Identification of genomic locus responsible for experimentallyinduced testicular teratoma 1 (ett1) on mouse Chr 18

Takehiro Miyazaki • Yoshie Ikeda • Ikue Kubo • Saeri Suganuma •

Nastumi Fujita • Makiko Itakura • Tae Hayashi • Shuji Takabayashi •

Hideki Katoh • Yukio Ohira • Masahiro Sato • Motoko Noguchi •

Toshinobu Tokumoto

Received: 4 April 2014 / Accepted: 12 June 2014

� Springer Science+Business Media New York 2014

Abstract Spontaneous testicular teratomas (STTs) com-

posed by various kinds of tissues are derived from pri-

mordial germ cells (PGCs) in the fetal testes of the mouse.

In contrast, intra-testicular grafts of the mouse strain (129/

Sv-Ter (?/?)) fetal testes possessed the ability to develop

the experimental testicular teratomas (ETTs), indistin-

guishable from the STTs at a morphological level. In this

study, linkage analysis was performed for exploration of

possible candidate genes involving in ETT development

using F2 intercross fetuses derived from [LTXBJ 9 129/

Sv-Ter (?/?)] F1 hybrids. Linkage analysis with selected

simple sequence length polymorphisms along

chromosomes 18 and 19, which have been expected to

contain ETT-susceptibility loci, demonstrated that a novel

recessive candidate gene responsible for ETT development

is located in 1.1 Mb region between the SSLP markers

D18Mit81 and D18Mit184 on chromosome 18 in the

129/Sv-Ter (?/?) genetic background. Since this locus is

different from the previously known loci (including Ter,

pgct1, and Tgct1) for STT development, we named this

novel gene ‘‘experimental testicular teratoma 1 (ett1)’’. To

resolve the location of ett1 independently from other sus-

ceptibility loci, ett1 loci was introduced in a congenic

strain in which the distal segment of chromosome 18 in

LTXBJ strain mice had been replaced by a 1.99 Mbp

genomic segment of the 129/Sv-Ter (?/?) mice. Congenic

males homozygous for the ett1 loci were confirmed to have

the ability to form ETTs, indicating that this locus contain

the gene responsible for ETTs. We listed candidate genes

included in this region, and discussed about their possible

involvement in induction of ETTs.

Introduction

Spontaneous testicular teratoma [STT; also called ‘‘testic-

ular germ cell tumors (TGCT)’’] in mice originate from

primordial germ cells (PGCs), normally the precursors of

the gametes in the developing fetus, around days 12.5 days

post-coitum (dpc) in fetal testes (Stevens 1967b; Stevens

and Little 1954). PGCs are then transformed through

unknown mechanisms to give rise to embryonic cells

resembling cells at egg cylinder stage, and then clusters of

foci derived from these transformed cells rupture the

seminiferous tubules from which they invade into the

interstitial space of a testis. These embryonal-like cells

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00335-014-9529-8) contains supplementarymaterial, which is available to authorized users.

T. Miyazaki � Y. Ikeda � I. Kubo � S. Suganuma � N. Fujita �M. Itakura � T. Hayashi � Y. Ohira � M. Noguchi �T. Tokumoto (&)

Biological Science Course, Graduate School of Science,

National University Corporation Shizuoka University, Ohya 836,

Suruga-ku, Shizuoka 422-8529, Japan

e-mail: sbttoku@ipc.shizuoka.ac.jp

S. Takabayashi � H. Katoh

Institute for Experimental Animals, Hamamatsu University

School of Medicine, 1-20-1, Handayama, Higashi-ku,

Hamamatsu, Shizuoka 431-3192, Japan

M. Sato

Section of Gene Expression Regulation, Frontier Science

Research Center, Kagoshima University, 8-35-1 Sakuragaoka,

Kagoshima, Kagoshima 890-8544, Japan

T. Tokumoto

Integrated Bioscience Section, Graduate School of Science and

Technology, National University Corporation Shizuoka

University, Ohya 836, Suruga-ku, Shizuoka 422–8529, Japan

123

Mamm Genome

DOI 10.1007/s00335-014-9529-8

form variety of cells resembling normal embryonic ecto-

derm, mesoderm, and endoderm, and then form STT

comprising of a variety of differentiated cells and tissues.

STT has been known to occur in human and birds, as well

as in mice (Stevens 1967b; Stevens and Little 1954).

