FOXP3 pathogenic variants cause male infertility through affecting the proliferation and apoptosis of human spermatogonial stem cells

FOXP3 PV in patients with NOA

To explore the functional importance of FOXP3 in human male infertility, we screened for potential PV of FOXP3 in 314 NOA patients and 14 OA controls (Figure 1A). Using exome sequencing, we identified the same variant in nine NOA patients (Patients 1–9) and another variant in one NOA patient (Patient 10) (Figure 1B and 1C); none of variant was detected in the OA controls (Figure 1B and 1C). The same variant was a homozygous missense PV in exon 1 (c.155 G > T (G52V)) (Figure 1B, left bottom panel, Figure 1C), while another variant was a heterozygous missense PV in exon 6 (c.691 C > A (Q231K)) (Figure 1B, right bottom panel, Figure 1C) in human FOXP3 gene (GenBank: NM_014009.3). A full-length FOXP3 protein model (amino acid 1-431) was constructed (Figure 1D, left panel). FOXP3 may be processed by proteolytic cleavage upon cell activation, which leads to a truncated FOXP3 form (amino acid 52-417) (Figure 1D, right panel).

Figure 1. Identification of FOXP3 genomic PV from patients with NOA. (A) A total of 314 patients with NOA and 14 OA patient controls were analyzed in this study. Ten PV of FOXP3 were identified in 314 NOA patients (3.18% incidence, 10/314), whereas no PV of FOXP3 was seen in the OA controls. (B) Chromatogram of the sequences in the mutant region of NOA patients and corresponding OA controls. (C) Comparison of the wild-type and mutant FOXP3 proteins. (D) FOXP3 structure model and stability prediction. (E) Pedigrees of Patients 1, 2 and 10 for their FOXP3 PV. Circles, females; squares, males. Filled symbols, affected individuals; open symbols, unaffected individuals; gray symbols, carriers.

To track these PV in NOA patient pedigrees, we further sequenced the families of Patients 1, 2 and 10. Notably, the mothers of Patient 1 and Patient 2 and the fertile sister of Patient 1 shared the same heterozygous G52V PV, while their fathers carried the wild-type sequence (Figure 1E, left and middle panels), which reflects a recessive inheritance model. In contrast, both parents of Patient 10 presented wild-type FOXP3 sequences (Figure 1E, right panel), indicating that Q231K arose de novo in this patient. These data suggest that both inheritable and de novo PV of FOXP3 affect male fertility, and c.155 G > T PV of FOXP3 is consistent with the X chromosome-linked inheritance patterns.

Phenotypic and morphological characteristics of FOXP3-mut NOA patients

We next evaluated the testicular morphology and determined whether intrinsic testicular defects occur in NOA patients when FOXP3 was mutated. H&E staining of OA testis tissues demonstrated the normal seminiferous tubules containing different-stage male germ cells from SSCs to mature sperms (Figure 2A, left panels; Supplementary Figure 1A, left panels). Conversely, in the NOA testis with the PV of either G52V or Q231K, the diameters of seminiferous tubule were apparently reduced with arrested spermatogenesis (Figure 2A, middle & right panels; Supplementary Figure 1A, middle & right panels). Although spermatogonia and spermatocytes were present in FOXP3-mut testis of NOA patients, neither round haploid spermatids nor elongated spermatids were observed. In addition, we compared the expression level of FOXP3 protein in FOXP3-mut patients and OA controls, and we found that FOXP3 protein was expressed at a lower level or undetected in spermatocytes (Figure 2B; Supplementary Figure 1B). Intriguingly, level of UCHL1, a hallmark for human SSCs, was significantly decreased in FOXP3-mut testis compared to OA controls (Figure 2C; Supplementary Figure 1C). These results implicate a functional disorder of SSCs and spermatocytes caused by mutant FOXP3 protein.

