This project (GMU2017012) was approved by the Ethics Committee of Gannan Medical University, according to the Declaration of Helsinki Principles. The study was conducted in accordance with the internationally accepted principles for laboratory animal use and care in the European Community guidelines (EEC Directive of 1986; 86/609/EEC). Thirty-six-week-old male rats were kept in an adaptive circumstance in an SPF-grade room with a 12 h light/dark cycle (Temperature: 21 −23°C; humidity: 45%−55%) for 1 week before further experiments. All rats were free to access water and food. Yttrium chloride (YCl₃, purity ≥ 99.99%) was purchased from Sigma-Aldrich (St. Louis, USA).

Rats were randomly divided into three groups of 10 rats each, including the negative control (NC) group (free access to blank water) and YCl₃-treated groups (free access to water with 12 and 24 mmol/L of YCl₃, respectively) (4). After 6 months, all rats were sacrificed. The testicular tissues were obtained for weight measurement, histopathological and immunohistochemical examination, and protein extraction.

The testicular weight and the body weight of each rat were determined. The gonadosomatic index was reached by the formula: gonadosomatic index = the testicular weight /the body weight × 100% (17).

The cauda epididymis was harvested and minced in the physiological saline at 36°C to prepare a sperm suspension. The sperms (100 sperms) moving in a straight line were counted using a blood cell counting plate, which was preheated at 36°C. To detect the concentration of sperm, the suspension (100 μl) was killed by a preheated water bath at 60°C. The sperm was counted using a blood cell counting plate. To examine the sperm deformity rate, the suspension (500 μl) was smeared and fixed with methanol. The sperm deformity was observed using a microscope.

A testosterone ELISA kit (Cat. no. KB3117, BOYAO, Shanghai, China) was employed to detect the concentrations of serum testosterone in rats. The procedures were conducted according to the instructions recommended by the kit manufacturers.

The collected testicular tissues were fixed with 4% paraformaldehyde. Then, they were embedded in the paraffin. The 5-μm thickness of sections was sliced and then stained with hematoxylin-eosin (H&E). Light microscopy was used to observe the pathological changes. For the immunohistochemical examination, the testicular tissues embedded in paraffin were sectioned for 5-μm thickness, deparaffinized in xylene, and rehydrated with different graded concentrations of ethanol on glass slides. The tissues were then interacted with by hydrogen peroxide for 10 min and incubated the primary antibodies (1:100) for 6 min and horseradish peroxidase (HRP)-conjugated secondary antibody for 8 min, respectively. After further incubation with 3,3-diaminobenzidine (DAB) for 10 min. Light microscopy was used to observe the immunized proteins (18, 19).

The CCK8 kit (Cat.no.C0038) (Beyotime, Shanghai, China) was used for testing the viability of Leydig cells (purchased from Procell Life Science&Technology Co. Ltd, Wuhan, China), according to the manufacturer's instructions. Leydig cells were grown in 96-well plates at a density of 5,000 cells/well for 24 h. Different concentrations of YCl₃ (0, 3, 10, 30, 100, and 300 μM) were used to pretreat the Leydig cells for 24 h. Then, CCK-8 solution (10 μl) was added into each well and incubated at 37°C for 4 h. After that, the absorbance was detected at the wavelength of 450 nm by using a microplate system (Leica microsystems, Germany).

TUNEL and DAPI staining kits were purchased from Abcam (Cambridge, MA, USA). Leydig cells were cultured and treated with YCl₃ for 48 h. Cells were harvested and then fixed with 4% paraformaldehyde. The apoptosis was analysis according to the instructions recommended by the manufacturer. The fluorescent images were observed using a Leica microsystem, and the florescence intensity was determined using ImageJ (an open-source image processing package).

The total protein was harvested, and the protein concentrations were detected by BCA protein assay kit (Beyotime). Each sample (30 μg) was subjected to SDS-PAGE and then transferred onto PVDF membranes. After being blocked, the membranes were co-incubated with the primary antibodies at 4°C overnight against IP3R1 (1: 1,000 dilution; Sigma), CaMKII (1: 1,000 dilution; Sigma), p-CaMKII (1: 1,000 dilution; Sigma), Bcl-2 (1: 1,000 dilution; Sigma), caspase-3 (1: 1,000 dilution; Sigma), cleaved-caspase-3 (1: 1,000 dilution; Sigma), and β-actin (1: 1,000 dilution; Sigma). Then, the secondary antibody conjugated with peroxidase (1: 5,000 dilution; Sigma) was employed. Protein bands were detected using the enhanced chemiluminescence detection system.

