Silica nanoparticles induce male reproductive toxicity via Crem hypermethylation mediated spermatocyte apoptosis and sperm flagella damage.

Exposure to silica nanoparticles (SiNPs) could causally contribute to malfunctioning of the spermatogenesis, but the underlying mechanism is rarely known. This study was designed to explore the mechanism of Crem hypermethylation in SiNP-induced reproductive toxicity. The male mice were exposure to SiNPs (0 and 20 mg/kg·bw) once every 5 days via intratracheal instillation for 35 days. After exposure stopped, half of each group was killed, and the rest were sacrificed after another 15-day feeding. GC-2 cells were treated with 0 and 20 µg/mL SiNPs. The results showed that SiNPs led to structure damage of spermatocyte and sperm, caused spermatocyte apoptosis, and decreased sperm quantity and quality. After 15 days of the withdrawal, the testicular tissue damage gradually recovered. Mechanistic study showed that SiNPs induced hypermethylation of the gene of cAMP responsive element modulator (Crem) in the promoter region. Downregulation of Crem inhibited the expression of outer dense fiber 1 (Odf1), resulting in abnormal sperm flagella structure; at the same time, Crem inhibited the expression of Bcl-xl, causing upregulation of cytochrome-C, cleaved-caspase-9/caspase-9, cleaved-caspase-3/caspase-3, resulting in mitochondrial dependent apoptotic pathway. However, 5-aza, DNA methylation inhibitor, could reverse the SiNP-induced downregulation of Crem and reverse the Crem/Bcl-xl-mediated mitochondrial dependent apoptotic pathway. These results suggested SiNPs could disrupt spermatogenesis by causing Crem hypermethylation to regulate the Odf1 and Bcl-xl in spermatocytes resulting in the sperm flagella structure and spermatocyte apoptosis. Our study provided new insights into the male reproductive toxicity mechanism of SiNPs; Crem demethylation may be a potential way to prevent reproductive dysfunction from SiNP exposure.

MiR-5622-3p inhibits ZCWPW1 to induce apoptosis in silica-exposed mice and spermatocyte cells.

Silica nanoparticles (SiNPs) could cause damage to spermatogenesis, and microRNAs were reported to be associated with male reproduction. This research was designed to explore the toxic impacts of SiNPs induced in male reproduction through miR-5622-3p. In vivo, 60 mice were randomized into the control group and SiNPs group, in which they were exposed to SiNPs for 35 days and then recovered for 15 days. In vitro, 4 groups were set: control group, SiNPs group, SiNPs + miR-5622-3p inhibitor group, and SiNPs + miR-5622-3p inhibitor negative control (NC) group. Our research indicated SiNPs caused the apoptosis of spermatogenic cells, increased level of γ-H2AX, raised the expressions of RAD51, DMC1, 53BP1, and LC8 which were DNA damage repair relative factors, and upregulated Cleaved-Caspase-9 and Cleaved-Caspase-3 levels. Furthermore, SiNPs also elevated the expression of miR-5622-3p but downregulated the level of ZCWPW1. However, miR-5622-3p inhibitor reduced the level of miR-5622-3p, increased the level of ZCWPW1, relieved DNA damage, and depressed the activation of apoptosis pathway, thus, alleviating spermatogenic cells apoptosis caused by SiNPs. The above-mentioned results indicated that SiNPs induced DNA damage resulting in activating of DNA damage response. Meanwhile, SiNPs raised the level of miR-5622-3p targeting inhibited expression of ZCWPW1 to suppress the repair process, possibly making DNA damage so severe that leading to the failure of DNA damage repair, finally inducing the apoptosis of spermatogenic cells.

Silica nanoparticles suppressed the spermatogenesis via downregulation of miR-450b-3p by targeting Layilin in spermatocyte of mouse.

