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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2011 Jul 18;108(31):12740–12745. doi: 10.1073/pnas.1109987108

MicroRNA-21 regulates the self-renewal of mouse spermatogonial stem cells

Zhiyv Niu a, Shaun M Goodyear a, Shilpa Rao b, Xin Wu a, John W Tobias b, Mary R Avarbock a, Ralph L Brinster a,1
PMCID: PMC3150879  PMID: 21768389

Abstract

MicroRNAs (miRs) play a key role in the control of gene expression in a wide array of tissue systems, where their functions include the regulation of self-renewal, cellular differentiation, proliferation, and apoptosis. However, the functional importance of individual miRs in controlling spermatogonial stem cell (SSC) homeostasis has not been investigated. Using high-throughput sequencing, we profiled the expression of miRs in the Thy1+ testis cell population, which is highly enriched for SSCs, and the Thy1− cell population, composed primarily of testis somatic cells. In addition, we profiled the global expression of miRs in cultured germ cells, also enriched for SSCs. Our results demonstrate that miR-21, along with miR-34c, -182, -183, and -146a, are preferentially expressed in the Thy1+ SSC-enriched population, compared with Thy1− somatic cells. Importantly, we demonstrate that transient inhibition of miR-21 in SSC-enriched germ cell cultures increased the number of germ cells undergoing apoptosis and significantly reduced the number of donor-derived colonies of spermatogenesis formed from transplanted treated cells in recipient mouse testes, indicating that miR-21 is important in maintaining the SSC population. Moreover, we show that in SSC-enriched germ cell cultures, miR-21 is regulated by the transcription factor ETV5, known to be critical for SSC self-renewal.

Keywords: male germline stem cells, small RNA

Spermatogonial stem cells (SSCs) are among the testis germ cells called undifferentiated type A spermatogonia, which are composed of Asingle, Apaired, and Aaligned spermatogonia. It is the Asingle spermatogonia that are considered to have stem cell potential. SSCs are essential for spermatogenesis, but only constitute about 1 in 3,000 cells in the adult mouse testis. Maintenance of the SSC depends on its capacity for self-renewal, which encompasses its ability to proliferate, differentiate, and undergo apoptosis (1, 2). The process of spermatogenesis is complex and involves numerous endocrine and paracrine signals to coordinate SSC self-renewal and differentiation of daughter cells to undergo mitosis, meiosis, and spermiogenesis to generate spermatozoa (1, 2). Recent studies have added a new layer of molecules associated with the intricate mechanisms of gene regulation, which include the expression of RNA-induced silencing complex (RISC) components as well as a number of microRNAs (miRs), suggesting that miRs are functionally important in the process of spermatogenesis (3, 4). Notably, the loss of the RISC component Dicer, in germ cells or Sertoli cells, perturbs germ cell development and leads to infertility, and highlights the need for miR function in regulating spermatogenesis (5, 6).

Several studies have reported the global expression of miRs in the murine testis, but few have examined miR expression in specific germ cell populations of the testis, particularly the SSC population (5, 7–10). Moreover, the functional importance of individual miRs controlling SSC homeostasis has not been investigated. In the studies presented here, the expression of miRs in the Thy1+ testis cell population, which is highly enriched for SSCs, and the Thy1− cell population, composed primarily of testis somatic cells, is profiled. Our results demonstrate that miR-21, along with miR-34c, -182, -183, -146a, -465a-3p, -465b-3p, -465c-3p, and -465c-5p, are preferentially expressed in the Thy1+ SSC-enriched population, compared with Thy1− testis somatic cells. Most important, we demonstrate that miR-21 is functionally important in maintaining the SSC population in vitro, and that the transcription factor ETV5, known to be critical for SSC self-renewal, is a direct regulator of miR-21 expression.

Results

Analysis of Small RNAs in Thy1+ and Thy1− Testis-Derived Cell Populations.

