ICR mice were purchased from the Experimental Animal Center, Chinese Academy of Sciences (Beijing, China). All experimental protocols were reviewed and approved by the Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Science.

SCs were isolated from the testes of 21-day-old mice as previous reports (12, 13). After decapsulating the testes, the collected seminiferous tubules were pooled and washed for three times with phosphate buffer saline. The tubules were digested for cell suspension, mainly containing SCs and type A spermatogonia. Cell suspension was then cultured at 35 ℃ with 5 % CO₂ incubator in Dulbecco modified Eagle medium/Ham F-12 (DMEM/F12) containing 10 % fetal calf serum, and 100 U/ml of penicillin. After 48 hr, cells were subjected to hypotonic treatment with 20 mM Tris (pH 7.4) to lyse the residual spermatogonia to obtain SCs.

Small interfering RNA (siRNA) molecules specifically targeting klf6 mRNA were purchased from GenePharma (Shanghai, China). The sequences of siRNAs for Klf6 and negative control were provided in Table 1. SCs were transfected with Klf6-targeted siRNA or negative control by lipofectamine RNAiMAX reagent (Invitrogen, CA, USA) following the manufacturer’s procedure at a final concentration of 0.1 μM. 72 hr after transfection, SCs were harvested for the mRNA quantification, Western blot, and immunofluorescence analysis.

Short hairpin RNA (shRNA) molecules specifically targeting klf6 mRNA were further constructed to make stable and long-term silencing effect on Klf6 gene. The effective siRNA (Klf6-984) was cloned into the pENTR/U6 for packaging lentivirus (14). The titer of lentivirus harboring klf6-shRNA was determined as 10⁹ IU/ml. SCs were infected with klf6-shRNA lentivirus and collected for further analysis after 72 hr.

Transfected SCs were collected and labeled with the Deadend^TM Fluorometric TUNEL System (Promega, CA, USA) following the manufacturer’s protocol. Briefly, the cells were fixed with 4 % paraformaldehyde on ice for 20 min and then incubated with 70 % ice-cold ethanol for 4 hr. After washing, the cells were resuspended in equilibration buffer for 5 min. Cells were incubated with reaction buffer containing equilibration buffer, nucleotide xix, and rTdT enzyme at 37 ^oC for 60 min. The reaction was terminated with 20 mM EDTA, and cells were further stained with DAPI (Beyotime, Shanghai, China) and analyzed with the FACSCalibur system (BD Biosciences, CA, USA).

Total RNA was extracted from both testes and cell samples with RNAprep Pure Micro Kit (TianGen, Beijing, China). Reverse transcription of purified RNA was performed with Reverse Transcription System (Abm, Beijing, China) following the manufacturer’s protocol. PCR was performed with Qingke mix (Beijing, China) and products were separated by 1.2 % agarose gel electrophoresis. Quantitative RT-PCR was performed with Power SYBR Green Master Mix (Applied Biosystems, CA, USA) and analyzed by QuantStudioTM 12 K Flex Real-Time PCR System (BioRad, MA, USA). Primer pairs of selected genes were listed in Table 2.

Proteins were collected from testes and SCs and then separated by 12 % SDS-PAGE and transferred to PVDF membranes (Millipore, CA, USA). PVDF membranes were blocked with 5 % skim milk for 1 hr and then incubated with primary antibodies at 4 ^oC overnight. Primary antibodies were listed as follows: Klf6 (1:1000, ab15580, Abcam), ZO-1 (1:1000, 339100, Invitrogen), Occludin (1:1000, ab31721, Abcam), claudin-3 (1:1000, 187340, Invitrogen), claudin-11 (1:1000, 364500, Invitrogen), vimentin (1:1000, 5741T, CST), and GAPDH (1:10000, MB001, Baode). The enzyme labeled secondary antibodies corresponding to the species of primary antibody were incubated with the PVDF membrane for 1 hr. Protein bands were developed with the Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific, CA, USA), and detected using ChemiDoc XRS system with Image lab software (Bio-Rad, MA, USA).

