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Institute for Laboratory Animal Science
Hannover Medical School
Dnd1: From Germ Cells to Teratomas in the Rat
THESIS
Submitted in partial fulfillment of the requirements for the degree
Doctor of Philosophy (PhD)
in
Laboratory Animal Science
at the University of Veterinary Medicine Hannover
by
Emily Northrup
(Nürnberg)
Hannover, Germany 2011
Supervisor: Prof. Dr. H.-J. Hedrich
Advisory committee: Prof. Dr. H.-J. Hedrich
Prof. Dr. U. Martin
Prof. Dr. I. Greiser-Wilke († 2010)
Prof. Dr. L. Haas
1st evaluation: Prof. Dr. H.-J. Hedrich (Hannover Medical School, Institute for
Laboratory Animal Science)
Prof. Dr. L. Haas (University of Veterinary Medicine Hannover,
Institute of Virology)
Prof. Dr. U. Martin (Hannover Medical School, Department of
Cardiothoracic, Transplantation and Vascular Surgery)
2nd evaluation: Prof. Dr. H.-J. Jacobsen (Leibniz Universität Hannover,
Institute for plant genetics)
Date of the oral examination: May 6, 2011
This study was funded by the German Cluster of Excellence REBIRTH
“From Regenerative Biology to Reconstructive Therapy”.
To my family
“It is a bad plan that
admits of no modification”
Publilius Syrus
Table of contents I
Table of contents
Publications ............................................................................................................. IV
List of abbreviations ............................................................................................... VI
1. Introduction ....................................................................................................... 1
2. Literature review ................................................................................................ 2
2.1. The germline ................................................................................................ 2
2.1.1. The germline: linking generations .......................................................... 2
2.1.2. Cell cycle, mitosis and meiosis ............................................................... 2
2.1.3. Germ cells during embryonic development ............................................ 3
2.1.4. Gametogenesis ...................................................................................... 5
2.2. Germ Cell Tumors ........................................................................................ 6
2.2.1. Classification and origin of germ cell tumors .......................................... 6
2.2.2. Teratomas and Teratocarcinomas ......................................................... 7
2.2.3. Incidence of germ cell tumors in humans ............................................... 8
2.2.4. Genetic and environmental factors in human GCTs ............................... 9
2.2.5. Infertility and TGCTs ............................................................................ 10
2.2.6. Teratomas in mice ................................................................................ 11
2.2.7. Teratomas in rats ................................................................................. 12
2.3. The Dead end homolog 1 gene (Dnd1) ...................................................... 13
2.3.1. Dead end (dnd) in anamnia .................................................................. 13
2.3.2. The Ter mutation in the mouse Dead end homolog 1 (Dnd1) .............. 14
2.3.3. Modulating the phenotype of Ter/Ter mice ........................................... 15
2.3.4. Dead end homolog 1 (DND1) in humans ............................................. 16
2.3.5. The RNA binding protein Dnd1 ............................................................ 17
2.3.6. Zebrafish dnd, miRNAs and target genes ............................................ 17
2.3.7. Mammalian Dnd1, miRNAs and target genes ...................................... 18
2.3.8. Interactions of Dnd1 with Apobec ......................................................... 19
2.3.9. Role of Dnd1 in germ cell cycle and gene expression .......................... 20
2.4. Pluripotent germ cells and their in vivo origin ............................................. 21
2.4.1. Primordial germ cell specification in the mouse ................................... 21
2.4.2. Identification of murine PGCs............................................................... 21
II Table of contents
2.4.3. Defining pluripotency ............................................................................ 23
2.4.4. Differentiation potential of PGCs .......................................................... 24
2.4.5. Embryonic stem cells ........................................................................... 24
2.4.6. Embryonic carcinoma cells................................................................... 25
2.4.7. Features of rodent embryonic germ cell derivation and culture ............ 26
2.4.8. The 2i-LIF media enabling culture of EGC and ESC ............................ 26
2.4.9. Epigenetic signature of PGCs and EGCs ............................................. 27
2.4.10. EGCs from different species ................................................................ 28
2.4.11. From ESC to PGCs .............................................................................. 29
3. Goals and objectives ....................................................................................... 30
4. The ter mutation in the rat Dnd1 gene causes gonadal teratomas and
infertility in both genders ............................................................................... 31
4.1. Abstract ...................................................................................................... 32
4.2. Introduction ................................................................................................ 32
4.3. Material and Methods ................................................................................. 35
4.4. Results ....................................................................................................... 39
4.5. Discussion .................................................................................................. 42
4.6. Figures ....................................................................................................... 46
5. Loss of Dnd1 facilitates the cultivation of genital ridge-derived rat
embryonic germ cells...................................................................................... 54
5.1. Abstract ...................................................................................................... 55
5.2. Introduction ................................................................................................ 55
5.3. Material and Methods ................................................................................. 57
5.4. Results ....................................................................................................... 62
5.5. Discussion .................................................................................................. 65
5.6. Figures ....................................................................................................... 69
5.7. Supplementary data ................................................................................... 75
6. Discussion and Conclusions ......................................................................... 79
6.1. The WKY-Dnd1ter/Ztm rat as a model for germ cell tumors ........................ 79
6.1.1. Highly conserved functions of Dnd1 in germ cell development ............ 80
6.1.2. Gonadal tumorigenesis and infertility in rodents ................................... 80
Table of contents III
6.1.3. Implications of the ter rat for human TDS and OGCTs ......................... 81
6.1.4. Gender and environment ..................................................................... 82
6.1.5. Genetic background ............................................................................. 83
6.1.6. DND1 in humans .................................................................................. 83
6.2. Derivation of pluripotent EGCs from the WKY-Dnd1ter/Ztm rat ................... 84
6.2.1. EGCs as a new approach to study rat Dnd1 ........................................ 85
6.2.2. The culture of EGCs ............................................................................. 86
6.2.3. Molecular networks of pluripotency in EGCs ........................................ 88
6.2.4. Germline competence of rat EGCs ...................................................... 89
6.3. Dnd1-related molecular mechanisms at work in germ cells ....................... 90
6.3.1. Interactions of Dnd1 and miRNAs ........................................................ 90
6.3.2. Effects of Dnd1 on EGC derivation and culture .................................... 90
6.3.3. Onset of germ cell loss and transformation .......................................... 92
6.3.4. Dnd1 in mitosis and meiosis................................................................. 92
6.4. Final conclusion and future perspectives ................................................... 93
7. Summary .......................................................................................................... 96
8. Zusammenfassung .......................................................................................... 99
9. References ..................................................................................................... 102
Acknowledgements .............................................................................................. 118
IV Publications
Publications
Research articles:
Northrup E, Zschemisch NH, Eisenblätter R, Glage S, Wedekind D, Cuppen E,
Dorsch M, Hedrich HJ. submitted. The ter mutation in the rat Dnd1 gene
causes gonadal teratomas and infertility in both genders.
