Cadmium exposure causes mouse spermatogonia apoptosis via inducing endoplasmic reticulum stress

Cadmium exposure causes mouse spermatogonia apoptosis via inducing endoplasmic reticulum stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cadmium exposure causes mouse spermatogonia apoptosis via inducing endoplasmic reticulum stress Jie Wu, Qizi Yin, Yi Wang, Rong Wang, Wenjing Gong, Yihang Chen, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3910028/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cadmium (Cd), which can cause testicular germ cell apoptosis, is a well-recognized male reproductive toxicant, but the underlying mechanism still needs investigation. To detect Cd toxicity on testicular germ cell, we treated mouse spermatogonia with CdCl 2 in vitro and investigated the responses from cells at both RNA and protein levels. After treating mouse-derived spermatogonia cell line GC-1 spg cells with 20 µM CdCl 2 for 24h, cell apoptosis was measured by TUNEL and flow cytometry assay. After then, the expressions of key genes and protein biomarkers involved in endoplasmic reticulum (ER) stress were detected by qPCR and western blot, respectively. Finally, untargeted metabolomics was performed to compare metabolic differences, and Illumina RNA sequencing was conducted to screen differentially expressed genes (DEGs). Our results indicated that Cd exposure caused cell apoptosis, DEGs were involved in several apoptosis-related pathways. Cd exposure apparently elevated the mRNA and protein expressions levels of both GRP78 and ATF6α, and disrupted the expression of many types of metabolites, especially for amino acids. Taken together, our study uncovers the pathway of Cd toxicity on mouse spermatogonia, provides deep understanding on Cd-induced testicular toxicity. cadmium spermatogonia apoptosis ATF6 transcriptome metabolomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Cd, a toxic heavy metal widely used as an important industrial raw material, is a ubiquitous environmental pollutant. It can be found in pigments, fluorescent dyes, pesticides, batteries, etc., and lead to health risk for both humans and animals[ 1 , 2 ]. Workers in Cd-related occupations are usually exposed to Cd at high level [ 3 , 4 ], while the general population is exposed to Cd via drinking water, food, and cigarette smoking at low level [ 5 ]. Since Cd is readily absorbed by the body and accumulates in the target organs (such as kidney, liver and bone, etc.), it can cause lesions even at low-level exposure [ 6 , 7 ]. As a result, Cd has been regarded as a carcinogen and exposure to Cd is associated with cancers of lung, prostate, kidney, and pancreas [ 8 – 10 ]. Recently, Cd-induced male reproductive toxicity has drawn the attention from many researchers [ 11 , 12 ]. Several studies have found that testes are exceptionally sensitive to Cd toxicity [ 13 , 14 ]. Both acute and chronic exposure to Cd can cause oxidative stress, resulting in germ cell apoptosis, testicular hemorrhage, necrosis, disruption of the blood-testes barrier (BTB) and infertility in different species [ 15 , 16 ]. Therefore, it is necessary and urgent to clarify the consequences of Cd exposure in the testis. Owing to the disrupted spermatogenesis in Cd-exposed men, studies focused on the decreased male fertility due to low sperm count and poor semen quality. Apoptosis of testicular germ cells is one of the reasons for the decrease of sperm number [ 17 , 18 ]. In line with this finding, our previous study found that Cd can cause endoplasmic reticulum (ER) stress, leading to mediate Cd-induced apoptosis of testicular germ cell in vivo [ 18 ]. However, the underlying mechanism of testicular germ cells responding to Cd stress still needs to be deeply investigated. Since testis is composed of a variety of cells, including spermatogenic cells, leydig cells, sertoli cells, peritubular muscle like cells and so on [ 19 ], it is difficult to evaluate the toxic effects of Cd on germ cells through in vivo experiments. The present study aimed at investigating Cd toxicity on mouse spermatogonia and underlying molecular mechanism because spermatogonia cells play a crucial role in spermatogenesis and have important physiological significance in maintaining normal sperm quantity. Materials and methods Reagents and antibodies CdCl 2 was purchased from Sigma-Aldrich, Inc. (St Louis, MO, USA). Fetal Bovine Serum (FBS) and DMEM with high glucose Medium (DMEM-H) were obtained from Hyclone (Logan, Utah, USA). The Cell Counting Kit-8, TUNEL C1090 and Annexin V-FITC Apoptosis Detection Kit were obtained from Beyotime Institute of Biotechnology (Shanghai, China). The Super-Signal West Femto was obtained from Thermo Fisher Scientific (Waltham, MA, USA). The PrimeScript RT reagent Kit and SYBR Premix Ex Taq II were obtained from Takara Bio. (Kyushu, Japan). Antibodies against ATF6α and GRP78 were obtained from Cell Signaling Technology (Beverley, MA, USA). Antibodies against β-actin was obtained from Santa Cruz Biotechnology (Dallas, Texas, USA). RNase-free DNase and primers were obtained from Sangon Biotech (Shanghai, China). Cell culture and treatment GC-1 spg cells (Mouse-derived spermatogonial cell line) were obtained from BeNa Culture Collection (Beijing, China). The cells were cultured in DMEM containing high glucose medium supplemented with 10% Fetal Bovine Serum and maintained at 37℃ in a humidified atmosphere containing 5% CO 2 . At 80% confluence, the cells were divided into 2 groups: control group treated with PBS and 20 µM CdCl 2 treated group (named Cd group). Cells were harvested for a serial of analyses after 24h incubation. Cell counting kit-8 assay CCK-8 assay was used to detect cell viability according to the instruction [ 17 ]. Briefly, cells were treated with 20 µM CdCl 2 for different time. After that, 10 µL of CCK-8 solution was added to the above mixture and incubated at 37℃ for 2h in the dark. The absorbance was determined at 450 nm wavelength using a microplate reader (Infinite 200 PRO Switzerland). Flow cytometry analysis About 1x10 5 cells were resuspended with 195 µL Annexin V-FITC binding solution, then 5µL of Annexin V-FITC and 10µL of propidium iodide (PI) were added in turn. After incubation at 37℃ for 20 min in the dark, cell apoptosis was measured using a flow cytometry (Beckman Coulter, CA, USA). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay Apoptosis was investigated using a One Step TUNEL Apoptosis Assay kit according to the manufacturer’s instruction. Briefly, cells were treated with 20 µM CdCl 2 for 24h, after that, cells were fixed with 4% paraformaldehyde for 30 min, then permeabilized in 0.1% Triton X-100 for 5 min and incubated with TUNEL assay reagents for 1h at 37 ℃. After washing with PBS, cells were stained with 4′,6-diamidino-2-phenylindole and antifade mounting medium. Images were captured using a fluorescence microscope. Isolation of total RNA and qPCR Total RNA was extracted from GC-1 spg cells using TRI reagent (Takara Bio Inc, Kusatsu, Shiga, Japan), followed by genomic DNA removing using DNase. Primer sequences were as follows: β-actin, forward, 5’-GGGAAATCGTGCGTGAC-3’ and reverse, 5’-AGGCTGGAAAAGAGCCT-3’; ATF6, forward, 5’-TCGCCTTTTAGTCCGGTTCTT-3’ and reverse, 5’-GGCTCCATAGGTCTGACTCC-3’; GRP78, forward, 5’-GATAATCAACCAACTGTTAC-3’ and reverse, 5’-GTATCCTCTTCACCAGTTGG-3’; The expression level of each gene was normalized to the β-actin, and the qPCR reactions were performed in triplicate. Western blot Western blot analysis was performed with reference to previous studies [ 20 ]. Briefly, total protein of the cells (20–50 µg per well) was separated in 8%-15% SDS-PAGE and transferred onto a polyvinylidencedifluoride membrane. After blocking the proteins using 5% fat-free milk, the membranes were incubated with the following antibodies: β-actin, ATF6α, GRP78. After washing, the membranes were incubated with the corresponding secondary antibodies. The signal was detected using ultra-sensitive ECL chemiluminescence kit (Thermo Fisher, Waltham, MA, USA). Non-targeted metabolomics profiling analysis To determine the metabolites that significantly changed in cells, the cells were rinsed thrice with ultrapure water in three replicates per group, and immediately frozen with liquid nitrogen for the extraction of metabolites. The metabolommic analysis was conducted by Shanghai Personal-bio Technology Co. Ltd. using liquid chromatography-mass spectrometry (LC-MS) system. Based on previous research [ 21 ], the raw data normalization was conducted on all samples using probabilistic quotient normalization algorithm. After that, Quality control (QC)-robust spline batch correction was conducted. The supervised PLS-DA was also performed to screen variables that discriminate more specific differences between the groups. RNA Sequencing and Data Analysis RNA extraction, quantification, and transcriptome sequencing were performed by Shanghai Personal-bio Technology Co. Ltd. The transcriptome libraries were generated on Illumina HiSeq 2500 platform. Raw reads were qualified by removing adapters, poly-N, and low-quality reads. All following analyses were based on these clean, high-quality reads. The level of gene expression was determined by the number of fragments per kilobase of the transcript sequence per million base pairs sequenced (FPKM). The analysis of the differentially expressed genes (DEGs) was conducted using DESeq R package. Gene ontology (GO) enrichment analysis of the DEGs was performed via GOseq R packages. The role of DEGs in some pathways was consulted to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Statistical analysis In the present study, all data were shown as mean ± standard deviation (SD). All statistical analysis was performed with SPSS 23.0. Differences between the groups were determined by one-way analysis of variance (ANOVA) or Student’s t -test. P < 0.05 was considered statistically significant. Results Cd exposure causes declined viability in GC-1 spg cells via apoptosis To verify Cd toxicity on GC-1 spg cells, we treated cells with 20 µM of CdCl 2 . As shown in Fig. 1 , cell viability in Cd group was significantly lower than that of the control group ( p < 0.05). To confirm that Cd exposure caused the decline of cell viability via apoptosis, the apoptosis of GC-1 spg cells was detected by TUNEL assay and flow cytometry, respectively. As shown in Fig. 2 A, the quantity of apoptotic cells in Cd group was significantly increased when compared with the control group, indicating that Cd exposure induced apoptosis in GC-1 spg cells. Meanwhile, the result from flow cytometry detection (Fig. 2 B) was consistent with TUNEL assay, showing that the percentage of apoptosis in Cd group was significantly higher than that in the control group. Interestingly, this pattern exhibited a time-dependent effect (Fig. 2 C). These results indicated that Cd exposure significantly reduced cell viability via apoptosis. Cd exposure induces ER stress via ATF6 signaling pathway After observing that Cd exposure decreased cell viability via apoptosis, next we are aimed to investigate the underlying mechanism. Since GRP78 is a target of ER chaperone and ATF6 signaling pathway, to test whether Cd exposure induces ER stress via ATF6 signaling pathway, we investigated the expression levels of mRNA and protein of GRP78, respectively. As shown in Fig. 3 A & C, the expression levels of mRNA and protein of GRP78 were significantly increased in Cd group. Cd exposure also up-regulated the expression levels of mRNA and protein of ATF6α (Fig. 3 B & D). These results indicated that Cd exposure activated ATF6 signaling pathway, leading to ER stress. Cd exposure alters the metabolic pattern of GC-1 spg cells To investigate metabolic phenotypes that possibly related to apoptosis, we conducted untargeted metabolomics in cells. Firstly, we performed PCA analysis to visualize an overview of the clustering information, and observed that the two groups were obviously distinguished, indicating the totally different metabolic compositions between the two groups (Fig. 4 ). Next, we screened differentially expressed metabolites (DEMs, VIP ≥ 1, p < 0.05) between the two groups, and harvested 580 up-regulated DEMs and 434 down-regulated DEMs. To better illustrate the expression pattern of these DEMs, we conducted hierarchical clustering analysis based on the similarity of DEMs abundance profile. As shown in Fig. 5 , these DEMs were mainly classified to lipid (28.1%), carbohydrates (22.7%), nucleotides (6.5%), amino acids (3.4%), and others. To identify the metabolic pathways involved by these DEMs, we conducted KEGG enrichment pathway analysis. As shown in Fig. 6 , seven significantly enriched KEGG pathways were identified when DEMs from the Cd-exposed cells were searched against the KEGG database. These KEGG pathways included thiamine metabolism, protein digestion and absorption, histidine metabolism, aminoacyl-tRNA biosynthesis, bile secretion, purine metabolism, and ABC transporters. According to their expression pattern, these DEMs were divided into two categories (Fig. 7 ): one was significantly increased under the stress of Cd, including 18 amino acids, 9 lipids, 2 carbohydrates and 1 nucleotide; another was significantly decreased, including 9 lipids and 1 nucleotide. Interestingly, we found that most of up-expressed metabolites are amino acids. Our results of qPCR and western blot have demonstrated that Cd imposes its toxicity on GC-1 spg cells through ATF6 signaling pathway, combining with the fact that this pathway plays an important role in protein metabolism, including protein synthesis, transport, folding, and etc. These results are consistent and indicating that these metabolites, especially amino acids, might play an important role in cells responding to Cd toxicity. Cd exposure changes the transcriptional pattern of GC-1 spg cells To deeply understand the molecular mechanism involved in Cd toxicity on GC-1 spg cells, we performed transcriptional sequencing using Illumina NovaSeq. After removing unqualified reads, we harvested an average of 43,023,796 and 42,143,998 clean reads from the two groups, respectively. To check the quality of the RNA sequencing data, we mapped these clean reads to the reference genome. Over 94% of the clean reads were mapped to the mouse reference genome. The detailed information is summarized in Table S1. These results demonstrated that our RNA-sequencing data was reliable. Next, we conducted