Sertoli cell‐derived exosomal MicroRNA‐486‐5p regulates differentiation of spermatogonial stem cell through PTEN in mice

Abstract Self‐renewal and differentiation of spermatogonial stem cell (SSC) are critical for male fertility and reproduction, both of which are highly regulated by testicular microenvironment. Exosomal miRNAs have emerged as new components in intercellular communication. However, their roles in the differentiation of SSC remain unclear. Here, we observed miR‐486‐5p enriched in Sertoli cell and Sertoli cell‐derived exosomes. The exosomes mediate the transfer of miR‐486‐5p from Sertoli cells to SSCs. Exosomes release miR‐486‐5p, thus up‐regulate expression of Stra8 (stimulated by retinoic acid 8) and promote differentiation of SSC. And PTEN was identified as a target of miR‐486‐5p. Overexpression of miR‐486‐5p in SSCs down‐regulates PTEN expression, which up‐regulates the expression of STRA8 and SYCP3, promotes SSCs differentiation. In addition, blocking the exosome‐mediated transfer of miR‐486‐5p inhibits differentiation of SSC. Our findings demonstrate that miR‐486‐5p acts as a communication molecule between Sertoli cells and SSCs in modulating differentiation of SSCs. This provides a new insight on molecular mechanisms that regulates SSC differentiation and a basis for the diagnosis, treatment, and prevention of male infertility.


| INTRODUC TI ON
Self-renewal and differentiation of Spermatogonial stem cells (SSCs) are the foundation for spermatogenesis. A delicate balance exists between self-renewal-commitment and differentiation of SSC.
Excessive self-renewal or differentiation of SSCs hinders spermatogenesis hence inducing male infertility. SSC is supported within niches (specialized microenvironments) which provide various endocrine and paracrine signals for regulating self-renewal and differentiation of SSC. 1,2 In mammalian testes, Sertoli and Leydig cells are the primary contributors to the SSC niches. Glial cell line-derived neurotrophic factor (GDNF), secreted by Sertoli cells, is a growth factor that was first identified as a crucial self-renewal factor of SSCs, 3 which is indispensable for self-renewal of SSC. Fibroblast growth factor 2 (FGF2), also secreted by Sertoli cell, is another growth factor essential for the self-renewal of SSCs. 4,5 Besides, Leydig cells produce cytokine colony-stimulating factor-1 (CSF-1) that regulates the selfrenewal of SSC and guides SSCs to their final position. 6 Regarding the differentiation of SSCs, it is well established that testosterone is a primary regulator of spermatogenesis. 7 Sertoli cell-secreted retinoic acid (RA), an active metabolite of vitamin A, induces meiotic entry of spermatogonia. 8 Also, activin A and bone morphogenic protein 4 (BMP4) enhances differentiation of SSC. 9,10 Nonetheless, factors and molecular mechanisms in differentiation of SSCs are largely unknown.
Exosomes are nanometre-sized (50-100 nm) vesicles. Increasing evidence showed that exosomes were detected in different body fluids, participate in intercellular communication through incorporation of their cargo into the target cells. 11 Various cells can release miRNAs via exosomes which function in a paracrine manner in the surrounding microenvironment, and promote cell development. 12 MSC exosomes mediate cartilage repair by promoting proliferation and attenuating apoptosis. 13 Fibroblast derived exosomes promote epithelial cell proliferation through the TGF-β2 signalling pathway. 14 Additionally, exosomes could mediate cardiac regeneration, which highlights the potential utility of exosomes as cell-free therapeutic candidates. 15 In our study, we observed Sertoli cell-derived exosomes (SC-EXO) promoted SSCs differentiation compared with Leydig cell-derived exosomes (LC-EXO). To obtain a better understanding of how SC-EXO regulate SSCs differentiation, we used a miRNA array and bioinformatics to analyse the miRNA landscape of SC-EXO and LC-EXO. And the top 10 miRNAs were analysed, among which miR-486-5p was found to positively regulate SSCs differentiation. miR-486-5p has been proven to regulate stem cell development and differentiation. Overexpression of miR-486-5p induces a premature phenotype and inhibits proliferation of MSCs, whereas inhibition of miR486-5p has the opposite effects. 16 Additionally, miR-486-5p promotes mammary gland development and location. 17 However, the role of exosome-derived miR-486-5p in SSCs differentiation remains largely unknown.
Here, we attempted to characterize the molecular mechanisms of miR-486-5p in SSCs differentiation. Results suggested that miR-486-enriched exosomes could transfer from Sertoli cells to SSCs and promote SSCs differentiation by targeting PTEN. Our findings revealed a mechanism in regulating the differentiation of SSC that might have an implication in male infertility.

