Animals
All animal experiments were approved by the Animal Research Ethics Committee of the Chinese University of Hong Kong (20-081-ECS). Eight-week-old male C57BL/6 mice were purchased from the laboratory animal service center (LASEC) of the Chinese University of Hong Kong.
Testis dissociation
The mice were sacrificed by cervical dislocation. For the one-step method, seminiferous tubules were suspended in the Accumax Cell Aggregate Dissociation Medium (20mg/ml) (Invitrogen; 00-4666-56) and incubated on a rocking platform at room temperature for 1 h. The dispersed cells were filtered through a 40-um-cell strainer (Corning; 352340) to a 50-ml falcon tube and washed with PBS. Filtered cells were pelleted at 300g for 5 min, and the EV-containing supernatant was further filtered through using a 0.8-um syringe filter (Millipore; SLAA033SB).
The two-step double-enzyme digest method was performed as described [26], and seminiferous tubules were suspended in PBS containing collagenase (1 mg/ml) (Sigma-Aldrich) and incubated at 37°C for 10 min. The cells were washed once with PBS and further incubated in Trypsin (0.25%) (Gibco) and Dnase I (5mg/ml) (Sigma-Aldrich) at 37°C for 10 min. Ten percent of FBS were added to inactivate the trypsin upon single-cell suspension, and the dispersed cells were washed with PBS and Dnase I before filtered through using a 40-um cell strainer. Filtered cells were pelleted at 300g for 5 min, and all EV-containing supernatants were collected and filtered through using a 0.8-um syringe filter.
Isolation of EVs
For the affinity column method, EV was isolated using exoEasy Maxi Kit (Qiagen, 76064) according to the manufacturer’s instructions and further concentrated by Ultracentrifugation (Hitachi CS-150GXII Micro UITRAcentrifuge), performed at 100,000g for 90 min at 4°C.
For the differential centrifugation method, MVs were isolated by centrifugation, at 10,000g for 30 min at 4°C using Beckman Avanti J-E Centrifuge. Exosomes were then isolated by centrifugation at 100,000g for 90 min at 4°C using Beckman Coulter Optima XPN-100 ultracentrifuge.
Transmission electron microscope
Morphology of testicular EVs was observed using a transmission electron microscope. EVs from two testes were resuspended in 20 μl PBS and fixed with 2% paraformaldehyde until use. Ten microliters of EVs were added onto the formvar grid (200 mesh) for 30–60 min, and excess fluid was removed with filter paper. EVs were fixed with 1% glutaraldehyde for 10 min, followed by negative staining with 2% uranyl acetate for 2 min, and images were captured using a Hitachi H-7700 transmission electron microscope.
For immunogold labeling, fixed EVs were applied to the formvar grids (200 mesh), after washing with PBS/50mM glycine to quench-free aldehyde groups for 3 min for a total of four washes, grids were blocked with PBS/5% BSA for 1 h, followed by primary antibody to Basigin (Abcam; ab188190, 1:20) in PBS/1% BSA and gold labelled secondary antibody (Boster; GA1013, 1:20) in PBS/1% BSA incubation for 1h. The grids were then stained with 2% uranyl acetate, and images were captured as described above.
Dynamic light scattering
Concentrated EVs were resuspended in 100 μl PBS, and 50 μl of EVs was diluted in 950 μl PBS for determination of size distribution using the dynamic light scattering (Zetasizer Nano-ZS system). Three independent measurements were performed for each sample.
Nanoparticle tracking analysis
The concentration of testicular EVs was measured by the Nanosight LM14C (Malvern) instrument. The samples were diluted with filtered PBS to obtain the optimal measurement condition of 5–15×108 particles/ml. Thirty second videos were recorded of each sample, and the concentration of testicular EVs was analyzed and calculated with nanosight software using the Stokes-Einstein equation.
Western blot
Protein lysate from EVs or cells was obtained by incubating with RIPA buffer with protease inhibitors. A total of 40 μg protein were electrophoresed under denaturing conditions on 12% polyacrylamide gels and transferred onto the PVDF membranes. After blocking with 5% non-fat milk for 1 h, the membranes were immunoblotted with primary antibodies to CD 81 (Santa Cruz; sc-166029, 1:1000), CD 63 (Santa Cruz; sc-365604, 1:1000), β-tubulin (Cell Signaling; 2146, 1:2000), Calnexin (Immnoway; YT0613, 1:1000), Golgin 97 (Santa Cruz; sc-59820, 1:1000), and BSG (Abcam; ab188190, 1:1000) overnight at 4 °C, followed by the relevant HRP-conjugated secondary antibodies incubation for 1 h at room temperature. Bands were visualized by Prime ECL.