Nils-Holger Zschemisch and Emily Northrup contributed equally to this work.
Northrup E, Eisenblätter R, Glage S, Rudolph C, Dorsch M, Hedrich HJ, Zschemisch
NH. submitted. Loss of Dnd1 facilitates the cultivation of genital ridge-derived
rat embryonic germ cells.
Held N, Smits BMG, Gockeln R, Schubert S, Nave H, Northrup E, Cuppen E, Hedrich
HJ, Wedekind D. 2011. A Mutation in Myo15 Leads to Usher-Like Symptoms
in LEW-ci2 rats. PlosOne 6(3): e15669.
Presentations at scientific meetings:
Northrup E, Eisenblätter R, Dorsch M, Hedrich HJ, Zschemisch NH; Loss of Dnd1
leads to immortalization of pluripotent rat primordial germ cells in vitro; Rat
Genomics and Models; December 2-5, 2009 in Cold Spring Harbor, New
York, USA
Zschemisch NH, Ji D, Wu Y, Northrup E, Dorsch M, McCoy A, Little L, Hedrich HJ,
Weinstein E, Cui X; Zinc-finger nuclease-mediated disruption of Rag-1 in the
rat; 9th Transgenic Technology Meeting; March 22-24, 2010 in Berlin,
Germany
Transgenic Research, 19:P355, 2010
Northrup E, Eisenblätter R, Dorsch M, Glage S., Rudolph C, Hedrich HJ, Zschemisch
NH; In vitro survival of pluripotent rat embryonic germ cells correlates with
Dnd1 deficiency; Vets for life, 11th Joint Scientific meeting of ESLAV, LAVA
and ComVet AFSTAL; September 26-28, 2010 in Toulouse, France
Publications V
Zschemisch NH, Eisenblätter R, Northrup E, Rudolph C, Dorsch M, Hedrich HJ;
Cultivation and characterization of embryonic stem cells from the WKY/Ztm
rats; The 18th International Workshop on Genetic Systems in the Rat;
November 30-December 3, 2010 in Kyoto, Japan
Northrup E, Eisenblätter R, Glage S, Rudolph C, Dorsch M, Hedrich HJ, Zschemisch
NH; Loss of Dnd1 facilitates the cultivation of genital ridge-derived rat
embryonic germ cells; The 18th International Workshop on Genetic Systems in
the Rat; November 30-December 3, 2010 in Kyoto, Japan
VI List of abbreviations
List of abbreviations
2i two inhibitors
+/+ wild type
μg microgram
μl microliter
μM micromolar
A adenine
AP alkaline phosphatase
Az. Aktenzeichen
bFGF basic fibroblast growth
factor
Bmp bone morphogenetic
protein
bp base pair
C cytosine
cDNA copy DNA
CDK cyclin-dependent kinase
cm centimeter
Chr chromosome
CIS carcinoma in situ
c-terminus carboxyl-terminus
CO2 carbon dioxide
d day
d pc day post coitum
DDX4 DEAD box polypeptide 4
(synonyms: Mvh, Vasa,
RVLG)
DNA deoxyribonucleic acid
dnd dead end
Dnd1 dead end homolog 1
EB embryoid body
EC embryonal carcinoma
ECC embryonic carcinoma cell
EGC embryonic germ cell
ESC embryonic stem cell
F filial generation
FCS fetal calf serum
FELASA Federation of European
Laboratory Animal
Science Associations
fig. figure
g gram
G guanine
G0, G1, G2 gap 0, gap 1, gap 2
GCT germ cell tumor
GSK-3 glycogen synthase
kinase-3
h hour
HE hematoxylin-eosin
HSA human chromosome
ICM inner cell mass
Ig immunoglobulin
iPS cell induced pluripotent stem
cell
ITGCN intratubular germ cell
neoplasia
(synonym: CIS)
LIF leukemia inhibitory factor
LOD logarithmic odds ratio
M mitosis (chapter 2 and 6)
M molar (chapter 4 and 5)
List of abbreviations VII
min minute
miRNA/miR microRNA
ml milliliter
mM millimolar
mm millimeter
MMU mouse chromosome
mRNA messenger RNA
Mvh mouse vasa homolog
ng nanogram
nm nanometer
nt nucleotide
OGCT ovarian germ cell tumor
p short chromosome arm
PBS phosphate buffered
saline
PCR polymerase chain
reaction
PGC primordial germ cells
pmol picomolar
q long chromosome arm
RBP RNA binding protein
RRM RNA recognition motif
rEGC rat EGC
RNA ribonucleic acid
RNO rat chromosome
RT reverse transcription
S synthesis
s second
SCF stem cell factor
SD standard deviation
SD Sprague Dawley
(chapter 6)
SNP single-nucleotide
polymorphism
Sry Sex-determining region Y
suppl. supplementary
T thymine
TDS testicular dysgenesis
syndrome
TGCT testicular germ cell tumor
UTR untranslated region
WHO World Health
Organization
YST yolk-sac tumor
Introduction 1
1. Introduction
Teratomas are germ cell tumors (GCTs) consisting of various tissues from all three
germ layers. The malignant version, the teratocarcinoma, also includes
undifferentiated embryonic carcinoma cells (ECCs). It has been confirmed that the
pluripotent ECCs are the actual cancer stem cells capable of differentiating into the
various tumor tissues (Kleinsmith & Pierce, 1964). ECCs are thought to result from
neoplastic transformation of the embryonic precursor cells of the gametes, the so-
called primordial germ cells (PGCs) (Stevens, 1967; Andrews, 1998). Testicular
GCTs are often associated with male infertility and although the molecular
mechanisms remain unknown, it is apparent that malfunctions in testis stem cell
regulation and DNA fidelity can lead to either. Both genetic and environmental factors
influence the incidence of GCTs as well as infertility (Hotaling & Walsh, 2009).
