Animals and study design
A total number of 10 male NMRI mice (3‐6 days‐old) underwent testicular surgery and were used as donners for SSC. 6-8weeks-old male NMRI mice (n=66) were used as recipients of SSC transplantation. All NMRI mice were obtained from the Pharmacy Faculty of Tehran University of Medical Sciences. Animals were preserved under standard conditions of 12 hours/12 hours’ light/dark. All surgical procedures were done under xylazine/ketamine (10/90 mg/kg, i.p.) anesthesia using sterile conditions. All procedures in this study were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (1996, published by National Academy Press, 2101 Constitution Ave. NW, Washington, DC 20055, USA), and also this study was approved by the Ethical Committee of Tehran University of Medical Sciences (IR.TUMS.MEDICINE.REC.1397.210). Forty-eight mice in four testicular torsion transplantation groups (12 mice in each group), 9 mice in the control group and 9 mice in the torsion group were considered as experimental groups. After testicular torsion model was confirmed, transplantation groups were divided as followed. The group that received SSCs (TT+Fresh), the group receiving SSCs after freezing (TT+Cryo), the group which received SSCs treated with PTX after freezing (TT+Cryo+PTX) and the group receiving SSCs with PTX (TT+Fresh+PTX). Also, control group received DMEM. The TT group had testicular torsion operation but did not undergo transplantation. Two weeks after testicular torsion, 3 mice from each of 6 groups were sacrificed to confirm the torsion by cervical dislocation. Two weeks after transplantation, 3 mice in each transplantation group were sacrificed to confirm the transplantation. Recipient mice were euthanized, and testes were collected and decapsulated 8 weeks after transplantation. Finally, 36 mice in all groups (6 groups of mice, 6 mice in each group) included four testicular torsion transplantation groups, control and torsion group (as a sham-transplanted testes) were used to evaluate the testes at the end of the study which was 8 weeks after transplantation (Supplementary Fig.2).
Testicular surgery, SSC isolation and enrichment
After anesthesia, testes were dissected and transferred into the phosphate‐buffered saline (PBS; “Sigma‐Aldrich)”. Then SSCs were isolated by a two‐step enzymatic digestion according to the protocol of Kanatsu-Shinohara. First, the testes were suspended in 3 mL Dulbecco modified Eagle medium (DMEM) containing 5 µg/mL DNase (Sigma‐Aldrich, Germany), 1 mg/mL collagenase type IV (Gibco, CA) and 1 mg/mL hyaluronidase (Sigma‐Aldrich). Then, they were transferred to an incubator for 20 min at 37°C until forming a cellular suspension. Interstitial cells were digested after pipetting and centrifuging at 15×g for 5 minutes. In the second step, cellular pellets were enriched by the same medium for 15 minutes. Cell detachment from seminiferous tubules occurred in this step. The suspension was centrifuged at 15×g for 5 minutes, and the cell pellets were transferred to the gelatinized dishes for further pelleting with a duration of 5 hours. For SSC enrichment, the cellular suspension was transferred into gelatin‐coated (Sigma‐Aldrich) culture dishes for 2 hours at 37°C. The non-adherent SSCs were cultured in minimal essential medium (Gibco‐Invitrogen, US) containing 2% Fetal bovine serum (FBS) (Life Technologies), 1000 U/mL Leukemia Inhibitory Factor (LIF; Sigma, Haverhill), 10 ng/mL basic fibroblast growth factor (Peprotech, Rocky Hill, NJ), 0.1 mM β‐mercaptoethanol (Sigma‐Aldrich), 1% non‐essential amino acids (Gibco, Invitrogen, UK), 10 μg/mL glial cell line‐derived neurotrophic factor (GDNF; Sigma‐ Aldrich, USA), 100 U/mL penicillin (Sigma‐Aldrich, Darmstadt) and 100 μg/mL streptomycin (Sigma, Germany). Afterward, the cells were transferred into an incubator (5% CO2, 37°C) for 2 weeks. The medium was changed every two days.
Alkaline phosphatase staining
Fast-Red TR/Naphthol AS‐MX tablets (Sigma‐Aldrich) were used for assessment of alkaline phosphatase activity. For this purpose, Tris buffer was added to the Fast‐Red TR/Naphthol AS‐MX tablet to form an alkaline dye and was observed under an inverted microscope (IX71, Olympus, Japan).