gene annotation and function analyses. All the identified genes were aligned to the public databases, including Gene Ontology (GO) and KEGG, and annotated using the GO database, followed by KEGG database. Finally, the DEGs were identified according to the RPKM ratio greater than two folds. The result of DEGs analysis revealed that many genes in GC-1 spg cells responded to Cd toxicity. Compared to the control group, 310 genes exhibited 2-fold or higher differential expression, including 272 up-regulated genes and 38 down-regulated genes. According to the result of KEGG pathway analysis, we found that these 272 up-regulated genes could be classified into four categories: the first category was involved in cell cycle, autophagy, and apoptosis. The second category was involved in several cell apoptosis-related signaling pathways, including p53 signaling pathway, FoxO signaling pathway, MAPK signaling pathway, and NF-κB signaling pathway. The third category was involved in several kinds of diseases. The fourth category was involved in protein digestion and absorption. Most of the 38 down-regulated genes were involved in four pathways, including Hippo signaling pathway, mTOR signaling pathway, Wnt signaling pathway, and ABC transporters. All of them are related to cell growth and proliferation. The detailed information is presented in Fig. 8 & Fig. 9 . Discussion Here, we found that caused the decline of mouse spermatogonial via activating ATF6 signaling pathway, leading to cell apoptosis. Moreover, some important metabolites, especially amino acids, were involved in the response to Cd toxicity. Meanwhile, the expression of several DEGs, relating to cell growth and proliferation, were disrupted. Our study reports a mechanism in the apoptosis of mouse spermatogonial caused by Cd exposure. Impact of Cd exposure on cell apoptosis In line with recent studies reporting that Cd can cause testicular germ cell apoptosis in rodent animals [ 18 , 22 ] and humans [ 23 ], our previous works also found that Cd exposure caused testicular spermatogonia in vitro [ 17 ]. In the present study, we found that Cd exposure remarkably reduced cell viability, and significantly elevated the percentage of apoptotic cells. These results suggested that apoptosis of mouse spermatogonia is one of the toxic effects imposed by Cd. ER is an important organelle and responsible for protein synthesis and transport, protein folding, lipid and steroid synthesis, carbohydrate metabolism and calcium homeostasis in eukaryotic cells [ 24 , 25 ]. Once unfolded and misfolded proteins are retained in the ER lumen, ER stress occurs, leading to the activation of the unfolded protein response (UPR) [ 26 ]. However, persistent or severe UPR activation can lead to apoptosis [ 27 ]. Many studies found that ER stress was related to male reproduction and infertility in animal models. Our previous study also reported ER stress in mice caused by Cd exposure and ER stress signaling mediate Cd-induced germ cell apoptosis in mouse testes[ 18 ]. Both ER stress and UPR are mediated by the three transmembrane ER proteins, including activating transcription factor 6 (ATF6), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and inositol-requiring enzyme 1α (IRE1α) [ 24 , 28 ]. ATF6 is mainly distributed in endoplasmic reticulum membrane under normal conditions. However, it is transferred to Golgi apparatus, cleaved and activated when exposed to stresses [ 28 , 29 ]. Therefore, ATF6 acts as an active transcription factor to increase the transcriptional levels of GRP78, GRP94 and other genes encoding endoplasmic reticulum chaperones [ 25 ]. Our result is in line with these reports, showing that the stress caused by Cd exposure upregulates ATF6 mRNA and protein expression levels in spermatogonia. Our finding demonstrate that CdCl 2 can activate ATF6 signaling pathway of UPR in spermatogonia. Metabolic response of GC-1 spg cells to Cd exposure By taking advantage of metabolomics, we deepened our understanding about the variations in the metabolic capacity of the cells under Cd stress. Notably, 1014 metabolites from 7 key metabolic pathways were enriched in Cd- treated cells. Most of these pathways were involved in protein digestion and absorption. At the same time, we observed an increment of amino acids. Amino acids are the product of protein digestion [ 30 ], and some intermediate metabolites can be used as signals for cells under the stress of heavy metals [ 31 , 32 ]. Our results imply that protein digestion was promoted in Cd-treated cells. Consistent with our finding, Lv et al. reported that amino acid metabolism was inhibited in spider exposed to Cd [ 33 ]. Thiamine plays an important role not only in the synthesis of carbohydrate, nucleic acids, adenosine triphosphate and nicotinamide adenine dinucleotide phosphate [ 34 ], but also in the relieving stress. A previous study reported that thiamine promotes the tolerance of strawberry to Cd toxicity [ 35 ]. It also can reduce hepatic and renal burden from Cd in rats [ 36 ]. We also observed high abundance of thiamine in Cd-treated cells. It was reported that histidine-rich Pseudomonas metallothionein displays higher binding capacity for cadmium than zinc [ 37 ]. Immobilizing Cd is one of the strategies in alleviating Cd toxicity [ 38 ]. The enrichment of histidine metabolism illustrated the strategy of GC-1 spg cells alleviating Cd toxicity. Transcriptional response of GC-1 spg cells to Cd exposure Transcriptional analysis allows us to explore the molecular mechanism of the cells to Cd stress. The possible function of DEGs can be deduced because the genes within the same pathway usually cooperate with each other to run their biological function [ 39 ]. Although many studies have demonstrated that Cd exposure has toxic impact on the reproductive system, leading to a decreased fertility [ 11 ], the underlying mechanism is still unclear. Our results based on the analysis of DEGs indicated that Cd exposure impacts GC-1 spg cells via multiple ways. Firstly, Cd exposure promotes the formation of free radicals and leads to cell apoptosis. This finding is consistent with the results of TUNEL assay and flow cytometry, and supported by the previous reports [ 40 , 41 ]. Liu et al . reported that BIP/GRP78 and the following PERKp-eIF2α-ATF4 pathway is activated by Cd exposure, leading to endoplasmic reticulum stress-induced ovarian granulosa cell damage [ 42 ]. Interestingly, we observed the activation of PERKp-eIF2α-ATF6 pathway by Cd exposure in GC-1 spg cells. We deduced that male & female mice-originated germ cell lines exhibit different responses to Cd toxicity. Meanwhile, we found that several DEGs were involved in p53 signaling pathway, FoxO signaling pathway, MAPK signaling pathway, and NF-κB signaling pathway. Our finding is consistent with the previous report [ 43 ], indicating that these pathways participate in Cd-induced cell apoptosis. In summary, our transcriptional results suggested that DEGs are involved in several apoptosis-related pathways in Cd exposed cells. Conclusion In this study, we demonstrated that Cd exposure induces ER stress via ATF6 signaling pathway, leading to apoptosis in vitro. The disrupted metabolism of amino acids and the activation of PERKp-eIF2α-ATF6 pathway may play important roles. Declarations Author Contributions: Conceptualization, Jie Wu, Qizi Yin, Yi Wang, Rong Wang and Wenjing Gong; methodology, Yihang Chen, Jie Wu and Mingming Zhang, project administration, Rong Wang; supervision, Yanli Ji; writing—original draft preparation, review and editing, Yanli Ji and Yehao Liu; funding acquisition, Yanli Ji and Yehao Liu. All authors have read and agreed to the published version of the manuscript. F unding: This research was funded by Natural Science Foundation of China (31571557), the Natural Science Foundation of Anhui Province (2208085MH263, 1908085MH287) and Innovation and Entrepreneurship Training Program for College Students of Anhui Medical University(S202110366089). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3910028","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":270462829,"identity":"fbe3a34b-01b2-4a40-b4ff-7475d6d9f8ad","order_by":0,"name":"Jie Wu","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Wu","suffix":""},{"id":270462830,"identity":"04a40254-59f0-4268-817e-13af365f161f","order_by":1,"name":"Qizi Yin","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qizi","middleName":"","lastName":"Yin","suffix":""},{"id":270462831,"identity":"dc141c96-60ec-40b2-afdf-faa74f391e05","order_by":2,"name":"Yi Wang","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Wang","suffix":""},{"id":270462832,"identity":"5a98bce4-2330-4826-9519-59044e96bfb1","order_by":3,"name":"Rong Wang","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Wang","suffix":""},{"id":270462833,"identity":"5884b70f-b991-4f9d-b532-355d333ca3b7","order_by":4,"name":"Wenjing Gong","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Gong","suffix":""},{"id":270462834,"identity":"0ba1fdbf-ed51-4c04-99fe-58fa5c6459a2","order_by":5,"name":"Yihang Chen","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yihang","middleName":"","lastName":"Chen","suffix":""},{"id":270462835,"identity":"0ea2653a-d541-4493-b2a0-74d75491e9db","order_by":6,"name":"Mingming Zhang","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mingming","middleName":"","lastName":"Zhang","suffix":""},{"id":270462836,"identity":"fadcf0cb-fdd6-4718-aee6-17a3dcdf3ce8","order_by":7,"name":"Yehao Liu","email":"","orcid":"","institution":"Anhui Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yehao","middleName":"","lastName":"Liu","suffix":""},{"id":270462837,"identity":"95af76a3-93ac-464b-acf9-51a94e49ed9e","order_by":8,"name":"Yanli Ji","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYDACdoaEAx8YDoDZEsRpYWZIODiDVC0MzDwkaTE4zPDwsE3NnWiDA8wHb/Mw2OURoyXhcM6xZ7kbDrAlW/MwJBcTqYXtMFALj5k00IWJDURpsfgH0sL/jQQtjG1gW9iI0yIJ1HKwt+9w7szDbMaWcwySCWvhO96T/OHHt8O5fcebH954U2FHWIvCAZ4ECIsZ7E5C6oFAvoH9ABHKRsEoGAWjYEQDAG0mQ7/MBNAnAAAAAElFTkSuQmCC","orcid":"","institution":"Anhui Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yanli","middleName":"","lastName":"Ji","suffix":""}],"badges":[],"createdAt":"2024-01-30 08:50:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3910028/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3910028/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50583124,"identity":"d38144ee-1c10-4ab5-ba07-741ee263ea43","added_by":"auto","created_at":"2024-02-02 20:23:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114664,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Cd exposure on GC-1 spg cells viability was detected by CCK-8 assay.\u003c/strong\u003e Cells were treated with 20 μM CdCl\u003csub\u003e2\u003c/sub\u003e for different time. All data were expressed as means ± SD (n = 6). **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3910028/v1/18597025df81bd5171041154.png"},{"id":50582962,"identity":"55282a7d-8ff2-4bf9-8c52-62566778b2c6","added_by":"auto","created_at":"2024-02-02 20:15:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":348376,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Cd exposure on GC-1 spg cells apoptosis.\u003c/strong\u003e GC-1 spg cells were treated with 20 μM CdCl\u003csub\u003e2\u003c/sub\u003e for different time. (A) Apoptosis was detected by TUNEL assay. DAPI stained the nucleus with blue fluorescence, and TUNEL stained apoptotic cells with red fluorescence. Magnification: 200×. (B)\u003cstrong\u003e \u003c/strong\u003eThe apoptosis was detected by flow cytometry with Annexin V-FITC/PI method. (C) The percentage of apoptotic cells. All data were expressed as means ± SD (n = 3). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3910028/v1/9a119aee86c63683d400b72e.png"},{"id":50583125,"identity":"73338086-0a88-4e2f-9185-b6500179bd74","added_by":"auto","created_at":"2024-02-02 20:23:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":235890,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of Cd exposure on ATF6 signaling pathway.\u003c/strong\u003e GC-1 spg cells were treated with 20 μM CdCl\u003csub\u003e2\u003c/sub\u003e for different time. (A and B) The mRNA expression levels of GRP78 and ATF6. (C and D) The protein expression levels of GRP78 and ATF6α. All data were expressed as means ± SD (n = 3). *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3910028/v1/573b2aa352cfae734ded5da7.png"},{"id":50582954,"identity":"d576ac5b-9cb9-4eb3-956c-349b5f301a09","added_by":"auto","created_at":"2024-02-02 20:15:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":68548,"visible":true,"origin":"","legend":"\u003cp\u003eThe enriched KEGG pathways based on the metabolites profile.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3910028/v1/e47c5cc7664c1452bf1f9017.png"},{"id":50582957,"identity":"ec46b3db-5885-41c3-b6cd-f038c1c3ad22","added_by":"auto","created_at":"2024-02-02 20:15:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":233889,"visible":true,"origin":"","legend":"\u003cp\u003eThe abundance profile of different types of detected metabolites.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3910028/v1/d6bab0cc090069892e6a28dd.png"},{"id":50583782,"identity":"4d44084f-a6c0-4cc6-9a66-bf9b45a5a963","added_by":"auto","created_at":"2024-02-02 20:31:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":77620,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe enriched KEGG pathways based on the metabolites profile.\u003c/strong\u003e * \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3910028/v1/59ad8ff98b1dbc3b1c1697b0.png"},{"id":50582959,"identity":"5c3f3fc7-ecc3-4daf-bcd5-73629f48b363","added_by":"auto","created_at":"2024-02-02 20:15:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":241952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression pattern of significantly enriched metabolites.\u003c/strong\u003eUP: up-regulated DEMs, down: down-regulated DEMs. Data were shown negative ion mode.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-3910028/v1/a0c73836d9ba4ce1b4b45bba.png"},{"id":50582963,"identity":"5678345d-9994-44dd-b245-6399a77e4c1d","added_by":"auto","created_at":"2024-02-02 20:15:47","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":160531,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistogram of the KEGG pathway enrichment annotations of the differentially expressed genes (DEGs) between the two groups.