STTs are exceedingly rare in mice with the exception of

the 129/Sv mouse strain, which is predisposed to STT fre-

quency of 1–7 % depending on the subline (Stevens 1975;

Stevens and Hummel 1957; Stevens and Little 1954). The

STT phenotype of 129/Sv strain males behaves as a recessive

trait with an estimated six to eight contributing loci (Lam

et al. 2007). The Ter mutation in chromosome (Chr) 18 has

been shown to increase the penetrance of the male STT

phenotype on the 129/Sv-Ter substrain (Asada et al. 1994;

Sakurai et al. 1994). Noguchi and Noguchi demonstrated

using STT-high sensitive 129/Sv-Ter mice that the Ter/Ter

genotype was associated with a lack of almost all PGCs in

both sexes and the development of STT at nearly100 %

incidence in males (Noguchi and Noguchi 1985). Males with

?/Ter and ?/? genotypes exhibited STT with frequencies of

17 and 1 %, respectively. Males homozygous for the Ter

mutation are sterile because of germ cell deficiency. This Ter

mutation is now recognized as a point mutation that occurred

in the coding region of Dnd1, the orthologue of the zebrafish

dead end (dnd) gene (Youngren et al. 2005), firstly identified

in zebrafish as a responsible gene for germ cell loss (Wei-

dinger et al. 2003). Although germ cell deficiency is asso-

ciated with teratocarcinogenesis, the Ter congenic strains did

not generate STTs (Noguchi et al. 1996). Thus, the mecha-

nisms by which genetic factors cause STTs remain elusive.

Grafting experiments of fetal 129/Sv subline testis from

embryo of 12.5 days old into an adult 129/Sv subline testis

caused development of experimental testicular teratomas

(ETTs) with high incidences (80–90 %) (Stevens 1964).

Noguchi et al. (1996) demonstrated that grafting of fetal

testis derived from congenic strain (termed C57BL/6J(B6)-

?/Ter) carrying Ter locus into adult B6 testis failed to

generate ETTs (Noguchi et al. 1996). In contrast, grafting

of fetal testis derived from 129/Sv-?/Ter (?/?) strain

(hereafter referred to as 129) lacking Ter locus resulted in

the development of ETTs with efficiencies of 80–90 %.

However, grafting of fetal testis from other strains such as

B6 and LTXBJ (hereafter referred to as LT), one of the

recombinant inbred (RI) strains of LT/Sv (Stevens and

Varnum 1974; Stevens et al. 1977), failed to develop ETTs.

These findings suggest the presence of a locus associated

with ETT development in the 129 genetic background.

Generation of ETTs with very high incidence after testis

grafting mentioned above facilitates a genetic linkage

analysis to identify the locus responsible for predisposing

the 129 strain to ETT development.

In this study, we conducted fine mapping of the locus

associated with generation of ETTs. As a result, it was

mapped on Chr 18, in which Ter gene is also present, but its

location was different from that of Ter gene. We therefore

designated this region as ‘‘experimental testicular teratoma

1 (ett1)’’. To study the effects of genetic background on

testicular teratocarcinogenesis and germ cell deficiency, we

established LT-ett1 congenic strains. Introduction of the ett1

gene into different genetic backgrounds revealed that ETTs

generate when the ett1 gene is in the homozygous state.

Materials and methods

Mice

129 strain has been established from STT-high permissive

129/terSv strain (Stevens 1973) by Noguchi (Noguchi and

Noguchi 1985). The 129 strain has a STT frequency of 1.4 %,

but exhibits ETT with efficiencies of 80–90 % after grafting of

fetal testes into adult syngenic testes (Noguchi et al. 1996). LT

mice (Stevens and Varnum 1974; Stevens et al. 1977), a

generous gift from Dr. Leroy Stevens (The Jackson Labora-

tory, Bar Harbor, ME, USA), were bred at the animal facilities

at Shizuoka University. The LT males exhibit normal sper-

matogenesis and generate neither STTs nor ETTs (Noguchi

et al. 1996). The LT females, however, generate ovarian ter-

atomas derived from a parthenogenesis event after the onset of

puberty with an efficiency of almost 100 %. For linkage

analysis for exploring ETT-susceptibility locus, F2 progeny

derived from intercross between (129 9 LT) F1 hybrids were

used. The recipient males receiving F2 fetal testis grafts are

males of the (129 9 LT) F1 hybrids. All animals were kept

under controlled conditions of temperature and humidity and

had free access to food and water. All animal procedures were

performed according to protocols approved by the Animal

Subject Committee at Shizuoka University.

Transplantation and histological analysis

For timed mating, females were inspected every morning for

the presence of vaginal plug, and noon on the day mice with

vaginal plug detected were considered embryonic day

(E) 0.5. The genital ridges were removed from E12.5, and

then only male gonads were selected. The host testes were

exposed through a median ventral incision in the skin and

body wall under sodium pentobarbital anesthesia. The grafts

were drawn into and expelled from glass capillary attached to

rubber tubing with a mouthpiece (Stevens 1964). Graft sites

were removed over 2 weeks after surgery. Tissues dissected

were fixed in Bouin’s solution, embedded in paraffin, and

finally subjected to serial sectioning at 6 lm in thickness.