Identification of SSC using flow cytometry
For the identification of SSCs using flow cytometry, Gfra1(1:100; Sigma Aldrich, USA), Id4 (PA5‐26976, Invitrogen, USA), rat polyclonal anti-α6-integrin (1:100; Sigma- Aldrich, USA) and rat polyclonal anti-β1-integrin (1:100; Sigma-Aldrich, USA) antibodies were added to a total number of 106 cells. Goat anti-rat labeled with fluorescein isothiocyanate (FITC) (1:200; Sigma-Aldrich, USA) was used as a secondary antibody for Id4 using flow cytometer (FACSCalibur, BD, Franklin Lakes, New Jersey, USA). The fluorescence intensity was measured between 464 and 601 nm for control-PE and between 515 and 545 nm for control-FITC using flow cytometry.
Single cells were suspended in 2.5×105 cells/mL of freezing medium containing Minimum Essential Medium Eagle - alpha modification (α -MEM) (Invitrogen) with 10% dimethyl sulfoxide (DMSO; 1.4 M, Sigma, Germany), MEM-α (Sigma, Germany), and 10% fetal bovine serum
(FBS; Sigma, Germany) without additional supplements (basal freezing medium) or basal freezing medium with the addition of 10 mM PTX (pH = 7.4, osmotic pressure 300 mOsm/kg, Purity(TLC) ≥ 98%, cat.no. P1784, Sigma-Aldrich), and were placed in 1.8-mL cryovials (Corning). Cryovials were frozen in a Nalgene freezing container (cat. no. Z359017, Sigma) at a rate of -1°C per minute to -80°C and stored overnight at -80°C. After overnight storage, cryovials were placed in liquid nitrogen for at least a week of storage. After removal from liquid nitrogen, samples were maintained at room temperature for 30 seconds, and then in a water bath at 37C for 2.5 minutes. The cryovial contents were transferred to a tube with prewarmed medium and diluted 1:10 with α-MEM containing 10% FBS in a drop-wise manner. The cells were washed two times with medium and centrifuged at 1200 × g for 5 minutes.
Cell viability assay
After thawing, cell viability was determined using Methyl thiazolyldiphenyl-tetrazolium bromide (MTT; Sigma-Aldrich) assay in control and treatment groups at doses of 5, 10, and 15 mM of PTX. 100 μl MTT reagent (5 mg/ml in PBS, pH 7.6) was added to each well of 96‐well plates, and plates were incubated in 5% CO2 for 2 hours at 37°C. After that, an equal volume of DMSO was added. The absorbance was measured at 570 nm with background subtraction at 630 nm.
Measurement of cellular reactive oxygen species
Cellular ROS was detected using specific ROS probes by flow cytometry. 10000 SSC cells were loading with 50 μm DCFH‐DA (ROS Assay Kit; Beyotime, Haimen, Jiangsu, China) according to the manufacture instructions. DCFH‐DA‐fluorescence was measured using logarithmic amplification in the flow cytometer (Becton Dickinson, USA). Data were reported as peak fluorescence intensity between 500 and 530 nm. Experiments were performed in triplicate and repeated three times. ROS production was calculated as the intensity in the fluorescence compared with the control group. The data was analyzed with Flowjo software [version 7.6.1].
Optimization of the mouse model for infertility
For this purpose, the scrotum was excised through a midline incision. The tunica vaginalis was opened, and the left testis was exposed to the surgical field. The left testis was rotated 720° in a clockwise direction and maintained in this torsion position by fixing the testicle to the scrotum with a 6-0 silk suture. Animals underwent 2 hours of unilateral testicular ischemia. Then, the suture was removed and the ischemic testis was untwisted and replaced in the scrotum, and the incision area was closed. For confirmation of model, 3 mice in each group (control, torsion and transplantation groups) were sacrificed and excluded from the study in order to examine the seminiferous tubules and confirm the torsion by cervical dislocation. After 2 weeks, TT and the testes of mice were removed, fixed in a Bouin’s solution for 48 hours, embedded in paraffin, sectioned at 5-μm thickness and finally stained with hematoxylin-eosin (H&E) and the testis tissue sections were examined using an optical microscope (Nikon, Japan).
Germ cell transplantation
To detect the transplanted cells and purify them from testicular endogenous cells, 2×105 /ml cells were exposed to a 2 μg di-alkyl indocarbocyanine (DiI, Eugene.OR, USA) before transplantation for 5 minutes. 1 ml of PBS preservative solution was placed at room temperature and then placed in a dark place for 20 min at 4 °C. After ensuring that the cells were stained under a fluorescent microscope (Japan, Olympus, LX71), the cell surface was washed with PBS and then isolated from petri dish by trypsin enzyme (25%) in 0.1% ethylenediamine tetraacetic acid (EDTA) (Sigma, USA). After washing 3 times in the medium, they were ready to be transplanted into the host testis.