\u003c/strong\u003eThe x-axis shows functional pathways and the y-axis shows statistical significance (\u003cem\u003ep\u003c/em\u003e-value).\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-3910028/v1/390f5518d5605b529b74bda4.png"},{"id":50582961,"identity":"827ff721-69af-4ab8-9f4e-49cae2a7e089","added_by":"auto","created_at":"2024-02-02 20:15:47","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":184523,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistogram of GO functional enrichment annotations of the differentially expressed genes (DEGs) between the two groups.\u003c/strong\u003e The x-axis shows top 10 terms in the three main categories, including cellular component (CC), biological process (BP), and molecular function (MF), and the y-axis shows statistical significance (\u003cem\u003ep\u003c/em\u003e-value).\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-3910028/v1/7302e4ef9822cc181eeec2aa.png"},{"id":51195703,"identity":"1ef3c52b-7ba1-4afc-9a89-41e84edda771","added_by":"auto","created_at":"2024-02-15 18:40:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1924528,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3910028/v1/131d60ad-0e73-4ee3-b0ce-35e434f37463.pdf"},{"id":50582956,"identity":"235681f6-ddf7-4280-b2a1-3096185c4b66","added_by":"auto","created_at":"2024-02-02 20:15:47","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":9071,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3910028/v1/67e14a0c0d25a8d4e1bc2a05.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cadmium exposure causes mouse spermatogonia apoptosis via inducing endoplasmic reticulum stress","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCd, a toxic heavy metal widely used as an important industrial raw material, is a ubiquitous environmental pollutant. It can be found in pigments, fluorescent dyes, pesticides, batteries, etc., and lead to health risk for both humans and animals[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Workers in Cd-related occupations are usually exposed to Cd at high level [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], while the general population is exposed to Cd via drinking water, food, and cigarette smoking at low level [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Since Cd is readily absorbed by the body and accumulates in the target organs (such as kidney, liver and bone, etc.), it can cause lesions even at low-level exposure [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As a result, Cd has been regarded as a carcinogen and exposure to Cd is associated with cancers of lung, prostate, kidney, and pancreas [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, Cd-induced male reproductive toxicity has drawn the attention from many researchers [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Several studies have found that testes are exceptionally sensitive to Cd toxicity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Both acute and chronic exposure to Cd can cause oxidative stress, resulting in germ cell apoptosis, testicular hemorrhage, necrosis, disruption of the blood-testes barrier (BTB) and infertility in different species [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, it is necessary and urgent to clarify the consequences of Cd exposure in the testis.\u003c/p\u003e \u003cp\u003eOwing to the disrupted spermatogenesis in Cd-exposed men, studies focused on the decreased male fertility due to low sperm count and poor semen quality. Apoptosis of testicular germ cells is one of the reasons for the decrease of sperm number [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In line with this finding, our previous study found that Cd can cause endoplasmic reticulum (ER) stress, leading to mediate Cd-induced apoptosis of testicular germ cell in vivo [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the underlying mechanism of testicular germ cells responding to Cd stress still needs to be deeply investigated. Since testis is composed of a variety of cells, including spermatogenic cells, leydig cells, sertoli cells, peritubular muscle like cells and so on [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], it is difficult to evaluate the toxic effects of Cd on germ cells through in vivo experiments. The present study aimed at investigating Cd toxicity on mouse spermatogonia and underlying molecular mechanism because spermatogonia cells play a crucial role in spermatogenesis and have important physiological significance in maintaining normal sperm quantity.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents and antibodies\u003c/h2\u003e \u003cp\u003eCdCl\u003csub\u003e2\u003c/sub\u003e was purchased from Sigma-Aldrich, Inc. (St Louis, MO, USA). Fetal Bovine Serum (FBS) and DMEM with high glucose Medium (DMEM-H) were obtained from Hyclone (Logan, Utah, USA). The Cell Counting Kit-8, TUNEL C1090 and Annexin V-FITC Apoptosis Detection Kit were obtained from Beyotime Institute of Biotechnology (Shanghai, China). The Super-Signal West Femto was obtained from Thermo Fisher Scientific (Waltham, MA, USA). The PrimeScript RT reagent Kit and SYBR Premix Ex Taq II were obtained from Takara Bio. (Kyushu, Japan). Antibodies against ATF6α and GRP78 were obtained from Cell Signaling Technology (Beverley, MA, USA). Antibodies against β-actin was obtained from Santa Cruz Biotechnology (Dallas, Texas, USA). RNase-free DNase and primers were obtained from Sangon Biotech (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and treatment\u003c/h2\u003e \u003cp\u003eGC-1 spg cells (Mouse-derived spermatogonial cell line) were obtained from BeNa Culture Collection (Beijing, China). The cells were cultured in DMEM containing high glucose medium supplemented with 10% Fetal Bovine Serum and maintained at 37℃ in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. At 80% confluence, the cells were divided into 2 groups: control group treated with PBS and 20 \u0026micro;M CdCl\u003csub\u003e2\u003c/sub\u003e treated group (named Cd group). Cells were harvested for a serial of analyses after 24h incubation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell counting kit-8 assay\u003c/h2\u003e \u003cp\u003eCCK-8 assay was used to detect cell viability according to the instruction [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Briefly, cells were treated with 20 \u0026micro;M CdCl\u003csub\u003e2\u003c/sub\u003e for different time. After that, 10 \u0026micro;L of CCK-8 solution was added to the above mixture and incubated at 37℃ for 2h in the dark. The absorbance was determined at 450 nm wavelength using a microplate reader (Infinite 200 PRO Switzerland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e \u003cp\u003eAbout 1x10\u003csup\u003e5\u003c/sup\u003e cells were resuspended with 195 \u0026micro;L Annexin V-FITC binding solution, then 5\u0026micro;L of Annexin V-FITC and 10\u0026micro;L of propidium iodide (PI) were added in turn. After incubation at 37℃ for 20 min in the dark, cell apoptosis was measured using a flow cytometry (Beckman Coulter, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay\u003c/h2\u003e \u003cp\u003eApoptosis was investigated using a One Step TUNEL Apoptosis Assay kit according to the manufacturer\u0026rsquo;s instruction. Briefly, cells were treated