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Keywords:

  • miRNA-20;
  • miRNA-106a;
  • Spermatogonial stem cells;
  • Self-renewal;
  • STAT3;
  • Ccnd1

Abstract

  1. Top of page
  2. A
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

Studies on spermatogonial stem cells (SSCs) are of unusual significance because they are the unique stem cells that transmit genetic information to subsequent generations and they can acquire pluripotency to become embryonic stem-like cells that have therapeutic applications in human diseases. MicroRNAs (miRNAs) have recently emerged as critical endogenous regulators in mammalian cells. However, the function and mechanisms of individual miRNAs in regulating SSC fate remain unknown. Here, we report for the first time that miRNA-20 and miRNA-106a are preferentially expressed in mouse SSCs. Functional assays in vitro and in vivo using miRNA mimics and inhibitors reveal that miRNA-20 and miRNA-106a are essential for renewal of SSCs. We further demonstrate that these two miRNAs promote renewal at the post-transcriptional level via targeting STAT3 and Ccnd1 and that knockdown of STAT3, Fos, and Ccnd1 results in renewal of SSCs. This study thus provides novel insights into molecular mechanisms regulating renewal and differentiation of SSCs and may have important implications for regulating male reproduction. Stem Cells 2013;31:2205–2217


Introduction

  1. Top of page
  2. A
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

MiRNAs have recently been identified as a novel class of short single-stranded endogenous small RNA molecules (∼22 nucleotides). Although microRNAs (miRNAs) were first discovered in Caenorhabditis elegans in 1993 [1], it was only 12 years ago that miRNAs were identified in mammals [2]. MiRNAs are highly conserved between animals and humans, and it has been estimated that miRNAs may regulate 30% of all genes in the human genome [3]. MiRNAs act as crucial regulators for post-transcriptional gene silencing by base-pairing with the 3′-untranslated regions of target mRNAs to form the RNA duplexes which lead to either endonucleolytic cleavage of the target mRNA or translation suppression. Recent studies indicate that miRNAs may have critical functions in diverse biological processes, including cell proliferation [4], differentiation [5, 6], and apoptosis [7].

Spermatogenesis is a complex process by which spermatogonial stem cells (SSCs; also called male germline stem cells) divide and differentiate into spermatozoa. Studies on SSCs are of paramount significance because they are the only stem cells that undergo renewal throughout life and transmit genetic information to subsequent generations. Furthermore, accumulating evidence indicates that SSCs can be cultured to become pluripotent embryonic stem cells (ESCs) that are able to differentiate into all cells of the three germ layers [8-13], highlighting potentially important applications of these cells for regenerative medicine. Gangaraju and Lin published an informative review on the role of miRNAs in stem cells [14] and underscored the functional importance of miRNAs in ESCs, germline stem cells, and somatic tissue stem cells. A recent study showed differential expression patterns of X-linked miRNAs in male germ cells [15]. Another report suggested that several miRNAs in the miRNA 17–92 cluster are highly expressed in gonocytes of mice at 3 days of age [16] and miRNA expression profiles have been shown in mouse SSCs, pre-meiotic germ cells, and meiotic male germ cells [17]. The role of miRNA-21 was recently shown to be important for regulating Thy1(+) enriched germ cells in the testis [18]. Thy1+ cells in mice contain the SSC population, but Thy1 is not a specific marker for SSCs. It has been reported that miRNA-221 and miRNA-222 are required for maintaining mouse spermatogonia in an undifferentiated state and the impaired function of these miRNAs leads to an in initial differentiation of SSCs into type A1–A4 spermatogonia [19]. MiRNA-146 has been shown to regulate the differentiation of mouse SSCs through the regulation of retinoic acid [20]. There are about 1,000 miRNAs present in the mouse and human genomes, and it is very likely that other miRNAs also regulate the fate of SSCs. Therefore, the function and mechanisms of individual miRNAs in regulating mammalian germline stem cell (SSC) fate determinations remain almost unknown and research on this topic is still in its infancy. Here we have for the first time explored the expression, function, and targets of miRNA-20 and miRNA-106a in mouse SSCs.

Materials and Methods

  1. Top of page
  2. A
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

Animals

BALB/c male mice at 8-day and 60-day-old, and mothers with 6-day-old male pups were obtained from the Charles River Laboratories, Inc (Wilmington, MA, http://www.criver.com). All animal care procedures were performed pursuant to the National Research Council's Guide for the Care and Use of Laboratory Animals, USA. Experimental protocols were approved by the Georgetown University Animal Care and Use Committee.

Cell Isolation and Culture

Seminiferous tubules were isolated from the testes of 6-day-, 8-day-, and 60-day-old mice using enzymatic digestion with collagenase IV (Sigma, St. Louis, MO, www.sigmaaldrich.com) and DNase I as described previously [21]. Germ cells and Sertoli cells were obtained using a second-step enzymatic digestion with collagenase IV, hyaluronidase (Sigma, St. Louis, MO, www.sigmaaldrich.com), trypsin (Sigma, St. Louis, MO, www.sigmaaldrich.com), and DNase [21]. Sertoli cells and germ cells were separated by differential plating [22]. GFRα1-positive and -negative spermatogonia (the non-stem cells) were further separated from germ cells of 6-day-old mice by magnetic-activated cell sorting (MACS) using an antibody to GFRα1 (Abcam, Cambridge, MA, www.abcam.com) pursuant to the procedure as previously described [23]. The c-kit-positive spermatogonia were separated from germ cells of 8-day-old mice by MACS using an antibody to c-kit as described [23]. We chose 6-old- and 8-day-old mice to isolate GFRα1-positive spermatogonia and c-kit-positive spermatogonia because As, Apr, and Aal spermatogonia are enriched in mice at 6 days of age [21], while differentiating spermatogonia (c-kit positive) predominate in mice at 8 days of age [24]. Adult germ cells were isolated from the testes of 60-day-old mice using a two-step enzymatic digestion and differential plating [21]. Germ cells were isolated from mouse testes after transplantation of SSCs transfected with miRNA mimic control or miRNA-20 and miRNA-106a mimics according to the procedure mentioned below.

The isolated mouse SSCs (GFRα1-positive spermatogonia) were cultured on plates coated with 0.1% gelatin in StemPro-34 SFM medium (Invitrogen, Carlsbad, CA) supplemented with 100 ng/ml GDNF (glial cell line-derived neurotrophic factor), 10 ng/ml human bFGF (basic fibroblast growth factor), 10 ng/ml LIF (leukemia inhibitory factor), 20 ng/ml EGF (epidermal growth factor), 30 ng/ml TGF-β (transforming growth factor-beta), and 100 ng/ml Nodal to maintain the characteristics of SSCs [25-27]. The StemPro-34 SFM medium consisted of a basal liquid medium and frozen supplement (StemPro-Nutrient Supplement from Invitrogen) that was added at the time of use. All cultures were maintained at 34°C in a humidified 5% CO2 incubator.