The inactivation of the gene dead end homolog 1 (Dnd1) can lead to reduced fertility
in various vertebrates and gonadal teratocarcinogenesis in mice. The Ter mutation in
the Dnd1 gene of the mouse results in a depletion of PGCs causing sterility in males
and reduced fertility in females. In males some of the PGCs dodge their demise and
are transformed into ECCs leading to a heightened incidence of testicular teratomas
in Ter mice (Noguchi & Noguchi, 1985; Youngren et al., 2005). Ordinarily, teratomas
have a rare occurrence in rodents. Nevertheless, a spontaneous mutation referred to
as ter has been described in the WKY/Ztm rat strain, which led to the formation of
congenital teratomas in the ovaries and testes along with sterility (Länger et al.,
2004). The first objective of this project was to identify the mutation and gather
further information on the phenotype of the mutant rat strain.
PGCs are highly specialized cells that eventually develop into mature oocytes or
sperm. However, at the same time they must maintain the ability exhibited by the
gametes to differentiate into all cell types after fertilization (McLaren, 2003).
Therefore, ontogenetic disorders cannot only lead to a loss of PGC that causes
infertility, but also to the development of teratoma-predecessors, or even to both as
seen in the WKY/Ztm-ter rat. ECC are not the only pluripotent cells derived from
PGCs. Through in vitro culture, PGCs can be reprogrammed into another type of
2 Literature review
pluripotent cells referred to as embryonic germ cells (EGCs). EGCs and to a certain
extent also ECCs possess similar characteristics as embryonic stem cells (ESCs)
(Matsui et al., 1992; Resnick et al., 1992). Culturing wild type and ter-mutant EGCs of
both genders from PGCs of the genital ridge was the second goal of this project. For
one, we wanted the proof of principal that genital ridge-derived EGC culture is
possible in the rat. For another, establishing the EGCs culture creates an ideal and
innovative tool to study teratocarcinogenesis.
2. Literature review
2.1. The germline
2.1.1. The germline: linking generations
In sexually reproducing organisms all the genetic information passed from parents to
their offspring is transmitted through cells of the germline. Early on in embryogenesis,
the first germ cells are specified and can be distinguished from surrounding somatic
cells. This is followed by a series of pre- and postnatal proliferation and differentiation
steps until the first fully differentiated gametes develop during puberty. The fusion of
oocyte and sperm at fertilization leads to a totipotent cell with the ability to create a
new organism by combining genes derived from two parents. The fertilized oocyte is
capable of differentiating into all cell types of both the somatic line and germline, as
well as any cell of the extraembryonic membranes. This brings the germline to a full
circle of specification, differentiation and fertilization that repeats itself over and over
(Seydoux & Braun, 2006; Western, 2009). Thus, the germ cell lineage is frequently
referred to as immortal and its DNA is passed from one generation to the next, while
the somatic cell line simply exists for one lifetime (Jones, 2007; Surani, 2007;
Nayernia, 2008).
2.1.2. Cell cycle, mitosis and meiosis
The cell cycle is an ordered set of events with complex control mechanisms by which
eukaryotic cells reproduce. The four stages are G1 (gap 1), S (synthesis of DNA), G2
Literature review 3
(gap 2: cells prepare for division) and M (mitosis). Mitosis can be subdivided into
prophase, prometaphase, metaphase, anaphase and telophase. It produces two
daughter cells that are identical to the parental cells (Wolgemuth et al., 2002). A cell
can exit the cell cycle at G1 to enter G0, where it maintains its specialized functions
but no longer undergoes cell division (Johnson & Walker, 1999). Gametes are
produced by meiosis, which involves two divisions producing a total of four
genetically different cells with a haploid chromosome set. Recombination of parental
chromosomes during meiosis is responsible for the genetic variety of the daughter
cells (Hochwagen, 2008). In contrast to mitosis, meiosis consists of two successive
meiotic M-phases without an intermediate S-phase (Brunet et al., 1999). Meiosis I
leads to a reduction of chromosomes and is followed by meiosis II in which cells
undergo a division without further chromosome reduction (Hochwagen, 2008).
Analogous to mitosis, each of the meiotic divisions subsist of prophase, metaphase,
anaphase and telophase. The first meiotic prophase distinguishes between the
stages leptotene, zygotene, pachytene, diplotene and diakinesis (Page & Hawley,
2003; Nicholas et al., 2009).
2.1.3. Germ cells during embryonic development
A common signaling and developmental pathway is very probable for mammals
during germ cell development (Donovan & de Miguel, 2003; Aflatoonian & Moore,
2006; De Miguel et al., 2010). Detailed information about the rat is hard to come by,
while their close relative, the mouse, has been extensively studied. In accordance
with the existing data for mouse and rat (Witschi, 1962; McLaren, 2003; Maatouk et
al., 2006; Leitch et al., 2010), it is likely that similar pathways exists for both species,
with the rat being one to three days behind in its embryonic development (Fujinaga et
al., 1992; Hill, 2010). Therefore, the following account and timeline of germ cell
development is that of the mouse, unless specified otherwise.
