive to search novel therapeutic targets.

Background
In recent years, the rapid development of the plastic industry leads to the serious pollution of plastic waste [1,2]. After wave action, UV radiation, photodegradation, and biodegradation, plastic waste is degraded into fragments and particles of different sizes [3,4]. Microplastics (MPs), with diameters less than 5 mm, have attracted broad attention [5,6], because of their small size, stable chemical properties and di culty in degradation [7]. In the wild, the main components of MPs are polyethylene terephthalate, polyethylene, polystyrene, polypropylene, and polyvinyl chloride [8,9]. MPs may induce biological health hazard, due to it is tend to accumulate in organisms and cause toxicity. What is worse, MPs can be easily transported into food chains and ultimately threaten human health [10,11]. Therefore, it warns us to pay special attention to the toxicity of MPs.
Previous researches have demonstrated that MPs are harmful to multiple organs in aquatic organisms and mammals, such as liver, kidney, gastrointestinal tract, brain [12,13,14,15,16,17,18,19,20,21]. Reproductive system plays a crucial role in an organism. Reproductive toxicity of MPs has been found in aquatic organisms, such as Daphnia, Hydra attenuate [21], medaka sh [22], and oyster [23]. So far, there are not many studies on the reproductive toxicity of MPs to mammals. Researchers have demonstrated that MPs induced decreased sperm quality, disordered hormone levels, and oxidative stress in testis following acute and short-term exposure [24,25,26]. However, it is of practical signi cance to study the toxic effect of long-term exposure to MPs under environmental concentrations on reproductive system of mammals.
Our previous study identi ed that following exposure to PS-MPs for 28 days, the testosterone level in serum and sperm quality were declined, resulting in male reproductive dysfunctions in mice. Nevertheless, the underlying mechanism of the decrease in testosterone levels caused by MPs remains unexplored. Testosterone, the most important androgen in males, plays an important role in spermatogenesis. [27] Testosterone is synthesized and secreted by Leydig cells [28]. Meanwhile, the speci c synthesis process is described below. LH, which is released by the anterior pituitary, binds to LH receptors (LHR) located on the membrane of Leydig cells, resulting in increased cAMP content. Then, protein kinase A (PKA) is activated and the levels of steroidogenic acute regulatory protein (StAR) and steroid synthase (P450scc, P450c17, 3β-HSD, 17β-HSD) are increased. Under the action of StAR, free cholesterol in cytoplasm is transported from the outer membrane of mitochondria to the inner membrane. P450scc on the intima converts cholesterol into pregnenolone. Pregnenolone sequentially enters the endoplasmic reticulum, where it is converted to testosterone by 3β-HSD, P450c17, and 17β-HSD [29]. A decreased testosterone level is strongly correlated with MPs-induced functional damage in the testes and Leydig cells.
In addition, previous studies reported that the toxicity of MPs in different tissues is closely related to its particle size. Deng et al. showed that 5µm and 20µm PS-MPs exposure induced disturbance of energy and lipid metabolism [16]. Jeong et al. demonstrated that 0.05 µm, 0.5 µm, and 6 µm PS-MPs treatment led to reduction in growth rate and fecundity, and pointed that the toxicity of MPs was size dependent and smaller microbeads were more poisonous [30]. Meanwhile, some researches elucidated that the toxicity of MPs is concentration dependent [17,19,31]. Therefore, we sought to investigate the effects of the different sizes and concentrations of MPs on the male reproductive system of mammals.
In present study, we exposed mice to MPs of different particle sizes (0.5 µm, 4 µm and 10 µm) at different environmental concentrations to explore male reproductive toxicity of MPs. In addition, we used primary Leydig cells model to investigate the underlying mechanism of the decrease in testosterone levels induced by MPs. Our ndings may provide a novel insight for preventing the reproductive toxicity of MPs.

Characterization of polystyrene microplastics (PS-MPs)
Confocal imaging was conducted to detect the morphology and sizes of PS-MPs used in this study. As shown in Fig. 1A, the MPs were spheres and the sizes of MPs met the design requirements. According to Raman spectra analysis, the monomer of MPs is polystyrene (Fig. 1B). The zeta potential values were exhibited in Supplementary Table S3.
Exposure to PS-MPs induced testicular tissue structure damage and sperm quality decrease in mice During the period of treatment, we monitored the body weights, consumption of food and water. The body weights and food consumption of mice were decreased (Supplementary Table S4). However, there was no remarkable change on water intake among different groups. Compared with control group, the testicular coeffcient ( Fig. 2A) and epididymal coe cient (Fig. 2B) decreased signifcantly in the 0.5 µm, 4 µm, and 10 µm group. Meanwhile, the testicular coe cient of 1000 µg/L PS-MPs with a diameter of 10 µm group decreased more obviously than that of 100 µg/L group. Following exposure to PS-MPs for 180 days, we detected the viability and morphology of sperms. Results revealed that PS-MPs exposure decreased the viability of sperm (Fig. 2C) and increased the rate of sperm deformity (Fig. 2D). In addition, we observed that PS-MPs induced derangement of cell layers and abscission of spermatogenic cells. And higher concentration of PS-MPs exposure could cause more severe damage of testicular structures (Fig.   2E).