RNA Extraction and RT-PCR

RNA was extracted from freshly isolated cells as mentioned above using Trizol (Invitrogen, Carlsbad, CA, URL: www.invitrogen.com). MiRNA was isolated from C18–4 cells and SSCs transfected with or without miRNA-20 or miRNA-106a mimics or inhibitors, or NIH 3T3 cells (a gift from Dr. Aykut Uren, Georgetown University Medical Center, http://www.gumc.georgetown.edu), using the mirVana miRNA Isolation Kit (Ambion, Austin, TX, www.invitrogen.com/ambion) and treated with DNase I to remove any genomic DNA contamination.

Total RNA was extracted from mouse SSCs transfected with or without miRNA-20 or miRNA-106a mimics or inhibitors using Trizol. Reverse transcription of purified RNA was performed using oligo(dT) priming and superscript II reverse transcription according to the manufacturer's instructions (Invitrogen). Polymerase chain reaction (PCR) reaction was performed according to the procedure as described previously [27]. The primer pairs of selected genes including Pcna, Kit, Plzf, Gfra1, Pou51, Ret, Stat3, Fos, and Gapdh were designed and listed in supporting information Table S2. The PCR reaction started with triplicates at 94°C for 2 minutes and was performed as follows: denaturation at 94°C for 30 seconds, annealing at a temperature (Tm) as indicated in supporting information Table S2 for 45 seconds, and elongation at 72°C for 45 seconds. After 35 cycles, the samples were incubated for an additional 7 minutes at 72°C. PCR products were separated by electrophoresis on 1.2% agarose gels. The gels were exposed to a Transilluminator (Fisher Scientific, Pittsburgh), and pictures were taken with a Photo-Documentation Camera (Fisher Scientific, Pittsburgh, PA, http://www.fishersci.com). The data were scanned with an Epson Perfection 3,200 PHOTO, and densitometric analyses were processed with Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA, www.adobe.com).

Real-Time PCR

RNA was extracted from the isolated GFRα1-positive spermatogonia and c-kit-positive spermatogonia, C18–4 cells, adult male germ cells, Sertoli cells, or NIH 3T3 cells, or male germ cells from recipient mice transplanted with SSCs transfected with or without miRNA-20 or miRNA-106a mimics or inhibitors using the mirVana miRNA Isolation Kit (Ambion, Austin, TX, www.invitrogen.com/ambion) and treated with DNase I to digest any contaminating genomic DNA. The primers and probes of miRNA-20, miRNA-106a, miRNA-106b, miRNA-92, miRNA-93, and miRNA16 used for real-time PCR were described previously [28]. All reverse transcriptase (RT) and real time-PCR reagents were purchased from Ambion. Real time PCR was performed as described previously [28] using miRNA-16 primers as internal control. MiRNA-16 was stably expressed in mouse testis and therefore it was used as housekeeping miRNA in this study. The relative expression level for each miRNA was calculated using the comparative Ct method (2ΔΔCt) after normalization to the level of miRNA-16. Individual samples were run in triplicate. All samples were run on the Applied Biosystems ABI Prism 7700 system (Grand Island, NY, www.appliedbiosystems.com).

MiRNA Microarray and cDNA Microarray

GFRα1-positive spermatogonia and GFRα1-negative spermatogonia (non-stem cells) were separated from testes of 6-day-old mice by MACS as described above. Total RNA was extracted from GFRα1-positive spermatogonia and GFRα1-negative spermatogonia using Trizol (Invitrogen), and a RNA clean-up was performed for all samples using the RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA, www.qiagen.com) following a specific protocol preserving the miRNA. The quality of total RNA was checked by gel and electropherogram (data not shown), and miRNA was further extracted from these cells for miRNA microarray. MiRNA microarrays were conducted on miRXplore Microarrays (Miltenyi Biotec, Auburn, CA, www.miltenyibiotec.com) with duplicates. Fluorescence signals of the hybridized miRXplore Microarrays were detected using a laser scanner from Agilent (Agilent Technologies, Santa Clara, CA, www.home.agilent.com). The cDNA microarrays of GFRα1-positive spermatogonia and GFRα1-negative spermatogonia were performed with duplicates according to the procedure as described previously [23].

In Situ Hybridization

Fluorescence in situ hybridization (FISH) in testes of 6-day- and 60-day-old mice was performed by adapting miRNA localization protocols [29]. The fluorescence probes to miRNA-20 and miRNA-106a were obtained from Exiqon (Woburn, MA, www.exiqon.com) and hybridized to mouse testis sections. All reagents and apparatus used were DEPC treated. Hybridizations were conducted overnight at 58°C. Sections were stained with 4′, 6′-diamidino-2-phenylindole (DAPI), and the in situ signal was visualized by confocal microscopy.

Transfection of Pre-miR MiRNA Precursors (mimics) and Anti-miR MiRNA Inhibitors into Mouse SSCs

Pre-miR miRNA precursors (mimics) and anti-miR miRNA inhibitors for miRNA-20 and miRNA-106a were purchased from Ambion. MiRNA mimics are chemically synthesized RNAs designed to mimic individual endogenous mature miRNAs. Mouse SSCs were cultured as mentioned above and they were transfected with miRNA mimic control, miRNA-20 mimic, miRNA inhibitor control, miRNA-20 inhibitor, miRNA-106a mimic, or miRNA-106 inhibitor, using siPORT NeoFX transfection agent (Ambion, Austin, TX, www.invitrogen.com/ambion). Optimization experiments were performed using various miRNA mimic or inhibitor concentrations. In this study, we used 100 nM miRNA-20, miRNA-106a mimics and inhibitors and found that this concentration was sufficient for their long-term biological effects. After 48 hours of culture, cells were harvested for the analysis of miRNA-20 and miRNA-106a expression and for determining phenotypes of various genes and proteins.

Proliferation Assays

Mouse SSCs were seeded at a density of 2,000 cells per well in 96-well microtiter plates coated with 0.1% gelatin in StemPro-34 SFM medium supplemented with 100 ng/ml GDNF and transfected without miRNA or with miRNA mimic control, miRNA-20 mimic or miRNA-106a mimic, miRNA inhibitor control, miRNA-20 inhibitor, or miRNA-106 inhibitor. After 5 days of culture, proliferation assays were performed using the Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, www.promega.com) as measured by the amount of absorbance at 490 nm according to the procedure as described previously [27].