Figure 2. Morphology and phenotype of FOXP3-mut NOA patients and OA controls. (A) H&E staining revealed that seminiferous tubule diameter was reduced and spermatogenesis was arrested in FOXP3-mut NOA patients. Scale bars = 40 μm and 20 μm, respectively. (B-C) Immunohistochemical staining demonstrated the expression of FOXP3 protein (B) and UCHL1 protein (C) in FOXP3-mut NOA patients (low panels) and OA controls (upper panels). Experiments were repeated for at least three times. Scale bars = 20 μm.

In light of the decreased seminiferous tubule sizes and disrupted spermatogenesis in FOXP3-mut patients, we further assessed the overall proliferation and apoptosis levels of their testis tissues. Immunohistochemistry of two generally recognized markers of cellular proliferation, namely PCNA and Ki67, revealed that the proliferation of male germ cells was obviously reduced in FOXP3-mut testis compared to OA controls (Figure 3A, 3B; Supplementary Figure 2A, 2B), whereas the percentage of TUNEL-positive cells was significantly increased in FOXP3-mut testis compared with OA controls (Figure 3C–3D; Supplementary Figure 2C). Collectively, these data indicate that FOXP3 PV results in abnormal testis morphology and spermatogenesis arrest in human by influencing the proliferation and apoptosis of male germ cells.

Figure 3. Cell proliferation and apoptosis in the testis of FOXP3-mut NOA patients and OA controls. (A, B) Immunohistochemical staining showed the levels of PCNA (A) and Ki67 (B), the hallmarks for cell proliferation, were decreased in FOXP3-mut NOA patients (lower panels) compared to the OA controls (upper panels). (C) TUNEL assay demonstrated the TUNEL-positive cells (red fluorescence) in FOXP3-mut NOA patients and OA controls. DAPI (blue fluorescence) was used to label cellular nuclei. Replacing the TdT enzyme with PBS was used as the negative control. Scale bars in A-C= 20 μm. (D) The percentages of apoptosis in male germ cells of FOXP3-mut NOA patients and OA controls were calculated using Student’s t-test. All values are means ± SD from three independent experiments. * indicated statistically significant differences (p<0.05).

FOXP3 expression in human testis tissues and SSCs

To determine cellular location of FOXP3 in human testis, immunohistochemistry was performed on human OA controls. FOXP3 protein was found to be extensively present in male germ cells, including SSCs, spermatocytes, and round spermatids of human testes, and the staining was especially strong in SSCs along the basement membrane (Figure 4A; Supplementary Figure 3A), which indicates functional importance of FOXP3 in mediating human SSC fate determination. Next, we verified the phenotype of our previously established human SSC line which expressed numerous genes for human germ cells, spermatogonia and SSCs, including VASA, GPR125, GFRA1, UCHL1, THY1, RET and PLZF, as shown by RT-PCR (Figure 4B; Supplementary Figure 3B). And the expression of SV40, GPR125, THY1, RET, DAZ2 and UCHL1 proteins have been previously demonstrated by Western blots and immunocytochemistry [19]. RT-PCR and Western blots showed that this SSC line expressed FOXP3 both at mRNA (Figure 4C) and protein (Figure 4D) levels, and immunocytochemistry identified FOXP3 location in the nuclei of these cells (Figure 4E; Supplementary Figure 3C).

Figure 4. Expression and location of FOXP3 proteins in human testes and human SSC line. Immuno-histochemistry revealed cellular localization of FOXP3 in human testes (left panel). Replacement of anti-FOXP3 with PBS was used as a negative control (right panel). Scale bars = 20 μm. (B) RT-PCR showed the transcripts of GPR125, THY1, PLZF, UCHL1, GFRA1, RET and VASA in human SSC line. Samples without cDNA (No cDNA) but PCR with gene primers were used as negative controls. ACTB served as loading controls of total RNA. (C) RT-PCR showed the mRNA level of FOXP3 in human SSC line. Samples without cDNA (No cDNA) but PCR with gene primers were used as negative controls, and ACTB served as a loading control of total RNA. (D) Western blots revealed the expression of FOXP3 protein in human SSC line. ACTB served as the control of the loading proteins. Human Sertoli cells were utilized as a positive control. (E) Immunocytochemistry revealed cellular localization of FOXP3 in human SSC line. Fluorescent signals of FOXP3 (red) and DAPI (blue) were imaged individually and merged under fluorescence microscope. Scale bars = 40 μm and 20 μm, respectively. All experiments were repeated for at least three times.

