The ter mutation responsible for germ cell deficiency but not testicular nor ovarian teratocarcinogenesis in ter / ter congenic mice

The ter (teratoma) gene causes germ cell deficiency and a high incidence of congenital testicular teratomas derived from primordial germ cells in 129/Sv- ter strain mice. Ovarian teratomas in LTXBJ mice originate from ovarian parthenotes. In order to study the function of the ter gene in germ cell development and teratocarcinogenesis, we examined the influence of a foreign genetic background on the ter action by introducing the ter gene of 129/Sv- ter strain mice into C57BL/6J, LTXBJ and C3H/HeJ genetic backgrounds by the backcross method and by thus establishing B6- ter , LTXBJ- ter and C3H- ter ter congenic strains, respectively. Histological analysis showed that germ cell deficiency occurred in both sexes of the ter mutants, through the fetal stages to adulthood, but that congenital testicular teratocarcinogenesis did not occur after the fifth backcross generation. The ter/ter gonads were smaller than normal (+/+ or +/ ter ). Experimental testicular teratomas never developed from intratesticular grafts of B6- ter genital ridges. LTXBJ- ter/ter females had no ovarian teratomas. It is concluded that the ter gene is solely responsible for germ cell deficiency, but not testicular teratocarcinogenesis, in ter congenic strains having background genes other than 129/Sv- ter and that the ter gene is not involved in ovarian teratocarcinogenesis.     

Premeiotic Origin of Teratomas: Is Meiosis Required for Differentiation into Mature Tissues?

By virtue of meiotic cell division, primordial germ cells with heterozygous alleles develop into postmeiotic germ cells with homozygous alleles. Female and male germ cells may develop tumors - so-called teratomas - with a unique co-existence of a variety of histological elements from all three embryonic germ layers. In particular, mature teratomas consist exclusively of developmentally mature tissues whereas immature teratomas contain variable amounts of mature and immature tissues. In this study, we report genetic analysis of individual tissue components from mature and immature teratomas. The majority of mature teratomas showed consistent and concordant homozygous alleles in all selectively procured tissue components. In a small subset of mature teratomas, we observed discordant homozygous alleles. In contrast, immature teratomatous tissue revealed a heterozygous genotype. Remarkably, mature tissue components within immature teratoma revealed homozygosity. The findings suggest that immature teratomas and at least a subset of mature teratomas may originate from premeiotic cells, and implicate that meiosis may be required for differentiation into mature tissues.     

Methylation Imprinting of H19 and SNRPN Genes in Human Benign Ovarian Teratomas

In humans, studies of female germ cells are very limited by ethics. The current study investigated the usefulness of benign ovarian teratomas as a substitute for ova in analyses of imprinted genes. Twenty-five human benign ovarian teratomas were typed with 45 microsatellite DNA markers and classified according to their genotypic features. Two oppositely imprinted genes, H19 and SNRPN, were then chosen for analysis of their methylation states in these tumors. These analyses revealed that benign ovarian teratomas consist of a mixture of genetically and epigenetically heterogeneous cell populations. In contrast to previous reports, we could document only one case rising from germ cells by meiosis-II nondisjunction. H19 and SNRPN were methylated in individual teratomas to various degrees, ranging from normal somatic cell to expected ovum levels. The allele with residual methylation of H19 was consistent with that methylated in the patient's blood DNA, thus being of paternal origin. Degrees of H19 hypomethylation and SNRPN hypermethylation increased as the cellular origin of the tumors advanced in oogenesis and were closely correlated in individual teratomas. These results could be best explained by the assumption that the primary imprinting is a progressively organized process and suggest that the establishment of primary imprints on different genes might be mechanistically linked, even when those genes are oppositely imprinted.     

Development of teratomas from yolk sac of genetically sterile embryos

Yolk sac-derived teratomas are composed of various well-differentiated tissues. These tissues must be derived from multipotent cells. To exclude a germ cell origin for these teratomas we used $\text{Steel}\text{-}\text{Sl}\text{+}$ females copulated with $\text{Sl}\text{+}$ males. The embryos generated from such mating comprise 25% $\text{Sl}\text{Sl}$ sterile embryos, deficient in primordial germ cells, 25% normal embryos (+/+), and 50% heterozygotes ($\text{Sl}\text{+}$). The results indicate that the genotype of the embryos does not influence the development of teratomas. Displaced yolks sacs belonging to genetically sterile embryos developed into teratomas as frequently as those from heterozygotes and from genetically normal embryos.     