Bromodeoxyuridine Incorporation Assay

The isolated mouse SSCs were grown on coverslips in six-well plates coated with 0.1% gelatin in StemPro-34 SFM medium supplemented with 100 ng/ml GDNF. The cells were transfected without miRNA or with miRNA mimic control, miRNA-20 mimic or miRNA-106a mimic, miRNA inhibitor control, miRNA-20 inhibitor, or miRNA-106 inhibitor, and then 30 µg/ml bromodeoxyuridine (BrdU; Sigma, St. Louis, MO, www.sigmaaldrich.com) were added to the medium. After 24 hours of culture, immunocytochemistry was performed using an antibody to BrdU (Sigma, St. Louis, MO, www.sigmaaldrich.com) according to the protocol as described previously [30].

Transplantation of Mouse SSCs Transfected with MiRNA-20 or MiRNA-106a Mimics or Inhibitors into Infertile Mice

Eighty male Ncr Swiss nude mice were treated with busulfan (44 mg/kg body weight) 2 months prior to transplantation to deplete endogenous germ cells. Viral vector AAV2-CMV-eGFP was shown to efficiently and stably label donor SSCs [31]. To distinguish transplanted cells from endogenous germ cells, SSCs were exposed to AAV2-CMV-eGFP (Vector core of the University of Pennsylvania, http://www.med.upenn.edu/gtp/vectorcore/) for 3 hours before transplantation. Mouse SSCs were transfected with miRNA mimic control, miRNA-20 mimic, or miRNA-106a mimic, miRNA inhibitor control, miRNA-20 inhibitor, or miRNA-106 inhibitor, and cultured for 1 day or 5 days with the media as mentioned above in proliferation assay, and 106 cells were transplanted to each testis of busulfan-treated nude mice (n = 6 each group) pursuant to the procedure for germ cell transplantation [32]. Recipient mice with Dulbecco's modified Eagle's medium injection but without mouse SSC transplantation served as controls. Two months after transplantation, recipient mice were sacrificed for determining phenotypic features of SSC markers in testicular cells.

Immunohistochemistry

Testes sections were prepared from mice transplanted with or without SSCs that were transfected with miRNA mimic control, miRNA-20 mimic, or miRNA-106a mimic, as mentioned above. For immunohistochemistry, 5 µm sections were dewaxed in xylene and rehydrated through a series of graded alcohols. Antigen retrieval was performed using the antigen retrieval citra plus solution (BioGenex Laboratories Inc., San Ramon, CA, www.biogenex.com), and the endogenous peroxidase activity was quenched with 3% hydrogen peroxide. After permeabilization with Triton X-100 and blocking with 10% normal serum, testis sections were incubated with primary antibodies, including rabbit polyclonal antibody to GFRA1 (Abcam Inc., Cambridge, MA, www.abcam.com) or VASA (Abcam Inc., Cambridge, MA, www.abcam.com) at a 1:100 dilution or mouse monoclonal antibody to PCNA (proliferating cell nuclear antigen) (EMD Biosciences Inc., San Diego, CA, www.emdmillipore.com) at a 1:200 dilution, pursuant to the procedures described previously [27]. Testis sections were incubated with secondary antibody to rhodamine-conjugated anti-rabbit or anti-mouse IgG at a 1:200 dilution for 30 minutes at 34°C and washed three times with PBS. All the secondary antibodies were purchased from Jackson ImmunoResearch laboratories. The DAPI was used to stain the nuclei of the cells in the testes, and the sections were observed for epifluorescence using an Olympus Fluoview 500 Laser Scanning Microscope (Olympus, Melville, NY, www.olympusamerica.com).

Immunofluorescence and Confocal Microscopy

Germ cells were isolated from mice after transplantation of mouse SSCs transfected with miRNA mimic control, miRNA-20 mimic, or miRNA-106a mimic, using a two-step enzymatic digestion and differential plating as described above [22]. Immunofluorescence by double staining was performed on these germ cells using antibody to GFRα1 (Abcam Inc., Cambridge, MA, www.abcam.com) and GCNA1 (a gift from Dr. George C. Enders, University of Kansas, http://www.kumc.edu) according to the procedure as described previously [30]. After three washes in PBS, the cells were incubated with the secondary antibody, including fluorescein (FITC)-conjugated donkey anti-rabbit IgG and Rhodamine-conjugated goat anti-rat IgG at a 1:200 dilution for 30 minutes at 34°C, and washed three times with PBS. All the secondary antibodies were purchased from the Jackson ImmunoResearch laboratories. The cells were observed for epifluorescence using an Olympus Fluoview 500 Laser Scanning Microscope (Olympus, Melville, NY, www.olympusamerica.com). Images were acquired and analyzed using the accompanying Fluoview software.

Western Blots

GFRα1-positive spermatogonia and c-kit-positive spermatogonia were isolated from testes of 6-day-old mice and 8-day-old mice respectively by MACS as described above [23]. Western blots of STAT3 expression in GFRα1-positive spermatogonia and c-kit-positive spermatogonia as well as STAT3 and Ccnd1 expression in mouse SSCs transfected with miRNA mimic control, or miRNA-20 mimic, miRNA-106a mimic, miRNA inhibitor control, miRNA-20 inhibitor, or miRNA-106 inhibitor, or with Stat3 small interfering RNAs (siRNAs) or Ccnd1 siRNAs, were carried out using antibody against STAT3 or Ccnd1 (Santa Cruz, Inc., Santa Cruz, CA, http://www.scbt.com) according to the protocol described previously [30]. The membranes were stripped with Restore Western Blot Stripping Buffer (Pierce Biotechnology, Inc., Rockford, IL, www.piercenet.com) and re-probed with antibody to ACTB (β-actin) (IMGENEX Corp., San Diego, CA, www.imgenex.com).

RNA Interference

SiRNAs against Stat3, Fos, and Ccnd1 and negative control siRNA were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China, http://www.genepharma.com) and their sequences were listed in supporting information Table S3. Mouse SSCs were isolated and cultured in the medium as mentioned above, and Stat3, Fos, or Ccnd1 siRNA, or negative control siRNA was transfected into the cells using Lipofectamine 2000 (Invitrogen) according to the procedure described previously [27]. RT-PCR analysis of Stat3, Fos, and Plzf and Western blots of STAT3 and Ccnd1 were performed at 48 hours after transfection according to the procedure mentioned above. Proliferation assay of SSCs with or without siRNA treatment was carried out after 5 days of culture.

Statistical Analysis

One-way analysis of variance (ANOVA) and Tukey post-tests using the SPSS statistical software were performed and the p value was calculated for each experiment. For all experiments with error bars, standard deviation was calculated to indicate the variation.