Silica nanoparticles (SiNPs) suppressed spermatogenesis leading to male reproductive toxicity, while the precise mechanism remains uncertain. Here, this study explored the role of miR-450b-3p in male reproductive toxicity induced by SiNPs. In vivo study, we found that SiNPs caused apoptosis of spermatocytes, decreased quantity and quality of sperms, up-regulated the cytoskeleton proteins (Layilin, Talin, and Vinculin), activated the Hippo pathway (Rho A, Yap, and p73), downregulated the expression of miR-450b-3p, damaged the compactness and density of desmosomes between spermatocytes and the basal of the testis. Moreover, in vitro study, we confirmed that SiNPs increased the expressions of cytoskeleton proteins, activated the Hippo pathway, and suppressed miR-450b-3p expressions. Meanwhile, miR-450b-3p mimic inhibited the up-regulation of cytoskeleton proteins, suppressed the activation of the Hippo pathway, and relieved the adhesion and traction stress. Eventually, atomic force microscopy (AFM) was performed to validate the traction stress and adhesion between GC-2spd cells enhanced by deregulation of miR-450b-3p. Taken together, we concluded that SiNPs suppressed spermatogenesis via inhibiting miR-450b-3p, in turn up-regulating the expression of cytoskeleton proteins, then inducing apoptosis via activating the Hippo pathway and enhancing the traction force and adhesion between GC-2spd cells. This work provides novel evidence for the study of reproductive toxicity and risk assessment of SiNPs.

BDE-209 induced spermatogenesis disorder by inhibiting SETD8/H4K20me1 related histone methylation in mice.

Past studies have observed that decabromodiphenyl ether (BDE-209) induces reproductive and developmental toxicity, but the specific mechanism remains unclear. Based on our previous work, male mice were orally given BDE-209 at 75 mg/kg/d via continuous exposure for one spermatozoon development period (50 days) and then stopping exposure for another 50 days. The mouse spermatocyte line GC-2spd was used to examine the toxic effects of BDE-209 on histone methylation and spermatogenesis. The findings indicated that BDE-209 damaged testis and epididymis structure, induced spermatogenic cell apoptosis, and decreased sperm quantity and quality after the 50-day exposure. Furthermore, BDE-209 lowered the levels of SETD8/H4K20me1 and activated the upstream signaling of DNA damage response (Mre11/Rad50/NBS1), thereby causing spermatogenic cell cycle arrest and apoptosis. Downregulation of meiotic promoter Stra8 was associated with a decrease in SETD8 after BDE-209 exposure. After stopping the exposure for 50 days, reproductive system damage and meiosis and cell cycle inhibition due to histone methylation did not improve. In vitro experiments revealed that Setd8 overexpression upregulated the histone methylation and Stra8 expression but did not promote the cell cycle in GC-2 cells. Therefore, BDE-209 exposure impaired spermatogenesis by affecting SETD8/H4K20me1-linked histone methylation and inhibiting meiosis initiation and cell cycle progression, thereby resulting in long-term male reproductive toxicity.

Dietary selenium excess affected spermatogenesis via DNA damage and telomere-related cell senescence and apoptosis in mice.

Selenium (Se) is a vital microelement for spermatogenesis and male fertility. The aim of this study was to investigate the effects of Se on the male reproductive function and possible mechanisms. Fourty male mice were randomly divided into 0, 0.1, 0.3 and 0.9 mg/kg Se supplementation groups and given with Se dietary intervention for 12 weeks. Our data showed that excessive Se intake damaged the tissue structure of testes and epididymides of the mice, resulting in decreased sperm quality and quantity. Moreover, excessive Se induced oxidative stress, causing DNA damage and activated DNA damage repair factors (Mre11/Rad50/Nbs1), and also disrupted telomere function by shortening telomere length and decreasing TERT expression. Se excess activated the senescence pathway p53/p21/p16, leading to germ cell senescence, and inhibited cell proliferation by suppressing the Sirt1/Foxo1/c-Myc pathway. All of this led to spermatogenic cell apoptosis, thereby causing a decrease of sperm quantity and quality. In conclusion, excessive Se caused reproductive toxicity via inducing telomere dysfunction due to DNA damage, leading to germ cellular senescence and apoptosis in the testes of male mice. Our research provide new proof to explain the underlying mechanism of male reproductive toxicity triggered by excessive Se intake.