Deparaffinized sections were stained with hematoxylin,

eosin, and alcian blue (H-E-A), and screened for the presence

of ETTs under a light microscope.

T. Miyazaki et al.: ett1 experimental testicular teratoma locus

123

PCR-simple sequence length polymorphisms (SSLP)

In this study, genomic locations of SSLP markers and genes

were matched for genome data (GCA_000001635.4.).

Genomic DNA was isolated from the dissected fetal tissues or

ear pieces of adult by the method of Blin and Stafford (1976).

SSLP genotyping was conducted using microsatellite markers

for genome wide genotyping and markers listed in Table S1

for fine mapping on Chr 18 and 19 (MapMaker ResGen

MapPairs, Invitrogen Co., Carlsbad, CA). PCR conditions

were based on the information on the Microsatellite Data Base

of Japan (http://www.shigen.nig.ac.jp/mouse/mmdbj/about.

jsp), and PCR was performed using the following basic con-

ditions: denaturation at 94 �C for 3 min followed by 30 cycles

of 94 �C for 20 s, 55 �C for 30 s, and 72 �C for 30 s and

subsequent extension at 72 �C for 5 min. PCR products were

separated by electrophoresis on 3 % agarose gels.

Linkage analysis and statistical analysis

Segregation data for markers on Chr 18 and 19 were ana-

lyzed by v2 test for intercross data [v2 = R[{(obsp- obsr)2/

obsr}] (Pearson 1900). To determine P values, an Internet-

accessible statistical program v2 calculator (http://www.

formilab.ch/rpkp/experiments/analysis/chiCal.html.) was

used. Linkage was defined by a v2 exceeding 5.99

(d.f. = 2) in the case of segregation ratios of 1:2:1 at

P = 0.05 %. Similarly, linkage was defined by a v2

exceeding 3.84 (d.f. = 1) in the case of segregation ratios

of 3:1 or 1:3 at P = 0.05 %.

Establishment of LT-ett1 congenic strain

A large segment of Chr 18 from the 129 strain was trans-

ferred onto the LT strain background through 13 consec-

utive backcross to generate the unique strain LT-ett1. After

13 backcross generations, congenic mice were mated to

generate F1 intercross offspring. Intercross proceeded more

than seven cycles. During the crossing, genotyping was

performed with PCR using D18Mit81 and D18Mit184

primer sets.

Results

ETT development after testis grafting

In a preliminary test, histological sections of ETTs derived

from grafting of 129 fetal testes carrying no Ter locus were

compared to those of STTs. When fetal testes from F2

progeny obtained by intercross between N1 (LTx129) were

transplanted into both testes of adult F1 males, ETTs were

seen in some of the grafts 2–4 weeks after surgery. They

were a mixture of various cells and tissues, which were

indistinguishable from STTs (Fig. 1a). In this case, fetuses

exhibiting ETT development in either testis of a recipient

male were considered as individuals predisposing to ETT

phenotype. Grafting of LT fetal testes resulted in genera-

tion of only small seminiferous tubules, probably differ-

entiated from the grafts (Fig. 1b). From these results, we

determined to use herein 129 as ETT-sensitive strain and

LT as ETT-resistant strain for exploration of ETT-sus-

ceptibility determining locus.

Comparison of polymorphisms on SSLPs among inbred

mice

To explore microsatellite (Mit) markers used for linkage

analysis for ETT-susceptibility determining locus, com-

mercially available Mit markers that are known to be

associated with Chr 18 and 19 were first searched whether

these markers had been correctly located on the sequence.

Fig. 1 ETT formed in the graft. Serial sections of host testis were

prepared and checked for teratoma formation. a ETT generated in the

graft derived from a 129/129 mouse. There are several differentiated

tissues such as neural ectodermal cells (Nec), secretory epithelium

(Se), and cartilage (Ca). b Normal seminiferous tubules (St) derived

from a LT/LT mouse. Seminiferous tubules of host 129/LT mouse

(Ht) was contained in this section. Scale bars 100 lm

T. Miyazaki et al.: ett1 experimental testicular teratoma locus

123

To examine the presence of possible SSLP polymorphism

between two inbred strains (129 and LT), we typed these

two strains for selected SSLPs along Chr 18 and 19. SSLPs

with distinguishable polymorphic size differences between

the 129 and LT strains were 39.1 % (43/110 tested) for Chr

18 and 37.1 % (26/70 tested) for Chr 19. F1 hybrid

(129 9 LT) gave rise to two bands showing heterozygosity

of 129 and LT. These results indicate that LT mice exhibit

quite different polymorphic patterns from 129 mice over

each chromosome, which will be very helpful for linkage

analysis. We used a total of 31 SSLPs that distinguish

between the 129 and LT strains (18 and 13 Mit markers

spanning through each of entire chromosome for Chr 18

and 19, respectively) (Table S1).