Results

  1. Top of page
  2. A
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

Identity of Spermatogonial Subpopulations Used in This Study

The GDNF/GFRα1 signaling system is the only known functional regulator of SSCs as shown initially by Meng and colleagues [25]. GFRα1, the receptor for GDNF, is expressed on the cell surface of mouse SSCs, and selection of mouse germ cells expressing GFRα1 is an excellent method to obtain a population of highly enriched SSCs in vitro (cell purity of greater than 95%) [33]. The starting cells we used in this study were GFRα1-positive SSCs. In a series of publications, Yoshida and colleagues confirmed that the Asingle (As) spermatogonia, the GFRα1-positive spermatogonia, are the actual SSCs. They also reported that Apaired (Apr) and Aaligned (Aal) spermatogonia could revert back to SSCs, and thus these cells would also have the GFRα1 receptor [34-36]. This spermatogonial dedifferentiation is almost identical to that reported earlier for the Drosophila male germline [37]. Thus, we used GFRα1-positive SSCs in this study. This population of cells contains As (mostly), Apr, and Aal spermatogonia, but they are almost all SSCs. In the early nineties, Yoshinaga and colleagues demonstrated that c-kit is a surface receptor marker for differentiated spermatogonia (A1 to A4) [38]. C-kit is not present in SSCs. We used the c-kit-positive cells for the experiments with differentiated spermatogonia.

Expression of MiRNA 17–92 Cluster in Mouse Germ Cells and Somatic Cells

We first probed the expression of the miRNA 17–92 cluster in various types of cells in adult mouse testes, including the SSCs. Among a number of miRNAs examined in the miRNA 17–92 cluster, we found distinct expression using real time PCR. Notably, we found that miRNA-20 and miRNA-106a were expressed at much higher levels in mouse SSCs (GFRα1-positive spermatogonia) and a mouse SSC line (C18–4 cells) [39], compared with differentiating spermatogonia (c-kit-positive spermatogonia) or adult male germ cells (Fig. 1A, 1B). There was no statistical difference in expression of miRNA-106b and miRNA-92 among mouse SSCs, the SSC line, the differentiating spermatogonia, and adult male germ cells (Fig. 1C; supporting information Fig. S1), while miRNA-93 was expressed at a lower level in SSCs compared with the differentiating spermatogonia (supporting information Fig. S1).

image

Figure 1. Distinct expression of microRNAs (miRNAs) in various types of cells and subcellular localization in adult mouse testes. (A–C): Real time polymerase chain reaction showed the expression of miRNA-20, miRNA-106a, and miRNA-106b in GFRα1-positive spermatogonia, c-kit-positive spermatogonia, C18–4 cells, adult male germ cells, NIH 3T3 cells, and Sertoli cells. House keeping miRNA-16 served as a loading control of total miRNAs. To compare the expression of miRNA-20, miRNA-106a and miRNA-106b in different types of cells, the expression of these miRNAs in c-kit-positive spermatogonia was set as 1. Compared with c-kit-positive spermatogonia, *, p < .05. (D–E): Fluorescein in situ hybridization displayed restricted expression of miRNA-20 (D) and miRNA-106a (E) only to spermatogonia at the basement membrane of the seminiferous tubules in adult mouse testes. 4′, 6′-Diamidino-2-phenylindole was used to show cell nuclei. Scale bars = 20 µm (D, E). Abbreviations: DAPI, 4′, 6′-diamidino-2-phenylindole; miRNA, microRNA.

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MiRNA Microarray and FISH Reveal that MiRNA-20 and MiRNA-106a Are Expressed Preferentially in SSCs

MiRNA microarray further reveled that miRNA-20 (also known as miRNA-20A) and miRNA-106a were expressed at higher levels in mouse SSCs (GFRα1-positive spermatogonia) compared with non-stem cells (GFRα1-negative spermatogonia) (supporting information Table S1). High-quality of the isolated RNA used for miRNA microarray was shown by gel imaging and electropherograms and reproducibility of the microarrays was verified (data not shown). Using FISH, we detected that miRNA-20 and miRNA-106a were largely confined to the spermatogonia along the basement membrane of adult mouse seminiferous tubules (Fig. 1D, 1E). FISH analyses also showed that miRNA-20 and miRNA-106a were expressed in spermatogonia of immature mice (supporting information Fig. S2A, S2B). Testis sections without miRNA probes in immature and adult mice (supporting information Fig. S2C, S2D) were used to verify specific expression of miRNA-20 and miRNA-106a in immature and adult mouse testes.

MiRNA-20 and MiRNA-106a Promote Proliferation and DNA Synthesis of SSCs

The “Holy Grail” of adult stem cell research is to modulate endogenous stem cells in vivo, possibly with small molecules specifically targeted to respective stem cell populations, as reviewed by Matzuk and colleagues [40]. Obviously, endogenous miRNAs regulating stem cell fate must be thoroughly understood before such in vivo studies can be initiated. We next explored the function of miRNA-20 and miRNA-106a in the regulation of SSC fate determinations using miRNA mimics and inhibitors (antagomirs). Pre-miR miRNA precursors are small chemically modified double-stranded RNA molecules designed to mimic endogenous mature miRNA. Anti-miR miRNA inhibitors are chemically modified single stranded nucleic acids designed to specifically bind to and inhibit endogenous miRNA. Pre-miR miRNA mimic controls and inhibitor controls were random sequence molecules that didn't produce any identifiable effects on known miRNA function. The transfection efficiency of miRNA-20 or miRNA-106a mimics or inhibitors in mouse SSCs was about 75% as assessed by co-transfection of fluorescently labeled miRNA. RT-PCR analysis showed that miRNA-20 expression was markedly increased (3.5-fold) in SSCs after miRNA-20 mimic transfection compared with cells treated with miRNA mimic control (one fold, Fig. 2A). Conversely, miRNA-20 expression was decreased by 80% in SSCs treated by miRNA-20 inhibitor transfection compared with SSCs with miRNA inhibitor control (100%, Fig. 2A). Likewise, miRNA-106a expression was enhanced (3.1-fold) in SSCs with miRNA-106a mimic transfection compared with the cells with miRNA mimic control (one fold, Fig. 2B). MiRNA-106a expression was reduced by 75% in SSCs with miRNA-106a inhibitor transfection compared with the cells with miRNA inhibitor control (100%, Fig. 2B). These data illustrated the effective specificity of miRNA mimics and inhibitors to miRNA-20 and miRNA-106a in mouse SSCs.

image

Figure 2. Differential expression of microRNA (miRNA)-20, miRNA-106a, Pcna, Kit, and Plzf in mouse spermatogonial stem cells (SSCs) transfected with or without miRNA-20 or miRNA-106a mimics and inhibitors. (A–B): Reverse transcriptase polymerase chain reaction (RT-PCR) revealed miRNA-20 (A) and miRNA-106a (B) in mouse SSCs transfected with or without miRNA-20 or miRNA-106a mimics or inhibitors. Compared with miRNA mimic control, *, p < .05. (C–D): Proliferation (C) and BrdU incorporation (D) assays of mouse SSCs transfected without miRNA or with miRNA mimic control, or miRNA-20 mimic or miRNA-106a mimics or inhibitors. Statistically significant differences (p < .05) between miRNA-20 mimic or miRNA-106a mimic or inhibitor-treated cells and miRNA mimic control were indicated by an asterisk. (E–F): RT-PCR showed Pcna (E), Kit (E), and Plzf (F) transcripts in mouse SSCs transfected with or without miRNA-20 or miRNA-106a mimics or inhibitors. Compared with miRNA mimic control, *,p < .05. Abbreviation: miRNA, microRNA.