Exposure to PS-MPs reduced the content of testosterone, LH and FSH in serum
To explore the effects of PS-MPs exposure on the concentration of reproductive hormones, we detected the levels of testosterone, LH and FSH in serum. The results indicated that LH levels were decreased apparently in a dose-dependent manner after treatment with PS-MPs (Fig. 3A). As shown in Fig. 3B, the concentrations of FSH in serum exhibited an appreciable decrease in all treatment groups, and the decrease was most obvious in the 0.5 µm PS-MPs treatment group. Meanwhile, the concentrations of testosterone in serum were markedly declined following exposure to various sizes of PS-MPs (Fig. 3C).

PS-MPs downregulated the expression of steroidogenic enzymes and StAR in mice
To assess whether PS-MPs may affect testosterone synthesis, we detected the levels of enzymes correlated with testosterone synthesis in the testis tissues. The results showed that the expression of P450scc, P450c17, 3β-HSD, and 17β-HSD was downregulated in the testis tissues ( Fig. 4A and B). And for the same particle size of PS-MPs, the decrease was more obvious in the 1000 µg/L exposure group than in the 100 µg/L exposure group. In addition, we detected StAR in testes of PS-MPs-treated mice by western blotting (Fig. 4A and B) and immuno uorescence analyses (Fig. 4C), and StAR was decreased apparently in PS-MPs treated group compared with control group.

Entering of PS-MPs into Leydig cells in vitro
Confocal imaging results revealed that PS-MPs attached to and became internalized by Leydig cells (Fig.  5).

PS-MPs inhibited the expression of testosterone, steroidogenic enzymes and StAR in primary Leydig cells
Testosterone synthesis is related to steroid synthase in Leydig cells. We exposed Leydig cells to various concentrations of PS-MPs, and examined the concentrations of testosterone in the supernatant by Elisa. Figure 6A showed that the contents of testosterone were declined following treatment with PS-MPs. RT-qPCR consequences veri ed that the mRNA levels of the above enzymes were all decreased (Fig. 6B). Subsequently, we detected the levels of steroidogenic enzymes in Leydig cells. A concentration-dependent decreasing trend of P450scc, P450c17, 3β-HSD, and 17β-HSD were observed following exposure to PS-MPs in Leydig cells ( Fig. 6C and D). Meanwhile, the expression of StAR in Leydig cells was decreased remarkably with increasing PS-MPs concentrations (Fig. 6B, C and D).

PS-MPs exposure inhibited the activation of AC/cAMP/PKA pathway in primary Leydig cells
In order to explore the role of AC/cAMP/PKA pathway in the expression of StAR, we rst observed that AC kinase activity was suppressed in PS-MPs-treated Leydig cells (Fig. 7A). And Elisa analysis revealed that the content of cAMP was declined following treatment with PS-MPs (Fig. 7B). Moreover, PKA kinase activity was dampened with the dose of PS-MPs-exposure (Fig. 7C), and PKA catalytic subunits (PRKACA and PRKACB) decreased with the concentration of PS-MPs-exposure ( Fig. 7D and E). These results elucidated that AC/cAMP/PKA pathway was involved in the inhibition of StAR expression by PS-MPs. To prove this assumption, we used 20 mM forskolin (cAMP agonist) and 0.1 mM 8-bromo-cAMP (PKA agonist) to treat cells. The results indicated that the reduction of StAR levels caused by PS-MPs was alleviated after treated by forskolin ( Fig. 7F and G) and 8-bromo-cAMP ( Fig. 7H and I). Collectively, these results implied that PS-MPs decreased StAR expression by inhibiting activation of AC/cAMP/PKA pathway.

Exposure to PS-MPs induced decrease of LHR level in primary Leydig cells
In general, LH binds to LHR located on the membrane of Leydig cells, resulting in activation of AC/cAMP/PKA pathway. To explore the speci c reason of AC/cAMP/PKA pathway inhibition induced by PS-MPs, we detected the expression of LHR in testes ( Fig. 8A and B) and Leydig cells ( Fig. 8C and D). Western blotting analysis revealed that PS-MPs induced a reduction of LHR level. RT-qPCR consequences showed that the mRNA level of LHR was decreased (Fig. 8E). Elisa results revealed that the transcriptional enhancer Sp1 was downregulated and the suppressor AP-2 was upregulated ( Fig. 8F and G). Regulation of LHR affected the level of StAR, steroidogenic enzymes and testosterone in PS-MPsexposed primary Leydig cells To determine the role of LHR on a decrease in testosterone levels induced by PS-MPs in Leydig cells, the cells were transfected with LHR by lentivirus. QRT-PCR and Western blotting analysis suggested that the level of LHR in the Leydig cells transfected with LHR by lentivirus was overexpressed ( Fig. 9A and C). The levels of testosterone and enzymes involved in testosterone synthesis under basal condition or treatment with PS-MPs were checked in Leydig cells infected with LHR or Vector with lentivirus. As shown in Fig. 9B and Fig. 9C, the overexpression of LHR could alleviate the reduction of testosterone and enzymes levels induced by PS-MPs.

