Silica nanoparticles inducing the apoptosis of spermatocyte cell through microRNA-450b-3p targeting MTCH2-mediating mitochondrial signaling pathway

Background: Silica nanoparticles (SiNPs) are found in environmental particulate matter and are proven to have adverse effects on fertility. The relationship and underlying mechanisms between miRNAs and apoptosis induced by SiNPs during spermatogenesis is currently ambiguous. Experimental design: The present study was designed to investigate the role of miRNA-450b-3p in the reproductive toxicity caused by SiNPs. In vivo, 40 male mice were randomly divided into control and SiNPs groups, 20 per group. The mice in the SiNPs group were administrated 20 mg/kg SiNPs by tracheal perfusion once every 5 days, for 35 days, and the control group were given the equivalent of a normal luminal saline. In vitro, spermatocyte cells were divided into 0 and 5 μg/mL SiNPs groups, after passaged for 30 generations, the GC-2spd cells in 5 μg/mL SiNPs groups were transfected with miRNA-450b-3p and its mimic and inhibitor. Results: In vivo, the results showed that SiNPs damaged tissue structures of testis, decreased the quantity and quality of the sperm, reduced the expression of miR-450b-3p, and increased the protein expressions of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, and Caspase-3 in the testis. In vitro, SiNPs obviously repressed the viability and increased the LDH level and apoptosis rate, decreased the levels of the miR-450b-3p, signicantly enhanced the protein expressions of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, Caspase-3; while the mimic of miR-450b-3p reversed the changes induced by SiNPs, but inhibitor further promoted the effects induced by SiNPs. Conclusion: The result suggested that SiNPs could induce the spermatocyte apoptosis by inhibiting the miR-450b-3p expression to target promoting the MTCH2 resulting in activating mitochondrial apoptotic signaling pathways in the spermatocyte cells.


Background
Silica nanoparticles (SiNPs) are found in environmental particulate matter (PM), and it is the major inorganic component of PM 2.5 (1). SiNPs are also one of the most widely used in engineered nanomaterials and listed as one of the top ve nanomaterials in consumer products (2). The unique physicochemical properties of SiNPs are extensively utilized in biomedical and biotechnological elds, such as drug delivery, gene therapy, diagnosis, and imaging (3)(4)(5). With increasing environmental pollution and the rapid application of SiNPs, their toxicity and toxic mechanisms have raised signi cant concerns, which will be helpful for risk assessment.
Environmental pollution has been proven to have an adverse effect on fertility. It was reported that atmospheric pollution was connected to the clinical and sub-clinical symbols of infertility, such as poor sperm quality (6). Infertility remains a major clinical problem that occurs in 10 to 15% of couples worldwide, and male factor infertility accounts for 40 to 50% of all infertility cases (7). Previous studies have shown that SiNPs could penetrate the blood-testis barrier (8) and decrease semen quality and count of sperm in mice (9). However, many of these studies focused on the pathways and proteins related to apoptosis and autophagy (10,11), and there are relatively few studies that focus on the epigenetic regulations of the toxic mechanisms of SiNPs on reproduction.
MicroRNAs (miRNAs) are noncoding RNAs with nearly 22-nucleotide-long (12). miRNA expression during spermatogenesis could regulate the expressions of mRNAs in the testis (13). It was reported that miR-34b/c and miR-449a/b/c clusters in the sperm are essential for fertilization and pre-implantation development in vivo (14). Mir-141, miR-429, and miR-7-1-3p increased in the seminal plasma of patients with non-obstructive azoospermia (15). SiNPs could induce apoptosis via loss of mitochondrial membrane potential and Caspase-3 activation, while miR-98 plays a key role in modulating this effect (16). Silica nanoparticles and MeHg co-exposure could induce expressions of miRNAs in zebra sh signi cantly (17). Exposure to Silica nanoparticles also exerted altered the expression of apoptosisassociated proteins in HaCaT cells (18). Our Previous study showed that SiNPs altered the miRNA pro le, and there were ve upregulated miRNAs and ten down-regulated miRNAs in spermatocyte cells, and miR-138 and miR-2861 were related to the death receptor pathways of apoptosis (10). However, the underlying mechanisms behind some miRNA in SiNPs induced male reproductive toxicity are still unclear.
It was reported that mitochondrial carrier homolog 2 (MTCH2) is a mitochondrial outer membrane protein that is essential for embryonic development (19). Recent research has shown that MTCH2 is highly expressed in rat testis and primarily related to the apoptosis of spermatocytes (20). Based on the above studies, the present study was designed to rstly investigate the effects of SiNPs on the expressions of miR-450b-3p, MTCH2 and mitochondrial apoptosis pathway in mice, further study the relationship between the miRNA and MTCH2 and the MTCH2 role in mitochondrial apoptosis pathway of mouse spermatocytes via transfecting mimic and inhibitor of miR-450b-3p in vitro, so as to clarify the role of miRNA in the reproductive toxicity caused by SiNPs.