Using high-throughput sequencing, we analyzed the small RNA populations in the testis of day 6 wild-type C57BL/6 mice. Specifically, small RNA libraries were generated from Thy1+ and Thy1− testis cells (referred to as Thy1+ SSC-enriched 1 and 2, and Thy1− somatic cell-enriched 1 and 2, respectively). A small RNA library was also sequenced from an established germ cell culture enriched for SSCs originally derived from Thy1+ testis cells (referred to as SSC-enriched germ cell culture). Sequencing analyses generated high-quality raw reads for each cell population, and these were processed and aligned to the miRBase database (release 12.0; http://www.mirbase.org) for detection of miRs (Fig. S1A). Based on annotations from the miRBase database, we observed that ∼50% of the raw reads from all five sequence libraries (Thy1+ SSC-enriched germ cell 1 and 2, Thy1− somatic cell-enriched 1 and 2, and SSC-enriched germ cell culture) were identified as known mature miRs, suggesting that miRs are the predominant small RNA species in the testis cell population from day 6 mice (Fig. S1A). Within the Thy1+ SSC-enriched libraries, 538 and 539 miRs were detected, and in the Thy1− somatic cell-enriched libraries, 548 and 532 miRs were identified. Sequencing of cultured germ cells enriched for SSCs detected 512 miRs (Fig. S1A and Table S1). In this study, only mature miRs were analyzed, and are therefore representative of transcriptionally active miRs in each library. To profile the overall chromosomal distribution of active miRs detected in the whole testis population, the read counts for each miR from both Thy1+ SSC-enriched and Thy1− somatic cell-enriched sequence libraries were consolidated and the cloning frequency (CF) of individual miRs was determined. The CF is reflective of individual miR abundance within the entire miR library and, using this parameter, only miRs with a CF >0.01% were considered. The miRs identified in the pooled population were mapped to their respective chromosomes using the miRBase database, and the total number of transcriptionally active miRs potentially encoded within each chromosome was enumerated. By comparing the total number of actively expressed miRs encoded within each chromosome with the total number of miRs encoded on each chromosome, a percentage of actively transcribed miRs per chromosome could be plotted (Fig. S1B). Approximately 30–80% of the total miRs encoded within individual chromosomes are actively transcribed, and in the combined Thy1+ and Thy1− sequence libraries (i.e., representative of the whole testis population), mature miRs detected and localized to chromosomes 1, 8, 9, and 19 constituted greater than 70% of the total miRs possibly encoded within these respective chromosomes. Conversely, less than 40% of the total miRs encoded within chromosomes 2, 3, 10, 12, and 18 are actively transcribed.

To better assess the differential expression of miRs between Thy1+ and Thy1− testis populations, the sequencing results for the miR expression level in Thy1+ cells were compared with the differential expression pattern of miRs between Thy1+ SSC-enriched germ cells and Thy1− somatic cell-enriched populations. The differential miR expression pattern between Thy1+ SSC-enriched and Thy1− somatic cells was plotted using the normalized miR read count number in the Thy1+ SSC-enriched library and plotted against the fold change of miR expression (Fig. 1A). This approach identified 139 miRs differentially expressed between Thy1+ SSC-enriched and Thy1− somatic cells, of which 84 miRs (highlighted in red) were considered “up-regulated” and 55 miRs (highlighted in blue) were considered “down-regulated” in the Thy1+ SSC-enriched cell population (padj < 0.05). The differential expression of miRs in the Thy1+ and Thy1− testis cell populations are described below, and additional characterization of miRs expressed in the Thy1+ SSC-enriched and Thy1− somatic cell-enriched libraries can be found in SI Results and Discussion.

Fig. 1.

Fig. 1.

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Detection of miRs differentially expressed between Thy1+ SSC-enriched and Thy1− somatic cell-enriched testis populations. (A) Differential expression of miRs in the Thy1+ SSC-enriched library. A log2 calculation was used to normalize the fold change (i.e., Thy1+/Thy1−) value of the read count for individual miRs (y axis). These values were plotted against the normalized log10 read counts of miRs from the Thy1+ SSC-enriched library (x axis). Statistical significance of miRs differentially expressed between the two libraries was determined using R package software analysis (padj < 0.05, where padj represents P values after adjustment for false discovery rate) (40). Red indicates differentially expressed miRs in the Thy1+ SSC-enriched cell population; blue indicates differentially expressed miRs in the Thy1− somatic cell-enriched population. (B) Chromosomal distribution of actively transcribed miRs in Thy1+ SSC-enriched and Thy1− somatic cell-enriched libraries. miRs identified as significantly expressed in A were mapped according to their respective chromosomal locations, and the sum of the cloning frequencies for these miRs was calculated. The sum of the CF of miRs localized to each chromosome suggests a profile of the chromosomes contributing to the active transcription of miRs in the Thy1+ SSC-enriched and Thy1− somatic cell-enriched libraries. In the cases where mature miRs were found to be possibly encoded in more than one chromosome, the CF for that miR was divided across the number of possible chromosomal locations (e.g., mature miR-let-7f is located on chromosome X or 13; therefore, the CF for miR-let-7f was divided by 2).

Characterization of miR Expression in Testis-Derived Thy1− Somatic Cells.

miRs that are both highly abundant and preferentially enriched in the Thy1− somatic cell-enriched population are shown in Table S2. Included in this profile are members of the let-7 family (i.e., let-7a/b/c/d/e/f), which are broadly expressed, found in multiple somatic cell types, and associated with differentiation in proliferating ES and cancer cells (11, 12). Highly abundant miRs displaying a significant degree of differential expression [i.e., n = 55 miRs with log2 fold change (Thy1+/Thy1−) < −1.68; padj < 0.05] were further mapped according to their individual chromosomal locations, and the sum of the CFs for miRs on each chromosome was determined (Fig. 1B). The CF sum provides a simplified means to examine chromosomes preferentially encoding for actively transcribed miRs. The chromosomal localization of expressed miRs in the Thy1− somatic cell-enriched population was more diverse in its distribution compared with the abundantly expressed miRs of the Thy1+ SSC-enriched population (Fig. 1B). In the Thy1− somatic cell population, highly abundant miRs were predominantly localized to chromosomes X, 2, 9, 13, 15, and 17. The CF sum for miRs associated with the Thy1− somatic cell population was greatest on chromosome 13, which expressed the highly abundant miR-let-7a (Table S2). Examination of chromosome X, which encoded for 32 of the detected miRs in the Thy1− somatic cell population, indicated that the CF sum was ∼6% of the actively transcribed miRs in the Thy1− somatic cell population (Fig. 1B). This is in marked contrast to the CF sum of ∼25% for actively transcribed miRs associated with the Thy1+ SSC-enriched testis cell population (Fig. 1B). Quantitative (q)RT-PCR profiling of selected miR expression further validated that the expression levels of miR-let-7a/c/f, -130, and -202-5p were higher in the Thy1− somatic cells than that of the Thy1+ SSC-enriched population or Thy1+ SSC-enriched cultured germ cells (Fig. S2).