Exploration of ETT-susceptibility locus on Chr 18

Of 360 grafts examined, 34.7 % (n = 125) exhibited ETT

development and 65.3 % (n = 235) did not. In this study,

114 of 125 fetal samples exhibiting ETT phenotype and

197 of 235 fetal samples exhibiting no ETT phenotype

were randomly selected for use in linkage analysis. A total

of 114 intercross F2 fetuses exhibiting ETT development

after testis grafting were analyzed individually with 18

SSLPs on Chr 18 that distinguish between the 129 and LT

mice. Three genotypes ([129/129], [129/LT], and [LT/LT])

were observed for all 18 Chr 18 SSLPs tested. Homozy-

gous 129 strain alleles were observed in 5 of 114 F2

intercross progeny at all 18 SSLPs. Similarly, heterozygous

[129/LT] alleles were observed in 6 of 114 F2 intercross

progeny at all 18 SSLPs. However, no evidence was found

for exclusive association of homozygous LT strain alleles

segregating with the ETT phenotype. If each maker on Chr

18 is not related to ETT formation, the genotype of all

ETT-generating samples would fit the expected 1:2:1

Mendelian ratios for [129/129]: [129/LT]: [LT/LT] geno-

types at any locus. To determine the statistical significance

of the over representation of homozygous 129 strain allele

at each marker among the ETT-generating mice, v2 ana-

lysis was performed (Table S1 A, B). For F2 progeny

exhibiting ETT formation after testis grafting, genotype

ratios were significantly skewed (v2 = 5.99; P \ 0.05)

over entire Chr 18 except the SSLP D18Mit64 (6.1 Mbp),

which is located near the centromere. Interestingly, segre-

gation was predominantly skewed toward homozygous 129

strain alleles with strongest association at D18Mit81

(66.5 Mbp) (v2 = 41.0; P = 0.13 9 10-8). The results are

presented as a graph of SSLP chromosomal position versus

v2 value for each SSLP (Fig. 2a). The graph shows the

deviation from Mendelian ratios at each locus, as measured

by portioned v2 values from the G-test for heterogeneity (in

this analysis, a v2 [ 3.55 suggests significance at

P \ 0.05). Greatest deviation was observed at the regions

spanning 60–80 Mbp around the D18Mit81 SSLP for [129/

129] and [LT/LT] genotypes. By contrast, there was no

obvious deviation for the [129/LT] genotype over Chr 18.

Similar analysis was made for F2 intercross progeny with

non-ETT phenotype using 18 Chr 18 SSLPs. The results

Fig. 2 ETT formation linked to ett1 locus. a v2 analysis of

distribution of microsatellite markers on Chr 18. v2 values of ETT-

bearing mice (closed circle) or non-bearing mice (open circle) are

plotted against the locus on Chr 18. Dashed lines indicate the border

of p value = 0.05 and 0.001. The loci of Ter and ett1 on Chr 18 are

indicated as closed and open squares, respectively. b Genotyping of

the ett1 locus. A representative result of SSLP analysis using

microsatellite markers is shown. c Results of genotyping of an LT-

ett1 congenic strain are summarized. Open square indicates a region

where the LT component has been replaced by the 129 one

T. Miyazaki et al.: ett1 experimental testicular teratoma locus

123

are shown as a map panel (Table S1). Of 114 tested, 9, 19,

and 8 exhibited, respectively [LT/LT], [129/LT] and [129/

129] alleles at all 18 SSLPs. When v2 analysis was per-

formed with significance at P \ 0.05, v2 values over 5.99

(P \ 0.05) were seen in 11 of 18 SSLPs tested. Segregation

was predominantly skewed toward homozygous LT strain

alleles. The highest v2 value was at D18Mit84 (33.8 Mbp)

(v2 = 14.42; P = 0.74 9 10-3). However, no statistical

significance was seen throughout the entire Chr 18. When

the graphs with relationship between the position of each

SSLP on Chr 18 and the v2 value for both ETT-generating

and non-generating groups were combined together, in

both groups highest v2 values were seen at regions span-

ning 60–80 Mbp including D18Mit81 (Fig. 2a). From these

results, there is a possibility for the presence of 129-derived

susceptibility locus near D18Mit81 (66.5 Mbp) on Chr 18

predisposing to ETT. We named this ETT-susceptibility

locus ‘‘experimental testicular teratoma 1 (ett1).’’