Discussion
In present study, we discovered that polystyrene MPs (PS-MPs) treatment induced the destruction of testicular histology, abnormal spermatogenesis, and interference of hormone secretion in serum. In those hormones, testosterone plays a crucial role in the process of spermatogenesis and male reproductive system, and its synthesis and secretion are mainly regulated via by LH-dependent signaling pathways in Leydig cells [28,29]. In this study, primary Leydig cells were used to discuss the mechanisms of the severely decreased testosterone levels that caused by PS-MPs treatment. Firstly, we con rmed that PS-MPs treatment affected the expression levels of steroidogenic enzymes. StAR is an important regulator of steroid hormone synthesis. And we discovered that following exposure to PS-MPs, the expression of StAR was obviously suppressed. Many factors affected the expression of StAR, such as cAMP/PKA signaling pathway, protein kinase C, and regulatory factors [32,33,34]. However, cAMP/PKA signaling pathway was the main factor affecting the expression of StAR. In our results, the inhibition of cAMP/PKA signaling pathway was observed, and the decrease of StAR expression induced by PS-MPs was alleviated following treatment of cAMP agonist and PKA agonist. In general, the cAMP/PKA signaling pathway was activated when LH binds to LHR. Meanwhile, we found that PS-MPs triggered a decrease of LHR protein levels, and overexpression of LHR could alleviate the reduction of testosterone levels induced by PS-MPs.
In conclusion, MPs reduced testosterone levels via regulating the testosterone synthesis pathway which was depended by LH, and the LHR was the critical initiator.
In this study, we had planned to try to explore the relationship between degrees of toxicity and particle size of MPs, so we chose three sizes (0.5 µm, 4 µm, 10 µm) of MPs to conduct the animal experiments in vivo. Whereas, we could not nd the signi cant difference among the three sizes in indexes related to reproductive ability. Well, these results were similar with some researches which regarded that PS-MPs of 0.5 and 50 µm were no difference to induce gut microbiota dysbiosis [35]. In addition, our results revealed that PS-MPs attached to and became internalized by Leydig cells. The results were similar with previous study which demonstrated that environmentally exposed MPs particles were internalized signi cantly into macrophages [36]. The doses of PS-MPs used in vitro experiments were selected for the purpose of building a cell model of PS-MPs-induced testosterone-reduction for subsequent studies on the mechanism.
It is the rst study to explore the reproductive effects of long-term MPs-exposure under the environmental pollution concentrations. In this study, the choice of MPs concentrations (100 µg/L and 1000 µg/L) based on both some previous studies and were close to those found in the natural contaminated areas [15,19,35]. We can nd there were concentration-dependent decreases among the sperm abnormality in 4 µm group, FSH level in 0.5 µm group, and several steroidogenic enzymes levels in three sizes groups. The doses of PS-MPs used in vitro experiments were selected for the purpose of building a cell model of PS-MPs-induced testosterone-reduction for subsequent studies on the mechanism.
In this study, we rstly demonstrated that long-term exposure to PS-MPs in environmental pollution concentrations caused damage to testicular tissue structure, decreased sperm quality and decreased testosterone levels, resulting in male reproductive toxicity in mice. In addition, we showed that the decrease of testosterone level induced by PS-MPs was achieved by inhibiting the LH-mediated LHR/cAMP/PKA/StAR pathway. Moreover, LHR may play a critical role in decrease of testosterone level induced by PS-MPs.

Conclusions
Taken together, destruction of testicular histology, abnormal spermatogenesis, and interference of hormone secretion in serum were observed in mice following 180-days-exposure to 0.

Sperm Viability and Sperm Abnormality Assessment
Sperm viability and sperm abnormality assessment were performed as previously described [37].
Quantitative Real-time PCR (qRT-PCR) Total RNA was extracted using Trizol reagent (Vazyme, Nanjing, China) according to the manufacturer's protocol. The cDNA was synthesized by using HiScript Q RT SuperMix (Vazyme, Nanjing, China). QRT-PCR were performed as previously described [38]. The primer sequences were exhibited in Supplementary  Table S1.

Immunofluorescence Staining
Immuno uorescence staining were performed as previously described [38]. The primary antibody rabbit anti-StAR (Cell Signaling Technology, USA) was employed. The secondary antibody Alexa Fluor 594conjugated goat anti-rabbit IgG was used in the study. Nuclei were stained with DAPI (Sigma). The images were captured using FV10i microscope (Olympus, Japan).

Statistical Analysis
GraphPad Prism 8 (USA) was applied for statistical analysis. Oneway ANOVA with Dunnett's t-test was used to analyze differences between groups. The data were shown as mean values ± SD. The value P < 0.05 was regarded as statistically signi cant.

Declarations
Ethics approval and consent to participate All experimental procedures were approved by the animal protocol number SYXK (Su) 2009-0017 by the Animal Care and Use Committee of Nanjing University.

Consent for publication
Not applicable.

Availability of data and materials
The datasets supporting the conclusions of this article are included within the article, Supplementary Materials, and cell imaging.