miR Signature in the Thy1+ SSC-Enriched Population.

We hypothesized that miRs with a high cloning frequency and/or those with a high degree of differential expression (i.e., fold change Thy1+/Thy1−) in the SSCs are likely to play an important role in regulating self-renewal and maintaining stem cell homeostasis. Therefore, we examined the 20 most abundant miRs in the Thy1+ SSC-enriched population that also possessed the highest degree of fold change (Thy1+/Thy1−) and observed that these miRs account for ∼26% of the total miR transcripts identified in the Thy1+ testis cell population (Table S3). Interestingly, 12 of these 20 miRs are predominantly expressed on the X chromosome, and miR-465a/-465b/-465c and miR-741/-880/-878/-881 are found as clusters on the X chromosome. The chromosomal distribution of actively transcribed miRs in the Thy1+ SSC-enriched population was again evaluated by determining the CF sum per chromosome (Fig. 1B). Abundant miRs showing significant differential expression within the Thy1+ SSC-enriched population were preferentially localized to chromosomes X, 6, and 11 (Fig. 1B). Among those miRs showing the greatest fold change (Thy1+/Thy1−) in the Thy1+ SSC-enriched population were members of the miR-291 family that have been previously shown to be important in maintaining an undifferentiated state in ES cells and may possibly have a similar role in maintaining the SSC population (Table S4) (13, 14).

Characterization of miR Expression in Thy1+ SSC-Enriched Germ Cell Cultures.

To better understand the role miRs have in regulating SSC self-renewal in vitro, we compared the sequence libraries of freshly isolated Thy1+ SSC-enriched testis cells to that of a germ cell culture established from Thy1+ SSC-enriched testis cells and observed that of the 512 known miRs identified in the cultured SSC-enriched germ cell population, 68 miRs were expressed at highly abundant levels (CF > 0.1%). The expression profile of miRs in cultured SSCs was very similar to the patterns of the two Thy1+ SSC testis cell libraries, with 62 of these 68 miRs observed as highly abundant in both the Thy1+ SSC-enriched libraries and SSC-enriched germ cell culture (Fig. 2 A and B and Table S6). These 62 miRs accounted for ∼95% of total miR transcripts in the Thy1+ SSC-enriched germ cell cultures, with the 10 most abundant miRs expressed in cultured SSCs accounting for 67.6% of all miR molecules, highlighting the possible importance of these miRs in maintaining stem cell activity in vitro (Table 1). In comparison with Thy1+ SSC-enriched libraries, we observed a higher cloning frequency for let-7f, miR-34c, and -21 in the SSC-enriched germ cell cultures (Table 1 and Table S1) which may reflect differences between in vitro and in vivo growth regulation. Results generated from the sequence analysis were further validated using qRT-PCR to assess the relative expression of miRs between Thy1+ SSC-enriched, Thy1− somatic cell-enriched, and SSC-enriched cultured germ cells (Fig. S2). In agreement with the sequencing results, the expression of miR-21, -146a, -378, -880-182, -183, -465a/b/c-3p, and -465c-5p was higher in Thy1+ SSC-enriched and SSC-enriched cultured germ cells compared with the Thy1− somatic cell-enriched population (Fig. S2). However, the relative expression of miR-21, -146, -378, -182, -183, -465a-3p, -465b-3p, -465c-3p, and -465c-5p was dramatically higher in SSC-enriched cultured germ cells compared with freshly isolated Thy1+ SSC-enriched testis cells. This difference in miR expression level can likely be attributed to the special characteristics for propagation and expansion of germ cells in vitro.

Fig. 2.

Fig. 2.

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Thy1+ SSC-enriched germ cell cultures and Thy1+ SSC-enriched testis cells show similarity in miR expression. (A) Comparison of relative miR abundance in sequence libraries derived from cultured germ cells from Thy1+ SSC-enriched testis cells and freshly isolated Thy1+ SSC-enriched testis cells (red, Thy1+ SSC-enriched library 1; blue, Thy1+ SSC-enriched library 2). The CF for individual miRs from the two Thy1+ SSC-enriched testis libraries was plotted against the CF of corresponding miRs in the germ cell culture library. (B) Venn diagram illustrating overlap of miR expression between freshly isolated Thy1+ SSC-enriched germ cells and cultured germ cells. Only miR expression in which the CF >0.1% is included.