Embryogenesis starts with fertilization and the formation of the zygote, leading to the
blastocyst around day 3.5 post coitum (d pc) in the mouse and one day later in the
rat (Schmidt & von Kreybig, 1965; Prather & First, 1988; De Miguel et al., 2010). The
first ancestors of the PGCs originate at d6.25pc in the posterior epiblast, in close
proximity to the extraembryonic ectoderm, after Bmp (bone morphogenetic protein)
4 Literature review
signaling (Lawson et al., 1999; Ying et al., 2001; McLaren & Lawson, 2005). During
the course of gastrulation the germ cell precursors move through the posterior
primitive streak and into the extraembryonic region (McLaren, 2003). Final
specification does not occur until d7.25pc in a group of about 40 progenitor cells
present in the extraembryonic mesoderm at the base of the allantois (Saitou et al.,
2002; Chuva de Sousa Lopes et al., 2007). At d8.5pc, the endoderm invaginates to
form the hind gut and the PGCs move along with the endodermal cells. Most of the
PGCs migrate out of the hind gut and into the dorsal body wall by d9.5pc.
PGCs enter the genital ridges from d10-11pc and remain stationary from this time on
(McLaren, 2003). As expected, the rat PGCs enter the genital ridge two days later
between d12pc and 13pc (Kemper & Peters, 1987), while human PGCs enter the
genital ridge around the 6th week of gestation (De Miguel et al., 2010). By the time
the PGCs are located in the genital ridge, they are referred to as late PGCs (De Felici
et al., 2009), gonocytes (McLaren, 2003) or as oogonia and prospermatogonia after
sexual differentiation (Richards et al., 1999; De Felici et al., 2004; Pepling, 2006).
The PGCs undergo several mitotic divisions during migration, with numbers
increasing in the mouse from around 100 at d8.5pc to 1000 at d10.5pc. This is
followed by two or three further rounds of mitosis after arrival in the genital ridge,
reaching a total of 25000 germ cells in each gonad at d13.5pc (Tam & Snow, 1981;
Saiti & Lacham-Kaplan, 2007). PGCs of both genders enter a premeiotic stage and
upregulate meiotic genes by d12.5pc (McLaren, 2003).
Male and female PGCs were indistinguishable up to this point in development and
only now, at d12.5pc, can the first sexual differences be identified (Adams &
McLaren, 2002; Western, 2009; Sabour et al., 2010). Meiosis is initiated by the
expression of the stra8 (stimulated by retinoic acid 8) gene and at d13.5pc female
germ cells start entering meiotic prophase (Koubova et al., 2006; Saiti & Lacham-
Kaplan, 2007), which is marked by germ cell arrest at the G2/M stage of the cell cycle
(Cook & Capel, 2010; Miles et al., 2010). Meiotic entry is blocked in the male by a
retinoid-degrading enzyme (Bowles et al., 2006; Saiti & Lacham-Kaplan, 2007).
Therefore, the male germ cells enter mitotic arrest in G0 between d12.5pc and
Literature review 5
d14.5pc (Adams & McLaren, 2002; Western et al., 2008) and differentiate into
prospermatogonia (McLaren, 1984, 2003; Durcova-Hills & Capel, 2008).
2.1.4. Gametogenesis
Gametogenesis is the process of cell division and differentiation, leading to the
mature haploid gamete.
In males the prospermatogonia in G0 mitotic arrest resume proliferation in the first
week after birth as spermatogonia (Chuma et al., 2005; Phillips et al., 2010). At
puberty the spermatogonia start entering meiosis, and mature sperm is formed
(Donovan, 1998). Spermatogenesis takes place in the seminiferous tubules of the
testis and is completed in about 9 days in the mouse, 13 days in the rat, and 16 days
in humans. The epithelium of the tubules consists of Sertoli cells, which provide
nourishment, different stimuli and protection for the developing sperm. During the
proliferation phase the spermatogonia undergo mitotic divisions to form
spermatocytes, which divide by meiosis and form the haploid spermatids.
Metamorphosis of the spermatids, including structural modifications and acrosome
formation, finally leads to the development of spermatozoa (Hermo et al., 2010). A
subpopulation of spermatogonia, the spermatogonial stem cells, retains the unique
ability of self-renewal as well as the capacity of differentiating to spermatozoa.
The process of gametogenesis is quite different in females. Although there has been
some recent controversy whether neo-oogenesis might exist, it is certain that for the
most part the replication of oocytes ceases before birth (Schnorr, 1996; Notarianni,
2011). The female oogonia only divide by mitosis during embryogenesis. After
entering the meiotic prophase, they differentiate into primary oocytes that arrest in
the diplotene stage of prophase I around the time of birth (McLaren, 2003). This
stage corresponds to the G2 phase of the cell cycle (Goren & Dekel, 1994). At the
end of prophase I, the oocytes and a surrounding layer of epithelial cells generate the
primordial follicles. The meiotic resting phase that then begins is called the dictyotene
and lasts until puberty, when the first oocytes complete meiosis I, resulting in a polar
body and the secondary oocyte. Meiosis II follows suit as it is initiated by the haploid
secondary oocyte. The oocyte is released from the follicle during ovulation (Schnorr,
1996). In most vertebrates meiosis is halted at the metaphase II stage at the time of
6 Literature review
ovulation until fertilization occurs and the second meiotic division is completed
(Verlhac et al., 1994; Eppig et al., 1996).
2.2. Germ Cell Tumors
2.2.1. Classification and origin of germ cell tumors
Tumors classified as germ cell tumors (GCTs) are derived from cells belonging to the
germ lineage (Looijenga & Oosterhuis, 1999). It is a heterogeneous group of
neoplasms with varying histopathological and clinical manifestations (Bahrami et al.,
2007). For the most part analogous tumors can be found in ovary and testes,
however, even though the morphology is similar, incidence and malignity differ
between the genders (Ulbright, 2005). The modern classification system for human
GCTs was first proposed by Oosterhuis and Looijenga in 2005 and is recognized by
the World Health Organization (WHO). The four major types of GCTs diagnosed in
the gonad are as follows: type I (teratomas and yolk-sac tumors of neonates and
infants); type II (seminomas/dysgerminoma and non-seminomas); type III
(spermatocytic seminomas of the testes; typically in older men); type IV (Dermoid
cysts of the ovary) (Oosterhuis & Looijenga, 2005).