Decabromodiphenyl ethane induces male reproductive toxicity by glycolipid metabolism imbalance and meiotic failure.

Decabromodiphenyl ethane (DBDPE) is a typical flame retardant found in various electrical and textile items. DBDPE is abundantly available in the surrounding environment and wild animals based on its persistence and bioaccumulation. DBDPE has been shown to cause apoptosis in rat spermatogenic cells, resulting in reproductive toxicity. However, the toxicity of DBDPE on the male reproductive system and the potential mechanisms are still unclear. This study evaluated the effect of DBDPE on the reproductive system in male SD rats and demonstrated the potential mechanisms of reproductive toxicity. DBDPE (0, 5, 50, and 500 mg/kg/day) was administered via gavage to male SD rats for 28 days. DBDPE caused histopathological changes in the testis, reduced sperm quantity and motility, and raised the malformation rate in rats, according to the findings. Furthermore, it caused DNA damage to rat testicular cells. It inhibited the expressions of spermatogenesis-and oogenesis-specific helix-loop-helix transcription factor 1 (Sohlh1), piwi-like RNA-mediated gene silencing 2 (MILI), cyclin-dependent kinase 2 (CDK2), and CyclinA, resulting in meiotic failure, as well as the expressions of synaptonemal complex proteins 1 and 3 (SYCP1 and SYCP3), leading to chromosomal association disorder in meiosis and spermatocyte cycle arrest. Moreover, DBDPE induced glycolipid metabolism disorder and activated mitochondria-mediated apoptosis pathways in the testes of SD rats. The quantity and quality of sperm might be declining due to these factors. Our findings offer further evidence of the harmful impact of DBDPE on the male reproductive system.

Maternal exposure to PM2.5 disrupting offspring spermatogenesis through induced sertoli cells apoptosis via inhibin B hypermethylation in mice.

Particulate Matter 2.5 (PM2.5) disrupts endocrine functions and may negatively affect sperm quality and quantity in males; however, the long-term effects and potential mechanisms of this effect are unknown. This study aimed to investigate the epigenetic mechanism of maternal exposure to PM2.5-induced inhibin B hypermethylation in male offspring. In this experiment design, pregnant C57BL/6 mice were treated with two doses of PM2.5 (4.8 and 43.2 mg/kg bw). The membrane control group was given a sampling membrane and the control group received nothing. Following the formation of the vaginal plug, intratracheal instillation of PM2.5 was administered every three days until delivery of the pups. To assess the effect of PM2.5 in vitro, TM4 cells, a Sertoli-like cell line, was treated with different concentrations (0, 25, 50, 100 µg/mL) of PM2.5 for 24 h. The results displayed that Sperm motility, as well as the number of adult offspring, was decreased in the PM2.5 exposed group relative to the untreated controls. Increased vacuolization was observed in the Sertoli cells of mice that were exposed to PM2.5 in utero. The levels of inhibin and testosterone were reduced and the levels of LH and FSH increased in the PM2.5 groups relative to the untreated controls. In vitro, PM2.5 resulted in cell cycle inhibition as well as increased apoptosis in TM4 cells. Moreover, PM2.5-induced inhibin B hypermethylation and activation of the p21/Cleaved Caspase-3 pathway resulted in TM4 cell apoptosis that was rescued through the use of a DNA methylation inhibitor. Together, our data suggest that prenatal exposure to PM2.5 results in inhibin B hypermethylation and can activate the p21/Cleaved Caspase-3 pathway, resulting in Sertoli cell apoptosis, aberrant secretion of androgen binding protein, and decreased testosterone, thus resulting in the inhibition of spermatogenesis.

