Male Kunming mice were purchased from the experimental animal center of Huazhong Agricultural University and housed in a controlled environment (temperature of 22 ± 2 °C, relative humidity of 50-60%, light/dark cycle of 12 h/12 h) with free accesses to food and water. All the animal procedures were approved by the Institutional Animal Care and Use Committee of Huazhong Agricultural University.
Cell culture and transfection
Primary murine SCs were isolated and purified from 18- to 21-day-old mouse testes . Murine SCs were cultured in DMEM/F12 (11320033, Gibco) supplemented with 10% fetal bovine serum (10099141C, Gibco), bovine insulin (5 μg/mL), human transferrin (5 μg/mL), and epidermal growth factor (2.5 ng/mL). The swine testicular (ST) cells (ATCC Cat# CRL-1746, RRID: CVCL_2204) that have been identified as immature Sertoli cells  were purchased from the Cell Bank of Wuhan University (Wuhan, China). The porcine ST cells were cultured in DMEM/High Glucose medium (SH30022.01, HyClone) supplemented with 10% fetal bovine serum (10099141C, Gibco) at 37 °C with 5% CO2.
The full-length Pafah1b1 cDNAs of mouse (NM_013625.4) and pig (NM_214250.1) were cloned into pcDNA3.1 vector using Trelief™ SoSoo Cloning Kit (TSV-S2, Tsingke Biotechnology). The miRNAs and siRNAs were designed and synthesized by GenePharma (Shanghai, China). The plasmids, miRNAs, or siRNAs were transfected into cells using Lipofectamine™ 3000 (L3000015, Invitrogen™) or RNAiMAX (13778030, Invitrogen™) transfection reagent. The oligo sequence information is listed in Supplementary Table 1.
Intratesticular injection with lentivirus of miR-181c/d
Lentiviral vectors carrying miR-181c/d and negative control were constructed by HANBIO Technology (Shanghai, China). For in vivo experiments, sexually immature male mice at age of 16 days were randomly divided into three groups (n = 12). Mice in Groups I, II, and III were injected intratesticularly with miR-181c overexpression lentivirus (abbreviated as “LV-miR-181c”), miR-181d overexpression lentivirus (abbreviated as “LV-miR-181d”), control lentivirus (abbreviated as “LV-control”), respectively. The mice were anesthetized with 5% chloral hydrate (0.5 mL/100 g body weight) and the scrotum was swabbed with ethyl alcohol. At age of 16 and 30 days, mice received a dose of lentivirus solution (10 μL and 20 μL per testis) via direct intratesticular injection using a 30-gauge needle as previously described [32, 33]. Two weeks after the final injection, the mice were sacrificed by cervical dislocation.
Indexes and histology of testis and epididymis
Murine testes and epididymides were isolated and weighted (n = 5). Testes and caput epididymides were fixed with 4% paraformaldehyde for 24 h, dehydrated for paraffin embedding, and transversely sectioned (5 μm thickness). Paraffin sections were stained using haematoxylin and eosin. Finally, the slides were observed under a light microscope (Olympus BX53, Japan).
Sperm count and morphological analysis
Sperms were isolated from cauda epididymis and suspended in 500 μL of TYH medium (M2050, Easycheck) for 30 min at 37 °C. The sperm counts were calculated using a cell counting plate. For sperm morphological analyses, cauda epididymal sperms were spread onto glass slides and stained with Giemsa (n = 5).
Transmission electron microscopy (TEM)
The freshly isolated testes (n = 3) and murine SCs were immersed and transferred into fresh TEM fixative solution at 4 °C. And then the samples were fixed with 1% OsO4 in phosphate-buffered saline (PBS). After removing OsO4, the samples were washed three times with PBS. The ultrathin sections were mounted on copper grids and then double-stained with 2% uranium acetate saturated alcohol solution and 2.6% lead citrate. The samples were examined with an 80 kV Transmission Electron Microscope (HT7800, HITACHI).