Yolk sac-derived rat teratomas are not of germ cell origin

In an attempt to verify the possibility that yolk sac-derived rat teratomas may be of germ cell origin, pregnant rats were treated with Busulphan. This drug, which is known to destroy the germ cells during their migratory phase, led to the complete absence of these cells in the gonads. However, Busulphan did not influence the number or the morphology of the yolk sacderived teratomas. Therefore it is concluded that these teratomas do not arise from germ cells.     

Ovarian teratomas associated with the insertion of an imprinted transgene

Ovarian teratomas are tumors that arise from female germ cells and are often a mixture of immature embryonal carcinoma cells and mature embryonic cells. Tissues derived from all three primary embryonic lineages (ectoderm, mesoderm, and endoderm) are typically found in the mature elements of a teratoma. In the case of the transgenic mouse line TG.KD, created with an imprinted transgene construct, malignant ovarian teratomas of a mixed germ cell tumor morphology occur in 15–20% of hemizygous female carriers of the transgene. The tumors frequently metastasize and can result in death of the mouse. Genetic analysis indicates that the tumors are associated with the transgene's integration site. Inbred FVB/N and female mice of other transgenic lines, also created in the inbred FVB/N strain with the same DNA construct as TG.KD, do not develop teratomas. In addition to teratomas, the integration of the transgene on Chromosome (Chr) 8 is associated with a perinatal lethality in homozygous transgenic carriers. The hemizygous genotypes of the teratomas suggest that they arise from early germ cells, prior to the completion of meiosis I. Received: 31 August 1995 / Accepted: 3 November 1995     

BAX-mediated cell death affects early germ cell loss and incidence of testicular teratomas in Dnd1 mice

A homozygous nonsense mutation (Ter) in murine Dnd1 (Dnd1^Ter/Ter) results in a significant early loss of primordial germ cells (PGCs) prior to colonization of the gonad in both sexes and all genetic backgrounds tested. The same mutation also leads to testicular teratomas only on the 129Sv/J background. Male mutants on other genetic backgrounds ultimately lose all PGCs with no incidence of teratoma formation. It is not clear how these PGCs are lost or what factors directly control the strain-specific phenotype variation. To determine the mechanism underlying early PGC loss we crossed Dnd1^Ter/Ter embryos to a Bax-null background and found that germ cells were partially rescued. Surprisingly, on a mixed genetic background, rescued male germ cells also generated fully developed teratomas at a high rate. Double-mutant females on a mixed background did not develop teratomas, but were fertile and produced viable off-spring. However, when Dnd1^Ter/Ter XX germ cells developed in a testicular environment they gave rise to the same neoplastic clusters as mutant XY germ cells in a testis. We conclude that BAX-mediated apoptosis plays a role in early germ cell loss and protects from testicular teratoma formation on a mixed genetic background.     

Genome Wide DNA Methylation Profiles Provide Clues to the Origin and Pathogenesis of Germ Cell Tumors

The cell of origin of the five subtypes (I-V) of germ cell tumors (GCTs) are assumed to be germ cells from different maturation stages. This is (potentially) reflected in their methylation status as fetal maturing primordial germ cells are globally demethylated during migration from the yolk sac to the gonad. Imprinted regions are erased in the gonad and later become uniparentally imprinted according to fetal sex. Here, 91 GCTs (type I-IV) and four cell lines were profiled (Illumina’s HumanMethylation450BeadChip). Data was pre-processed controlling for cross hybridization, SNPs, detection rate, probe-type bias and batch effects. The annotation was extended, covering snRNAs/microRNAs, repeat elements and imprinted regions. A Hidden Markov Model-based genome segmentation was devised to identify differentially methylated genomic regions. Methylation profiles allowed for separation of clusters of non-seminomas (type II), seminomas/dysgerminomas (type II), spermatocytic seminomas (type III) and teratomas/dermoid cysts (type I/IV). The seminomas, dysgerminomas and spermatocytic seminomas were globally hypomethylated, in line with previous reports and their demethylated precursor. Differential methylation and imprinting status between subtypes reflected their presumed cell of origin. Ovarian type I teratomas and dermoid cysts showed (partial) sex specific uniparental maternal imprinting. The spermatocytic seminomas showed uniparental paternal imprinting while testicular teratomas exhibited partial imprinting erasure. Somatic imprinting in type II GCTs might indicate a cell of origin after global demethylation but before imprinting erasure. This is earlier than previously described, but agrees with the totipotent/embryonic stem cell like potential of type II GCTs and their rare extra-gonadal localization. The results support the common origin of the type I teratomas and show strong similarity between ovarian type I teratomas and dermoid cysts. In conclusion, we identified specific and global methylation differences between GCT subtypes, providing insight into their developmental timing and underlying developmental biology. Data and extended annotation are deposited at GEO (GSE58538 and GPL18809).     