Fat mass and obesity-associated gene (FTO) hypermethylation induced by decabromodiphenyl ethane causing cardiac dysfunction via glucolipid metabolism disorder.

Decabromodiphenyl ethane (DBDPE) is a major alternative to BDE-209 owing to its lower toxicity. However, the mass production and increased consumption of DBDPE in recent years have raised concerns related to its adverse health effects. However, the effect and mechanism of DBDPE on cardiotoxicity have rarely been studied. In the present study, we investigated the impacts of DBDPE on the cardiovascular system in male SD rats and then explored the underlying mechanisms to explain the cardiotoxicity of DBDPE using AC16 cells. Under in vivo conditions, male rats were administered with an oral dosage of DBDPE at 0, 5, 50, and 500 mg/kg/day for 28 days, respectively. Histopathological analysis demonstrated that DBDPE induced cardiomyocyte injury and fibrosis, and ultrastructural observation revealed that DBDPE could induce mitochondria damage and dissolution. DBDPE could thus decrease the level of MYH6 and increase the level of SERCA2, which are the two key proteins involved in the maintenance of homeostasis during myocardial contractile and diastolic processes. Furthermore, DBDPE could increase the serum levels of glucose and low-density lipoprotein but decrease the content of high-density lipoprotein. In addition, DBDPE could activate the PI3K/AKT/GLUT2 and PPARγ/RXRα signaling pathways in AC16 cells. In addition, DBDPE decreased the UCP2 level and ATP synthesis in mitochondria both under in vitro and in vivo conditions, consequently leading to apoptosis via the Cytochrome C/Caspase-9/Caspase-3 pathway. Bisulfite sequencing PCR (BSP) identified the hypermethylation status of fat mass and obesity-associated gene (FTO). 5-aza exerted the opposite effects on the PI3K/AKT/GLUT2, PPARγ/RXRα, and Cytochrome C/Caspase-9/Caspase-3 signaling pathways induced by DBDPE in AC16 cells. In addition, the DBDPE-treated altered levels of UCP2, ATP, and apoptosis were also found to be significantly reversed by 5-aza in AC16 cells. These results suggested that FTO hypermethylation played a regulative role in the pathological process of DBDPE-induced glycolipid metabolism disorder, thereby contributing to the dysfunction of myocardial contraction and relaxation through cardiomyocytes fibrosis and apoptosis via the mitochondrial-mediated apoptotic pathway resulting from mitochondrial dysfunction.

Decabromodiphenyl ether-induced PRKACA hypermethylation contributed to glycolipid metabolism disorder via regulating PKA/AMPK pathway in rat and L-02 cells.

BDE-209 is the most prevalent congener of polybrominated diphenyl ethers and has high bioaccumulation in humans and animals. BDE-209 has been reported to disrupt glycolipid metabolism, but the mechanisms are still unclear. In this study, we found that BDE-209 induced liver tissue injury and hepatotoxicity, increased the glucose and total cholesterol levels in the serum of rats, and increased glucose and triglyceride levels in L-02 cells. BDE-209 exposure changed the PKA, p-PKA, AMPK, p-AMPK, ACC, and FAS expression in rats' liver and L-02 cells. Moreover, BDE-209 induced PRKACA-1 hypermethylation in L-02 cells. AMPK activator (AICAR) inhibited the changes of p-AMPK, ACC, and FAS expression and elevation of glucose and triglyceride levels induced by BDE-209. DNA methylation inhibitor (5-Aza-CdR) reversed BDE-209 induced alters of PKA/AMPK/ACC/FAS signaling pathway. These results demonstrated that BDE-209 could disrupt the glycolipid metabolism by causing PRKACA-1 hypermethylation to regulate the PKA/AMPK signaling pathway in hepatocytes.

DNA methylation changes induced by BDE-209 are related to DNA damage response and germ cell development in GC-2spd.