Characterization of the silica nanoparticles
The shape of SiNPs was near-spherical, and the size was uniform (Fig. 1). The average diameter of the SiNPs was 65.72 ± 7.29 nm. The SiNPs exhibited remarkable monodispersity and stability in ultrapure water, DMEM, and saline. Our data demonstrated that SiNPs have good dispersibility in testing medium.
The characterization of SiNPs was performed, which is similar to our previous study (10). The results of Zeta potential and surface area of the SiNPs in the ultrapure water, DMEM, and saline are shown in Table 1. Changes of the quality and quantity of sperm caused by the SiNPs.
The results showed that sperm morphology in the control group is normal (Fig. 2a), while in the SiNPs group had different morphological defects in the head, neck, and tail of the sperm (Fig. 2a). Meanwhile, the sperm abnormalities in the SiNPs group obviously increased when compared with the control group (p < 0.05) (Fig. 2b). The sperm motility and sperm concentrations in the SiNPs groups were signi cantly lower than those of the control groups (p 0.05) (Fig. 2c, d).
Effects of SiNPs on the tissue structure and spermatogenic cell apoptosis in testis The tissue structures of testis in the control groups were normal morphology, the seminiferous tubules with spermatogenic cells arranged regularly, the lumens were lled with amounts of sperms; while in the SiNPs groups, the damages of the seminiferous tubules were observed, there were the depletion and exfoliation of spermatogenic cells and vacuolation in lumens (Fig. 3a).
The data from the TUNEL staining showed that the relative uorescence intensity in the SiNPs group was increased compared with their control group, which means spermatogenic cell apoptosis in lumens of testis was obviously higher than that of control group (p 0.05) (Fig. 3b, 3c).
The protein expressions of changes of MTCH2 and mitochondrial apoptotic signaling pathway in testis The present results showed that SiNPs signi cantly affected the protein expressions of MTCH2 and mitochondrial apoptotic signaling pathways in testes of mice (p 0.05) (Fig. 4). The protein expression level of MTCH2 in the SiNPs group was higher than that of the control group (p 0.05) (Fig. 4a, b). The protein expressions of BID, BAX, Cytochrome C, Caspase-9, and Caspase-3 in mitochondrial apoptosis signaling pathway in the SiNPs group signi cantly increased when compared to control group (p 0.05) (Fig. 4a, b).
Prediction and veri cation of the miR-450b-3p and veri cation of the target genes of the miR-450b-3p in vivo and in vitro Results from the document searching showed that the MTCH2 might be the target genes of miR-450b-3p ( Fig. 5a, Table S1). Starbase was used to predict the relationships between the miR-450b-3p and MTCH2 (Fig. 5a). We found that the combination of the mir-450b-3p and MTCH2 is 6mer and identi ed its binding region on the chromosome. Bottini et al. veri ed the targeting relationship between the mir-450b-3p and MTCH2, through the Ago-CLIP experiment (21). To verify the relationship between the miR-450b-3p and MTCH2,we rstly examined the levels of miR-450b-3p and MTCH2 mRNA in testis by RT-PCR. The results showed that the levels of miR-450b-3p in the SiNPs group was lower than that of the control group (p 0.05), while the level of MTCH2 mRNA was higher than that of the control group (p 0.05) (Fig. 5b, c).
To further verify the relationship among the miR-450b-3p, MTCH2, and mitochondrial apoptotic signaling pathway, the mimic and inhibitor of miR-450b-3p were used in vitro via transfection. The results showed that the level of the miR-450b-3p was downregulated after exposure of the GC-2spd to the SiNPs for 30 generations when compared to that in the control group (p 0.05) (Fig. 5d). While the inhibitor of miR-450b-3p further decreased the expression level of miR-450b-3p, and the mimic of miR-450b-3p antagonized the expression decrease of miR-450b-3p caused by SiNPs when compared to those in 5 µg/mL SiNPs group (p 0.05) (Fig. 5d). Besides, there were no signi cant differences in miR-450b-3p level among the 5 µg/mL SiNPs group and 5 µg/mL SiNPs with two negative control groups (Fig. 5d), which means that the solvents of mimic and inhibitor had no signi cant effect on the levels of miR-450b-3p (Fig. 5d). The mRNA levels of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, and Caspase-3 in the SiNPs group signi cantly increased when compared to control group (p 0.05); while in the mimic of miR-450b-3p group, the mRNA levels of MTCH2, BAX, Cytochrome C, Caspase-9, and Caspase-3 obviously decreased when compared to the 5 µg/mL SiNPs group, but BID had no signi cant difference than that in the 5 µg/mL SiNPs. In addition, the mRNA levels of MTCH2, BID, BAX, Cytochrome C, Caspase-9, and Caspase-3 in the inhibitor of the miR-450b-3p group were higher than those in the 5 µg/mL SiNPs (p 0.05) (Fig. 5e).