Genetics and biology of human ovarian teratomas. I. Cytogenetic analysis and mechanism of origin.

One hundred and two benign, mature ovarian teratomas and two immature, malignant teratomas were karyotyped and scored for centromeric heteromorphisms as part of an ongoing project to determine the chromosomal karyotype and the genetic origin of ovarian teratomas and to assess their utility for gene-centromere mapping. Karyotypic analysis of the benign cases revealed 95 46,XX teratomas and 7 chromosomally abnormal teratomas (47,XXX, 47,XX,+8 [two cases], 47,XX,+15, 48,XX,+7,+12 91,XXXX,-13 [mosaic], 47,XX,-15,+21,+mar). Our study reports on the first cases of tetraploidy and structural rearrangement in benign ovarian teratomas. The two immature cases had modal chromosome numbers of 78 and 49. Centromeric heteromorphisms that were heterozygous in the host were homozygous in 65.2% (n = 58) of the benign teratomas and heterozygous in the remaining 34.8% (n = 31). Chromosome 13 heteromorphisms were the most informative, with 72.7% heterozygosity in hosts. The cytogenetic data indicate that 65% of teratomas are derived from a single germ cell after meiosis I and failure of meiosis II (type II) or endoreduplication of a mature ovum (type III); 35% arise by failure of meiosis I (type I) or mitotic division of premeiotic germ cells (type IV).     

Germ cell pluripotency, premature differentiation and susceptibility to testicular teratomas in mice

Testicular teratomas result from anomalies in germ cell development during embryogenesis. In the 129 family of inbred strains of mice, teratomas initiate around embryonic day (E) 13.5 during the same developmental period in which female germ cells initiate meiosis and male germ cells enter mitotic arrest. Here, we report that three germ cell developmental abnormalities, namely continued proliferation, retention of pluripotency, and premature induction of differentiation, associate with teratoma susceptibility. Using mouse strains with low versus high teratoma incidence (129 versus 129-Chr19(MOLF/Ei)), and resistant to teratoma formation (FVB), we found that germ cell proliferation and expression of the pluripotency factor Nanog at a specific time point, E15.5, were directly related with increased tumor risk. Additionally, we discovered that genes expressed in pre-meiotic embryonic female and adult male germ cells, including cyclin D1 (Ccnd1) and stimulated by retinoic acid 8 (Stra8), were prematurely expressed in teratoma-susceptible germ cells and, in rare instances, induced entry into meiosis. As with Nanog, expression of differentiation-associated factors at a specific time point, E15.5, increased with tumor risk. Furthermore, Nanog and Ccnd1, genes with known roles in testicular cancer risk and tumorigenesis, respectively, were co-expressed in teratoma-susceptible germ cells and tumor stem cells, suggesting that retention of pluripotency and premature germ cell differentiation both contribute to tumorigenesis. Importantly, Stra8-deficient mice had an 88% decrease in teratoma incidence, providing direct evidence that premature initiation of the meiotic program contributes to tumorigenesis. These results show that deregulation of the mitotic-meiotic switch in XY germ cells contributes to teratoma initiation.     

Sequential morphological study of teratomas derived from displaced yolk sac.

The sequential histological and ultrastructural morphology of benign teratomas derived from displaced visceral yolk sac is described. The appearance and the differentiation of all the tissues formed in these teratomas is compared to the differentiation of normal embryonic tissues and to the development and differentiation observed in experimental teratocarcinomas. The presence in the teratomas of adult well-differentiated tissues derived from all three germ layers is discussed in relation to the multipotentiality of cells from the extra-embryonic parts of the embryo.     

Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture

Embryonic stem (ES) cell lines derived from human blastocysts have the developmental potential to form derivatives of all three embryonic germ layers even after prolonged culture. Here we describe the clonal derivation of two human ES cell lines, H9.1 and H9.2. At the time of the clonal derivation of the H9.1 and H9.2 ES cell lines, the parental ES cell line, H9, had already been continuously cultured for 6 months. After an additional 8 months of culture, H9.1 and H9.2 ES cell lines continued to: (1) actively proliferate, (2) express high levels of telomerase, and (3) retain normal karyotypes. Telomere lengths, while somewhat variable, were maintained between 8 and 12 kb in high-passage H9.1 and H9.2 cells. High-passage H9.1 and H9.2 cells both formed teratomas in SCID-beige mice that included differentiated derivatives of all three embryonic germ layers. These results demonstrate the pluripotency of single human ES cells, the maintenance of pluripotency during an extended period of culture, and the long-term self-renewing properties of cultured human ES cells. The remarkable developmental potential, proliferative capacity, and karyotypic stability of human ES cells distinguish them from adult cells.     