Type I GCTs are diagnosed before puberty and the biparental, partially erased
patterns of genomic imprinting suggest that the precursor cells are earlier
PGCs/gonocytes (Stevens, 1967; Oosterhuis & Looijenga, 2005). These tumors lack
typical precursor lesions (carcinoma in situ), and the PGCs are directly transformed
into ECCs (Bahrami et al., 2007; Kristensen et al., 2008). Teratomas are
karyotypically normal (Anderson et al., 2009) while the yolk-sac tumors (YST) may be
aneuploid (Looijenga & Oosterhuis, 1999).
GCTs type II are divided into two histological subgroups with different biological and
clinical features: seminomas/dysgerminoma and non-seminomas (von Eyben, 2004;
Hussain et al., 2008; McIntyre et al., 2008). Some GCTs contain elements of both
forms (McIntyre et al., 2008). Pure seminomas occur in the testes and consist of a
homogeneous cell population that has undergone gonadal differentiation and
resembles PGCs and gonocytes (McIntyre et al., 2008). Dysgerminomas are ovarian
germ cell tumors (OGCT) with an identical morphology as the seminoma (Ulbright,
Literature review 7
2005). The more malignant non-seminomas are a very heterogeneous group of
tumors that can contain different histological elements including teratoma, embryonal
carcinoma (EC), YST and choriocarcinoma (Lee-Jones, 2003; Oosterhuis &
Looijenga, 2005). Pure forms of non-seminomas are relatively rare and, instead,
mixed GCTs with a combination of components are more frequent (Lee-Jones, 2003;
Chieffi et al., 2009). Genomic imprinting is erased, and the development of the
aneuploid type II GCTs probably involves late PGCs/gonocytes that are blocked from
their physiological differentiation (Oosterhuis et al., 1989; Oosterhuis & Looijenga,
2005; van de Geijn et al., 2009).
All TGCTs type II stem from the precursor lesion carcinoma in situ (CIS) (known as
intratubular germ cell neoplasia, in short ITGCN, according to the WHO) localized in
the seminiferous tubules that progresses towards invasiveness. The CIS gives rise to
either seminoma or ECCs (Andrews, 1998; Leendert et al., 1999; Krausz &
Looijenga, 2008; Kristensen et al., 2008). Cells of the EC are the actual pluripotent
cancer stem cells (Kleinsmith & Pierce, 1964; Wicha et al., 2006) and have the ability
to differentiate into embryonal or adult tissue (teratoma) as well as extraembryonal
tissue (YST and choriocarcinoma) (von Eyben, 2004; van de Geijn et al., 2009).
In contrast to the TGCTs, little is known for certain about the pathogenesis of OGCTs
(Faulkner & Friedlander, 2000). The malignant dysgerminomas are expected to stem
from PGCs before meiosis of the oocytes (Hoei-Hansen et al., 2007), while the
dermoid cysts, also known as type IV benign ovarian teratomas, seem to stem from
germ cells having completed meiosis I (Ulbright, 2005). Postpubertal testis tumors
are often aneuploid and have a consistent chromosomal abnormality of HSA12p
(Oosterhuis & Looijenga, 2005; Ulbright, 2005; Bahrami et al., 2007) also seen in
malignant OGCTs. The histological, genetic, and biological similarities found in
malignant GCTs of ovary and testes suggest a shared pathogenesis (Faulkner &
Friedlander, 2000).
2.2.2. Teratomas and Teratocarcinomas
Teratomas develop from pluripotent ECCs and therefore contain normal derivatives
from all three germ layers: ectoderm, mesoderm and endoderm. They consist of an
assortment of tissues such as cartilage, muscle, epithelium and neuronal tissue and
8 Literature review
can include organoid structures such as teeth or bone fragments (Andrews, 1998;
Donovan, 1998; Bulic-Jakus et al., 2006).
Dermoid cysts of the ovary are usually classified as benign teratomas, albeit the fact
that they only contain derivatives of mesoderm and endoderm (Akgul Ozmen et al.,
2009). In some cases tumors consisting of one germ layer have been defined as
monodermal teratomas (Bahrami et al., 2007). A teratoma is considered immature
when it contains embryonal or other immature structures and mature when it consists
of differentiated, adult-like cells (Bosl & Motzer, 1997). Incomplete somatic
differentiation of the pluripotent cells leads to malignant teratocarcinomas, which
retain clusters of pluripotent ECC alongside the differentiated tissue (Andrews, 1998,
2002; Yu & Thomson, 2008). As teratocarcinomas consist of more than one germ cell
component, they belong to the group of mixed germ cell tumors (Stamatiou et al.,
2009).
Pure, mature human teratomas found in the ovaries and prepubertal testes are
generally benign while teratomas found in the postpubertal testes and immature
teratomas of the adult ovaries are often malignant (Ulbright, 2005). Human teratomas
are extragonadal or gonadal tumors and the main sites are the sacrococcygeal
region, the ovaries and the testes (Harms et al., 2006).
2.2.3. Incidence of germ cell tumors in humans
Type I GCTs include mature and immature teratomas as well as yolk sac tumors and
have a very low incidence of 0.5-2.0 of 100 000 (Nerli et al.). Although even the most
frequent of all TGCTs, type II TGCTs, are rare tumors and comprise only 1% of all
cancers in men, their importance derives from the fact that with 60% it is the most
common malignant tumor in 20-40 year old Caucasian men (Jemal et al., 2004;
Krausz & Looijenga, 2008; Greene et al., 2010). Furthermore, the incidence is on the
rise and has more than doubled over the past 50 years (Huyghe et al., 2003; Jemal
et al., 2004). The reasons for this development are still unclear.
GCTs account for 95% of all testicular tumors, split evenly between seminomas and
non-seminomas with a small fraction being a combination of both (Looijenga &
Oosterhuis, 1999; Hussain et al., 2008). According to Ulbright, pure teratomas
account for 4% of all TGCTs, while embryonal carcinomas make up 10%, and 33%
Literature review 9
are mixed germ cell tumors (Ulbright, 2005). The most frequent mixed GCT are
teratocarcinomas, which are responsible for 25% of all TGCTs (Stamatiou et al.,
2009).