Effect of FOXP3 on proliferation and apoptosis of human SSCs

To further explore the function of FOXP3 in mediating the fate decision of human SSCs, we designed three pairs of small siRNAs targeting different regions of FOXP3 mRNA. FAM-labeled siRNA oligonucleotides indicated that the transfection efficiency of siRNA in human SSC line was around 80% (Figure 5A; Supplementary Figure 4A). Real-time PCR and Western blots revealed that both FOXP3 mRNA and FOXP3 protein were significantly decreased in human SSC line by FOXP3-siRNA 1 and FOXP3-siRNA 3 (Figure 5B, 5C; Supplementary Figure 4B, 4C). Proliferation ability of human SSC line after knockdown of FOXP3 was determined by EDU and CCK-8 assays. The percentage of EDU-positive cells was diminished in human SSC line by FOXP3-siRNA 1 and FOXP3-siRNA 3 (Figure 5D; Supplementary Figure 4E). Consistently, CCK-8 assay showed that FOXP3-siRNA 1 and FOXP3-siRNA 3 inhibited the proliferation of human SSCs in a time-dependent manner (Figure 5E; Supplementary Figure 4D). Western blots displayed the decrease in PCNA level after knockdown of FOXP3 (Supplementary Figure 5). These data indicated that FOXP3 promotes proliferation and DNA synthesis of human SSCs. The apoptosis of SSCs after transfection of FOXP3-siRNA 1 and FOXP3-siRNA 3 was assessed by Annexin V-FITC/PI staining and flow cytometry demonstrating that FOXP3 silencing led to an increase of both early and late apoptosis of human SSC line, especially the early apoptosis (Figure 5F–5G; Supplementary Figure 4F). Taken together, these results suggest that FOXP3 promotes the proliferation and DNA synthesis whereas inhibits the apoptosis of human SSCs.

Figure 5. Influence of FOXP3 Knockdown on the proliferation, DNA synthesis and apoptosis of human SSC line. (A) Fluorescence microscope and phase-contrast microscope revealed transfection efficiency of FOXP3-siRNAs using the FAM-labeled miRNA oligonucleotides. Scale bars = 40 μm. (B) Western blots showed the protein changes of FOXP3 by FOXP3-siRNAs in human SSC line. (C) Real-time PCR displayed the mRNA changes of FOXP3 by FOXP3-siRNAs in human SSC line. (D) EDU incorporation assay showed the percentages of EDU-positive cells affected by control siRNA, FOXP3-siRNA 1 and FOXP3-siRNA 3 in human SSC line. Scale bars = 40 μm. Values are means ± SD from three independent experiments. (E) CCK-8 assay demonstrated the proliferation of human SSC line after transfection with FOXP3-siRNA 1 and FOXP3-siRNA 3. (F–G) Annexin-V/PI staining and flow cytometry displayed the percentages of early apoptosis and late apoptosis of human SSC line transfected with FOXP3-siRNA 1 and control siRNA. All values are means ± SD from three independent experiments. * indicated statistical significance (p<0.05) between FOXP3-siRNAs and control siRNA treatments. All experiments were repeated for at least three times.

Human NOA and OA patients as well as peripheral blood and testis samples

In total, 314 patients with NOA were recruited from 4 hospitals, including Shanghai General Hospital affiliated to Shanghai Jiao Tong University, Reproductive and Genetic Hospital of CITIC-Xiangya, Oriental Jinghua Hospital of Shenyang, and The Third Affiliated Hospital of Guangzhou Medical University. Additionally, 14 obstructive azoospermia (OA) patients with normal spermatogenesis were recruited from Shanghai General Hospital affiliated to Shanghai Jiao Tong University. The written informed consent was obtained from all participants. Genomic DNA was extracted from the peripheral blood samples of NOA and OA patients. Testicular biopsies of patients identified with FOXP3 mutant were further obtained to verify their PV in testicular genomic DNA.