Table 1 Identity and features

of the genetic determinants

within the ett1 locus

Gene Description GO Location on

Chr 18 (bp)

Strand Gene type

Gm15958 Predicted gene 15958 ND 66291220 ? Novel

processed

transcript

Gm20294 Predicted gene 20294 66417335 ? Pseudogene

Pmaip1 Phorbol-12-myristate-

13-acetate-

induced protein 1

Protein binding 66458604 ? Protein

coding

Gm9926 Predicted gene 9926 66504178 ? Non-coding

RNA

LOC102639886 U6 snRNA-associated

Sm-like

protein LSm7-like

66561615 - Pseudogene

LOC102632095 Uncharacterized

LOC102632095

66672212 ? Non-coding

RNA

LOC102632009 Uncharacterized

LOC102632009

66673085 - Non-coding

RNA

Mc4r Melanocortin 4 receptor Melanocortin

receptor

activity

66857705 - Protein

coding

Gm19784 Predicted gene 19784 ND 66857705 - Non-coding

RNA

Gnal Guanine nucleotide

binding protein, astimulating, olfactory

type

G-protein-coupled

receptor binding

67088336 ? Protein

coding

Chmp1b Charged multivesicular

body protein 1B

Protein domain

specific

binding

67205359 ? Protein

coding

Mppe1 Metallophosphoesterase

1

ND 67225048 – Protein

coding

Impa2 Inositol (myo)-1(or 4)-

monophosphatase 2

Inositol

monophosphate

phosphatase

activity

67289223 ? Protein

coding

LOC102632177 Uncharacterized

LOC102632177

67294293 – Non-coding

RNA

B430212C06Rik RIKEN cDNA

B430212C06 gene

ND 67321209 – Non-coding

RNA

Cidea Cell death-inducing

DNA fragmentation

factor, alpha subunit-

like effector A

Protein

homodimerization

activity

67343564 – Protein

coding

Tubb6 Tubulin, b6 class V GTPase activity 67390731 ? Protein

coding

T. Miyazaki et al.: ett1 experimental testicular teratoma locus

123

We also typed F2 intercross progeny for selected SSLPs

along Chr 19 and compared the genotypes of ETT-generating

and non-generating mice. As a result, there was no obvious

region associated with ETT development (Table S1 C, D).

Since the ett1 locus exists in a region spanning 1.1 Mbp

(66.3 * 67.4 Mbp which include peak markers D18Mit81

and D18Mit184), computer-aided search was performed for

possible known genes with the mouse genome data [NCBI

http://www.ncbi.nlm.nih.gov/pubmed, Ensembl http://asia.

ensembl.org/index.html]. The 1.1 Mbp ett1 interval con-

tains 17 unique annotated genetic determinants, including

eight protein-coding genes, one processed transcript, six

non-coding RNA genes, and one pseudogene. Table 1

summarizes the features of the 17 annotated genes in the

ett1 interval. Eight characterized protein-coding genes

within the ett1 region are found by current annotations:

Pmaip1, Mc4r, Gnal, Chmp1b, Mppe1, Impa2, Cidea, and

Tubb6.

Establishment of ett1 congenic strain

To identify the genomic region containing ett1, we estab-

lished LT-ett1 congenic strains. Successive backcrosses

were performed to generate congenic lines with the ett1

region in the background of the LT strain. During the

crossing, genotyping was performed with PCR using

D18Mit81 and D18Mit184 primer sets (Fig. 2b). To check

the ETT forming ability of the establishing strain, trans-

plantation experiments were conducted for mice after

several generations of inbreeding. Table 2 shows a repre-

sentative result of transplantation performed at between N4

and N1. When fetal testis from N4 mice was transplanted

to N1 adult testis, ett1 locus-dependent ETT formation was

observed. Mice carrying ett1 region in the LT strain

background ([13 backcross generations) were then mated

to generate F1 intercross offspring. Intercross proceeded

eight cycles at present. Finally, we obtained congenic strain

which carrying the 1.9 Mbp region of 129 strain including

ett1 loci (ett1 congenic strain: LTXBJ.129/Sv-Ter/?(?/?)-

ett1 designated as LT-ett1) (Fig. 2c). The strain does not

carry Ter locus and is fertile. To show the ETT forming

ability of the strain, transplantation was conducted.