| Animals
Mice used in experiments were purchased from the Experimental Animal Center of Guangdong Province, China. Animals were maintained under a 12 hours light/dark cycle and a controlled temperature (24 ± 2°C) with relative humidity (50%-60%). The standard rodent diet and drinking water were freely accessible. All experiments were conducted according to the National Institute of Health guidelines for the care and use of animals and approved by the Institutional Animal Care and Use Committee of Jinan University.

| Spermatogonial stem cell (SSC) isolation and culture
Spermatogonial stem cell derived from testes of six-day-old male mice. To obtain SSCs suspension from the testicular tissue, a twostep enzymatic digestion protocol was applied. 18 Briefly, decapsulated testes were treated with collagenase type IV (1 mg/mL) for 15 minutes at 37℃, followed by digestion in 0.25% trypsin and 1 mmol/L EDTA for 10 minutes at 37°C. The singly dissociated cells were incubated overnight in dished to removed somatic cells. Nonadherent and weakly adherent cells were collected and then labelled these cells with CD326 (EpCAM) MicroBeads by Mini MACS Starting Kit (Miltenyi Biotec) according to the manufacturer's protocol. Cells were rinsed with PBS containing 0.5% BSA (Sigma), and the CD326positive cells were collected. Then, the CD326-positive cells were plated onto laminin (2 mg/mL) coated plates with SSC culture medium. The SSC medium was composed of DMEM, 15% foetal bovine serum (FBS, Thermo Fisher Scientific), 50 µmol/L-mercaptoethanol (Thermo Fisher Scientific), 1 × minimal essential medium (MEM) non-essential amino acids (Thermo Fisher Scientific), and 10 ng/mL mouse GDNF (Peprotech).

| Isolation of Sertoli cells and Leydig cells
Sertoli cells and Leydig cells were isolated from the testes of 21-day-old mice. Briefly, the testes denuded of tunica albuginea were incubated with 1 mg/mL collagenase type IV in DMEM (Gibco) for 15 minutes at 37°C on a shaker and then filtered through 100 μm cell strainer (Falcon) to isolate Leydig cells. The filtered seminiferous tubules were further digested with 0.25% trypsin-EDTA (Gibco) for 15 minutes at 37°C on a shaker and then filtered through 40 μm cell strainer (Falcon).
Cells in the filtrate were collected by centrifugation (250 g, 5 minutes) and resuspended in DMEM medium (Gibco) with 10% FBS (Life Technologies). Subsequently, the cell suspensions containing primary Sertoli cells and spermatogonium were cultured in cell culture flasks at 37°C with 5% CO 2 . After 24 hours, the culture supernatant was collected to obtain spermatogonium and the adherent cells were treated with a hypotonic solution (20 mmol/L Tris, pH 7.4) for 2 minutes to obtain pure Sertoli cells. Sertoli cells and Leydig cells were cultured in DMEM supplemented with 10% FBS at 37°C with 5% CO 2 .

| Extraction of exosomes from Sertoli cells and Leydig cells
For exosome isolation, equal numbers of Leydig cells and Sertoli cells were transplanted into T75 flasks and maintained in fresh DMEM with 10% Exosome-depleted FBS Media Supplement (SBI).
After 48 hours, culture medium was collected and filtrated through 0.22μm filters (Millipore). Exosomes were collected by differential centrifugation. Briefly, the supernatant was centrifuged as follows: 300 g for 10 minutes, 10 000 g for 30 minutes and 100 000 g for 70 minutes (all the steps were performed at 4°C). For exosomes purification, the pellets were followed by an additional washing step with PBS at 160 000 g for 1 hour. Exosomes were resuspended in basal medium and stored at −80°C.

| Nanoparticle tracking analysis
Exosomes from Sertoli and Leydig cells were suspended in PBS, respectively, to maintain their concentrations within the measurable concentration range of NanoSight instrument. Each sample was performed for four recordings.

| Transmission electron microscope
The obtained exosomes were fixed with fixative buffer containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 mol/L PBS. After embedding, samples were cut into 0.12 μm sections and stained with 0.2% lead citrate and 1% uranyl acetate. The images were photographed under an electron microscope.