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Next, we explored the effects of mimics of miRNA-20 and miRAN-106a in controlling the renewal and/or differentiation of the mouse SSCs. MiRNA mimic and inhibitor controls were used to assure that transfection of the mimics or inhibitors didn't affect SSC maintenance. Proliferation assays demonstrated that miRNA-20 mimic and miRNA-106a mimic induced a 5.1- and 4.8-fold increase of cell number in mouse SSCs after culture for 5 days, respectively, compared with miRNA mimic control or without miRNA transfection (one fold, Fig. 2C). Mouse SSCs transfected with miRNA-20 or miRNA-106a mimics could be cultured for more than 45 days with an increase of cells by 452- and 364-fold, respectively, compared with miRNA mimic controls. In contrast, miRNA-20 and miRNA-106a inhibitors resulted in a 23% and 26% reduction of cell number in mouse SSCs, respectively, compared with miRNA inhibitor control or without miRNA transfection (Fig. 2C). DNA synthesis in mouse SSCs by miRNA-20 and miRNA-106a mimics was assessed immunocytochemically using BrdU incorporation assays. An increase in the BrdU-positive cells was seen in SSCs with miRNA-20 mimic (3.2-fold) and miRNA-106a mimic (3.1-fold) compared with miRNA mimic control (one fold, Fig. 2D). Conversely, miRNA-20 and miRNA-106a inhibitors led to a 25% and 29% reduction of BrdU-positive cells in mouse SSCs, respectively, compared with miRNA inhibitor control or without miRNA transfection (Fig. 2D). These results implicate the role of miRNA-20 and miRNA-106a in promoting proliferation and DNA synthesis of SSCs.

Pcna, Kit, and Plzf Expression in SSCs Transfected with MiRNA-20 or MiRNA-106a Mimics and Inhibitors

We determined the phenotypic changes of SSCs transfected with miRNA-20 or miRNA-106a mimics and inhibitors. Pcna (Fig. 2E) and Plzf (Fig. 2F) mRNA was markedly elevated in SSCs with miRNA-20 or miRNA-106a mimic transfection compared with the cells with miRNA mimic control. Conversely, Pcna and Plzf transcripts were reduced in SSCs with miRNA-20 or miRNA-106a inhibitor transfection compared with the cells with miRNA inhibitor control (Fig. 2E, 2F). In direct contrast to Pcna and Plzf transcripts, Kit mRNA (Fig. 2E) was reduced in SSCs with miRNA-20 or miRNA-106a mimic transfection compared with the cells with miRNA mimic control. Kit mRNA was enhanced in SSCs with miR-20 or miRNA-106a inhibitor transfection compared with the cells with miRNA inhibitor control (Fig. 2E). PCNA has been used extensively in the identification of proliferating spermatogonia in the testes of rodents and primates [41], while Plzf is required for renewal of mouse SSCs [42]. Kit has been used as a marker for differentiating spermatogonia, and type A1-A4 spermatogonia divisions are known to be Kit-dependent [24]. Considered together, our results suggest that miRNA-20 and miRNA-106a are required for renewal of mouse SSCs.

MiRNA-20 or MiRNA-106a Mimics Enhance Colony Formation of SSCs and Induce Expression of GFRα1, PCNA, Pou5f1, Plzf, and Ret in Mouse Testes In Vivo

To address the function of miRNA-20 and miRNA-106a in regulating SSCs in vivo, we transplanted fresh and cultured mouse SSCs with miRNA mimic control, or transfection with miRNA-20 mimic or inhibitor, or with miRNA-106a mimic or inhibitor into seminiferous tubules of sterile busulfan-treated nude mice. Two months after busulfan treatment, no male germ cells remained in the testes of recipient null mice (Fig. 3A, 3B). AAV2-CMV has been shown to be effective in transducing mouse SSCs for 12 months [31] and pig SSCs for 5 and 7 months [43]. To visualize donor-derived colonization in recipient testis, we exposed SSCs (GFRα1-positive spermatogonia) to AAV2-CMV-eGFP before transplantation and transfection efficiency of AAV2-CMV-eGFP in mouse SSCs was around 80% (supporting information Fig. S3A). Transfection of miRNA inhibitors or mimics into SSCs could have long-term effects on SSC self-renewal and differentiation in vivo, as evidenced by recent findings that transfection of miRNA-21 inhibitor reduces colony number of SSCs at 2 months after transplantation [18]. Two months after transplantation, colony numbers of SSCs were enhanced in fresh SSCs treated with miRNA-20 mimic (Fig. 3F, 3L) or miRNA-106a mimic (Fig. 3G, 3L) and in cultured SSCs with miRNA-20 mimic (Fig. 3H, 3L) or miRNA-106a mimic (Fig. 3I, 3L) compared with miRNA mimic control (Fig. 3D, 3L). In contrast, colony formation was reduced in SSCs treated with miRNA-20 inhibitor (Fig. 3J–3L) or miRNA-106a inhibitor (Fig. 3K, 3L), compared with miRNA inhibitor control (Fig. 3E, 3L). These results suggest that miRNA-20 and miRNA-106a are essential for renewal of SSCs. Immunohistochemistry demonstrated that miRNA-20 and miRNA-106a mimics resulted in a remarkable increase of male gem cells that were positive for GFRA1 (Fig. 4A) and PCNA (Fig. 4B), a hallmark for proliferating spermatogonia [41], whereas miRNA-20 and miRNA-106a inhibitors led to rare cells expressing GFRA1 (Fig. 4A) and PCNA (Fig. 4B). To probe whether miRNA-20 and miRNA-106a have an effect on the differentiation of SSCs, we determined the expression of VASA, a marker for germ cells ranging from spermatogonia to spermatids [44]. Immunohistochemistry with antibody to VASA revealed that there was a statistical decrease in the differentiation of SSCs into round spermatids by miRNA-20 and miRNA-106a mimics or inhibitors compared with miRNA mimic or inhibitor controls (Fig. 5A–5G).