Ovarian Germ Cell Tumors (OGCT) are mainly diagnosed in young women and
account for over 60% of all ovarian neoplasms in children and adolescents and for
15-20% in all age groups (Lee-Jones, 2003). The majority of OGCTs are benign and
the most frequent are the benign teratomas known as dermoid cyst, with an
incidence of 95% (Faulkner & Friedlander, 2000; Ulbright, 2005). Malignant OGCTs
account for the remaining 5% (Faulkner & Friedlander, 2000) and, with an incidence
of only 2%, dysgerminomas are the second most common OGCT (Ulbright, 2005).
2.2.4. Genetic and environmental factors in human GCTs
Multiple observations suggest that genetic factors play an important role in the
development of GCTs and could be a contributing factor in over a quarter of all TGCT
patients (Nicholson & Harland, 1995). Familial clusters of OGCTs, TGCTs or both
have been identified (Stettner et al., 1999; Giambartolomei et al., 2009; Greene et al.,
2010; Kratz et al., 2010), and family history is a significant risk factor increasing the
development of TGCT (Greene et al., 2010). Prevalence of TGCTs varies among
different races and geographic locations and is highest in Caucasians, especially in
the residents of Scandinavia, New Zealand and Germany (Bahrami et al., 2007). The
incidence of OGCT in the United States is elevated in Hispanics and Asian-Pacific
Islanders (Smith et al., 2006).
Pre-natal or post-natal environmental factors play a role in TGCT development as
immigrants to Sweden were shown to retain the risk of their native country, while the
incidence seen in their children is comparable to that of native Swedes (Hemminki &
Li, 2002). These factors have not been determined yet, but high maternal estrogen,
organic pollutants in the mother‟s blood or maternal smoking might affect testicular
development and increase chances of TGCTs in sons (Skakkebaek et al., 2007;
Chieffi et al., 2009). The risk is also augmented in some professions or by low birth
weight and small testes (Bosland, 1996; Sharpe, 2006). High maternal body mass
and the use of exogenous hormones during pregnancy raises the risk for OGCTs
(Walker et al., 1988).
10 Literature review
However, low-risk groups such as African Americans maintain their low TGCT risk
even when living in high-incidence areas, showing a case of genetics over
environment (Gajendran et al., 2005). Linkage analysis and simulation analysis
showed that multiple loci and not one major gene are responsible for TGCTs
(Crockford et al., 2006; Zhu et al., 2007a; Krausz & Looijenga, 2008). The overall
mutation rate is low in TGCTs in comparison to other solid cancers. Activating
mutations have been found in the proto-oncogene BRAF, in KRAS-2, c-kit and the
microRNA cluster 371-373 (Voorhoeve et al., 2006; Krausz & Looijenga, 2008).
2.2.5. Infertility and TGCTs
The exact molecular mechanisms remain unknown, but infertility is associated with
testicular cancer, and disturbances in DNA fidelity or stem cell regulation can lead to
either. Organized, high-speed cell divisions are essential in spermatogenesis and
precisely these are responsible for its high sensitivity to interruptions of germ cell
development by different genetic, hormonal and environmental factors (Hotaling &
Walsh, 2009). Infertile men exhibit an increased risk of developing testicular cancer;
furthermore declining fertility might be the first marker for TGCTs (Hotaling & Walsh,
2009; Walsh et al., 2009; Burns et al., 2010).
Skakkebaek and colleagues have suggested that poor semen quality, infertility,
testicular cancer, cryptorchidism and hypospadias are different symptoms belonging
to one entity named the testicular dysgenesis syndrome (TDS) (Skakkebaek et al.,
2001; Skakkebaek et al., 2003; Skakkebaek et al., 2007). Evidence for this is found
in the rising incidence and the similar geographic distributions of the different
symptoms (Hemminki & Li, 2002; Skakkebaek et al., 2007; Hotaling & Walsh, 2009).
The hypothesis is that disruptions in embryonal programming or gonadal
development occur during fetal life, and can result in any of the symptoms belonging
to TDS (Skakkebaek et al., 2001; Walsh et al., 2009; Burns et al., 2010).
The situation in women is a different one as, so far, there is no evidence linking
infertility to the development of GCTs in the ovary.
Literature review 11
2.2.6. Teratomas in mice
To date, all teratomas observed in mice most closely resemble prepubertal GCTs.
GCTs of mice and the infantile testis both arise from PGCs and lack CIS as well as
the karyotypic abnormalities (e.g. in HSA12p) found in adult TGCTs (Walt et al.,
1993; Anderson et al., 2009). Therefore, they are most likely a model for type I
GCTs, which also means that there are no animal models available for type II GCTs
(Looijenga & Oosterhuis, 1999; Oosterhuis & Looijenga, 2005). It has been
suggested that the lack of type II GCTs in rodents is related to their rapid embryonic
development and brief puberty not allowing the neoplastic germ cells to accumulate
further genetic and epigenetic changes (Zhu et al., 2007a). In general, GCTs are
uncommon in rodents (Stevens, 1980). Parthenogenetic oocytes arrested in the
metaphase of meiosis I can lead to ovarian teratomas in the inbred LT/Sv mouse
strain (Eppig et al., 1996). Only mice of the inbred strain 129/Sv develop testicular
teratomas spontaneously at a rate of 1-10% (Stevens & Hummel, 1957; Youngren et
al., 2005). The incidence of teratocarcinomas is considerably increased through the
Ter mutation of the Dnd1 gene in males of this, and only this, strain, while fertility is
reduced in both genders of numerous strains (Noguchi & Noguchi, 1985; Zhu et al.,
2007a). Loss of Dmrt1 causes a high incidence of teratomas in males of the same
129/Sv mouse strain, with only testicular dysgenesis and no tumors found in the
C57BL/6J as background strain (Krentz et al., 2009). The pgct1 locus on
chromosome (Chr) 13 segregates with the male teratoma phenotype of the 129/Sv
strain (Muller et al., 2000). Furthermore, the chromosome substitution strain
129.MOLF-Chr19 in which Chr 19 of 129/Sv+/+ was replaced by its MOLF-derived
homologue shows a high frequency (70-80%) of GCTs in the testis. The PGC-
specific knockout of Pten (phosphatase and tensin homolog) causes teratomas in all
male and even in some female mice and coincides with PGC loss (Kimura et al.,
2003). Another tumor suppressor whose absence leads to TGCTs is p53, which may
play a role in promoting cell cycle arrest in PGCs around d13.5pc (Matin, 2007).