Table 1.

Top 10 most abundantly expressed miRs in SSC-enriched germ cell cultures

ID Read count Cloning frequency # ETS binding sites in 5kb
mmu-miR-34c 1001090 18.36 1
mmu-let-7f 632879 11.61 2 ; 6
mmu-miR-21 607738 11.14 3
mmu-let-7a 448696 8.23 2 ; 1
mmu-let-7c 360981 6.62 3
mmu-miR-143 193927 3.56 1
mmu-miR-199a-3p 123617 2.27 2 ; 5
mmu-miR-199b 123617 2.27 10
mmu-miR-465a-3p 97726 1.79
mmu-miR-465b-3p 97726 1.79
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The cloning frequency was used to rank the most abundantly expressed mature miRs in the SSC-enriched germ cell culture library. The cloning frequency was calculated as the percent abundance of the read counts for individual miRs over the sum of all read counts in the SSC-enriched germ cell culture library. Promoter software (TraFac) analysis found that 13 of the 20 miRs present possessed one or more putative ETS binding sites within the enhancer region of chromosome encoding for respective miRs. Profiling of ETS binding sites was performed to incorporate 5kb upstream of the mature miR sequence. In some cases, miRs are encoded on more than one chromosome and a semi-colon (;) is used to separate the number of putative ETS binding sites on each chromosome.

ETV5 Directly Regulates miR-21 Expression in Mouse SSCs.

The glial cell line-derived neurotrophic factor (GDNF) pathway plays a critical role in regulating the self-renewal and maintenance of the SSC population, both in vivo and in vitro (15–17). Important downstream effectors of GDNF signaling include ETV5, BCL6B, POU3f1, and LHX1, all of which are critical to maintaining stemness (18, 19). Similarly, ETV5 may control miR expression as one means of regulating SSC homeostasis. Using a comparative genomic transcription factor binding site analysis program [TraFac (20)], we observed more than 260 miRs that possessed ETS binding sites that are highly conserved between human and mouse, with 8 of the 10 most abundant miRs expressed in the SSC-enriched germ cell cultures possessing multiple ETS (E-twenty six) binding sites (Table 1). This observation suggests a potential role for GDNF-ETV5 signaling in regulating these and other miRs. In particular, miR-21 has been shown to be an important antiapoptotic factor that greatly enhances tumor progression, and additional studies have shown that miR-21 can prevent apoptosis in mouse periovulatory granulosa cells (21, 22). The multiple conserved ETS-binding motifs identified on the miR-21 promoter region (Fig. 3A and Table 1), along with the high levels of miR-21 expression observed in SSC-enriched germ cell cultures (Fig. S2), strongly suggest an important in vitro function. To verify possible ETV5 regulation of miR-21, chromatin immunoprecipitation (ChIP) using mouse germ cell cultures was performed. PCR amplification using primers flanking two of the predicted ETS-binding motifs each produced a band for DNA coprecipitated with ETV5 and ETV5 antibody but not in the isotype antibody controls (Fig. 3B). These results indicate that ETV5 binds to the miR-21 enhancer. To further investigate the putative regulation of miR-21 expression by ETV5, germ cell cultures were infected with a lentivirus vector expressing mouse ETV5 cDNA under the control of the EF1α promoter. In transduced germ cells, Etv5 expression was significantly elevated to greater than twofold compared with controls, and this was accompanied by a significant, 1.78-fold, increase in the expression of miR-21 (Fig. 3C). These results strongly suggest that ETV5 has a role in regulating miR-21 expression in SSC-enriched germ cells. We also observed putative STAT3 and POU3f1 binding sites within the miR-21 enhancer region (Fig. 3A). Previous reports have demonstrated that STAT3 promotes differentiation of SSCs (23), whereas POU3f1, a GDNF-regulated transcription factor, is shown to be critical in regulating the SSC population in vitro (24), suggesting that several transcription factors may be involved in the regulation of miR-21 expression.

Fig. 3.

Fig. 3.

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ETV5 directly regulates miR-21 expression in Thy1+ SSC-enriched germ cell cultures. (A) ETV5, as well as POU3f1 and STAT3, possess multiple binding sites within the miR-21 enhancer region. (B) ChIP of ETV5 binding to the miR-21 enhancer. ChIP #1 and ChIP #2 represent replicate experiments for two enhancer regions located 236 and 330 bp upstream of the miR-21 transcriptional start site. (C) Increased expression of ETV5 from 0.99 ± 0.001 to 2.63 ± 0.36 following transduction of germ cell cultures with a lentiviral (pWPI) construct constitutively expressing ETV5 cDNA. Compared with empty vector controls (1.00 ± 0.02), the overexpression of ETV5 resulted in a significant increase in miR-21 expression (1.78 ± 0.27). The asterisk denotes significance where P < 0.05 (mean ± SEM, n = 5).