Immunofluorescent staining was performed as our previous report (6). Briefly, testes were freshly collected, fixed in 4 % PFA and embedded in paraffin. The paraffin sections of 5 µm were obtained with a microtome. Sections were incubated with the primary antibody at 4°C overnight. Antibodies were obtained commercially as follows: Klf6 (1:100, ab15580, Abcam), Sox9 (1:200, AB5535, Millipore), Zo-1 (1:200, 339100, Invitrogen), Wt1 (1:200, ab89901, Abcam), vimentin (1:200, 5741T, CST). After washing, sections were treated with FITC or TRITC-conjugated secondary antibodies (Jackson ImmunoResearch, PA, USA) for 45 min. Nuclei were stained with DAPI (Beyotime, Shanghai, China). Images were acquired using a Nikon Eclipse 80i fluorescence microscope (Tokyo, Japan) with Nikon CCD camera.

SCs would assemble an intact epithelium with a functional tight junction barrier, which was able to resist the conductivity of electrical current delivered by a Millicell Electrical Resistance System (Millipore, MA, USA). Briefly, SCs were firstly plated on matrigel-coated bicameral units (diameter 12 mm; pore size 0.45 μm, effective surface area 0.6 cm²; Millipore) with the cell density of 1.0×10⁶ cells/cm². Each bicameral unit was placed inside the well of a 24-well dish with 0.5 ml DMEM/F12 each in the apical and the basal compartments. The resistance was then quantified as the trans-epithelial electrical resistance (TER) in ohms (Ω) by placing two electrodes across the SC epithelium, one in the apical and one in the basal compartment of the bicameral units.

Testes were firstly fixed with 2.5 % glutaraldehyde in 0.2 M cacodylate buffer overnight. Then, testes were cut into small pieces (1 mm³) and immersed in 1 % OsO₄ in 0.2 M cacodylate buffer for 2 hr at 4°C. After fixation, ethanol gradient dehydration was performed and samples were embedded in resin. Ultrathin sections were obtained with ultramicrotome (Leica, Germany). Sections were then stained with uranyl acetate and lead citrate, and observed with a JEM-1400 transmission electron microscope (JEOL, Japan).

Kfl6 cDNA in eukaryotic expressing vector pFlag-CMV4 were obtained from Addgene (MA, USA). Promoters of claudin-3, vimentin and ZO-1 were amplified from mouse genomic DNA and then cloned into PGL4.17-Luciferase vector (Addgene, E6721). Klf6-expressing plasmids, promoter-luciferase plasmids, and internal control pRL-TK-Renilla (Promega, CA, USA) constructs were co-transfected into HEK293T cells on 96-well plates using lipofectamine 2000 (Invitrogen, CA, USA) following the manufacturer’s protocol. Cell extracts were prepared 48 hr after transfecting with the lysis buffer provided in the Dual-Luciferase Reporter Assay System kit (Vigorous Biotechnology, Beijing, China). Luciferase activity was measured on a Synergy Neo2 Multi-Mode Microplate Reader instrument (Bio-Tek, MA, USA) and the Renilla luciferase activity was applied to normalize the firefly luciferase activity.

All experiments were independently repeated at least three times. Data was presented as mean ± SEM (standard errors). Statistical analysis was performed with student’s t-test. All statistical tests were performed with SPSS 13.0 software (SPSS, CA, USA). *P<0.05 was considered as significance.

The expression profile of Klf6 in various tissues of mice was examined using RT-PCR. Klf6 mRNA was universally expressed in lung, kidney, skin, brain, testis, ovary, liver, and heart (Figure 1A). Furthermore, the expression of klf6 mRNA and protein was examined during testis development. Klf6 mRNA level in testes was gradually increased from 2 to 7 day-post-partum (dpp), while it was decreased from 7 dpp to 56 dpp (Figure 1B). Western blot analysis revealed that the Klf6 protein level in testes was increased from 6 to 56 dpp (Figure 1C). The expression of Klf6 in Sertoli cells and Leydig cells of adult mouse testes was confirmed by Western blot (Figure 1D) and immunofluorescent staining (Figure 1E). Collectively, transcription factor Klf6 was consistently expressed during testis development from neonate to adulthood in mice.

Figure 1.

Expression of Klf6 in mouse testes. (A) Klf6 mRNA in various tissues of mice was analyzed with RT-PCR. Klf6 mRNA (B) and protein (C) level during testis development, from birth to adulthood (0-56 dpp). Gapdh mRNA and Gapdh protein was applied as internal control. (D) Klf6 protein level in Leydig cells and Sertoli cells. (E) Klf6 in adult mouse testes was examined by immunofluorescence staining. DAPI indicated the nucleus. Scale bar, 100 μm.

