Xuelian Zhang,Ming Li,Wenjie Li,Linxiu Yue,Taiyuan Zhang,Qi Tang,Naidan Zhang,Xianyong Lan,Chuanying Pan
Chlorpyrifos induces male infertility in pigs through ROS and PI3K-AKT pathway
Toxicology,Reproductive medicine,Chemistry,Biological sciences,Environmental toxicology
Summary Chlorpyrifos (CPF) has been shown to have male reproductive toxicity in mice and rat. However, the association of CPF and male reproduction in pigs remains unknown. Therefore, this study attempts to investigate the damage of CPF on male reproduction in pigs and its potential molecular mechanisms. First, ST cells and porcine sperms were treated with CPF and then cell proliferation, apoptosis, motility of sperm, and oxidative stress levels were examined, respectively. Meanwhile, RNA sequencing was performed on ST cells before and after the treatment of CPF. The results of experiments in vitro showed that CPF had broad-spectrum toxic effects on ST cells and porcine sperms. The RNA-sequencing data and WB results indicated that CPF may regulate cell survival through the PI3K-AKT pathway. In conclusion, this study may lay the foundation for improving male fertility in pigs and provide theoretical information for human infertility.
Chlorpyrifos induces male infertility in pigs through ROS and PI3K-AKT pathway

Chlorpyrifos (CPF) has been shown to have male reproductive toxicity in mice and rat. However, the association of CPF and male reproduction in pigs remains unknown. Therefore, this study attempts to investigate the damage of CPF on male reproduction in pigs and its potential molecular mechanisms. First, ST cells and porcine sperms were treated with CPF and then cell proliferation, apoptosis, motility of sperm, and oxidative stress levels were examined, respectively. Meanwhile, RNA sequencing was performed on ST cells before and after the treatment of CPF. The results of experiments in vitro showed that CPF had broad-spectrum toxic effects on ST cells and porcine sperms. The RNA-sequencing data and WB results indicated that CPF may regulate cell survival through the PI3K-AKT pathway. In conclusion, this study may lay the foundation for improving male fertility in pigs and provide theoretical information for human infertility.

In recent years, global semen quality is declining, including declines in sperm concentration, mean seminal volume, and sperm motility. This will certainly lead to reduced male fertility. Although genetic differences may play a detrimental role in male fertility, numerous studies indicated that environmental factors are potential contribution of it. Montano et al. showed that organic chemical pollutants have a great toxic effect on male reproduction., Meanwhile, heavy metal pollution in the environment is also an important causal factor for male sterility., Furthermore, in order to guarantee agricultural demand and high and stable crop production due to the increase in the population, increasing use of pesticides has become uncontrollable. According to the latest Worldometer data, the use of pesticides worldwide has reached 4 million tons (searched on October 13, 2022)., Along with the use of organic compounds such as pesticides, their residues will lead to massive damage to human and animal health, biodiversity, and ecosystems., Chlorpyrifos (CPF), a widely used organophosphorus pesticide, is a nerve-touch pesticide. In recent years, the household purpose of eliminating cockroaches or termites has been banned in America, whereas, from 1992 to 2017, approximately 100,000 tons of CPF was still used in agricultural production. Until now, CPF is still widely utilized worldwide. The damaging effects of CPF have been identified in a variety of organisms, and its toxic manifestations are diverse, including neurotoxicity,, imbalance of intestinal flora, impaired intestinal transport, immune impairment and inflammatory damage, mitochondrial damage and oxidative stress, embryotoxicity, and metabolic disorders., Notably, growing evidence identifies that CPF can pose reproductive toxicity and seriously threaten the physiological health of animals. On the one hand, CPF affects ovarian steroid synthesis and oxidative stress levels in female rats, leading to changes in the normal cyclicity of the animals,, and causes spindle disruption and mitochondrial dysfunction in porcine oocytes during maturation in vitro. On the other hand, in rats or mice, CPF induces oxidative stress in the male reproductive organs and germ cell apoptosis., It also suppresses spermatogenesis, sperm motility, and steroid hormone synthesis.,, Of note, it can alter sperm and progeny methylation patterns and reduce male fertility., However, until now, no studies on CPF and male reproduction in pigs have been seen. Currently, mice and rats are the most commonly used mammals in toxicology experiments, but the distinct differences from humans, especially the inconsistent body size, have limited their usage as a model for human disease. As a result, increasing attention has been altered to larger animals such as pigs. Furthermore, pork is the main meat consumed globally and is an important part of the diet. More importantly, 90% of pigs are produced through artificial insemination of boar sperm, and in this way, improving boar sperm quality will maximize future production. Therefore, studying male reproductive regulation in pigs could greatly improve the productivity of the pork industry and meet future demand for food. In summary, the aim of this study was to investigate the potential molecular mechanisms of CPF affecting male reproduction in pigs. Therefore, ST cells were cultured in vitro, cell cycle and apoptosis were detected, oxidative stress levels were examined, and RNA sequencing was used. This study, taken together, lays the foundation for improving male fertility in pigs and provides theoretical information for human infertility.