Cytotoxicity of the SiNPs on the spermatocyte cells in vitro
To determine the cytotoxicity of the SiNPs on the spermatocyte cells through modi ed expression levels of the miR-450b-3p, cell viability, and LDH activity were determined. The results showed that the viability of the GC-2spd cell in the 5 µg/mL SiNPs group was lower than that in the control group (p 0.05) (Fig. 6a), the LDH level and apoptosis rate in 5 µg/mL SiNPs group were higher than those in the control group (p 0.05) (Fig. 6b, c d). While the mimic of the miR-450b-3p group signi cantly reversed the decrease of cell viability and relieved the increases of LDH level and apoptosis rate caused by SiNPs when compared to the 5 µg/mL SiNPs group (p 0.05); but the inhibitor of the miR-450b-3p further repressed the decrease of cell viability, and promoted the increases of LDH level and apoptosis rate induced by SiNPs compared to the 5 µg/mL SiNPs group (p 0.05) (Fig. 6).

Changes of protein expressions of in the MTCH2 signaling pathways in vitro
The protein expression of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, and Caspase-3 were measured by western blot analysis. In vitro, compared with the control group, the expression of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, and Caspase-3 were signi cantly increased in the SiNPs group (p 0.05), while the mimic of miR-450b-3p relieved the increases induced by SiNPs (p 0.05). The inhibitor of miR-450b-3p further promoted the protein expression increases of MTCH2, BID, BAX, Caspase-9, and Caspase-3 caused by SiNPs (p 0.05) compared to 5 µg/mL SiNPs group, while Cytochrome C has no signi cant change between the 5 µg/mL SiNPs group and 5 µg/mL SiNPs group with inhibitor group although there was an increasing tendency (p 0.05) (Fig. 7a-c).