with 20 \u0026micro;M CdCl\u003csub\u003e2\u003c/sub\u003e for 24h, after that, cells were fixed with 4% paraformaldehyde for 30 min, then permeabilized in 0.1% Triton X-100 for 5 min and incubated with TUNEL assay reagents for 1h at 37 ℃. After washing with PBS, cells were stained with 4\u0026prime;,6-diamidino-2-phenylindole and antifade mounting medium. Images were captured using a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of total RNA and qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from GC-1 spg cells using TRI reagent (Takara Bio Inc, Kusatsu, Shiga, Japan), followed by genomic DNA removing using DNase. Primer sequences were as follows: β-actin, forward, 5\u0026rsquo;-GGGAAATCGTGCGTGAC-3\u0026rsquo; and reverse, 5\u0026rsquo;-AGGCTGGAAAAGAGCCT-3\u0026rsquo;; ATF6, forward, 5\u0026rsquo;-TCGCCTTTTAGTCCGGTTCTT-3\u0026rsquo; and reverse, 5\u0026rsquo;-GGCTCCATAGGTCTGACTCC-3\u0026rsquo;; GRP78, forward, 5\u0026rsquo;-GATAATCAACCAACTGTTAC-3\u0026rsquo; and reverse, 5\u0026rsquo;-GTATCCTCTTCACCAGTTGG-3\u0026rsquo;; The expression level of each gene was normalized to the β-actin, and the qPCR reactions were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eWestern blot analysis was performed with reference to previous studies [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Briefly, total protein of the cells (20\u0026ndash;50 \u0026micro;g per well) was separated in 8%-15% SDS-PAGE and transferred onto a polyvinylidencedifluoride membrane. After blocking the proteins using 5% fat-free milk, the membranes were incubated with the following antibodies: β-actin, ATF6α, GRP78. After washing, the membranes were incubated with the corresponding secondary antibodies. The signal was detected using ultra-sensitive ECL chemiluminescence kit (Thermo Fisher, Waltham, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eNon-targeted metabolomics profiling analysis\u003c/h2\u003e \u003cp\u003eTo determine the metabolites that significantly changed in cells, the cells were rinsed thrice with ultrapure water in three replicates per group, and immediately frozen with liquid nitrogen for the extraction of metabolites. The metabolommic analysis was conducted by Shanghai Personal-bio Technology Co. Ltd. using liquid chromatography-mass spectrometry (LC-MS) system.\u003c/p\u003e \u003cp\u003eBased on previous research [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], the raw data normalization was conducted on all samples using probabilistic quotient normalization algorithm. After that, Quality control (QC)-robust spline batch correction was conducted. The supervised PLS-DA was also performed to screen variables that discriminate more specific differences between the groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA Sequencing and Data Analysis\u003c/h2\u003e \u003cp\u003eRNA extraction, quantification, and transcriptome sequencing were performed by Shanghai Personal-bio Technology Co. Ltd. The transcriptome libraries were generated on Illumina HiSeq 2500 platform. Raw reads were qualified by removing adapters, poly-N, and low-quality reads. All following analyses were based on these clean, high-quality reads. The level of gene expression was determined by the number of fragments per kilobase of the transcript sequence per million base pairs sequenced (FPKM). The analysis of the differentially expressed genes (DEGs) was conducted using DESeq R package.\u003c/p\u003e \u003cp\u003eGene ontology (GO) enrichment analysis of the DEGs was performed via GOseq R packages. The role of DEGs in some pathways was consulted to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eIn the present study, all data were shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). All statistical analysis was performed with SPSS 23.0. Differences between the groups were determined by one-way analysis of variance (ANOVA) or Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCd exposure causes declined viability in GC-1 spg cells via apoptosis\u003c/h2\u003e \u003cp\u003eTo verify Cd toxicity on GC-1 spg cells, we treated cells with 20 \u0026micro;M of CdCl\u003csub\u003e2\u003c/sub\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, cell viability in Cd group was significantly lower than that of the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm that Cd exposure caused the decline of cell viability via apoptosis, the apoptosis of GC-1 spg cells was detected by TUNEL assay and flow cytometry, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the quantity of apoptotic cells in Cd group was significantly increased when compared with the control group, indicating that Cd exposure induced apoptosis in GC-1 spg cells. Meanwhile, the result from flow cytometry detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) was consistent with TUNEL assay, showing that the percentage of apoptosis in Cd group was significantly higher than that in the control group. Interestingly, this pattern exhibited a time-dependent effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These results indicated that Cd exposure significantly reduced cell viability via apoptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCd exposure induces ER stress via ATF6 signaling pathway\u003c/h2\u003e \u003cp\u003eAfter observing that Cd exposure decreased cell viability via apoptosis, next we are aimed to investigate the underlying mechanism. Since GRP78 is a target of ER chaperone and ATF6 signaling pathway, to test whether Cd exposure induces ER stress via ATF6 signaling pathway, we investigated the expression levels of mRNA and protein of GRP78, respectively. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u0026amp; C, the expression levels of mRNA and protein of GRP78 were significantly increased in Cd group. Cd exposure also up-regulated the expression levels of mRNA and protein of ATF6α (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB \u0026amp; D). These results indicated that Cd exposure activated ATF6 signaling pathway, leading to ER stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCd exposure alters the metabolic pattern of GC-1 spg cells\u003c/h2\u003e \u003cp\u003eTo investigate metabolic phenotypes that possibly related to apoptosis, we conducted untargeted metabolomics in cells. Firstly, we performed PCA analysis to visualize an overview of the clustering information, and observed that the two groups were obviously distinguished, indicating the totally different metabolic compositions between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we screened differentially expressed metabolites (DEMs, VIP\u0026thinsp;\u0026ge;\u0026thinsp;1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between the two groups, and harvested 580 up-regulated DEMs and 434 down-regulated DEMs. To better illustrate the expression pattern of these DEMs, we conducted hierarchical clustering analysis based on the similarity of DEMs abundance profile. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, these DEMs were mainly classified to lipid (28.1%), carbohydrates (22.7%), nucleotides (6.5%), amino acids (3.4%), and others.