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Figure 3. Effects of microRNA (miRNA)-20 and miRNA-106a mimics or inhibitors on colony formation in recipient mice. (A-B): Testis morphology of mice at 2 months after busulfan treatment. Only Sertoli cells remained in the seminiferous tubules. Scale bar = 20 µm (A), = 10 µm (B). (C–K): Expression of eGFP from seminiferous tubules of recipient mice without spermatogonial stem cell (SSC) transplantation (C), or from seminiferous tubules after transplantation of SSCs with miRNA mimic control (D), or with miRNA inhibitor control (E), or with miRNA-20 mimic (F), or with miRNA-106a mimic (G), or with miRNA-20 inhibitor (J); or with miRNA-106a inhibitor (K). (H, I): Transplantation of cultured SSCs for 5 days with miRNA-20 mimic (H) or miRNA-20 mimic (I). Scale bars = 100 µm (C–K). (L): Colony number of recipient mouse testes without SSC transplantation, or from transplantation of SSCs with miRNA mimic control, or miRNA inhibitor control, or miRNA-20 mimic, or miRNA-106a mimic, or miRNA-20 inhibitor, or miRNA-106a inhibitor, or from transplantation of cultured SSCs for 5 days with miRNA-20 mimic, or miRNA-106a mimic. Compared with miRNA mimic control, *, p < .05. Abbreviations: eGFP, enhanced green fluorescence protein; miRNA, microRNA; SSC, spermatogonial stem cell.

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Figure 4. Biochemical phenotypes of mouse testes transplanted with or without spermatogonial stem cells that were transfected with microRNA (miRNA) mimic control, or miRNA-20 mimic, or miRNA-106a mimic, or miRNA-20 inhibitor, or miRNA-106a inhibitor. (A–B): Immunohistochemistry showed that GFRA1 (A) and PCNA (B) expression was increased in mouse testes transplanted with miRNA-20 or miRNA-106a mimics. Scale bars = 10 µm (A, B). Abbreviations: DAPI, 4′, 6′-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; GFRA1, (GDNF family receptor alpha 1); miRNA, microRNA; SSC, spermatogonial stem cell.

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Figure 5. VASA expression in recipient mouse testes. (A–F): Immunohistochemistry revealed VASA expression in mouse testes transplanted without spermatogonial stem cells (SSCs) (A) or with SSCs that were transfected with microRNA (miRNA) mimic control (B), or miRNA-20 mimic (C), or miRNA-106a mimics (D), or miRNA-20 inhibitor (E), or miRNA-106a inhibitor (F). Scale bars = 20 µm. (G): Number of tubule cross sections with full spermatogenesis in recipient mouse testes without SSC transplantation, or from transplantation of SSCs with miRNA mimic control, or miRNA inhibitor control, or miRNA-20 mimic, or miRNA-106a mimic, or miRNA-20 inhibitor, or miRNA-106a inhibitor. Compared with miRNA mimic control, *, p < .05. Abbreviations: DAPI, 4′, 6′-diamidino-2-phenylindole; miRNA, microRNA; SSC, spermatogonial stem cell; VASA, member of the DEAD-box protein family.

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We checked the expression of miRNA-20 and miRNA-106a in male germ cells recovered from recipient mouse testes 2 months after transplantation of SSCs transfected with miRNA mimic control or miRNA-20 mimic, or miRNA-106a mimic, or with miRNA inhibitor control or miRNA-20 inhibitor, or miRNA-106a inhibitor. Real time PCR analysis revealed that miRNA-20 and miRNA-106a expression was markedly increased in male germ cells from recipient mice transplanted with SSCs after miRNA-20 or miRNA-106a mimic transfection compared with cells treated with miRNA mimic control (supporting information Fig. S3B, S3C). In contrast, miRNA-20 and miRNA-106a expression was reduced in male germ cells recovered from recipient mice transplanted with SSCs after miRNA-20 or miRNA-106a inhibitor transfection compared with cells treated with miRNA inhibitor control (supporting information Fig. S3B, S3C). Confocal microscopy by doubling immunostaining using anti-GFRA1 and anti-GCNA1 (germ cell nuclear antigen 1, a marker for germ cells) showed that GFRA1 was expressed in very few spermatogonia but not in other germ cells (e.g., pachytene spermatocytes and round spermatids) from mice with transplantation of mouse SSCs transfected with miRNA mimic control (Fig. 6A). Importantly, many more (>90% of the germ cells) spermatogonia were positive for GFRA1 in the mice with transplantation of mouse SSCs transfected with miRNA-20 mimic (Fig. 6B) or miRNA-106a mimic (Fig. 6C). Collectively, these data indicate that overexpression of miRNA-20 and miRNA-106a leads to an accumulation of undifferentiated SSCs in vivo.

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Figure 6. Phenotypic characteristics of testicular cells from mice transplanted with or without spermatogonial stem cells (SSCs) that were transfected with microRNA (miRNA) mimic control, or miRNA-20 mimic, or miRNA-106a mimic. (A–C): Confocal microscopy showed GFRA1 (green fluorescence) and GCNA1 (red fluorescence) expression in germ cells from mice with transplantation of mouse SSCs transfected with miRNA mimic control (A), or miRNA-20 mimic (B), or miRNA-106 mimic (C): Note: spermatogonia (arrowheads); pachytene spermatocytes (asterisk); round spermatids (arrow). Scale bars = 10 µm (A–C). (D): Reverse transcriptase polymerase chain reaction (RT-PCR) showed expression of Gfra1, Pou5f1, Plzf, Ret, and Pcna in germ cells and Sertoli cells from mice with or without SSCs that were transfected with miRNA mimic control, or miRNA-20 mimic, or miRNA-106a mimics. Total RNA without reverse transcription (RT-) but with PCR served as negative controls. (E): Western blots showed STAT3 expression in GFRA1-positive spermatogonia and c-kit-positive spermatogonia. (F–K): RT-PCR and Western blots revealed Stat3 (F, G), STAT3 (H, I), Fos (J), and Ccnd1 expression (K) in mouse SSCs transfected with or without miRNA-20 and miRNA-106a mimics or inhibitors. Compared with miRNA mimic control, *, p < .05. Abbreviations: ACTB, actin beta; miRNA, microRNA.