In order to research the toxic effect of CPF, it was first added to the medium of ST cells for 24 and 48 h in vitro. The results showed that after the treatment of CPF25 and CPF50, the number of dead cells floating in the ST cell culture medium increased and the number of adherent cells decreased (Figure 1A). In line with this, further CCK8 results also reflected that exposure to CPF for 24 or 48 h could significantly reduce the cell viability of ST cells (Figures 1B and 1C).

There may be two reasons for the decrease of adherent cells. One is to inhibit the cell cycle, and the other is to promote apoptosis. Therefore, we first examined changes in the ST cell cycle after CPF treatment. The results showed that after CPF treatment for 24 h, the cells in the DMSO, CPF25, and CPF50 groups were 49.00 ± 0.28, 69.48 ± 1.24, and 66.82 ± 0.93 in the G1 phase, 23.67 ± 1.46, 9.38 ± 0.41, and 10.24 ± 0.32 in the S phase, and 27.33 ± 1.38, 21.14 ± 1.41, and 22.97 ± 0.65 in the G2 phases, respectively (Figures 2A–2C). Overall, ST cells were arrested in the G1 phase, while cells in S and G2 phases were reduced (Figure 2D). In addition, the expression of PCNA also decreased in a dose-dependent manner after CPF treatment of ST cells for 24 h (Figure 2E).

Next, the apoptosis of ST cells after CPF treatment was examined. The results pointed out that after ST cells were treated with 50 μM CPF for 24 h, the proportion of early apoptotic cells was significantly elevated, while the number of live cells was significantly diminished (Figures 3A–3C). Consistent with this, the mRNA expression of Caspase3 increased significantly (Figure 3D).

Most apoptosis in the testis results from dysregulated levels of intracellular oxidative stress. Therefore, according to the previously described results, the level of oxidative stress in ST cells after CPF treatment was further evaluated. The results showed that after CPF treatment for 24 h, the level of reactive oxygen species (ROS) in ST cells was significantly increased (Figure 4A), and the MMP and total antioxidant capacity were significantly decreased (Figures 4B and 4C). Meanwhile, after CPF treatment for 48 h, the level of ROS in ST cells also increased significantly in a dose-dependent manner (Figure 4D). We speculate that CPF may induce apoptosis by increasing cellular oxidative stress.

