A total of 10 C57 male mice weighing 18–21 g were purchased and raised in Guangzhou Medical University Animal Center (Guangzhou, China). All mice were housed under standard conditions (21±2°C, 40% humidity and 12-h light/dark cycle), and had free access to water and food. The procedures were performed as described previously (15,16). Briefly, mouse models of hypospermatogenesis (N=5) were established by a single intraperitoneal injection of busulfan (30 mg/kg). Normal controls (N=5) didn't receive any treatment. Following 2 weeks, fresh testis tissues were collected and stored in liquid nitrogen or fixed with 1.25% Bouin solution for 6 h at room temperature. The current study was approved by the Guangzhou Medical University Institutional Animal Care and Use Committee.

Testis tissues were fixed with Bouin's solution for 6 h at room temperature and embedded in paraffin. Sections (2-µm-thick) were heated at 65°C for 4 h, deparaffinized with dimethylbenzene, rehydrated in gradient ethanol series and stained with HE for 5 min at room temperature. Under the light microscope, reduced production of spermatozoid in seminiferous tubules was easily observed in mouse models of hypospermatogenesis by counting the number of sperm cells.

Total RNA was extracted from testis tissues and spermatogenic cells using RNA TRIzol reagent (Takara Bio, Inc., Otsu, Japan) and synthesized using the PrimeScript™ RT Reagent kit (Takara Bio, Inc.) at 37°C for 15 min. qPCR analysis was performed using the SYBR Green RT-PCR kit (Takara Bio, Inc.) according to the manufacturer's protocol. The primers of PGAM1 and GAPDH were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The following primers were used: PGAM1, forward (F) 5′-ATGCTAAGCCATGACCAGTGAG-3′ and reverse (R) 5′-ATCACCACGCAGGTTACATTCG-3′; GAPDH, F 5′-AGGTCGGTGTGAACGGATTTG-3′ and R 5′-TGTAGACCATGTAGTTGAGGTCA-3′. Cycling conditions were 95°C for 5 min, followed by 40 cycles at 95°C for 40 sec, 60°C for 30 sec and 72°C for 30 sec. Experiments were repeated three times for each reaction. The 2^−ΔΔCq method was used to determine the relative mRNA expression levels (16).

Total protein lysates were extracted from testis tissues and spermatogenic cells using cell lysis buffer (cat. no. P0013; Beyotime Institute of Biotechnology, Shanghai, China) containing a protease inhibitor cocktail (cat. no. P1006; Beyotime Institute of Biotechnology) and 100 nM phenylmethanesufonyl fluoride. Protein concentration was determined by bicinchoninic acid assay. Subsequently, 50 µg protein was separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were dipped into PBS with 0.1% Tween 2000 (BestBio, Shanghai, China) solution and blocked with 5% skimmed milk at room temperature for 2 h, and then incubated with rabbit monoclonal PGAM1 antibody (cat. no. ab129191; 1:1,000; Abcam, Cambridge, MA, USA), rabbit polyclonal P53 antibody (cat. no. ab16033; 1:2,000; Abcam), rabbit polyclonal Caspase 3 antibody (cat. no. ab13847; 1:500; Abcam), rabbit polyclonal Caspase 6 antibody (cat. no. ab231349; 1:500; Abcam), rabbit polyclonal Caspase 9 antibody (cat. no. ab52298; 1:500; Abcam) and monoclonal tubulin antibody (cat. no. ab6160; 1:2,000; Abcam), respectively. Finally, polyvinylidene difluoride membranes were incubated with a rabbit anti-goat horseradish peroxidase-conjugated secondary antibody (cat. no. ZB-2306; 1:1,000; OriGene Technologies, Inc., Beijing, China) at room temperature for 30 min. Signals were detected using an EasyBlot ECL kit (cat. no. C506668-0050; Sangon Biotech Co., Ltd., Shanghai, China) and an enhanced chemiluminescence detecter (ProteinSimple; Bio-Techne, Minneapolis, MN, USA).

Testis tissues were fixed with Bouin's solution for 6 h at room temperature and embedded in paraffin. Sections (2-µm-thick) were deparaffinized with dimethylbenzene and rehydrated with gradient ethanol. Antigen retrieval was performed with 0.01 mol/l sodium citrate buffer (pH 7.0). The sections were incubated with rabbit monoclonal PGAM1 antibody (cat. no. ab129191; 1:150; Abcam) for 2 h, followed by incubation with 50 µl horseradish peroxidase-labeled goat anti-mouse/rabbit IgG polymer (cat. no. PV-6000; OriGene Technologies, Inc.) for 30 min at room temperature. Finally, sections were reacted with diaminobenzidine at room temperature for 1–3 min and stained with hematoxylin for 3 min at room temperature. Negative controls were performed by replacing the primary antibody with PBS. Sections with positive expression of PGAM1 were treated as positive controls.