Interruptions in germ cell development can also cause infertility without teratoma
development. This is seen in mice carrying the germ cell-deficient mutation caused
by the deletion of the Fancl gene (Pellas et al., 1991; Agoulnik et al., 2002), as well
12 Literature review
as in mice with the homozygous dominant white spotting or steel mutation of the loci
encoding for the c-kit receptor or its ligand the stem cell factor (also known as SCF or
Kitl), respectively. However, teratoma incidence is increased in heterozygous
steel/wild type males (Stevens, 1981; Noguchi & Noguchi, 1985; Donovan & de
Miguel, 2003).
2.2.7. Teratomas in rats
Teratomas rarely occur in rats. A few isolated cases of spontaneous teratogenesis
have been observed in the adrenal gland, kidney, central nervous system and
abdomen of Sprague-Dawley and Wistar rats (Schardein & Fitzgerald, 1977;
Ninomiya, 1983; Itoh et al., 1985). Hereditary teratomas of the ovary and testes have
been found in the Csk:Wistar-Imamichi strain in Japan. The strain was denominated
Tera, and a mutation with a recessive mode of inheritance was thought to cause the
teratomas (Miwa et al., 1987). However, this strain no longer exists and the
underlying mutation was never identified. Länger and colleagues have described a
spontaneous mutation in the inbred WKY/Ztm rat strain leading to teratocarcinomas
of the ovary and testes (Länger et al., 2004). They established a coisogenic mutant
strain by mating the ancestors of the affected animals. Analogous to the Ter mutation
in the mouse and based on the hypothesis of a single recessive ter allele causing the
teratomas, this new animal model for teratomas was denominated WKY/Ztm-ter.
Unlike the Ter mouse, where tumors only arise in males, the ter rat developed
teratomas in both genders with an ongoing high incidence of 25%. However, tumor
progression was influenced by gender, and the females developed mostly bilateral
tumors between day 21 and 63, while the males developed tumors between day 14
and 224, with 50% being unilateral. The partially undifferentiated teratocarcinomas
consisted of derivatives from all three germ layers, approximately 10%
undifferentiated cells and also included immature tissue resembling embryonic
structures (Länger et al., 2004).
Literature review 13
2.3. The Dead end homolog 1 gene (Dnd1)
The dead end genes play an important role in survival, migration and development of
primordial germ cells in different species. The dead end homolog 1 genes in mice
(Dnd1), rats (Dnd1) and humans (DND1) are orthologs of the dead end (dnd) gene
expressed in zebrafish and African clawed frog (Youngren et al., 2005).
2.3.1. Dead end (dnd) in anamnia
In some non-mammalian species, such as zebrafish (Danio rerio) and the African
clawed frog (Xenopus laevis), the germ cell lineage is established by the assembly of
germ plasm in the cytoplasm of the oocyte. Cells inheriting the preformed germ
plasm, which is rich in RNAs, RNA-binding proteins and ribosomes, ultimately give
rise to the germ cell lineage (Fox et al., 2007; Richardson & Lehmann, 2010).
Therefore, the germ plasm is one of the unique features of PGCs (Kedde & Agami,
2008), and several germ plasm components involved in the early PGC development
in lower organisms are also expressed in late mammalian PGCs of the genital ridge
(Weidinger et al., 2003). Dead end (dnd) is specifically expressed in the germ plasm
of zebrafish and the African clawed frog throughout embryogenesis (Weidinger et al.,
2003; Horvay et al., 2006). The 3‟untranslated region (UTR) of dnd in zebrafish is
responsible for the germ cell specific expression as 3‟UTR-dnd injected into the 1-cell
stage is degraded in somatic cells and protected in germ cells (Slanchev et al.,
2009).
Knockdown of dnd in zebrafish initially allows normal PGC specification, however, it
then blocks the restriction of PGCs to the deep blastoderm and leads to large
numbers of ectopic PGCs in the outermost cell layer. Furthermore, PGCs no longer
exhibit a motile behavior, and failure to migrate is followed by the demise of PGCs on
the first day of development (Weidinger et al., 2003). PGCs of the African clawed frog
disappear at the tadpole stage of the embryos as a consequence of inhibiting the dnd
translation. Early specification does not appear to be affected, but PGCs do not
migrate dorsally and cease to exist (Horvay et al., 2006).
Consequently, Dnd is required for PGC migration in both species, with no effects
observed in PGC specification or somatic cells. The similar phenotype observed in
14 Literature review
anamnia, along with the reciprocal functional redundancy of mouse and zebrafish
Dead end indicates an evolutionary conserved role of dnd in germ cell development
in vertebrates (Kedde et al., 2007; Slanchev et al., 2009).