Subsequently, data analysis was performed based on the ST cell RNA-sequencing results. The sample expression violin plot showed that the quality of the sequencing data was up to standard (Figure 5A). The results of principal component analysis and heatmap displayed that the consistency within the group was good, and the samples were qualified for subsequent experiments (Figures 5B and 5C). The statistical chart of differential genes represented that after 25 μM CPF treated ST cells for 24 h, compared with the DMSO group, a total of 1117 differential genes, of which 398 were upregulated and 719 were downregulated; in the DMSO vs. CPF50 group, 2189 differential genes, of which 1076 were upregulated and 1113 were downregulated. Compared with CPF25, CPF50 had fewer differential genes, with a total of 709 genes, of which 497 were upregulated and 212 were downregulated (Figure 5D). The results of the comparative analysis of the differential genes showed that 91 overlaps in the three groups of differential genes, and this part of the differential genes will be considered in later experiments (Figure 5E). Gene Ontology (GO) enrichment was performed on the differential genes, cell process, reproduction, growth, antioxidant activity, cell adhesion and cell immunity have changed in CPF25 and CPF50 groups, compared with the DMSO groups (Figures 5F and 5G). And the results of differential gene GO enrichment compared to CPF25 and CPF50 were similar to the previously described results (Figure 5H). Kyoto Encyclopedia of Genes and Genomes (KEGG) results revealed that compared with the DMSO groups, the differential genes in the CPF25 groups were mainly enriched in IL17, TNF-α, PI3K-AKT, protein design, and absorption and metabolism pathways, whereas those in the CPF50 groups were mainly enriched in IL17, TNF-α, PI3K-AKT, steroid biosynthesis, JAK-STAT, metabolism pathways, etc (Figures 5I and 5J). CPF25 compared with CPF50 groups. IL17, TNF-α, JAK-STAT, cell cycle, and P53 pathways were significantly changed (Figure 5K). The results of Reactome analysis showed that the differential genes were related to cell metabolism, hormone synthesis, and cell cycle (Figures 5L–5N). These sequencing results were all consistent with our observed cellular phenotypes.

Based on the previously described differential gene analysis data, we selected and verified the expression of inflammatory response-related genes (IL19 and IL20), redox-related genes (PTGS2, GSTA4, and TXNIP), reproduction-related genes (HSD3B1 and NPC1L1), and cycle-related genes (CDKN1A). The results demonstrated that after CPF treatment, the expressions of IL19, IL20, CDKN1A, and PTGS2 in ST cells were significantly upregulated (Figures 6A–6D); the expressions of HSD3B1, GSTA4, and TXNIP were significantly downregulated (Figures 6E–6G). NPC1L1 expression was inhibited but not significantly (Figure 6H). The previously described results were equal to the RNA-sequencing analysis.

Based on the sequencing results and previous articles, the PI3K-AKT pathway was first verified by western blot. The results indicated no significant change in total PI3K and AKT expression (Figures 7A, 7B, 7D, and 7E). In contrast, the expression of the downstream effector protein p-AKT was significantly reduced (Figures 7C and 7F).

To get further insight into the previously described results, the changes in sperm motility after CPF incubation in vitro were examined using boar sperms. The results illustrated that, compared with the NC group, the sperm motility of CPF12.5, CPF25, and CPF50 groups decreased to varying degrees after incubation for 6 h (Figure 8A). This difference continued to widen up to 6 days of treatment (Figures 8B–8F). Then the results of ROS detection showed that CPF incubation for 1 day or 2 days did not change the level of ROS in sperm (Figures 8G and 8H); but after 3 days, the level of ROS in boar sperm increased significantly (Figure 8I). The results of CPF treatment for 6 days were consistent with those of 3 days (Figure 8J). In addition, CPF incubation for 3 days, MMP and total antioxidant capacity were significantly reduced (Figures 8K and 8L). Overall, the results of sperm were the same as the results of ST cells.