| PKH26-labelled exosome transfer
Exosomes from Sertoli cells were labelled using a PKH26 red fluorescent labelling kit (Sigma) according to the manufacturer's protocol. Briefly, the exosomes were incubated with the PKH26 dye for 4 minutes, and the reaction was terminated by adding exosomedepleted FBS Media Supplement (SBI). Then, the exosomes were washed three times and excess PKH26 dye removed by 100 kD Amicon Ultra-4 (Millipore), then incubated with SSCs. The rate of uptake of the exosomes into cells was measured by flow cytometry.

| Western blotting
After the cell samples were lysed and the protein concentration was determined by BCA. The samples were separated by electrophoresis on 10% SDS-PAGE gel and transferred to PDVF membrane. After 1 hour of sealing with 5% skimmed milk, PTEN (Abcam), STRA8 (CST) antibodies were added and incubated overnight at 4°C. Membranes were then rinsed six times (7 minutes each) with TBST and incubated with an HRP-conjugated secondary antibody (1:5000) at room temperature for 1 hour. Membranes were rinsed six times (7 minutes each) with TBST, and immunoreactions were detected by enhanced chemiluminescence (ECL) detection. The protein expression levels were normalized to GAPDH.

| Immunofluorescence
Cells were fixed in 4% paraformaldehyde for 30 minutes and washed three times with PBS. The cells were treated in 0.2% Triton-X for 10 minutes. Non-specific adhesion sites were blocked with 3% bovine serum albumin (BSA; Sigma, Poole, Dorset, UK) for 30 minutes at room temperature. The primary and secondary antibodies were diluted in a solution of PBS containing 3% BSA, 1% horse serum and 0.1% Triton X-100. Cells were incubated with primary antibodies SYCP3 (Abcam) overnight at 4°C, followed by incubation with secondary antibodies for 2 hours at room temperature. Nuclei were stained with DAPI (Thermo Fisher Scientific).
Stained samples were then visualized, and images were captured using a LSM710 confocal microscope (Zeiss) and analysed by the Image J software.

| Total RNA extraction by qRT-PCR
Total RNA was extracted from exosomes or cells using Trizol (Invitrogen) according to the manufacturer's instructions. Briefly, Trizol (Invitrogen) was added to the exosome or cells precipitation, and homogenized at room temperature for 5 minutes. Then, trichloromethane was added and mixed thoroughly, followed by  Table 1.

TA B L E 1 Primer sequences
Reverse transcription and qRT-PCR for exosomal miRNA, as well as internal reference U6, were performed using miRNA  Table 1.

| Cell transfection
miR486-5p mimic, inhibitor and their corresponding negative controls were all purchased from Genepharma. All cells were seeded in 6-well plates at a density of 1 × 10 6 cells/well and grown to 70% These images were obtained from three independent experiments, and data were presented as mean ± SD, ***P < 0.001; ****P < 0.0001 were, respectively, diluted with 250 μL serum-free medium Opti-MEM and incubated at room temperature for 5 minutes. Then, the two solutions were mixed and incubated at room temperature for 20 minutes. A total of 500 μL miRNA mimics-RNAiMAX mixture was added to each well which was supplemented with DMEM complete medium without antibiotics to a total volume of 2 mL. Finally, the culture plates were incubated. After 48 hours, the cell samples were collected. Western blotting was used to detect the expression of related proteins.

| Statistical analyses
All experiments were repeated at least three times, and data were expressed as the mean ± one standard deviation around the mean (SD). Statistical analyses were performed by Prism software (GraphPad Software). Statistical analyses were performed with an unpaired Student's t test or one-way ANOVA for more than two groups. A two-tailed value of P < 0.05 was considered statistically significant. SSCs labelled with PKH26 were used as positive control. B, Representative fluorescent confocal images of SSCs that were exposed to PKH26-labelled exosomes (red) from Sertoli cells for 24 h. Nuclei were stained with DAPI (blue). Scale bars, 20 μm. C-D, RT-qPCR analysis of Stra8 and Sycp3 expression in SSCs treated with SC-EXO at different concentrations for 24 h. SSCs without exosome treatment were used as control. EXO-1:4.44 × 10 9 particles/mL, EXO-2:8.88 × 10 9 , EXO-3:1.33 × 10 10 particles/mL. The copy number of mRNA of each gene was normalized with Gapdh, and the data were obtained from three independent experiments and are presented as mean ± SD; *P < 0.05 The SC was transfected with FITClabelled miR-486, followed by treatment with 10 μmol/L GW4869, an inhibitor of neutral sphingomyelinase. The copy number of miR-486-5p was normalized with U6, and the data were obtained from three independent experiments and are presented as mean ± SD. **P < 0.01, ***P < 0.001 markers, including CD63, CD9 and CD81, was detected in SC-EXO and LC-EXO, whereas calnexin, an integral protein not expressed in exosomes, was barely detected ( Figure 1G). The size range and concentration of the particles were measured by nanoparticle tracking analysis (NTA). The diameters of almost all particles were between 50 and 100 nm. The mean diameter was 84.2 nm in LC-EXO and 82.2 nm in SC-EXO ( Figure 1H,I). These data demonstrated that the isolated particles were exosomes.