Short-term serum-free culture reveals that inhibition of Gsk3β induces the tumor-like growth of mouse embryonic stem cells

Here, we present evidence that the tumor-like growth of mouse embryonic stem cells (mESCs) is suppressed by short-term serum-free culture, which is reversed by pharmacological inhibition of Gsk3β. Mouse ESCs maintained under standard conditions using fetal bovine serum (FBS) were cultured in a uniquely formulated chemically-defined serum-free (CDSF) medium, namely ESF7, for three passages before being subcutaneously transplanted into immunocompromised mice. Surprisingly, the mESCs failed to produce teratomas for up to six months, whereas mESCs maintained under standard conditions generated well-developed teratomas in five weeks. Mouse ESCs cultured under CDSF conditions maintained the expression of Oct3/4, Nanog, Sox2 and SSEA1, and differentiated into germ cells in vivo. In addition, when mESCs were cultured under CDSF conditions supplemented with FBS, or when the cells were cultured under CDSF conditions followed by standard culture conditions, they consistently developed into teratomas. Thus, these results validate that the pluripotency of mESCs was not compromised by CDSF conditions. Mouse ESCs cultured under CDSF conditions proliferated significantly more slowly than mESCs cultured under standard conditions, and were reminiscent of Eras-null mESCs. In fact, their slower proliferation was accompanied by the downregulation of Eras and c-Myc, which regulate the tumor-like growth of mESCs. Remarkably, when mESCs were cultured under CDSF conditions supplemented with a pharmacological inhibitor of Gsk3β, they efficiently proliferated and developed into teratomas without upregulation of Eras and c-Myc, whereas mESCs cultured under standard conditions expressed Eras and c-Myc. Although the role of Gsk3β in the self-renewal of ESCs has been established, it is suggested with these data that Gsk3β governs the tumor-like growth of mESCs by means of a mechanism different from the one to support the pluripotency of ESCs.     

β1 Integrin Is Essential for Teratoma Growth and Angiogenesis

Teratomas are benign tumors that form after ectopic injection of embryonic stem (ES) cells into mice and contain derivatives of all primitive germ layers. To study the role of β1 integrin during teratoma formation, we compared teratomas induced by normal and β1-null ES cells. Injection of normal ES cells gave rise to large teratomas. In contrast, β1-null ES cells either did not grow or formed small teratomas with an average weight of <5% of that of normal teratomas. Histological analysis of β1-null teratomas revealed the presence of various differentiated cells, however, a much lower number of host-derived stromal cells than in normal teratomas. Fibronectin, collagen I, and nidogen were expressed but, in contrast to normal teratomas, diffusely deposited in β1-null teratomas. Basement membranes were present but with irregular shape and detached from the cell surface.

Normal teratomas had large blood vessels with a smooth inner surface, containing both host- and ES cell–derived endothelial cells. In contrast, β1-null teratomas had small vessels that were loosely embedded into the connective tissue. Furthermore, endothelial cells were always of host-derived origin and formed blood vessels with an irregular inner surface. Although β1- deficient endothelial cells were absent in teratomas, β1-null ES cells could differentiate in vitro into endothelial cells. The formation of a complex vasculature, however, was significantly delayed and of poor quality in β1-null embryoid bodies. Moreover, while vascular endothelial growth factor induced proliferation of endothelial cells as well as an extensive branching of blood vessels in normal embryoid bodies, it had no effect in β1-null embryoid bodies.