Exome sequencing

Exome sequencing was performed on genomic DNA of the OA and NOA patients with their families by Genewiz Co., Ltd. Primers designed to amplify each exon of the FOXP3 gene were listed in Table 1. The TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) was used for gDNA capture, and the PCR products were detected by agarose gel electrophoresis. Paired-end sequencing was conducted using ABI Prism^® 3730 instrument (Applied Biosystems, Foster City, CA, USA).

Immunohistochemistry

Human testis paraffin sections were routinely deparaffinized and hydrated. After antigen retrieval and endogenous peroxidase blocking, the sections were blocked with 5% BSA (Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at room temperature and incubated with primary antibodies, including anti-FOXP3 (Abcam, catalogue: ab22510), anti-UCHL1 (Abcam), anti-PCNA (Abcam), and anti-Ki67 (Abcam), at 4°C overnight. Antibodies binding were detected with HRP conjugated second antibodies, stained by DAB (Vector Lab, Burlingame, USA) and counterstained with hematoxylin, or detected by Alexa Fluor-conjugated second antibody (Invitrogen, USA) (1:200, room temperature, 1 h). DAPI was utilized for staining cell nuclei. The images were captured using the fluorescence microscope (Nikon, Tokyo, Japan).

TUNEL

For testis paraffin sections, the general levels of apoptosis were evaluated via TUNEL Apoptosis Detection Kit (Alexa Fluor 647) (YEASEN, Shanghai, China) according to manufacturer’s instruction. After being deparaffinized and hydrated, human testis sections were permeabilized (Proteinase K, 20 min), equilibrated (1x Equilibration, room temperature, 30 min), incubated with TdT buffer (Alexa Fluor 647-dUTP Labeling Mix and TdT enzyme, 37°C, 1 h, in the dark), and counterstained with DAPI (room temperature, 5 min). Replacement of the TdT enzyme with PBS was used as the negative control, and the images was captured under fluorescence microscope.

Cell culture and RNA interference (RNAi) of FOXP3

Human spermatogonial stem cell (SSC) line was established previously in our laboratory by transfecting primary human SSCs with a plasmid expressing the SV40 large T antigen [19], and this SSC line has been characterized with unlimited proliferation potentials and positive for a number of hallmarks for human spermatogonia and SSCs [19, 20]. Human SSC line was cultured (37°C, 5% CO₂) in DMEM/F12 (Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA).

The FOXP3-siRNAs and control siRNA were synthesized by GenePharma (Suzhou, China), and the sequences of siRNAs were listed in Table 2. Cells were seeded at 0.8 x 10⁵/well density to 6-well plates, and transfection was performed using lipofectamine 3000 (Life technologies, Carlsbad, USA) according to manufacturer’s instruction. For each transfection, 4 μl of lipofectamine 3000 transfection reagent in 200 μl Opti-MEM (Invitrogen) were added to 200 μl of Opti-MEM predissolved with 120 pmol of siRNAs and incubated for 15 min at room temperature. The transfection efficiency was detected 6 h later using Nikon fluorescence microscope, and transfected cells were harvested at 24 h or 48 h for mRNA or protein analysis, respectively.

RNA extraction, RT-PCR and quantitative real-time PCR

Total RNA was extracted from human SSC line using RNAiso Plus reagent (Takara, Kusatsu, Japan), and the concentrations and quality of isolated RNA were determined by Nanodrop (Thermo Scientific, Massachusetts, USA). Reverse transcription (RT) was conducted by the HiScript^@II 1^st Strand cDNA Synthesis Kit (Vazyme, R212-02) according to the manufacturer’s instruction. The primers of genes for RT-PCR and real-time PCR were listed in Table 3. The PCR reactions were carried out using Premix Taq™ (TaKaRa Taq™ Version 2.0 plus dye), and the products were analyzed by agarose gels stained with GenGreen nucleic acid gel stain (Genview) and Image Analyzer ChemiDoc XRS⁺ (Bio-Rad).