Cell line
The cell lines used in this study were C18-4 mouse undifferentiated spermatogonia cell line, GC1-spg mouse spermatogonia cell line, and TM4 mouse Sertoli cell line. C18-4 cells were maintained in DMEM medium supplemented with 10% FBS, 1mM sodium pyruvate, 1% l-glutamine, and 1 x nonessential amino acids at 35 °C with 5% CO2. GC1-spg cells and TM4 cells were cultured in DMEM or DMEM/F12 medium respectively containing 10% FBS at 37 °C with 5% CO2. C18-4, GC1-spg, and TM4 cells were seeded on five 15-mm dishes containing 20 ml of cell medium with exosome-depleted FBS (Thermo Fisher, A2720803), and cell medium was collected for EV isolation when cells were grown to 95% confluency.
Uptake of EVs
Concentrated EVs were labeled with PKH67 Green Fluorescent Cell Linker Mini Kit (Sigma-Aldrich, MINI67-1KT) according to the manufacturer’s instructions. EV concentrations were determined by protein assay. To study the EV uptake in testicular cells or somatic cells in vitro, indicated amount of labeled EVs or vehicle controls were incubated with C18-4, TM4, and GC1-spg cell line when cells reached 60–70% of confluency in 24-well plates for indicated time-points. The uptake of EVs in cells or spermatozoa was analyzed by immunofluorescence staining or flow cytometry.
To study the EV uptake in the mouse testis, mice were fully anesthetized with 100mg/kg ketamine and 10 mg/kg xylazine mix and labeled EVs or vehicle controls (PKH67 dye) were injected to the seminiferous tubules via efferent duct or interstitium of the mouse testes [45,46,47]. After 24 h, mice were sacrificed and the testes were collected. Snap-frozen testicular sections (5 μm) were fixed with 4% paraformaldehyde for 15 min, and slides were mounted in an antifade mounting medium with DAPI.
For the study of EV uptakes in primary testicular haploid germ cells, the haploid germ cells close to the lumen of the seminiferous tubules were isolated by manual pipetting of the seminiferous tubules followed by a Percoll gradient purification. Briefly, the seminiferous tubules were spread and rocked (~80 rpm) in 1 x HBSS at a volume of 1 ml per 20 mg tissue at room temperature for 1 h. The cells were dispersed by pipetting 20 times once every 30 min. Then, the cell suspension was filtered through a 40-μm cell strainer and pelleted by spinning at 500 x g for 5 min. The primary germ cells were purified by 22.5–45% (w/v 1 x HBSS) Percoll gradients. The 22.5% percoll gradient layer was obtained, and 15 mL of 1 x PBS was added followed by centrifugation at 500 x g for 5 min to remove the residual Percoll solution. The primary germ cells enriched in haploid population cell pellets were counted, and 1 x 106 cells were used for tEV treatment. Staging of the haploid germ cells was performed by PNA staining after the tEV uptake (see below).
Thy1+ isolation with MACS
1x107 cells from one-step and two-step double-enzyme digest methods were resuspended in 200ul 2% FBS DMEM and incubated with 20ul of Biotinylated Thy1.2 CD90.2 primary antibody (BD IMag™, 551518) on ice for 15 min on a slow-rocking platform. Cells were washed with PBS and resuspended with 500ul 2% FBS DMEM before loaded into the pre-calibrated MS column (Miltenyi Biotec, 130-042-201) to allow Thy1- cells to flow through by gravity. Columns were washed twice with 500ul 2% FBS DMEM, and finally, Thy1+ cells were eluted with 1ml 2% FBS DMEM.
Flow cytometry
Cells collected from one-step and two-step double-enzyme digest methods were washed once with PBS and permeabilized overnight with 70% ethanol at −20°C. Samples were stained with 40ug/ml PI in PBS and 100ug/ml Rnase A and incubated at 37°C for 30 min in dark. PI-stained cells were analyzed with BD LSR Cell Analyzer, counting 10,000–20,000 events per sample.
Flow cytometry was used to determine the percentage of EV uptake in cell lines. After the incubation period with labeled EVs or vehicle control, cells were washed with PBS once and trypsinized, pelleted by centrifugation at 1000 rpm for 5 min, and resuspended in 300 μl of cell medium. Fluorescence-positive cells were analyzed BD LSR Fortessa Cell Analyzer, counting 50,000 cells for each sample.
