Abstract
BackgroundTestis-derived male germ-line stem (GS) cells, the in vitro counterpart of spermatogonial stem cells (SSC), can acquire multipotency under appropriate culture conditions to become multipotent adult germ-line stem (maGS) cells, which upon testicular transplantation, produce teratoma instead of initiating spermatogenesis. Consequently, a molecular marker that can distinguish GS cells from maGS cells would be of potential value in both clinical and experimental research settings.Methods and FindingsUsing mouse as a model system, here we show that, similar to sperm, 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 embryonic stem (ES) cells. DNA methylation analyses of imprinting control regions (ICR), that control the expression of all imprinted miRNAs in respective gene clusters (Gnas-Nespas DMR, Igf2-H19 ICR and Dlk1-Dio3 IG-DMR), confirmed that imprinted miRNAs were androgenetic in GS cells. On the other hand, DNA methylation of imprinted miRNA genes in maGS cells resembled those of ES cells but the expression pattern of the imprinted miRNAs was intermediate between those of GS and ES cells. The expression of imprinted miRNAs in GS and maGS cells were also altered during their in vitro differentiation and varied both with the differentiation stage and the miRNA.ConclusionsOur data suggest that GS cells have androgenetic DNA methylation and expression of imprinted miRNAs which changes to ES cell-like pattern upon their conversion to maGS cells. Differential genomic imprinting of imprinted miRNAs may thus, serve as epigenetic miRNA signature or molecular marker to distinguish GS cells from maGS cells.
Highlights
Germ-line stem (GS) cells, the in vitro counterpart of spermatogonial stem cells (SSC) in the testis, can self-renew in vitro for more than two years and, when transplanted into the seminiferous tubules of an infertile male mouse, can establish donor-derived spermatogenesis to transmit the donor haplotype to progeny [1,2,3,4]
Our data suggest that germ-line stem (GS) cells have androgenetic DNA methylation and expression of imprinted miRNAs which changes to embryonic stem (ES) cell-like pattern upon their conversion to multipotent adult germ-line stem (maGS) cells
We recently showed that GS and maGS cells show differential expression of Let-7 and miR-294 miRNAs which may serve as miRNA signature to distinguish GS cells from maGS or ES cells [29]
Summary
Germ-line stem (GS) cells, the in vitro counterpart of spermatogonial stem cells (SSC) in the testis, can self-renew in vitro for more than two years and, when transplanted into the seminiferous tubules of an infertile male mouse, can establish donor-derived spermatogenesis to transmit the donor haplotype to progeny [1,2,3,4]. At any particular time point, in vitro cultured GS cells may contain some contaminating mGS or maGS cells which may produce teratoma instead of initiating spermatogenesis upon their transplantation into recipient testis [5,6,11]. A molecular marker that can distinguish GS cells from mGS or maGS cells would be of potential value in both clinical and experimental research settings. Testis-derived male germ-line stem (GS) cells, the in vitro counterpart of spermatogonial stem cells (SSC), can acquire multipotency under appropriate culture conditions to become multipotent adult germ-line stem (maGS) cells, which upon testicular transplantation, produce teratoma instead of initiating spermatogenesis. A molecular marker that can distinguish GS cells from maGS cells would be of potential value in both clinical and experimental research settings
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