About 15% of couples worldwide suffer from infertility problems, and male fertility accounts for about 50%–60% of them. According to two meta-analyses in 1992 and 2000, the quality of male semen worldwide has declined over time., Environmental factors are considered to be one of the major factors leading to the decrease in sperm quality. CPF, as an endocrine disruptor, can pose a threat to male reproduction., However, most of the current studies on CPF have focused on aquatic organisms and laboratory mice or rats,,, and only a paucity of studies have focused on large experimental animal models. As a vital model organism, pigs share many similarities to humans. Taking full advantage of this, the present study aimed to reveal the potential molecular mechanisms of CPF damage to male reproduction in pigs and to provide the theoretical basis for CPF damage to male reproduction. The ST cell line (a porcine testicular mixed cell line) was used to explore the potential mechanism of CPF-impaired male reproduction in pigs. Cell culture brightfield plots revealed that the number of adherent cells was significantly reduced upon CPF treatment, as indicated by declined cell proliferation and increased cell death (Figure 1). Given that cell proliferation depends on the robustness of the cell cycle, the cell cycle of ST cells after CPF treatment was examined. The results showed that cell arrest occurred in ST cells after CPF treatment compared to the control group and the percentage of cells in the G1 phase increased significantly (Figure 2). This may be due to the presence of DNA damage that prevents the cells from passing through the G1/S cell cycle transition. Of note, the increased early apoptotic cells after CPF treatment also provided support for the decrease in apposed ST cells (Figure 3). ROS have an essential role in male reproduction, and moderate amounts of ROS facilitate sperm energy metabolism, motility, and fertilization. However, due to the specific differentiation of spermatozoa, mature sperms have limited antioxidant capacity. When excessive ROS are produced, they will hardly be removed by sperm in an efficient and rapid way, thereby negatively affecting sperm motility, fertilization, etc. In addition, certain proteins such as sperm nuclear basic proteins can also exacerbate DNA damage and thus intracellular oxidative damage in the presence of adversity. It has been well documented that environmental pollutants can impair male reproduction by affecting oxidative stress. For example, there is a positive correlation exists between Ni concentration in gonad of Mytilus galloprovincialis and π-gst; pyriproxyfen induced male reproductive disorders in zebrafish by continuously affecting the antioxidant mechanism of spermatogenesis; abamectin caused apoptosis and autophagy in TM3 cells through ROS accumulation; fluorochloridone affected TM4 cell autophagy via the ROS-mediated AKT/mTOR pathway. And our previous studies have also shown that CPF can lead to male infertility in mice via ROS., Therefore, in this study, oxidative stress levels were measured in ST cells and porcine sperm after CPF treatment, and partial results were consistent with previous mouse studies (Figures 4 and 8). GO enrichment results also displayed that CPF treatment affected cellular antioxidants (Figure 5 F, G, and H); the alternations in the expression of redox-related genes (PTGS2, GSTA4, and TXNIP) were also observed (Figure 6). In brief, these results suggest that CPF can induce male reproductive disorders in pigs by promoting ROS accumulation. Combining the results of KEGG enrichment of ST cells and sequencing data of CPF-treated mouse cells (data unpublished), the PI3K-AKT pathway was selected for subsequent experiments. The PI3K-AKT pathway can be involved in a variety of cellular processes, such as protein synthesis, survival, migration, proliferation, glucose metabolism, and neuroscience. The results illustrate that p-AKT expression was reduced in ST cells after CPF treatment (Figure 7), which is consistent with the findings of Wang and Yang et al., High levels of ROS can inhibit AKT activation via PI3K, which explained that the treatment of CPF induces oxidative stress and thus inhibits AKT activation. In addition, the PI3K-AKT pathway can directly regulate cell survival and cell cycle through P21, cyclin D, etc., which may be a potential contribution to cell cycle distribution in ST cells; it can also directly act on Bad, cleaved Caspase9, etc. to regulate apoptosis,, which could be one of the potential causes of apoptosis in ST cells. In summary, the treatment of CPF induced oxidative stress in porcine male germ cells, which in turn inhibited PI3K-AKT pathway activity, arrested the cell cycle, increased apoptosis, and reduced male fertility. Moreover, in the results, both GO enrichment and KEGG enrichment showed that protein design and absorption and protein binding were altered after CPF treatment of ST cells. Since CPF can affect epigenetic modifications of a variety of cells, does this led to altered epigenetics in ST cells? Furthermore, environmental toxicants have a great impact on sperm epigenetic modifications. Could CPF affect sperm epigenetic modifications (including histone deacetylation, histone acetylation, etc.)? This issue, being of great significance, still needs to be addressed in more depth. In conclusion, this study revealed that CPF could detrimentally impact the viability, cell cycle, apoptosis, ROS, and antioxidant capacity of ST cells. Meanwhile, CPF reduced the motility of porcine sperm and induced oxidative stress. RNA sequencing indicated that the previously described results were plausible and CPF can regulate ST cell survival through the PI3K-AKT pathway. Our results decipher the mechanism of CPF in regulating pig reproduction and provide a theoretical basis for the treatment of reproductive diseases.