Immunofluorescence staining
Cell lines or primary testicular germ cells were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min followed by incubation with Alexa Fluor 568 Phalloidin (dilution 1:200) for 20 min or Lectin PNA (Invitrogen; L32458, 1 μg/ml) for 1 h. Cells were counterstained with Hoechst 33342 (1:2000) or DAPI for 10 min. The fluorescence images were captured with Leica TCS SP8 confocal microscope, and Z-stack images were analyzed with Imaris Cell Imaging Software.
Alkali/detergent and nuclease treatment of testis EV
Prior to RNA extraction, EV/sperm samples were treated with or without 1% Triton X-100 (Boehringer Mannheim, 1332481) and incubated at room temperature for 30 min to disrupt the membrane. The samples were then treated with 0.05ug/ul Proteinase K (Qiagen, 1014023) at 37°C for 10 min, inactivated with 5-mM PMSF (Sigma-Aldrich, P7627) at room temperature for 10 min and then treated with 0.5ug/ul Rnase A (Thermo Scientific™, EN0531) at 37°C for 20 min. Alkaline hydrolysis was performed as described [48]. Briefly, the samples were first incubated at 65°C for 1 h in 0.1M Tris (pH 8.0) and 1M NaOH, then neutralized with 1M HCl at a ratio of 2.33:1:1.
RNA extraction, reverse transcription and real-time PCR
The total RNA was extracted using the miRNeasy Mini Kit (Qiagen, 217004) with RNase-Free DNase Set (Qiagen, 79254) according to the manufacturer’s instructions. A 10pg of mirVana miRNA mimic miR-199a-5p (ThermoFisher, Assay ID MC10893 and 002304) was added to 0.1–0.5ug of the total RNA as a spike-in control for RT-qPCR normalization. Reverse transcription was performed using PrimeScript™ RT Master Mix (TaKaRa, RR036A) for novel miRNA and TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems, 4366597) for the candidate miRNAs let-7b-5p (ThermoFisher, Assay ID 000378) and miR-34a-5p (ThermoFisher, Assay ID 000426). One microliter of cDNA was used for a real-time PCR using TaqMan™ Universal PCR Master Mix (Applied Biosystems, 4364340) on the ABI QuantStudio 7 Flex Real-Time RCR system. Primers and probes used are listed in the Additional file 5: Table S4. Results were calculated with 2(−ΔΔCt).
Small RNA sequencing
EV RNA after treatment with RNase A and proteinase K was extracted as described above and used for small RNA-Seq which was performed by BGI (Shenzhen, China). Small-RNA libraries were prepared, and the PCR products were sequenced using BGISEQ-500 technology. After elimination of low-quality reads, clean reads were mapped to reference genome and to other sRNA databases using Bowtie2 and cmsearch [49, 50]. Classification of sRNA follow the priority rule: MiRbase> pirnabank> snoRNA(human/plant)> Rfam> other sRNA to ensure unique map of each entry. Novel miRNAs and piRNAs were predicted using miRDeep2 and Piano, respectively [51, 52].
LC-MS/MS
Testicular EV proteins were extracted with RIPA buffer, and LC-MS/MS proteomics analyses were performed by Winninnovate Bio (Shenzhen, China). Gene ontology (GO) enrichment and KEGG pathway enrichment were analyzed. To map, the cell types that express the identified proteins, we analysed the expression of genes encoding the identified proteins by taking the average expression level of these genes in different cell clusters from single cell RNA sequencing analysis of adult mouse testes [31].
GW4869 Treatment
To study the effect of exosome generation inhibitor GW4869 (Sigma-Aldrich, D1692) on spermatogenesis in mouse, GW4869 dissolved in DMSO (20 μM, 20μl/testis) or vehicle (DMSO) were injected into the mice seminiferous tubules or interstitium of testes as described above. Treatments were conducted once or every other day for three times. After the indicated treatment time, mice were sacrificed and testes were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. The TUNEL assay was performed on the testis sections (5 μm) with ApopTag plus peroxidase in situ apoptosis detection kit (Millipore, S7101). The total number of seminiferous tubules and the number of tubules with TUNEL-positive cells were counted in at least 10 random fields of the testis sections. The percentage of tubules with positive TUNEL cells were then calculated.
Statistical analysis
Statistical analysis was carried out using GraphPad Prism version 8.02. Student’s t test was used to compare group means. Ordinary one-way analysis of variance (ANOVA) with Dunnett’s post hoc test was used for analysis involving three or more groups of samples. A P value of <0.05 was considered significant.