     

Innervation and maturation of muscular tissue in testicular teratomas in strain 129/Sv-ter mice

In strain 129/Sv-ter mice, teratomas develop spontaneously during the 13th day of gestation. These testicular germ cell tumors exhibit characteristics of different germ layers closely resembling normal embryonic tissue. We investigated the interrelationship between nervous and muscular tissues (often found side by side) in teratomas of 4-week-old 129/Sv-ter mice. In well-differentiated mouse teratomas, histochemically and immunohistochemically distinct muscle fiber types could be distinguished, but not with all reactions. According to its aerobic oxidative capacity, teratoma muscle tissue was comparable with normal muscles. However, with respect to myosin-related properties, fiber type differentiation was incomplete. The muscle fibers - generally arranged in bundles - contained one centrally located endplate which was contacted mostly by a single nerve terminal. From this, proper endplate zones within the fiber bundles were formed. Occasionally "type grouping" was encountered, suggesting collateral axonal branching paralleled by synapse elimination. Together with the earlier in vivo observation of muscular contractions, we assume that teratoma muscle fibers are innervated by nerve cells (within the nervous tissue compartments) corresponding to spinal motoneurons. Thus, myogenesis, maturation and innervation of skeletal muscular tissue in mouse teratomas are largely comparable to normal development.     

Mitotic arrest in teratoma susceptible fetal male germ cells

Formation of germ cell derived teratomas occurs in mice of the 129/SvJ strain, but not in C57Bl/6 inbred or CD1 outbred mice. Despite this, there have been few comparative studies aimed at determining the similarities and differences between teratoma susceptible and non-susceptible mouse strains. This study examines the entry of fetal germ cells into the male pathway and mitotic arrest in 129T2/SvJ mice. We find that although the entry of fetal germ cells into mitotic arrest is similar between 129T2/SvJ, C57Bl/6 and CD1 mice, there were significant differences in the size and germ cell content of the testis cords in these strains. In 129T2/SvJ mice germ cell mitotic arrest involves upregulation of p27(KIP1), p15(INK4B), activation of RB, the expression of male germ cell differentiation markers NANOS2, DNMT3L and MILI and repression of the pluripotency network. The germ-line markers DPPA2 and DPPA4 show reciprocal repression and upregulation, respectively, while FGFR3 is substantially enriched in the nucleus of differentiating male germ cells. Further understanding of fetal male germ cell differentiation promises to provide insight into disorders of the testis and germ cell lineage, such as testis tumour formation and infertility.     

The plasticity of germ cell cancers and its dependence on the cellular microenvironment

So far, the understanding of germ cell cancer (GCC) pathogenesis is based on a model, where seminomas and non‐seminomas represent distinct entities although originating from a common precursor termed germ cell neoplasia in situ (GCNIS). Embryonal carcinomas (ECs), the stem cell population of the non‐seminomas, is pluri‐ to totipotent and able to differentiate into cells of all three germ layers, giving rise to teratomas or tumours mimicking extraembryonic tissues (yolk sac tumours, choriocarcinomas). With regard to gene expression, (epi)genetics and histology, seminomas are highly similar to GCNIS and primordial germ cells, but limited in development. It remains elusive, whether this block in differentiation is controlled by cell intrinsic mechanisms or by signals from the surrounding microenvironment. Here, we reviewed the recent literature emphasizing the plasticity of GCCs, especially of seminomas. We propose that this plasticity is controlled by the microenvironment, allowing seminomas to transit into an EC or mixed non‐seminoma and vice versa. We discuss several mechanisms and routes of reprogramming that might be responsible for this change in the cell fate. We finally integrate this plasticity into a new model of GCC pathogenesis, allowing for an alternative view on the dynamics of GCC development and progression.     

The stabilization of beta-catenin leads to impaired primordial germ cell development via aberrant cell cycle progression

Primordial germ cells (PGCs) are germ cell precursors that are committed to sperm or oocytes. Dramatic proliferation during PGC development determines the number of founder spermatogonia and oocytes. Although specified to a germ lineage, PGCs produce pluripotent embryonic germ (EG) cells in vitro and testicular teratomas in vivo. Wnt/beta-catenin signaling regulates pluripotency and differentiation in various stem cell systems, and dysregulation of this signaling causes various human cancers. Here, we examined the role of Wnt/beta-catenin signaling in PGC development. In normal PGC development, Wnt/beta-catenin signaling is suppressed by the GSK3beta-mediated active degradation of beta-catenin and the low expression of canonical Wnt molecules. The effects of aberrant activation of Wnt/beta-catenin signaling in PGCs were analyzed using mice carrying a deletion of the exon that encodes the GSK3beta phosphorylation sites in the beta-catenin locus. Despite the potential activity of Wnt/beta-catenin signaling in stem cell maintenance and carcinogenesis in various cell lineages, teratomas were not induced in the mice expressing the nuclear-localized beta-catenin in PGCs. Instead, the mutant mice showed germ cell deficiency caused by the delayed cell cycle progression of the proliferative phase PGCs. Our results show that the suppression of Wnt/beta-catenin signaling is a prerequisite for the normal development of PGCs.