Biotin tracer studies
The integrity of BTB was tested using a biotin tracer, as previously described . Briefly, two weeks after the final administration, mice were anesthetized with 5% chloral hydrate (0.5 mL/100 g body weight) (n = 3). Thirty microliters of EZ-Link Sulfo-NHS-LC-Biotin solution (10 mg/mL in PBS) were injected into the testicular interstitium. After 30 min, the mice were euthanized. The testes were collected, embedded in Tissue-Tek O. C. T Compound (Sakura Finetek, Japan), and frozen at −80 °C until use. Frozen sections (6 μm thickness) were fixed with 4% paraformaldehyde for 15 min and incubated with Streptavidin-FITC (S3762, Sigma-Aldrich). The cell nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI) (D9542, Sigma-Aldrich). Fluorescence images were visualized using an epifluorescence microscope (Olympus BX53, Japan). CdCl2 is known to induce BTB disruption , and mice injected intraperitoneally with CdCl2 (1 mg/kg) continuously for three days were used as positive controls. Randomly selected fields from each testis tissue section were evaluated. For semi-quantify the extent of BTB damage, we measured the distance traveled by biotin in the tubule (DBiotin) and the radius of the same tubule (DRadius). For an oval-shaped tubule, the radius is the average of the shortest and the longest distance of the tubule. The extent of the BTB damage can be expressed in percentage as: E = [DBiotin/DRadius] × 100% . The relative distance of fluorescence distribution was quantified using Image J software.
Immunofluorescence and F-actin staining
Immunofluorescence staining was performed as previously described [37, 38]. Briefly, frozen sections of testes (n = 3) or freshly isolated murine SCs cultured on coverslips were fixed with 4% paraformaldehyde for 15 min and then washed with PBS. The samples were incubated with primary antibodies and secondary antibodies. Cell nuclei were stained with DAPI. The following antibodies were used: Ki67 (A2094, ABclonal; 1:100), N-cadherin (33-3900, Invitrogen; 1:100), Occludin (71–1500, Invitrogen; 1:100), PAFAH1B1 (sc-374586, SantaCruz; 1:100), ZO-1 (61-7300, Invitrogen; 1:100), β-catenin (71-2700, Invitrogen; 1:100), WT1 (ab89901, Abcam; 1:50), FITC Goat Anti-Mouse IgG (F0257, Sigma; 1:200), FITC Goat Anti-Rabbit IgG (F0382, Sigma; 1:200), CY3 Goat Anti-Rabbit IgG (SA00009-2, Proteintech; 1:200), and CY3 Goat Anti-Mouse IgG (SA00009-1, Proteintech; 1:200). Randomly selected fields from each testis tissue section were evaluated. The relative distance of fluorescence distribution was quantified using Image J software.
For F-actin staining, testis sections (n = 3) or murine SCs were incubated with Alexa Fluor 594 phalloidin (A12381, Invitrogen) or Alexa Fluor 488 phalloidin (A12379, Invitrogen). Cell nuclei were stained with DAPI. Fluorescence images were visualized using an epifluorescence microscope (Olympus BX53, Japan) or a confocal laser scanning microscope (Zeiss LSM 800, Carl Zeiss Imaging, Germany).
Assessment of the permeability of Sertoli cell barrier in vitro
Murine SCs were plated on Matrigel-coated Millicell bicameral units (diameter, 12mm; pore size, 0.45 μm; effective surface area, 0.33 cm2, Millipore Corp) in 24-well plates containing 0.5 mL F12/DMEM. The permeability of the Sertoli cell barrier can be assessed in vitro by quantifying the trans-epithelial resistance (TER) with the Millicell ERS system (Millipore Corp) . TER value was measured at three different areas in each bicameral culture. TER values of each sample were calculated as TER sample (Ω cm2) = (R sample – R blank) (Ω) × effective membrane area (cm2).
The permeability of Sertoli cell barrier was also assessed in vitro using sodium fluorescein (Na-F) . The Na-F concentration in the basal chamber of the control group before treatment was arbitrarily set as 100% for the experiment.
Cell Counting Kit-8 assay
The cell viability was assessed using Cell Counting Kit-8 (CCK-8; CK04, Dojindo). Ten microliters of CCK-8 reagent were added to each well and incubated at 37 °C for 2 h. The data of optical density value at 450 nm was measured by a microplate reader (Bio-Rad, USA).