Decabrominated diphenyl ether (BDE-209) is generally utilized in multiple polymer materials as common brominated flame retardant. BDE-209 has been listed as persistent organic pollutants (POPs), which was considered to be reproductive toxin in the environment. But it still remains unclear about the effects of BDE-209 on DNA methylation and the induced-male reproductive toxicity. Due to the extensive epigenetic regulation in germ line development, we hypothesize that BDE-209 exposure impacts the statue of DNA methylation in spermatocytes in vitro. Therefore, the mouse GC-2spd (GC-2) cells were used for the genome wide DNA methylation analysis after treated with 32 µg/mL BDE-209 for 24 hr. The results showed that BDE-209 caused genomic methylation changes with 32,083 differentially methylated CpGs in GC-2 cells, including 16,164 (50.38%) hypermethylated and 15,919 (49.62%) hypomethylated sites. With integrated analysis of DNA methylation data and functional enrichment, we found that BDE-209 might affect the functional transcription in cell growth and sperm development by differential gene methylation. qRT-PCR validation demonstrated the involvement of p53-dependent DNA damage response in the GC-2 cells after BDE-209 exposure. In general, our findings indicated that BDE-209-induced genome wide methylation changes could be interrelated with reproductive dysfunction. This study might provide new insights into the mechanisms of male reproductive toxicity under the environmental exposure to BDE-209.

Decabromodiphenyl ether induces male reproductive toxicity by activating mitochondrial apoptotic pathway through glycolipid metabolism dysbiosis.

Decabromodiphenyl ether (BDE-209), an extensively used flame retardant, exists widely in the environment. Although male reproductive toxicity induced by BDE-209 has been reported, its mechanisms remain unclear. To explore the role of glycolipid metabolism in male reproductive toxicity and the potential mechanisms, forty male SD rats were divided into four groups and given gavage with BDE-209 at 0, 5, 50, and 500 mg/kg/d for 28 days. In vitro, the spermatogenic cell lines GC-2spd cells were divided into four groups: the control group, 32 µg/mL BDE-209 group, 32 µg/mL BDE-209 + 0.4 µM Fatostatin (the inhibitor of SREBP-1) group, and 0.4 µM Fatostatin group. Our results showed that BDE-209 decreased sperm quality and quantity, which was correlated with glycolipid metabolism dysbiosis of testis. The levels of glucose, triglyceride, and total cholesterol were negatively correlated with sperm concentration, and triglyceride and total cholesterol levels were negatively correlated with sperm motility, while positively correlated with the sperm malformation rate. Moreover, BDE-209 exposure activated the glycolipid metabolism pathways (PPARγ/RXRα/SCAP/SREBP-1) and mitochondrial apoptotic pathway, thereby inducing the apoptosis of spermatogenic cells. In vitro, BDE-209 caused triglyceride and total cholesterol disorder and apoptosis of GC-2spd cells, the lipid metabolism pathways inhibitor fatostain downregulated the elevation of triglyceride and total cholesterol concentrations, and suppressed apoptosis and the activation of the mitochondrial apoptotic pathway in GC-2spd cells caused by BDE-209. Our results indicated that BDE-209 induced male reproductive toxicity by causing glycolipid metabolism dysbiosis of testis resulting in activating of the mitochondrial apoptotic pathway in spermatogenic cells. The study provides new insight into the mechanisms of male reproductive toxicity caused by BDE-209.

Silica nanoparticles inhibiting the differentiation of round spermatid and chromatin remodeling of haploid period via MIWI in mice.