This study reveals that CPF can cause ST cell-cycle arrest and apoptosis via the ROS/PI3K-AKT signaling pathway, leading to a decrease in male fertility in pigs. However, further research is necessary to fully understand the effects of CPF on male reproduction. Firstly, it is essential to investigate if there are other mechanisms or pathways through which CPF impacts male fertility. Secondly, we must explore the potential target genes of CPF-induced reduction in male fertility. Moreover, it is crucial to examine the residual amount of CPF in the testis of humans and validate the findings mentioned previously.



Further information and requests for resources and reagents should be directed to and will be fulfilled by the Chuanying Pan (Chuanyingpan@126.com).

There was no newly generated material in this study. ST cell line in this study is listed in the key resources table and available from the lead contact on request.

The cell culture medium contained 86% DMEM/HIGH GLUCOSE, 10% fetal bovine serum, 1% Penicillin-Streptomycin, 1% GlutaMAX, 1% AdvanceSTEM ES Qualified Non-Essential Amino Acids (100X) and 1% Sodium Pyruvate. The densities of the cells on 6-well plates were 30 × 104 cells. Treatments were conducted after 12 h of cell culture. CPF is dissolved in DMSO. According to the published articles, previous experimental results in mouse and pre-experimental results, in the ST cell line, 25 μM CPF (CPF25) and 50 μM CPF (CPF50) were chosen and treated for 24h or 48h and then tested. Simultaneously, the same volume of DMSO (vehicle) was added as a control group. All experimental animals and experimental procedures in the experiment were approved by the Faculty of Animal Policy and Welfare Committee of Northwest A&F University, Xianyang, Shaanxi, China and fully complied with local animal welfare laws, guidelines, and policies. The semen was collected from boar breeding farms around the school. The processes of semen collection did not have any adverse effects on the boar. The collected fresh semen was placed in a bath at 37°C and sent to the laboratory within 30 min. The motility and density of the semen were tested by CASA after dilution. Semen samples with total motility greater than 80% will be used for subsequent experiments. The semen was diluted to 1×109/mL with Modena medium, and 0, 6.25, 12.5, 25 and 50 μM CPF were added. The recipe of Modena was detailed in the article by Li et al. Then the samples were incubated at 17°C incubator. Finally, motility and oxidative stress assays were performed after CPF treatment at different times.

The viability of the ST cells was evaluated using the Cell Counting Kit 8 (CCK-8) according to the manufacturer's protocol. A total of 10 μL of CCK-8 solution was added per 100 μL of cell culture medium. After incubation for 2 h in a cell culture incubator, the absorbance was measured at 450 nm using a microplate reader.

After ST cells were treated with CPF (0, 25, and 50 μM) for 24h, dead cells were removed. Live cells were collected by digestion and centrifugation. Subsequently, the live cells were resuspended and washed using PBS to remove cell debris. Next, the cells were fixed with absolute ethanol and PBS (absolute ethanol : PBS = 2 : 1) at 4 C for 30min. After fixation, cells were washed with PBS to remove ethanol and cell debris, followed by the addition of RNase to remove RNA and staining with Propidium Iodide (PI). Filtration to obtain individual suspension cells. Finally, each cell cycle was detected via flow cytometry and plotted using ModFit Software.

