Technical Report for Research Unit FOR-1511

This technical report presents the interim results of the DFG research unit FOR1511 "Protection and Control Systems for Reliable and Secure Operation of Electrical Transmission Systems"

The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders

A spontaneous mutation leading to the formation of congenital ovarian and testicular tumors was detected in the WKY/Ztm rat strain. The histological evaluation revealed derivatives from all three germ layers, thereby identifying these tumors as teratomas. Teratocarcinogenesis was accompanied by infertility and the underlying mutation was termed ter. Linkage analysis of 58 (WKY-ter×SPRD-Cu3) F2 rats associated the ter mutation with RNO18 (LOD = 3.25). Sequencing of candidate genes detected a point mutation in exon 4 of the dead-end homolog 1 gene (Dnd1), which introduces a premature stop codon assumed to cause a truncation of the Dnd1 protein. Genotyping of the recessive ter mutation revealed a complete penetrance of teratocarcinogenesis and infertility in homozygous ter rats of both genders. Morphologically non-tumorous testes of homozygous ter males were reduced in both size and weight. This testicular malformation was linked to a lack of spermatogenesis using immunohistochemical and histological staining. Our WKY-Dnd1ter/Ztm rat is a novel animal model to investigate gonadal teratocarcinogenesis and the molecular mechanisms involved in germ cell development of both genders

Immortalized tumor derived rat fibroblasts as feeder cells facilitate the cultivation of male embryonic stem cells from the rat strain WKY/Ztm

Feeder cells are essential for the establishment and culture of pluripotent rat embryonic stem cells (ESC) in vitro. Therefore, we tested several fibroblast and epithelial cell lines derived from the female genital tract as feeder cells to further improve ESC culture conditions. The immortalized tumor derived rat fibroblast TRF-O3 cells isolated from a Dnd1-deficient teratoma were identified as optimal feeder cells supporting stemness and proliferation of rat ESC. The TRF-O3 cells were characterized as myofibroblasts by expression of fibroblast specific genes alpha-2 type I collagen, collagen prolyl 4-hydroxylase alpha (II), vimentin, S100A4, and smooth muscle α-actin. Culture of inner cell masses (ICM) derived from WKY/Ztm rat blastocysts in 2i-LIF medium on TRF-O3 feeder cells lacking LIF, SCF and FGF2 expression resulted in pluripotent and germ-line competent rat ESC lines. Therein, genotyping confirmed up to 26% male ESC lines. On the other hand the TRF-O3 specific BMP4 expression was correlated with transcriptional activity of the mesodermal marker T-brachyury and the ectoderm specific nestin in the ESC line ES21 demonstrating mesodermal or ectodermal cell lineage differentiation processes within the ESC population. Substitution of 2i-LIF by serum-containing YPAC medium supplemented with TGF-β and rho kinase inhibitors or by 4i medium in combination with TRF-O3 feeder cells led to enhanced differentiation of ES21 cells and freshly isolated ICMs. These results suggest that the ESC culture conditions using TRF-O3 feeder cells and 2i-LIF medium supported the establishment of male ESC lines from WKY/Ztm rats, which represent a favored, permissive genetic background for rat ESC culture. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/2193-1801-3-588) contains supplementary material, which is available to authorized users

Non-tumorous ovaries.

<p>(<b>A</b>) Box plot analysis of non-tumorous ovaries from WKY/Ztm and WKY-<i>Dnd1^ter</i>/Ztm with wild type (+/+), heterozygous (<i>ter</i>/+) and homozygous (<i>ter/ter</i>) genotype (**: p<0.01; ***: p<0.001). (<b>B</b>) HE staining of wild type (+/+, top panel) and mutant ovaries (<i>ter/ter</i>, bottom panel) of WKY-<i>Dnd1^ter</i>/Ztm rats at 6 weeks of age. While different stages of follicle maturation (top panel, left) and primary follicles with a central oocyte (top panel, right) were evident in the wild type ovary, few follicle-like structures without germ cells (bottom panel, right, arrows) remained in the <i>ter/ter</i> ovary.</p

Immunohistochemical staining of testes.

<p>(<b>A</b>) anti c-kit staining and (<b>B</b>) anti DDX4/MVH staining of paraffin sections from homozygous <i>ter/ter</i> and wild type testes of WKY-<i>Dnd1^ter</i>/Ztm rats at 10 days, 3 weeks and 6 weeks of age (black arrows: clusters of surviving c-kit positive germ cell). Abundance of germ cells detectable in +/+ testes, while few scattered clusters of positive cells remain in <i>ter/ter</i> testes.</p

Non-tumorous testes.

<p>(<b>A</b>) Box plot analysis of the size of non-tumorous testes in WKY/Ztm and WKY-<i>Dnd1^ter</i>/Ztm with wild type (+/+), heterozygous (<i>ter/+</i>) and homozygous (<i>ter/ter</i>) genotype at 3, 6 and 9 weeks of age. <i>ter/ter</i> testes are degenerated and significantly smaller than +/+ and <i>ter</i>/+ testes (***: p<0,001). (<b>B</b>) HE staining of wild type and homozygous <i>ter/ter</i> testes of WKY-<i>Dnd1^ter</i>/Ztm rats from 3 to 9 weeks of age.</p

Genotyping, survival and tumor progression.

<p>(<b>A</b>) Genotyping. The <i>ter</i> mutation disrupts a <i>Kpn</i>I restriction site used for genotyping of the <i>ter</i> allele performing PCR amplification and restriction digest. <i>Kpn</i>I digest (black arrows) cleaved the PCR product of 560 bp into 380 bp and 180 bp fragments, N negative control. (<b>B</b>) Kaplan-Meyer survival analysis of male and female WKY-<i>Dnd1^ter</i>/Ztm rats carrying heterozygous and homozygous <i>ter</i> alleles compared to wild type animals. (<b>C</b>) Sizes of TGCTs and OGCTs after 3, 6 and 9 weeks of age in homozygous WKY-<i>Dnd1^ter</i>/Ztm rats.</p