Transient Inhibition of miR-21 Increases Germ Cell Apoptosis and Affects Stem Cell Activity.

Because the above findings suggested that ETV5 in part regulates miR-21, and miR-21 expression accounts for ∼11% of the miR molecules in SSC-enriched cultured germ cells, the biological function of miR-21 in regulating SSC homeostasis in vitro was investigated. SSC-enriched germ cell cultures were transiently transfected with a prevalidated miR-21 inhibitor (anti–miR-21) oligonucleotide (oligos), and its effect on germ cell expansion and stem cell activity was evaluated. The doubling time for mouse SSCs is ∼5.6 d (25); for that reason, following exposure to anti–miR-21 oligos or nontargeting control oligos, germ cells were plated onto new feeders and cultured for an additional 7 d posttransfection before being collected and counted. The transient inhibition of miR-21 significantly decreased the number of germ cells in vitro compared with nontargeting controls (0.98 ± 0.14 × 105 cells vs. 1.56 ± 0.21 × 105 cells, respectively; Fig. 4A). The role of miR-21 as an antiapoptotic factor has been reported for several systems (21, 22), and therefore Annexin V staining was used to assess the level of apoptosis caused by the inhibition of miR-21. At 20 h posttransfection, the apoptotic index for germ cells treated with Lipofectamine alone or nontargeting control oligos was 3.87 ± 1.06% and 3.67 ± 0.34%, respectively (Fig. 4B). In comparison, inhibition of miR-21 significantly increased apoptosis in germ cell cultures to 7.07 ± 0.57% (Fig. 4B).

Fig. 4.

Fig. 4.

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Biological importance of miR-21 in Thy1+ SSC-enriched germ cell cultures. (A) At the start of the experiment, 1.0 × 105 germ cells in culture were transiently transfected with anti–miR-21 oligonucleotides or nontargeting control oligo. After 7 d of being maintained in culture posttransfection, the total number of germ cells in each treatment was counted. The number of anti–miR-21-treated germ cells was significantly reduced to 0.98 × 105 ± 0.14 cells compared with germ cells treated with nontargeting control oligos (1.57 × 105 ± 0.21 cells). (B) Transient inhibition of miR-21 promotes apoptosis in germ cell cultures. Following 20 h posttransfection with anti–miR-21 oligos, nontargeting control oligos, or Lipofectamine alone, germ cell cultures were collected, washed, and incubated with Annexin V antibody and 7-aminoactinomycin D reagent. The apoptosis index was determined by comparing the number of apoptotic germ cells with the total number of germ cells. Compared with germ cells treated with Lipofectamine alone (3.87 ± 1.06%) or nontargeting control oligos (3.67 ± 0.34%), the number of germ cells undergoing apoptosis was significantly increased by treatment with anti–miR-21 oligos (7.07 ± 0.57%). (C) Inhibition of miR-21 activity decreases the in vivo colony formation ability of treated germ cells. The average number of colonies formed in recipient testes from 105 cells transplanted to recipient testes was determined for germ cell cultures treated with nontargeting control oligos or anti–miR-21 oligos. Treated cells were maintained for 7 d posttransfection before being transplanted into the testes of recipient mice. Two months after transplantation, the number of donor-derived colonies was counted. Inhibition of miR-21 activity caused the number of donor-derived colonies to significantly decrease from 178 ± 20.9 colonies for control nontargeting oligo-treated germ cells to 108 ± 23.2 colonies for anti–miR-21-treated germ cells. All data are representative of three independent replicate cultures (mean ± SEM), and for transplantation studies this resulted in 16 testes (n = 8 mice) per treatment. The asterisk denotes significant differences between treatment means using Student’s t test (P < 0.05).

The in vitro impact of miR-21 inhibition on SSC survival and proliferation was evaluated with an in vivo transplantation assay. Transplantation of donor germ cells into the testes of busulfan-treated recipient mice is the only functional assay to quantify the number of SSCs in any cell population, and provides an unequivocal means to determine whether a reduction in the number of SSCs occurred as a result of varying treatments to the in vitro SSC-enriched germ cell culture (19, 26). Following 7 d in culture posttransfection with anti–miR-21 oligos or nontargeting control oligos, germ cells were transplanted into recipient testis. The transient inhibition of miR-21 decreased the number of donor colonies formed from 178 ± 20.9 per 105 germ cells in the nontargeting oligo control group to 108 ± 23.2 per 105 germ cells in anti–miR-21-treated germ cell cultures (Fig. 4C; P < 0.05). These results demonstrate a requirement for miR-21 expression and function in the in vitro maintenance of the mouse SSC population, and suggest that one mechanism of miR-21 effect is through the regulation of apoptosis.