According to the manufacturer’s protocol, ST cells that were treated with CPF (0 and 50 μM) were stained with Annexin-V-FLUOS and PI using an apoptosis kit. The cells per tube were more than 10,000. The live cells could not be stained by Annexin V and PI; when the cells are in the early apoptosis stage, phosphatidylserine turns to the outer side of the cell membrane, and Annexin V can then bind to it and emit green fluorescence; while in late-apoptotic cells and necrotic cells, the cell membrane is incomplete, and Annexin V and PI can enter into the cells and emit red and green fluorescence at the same time. After 30 min incubation at room temperature in dark, the staining of cells was detected by flow cytometry and plotted as previously described. The excitation wavelength is 488 nm and the emission wavelength is 530 nm.

ST cells treated with CPF were lysed by RNAiso Plus. Then, chloroform, isopropanol, and ethanol were used to remove impurities. Finally, the pure RNA is resuspended in DNase/RNase-Free Water. The specific steps were referred to the manufacturer's protocol. The RNA purity and quality were assessed using the NanoDrop1000 Spectrometer. Qualified RNA samples were placed in a -80°C refrigerator for subsequent experiments.

The synthesis of first-strand cDNA was performed using Hifair® 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) according to the manufacturer’s protocol. Quantitative real-time PCR (qRT-PCR) primers were designed via NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (Table S1). The housekeeping gene, GAPDH, was used as an internal standard. The qRT-PCR was performed using ChamQ SYBR qPCR Master Mix according to the reaction and protocol described by Tang and Zhang et al., In brief, the 12 μL PCR reaction system includes 6 μL SYBR, 0.5 μL upstream primer, 0.5 μL of downstream primer, and 5 μL diluted cDNA. The PCR amplification was 95°C for 30 min for 1 cycle, 40 cycles at 95°C for 15 s, 60°C for 30 s and then 72°C for 30 s.

A ROS Assay Kit and mitochondrial membrane potential assay kit with JC-1 were used to measure the reactive oxygen species (ROS) levels and mitochondrial membrane potential (MMP) following the manufacturer's protocol, respectively. The specific experimental methods were according to Zhang et al. and Chen et al., The total antioxidant capacity assay kit with the ABTS method uses Trolox as a reference for the total antioxidant capacity of other antioxidants. ABTS is oxidized to green ABTS+ under the action of appropriate oxidants, and the production of ABTS+ will be inhibited in the presence of antioxidants. The total antioxidant capacity of the samples was determined and calculated by a multi-detection microplate reader to measure the absorbance of ABTS+ at 734nm. The total antioxidant capacity of ST cells and sperm was tested according to the manufacturer's protocol.

The extracted RNA was enriched by Oligo (dT) beads, fragmented into short fragments of mRNA, and transcribed reversely into cDNA. The cDNA was end-repaired, A base added, ligated to Illumina sequencing adapters, fragments were screened, and finally, PCR library enrichment and purification were performed. The resulting cDNA library was sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China). The obtained data were screened, compared with ribosome RNA (rRNA) and the reference genome, and quantified for gene abundance. Finally, principal component analysis (PCA) and differentially expressed genes (DEGs), including GO, KEGG and Reactome enrichment, were performed.

ST cells were washed with DPBS, then lysed with RIPA containing 1 mmol/L PMSF and phosphatase inhibitor (50X). Next, 10%-15% SDS-PAGE was used to separate the lysates, which were then transferred to PVDF membranes. The PVDF membranes were sealed using skimmed milk powder. Subsequently, antibody like PI3K was separately added and incubated overnight at 4°C. After washing and removing the antibody using TBST, the HRP-conjugated Goat Anti-Rabbit IgG or HRP-conjugated Goat Anti-mouse IgG was added and incubated for 2 h at room temperature. The secondary antibody is removed again by using TBST. Finally, the PVDF membranes were stained with reagents from the Western Bright ECL Kit and then visualized using Bio-Rad Chemidoc.

SPSS 23.0 software was used for statistical analysis of the data. Differences between groups were tested using independent samples t-test (t-test) or one-way analysis of variance (ANOVA) and were considered significant or extremely significant when P < 0.05 or < 0.01, respectively. All data are expressed as mean ± standard error of the mean (SEM). And all biological replicates in the experiments were more than or equal to 3 (n ≥ 3).