Neighboring SCs form BTB structure to support testis development and spermatogenesis (1-3). Here, we isolated and cultured mouse primary SCs to explore the roles of Klf6 in vitro. Isolated cells were confirmed to be authentic Sertoli cells using specific Sertoli cell markers Sox9 and Wt1; as expected, they were expressed in the nucleus of >90% of isolated cells (Figure 2A, B). Furthermore, tight junction protein Zo-1 (6) was expressed in the cortical zone of neighboring SCs (Figure 2C). Vimentin, a cytoskeletal protein, was localized in the cytoskeleton of cultured SCs (Figure 2D). Notably, immunofluorescent staining indicated that Klf6 protein was localized in the nucleus of these primary SCs (Figure 2E), consistent with earlier reports (6, 13). We suggest that transcription factor Klf6 was expressed in the nucleus of SCs and hypothesize that Klf6 may play some roles in the transcription regulatory network within SCs.

Figure 2.

The expression of Klf6 in isolated SCs. (A, B) Isolated cells were characterized with SC specific markers Wt1 and Sox9 that were localized in the nucleus. (C) Immunostaining of tight junction marker Zo-1. (D) Immunostaining of cytoskeletal marker Vimentin. (E) Klf6 protein was localized in the nucleus of isolated primary SCs. Scale bar, 100 μm.

Klf6 plays important roles in the proliferation and differentiation of embryonic stem cells (15). To determine the potential roles of Klf6 in the regulation of SCs, Klf6 was knocked down with its specific siRNA duplexes and the proliferative activity was examined. After the transfection of klf6 siRNA, the number of SCs was considerably reduced as compared with negative control group (Figure 3A, B). RT-PCR and Western blot analyses revealed that Klf6 was successfully silenced by siRNA approach. The knockdown efficiency was ~50% (Figure 3C, D). Moreover, cell apoptosis was examined by TUNEL assay. The apoptosis of SCs was considerably increased by Klf6 knockdown (Figure 3E, F). Collectively, these data suggest that Klf6 was indispensable for the proliferation and cell viability of SCs.

Figure 3.

Knockdown of Klf6 and the proliferation and apoptosis of SCs. SCs were transfected with Klf6 siRNA or negative control siRNA for 72 hr. (A, B) Proliferation of SCs was reduced after transfection with Klf6 siRNA. (C, D) Klf6 mRNA and protein levels were quantified with quantitative RT-PCR and Western blot, respectively. Figure (E) was the statistical result of Figure (D). All the data were obtained from three independent experiments and analyzed by student’s t-test. **P<0.01, *P<0.05.

A lentiviral vector harboring Klf6-silenced shRNA was constructed, with higher transfection efficiency of approximately 70 % (Figure 4). SCs were infected with lentivirus and the integrity and permeability of BTB among neighboring SCs was further examined with electron microscopy. The typical BTB structure was observed among SCs infected with negative control lentivirus; however, BTB structure was not complete in klf6-shRNA lentivirus-infected SCs, exhibiting irregular cell edge (Figure 5A). SC tight junction permeability was examined daily by trans-epithelial electrical resistance assay (Figure 5B). Based on the resistance values (Ω), we found that the permeability of SCs tight junction was significantly disrupted after Klf6 silencing. Collectively, these data suggest that Klf6 played a critical role in the formation and/or maintain of tight junction and BTB structure of SCs.

Figure 4.

Knockdown of Klf6 by shRNA lentivirus infection. (A, B) SCs were infected with NC or Klf6-shRNA lentivirus for 72 hr. (C) Relative mRNA level of Klf6 was determined by quantitative RT-PCR. Gapdh mRNA was served as internal control. (D) Proliferation of SCs was reduced after transfection with Klf6 shRNA. Data was expressed as mean±SEM (n=3) and analyzed by student’s t-test. **P<0.01, *P<0.05.

Figure 5.