Real-time PCR reactions were conducted using PowerUp^TM SYBR^® Green Master Mix (Applied Biosystems, Woolston Warrington, UK) and StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). The relative expression levels of indicated genes were analyzed via the C_T (threshold cycle) value by normalizing with the housekeeping gene ACTB [ΔC_T=C_T(GENE)−C_T(ACTB)], and the changes of gene expression after treatment compared to the control was calculated by formula 2^−ΔΔCT [ΔΔC_T =ΔC_T(treated)−ΔC_T(control)].

Western blots

Human SSC line was collected 48 h after transfection with control siRNAs or FOXP3-siRNAs. After being lysed with RIPA buffer (BiotechWell, Shanghai, China) containing protease inhibitor cocktail (MedChemExpress, New Jersey, USA) for 30 min on ice, lysates were centrifugated (12,000 rpm, 4°C, 15 min), separated using 10% SDS-PAGE and transferred to PVDF membranes. The blots were probed with primary antibodies against FOXP3 (Abcam, catalogue: ab22510) or PCNA (Abcam) and incubated with HRP-labeled secondary antibodies (Abcam). HRP-labeled ACTB (Proteintech, catalogue: HRP-60008) was used as control. The blots were detected by Chemi-Doc XRS system (Bio-Rad Laboratories, Hercules, USA).

Immunocytochemistry

Human SSC line was fixed with 4% paraformaldehyde (PFA, room temperature, 30 min), permeabilized with 0.5% Triton X-100 (Sigma), blocked with 5% BSA (room temperature, 1 h) and incubated with the primary antibodies against FOXP3 (Santa Cruz, sc-166212) at a dilution of 1:50 at 4°C overnight. Antibody binding was detected with Alexa Fluor-conjugated second antibody (Invitrogen, USA) (1:200, room temperature, 1 h) and the nuclei were stained with DAPI (room temperature, 5 min). After extensive washes with PBS, cells were observed under the fluorescence microscope.

CCK-8 assay

Human SSC line was seeded at a density of 4,000 cells/well in 96-well microtiter plates and transfected with various kinds of siRNAs as mentioned above. The proliferation of the cells was detected by CCK-8 assay (Dojin Laboratories, Kumamoto, Japan) according to the manufacturer’s instruction. In brief, 10 μl of CCK-8 reagents and 100 μl of DMEM/F12 medium were mixed before being added to each well. After three hours’ incubation, the absorbance at the wavelength of 450 nm was measured by microplate reader (Thermo Scientific).

EDU incorporation assay

After being transfected with FOXP3 siRNAs in 96-well plate for 24 h, human SSC line was incubated with 50 μM EDU (RiboBio, Guangzhou, China) for 12 h, fixed (4% PFA, room temperature, 30 min), neutralized (2 mg/ml glycine) and permeabilized (0.5% Triton X-100 room temperature, 10 min). After being washed with 0.5% tritonx-100 in PBS and incubated with 100 μl of 1x Apollo-Fluor (RiboBio, Guangzhou, China) (room temperature, 30 min, in the dark), cellular nuclei were stained with DAPI for 5 min. The percentages of EDU-positive cells were counted from more than 500 cells and three independent experiments were performed.

Annexin-V/PI staining and flow cytometry

The apoptosis rates of transfected human SSC line was determined by the APC Annexin V Apoptosis Detection Kit with PI (Biolegend, London, UK) and detected by flow cytometry pursuant to the manufacturer’s instruction. Cells were seeded at 0.8x10⁵ cells/well in 6-well plates and harvested after transfection. Simultaneous staining with APC Annexin V and PI distinguished healthy cells (FITC^-PI^-), early apoptotic (FITC⁺PI^-) and late apoptotic or necrotic cells (FITC⁺PI⁺).

Statistical Analysis

All data were presented as mean ± SD from at least three independent experiments and analyzed by Student’s t-test using GraphPad Prism 6.01 Software. Normality and homogeneity of variances were checked prior to conduct Student’s t-test, and P < 0.05 was considered statistically significant.