Cell apoptosis assays
Cell apoptosis analysis was performed using an Annexin V-FITC Apoptosis Detection Kit (AD10, Dojindo) with FACS Calibur Flow Cytometry (Beckman Coulter, Brea, USA). For testis sections (n = 3), apoptotic cells were detected using the TUNEL Apoptosis Assay Kit (C1086, Beyotime). The testis sections were incubated with TUNEL reaction mixture for 60 min at 37 °C, then washed with PBS. Cell nuclei were stained with DAPI. Fluorescence images were visualized using an epifluorescence microscope (Olympus BX53, Japan).
Dual-luciferase reporter assay
The fragments of Pafah1b1 3’ untranslated region (3’ UTR) containing the wild-type or mutated miR-181c/d binding sites were amplified and cloned into the pmirGLO dual-luciferase vector (Promega). Primers used in the experiment are listed in Supplementary Table 1. The recombinant construct plasmids were co-transfected with miR-181c/d mimics or mimics NC into porcine ST cells and murine SCs. Luciferase activity was measured with the Dual-Luciferase Reporter Assay System (E1960, Promega). Firefly luciferase activity was normalized to Renilla luciferase activity for each sample.
Real-time quantitative PCR (RT-qPCR)
Total RNA was extracted using the TRIzol™ Reagent (15596026, Invitrogen). RT-qPCR analysis was performed using the iTaq™ Universal SYBR® Green Supermix (1725121, Bio-Rad) on a CFX384 Touch™ Real-Time PCR Detection System (Bio-Rad, USA). RT-qPCR primers are listed in Supplementary Table 1. U6 and β-actin were used as internal controls for the miR-181c/d and coding genes, respectively. The relative expression of miRNAs or genes was calculated using the 2 -△△Ct method.
Protein samples were transferred to polyvinylidene difluoride membrane (ISEQ00010, Millipore). The blots were blocked with 5% nonfat milk for 2 h and then incubated with primary antibodies and secondary antibodies. The Clarity Western ECL Substrate Kit (170-5061, Bio-rad) was used to visualize the immunoreactive bands. Images were captured with an Image Quant LAS4000 system (GE Healthcare Life Sciences, Piscataway, NJ, USA). β-actin served as a protein loading control. The following antibodies were used: BAX (A0207, ABclonal; 1:1000), BCL2 (60178-1-Ig, 1:3000; Proteintech,), CDC42 (ab187643, Abcam; 1:20000), Cofilin (A1704, ABclonal; 1:1000), IQGAP1 (sc-376021, SantaCruz; 1:500), LIMK1 (ab108507, Abcam; 1:5000), N-cadherin (33-3900, Invitrogen; 1:500), Occludin (71–1500, Invitrogen; 1:500), PAK1 (A19608, ABclonal; 1:1000), PAFAH1B1 (ab109630, Abcam; 1:5000), PCNA (A12427, ABclonal; 1:1000), p-Cofilin (AP0178, ABclonal; 1:1000), ZO-1 (61-7300, Invitrogen; 1:500), β-actin (AC028, ABclonal; 1:100000), β-catenin (71-2700, Invitrogen; 1:500), HRP Goat Anti-Mouse IgG (AS003, ABclonal; 1:3000), and HRP Goat Anti-Rabbit IgG (AS014, ABclonal; 1:3000).
Sixty microlitres of Protein G magnetic beads (1614023, Bio-Rad) were incubated with antibodies for 2 h at room temperature. Then, the protein extracts were added to the beads and incubated overnight at 4 °C with rotation. The beads were washed with 1×PBST. The proteins bound to the beads were eluted in standard 1×SDS buffer and heated at 90 °C for 10 min. Finally, proteins were electrophoresed on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membrane for the immunoblot analysis. IQGAP1 (sc-376021, SantaCruz; 1:50) and PAFAH1B1 (sc-374586, SantaCruz; 1:50) were used as the precipitating antibodies.
The potential binding sites of miR-181c/d within Pafah1b1 3’ UTR were predicted by Targetscan (http://www.targetscan.org/) online software. The three-dimensional structure was predicted by I-TASSER (https://zhanggroup.org//I-TASSER/). The protein-protein interaction was performed by the ZDOCK server (https://zdock.umassmed.edu/).
All data are presented as the mean ± standard deviation (SD). At least three independent experiments were performed and quantified. A two-tailed Student's t-test was used for comparison between two groups. p < 0.05 was considered statistically significant.