296-3p, miR-296-5p, miR-483) and lower (P,0.001) expression of imprinted and maternally expressed miRNAs (miR-127, miR-127- 5p) than those observed with somatic cells (Figure 1B). Similar expression patterns of imprinted miRNAs were also observed in testicular tissue although the deviation in miRNA levels, from those of somatic cells, was less prominent than those observed with sperm, possibly due to the presence of both somatic and germ cells in the testicular tissue. Similar to sperm, the expression of imprinted and paternally expressed miRNAs (miR-296-3p, miR- 296-5p, miR-483) were consistently higher (P,0.001) while those of imprinted and maternally expressed miRNA (miR-127, miR- 127-5p) were consistently lower (P,0.001) in GS cells than in control ES cells (Figure 2). These data suggest that the expression pattern of imprinted miRNA in mouse GS cells is likely androgenetic and therefore, may possibly form an epigenetic signature on testes-derived male germ-line stem cells. To further confirm this, we performed DNA methylation analysis of ICRs that control the expression of all imprinted miRNAs in respective gene clusters [16,20,30,31,32,36]. We found that, 0.0, 99.6 and 95.0% of CpGs in Gnas-Nespas DMR, Igf2-H19 ICR and Dlk1-Dio3 IG-DMR, respectively were methylated in GS cells and were similar to 0.0, 99.3 and 90.0% methylation observed for respective sites in sperm (Figure 3). Thus, genomic imprinting of imprinted miRNAs is androgenetic in GS cells. Imprinted miRNA Signature in Male GS Cells On the other hand, maGS cells resembled ES cells for the DNA methylation of imprinted miRNA genes as has also been reported earlier for several other imprinted genes [6,9]. However, expression pattern of the imprinted miRNAs in maGS cells was intermediate between GS and ES cells and did not completely resemble the ES cell-like expression pattern (Figure 2). Similar results were also observed for the expression pattern of mRNA transcripts encoded by these imprinted gene clusters (Supplemen- tary Figure S3) except for the Igf2-H19 as we had shown previously [10,11,29]. These data confirm the conversion of androgenetic GS cells to multipotent maGS cells [1,6,7,11] but also suggest that the acquisition of ES cell-like characteristics in maGS cells was likely incomplete or partial [6,7,11]. Indeed, previous studies have also shown that genomic imprinting in maGS cells stand in between GS and ES cells [6,7]. The maGS cells also expressed higher level of germ cell markers characteristic of primordial germ cells and spermatogonia compared with ES cells [28], although the expression pattern of the ES cell-specific miR-290 and miR-302 cluster of miRNA in maGS cells resembled that of the ES cells [27]. Thus, in case of maGS cells, which show discrepancy between DNA methylation and transcription pattern of imprinted miRNAs, it appears that expression of individual imprinted miRNAs may be leaky [37,38] or may be modified by in vitro manipulation, physiological state, level of differentiation, in vitro Figure 3. DNA methylation status of imprinting control regions that control the expression of imprinted miRNAs at Gnas-Nespas (Gnas-Nespas DMR), Dlk1-Dio3 (Dlk-Dio3 IG-DMR) and Igf2-H19 (Igf2-H19 ICR) loci in male germ-line (GS) and multipotent adult germ- line (maGS) stem cells. Sperm, oocyte and embryonic stem (ES) cells were used as control for comparison. Each line represents a separate clone and each CpG site is shown by its sequence position in the GenBank sequence. Each circle indicates individual CpG residues within the areas amplified. Open and closed circles indicate unmethylated and methylated CpGs, respectively. doi:10.1371/journal.pone.0022481.g003 PLoS ONE | www.plosone.org 5 July 2011 | Volume 6 | Issue 7 | e22481