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We further probed the biochemical phenotypes of the testicular cells isolated from mice with or without SSCs transfected with miRNA-20 or miRNA-106a mimics. Much higher levels of Gfra1 mRNA expression were observed in the cell mixture containing germ cells and Sertoli cells from the mice transplanted with SSCs and miRNA-20 mimic or miRNA-106a mimic, compared with the cells from the mice transplanted with SSCs and miRNA mimic control (Fig. 6D). Gfra1 mRNA was not detected in the cells from the mice without SSC transplantation (Fig. 6D). Importantly, higher levels of Pou5f1 (Oct4), Plzf, Ret, and Pcna, makers for proliferation and maintenance of rodent SSCs [30, 33, 45], were seen in male germ cells and Sertoli cells from the mice transplanted with SSCs transfected with miRNA-106a mimic (Fig. 6D). These data, together with GFRA1 and GCNA1 expression, further suggest that miRNA-20 and miRNA-106a are required for renewal of mouse SSCs in vivo.

STAT3 and Ccnd1 Are Direct Targets of MiRNA-20 and MiRNA-106a in SSCs

To gain an insight into the molecular mechanisms of miRNA-20 and miRNA-106a in SSCs, we identified the targets of these miRNAs. Using different miRNA prediction software programs [3] we predicted that STAT3 was a potential target of the miRNA 17–92 cluster in mouse SSCs. Western blots showed that STAT3 was expressed at a lower level in mouse SSCs (GFRα1-positive spermatogonia) compared with the differentiating spermatogonia (c-kit-positive spermatogonia) (Fig. 6E). RT-PCR showed that Stat3 was significantly decreased in mouse SSCs transfected with miRNA-20 or miRNA-106a mimics compared with the cells with miRNA mimic controls (Fig. 6F, 6G). Conversely, Stat3 mRNA was increased markedly in SSCs with miRNA-20 and miRNA-106a inhibitor transfection compared with the cells with miRNA inhibitor controls (Fig. 6F, 6G). Likewise, Western blots revealed that STAT3 protein was significantly decreased in mouse SSCs transfected with miRNA-20 or miRNA-106a mimics compared with the cells with miRNA mimic controls (Fig. 6H, 6I), whereas STAT3 protein was increased markedly in SSCs with miRNA-20 or miRNA-106a inhibitor transfection compared with the cells with miRNA inhibitor controls (Fig. 6H, 6I). Thus, STAT3 is a binding target of miRNA-20 and miRNA-106 in mouse SSCs. To define downstream targets of miRNA-20 and miRNA-106, we examined expression of cell cycle regulators. We observed that miRNA-20 or miRNA-106a mimics reduced the transcription of Fos and translation of Ccnd1 (Fig. 6J, 6K), whereas miRNA-20 or miRNA-106a inhibitors increased Fos and Ccnd1 expression (Fig. 6J, 6K), indicating that Ccnd1 is a direct target of miRNA-20 or miRNA-106a.

We further explored the function of STAT3, Fos, and Ccnd1 in regulating renewal of mouse SSCs. Using small interfering RNA (siRNA), we found that siRNAs against Stat3, Fos, and Ccnd1 specifically knocked down expression of Stat3 mRNA (Fig. 7A, 7B), STAT3 protein (Fig. 7D, 7E), Fos (Fig. 7G, 7H), and Ccnd1 (Fig. 7J, 7K). Stat3 silencing led to reduction of Plzf transcription of SSCs (Fig. 7A, 7C). Significantly, STAT3, Fos and Ccnd1 knockdown resulted in an obvious increase of SSCs (Fig. 7F, 7I, 7L), suggesting that STAT3 and Ccnd1 promote renewal of SSCs.

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Figure 7. Knockdown of Stat3, Fos, and Ccnd1 and its effects on proliferation of spermatogonial stem cells (SSCs). (A): Reverse transcriptase polymerase chain reaction (RT-PCR) showed the expression of Stat3 and Plzf after Stat3 silencing. Gapdh served as a loading control of total RNA. (B): Relative expression of Stat3 to Gapdh after silencing of Stat3. Compared with negative control small interfering RNA (siRNA), *, p < .05. (C): Relative expression of Plzf to Gapdh after silencing of Stat3. Compared with negative control siRNA, *, p < .05. (D): Western blots revealed STAT3 expression after Stat3 silencing. ACTB served as a loading control of total protein. (E): Relative expression of STAT3 to ACTB after silencing of Stat3. Compared with negative control siRNA, *, p < .05. (F): Proliferation assay displayed cell number of SSCs by Stat3 siRNAs. Compared with negative control siRNA, *, p < .05. (G): RT-PCR showed the expression of Fos after Fos silencing. Gapdh was used as a loading control of total RNA. (H): Relative expression of Fos to Gapdh after silencing of Fos. Compared with negative control siRNA, *, p < .05. (I): Proliferation assay displayed cell number of SSCs by Fos siRNAs. Compared with negative control siRNA, *, p < .05. (J): Western blots revealed Ccnd1 expression after Ccnd1 silencing. ACTB served as a loading control of total protein. (K): Relative expression of Ccnd1 to ACTB after silencing of Ccnd1. Compared with negative control siRNA, “*, p < .05. (L): Proliferation assay displayed cell number of SSCs by Ccnd1 siRNAs. Abbreviations: ACTB, (actin beta); siRNA, small interfering RNA.

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Discussion

  1. Top of page
  2. A
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

Several lines of evidence indicate that SSCs constitute one of the most important stem cells systems in view of a number of their unique characteristics. First, SSCs can be considered “immortal” germ cells because they are present from birth to death and they produce sperm that transmit genetic information to subsequent generations. Thus, they represent a very valuable resource for experimental modification of the mammalian genome [46]. Second, SSCs self-renew throughout life and they differentiate into spermatocytes, spermatids, and eventually mature spermatozoa, and thus they can be used as an excellent model to elucidate the molecular mechanisms governing stem cell renewal versus differentiation. Third there is the clear potential to use SSC transplantation to restore fertility in cancer patients after chemotherapy or irradiation therapy [47]. Finally, recent studies have demonstrated that SSCs can acquire pluripotency to become ESCs that are able to differentiate into all cell lineages of the three germ cell layers [8-13]. Therefore, SSCs have great potential for cell-based and autologous organ regeneration therapy for human diseases without the associated ethical issues and immune rejection. The regulation of SSCs requires the actions of intrinsic factors and extrinsic signals, and it has been suggested that SSC renewal is regulated primarily by intrinsic factors [18]. Thus, it is imperative to uncover intrinsic molecules regulating the renewal or differentiation of SSCs before the cells can be used in the clinic.

