Fiction Fix 11

https://digitalcommons.unf.edu/fiction_fix/1001/thumbnail.jp

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

A Mutation in Myo15 Leads to Usher-Like Symptoms in LEW/Ztm-ci2 Rats

The LEW/Ztm-ci2 rat is an animal model for syndromal deafness that arose from a spontaneous mutation. Homozygous animals show locomotor abnormalities like lateralized circling behavior. Additionally, an impaired vision can be observed in some animals through behavioral studies. Syndromal deafness as well as retinal degeneration are features of the Usher syndrome in humans. In the present study, the mutation was identified as a base substitution (T->C) in exon 56 of Myo15, leading to an amino acid exchange from leucine (Leu) to proline (Pro) within the carboxy-terminal MyTH4 domain in the proteins' tail region. Myo15 mRNA was expressed in the retina as demonstrated for the first time with the help of in-situ hybridization and PCR. To characterize the visual phenotype, rats were examined by scotopic and photopic electroretinography and, additionally, histological analyses of the retinas were conducted. The complete loss of sight was detected along with a severe degeneration of photoreceptor cells. Interestingly, the manifestation of the disease does not solely depend on the mutation, but also on environmental factors. Since the LEW/Ztm-ci2 rat features the entire range of symptoms of the human Usher syndrome we think that this strain is an appropriate model for this disease. Our findings display that mutations in binding domains of myosin XV do not only cause non-syndromic hearing loss but can also lead to syndromic disorders including retinal dysfunction

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

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 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

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

Teratomas and gonads in <i>ter/ter</i> rats.

<p>(<b>A</b>) Left: Bilateral GCTs in a 6 week old female rat (black arrows: OGCTs). Right: Unilateral GCT in male rat (white arrow: non-neoplastic testes; red arrow: left TGCT). (<b>B</b>) HE staining of teratomas. Top left: ectodermal tissues in the ovarian teratoma of a 5-week-old female; neural tube formation (black star) and squamous epithelium (black arrow). Top right: mesodermal and endodermal tissues in the ovarian teratoma of a 9-week-old female; cartilage (white star), glandular structures (white arrow) and skeletal muscle (black arrow). Bottom left: heart muscle-like tissue from a contractile ovarian teratoma. Bottom right: immature neuronal tissue from a testicular teratoma of a 6-week-old male. (<b>C</b>) Rat gonads from 3-week-old siblings. Left: non-tumorous <i>ter/ter</i> testes are significantly smaller than wild type. Right: physiological wild type ovaries compared to an ovarian teratoma and an abnormal, cystic ovary (confirmed by histological section and HE staining).</p

HE staining of early tumorigenesis and metastasis.

<p>(<b>A</b>) Male. Tumor formation in the testis of a 1-day-old <i>Dnd1</i>-deficient male. 5× and 40× (<b>B</b>) Female. Neoplastic transformation in the ovary of <i>ter/ter</i> females at 5 and 10 days post partum. Top: 10× and 40×. Bottom: 2.5× and 40×. (<b>C</b>) Metastasis. Teratoma in adjunction to the vertebral column (white star). Tissue of ectodermal (squamous epithelium, black arrow), mesodermal (cartilage, gray arrow) and endodermal (glandular structures, white arrow) origin was identified. 2.5× and 10×.</p