Discussion

Using high-throughput sequencing, we identified an miR signature that was common to Thy1+ SSC-enriched testis cells and germ cell cultures, and a high degree of similarity was observed between the two libraries. Notably, some cloning frequencies of SSC-associated miRs were dramatically higher in SSC-enriched germ cell cultures compared with Thy1+ testis cells. The higher miR expression in vitro may reflect a more active role of miRs in regulating SSC self-renewal and/or early differentiation steps in vitro. However, an important observation is that in the absence of the physiological in vivo niche influences, the cultured SSCs and undifferentiated type A spermatogonia maintain a remarkably similar miR profile to the Thy1+ SSC-enriched testis cells isolated directly from the testes. These similarities in miR expression suggest that regulation of SSC self-renewal and the undifferentiated type A spermatogonia are primarily regulated by the germ cell program rather than by external cues and may require mostly a permissive environment, such as the in vivo niche and an appropriate culture milieu. A similar conclusion regarding germ cell program dominance versus environmental cues is suggested by the finding that rat spermatogenesis is supported in vivo by mouse Sertoli cells at the exact timing and organizational arrangement of the rat and not mouse (27). Therefore, the mouse Sertoli cell environment cannot change the length of the rat cycle (52 d) in the seminiferous epithelium to that of the mouse cycle [35 d (27, 28)]. These findings support the use of cultured germ cells, enriched for SSCs, as a powerful model to examine the expression and function of miRs and genes in this germ cell population, which contains almost exclusively undifferentiated spermatogonia that are uniform in morphological characteristics and a portion of which may convert to SSCs under certain conditions (29, 30).

The transcription factor ETV5 is one of the downstream targets of GDNF signaling, and is essential for SSC self-renewal (18, 31–33). In the testis, ETV5 is expressed in Sertoli cells and in the SSC-enriched cell populations (18, 31, 34). In Etv5−/− mice, Sertoli cell function is compromised, resulting in the complete loss of adult germ cells following the first wave of spermatogenesis (31). We examined putative ETS binding sites within the enhancer/promoter regions of highly abundant miRs and found that 8 of 10 of the most abundant miRs in the Thy1+ SSC-enriched population contained ETS binding sites in their enhancer regions, including miR-21. We observed that ETV5 is capable of regulating miR-21 expression and, because ETV5 is a known downstream effecter of GDNF signaling, it is likely that GDNF has a role in the regulation of miRs. This is supported by a report demonstrating that miR-21 expression is induced by GDNF in BE(2)-C neuronal cells (35). Importantly, we observe that inhibition of miR-21 increased apoptosis in SSC-enriched germ cell cultures, suggesting it has a role in maintaining SSC survival. Because apoptosis is a major regulator of spermatogenesis (36), miR-21 may play a pivotal role in the early stages of spermatogenesis by regulating apoptosis in the undifferentiated spermatogonial cell population, including SSCs.

The stringency of the profiling used to analyze results from high-throughput sequencing for both the Thy1+ SSC-enriched cell population and SSC-enriched germ cell cultures strongly suggests that the in vitro culture system provides an ideal model to functionally examine the role of miRs in regulating SSC fate decisions. Importantly, significant advances in understanding the cellular and molecular mechanisms regulating SSC self-renewal have been made using culture systems that are able to support proliferation of SSC-enriched germ cells in vitro (25), and a robust transplantation assay that is capable of determining the biological activity of donor-derived SSCs in the seminiferous tubules of recipient testis provides the ultimate test of various treatment effects (37, 38). The combination of these two systems provides a method of analyzing the biological activity of SSCs by unequivocally measuring SSC number, and in this report has provided the foundation to demonstrate the validity of using germ cell cultures to examine the function of miRs in our profile and the means to demonstrate the specific effect of miR-21 on SSC self-renewal.

Materials and Methods

Isolation of SSC-Enriched Germ Cells.

Testes of day 6 postnatal mice were harvested from two strains: C57BL/6 mice (Jackson Laboratory stock no. 000664) or Escherichia coli β-galactosidase (LacZ)-expressing mice (B6.129S7-Gtrosa26, designated ROSA; Jackson Laboratory stock no. 002073). miR profiling studies were conducted using the testes derived from C57BL/6 mouse pups, whereas the testes of ROSA mouse pups, expressing the LacZ gene, were used to derive germ cell cultures for transplantation assays (see below). Digestion of testis and isolation of Thy1+ and Thy1− testis cell populations were carried out as previously described (26, 39). Thy1+ SSC-enriched germ cells and Thy1− somatic cell-enriched testis cells were further processed for isolation of small RNAs or germ cell culture. Establishment of germ cell cultures was carried out using Thy1+ SSC-enriched germ cells as previously described (26, 39). A detailed method of maintaining SSC-enriched germ cell cultures can be found in SI Materials and Methods. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

Small RNA Library Construction and Sequencing.

Small RNA libraries from Thy1+ SSC-enriched and Thy1− somatic cell-enriched testis cell populations or germ cell cultures were isolated using TRIzol (Invitrogen) and purified using the Illumina manufacturer protocols “Preparing Samples for Analysis of Small RNA” and “Preparing Samples for Small RNA Sequencing Using the Alternative v1.5 Protocol.” The final libraries of small RNAs for each sample were gel-purified and measured using an Agilent Bioanalyzer. Sequencing of small RNA libraries was conducted in the Penn Microarray Facility using an Illumina Genome Analyzer. Bioinformatics analysis and quantitative real-time PCR validation are described in SI Materials and Methods.