Discussion
This investigation has shown that SiNPs destroyed the histological structures of the testis, decreased sperm numbers and motility, increased sperm malformation rates, and led to spermatogenic cell apoptosis of the testis in mice; the results in vitro also showed that the SiNPs caused the apoptosis and a viability decrease in the GC-2 spermatocyte line. The current results from both the in vivo and in vitro suggested that the SiNPs exposure could affect the sperm quality and quantity by damaging the histological structures of the testis and inducing the adverse effects on spermatogenesis. The present results are similar to the study from Xu et al. (22), who found that SiNPs could cause reversible damages to the sperms and testicular structures. The apoptosis of the sperm cells induced by the SiNPs was also previously reported by Özgür ME (16,23).
To further explore the mechanisms of reproduction toxicity induced by SiNPs, we measured the protein expressions of the MTCH2 mitochondrial apoptosis signaling pathway in the testicular tissues of mice.
The results showed that the SiNPs signi cantly increased the protein expression of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, and Caspase-3, which suggested that the SiNPs activated the MTCH2 and mitochondrial apoptosis signaling pathway in vivo. MTCH2, a mitochondrial outer membrane protein (24), was mostly related to the apoptosis of spermatocytes (20). It was reported that MTCH2 could facilitate the recruitment of the truncated BID to mitochondria (24). BID could indirectly activate BAX/BAK and thus allowing them to undergo unimpeded, spontaneous activation in the mitochondrial outer membrane, leading to apoptosis initiation (25). Studies have suggested that, when activated, Bak and Bax, create discontinuity or pores in the outer mitochondrial membrane, to mediate the release of Cytochrome C(26). Caspase-9 is a vital apoptosis initiator, while Caspase-3 is an apoptosis executioner (27). Therefore, SiNPs could activate the MTCH2-mediating the mitochondrial signaling pathway and therefore lead to apoptosis of the spermatocyte.
miRNAs play a crucial role in SiNPs -induced male reproduction toxicity. A previous study from Xu et al.
showed that miR-98 exerts an important role in the apoptosis of germ cells caused by SiNPs (16). Our previous study found that SiNPs could change the miRNA pro le of the spermatocytes exposed to the SiNPs for 30 generations and lead to the expression changes of 15 miRNAs, including 5 upregulated miRNAs and 10 downregulated miRNAs. Among the 15 miRNAs, the expressions of the miRNA-450b-3p were downregulated (10). The miRNA database prediction tool Starbase was used to predict the relationships between the miR-450b-3p and MTCH2 (21,28), which showed that the target gene of the miR-450-3p is MTCH2. The present results showed that the MTCH2 could mediate the mitochondrial apoptosis pathway of the spermatocytes. The above results indicated that the effects of the SiNPs on reproduction toxicity might be induced by miRNA and that miRNA-450b-3p might play an important role in the apoptosis of the spermatocytes via the mitochondrial apoptosis pathway. Therefore, the miRNA-450b-3p was chosen for further investigation of its functions in the toxicity of the spermatocytes caused by SiNPs. Moreover, miRNAs are thought to be functionally important in regulating apoptosis. Their abnormal expression could alter the expression in target genes. miR-450b-3p exists in abundance in the testis in mice. Therefore, miRNA-450b-3p might be considered as the biomarker of the SiNPs-induced apoptosis of the spermatocyte cells, as determined by the mitochondrial signaling pathways in this investigation, which may be useful for the early detection and early warning of the reproductive toxicity of SiNPs.

Conclusion
SiNPs reduced the expressions of miR-450b-3p, increased the protein expressions of the MTCH2, BID, BAX, Cytochrome C, Caspase-9, and Caspase-3 in testis and GC-2spd cell in vitro, while the mimic of miR-450b-3p reversed the changes induced by SiNPs, but inhibitor further promoted the effects induced by SiNPs. The results suggested that miR-450b-3p could target and regulate the expression of MTCH2, SiNPs induce the spermatocyte apoptosis by inhibiting the expression of the miR-450b-3p to target promoting the expression of MTCH2, thereby resulting in activating the mitochondrial apoptosis signaling pathways of the spermatocyte cells. This suggested that miR-450b-3p might play important roles in the apoptosis of spermatogenic cells via MTCH2-mediating the mitochondrial signaling pathways induced by the SiNPs. The miRNA-450-3p might be considered as the biomarker for male reproductive toxicity induced by SiNPs.