MiRNAs play crucial roles in the regulation of cellular proliferation [4], differentiation [5, 6], and apoptosis [7]. Generating expression profiles of miRNAs in male germ cells is a prerequisite for a thorough understanding of their roles in regulating SSC fate decisions. Although several studies show the expression patterns of miRNAs in mouse germ cells [15] and mouse gonocytes [16], the expression, function, and the targets of individual miRNAs in controlling SSC fate determinations remain mostly unknown. We found using real time PCR and miRNA microarray that miRNA-20 and miRNA-106a were expressed preferentially in mouse SSCs. Our results are consistent with a recent report by Smorag and colleagues showing that miRNA-20 (also known as miRNA-20a) is highly expressed in mouse SSCs [17]. However, the same group also showed that miRNA-20 is present at a high level in other spermatogonia and in meiotic germ cells. This is probably due to the fact that the more differentiated spermatogonia and the isolated meiotic germ cells from Sycp3/DsRed transgenic mice may contain SSCs. Purity of the isolated cell fractions was not presented in the Smorag paper. To some extent, our results are also consistent with the report showing that miRNA-106a is present in type A spermatogonia but absent in the differentiated type B spermatogonia [15]. We further revealed cellular localization of miRNA-20 and miRNA-106a in the spermatogonia. Considered together, the expression profiles and subcellular localization of miRNA-20 and miRNA-106a in adult mouse testes suggest that miRNA-20 and miRNA-106a may play essential roles in regulating the renewal of mouse SSCs.

Interestingly, our miRNA and cDNA microarray revealed an inverse relationship in the expression of Lin28 mRNA and miRNAs Let-7G and Let-7I between mouse SSCs (GFRα1-positive spermatogonia) and the non-stem cells (GFRα1-negative spermatogonia) (supporting information Table S1), suggesting that Let-7G/Let-7I/Lin28 form a regulatory loop to regulate fate decisions of SSCs. It has been shown that Lin28 is specifically expressed in mouse As, Apr, and Aal spermatogonia and that Lin28-positive spermatogonia comprise two cell subpopulations including high stem cell potential and high differentiation commitment [48]. Taken together, Lin28 is present preferentially in spermatogonia with high differentiation commitment. Notably, our microarray data on the expression of Let-7G, Let-7I, and Lin28 are in contrast to the finding that down-regulation of Let-7 and higher levels of Lin28 are required for maintenance of ESC pluripotency [49], highlighting that SSCs are distinct from ESCs in miRNA signatures and gene regulation networks, although SSCs can acquire pluripotency in vitro to become ESCs [8-13]. Moreover, our miRNA microarray showed that miRNA-21 was over-expressed (1.46-fold) in mouse SSCs (GFRα1-positive spermatogonia) compared with the non-stem cells (GFRα1-negative spermatogonia), suggesting that miRNA-21 may regulate the renewal of GFRα1-positive SSCS. We also identify a list of miRNAs that are enriched in mouse SSCs compared with non-stem cells. These miRNAs may play important roles in the maintenance of SSCs.

Using miRNA mimics and inhibitors, we revealed that miRNA-20 and miRNA-106a promote renewal (proliferation and DNA synthesis) of SSCs. More importantly, functional assays by miRNA mimics and inhibitors show that these two miRNAs are essential for renewal of SSCs, as evidenced by the phenotypic changes of Pcna, Plzf, and Kit in vitro. We further demonstrated that miRNA-20 and miRNA-106a are required for renewal of mouse SSCs in vivo, as shown by the enhancement of colony formation and Gfra1, PCNA, Pou5f1, Plzf, and Ret expression. Our ability to identify miRNA-20 and miRNA-106a as endogenous regulators promoting mouse SSC renewal and proliferation may be applicable in humans to obtain sufficient numbers of SSCs for therapeutic applications, as human SSCs are very rare (only one or two SSCs in each 5 µm cross section) [50].

The transcription factor ETV5 is a regulator for miRNA-21 in SSCs [18]. However, nothing is known about the targets of miRNAs regulating SSCs. We predicted that Stat3 is a target of miRNA 17–92 cluster, using three different miRNA prediction programs, namely: (1) the miRBase website employing the miRanda algorithm to predict miRNA: mRNA pairs; (2) the microInspector website using a different algorithm that allows for G:U wobbles in seed matches; and (3) custom made miRNA prediction program utilizing a variation of the TargetScans algorithm [3]. Importantly, we have for the first time identified STAT3 and Ccnd1 as targets of miRNAs, specifically for miRNA-20 and miRNA-106a, which offers a novel insight into mechanism by which miRNAs act on SSC regulation. Our data are consistent with the recent finding showing that STAT3 promotes the differentiation of mouse SSCs [51]. Significantly, we demonstrate for the first time that silencing of Stat3, Fos, and Ccnd1 stimulates the renewal of SSCs.

Conclusion

  1. Top of page
  2. A
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

In summary, we identified miRNA-20 and miRNA-106a as novel intrinsic RNA molecules that promote renewal of mouse SSCs at the post-transcriptional level via targeting STAT3 and Ccnd1. This study thus provides novel mechanisms regulating SSC fate determinations. MiRNAs are highly conserved between animals and humans [3]. Given the functional importance of miRNA-20 and miRNA-106a in spermatogenesis, these miRNAs could have great implications in offering novel therapeutic targets for treating male infertility or for male contraception. MiRNA inhibitors can be taken up by live cells and effectively exert their suppressive effects via binding the target miRNAs with high affinity and specificity [52]. Inhibitors for miRNA-20 and miRNA-106a are expected to satisfy the demand for a male contraceptive with high specificity and safety [53], as these miRNAs are expressed abundantly in SSCs in testis. Alternatively, the identification of specific miRNAs regulating SSC renewal may eventually be used to produce sufficient quantities of human SSCs and then use these adult stem cells to obtain a high yield of pluripotent ESCs for regenerative medicine.

Acknowledgments

  1. Top of page
  2. A
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

We thank Dr. George C. Enders, Department of Anatomy and Cell Biology, University of Kansas Medical Center, for providing an antibody to GCNA1. We also thank Dr. Aykut Uren, Georgetown University Medical Center, for providing NIH 3T3 cells. This work was supported by NIH R01 grant HD33728 (to M.D.). This work was also supported by start-up funds from the Renji Hospital, Shanghai Jiao Tong University School of Medicine and by the National Natural Science Foundation of China (31230048), Chinese Ministry of Science and Technology (2013CB947901), and the Shanghai Municipal Science and Technology Commission (12JC1405900) (to Z.H.). Z.H. is currently affiliated with the Clinical Stem Cell Research Center, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

References

  1. Top of page
  2. A
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

Supporting Information

  1. Top of page
  2. A
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. Disclosure of Potential Conflicts of Interest
  10. References
  11. Supporting Information

Additional Supporting information may be found in the online version of this article.

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stem1474-sup-0001-suppfigs1.tif95KSupporting Information Figure S1
stem1474-sup-0002-suppfigs2.tif898KSupporting Information Figure S2
stem1474-sup-0003-suppfigs3.tif14160KSupporting Information Figure S3
stem1474-sup-0004-supptabs1.docx92KSupporting Information Tables

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