Two weeks after teticular torsion, the recipient mice in four transplantation groups, as described above (n=36) received transplantation. By clipping the abdominal hair and disinfection of the area with cedium chlorhexidini alcoholicus 0.5% (BE351513; Laboratoires Gifrer Barbezat, Décines-Charpieu, France), the surgical area was ready for use. The testes were exteriorized by incision of abdomen. Afterward, the fatty tissue around the efferent ducts was gently removed, and 12 μl of the cell suspension containing 2×105 cells/ml was injected into the efferent ducts using a microinjection needle under a stereomicroscope. If the tracking dye (trypan blue at the tip of the pipette) along with the injected solution entered the seminiferous tubules, the transplantation was considered as “successful”.
Immunofluorescent Staining of Testis Cross Sections and Selected Germ Cells
The immunofluorescent analysis was used to explore the expression of Ddx4 and C-Kit in mice testes. After dissection of testes from the mice, testes were fixed in 4% paraformaldehyde for 12 h at 4°C, embedded in paraffin, and sectioned. Sodium citrate buffer (10 mM sodium citrate [pH 6.0]) was used for antigen retrieval by boiling the sections for 15 minutes. Endogenous peroxidase activity was blocked using blocking solution for 10 minutes at room temperature. Sections were then blocked with 10% normal goat serum, followed by incubation with respective primary antibodies of Ddx4 Ddx4 (1:500, ab13840, Abcam, UK) and C-Kit (1:500, ab5506, Abcam, UK) diluted in PBS containing 0.5% Bovine Serum Albumin (BSA) at 4°C overnight, followed by incubation with the appropriate Alexa Fluor dye-conjugated secondary antibodies FITC Goat anti‐rabbit IgG H&L (ab6717, Abcam, UK). The sections were washed and incubated with DAPI (4,6-diamidino-2-phenylindole dihydrochloride hydrate) (Vector Laboratories, CA, USA) in PBS to label the cell nuclei, followed by a fluorescence microscope with magnitude of x40. All image analysis was done by ImageJ 1.5 software.
All of protein was extracted from tissue using a TriPure Isolation Reagent (Roche, Germany). 20 µg of the extracted proteins were loaded on 12% SDS-polyacrylamide gels (BioRad Laboratories, Hercules, CA) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Roche, Germany). The nonspecific binding was blocked by PBS buffer containing 0.1% Tween-20, 2% BSA, and 5% nonfat dry milk (Carnation, CA). Afterward, samples were incubated by primary antibodies against Id4 (1:1000, Invitrogen, US), Sycp3 (1:1000, Abcam, UK) and β‐actin (1:3000, Abcam, UK) as internal control for an overnight at 4°C, and then incubated using secondary antibodies goat anti‐mouse IgG H&L‐HRP conjugate (1:5000, Bio-Rad, US) and HRP‐ conjugated goat anti‐rabbit IgG H&L (Abcam, USA). Finally, rates of expression for these proteins were evaluated using enhanced chemiluminescence.
Quantitative real‐time PCR
Expressions of Tnp1, Tnp2, and Prm1 genes was assessed by quantitative real‐time PCR. Extraction of total RNA from tissue samples was performed using a Trizol reagent (Roche, Germany). Reverse transcription of 500 ng of the extracted RNA into complementary DNA (cDNA) was carried out by a cDNA synthesis Kit (PrimeScript™ RT Reagent Kit Fast, RR037A, TaKaRa, Japan). PCR assay was performed using a thermocycler (Bio-Rad Laboratories) and aSYBR Green master mix (SYBR Premix Ex Taq II (Tli Plus), TaKaRa‐ RR820L). Samples were undergoing an initial melting stage for 5 minutes at 95°C followed by melting stage (40 cycles) for 5 seconds at 95°C and synthesis for 30 seconds at 60°C (n=3). The gene expression cycle threshold (∆∆Ct) values were calculated after normalizing with Hprt internal control. Sequences of primers are listed in the Table 1.
The normality of the distribution of variances was tested by the Kolmogorov‐Smirnov test. Data were presented as the mean ± standard deviation (SD). two-group comparison was analyzed using student t-test. Statistical analysis was performed using GraphPad Prism 8.3.0 (GraphPad Software, Inc. La Jolla, California). Statistical significance was set at P-value less than 0.05.