Animals and treatment
Forty-ve-week-old clean grade male ICR mice whose weights ranged from 20-22g were obtained from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). The mice put into standard plastic boxes (26 cm ×15 cm ×15 cm), 5 mice per box, with hygienic laboratory conditions at 20 ± 2 ℃ with a 12 h photoperiod and relative humidity of 60 ± 10%, with a 12:12 hour light/dark cycle. Animals were fed pellets food and water ad libitum, following the procedures of the Animal Experiments and Experimental Animal Welfare Committee of Capital Medical University (ethical review number: AEEI-2019-003).
After adapting to the lab conditions for one week, 40 healthy adult male mice were randomly divided into either a saline control group or SiNPs group and there were 20 mice per group. The mice in the SiNPs group were administrated with 20mg/kg SiNPs by tracheal perfusion once every 5 days for a total of 35 days. The dosage of 20 mg/kg was based on previous studies of acute toxicity studies (22). The mice in the saline control group were given equivalently volumes of normal saline. After 35 days, the mice were sacri ced using tribromoethanol anesthesia, and the blood, testis, and epididymides collected from each animal for analysis. After 1 h at room temperature, the blood samples were centrifuged for 15 min at 3000 rpm (Eppendorf, 5415D, USA), then the serum samples were amassed and stored at -80℃ for future analysis, one side of the testis was xed for the histopathological experiment, and the other side was stored at -80℃ and liquid nitrogen.

Determination of the SiNPs characterization
The con guration of the SiNPs was veri ed using the transmission electron microscope (TEM) (JEOL JEM 2100, Japan), and their hydrodynamic size and zeta potential of SiNPs were detected in distilled water, saline, and Dulbecco's modi ed eagle's medium (DMEM) using a Zetasizer (Nano ZS90; Malvern, UK). Before addition of the SiNPs to the culture medium, a sonicator (160 W, 20 kHz, 5 min) (Bioruptor UDC-200, Belgium) was used to suspend SiNPs to minimize their aggregation.
Analysis of the sperm quality and quantity Sperms were extracted from the epididymides and immediately incubated in Dulbecco's Modified Eagle Medium (2 mL) at 37 ℃ for 5 min, then their concentrations and motility were measured using semen analyzer (Hamilton Thorne IVOS-II; Hamilton Thorne Research, Beverly, MA, USA). A smear of the sperm suspension was made on the glass. To determine the sperm deformation, the number 1000 sperms under a high magnification microscope (Olympus BX53, Japan) by counting. Sperm were scored as normal or abnormal using the Kruger strict criteria. Sperm morphology was observed and imaged under a light microscope (Olympus BX53, Japan).
The sperm abnormality=The abnormality sperm number/1,000×100% Histopathological analysis of the testis After animals were sacrificed with tribromoethanol anesthesia, the testis from a side of the mice in all groups were fixed in 4 % paraformaldehyde, embedded in paraffin blocks, sectioned and stained with hematoxylin and eosin (HE) for histological examination. The testis sections were observed using the Qupath (The University of Edinburgh, Scotland) software.