The experimental procedures were conducted according to the instructions of the kit's manufacturer (HL10153.1, Shanghai Haling Biological Technology, Shanghai, China). Leydig cells were incubated with Rhod-2/AM for 1 h at 37°C. Analysis was conducted by a fluorescence microplate reader (Varioskanlux, Thermo Fisher Scientific, Waltham, MA, USA) with the parameters, including the wavelength of 550 nm (excitation) and 590 nm (emission).

All experiments were performed three times independently and data are expressed as the mean ± standard deviation (SD). SPSS 20.0 software was employed for statistical analysis. One-way ANOVA and Turkey's post hoc test were used to analyze the differences between multiple groups. P < 0.05 indicated a significant difference statistically.

Rats were free to access water containing 12 and 24 mmol/L of YCl₃, respectively, for 6 months, and the chronic effects of YCl₃ on the testis in rats were investigated. The results showed that YCl₃ could dose-dependently reduce the semen quality in rats, as indicated by decreased sperm motility (Figure 1A) and concentrations (Figure 1B) and increased sperm deformity (Figure 1C). In addition, the gonadosomatic index (Figure 1D) and the levels of serum testosterone (Figure 1E) were also decreased by YCl₃ treatment in a dose-dependent manner. Histopathological examination by HE staining (Figure 1F) in the YCl₃-treated group indicated that the arrangement of spermatogenic cells at all levels was loose and disordered and the number of mature spermatozoa was reduced, compared with those in the negative control group. Furthermore, the anatomic structure of seminiferous tubules was damaged and the distance among the seminiferous tubules was enlarged. These suggested that YCl₃ treatment induced significant toxicity and damage to the testis.

To further investigate how YCl₃ treatment induced testis damage, the protein expression of Bcl-2 and caspase-3 in the testis organs was detected. YCl₃ treatment could down regulate Bcl-2 expression (Figures 2A, B) and up regulate caspase-3 expression (Figures 2A, C), indicating induction of apoptosis. In addition, the in vitro study on the cytotoxicity of YCl₃ on the cultured Leydig cells was detected. The results from the cellular viability detection showed that YCl₃ at the doses of 300 μM indicated almost the greatest cytotoxicity (Figure 2D). Additionally, the expression of cleaved caspase-3 was up regulated, and the expression of Bcl-2 was down regulated (Figures 2E–G). In the TUNEL/DAPI staining assays, YCl₃ significantly increased cell apoptosis (Figure 2H). Collectively, the induction of testis damage by YCl₃ was associated with increased apoptosis.

To further explore the possible mechanism of YCl₃ in the regulation of apoptosis, the expression of IP3R1 was detected. The in vivo immunohistochemical study (Figures 3A, B) showed that the expression of IP3R1 in YCl₃-treated rats was increased. In YCl₃-treated Leydig cells in vitro, the protein expression of IP3R1, p-CaMKII, and CaMKII (Figures 3C–F) was up regulated. The protein expression of Bcl-2 (Figures 3C, G) was increased, and the expression of cleaved caspase-3 (Figures 3C, H) was decreased. In addition, the concentration of cytosolic Ca²⁺ (Figure 3I) was significantly enhanced. The apoptosis rate (Figure 3J) was elevated. The stimulating effects of YCl₃ were blocked by co-treatment with IP3R1 inhibitor 2-APB (1 μM). These suggested that YCl₃ might induce cell apoptosis by up regulating the expression of Ca²⁺/IP3R1 signaling in Leydig cells.

To further explore the possible mechanism of YCl₃ in the regulation of cell apoptosis, the activity of CaMKII was determined. The expression of CaMKII (Figures 4A, B) in YCl₃-treated rats was investigated by an in vivo immunohistochemical study, and it was significantly increased. The expression of CaMKII and p-CaMKII in YCl₃-treated Leydig cells in vitro was also up regulated. After co-treatment with CaMKII inhibitor KN93 (1.48 μM), the increased expression of p-CaMKII and cleaved caspase-3 and the decreased expression of Bcl-2 were reversed (Figures 4C–G). Similarly, the changes in cell apoptosis (Figure 4H) were rescued. Collectively, YCl₃ induced cell apoptosis by up regulating the activity of CaMKII in Leydig cells.