Additional Methods.

Detailed descriptions of methods for functional analysis of miR-21 in germ cell cultures are available in SI Materials and Methods.

Supplementary Material

Supporting Information
supp_108_31_12740__index.html (1.4KB, html)

Acknowledgments

We thank Drs. R. Behringer and M. Kotlikoff for critical evaluation of the manuscript, and C. Freeman and R. Naroznowski for assistance with animal maintenance. This study was supported by National Institute of Child Health and Human Development Grant HD 052728 (to R.L.B.) and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation (R.L.B.).

Footnotes

The authors declare no conflict of interest.

Data deposition: MicroRNA profiling data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE29613).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1109987108/-/DCSupplemental.

References

  • 1.Oatley JM, Brinster RL. Regulation of spermatogonial stem cell self-renewal in mammals. Annu Rev Cell Dev Biol. 2008;24:263–286. doi: 10.1146/annurev.cellbio.24.110707.175355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Brinster RL. Male germline stem cells: From mice to men. Science. 2007;316:404–405. doi: 10.1126/science.1137741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.González-González E, López-Casas PP, Del Mazo J. Gene silencing by RNAi in mouse Sertoli cells. Reprod Biol Endocrinol. 2008;6:29. doi: 10.1186/1477-7827-6-29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Kotaja N, et al. The chromatoid body of male germ cells: Similarity with processing bodies and presence of Dicer and microRNA pathway components. Proc Natl Acad Sci USA. 2006;103:2647–2652. doi: 10.1073/pnas.0509333103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Hayashi K, et al. MicroRNA biogenesis is required for mouse primordial germ cell development and spermatogenesis. PLoS One. 2008;3:e1738. doi: 10.1371/journal.pone.0001738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Papaioannou MD, Nef S. microRNAs in the testis: Building up male fertility. J Androl. 2010;31:26–33. doi: 10.2164/jandrol.109.008128. [DOI] [PubMed] [Google Scholar]
  • 7.Buchold GM, et al. Analysis of microRNA expression in the prepubertal testis. PLoS One. 2010;5:e15317. doi: 10.1371/journal.pone.0015317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Jung YH, Gupta MK, Shin JY, Uhm SJ, Lee HT. MicroRNA signature in testes-derived male germ-line stem cells. Mol Hum Reprod. 2010;16:804–810. doi: 10.1093/molehr/gaq058. [DOI] [PubMed] [Google Scholar]
  • 9.Ro S, Park C, Sanders KM, McCarrey JR, Yan W. Cloning and expression profiling of testis-expressed microRNAs. Dev Biol. 2007;311:592–602. doi: 10.1016/j.ydbio.2007.09.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Yan N, et al. A microarray for microRNA profiling in mouse testis tissues. Reproduction. 2007;134:73–79. doi: 10.1530/REP-07-0056. [DOI] [PubMed] [Google Scholar]
  • 11.Büssing I, Slack FJ, Grosshans H. let-7 microRNAs in development, stem cells and cancer. Trends Mol Med. 2008;14:400–409. doi: 10.1016/j.molmed.2008.07.001. [DOI] [PubMed] [Google Scholar]
  • 12.Roush S, Slack FJ. The let-7 family of microRNAs. Trends Cell Biol. 2008;18:505–516. doi: 10.1016/j.tcb.2008.07.007. [DOI] [PubMed] [Google Scholar]
  • 13.Lichner Z, et al. The miR-290-295 cluster promotes pluripotency maintenance by regulating cell cycle phase distribution in mouse embryonic stem cells. Differentiation. 2011;81:11–24. doi: 10.1016/j.diff.2010.08.002. [DOI] [PubMed] [Google Scholar]
  • 14.Zovoilis A, et al. Embryonic stem cell-related miRNAs are involved in differentiation of pluripotent cells originating from the germ line. Mol Hum Reprod. 2010;16:793–803. doi: 10.1093/molehr/gaq053. [DOI] [PubMed] [Google Scholar]
  • 15.Kubota H, Avarbock MR, Brinster RL. Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells. Biol Reprod. 2004;71:722–731. doi: 10.1095/biolreprod.104.029207. [DOI] [PubMed] [Google Scholar]
  • 16.Meng X, et al. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science. 2000;287:1489–1493. doi: 10.1126/science.287.5457.1489. [DOI] [PubMed] [Google Scholar]