Researches have shown that silica nanoparticles (SiNPs) could reduce both the quantity and quality of sperm. However, the mechanism of toxicity induced by SiNPs in the male reproductive system is still unclear. In this study, male mice were randomly divided into a control group, and SiNPs treated group (20 mg/kg dose; n = 30 per group). Half of the mice per group were sacrificed on 35 days and the remaining on 50 days of the SiNPs exposure. SiNPs were found to decrease sperm count and mobility, increase the sperm abnormality rate, and damage the testes' structure. Furthermore, SiNPs decreased the protein levels of Protamine 1(PRM1) and elevated the histones' levels and suppressed the chromatin condensation of sperm. There was a significant reduction of the ubiquitinated H2A (ubH2A)/H2B (ubH2B) and RING finger protein 8 (RNF8) levels in the spermatid nucleus, while the RNF8 level in the spermatid cytoplasm increased evidently. The protein expression levels of PIWI-like protein 1(MIWI) in the late spermatids significantly increased on day 35 of SiNPs exposure. After 15 days of the withdrawal, the sperm parameters and protamine levels, and histones in the epididymal sperm were unrecovered; however, the changes in testis induced by SiNPs were recovered. Our results suggested that SiNPs could decrease the RNF8 level in the nucleus of spermatid either by upregulating of the expression of MIWI or by inhibiting its degradation. This resulted in the detention of RNF8 in the cytoplasm that maybe inhibited the RNF8-mediated ubiquitination of ubH2A and ubH2B. These events culminated in creating obstacles during the H2A and H2B removal and chromatin condensation, thereby suppressing the differentiation of round spermatids and chromatin remodeling, which compromised the sperm quality and quantity.

The effect of SiNPs on DNA methylation of genome in mouse spermatocytes.

Silica nanoparticles (SiNPs), which are the main inorganic components of atmospheric particulate matter, have been proved to have certain male reproductive toxicity in previous studies. Spermatogenesis involves complex epigenetic regulation, but it is still unclear if SiNPs exposure will interfere with the DNA methylation patterns in mouse spermatocytes. The present study was designed to investigate the effects of SiNPs on DNA methylation in the mouse spermatocyte GC-2spd(ts). GC-2 cells were treated with 0 and 20 µg/mL SiNPs for 24 h. MeDIP-seq assay was then performed to analyze the differentially methylated genes related to spermatogenesis. The results showed that SiNPs induced extensive methylation changes in the genome of GC-2 cells, and 24a total of 428 hyper-methylated genes and 398 hypo-methylated genes were identified. Gene Ontology and pathway analysis showed that differential DNA methylation induced by SiNPs was probably involved with abnormal transcription and translation, mitochondrial damage, and cell apoptosis. Results from qRT-PCR verification showed that the expression of spermatogenesis-related genes Akap1, Crem, Spz1, and Tex11 were dysregulated by SiNPs exposure, which was consistent with the MeDIP-seq assay. In general, this study suggested that SiNPs caused genome-wide DNA methylation changes in GC-2 cells, providing valuable reference for the future epigenetic studies in SiNPs-induced male reproductive toxicity.

Silica nanoparticles inducing the apoptosis via microRNA-450b-3p targeting MTCH2 in mice and spermatocyte cell.

Silica nanoparticles (SiNPs) could cause reproductive toxicity. The role of miRNAs in reproductive toxicity induced by SiNPs is still ambiguous. The present study was designed to investigate the role of miRNA-450 b-3p. In vivo, 40 male mice were randomly divided into control, and 20 mg/kg SiNPs groups. The mice were administrated by tracheal perfusion for 35 days. In vitro, spermatocyte cells (GC-2spd cells) were divided into 6 groups: 0 µg/mL SiNPs groups, 5 µg/mL SiNPs groups, 5 µg/mL SiNPs + miRNA-450 b-3p mimic transfection group, 5 µg/mL SiNPs + miRNA-450 b-3p mimic negative control group, 5 µg/mL SiNPs + miRNA-450 b-3p inhibitor transfection group, and 5 µg/mL SiNPs + miRNA-450 b-3p inhibitor negative control group. The results showed that SiNPs induced the apoptosis of spermatogenic cells, decreased the quantity and quality of the sperm, reduced the expressions of miR-450 b-3p, and increased the protein expressions of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, and Caspase-3 in the testis. In vitro, the mimic of miRNA-450 b-3p reversed the decrease of viability and the increase of apoptosis rate and significantly antagonized the expression enhancements of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, Caspase-3 induced by SiNPs, while inhibitor of miRNA-450 b-3p further promoted the effects induced by SiNPs. The result suggested that SiNPs could inhibit the miR-450 b-3p expression resulting in activation of the mitochondrial apoptosis signaling pathways by regulating the MTCH2 in the spermatocyte cells and, thus, induce the reproductive toxicity.