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify the metabolic pathways involved by these DEMs, we conducted KEGG enrichment pathway analysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, seven significantly enriched KEGG pathways were identified when DEMs from the Cd-exposed cells were searched against the KEGG database. These KEGG pathways included thiamine metabolism, protein digestion and absorption, histidine metabolism, aminoacyl-tRNA biosynthesis, bile secretion, purine metabolism, and ABC transporters. According to their expression pattern, these DEMs were divided into two categories (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e): one was significantly increased under the stress of Cd, including 18 amino acids, 9 lipids, 2 carbohydrates and 1 nucleotide; another was significantly decreased, including 9 lipids and 1 nucleotide. Interestingly, we found that most of up-expressed metabolites are amino acids. Our results of qPCR and western blot have demonstrated that Cd imposes its toxicity on GC-1 spg cells through ATF6 signaling pathway, combining with the fact that this pathway plays an important role in protein metabolism, including protein synthesis, transport, folding, and etc. These results are consistent and indicating that these metabolites, especially amino acids, might play an important role in cells responding to Cd toxicity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCd exposure changes the transcriptional pattern of GC-1 spg cells\u003c/h2\u003e \u003cp\u003eTo deeply understand the molecular mechanism involved in Cd toxicity on GC-1 spg cells, we performed transcriptional sequencing using Illumina NovaSeq.\u0026nbsp;After removing unqualified reads, we harvested an average of 43,023,796 and 42,143,998 clean reads from the two groups, respectively. To check the quality of the RNA sequencing data, we mapped these clean reads to the reference genome. Over 94% of the clean reads were mapped to the mouse reference genome. The detailed information is summarized in Table S1. These results demonstrated that our RNA-sequencing data was reliable. Next, we conducted gene annotation and function analyses. All the identified genes were aligned to the public databases, including Gene Ontology (GO) and KEGG, and annotated using the GO database, followed by KEGG database. Finally, the DEGs were identified according to the RPKM ratio greater than two folds.\u003c/p\u003e \u003cp\u003eThe result of DEGs analysis revealed that many genes in GC-1 spg cells responded to Cd toxicity. Compared to the control group, 310 genes exhibited 2-fold or higher differential expression, including 272 up-regulated genes and 38 down-regulated genes. According to the result of KEGG pathway analysis, we found that these 272 up-regulated genes could be classified into four categories: the first category was involved in cell cycle, autophagy, and apoptosis. The second category was involved in several cell apoptosis-related signaling pathways, including p53 signaling pathway, FoxO signaling pathway, MAPK signaling pathway, and NF-κB signaling pathway. The third category was involved in several kinds of diseases. The fourth category was involved in protein digestion and absorption. Most of the 38 down-regulated genes were involved in four pathways, including Hippo signaling pathway, mTOR signaling pathway, Wnt signaling pathway, and ABC transporters. All of them are related to cell growth and proliferation. The detailed information is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e \u0026amp; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we found that caused the decline of mouse spermatogonial via activating ATF6 signaling pathway, leading to cell apoptosis. Moreover, some important metabolites, especially amino acids, were involved in the response to Cd toxicity. Meanwhile, the expression of several DEGs, relating to cell growth and proliferation, were disrupted. Our study reports a mechanism in the apoptosis of mouse spermatogonial caused by Cd exposure.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eImpact of Cd exposure on cell apoptosis\u003c/h2\u003e \u003cp\u003eIn line with recent studies reporting that Cd can cause testicular germ cell apoptosis in rodent animals [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and humans [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], our previous works also found that Cd exposure caused testicular spermatogonia in vitro [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In the present study, we found that Cd exposure remarkably reduced cell viability, and significantly elevated the percentage of apoptotic cells. These results suggested that apoptosis of mouse spermatogonia is one of the toxic effects imposed by Cd.\u003c/p\u003e \u003cp\u003eER is an important organelle and responsible for protein synthesis and transport, protein folding, lipid and steroid synthesis, carbohydrate metabolism and calcium homeostasis in eukaryotic cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Once unfolded and misfolded proteins are retained in the ER lumen, ER stress occurs, leading to the activation of the unfolded protein response (UPR) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, persistent or severe UPR activation can lead to apoptosis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Many studies found that ER stress was related to male reproduction and infertility in animal models. Our previous study also reported ER stress in mice caused by Cd exposure and ER stress signaling mediate Cd-induced germ cell apoptosis in mouse testes[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBoth ER stress and UPR are mediated by the three transmembrane ER proteins, including activating transcription factor 6 (ATF6), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and inositol-requiring enzyme 1α (IRE1α) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. ATF6 is mainly distributed in endoplasmic reticulum membrane under normal conditions. However, it is transferred to Golgi apparatus, cleaved and activated when exposed to stresses [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, ATF6 acts as an active transcription factor to increase the transcriptional levels of GRP78, GRP94 and other genes encoding endoplasmic reticulum chaperones [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Our result is in line with these reports, showing that the stress caused by Cd exposure upregulates ATF6 mRNA and protein expression levels in spermatogonia. Our finding demonstrate that CdCl\u003csub\u003e2\u003c/sub\u003e can activate ATF6 signaling pathway of UPR in spermatogonia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMetabolic response of GC-1 spg cells to Cd exposure\u003c/h2\u003e \u003cp\u003eBy taking advantage of metabolomics, we deepened our understanding about the variations in the metabolic capacity of the cells under Cd stress. Notably, 1014 metabolites from 7 key metabolic pathways were enriched in Cd- treated cells. Most of these pathways were involved in protein digestion and absorption. At the same time, we observed an increment of amino acids. Amino acids are the product of protein digestion [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], and some intermediate metabolites can be used as signals for cells under the stress of heavy metals [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our results imply that protein digestion was promoted in Cd-treated cells. Consistent with our finding, Lv et al. reported that amino acid metabolism was inhibited in spider exposed to Cd [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Thiamine plays an important role not only in the synthesis of carbohydrate, nucleic acids, adenosine triphosphate and nicotinamide adenine dinucleotide phosphate [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], but also in the relieving stress. A previous study reported that thiamine promotes the tolerance of strawberry to Cd toxicity [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. It also can reduce hepatic and renal burden from Cd in rats [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. We also observed high abundance of thiamine in Cd-treated cells. It was reported that histidine-rich \u003cem\u003ePseudomonas metallothionein\u003c/em\u003e displays higher binding capacity for cadmium than zinc [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Immobilizing Cd is one of the strategies in alleviating Cd toxicity [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The enrichment of histidine metabolism illustrated the strategy of GC-1 spg cells alleviating Cd toxicity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptional response of GC-1 spg cells to Cd exposure\u003c/h2\u003e \u003cp\u003eTranscriptional analysis allows us to explore the molecular mechanism of the cells to Cd stress. The possible function of DEGs can be deduced because the genes within the same pathway usually cooperate with each other to run their biological function [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Although many studies have demonstrated that Cd exposure has toxic impact on the reproductive system, leading to a decreased fertility [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], the underlying mechanism is still unclear. Our results based on the analysis of DEGs indicated that Cd exposure impacts GC-1 spg cells via multiple ways. Firstly, Cd exposure promotes the formation of free radicals and leads to cell apoptosis. This finding is consistent with the results of TUNEL assay and flow cytometry, and supported by the previous reports [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Liu \u003cem\u003eet al\u003c/em\u003e. reported that BIP/GRP78 and the following PERKp-eIF2α-ATF4 pathway is activated by Cd exposure, leading to endoplasmic reticulum stress-induced ovarian granulosa cell damage [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Interestingly, we observed the activation of PERKp-eIF2α-ATF6 pathway by Cd exposure in GC-1 spg cells. We deduced that male \u0026amp; female mice-originated germ cell lines exhibit different responses to Cd toxicity. Meanwhile, we found that several DEGs were involved in p53 signaling pathway, FoxO signaling pathway, MAPK signaling pathway, and NF-κB signaling pathway. Our finding is consistent with the previous report [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], indicating that these pathways participate in Cd-induced cell apoptosis. In summary, our transcriptional results suggested that DEGs are involved in several apoptosis-related pathways in Cd exposed cells.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we demonstrated that Cd exposure induces ER stress via ATF6 signaling pathway, leading to apoptosis in vitro. The disrupted metabolism of amino acids and the activation of PERKp-eIF2α-ATF6 pathway may play important roles.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, Jie Wu, Qizi Yin, Yi\u0026nbsp;Wang, Rong Wang and Wenjing Gong; methodology, Yihang Chen, Jie Wu and Mingming Zhang, project administration, Rong Wang; supervision, Yanli Ji; writing\u0026mdash;original draft preparation, review and editing, Yanli Ji and Yehao Liu; funding acquisition, Yanli Ji and Yehao Liu. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003cstrong\u003eunding:\u003c/strong\u003e This research was funded by Natural Science Foundation of China (31571557), the Natural Science Foundation of Anhui Province (2208085MH263, 1908085MH287) and Innovation and Entrepreneurship Training Program for College Students of Anhui Medical University(S202110366089).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement: \u0026nbsp;\u003c/strong\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e Data is contained within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e\u0026Aring;kesson A, Barregard L, Bergdahl IA, Nordberg GF, Nordberg M, Skerfving S (2014) Non-renal effects and the risk assessment of environmental cadmium exposure. 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Food Chem (Oxf) 4:100095. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.fochms.2022.100095\u003c/span\u003e\u003cspan address=\"10.1016/j.fochms.2022.100095\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cadmium, spermatogonia, apoptosis, ATF6, transcriptome, metabolomics","lastPublishedDoi":"10.21203/rs.3.rs-3910028/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3910028/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCadmium (Cd), which can cause testicular germ cell apoptosis, is a well-recognized male reproductive toxicant, but the underlying mechanism still needs investigation. To detect Cd toxicity on testicular germ cell, we treated mouse spermatogonia with CdCl\u003csub\u003e2\u003c/sub\u003e in vitro and investigated the responses from cells at both RNA and protein levels. After treating mouse-derived spermatogonia cell line GC-1 spg cells with 20 \u0026micro;M CdCl\u003csub\u003e2\u003c/sub\u003e for 24h, cell apoptosis was measured by TUNEL and flow cytometry assay. After then, the expressions of key genes and protein biomarkers involved in endoplasmic reticulum (ER) stress were detected by qPCR and western blot, respectively. Finally, untargeted metabolomics was performed to compare metabolic differences, and Illumina RNA sequencing was conducted to screen differentially expressed genes (DEGs). Our results indicated that Cd exposure caused cell apoptosis, DEGs were involved in several apoptosis-related pathways. Cd exposure apparently elevated the mRNA and protein expressions levels of both GRP78 and ATF6α, and disrupted the expression of many types of metabolites, especially for amino acids. Taken together, our study uncovers the pathway of Cd toxicity on mouse spermatogonia, provides deep understanding on Cd-induced testicular toxicity.\u003c/p\u003e","manuscriptTitle":"Cadmium exposure causes mouse spermatogonia apoptosis via inducing endoplasmic reticulum stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-02 20:15:42","doi":"10.21203/rs.3.rs-3910028/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"abf15978-6ef7-410d-8798-1fed6691a545","owner":[],"postedDate":"February 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-15T18:32:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-02 20:15:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3910028","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3910028","identity":"rs-3910028","version":["v1"]},"buildId":"zQwnuV7TCBrMSSSToR1PI","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}