Testes of embryo from a F7 (seven intercross genera-

tions) LT-ett1 9 LT-ett1 were grafted to the testes of LT-

ett1 congenic strain and LT strain. 60 % (n = 3/5) of the

ett1 homozygous [129/129] fetus testes developed terato-

mas in hosts testes, where grafts from LT did not (0 %,

n = 0/6). The tumor composed of secretary epithelium

(Se), cartilage (Ca), and bone (Bo) (Fig. 3). These results

Table 2 Summary of ETT formation efficiency in the fetal testicular

grafts with different genetic backgrounds when N1 hybrid (129/LT)

male mice are used as a host

Donor at N4a % (no.) of ETTsb

129/129 88.9 (8/9)

129/LT 36.8 (7/19)

LT/LT 0 (0/8)

a Numbers of generations during LT-ett1 9 LT backcrossb The male genital ridges on E12.5 were grafted within a host testis

using a method shown in Materials and methods. Graft sites were

removed over 2 weeks after surgery and subjected to histological

inspection. Grafts carrying teratomas are defined as ETTs

Fig. 3 ETT formed in the grafts derived from LT-ett1 congenic fetal

testes. Serial sections of host testis were prepared and checked for

teratoma formation. a A representative ETT carrying a variety of

differentiated tissues such as bone (Bo), cartilage (Ca), and secretory

epithelium (Se). b–d Higher magnification views of differentiated

tissues shown in (a): neural tube for (b), bone tissues with bone

marrow (Ma) and cartilage (Ca) for (c) and secretory epithelium for

(d). Scale bars 100 lm

T. Miyazaki et al.: ett1 experimental testicular teratoma locus

123

demonstrated that we established a LT-ett1 congenic strain

which possesses inducing gene of ETT.

Discussion

In this study, we found a strong association between ETT

formation and Chr 18 in the 129 genetic context. Linkage

analysis in more than 300 F2 fetuses suggested a possible

presence of 129-derived susceptibility locus near D18Mit81

(66.5 Mbp) on Chr 18 predisposing to ETT. Notably, a

gradual increase in v2 value from both ends of Chr 18 toward

the D18Mit81 locus was seen in the samples exhibiting ETT

development, suggesting the possibility that loci relating to

ETT development may be present near D18Mit81 locus.

Since there is no evidence for transmission distortion around

the D18Mit81 locus, we named this locus ett1.

The ett1 locus is present far from other Chr 18-linked loci

including the Ter locus (ca. 20 cM) (Sakurai et al. 1994) and

Ter locus near D18Mit62 (27.6 Mbp; 16.0 cM), which has

been known to increase the susceptibility of STT (Asada

et al. 1994). The same locus on Chr 18 related to suscepti-

bility of STT was previously reported by establishing con-

genic strains between 129 and MOLF (Anderson et al. 2009).

We could narrow the ett1 locus in a region spanning from ca.

1.1 Mbp around D18Mit81 (66.5 Mbp) to D18Mit184

(66.8 Mbp). In this region, eight functionally characterized

protein coding genes within the ett1 region are found on

current annotations: Pmaip1/Noxa, Mc4r, Gnal, Chmp1b,

Mppe1, Impa2, Cidea, and Tubb6. Since our goal is to

identify the molecular mechanism underlying generation of

ETTs, these listed genes appear to be of significant interest

for exploring candidate gene(s) for ETTs.

Pmaip1/Noxa

Pmaip1 coding for phorbol-12-myristate-13-acetate-

induced protein 1 (PMAIP1), which is also known as Noxa

(Latin for damage), was identified as one of the candidates

for tumor suppressor. Overexpression of PMAIP1 caused

suppression of cell proliferation in human pancreatic cancer,

and RNAi-mediated knockdown of PMAIP1 rescued the

suppressed cell proliferation (Ishida et al. 2008). Noxa

knock-out mice showed resistance to X-ray irradiation-

induced gastrointestinal death, which was accompanied by

impaired apoptosis of the epithelial cells of small intestinal

crypts, indicating the contribution of Noxa to the p53

response in vivo (Shibue et al. 2003). Double knock-out mice

lacking expression of both Noxa and its related gene p53

upregulated modulator of apoptosis (Puma) were unable to

undergo p53-mediated apoptosis, G1/S cell-cycle arrest, and

senescence (Valente et al. 2013). All these report

demonstrate that Pmaip1/Noxa is responsible for tumor

suppression, and suggest that it is a possible candidate for

ett1.