BDE-209 and DBDPE induce male reproductive toxicity through telomere-related cell senescence and apoptosis in SD rat.

Decabrominated diphenyl ether (BDE-209) and decabromodiphenyl ethane (DBDPE) are common flame retardants utilized in many kinds of electronic and textile products. Due to their persistence and bioaccumulation, BDE-209 and DBDPE extensively exist in the surrounding environment and wild animals. Previous studies have indicated that BDE-209 could induce male reproductive toxicity, whereas those of DBDPE remains relatively rare. In this study, we investigated the effects of both BDE-209 and DBDPE on reproductive system in male SD rats, and explored the potential mechanisms under the reproductive toxicity of BDE-209 and DBDPE. Male rats were orally administered with BDE-209 and DBDPE (0, 5, 50 and 500 mg/kg/day) for a 28-day exposure experiment. The current results showed that BDE-209 and DBDPE led to testicular damage in physiological structure, decreased the sperm number and motility, and increased the sperm malformation rates in rat. Moreover, BDE-209 and DBDPE could damage the telomeric function by shortening telomere length and reducing telomerase activity, which consequently caused cell senescence and apoptosis in testis of rat. This could contribute to the decline of sperm quality and quantity. In conclusion, BDE-209 and DBDPE led to reproductive toxicity by inducing telomere dysfunction and the related cell senescence and apoptosis in testis of SD rat. Comparatively, BDE-209 had more severe effects on male reproduction. Our findings may provide new insight into the potential deleterious effects of BFRs on male reproductive health.

The alterations of miRNA and mRNA expression profile and their integration analysis induced by silica nanoparticles in spermatocyte cells.

Air pollution and the application of Silica nanoparticles (SiNPs) have increased the risk of human exposure to SiNPs. SiNPs are known to induce cytotoxicity in spermatocyte cells (GC-2spd cells) of mice and male reproductive system damage. However, the expression profiles of miRNA and mRNA and the molecular mechanism of miRNA-mRNA integration in reproductive toxicity induced by SiNPs in GC-2spd cells are still unclear. Therefore, GC-2spd cells were divided into 0 µg/mL and 5 µg/mL SiNPs groups, and the cells were collected and analyzed after passaging for 30 generations using miRNA microarray and Illumina high-throughput sequencing (Illumina HiSeq) for the integrated analysis of miRNA and mRNA expression. Both miRNA Microarray and Illumina Hiseq identified 15 significant differentially expressed miRNAs and 1648 significant differentially expressed mRNAs. Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and miRNA-gene-pathway-network analysis revealed 15 significant differentially expressed miRNAs that could regulate the DNA replication and the fatty acid metabolism, respectively. Furthermore, the mRNA-mRNA regulatory network analysis revealed that Pkfl (phosphofructokinase, liver, B-type) and DHCR24 (24-dehydrocholesterol reductase) were highly expressed, but also affected DNA replication and fatty acid metabolism in SiNPs-treated GC-2spd cells. Additionally, miRNA-mRNA integration analysis revealed that miRNA-138-1-3p might have a regulatory relationship with fatty acid metabolism and DNA replication. It is confirmed that SiNPs could decrease the expression of 10 miRNAs and increase the expression of 5 miRNAs. These findings suggest that the cytotoxicity of GC-2spd cells induced by SiNPs depends on the deregulation of multiple miRNAs, which regulate the DNA replication and fatty acid metabolism. Our results are the first to establish an integrated analysis of miRNA-mRNA interactions and mRNA-mRNA and defines multiple pathways involved in SiNPs-treated GC-2spd cells.