Detection of spermatozoa cell apoptosis in the testis
Cell apoptosis in the testis was analyzed using the method of terminal deoxyribonucleotide transferasemediated nick end labeling (TUNEL). Each group of testis sections were stained using the TUNEL assay kit (KeyGen, Nanjing, China). In apoptotic cells, Biotin-labeled dUTP could be linked to the 3′-OH end of the DNA fragments with the TdT enzyme. Fluorescence microscopy can detect uorescein-isothiocyanate (FITC), which the labeled streptavidin binds to biotin during the biotin-streptavidin ampli cation. The testis sections were rst dewaxed, and then the sections were dealt with Proteinase-K working uid for permeabilization and then reacted with the TdT working solution (mixture of dUTP and the TdT-enzyme). The testis sections were stained with streptavidin uorescein; meanwhile, the nucleus were stained by DAPI (ThermoFisher, USA). The testis sections of each group were imaged using laser scanning confocal microscopy (A1R HD25, Nikon, Japan). Ten visual elds from each group were selected randomly to examine the relative uorescence intensity and count the number of TUNEL-positive cells per tubule. The relative uorescence intensity was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Protein expressions detection of MTCH2 and apoptotic signaling pathway in the testis To expound the in uence of the SiNPs on the expression of the cellular factors involved in the MTCH2 and apoptotic signaling pathway, the protein levels of MTCH2, BID, BAX, Cytochrome C, Caspase-9, and Caspase-3 in the testis were determined with western blot analysis.
The total protein of the testis tissues was extracted using a bicinchoninic acid (BCA) protein assay. Equal amounts of lysate proteins (40 μg per testis tissue) were electrophoresed in SDS-polyacrylamide gels (12% separation gels) and transferred to nitrocellulose membranes (Millipore, USA). After being blocked with 5% bovine serum albumin (BSA) (Epsilon, USA) and being dissolved in Tris-buffered saline (TBS) containing 0. dishes at a density of 1 × 10 5 cells/mL. The spermatocyte cells were divided into two groups, those with 0 mg/mL SiNPs and those with 5 mg/mL SiNPs and the cells in each group were serially passaged for 30 generations. At each generation, the cells in the 5 mg/mL SiNPs group were exposed to SiNPs in a culture medium for 24 h after cell attachment, and the cells in the 0 mg/mL SiNPs group were exposed to an equivalent volume of culture medium without the SiNPs. To evaluate the function of the key miRNAs associated with apoptosis, the spermatocyte cells were transfected with mimic and inhibitor. The mimic can increase the activity of the miRNAs, whereas the inhibitor can restrain its activity. The transfection experiment of the mimic and inhibitor involved 6 groups: control group, SiNPs group, mimic transfection group, mimic negative control group, inhibitor transfection group, and inhibitor negative control group.  Detection of cell viability and lactate dehydrogenase After being exposed to 5μg/mL of SiNPs for 30 generations, the GC-2spd cells were seeded into 96 wells at a density of 7000 cells per well. Then, the mimic, mimic NC, inhibitor, and inhibitor NC of the miRNA-450b-3p were transfected for 24 h. After culturing for 24 h, 10 mL of Cell Counting Kit-8(CCK8) (Dojindo, Japan)was added to the cells, and then, they were incubated for 4 h. The absorbance was detected at 450nm using a microplate reader (Thermo Multiskan MK3, USA). In order to indicate whether the cell membrane was damaged, lactate dehydrogenase (LDH) leakages were detected using the LDH Kit (KeyGEN BioTECH, Nanjing, China), as its instructions. After GC-2spd cells were transfected, we assessed the supernatants by detecting their absorbance at 440nm using a microplate reader (Thermo Multiskan MK3, USA).

Detection of the GC-2spd cells apoptosis in testis
After being exposed to 5μg/mL of SiNPs for 30 generations, the GC-2spd cells were seeded into 6 wells at a density of 1 × 10 5 cells per well. Then, we transfected the mimic, mimic NC, inhibitor, and inhibitor NC of the miRNA-450b-3p for 24 h. Using the PBS washing GC-2spd cells for three times and then centrifuging the cells at 1500 rpm for 5 min and then they were suspended in 500 μL of binding buffer. There was 5 μL

Statistical analysis
All the experimental data were analyzed by using the SPSS 17.0 software. Independent-sample t tests was used to analyze the data in the experiments. The values of p < 0.05 was considered as statistically signi cant. Data were expressed as means ± standard deviation (S.D.).

Declarations
Funding Figure 1 Characterization of SiNPs.