Mc4r

The melanocortin system consists of several agonists, two

antagonists, and five receptors (Gantz et al. 1993a; Gantz

et al. 1993b; Gantz et al. 1994; Mountjoy et al. 1992). The

agonists, including a-MSH, b-MSH, c-MSH, and ACTH, are

all derived from tissue-specific posttranslational processing

of a pre-prohormone, pro-opimelanocortin (POMC) (Ber-

tagna 1994; Smith and Funder 1988). Mc4r coding for

melanocortin four receptor (MC4R) is known to involve in

the regulation of both food intake and energy expenditure

(Kublaoui et al. 2006). MC4R is by coupling to heterotri-

meric stimulatory G-protein (Gs). Receptor activation leads

to increased cAMP production, consequently protein kinase

A (PKA) activation and inhibits the c-Jun N-terminal kinase

(JNK) (Tao 2010). Generally, Mc4r is known as a gene

related to the regulation of energy balance and energy

dynamic balance, because disruption of Mc4r by gene tar-

geting resulted in maturity onset obesity syndrome of mice

(Huszar et al. 1997). However, there are some reports that

Mc4r is associated with tumors. 129/Sv-Ay (Ay/?) males

have one tenth as many testicular tumors as their wild-type

littermates (Stevens 1967a). Ay is dominant mutation of the

agouti gene, which results in ectopic expression of agouti

gene product and leads to chronic inhibition of signal

transduction mediated through the melanocortin one recep-

tor (MC1R) and MC4R (Bultman et al. 1992; Dinulescu and

Cone 2000; Miller et al. 1993). Although ectopic expression

of agouti in transgenic did not affect the incidence of TGCT

(Heaney et al. 2009). When a small molecule MC4R inverse

agonist was administrated to mice grafted with Lewis lung

carcinoma tumors, it reached the central nervous system and

functioned to reduce tumor-induced weight loss in those

mice (Nicholson et al. 2006). These results support the notion

that Mc4r might function as a mediator of signal transduction

leading to generation of ETTs.

Gnal

Gnal encodes guanine nucleotide-binding protein (G-pro-

tein) olfactory type (GNAL), which is also termed het-

erotrimeric G-protein stimulatory alpha subunit, Golf

(Strathmann et al. 1989). Several reports have identified

disturbance in G-protein activity and/or expression in

bipolar affective disorder, as well as effect of subchronic

dosing of lithium on Golf (Corradi et al. 2005). Unfortu-

nately, possible connection between Gnal and ETT remains

unknown at present.

T. Miyazaki et al.: ett1 experimental testicular teratoma locus

123

Chmp1b

Chmp1b encodes charged multivesicular body protein 1B

(CHMP1B) protein, which is one of the component of

endosomal sorting complex required for transport (ESC-

RT)-III complex (Tanaka et al. 2008). The dysregulation of

ESCRT proteins results in development of various human

diseases, including many types of cancers and neurode-

generative disorders. Thus, Chmp1b is also a likely can-

didate for ett1.

Mppe1

Mppe1encodes metallophosphoesterase 1 (MPPE1) protein

that contains metal binding and active sites similar to

serine/threonine phosphoprotein phosphatase catalytic

subunits. MPPE1 is known to participate in the addition of

the glycosylphosphatidylinositol (GPI) moiety to proteins

(Fujita et al. 2009). Although there is no direct evidence for

correlation between Mppe1 and tumorigenesis at present,

studies on Mppe1 expression have demonstrated that down-

regulation of Mppe1 mRNA expression occurs in mela-

noma (Hoek 2007). Thus, Mppe1 might be one of the

candidates for ett1.

Impa2

Impa2 encodes inositol monophosphatase 2 (IMPA2) that

belongs to the superfamily of Mg2?-dependent, lithium-

blockable phosphatase (Sjoholt et al. 2000). IMPA2 KO

mice have lower kidney inositol levels, suggesting that

Impa2 contributes to kidney inositol monophosphatase

activity (Cryns et al. 2007). We could not find out any

connection between Impa2 and ETT.

Cidea

Cidea encodes CIDE-A, one (A) of three members (A, B,

and C) of the cell death-inducing DNA fragmentation

factor-45-like effector (CIDE) family of proteins, that is

highly enriched in brown adipose tissue, and known to play

a critical role in adaptive thermogenesis and fat accumu-

lation. Cidea-null mice have increased energy expenditure

with resistance to high fat diet-induced obesity and dia-

betes (Zhou et al. 2003). It is also suggested that CIDE-A

functions as DNA fragmentation factor (DFF45)-inhibita-

ble effectors that promote cell death and DNA fragmen-

tation (Inohara et al. 1998). Thus, CIDE-A involves in

apoptosis. Also, CIDE-A is known as novel hypermethy-

lated markers in endometrial cancer (Huang et al. 2010).

Since ETT is closely related to apoptosis and cancer, Cidea

can be one of the candidates for ett1.

Tubb6

b-tubulins are microtubule components encode by a multi-

gene family, which produces slightly different proteins with

complex expression patterns (Cleveland 1987; Hiser et al.

2006). Tubb6 encodes tubulin b6 isotype (TUBB6) belong-

ing to class V that is ubiquitously distributed in various tis-

sues, although TUBB6 is abundantly expressed in heart.

