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

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.

Fig. 1.

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 (p_adj < 0.05, where p_adj 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).

Fig. 2.

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.

Fig. 3.

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).

Fig. 4.

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).