| SC-derived exosomes promote SSC differentiation
We examined whether LC-EXO and SC-EXO influence differen- Immunofluorescence results revealed that the percentage of SYCP3-positive cells was 45.3% in the SC-EXO treatment group, which was significantly higher than in the LC-EXO ( Figure 2B-C).
These results suggested that SC-EXO promotes differentiation of SSC.

| Exosomes mediate the transfer of miR-486 from SC to SSCs
To further understand the mechanism of SC-EXO in promoting SSC differentiation, the expression of miRNAs in both SC-EXO and LC-EXO was determined. According to the differential analysis, we found miR-486-5p was one of the most prominently upregulated miRNAs in SC-EXO compared with LC-EXO ( Figure 4A).
qRT-PCR further revealed that the expression of miR-486-5p in SC-EXO was significantly higher than in LC-EXO ( Figure 4B). In addition, the expression level of miR-486-5p was also significantly up-regulated in Sertoli cells compared to Leydig cells. In contrast, the expression of miR-486-5p in SSC was very low ( Figure 4C).
Interestingly, when the SSC was treated with conditioned medium from LC or SC for 48 hours, the conditioned medium from SC elevated the miR-486 expression in SSC ( Figure 4D). To assess whether SC-EXO transport miR-486 to SSC, SCs transfected with fluorescein isothiocyanate (FITC)-labelled miR-486 were placed in the upper chamber of a transwell co-culture system, and SSCs were seeded in the lower chamber ( Figure 4E). After 24 hours of co-culture, SSCs were surrounded by fluorescently labelled miR-486 mimics. However, the pharmacological inhibition of sphingomyelinase GW4869, which is known to inhibit exosome generation, attenuated the transfer of FITC-miR-486 to SSC ( Figure 4F), indicating that the miR-486-5p transfer was mediated by exosomes. qRT-PCR analysis further confirmed that the expression of miR-486 in SSC was significant increase when SSCs were co-cultured with SCs, while this effect was blunted when SCs were treated with GW4869 ( Figure 4G). These findings suggested that SC-derived exosomes were able to transport miR-486-5p to SSC.

| miR-486 directly targets Pten and promotes SSC differentiation
TargetScan was applied to predict the possible target genes.
Among hundreds of genes that were predicted as potential tar- EdU-positive (green) rate was remarkably decreased when miR-486-5p was overexpressed in SSCs. The percentages of EdU-positive cells were counted out of 500 total cells from independent ten images. These images were obtained from three independent experiments, and data were presented as mean ± SD, ***P < 0.001 significantly elevated ( Figure 5C-E). These results suggested that miR-486-5p increased STRA8 expression level by targeting PTEN in SSC.
STRA8 plays a key role in the initiation of meiosis in mammals.
In addition, EdU incorporation assays showed that the EdU-positive rate was remarkably decreased when miR-486-5p was overexpressed in SSC, suggesting that miR-486-5p inhibited SSC proliferation ( Figure 5H,I). Collectively, these results demonstrated that miR-486-5p inhibited the self-renewal of SSC and promoted SSC differentiation by targeting PTEN.

| D ISCUSS I ON
In testis, Sertoli cell provides an appropriate microenvironment or niche for the development of male germ cells, which is indispensable for maintaining normal spermatogenesis. Therefore, it is necessary to determine the potential molecular mechanisms of Sertoli cell during spermatogenesis, which will offer novel insights into the aetiology of infertility and provide new targets for gene therapy in male infertility. Exosomes are critical components in the crosstalk between cells.
The biological role of exosomes on target cells depends on the selectively packages of miRNA content. 19 In this study, we demonstrated can be utilized as a potential biomarker for the diagnosis of male infertility.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.