Tubb6 expression was largely decreased in tumors derived

from various tissues, except for renal tumors (Leandro-

Garcia et al. 2010). Mouse TUBB6 has been shown in vitro to

exhibit its unique properties concerning microtubule-based

organizations, such as dose-dependent disruption of micro-

tubule organization, increased microtubule fragmentation,

and concomitant reduction in cellular microtubule polymer

levels. These changes also cause disruption of mitotic spin-

dle assembly and blockade of cell proliferation (Bhattach-

arya and Cabral 2004). There is a possibility that changes in

cell proliferation caused by TUBB6 expression may affect

ETT development.

To correlate those listed genes with ETT development,

experiments using RT- or q-PCR are now underway to seek

possible difference in the levels of mRNA expression in

fetal testis between LT-ett1 congenic and LT strains.

How does the ett1 act in transformation of PGC to ETTs

upon testicular grafting of fetal testes? Normally, PGCs in

the 129 males can differentiate into male germ cells and

finally to mature sperm. Furthermore, EC (embryonal car-

cinoma) cells derived from STTs developed in 129/Sv-?/Ter

strain can be normalized after injection into the normal

blastocysts (Hanaoka et al. 1991). These findings suggest

that the coding region of the ett1 gene in the genome of 129

strain may be normal. It seems that mutation in the non-

coding region of ett1 gene, which would elicit a defect in

gene expressions such as abnormal splicing of ett1 mRNA,

or mutations in silencer or enhancer of ett1 gene, may cause

transformation of PGC into ETTs upon testicular grafting.

Analysis by q-PCR for exploration of ett1 mRNA would thus

be a key factor for answering the above question. As indi-

cated previously, the PGCs in the 129 strain tend to be

transformed into undifferentiated tumor cells like embryonic

stem (ES) cells under specific conditions such as ectopic

grafting of fetal testes and in vitro culturing of PGCs. In this

process, expression of differentiation/proliferation-related

genes that would force PGCs to change into ES cells may

commence. The product of ett1 gene may involve in such

process as a modifier. Generally, the fate of PGCs is affected

by several factors through interaction between PGCs and

their surrounding somatic cells (Kierszenbaum and Tres

2001; Skinner 1991). The ett1 may act as signal-like factor

that mediates interaction between PGCs and their sur-

rounding somatic cells, and overproduction of ett1 product

T. Miyazaki et al.: ett1 experimental testicular teratoma locus

123

would stimulate proliferation of PGCs. From this point of

view, Pmaip1, Mc4r, Gnal, Mppe1, and Impa2 could be

promising candidates of ett1.

In fact, we have obtained experimental data supporting

the above notion using testes reconstituted with PGCs and

testicular somatic cells. When PGCs from LT strain are

mixed with the somatic cells from fetal testes of 129 strain,

PGCs in the resulting reconstituted testes developed to

ETTs. In contrast, mixture between the 129 strain-derived

PGCs and the LT strain-derived testicular somatic cells

resulted in normal spermatogenesis and did not elicit ETTs

(Noguchi et al. unpublished). These findings suggested that

testicular somatic cells of 129 strain produce factors forc-

ing PGCs to develop into teratomas (Takabayashi et al.

2001). However, opposed result reported that ETT only

developed from PGCs of 129 strain (Regenass et al. 1982).

Ability of PGCs to form ETT is largely dependent on the

conditions before the transplantation. Changes of gene

expression patterns between the strains and during the

course of transplantation should be analyzed

Regarding Chr 19, Tgct1 (testicular germ cell tumor 1)

locus has been reported as a STT resistant one located near

D19Mit5 (35.3 Mbp, 34.0 cM) in the 129 strain back-

ground (Matin et al. 1999; Youngren et al. 2003). In an

attempt to examine whether the D19Mit5 locus can affect

the efficiency of ETT development, we examined SSLP in

the ETT-generating and non-generating mice. v2 values at

regions near D19Mit5 including D19Mit39 (29.2 Mbp,

24.0 cM) and D19Mit119 (41.2 Mbp, 27.5 cM) were very

low, indicating no linkage between ETT development and

D19Mit5 locus (Table S1 C, D). Thus, the Tgct1 locus

appears not to involve in ETT formation or the ETT

resistance phenotype.

Acknowledgments This work was supported by JSPS KAKENHI

Grant Number 12680809, 14380381 and 16650092 (to MN). This

work also supported by Grants-in-Aid for Scientific Research in

Priority Areas from the Ministry of Education, Culture, Sports, Sci-

ence and Technology of Japan for preservation of special lines of

mice to Shizuoka University.

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