Identification and Characterization of Germ Cell Genes Expressed in the F9 Testicular Teratoma Stem Cell Line

Abstract

The F9 cell line, which was derived from a mouse testicular teratoma that originated from pluripotent germ cells, has been used as a model for differentiation. However, it is largely unknown whether F9 cells possess the characteristics of male germ cells. In the present study, we investigated spermatogenic stage- and cell type-specific gene expression in F9 cells. Analysis of previous microarray data showed that a large number of stage-regulated germ cell genes are expressed in F9 cells. Specifically, genes that are prominently expressed in spermatogonia and have transcriptional regulatory functions appear to be enriched in F9 cells. Our in silico and in vitro analyses identified several germ cell-specific or -predominant genes that are expressed in F9 cells. Among them, strong promoter activities were observed in the regions upstream of the spermatogonial genes, Dmrt1 (doublesex and mab-3 related transcription factor 1), Stra8 (stimulated by retinoic acid gene 8) and Tex13 (testis expressed gene 13), in F9 cells. A detailed analysis of the Tex13 promoter allowed us to identify an enhancer and a region that is implicated in germ cell-specificity. We also found that Tex13 expression is regulated by DNA methylation. Finally, analysis of GFP (green fluorescent protein) TEX13 localization revealed that the protein distributes heterogeneously in the cytoplasm and nucleus, suggesting that TEX13 shuttles between these two compartments. Taken together, our results demonstrate that F9 cells express numerous spermatogonial genes and could be used for transcriptional studies focusing on such genes. As an example of this, we use F9 cells to provide comprehensive expressional information about Tex13, and report that this gene appears to encode a germ cell-specific protein that functions in the nucleus during early spermatogenesis.

TEX13 is a novel male germ cell‐specific nuclear protein potentially involved in transcriptional repression

“…We previously reported that GFP-TEX13 can shuttle between the nucleus and cytoplasm in NIH 3T3 and F9 cells [19], and our present immunostaining results confirmed the nuclear localization of TEX13 in vivo (Fig. 2).…”

“…B). This contrasts with the mRNA expression of Tex13 , which reportedly begins at postnatal day 8, corresponding to the appearance of spermatogonia . Because of meiotic sex chromosome inactivation (MSCI), a majority of sex‐linked genes are transcriptionally suppressed from primary meiotic germ cells .…”

Regulation of Translocator Protein 18 kDa (TSPO) Expression in Rat and Human Male Germ Cells

“…Similar effects were observed in F9 mouse embryonal carcinoma cells, a cell line issued from a mouse testicular teratoma, which shares a number of pluripotency markers with embryonic stem cells as well as gonocytes [ 15 , 16 , 29 , 30 ]. In F9 cells, which express genes from both the somatic and germline lineages, RA treatment can induce the expression of markers of somatic (e.g., collagen IV) and germ cell (e.g., Stra8) fates, depending of the culture conditions and downstream pathway activated [ 16 , 30 ]. All TGCTs, including embryonal carcinoma, are believed to derive from a common precursor, the carcinoma in situ, itself resulting from the failed differentiation of gonocytes [ 18 ].…”

Profiling of testis-specific long noncoding RNAs in mice

“…F9 embryonal carcinoma cells are stem cells of testicular carcinoma, and have the ability to differentiate into three embryonic layers [ 33 ]. Although F9 cells are considered to be similar to embryonic cells, the former can transcribe germ cell-specific genes and possess the characteristics of male germ cells [ 34 ]. Immortalized GC-1 cells exhibit the characteristics of a stage between type B spermatogonia and primary spermatocytes [ 35 ], while GC-2 cells were derived from cells arrested at a premeiotic stage and exhibit the characteristics of spermatocytes [ 36 ].…”