Klf6 regulated the integrity and permeability of BTB in SCs. (A) Apoptosis of SCs was increased after Klf6 silencing by flow cytometry. Figure (C) was the statistical result of Figure (A). (B) Electron microscopy assay indicated the complete BTB structure in the control group (White braces). The structure was disrupted in the Klf6-shRNA group and the edge of SCs appeared irregular. Scale bar, 1 μm. (D) The function of SC tight junction was examined daily by trans-epithelial electrical resistance (TER) from day 1 to 8 of culture. The solid circular was the negative control, and the solid cube was the Klf6-shRNA group, which was treated with Klf6 shRNA lentivirus in vitro. Data was expressed as mean±SEM (n=3) and analyzed by student’s t-test. **P<0.01, *P<0.05.

Loss of BTB integrity is always accompanied by disordered expression of BTB-associated proteins (e.g., adhesion proteins and tight junction molecules) (1-3). To explore the underlying mechanism of Klf6 regulation of BTB permeability, we examined the expression of BTB-associated markers, including Zo-1, Occludin, Claudin-3, Claudin-11, and Vimentin. The mRNA levels of Zo-1, Claudin-11, and Vimentin were significantly declined while Claudin-3 expression was significantly increased after Klf6 silencing. Occludin mRNA level was decreased in Klf6 knockdown group, while the result was not significant (Figure 6A). In consistent with RT-PCR results, Western blot analysis indicated that the protein levels of Zo-1, Claudin-11, and Vimentin were considerably decreased, and Claudin-3 protein was up-regulated (Figure 6B).

Figure 6.

Klf6 regulated the expression of BTB-related proteins in SCs. (A) After transfection with negative control lentivirus and Klf6-shRNA lentivirus for 72 hr, mRNA was extracted from SCs and quantitative RT-PCR was conducted for Zo-1, Occludin, Claudin-11, Claudin-3, and Vimentin. (B) Proteins were extracted and Western blot were performed for Zo-1, Occludin, Claudin-11, Claudin-3, and Vimentin. Gapdh mRNA and Gapdh protein were applied as internal control. Figure (C) was the statistical result of Figure (B). Data was expressed as mean±SEM (n=3) and analyzed by student’s t-test. ***P<0.001, **P<0.01, *P<0.05.

Candidate target genes of transcription factor Klf6 were determined with dual-luciferase assay. The binding motif sequence of Klf6 was identified and the potential Klf6 binding sites in promoter of Vimentin, Claudin-3 and Zo-1 were predicted with JASPAR (http://jaspar.genereg.net/) (16, 17). The predicted binding sites were further screened according to previous studies (11, 18, 19). It indicated that Klf6 bound to ‘CACCC’ and ‘GC box’ DNA sequences (Figure 7A, B).

Figure 7.

Identification of Klf6 target genes with dual-luciferase assay. (A, B) Scheme of the proximal promoters containing predicted Klf6-binding sites for Vimentin (from -519 to -40 of the TSS), Claudin-3 (from -566 to -141 of the TSS), and Zo-1 (from -700 to -1 of the TSS). Sequences of predicted Klf6 motif and potential Klf6-binding sites in Vimentin (M1, M2, M3), Claudin-3 (M4, M5, M6, M7), and Zo-1 (M8, M9, M10) (in red) were listed. TSS: transcription start site. (C-E) Dual-luciferase assay indicated the significant activation of Claudin3 and Zo-1 promoter by transcription factor Klf6. Data was expressed as mean±SEM (n=3) and analyzed by student’s t-test. *P<0.05.

The vectors of proximal promoters containing predicted Klf6-binding sites for Vimentin (M1, M2, M3; from -519 to -40 of the transcription start site), Claudin-3 (M4, M5, M6, M7; from -566 to -141 of the transcription start site), and Zo-1 (M8, M9, M10; from -700 to -1 of the transcription start site) were constructed. Klf6-expressing plasmids, promoter-luciferase plasmids, and pRL-TK-Renilla (internal control) constructs were co-transfected into HEK293T cells. We found that vimentin promoter luciferase activity was not obviously affected by Klf6 expression (Figure 7C). Notably, Klf6 significantly increased the luciferase activity of claudin-3 and ZO-1 promoters (Figure 7D, E). Collectively, transcription factor Klf6 may regulate the tight junction and BTB structure of SCs via targeting BTB protein Zo-1 and Claudin-3.