The procedures were performed as described previously (17). Testis tissues were fixed with Bouin's solution for 12 h at room temperature and embedded in paraffin. Sections (3-µm-thick) were deparaffinized with dimethylbenzene and rehydrated with gradient ethanol. Then, sections were incubated with 1% proteinase K (20 mg/ml) at 37°C for 30 min. Following washing with PBS, sections were incubated with TUNEL mix (45 µl Equilibration Buffer, 1.0 µl biotin-11-dUTP, 4.0 µl TdT enzyme and 50 µl reaction buffer) at 37°C for 60 min. Finally, sections were stained with DAPI for 5 min at room temperature and mounted in 50% glycerin diluted in water. A total of 10 randomly chosen microscopic fields were analyzed under a fluorescence microscope (magnification, ×40). The nuclei of apoptotic cells were marked as red under fluorescence microscopy at 570 nm.

GC-1 spg cells (a mouse spermatogonia cell line) and GC2 cells (a mouse spermatocyte cell line) were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a 5% CO₂ incubator. Cells at 40–60% confluence were transfected with PGAM1 short hairpin (sh)RNA (sense, 5′-ATGCTAAGCCATGACCAGTGAG-3′ and anti-sense, 5′-ATCACCACGCAGGTTACATTCG-3′) lentivirus and recombinant PGAM1 lentivirus, respectively. The negative control was transfected with scrambled shRNA lentivirus (sense, 5′-UUCUCCGAACGUGUCACGU-3′ and anti-sense, 5′-AAGAGGCUUGCACAUGCA-3′).

At 48 h following transfection, 1×10⁶ GC1 and GC2 cells were collected and washed with PBS. Then, cells were incubated with propidium iodide (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and Annexin V-FITC (BestBio) at room temperature for 15 min. Cell apoptosis was analyzed using a BD FACSCalibur™ flow cytometer and BD CellQuest™ software version 5.1 (BD Biosciences, Franklin Lakes, NJ, USA).

All experiments were repeated three times and the data are presented as the mean ± standard deviation. All data were analyzed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). Independent samples t test was used to compare the difference between two groups. All tests were two-sided and P<0.05 was considered to indicate a statistically significant difference.

To investigate the association between PGAM1 expression and hypospermatogenesis, the expression of PGAM1 was detected in mouse models of busulfan-induced hypospermatogenesis by western blotting, RT-qPCR and IHC (Fig. 1). Western blotting (Fig. 1A) and RT-qPCR (Fig. 1B) results revealed that PGAM1 expression levels were significantly decreased compared with normal controls (P<0.001). HE staining demonstrated that the number of spermatogonia, spermatocytes and spermatozoid in seminiferous tubules was decreased in mouse models of busulfan-induced hypospermatogenesis compared with the controls (Fig. 1C and D). IHC results demonstrated that positive PGAM1 expression, visible as brown staining, was present in spermatogonia, spermatocytes and Leydig cells in the testis tissues with normal spermatogenesis (Fig. 1E), while expression was hardly detected in mouse models of busulfan-induced hypospermatogenesis (Fig. 1F). These data indicated that PGAM1 downregulation was associated with busulfan-induced hypospermatogenesis.

To investigate the association between PGAM1 expression and spermatogenic cell apoptosis in a mouse model of busulfan-induced hypospermatogenesis, a TUNEL assay was performed. As presented in Fig. 2, the apoptosis rate of spermatogenic cells was accelerated in mouse models of hypospermatogenesis compared with normal spermatogenesis. These data indicated that PGAM1 downregulation may be associated with spermatogenic cell apoptosis in mouse models of busulfan-induced hypospermatogenesis.

To further investigate the association between PGAM1 expression and spermatogenic cell apoptosis in vitro, a flow cytometry assay was performed. Firstly, spermatogenic cells were infected with PGAM1 shRNA lentivirus or recombinant PGAM1 lentivirus. As presented in Fig. 3, western blotting results indicated that PGAM1 was successfully knocked down and upregulated following transfection, respectively. Then, cell apoptosis was measured by flow cytometry (Fig. 4). The rate of apoptosis was 15.7±2.3% in GC1 cells with PGAM1 downregulation, which was significantly higher compared with the negative controls (4.8±0.9%; P<0.001). The rate of apoptosis was 2.2±0.6% in GC1 cells with PGAM1 upregulation, which was significantly reduced compared with negative controls (4.8±0.9%; P<0.001). In addition, the rate of apoptosis was 16.4±2.8% in GC2 cells with PGAM1 knockdown, which was significantly greater compared with negative controls (5.9±1.1%; P<0.001). When PGAM1 expression was upregulated in GC2 cells, the rate of apoptosis was 2.3±0.5%, which was significantly reduced compared with negative controls (5.9±1.1%; P<0.001). These data demonstrated that PGAM1 knockdown contributed to spermatogenic cell apoptosis.

To investigate the mechanism of PGAM1, the expression levels of P53, Caspase 3, Caspase 6 and Caspase 9 were detected by western blotting. As presented in Fig. 3, the expression levels of P53, Caspase 3, Caspase 6 and Caspase 9 were significantly elevated following PGAM1 knockdown. However, PGAM1 upregulation produced the opposite effects. These data indicated that PGAM1 knockdown activated the P53/Caspase 3/Caspase 6/Caspase 9 signaling pathway, which may result in spermatogenic cell apoptosis.