  • 17.Ryu B-Y, Kubota H, Avarbock MR, Brinster RL. Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat. Proc Natl Acad Sci USA. 2005;102:14302–14307. doi: 10.1073/pnas.0506970102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Oatley JM, Avarbock MR, Brinster RL. Glial cell line-derived neurotrophic factor regulation of genes essential for self-renewal of mouse spermatogonial stem cells is dependent on Src family kinase signaling. J Biol Chem. 2007;282:25842–25851. doi: 10.1074/jbc.M703474200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Oatley JM, Avarbock MR, Telaranta AI, Fearon DT, Brinster RL. Identifying genes important for spermatogonial stem cell self-renewal and survival. Proc Natl Acad Sci USA. 2006;103:9524–9529. doi: 10.1073/pnas.0603332103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Jegga AG, et al. Detection and visualization of compositionally similar cis-regulatory element clusters in orthologous and coordinately controlled genes. Genome Res. 2002;12:1408–1417. doi: 10.1101/gr.255002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Carletti MZ, Fiedler SD, Christenson LK. MicroRNA 21 blocks apoptosis in mouse periovulatory granulosa cells. Biol Reprod. 2010;83:286–295. doi: 10.1095/biolreprod.109.081448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Krichevsky AM, Gabriely G. miR-21: A small multi-faceted RNA. J Cell Mol Med. 2009;13:39–53. doi: 10.1111/j.1582-4934.2008.00556.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Oatley JM, Kaucher AV, Avarbock MR, Brinster RL. Regulation of mouse spermatogonial stem cell differentiation by STAT3 signaling. Biol Reprod. 2010;83:427–433. doi: 10.1095/biolreprod.109.083352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Wu X, et al. The POU domain transcription factor POU3F1 is an important intrinsic regulator of GDNF-induced survival and self-renewal of mouse spermatogonial stem cells. Biol Reprod. 2010;82:1103–1111. doi: 10.1095/biolreprod.109.083097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Kubota H, Avarbock MR, Brinster RL. Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc Natl Acad Sci USA. 2004;101:16489–16494. doi: 10.1073/pnas.0407063101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Oatley JM, Brinster RL. Spermatogonial stem cells. Methods Enzymol. 2006;419:259–282. doi: 10.1016/S0076-6879(06)19011-4. [DOI] [PubMed] [Google Scholar]
  • 27.França LR, Ogawa T, Avarbock MR, Brinster RL, Russell LD. Germ cell genotype controls cell cycle during spermatogenesis in the rat. Biol Reprod. 1998;59:1371–1377. doi: 10.1095/biolreprod59.6.1371. [DOI] [PubMed] [Google Scholar]
  • 28.Russell LD. The Sertoli Cell, eds Russell LD, Griswold MD. Clearwater, FL: Cache River; 1993. pp. 365–390. [Google Scholar]
  • 29.Kubota H, Avarbock MR, Brinster RL. Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells. Proc Natl Acad Sci USA. 2003;100:6487–6492. doi: 10.1073/pnas.0631767100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Nakagawa T, Sharma M, Nabeshima Y-i, Braun RE, Yoshida S. Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment. Science. 2010;328:62–67. doi: 10.1126/science.1182868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Chen C, et al. ERM is required for transcriptional control of the spermatogonial stem cell niche. Nature. 2005;436:1030–1034. doi: 10.1038/nature03894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Lu BC, et al. Etv4 and Etv5 are required downstream of GDNF and Ret for kidney branching morphogenesis. Nat Genet. 2009;41:1295–1302. doi: 10.1038/ng.476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Tyagi G, et al. Loss of Etv5 decreases proliferation and RET levels in neonatal mouse testicular germ cells and causes an abnormal first wave of spermatogenesis. Biol Reprod. 2009;81:258–266. doi: 10.1095/biolreprod.108.075200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Yoon K-A, Chae Y-M, Cho J-Y. FGF2 stimulates SDF-1 expression through the Erm transcription factor in Sertoli cells. J Cell Physiol. 2009;220:245–256. doi: 10.1002/jcp.21759. [DOI] [PubMed] [Google Scholar]
  • 35.Yoong LF, Wan G, Too H-P. Glial cell-line derived neurotrophic factor and neurturin regulate the expressions of distinct miRNA precursors through the activation of GFRα2. J Neurochem. 2006;98:1149–1158. doi: 10.1111/j.1471-4159.2006.03959.x. [DOI] [PubMed] [Google Scholar]
  • 36.Shaha C, Tripathi R, Mishra DP. Male germ cell apoptosis: Regulation and biology. Philos Trans R Soc Lond B Biol Sci. 2010;365:1501–1515. doi: 10.1098/rstb.2009.0124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Brinster RL, Avarbock MR. Germline transmission of donor haplotype following spermatogonial transplantation. Proc Natl Acad Sci USA. 1994;91:11303–11307. doi: 10.1073/pnas.91.24.11303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci USA. 1994;91:11298–11302. doi: 10.1073/pnas.91.24.11298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Kubota H, Brinster RL. Culture of rodent spermatogonial stem cells, male germline stem cells of the postnatal animal. Methods Cell Biol. 2008;86:59–84. doi: 10.1016/S0091-679X(08)00004-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Anders S, Huber W. Differential expression analysis for sequence count data. Genome Biol. 2010;11:R106. doi: 10.1186/gb-2010-11-10-r106. [DOI] [PMC free article] [PubMed] [Google Scholar]

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