2.3.2. The Ter mutation in the mouse Dead end homolog 1 (Dnd1)
The spontaneous ter mutation that took place in the 129/Sv strain some 40 years ago
(Stevens, 1973) has been traced to the inactivation of the Dead end homolog 1
(Dnd1) gene on mouse Chr 18. A single base change (cytosine to thymine)
introduces a termination codon in the coding region of Dnd1 and ends up causing
germ cell deficiency in both genders along with testicular teratomas (Asada et al.,
1994; Matin & Nadeau, 2005; Youngren et al., 2005). The teratoma incidence in male
129/Sv-Ter mice was 94% in homozygous Ter/Ter and 17% in heterozygous Ter/+
mice in comparison to the 1.4% in wild type (+/+) mice. Teratocarcinogenesis in
Ter/Ter males was unilateral in 25% of the cases and associated with reduced size
and weight of non-tumorous testis (Noguchi & Noguchi, 1985). No germ cells are
visible in Ter/Ter males after birth as all PGCs either perish or transform into ECC
(Zhu et al., 2007a). Germ cell number is not affected in Ter/+ as there were no signs
of germ cell deficiency in mice presumed to be Ter/+ (Noguchi & Noguchi, 1985), and
the number of PGCs in Ter/+ embryos did not differ from that in +/+ embryos
(Sakurai et al., 1995). Although all homozygous males are sterile, a number of the
females remain fertile. Decreased fertility in Ter/Ter females is linked to smaller
ovaries containing only 10% of the oocytes found in wild type 129/Sv mice (Stevens,
1973; Noguchi & Noguchi, 1985). Transfer of Ter onto other genetic backgrounds
resulted in germ-cell deficient mice with small gonads, but without
teratocarcinogenesis (Noguchi & Noguchi, 1985; Sakurai et al., 1995). Therefore, the
primary effect of the Dnd1 mutation in homozygous mice is the progressive loss of
PGCs in Ter/Ter animals during embryonic development. PGC reduction starts at
d8pc in all congenic strains and can be seen throughout the migratory and
proliferative phase (Sakurai et al., 1995). The migration of the PGCs to the genital
ridge does not seem to be affected by the Dnd1 deficit (Sakurai et al., 1995;
Youngren et al., 2005). Tumor development is presumed to start around embryonic
d12.5pc and the PGCs are transformed into EC cells that are first visible in the gonad
Literature review 15
at d14.5pc (Noguchi & Stevens, 1982; Rivers & Hamilton, 1986; Matin, 2007). They
proliferate within the developing seminiferous cord, rupture the basement membrane
to invade the interstitial space and proliferate in the gonads after birth. At about 5
days after birth most EC cells differentiate (Oosterhuis & Looijenga, 2005; Zhu et al.,
2007a) and nearly all tumors can be detected macroscopically by 3-4 weeks of age
(Heaney & Nadeau, 2008).
At d6.75pc Dnd1 is expressed in PGC precursors and somatic neighbors at a specific
frequency, whereas this gene is markedly upregulated only in PGCs after d7.25pc
(Yabuta et al., 2006). Furthermore, Dnd1 expression was detected in the genital
ridges of d11.5pc embryos and it was upregulated in the male gonad between d12.5
and 14.5pc, while it was downregulated in the female gonads during this period
(Youngren et al., 2005). Dnd1 is detectable in embryonic germ cells (EGC) and
embryonic stem cells (ESC) from the mouse (Bhattacharya et al., 2007; Zhu et al.,
2007a), but not in Sertoli cell lines (Youngren et al., 2005) or embryonic Sertoli cells
(Cook et al., 2009). In the adult mouse Dnd1 is expressed in the heart and testis, but
not in other tissues such as brain, spleen, lung, kidney or liver (Youngren et al., 2005;
Bhattacharya et al., 2007).
2.3.3. Modulating the phenotype of Ter/Ter mice
Genetic background and gender are the decisive factors in teratoma development
but only the latter plays a role in germ cell development. Bax-dependent apoptosis is
a physiological pathway to remove extragonadal PGCs in the mouse (Stallock et al.,
2003) and may be one pathway eliminating Ter/Ter germ cells from the fetal testis
prior to neoplastic transformation (Cook et al., 2009). Crossing of Ter/Ter to a Bax-
null background resulted in a partial rescue of germ cells in the embryo (Cook et al.,
2009). Furthermore, teratomas developed in Ter/Ter males on a mixed genetic
background of C57BL/6J and 129/Sv in homozygous (90%) or heterozygous Bax
(44%) mutants (Cook et al., 2009). Complete backcrossing to the C57BL/6J led to
survival of the germ cells without teratocarcinogenesis (Cook et al., 2011). Double-
mutant females on a mixed background did not develop teratomas, but were fertile
and maintained an increased number of oocytes (Cook et al., 2009).
16 Literature review
Extrinsic factors, such as the environment provided by somatic cells of the gonads,
play a role in Dnd1-related teratoma formation (Regenass et al., 1982; Matin, 2007).
This is substantiated by the fact that Ter/Ter XX germ cells that develop in a
testicular environment give rise to the same neoplastic clusters as mutant XY germ
cells in a testis (Cook et al., 2009).
Chr X also modulates the teratoma incidence in 129/Sv males, while Chr Y has no
effect. Chr X and Y from the 129/Sv-Ter strain were substituted with those from the
C57BL/6J. Ter/Ter males with a C57BL/6J Chr X were sterile but developed less
teratomas than their counterparts. This is either due to unidentified C57BL/6J tumor
suppressor genes or 129/Sv tumor susceptibility genes on Chr X (Hammond et al.,
2007).
Furthermore, considerable interactions are seen between Ter and six TGCT
susceptibility modifiers, including mutations in p53 and SCF (KitlSl-J) as well as
segments from Chr 19 of the MOLF strain. Trans-generational epistasis affects tumor
development and the presence of one of the six genetic variants in either parent
modifies the tumor rate found in male Ter/+ offspring (Lam et al., 2007).
2.3.4. Dead end homolog 1 (DND1) in humans
Information on DND1 in humans is scarce and its physiological relevance has not
been identified. The gene is shown to be expressed in a range of normal tissues,
including testicular germ cells, Leydig cells, interstitial cells and cells from the
seminiferous tubules (Sijmons et al., 2010). To date, it is known that DND1 maps to
the chromosomal region 5q31.1 and deletions in this region have been found in germ
cell tumor tissue and cell lines (Sijmons et al., 2010). Two studies have been
performed to identify mutations in human DND1 in TGCTs. Linger and colleagues
found a rare heterozygous variant, p. Glu86Ala, with unknown effect in DND1 from 1
of 263 GCTs (Linger et al., 2008). Sijmons and colleagues screened DND1 in 272
men with TGCT and observed only one case of a nucleotide substitution c.657C > G
(p.Asp219Glu) predicted to be non-pathogenic (Sijmons et al., 2010). Therefore,
DND1 mutations are unlikely to contribute to the majority of human TGCTs. DND1 is
upregulated in some cancers, such as adenocarcinoma, leukemia or prostate cancer
Literature review 17
(Sijmons et al., 2010). Upregulation of microRNA-24 causes a reduced DND1
expression and is observed in the tongue squamous cell carcinoma (Liu et al., 2010).
2.3.5. The RNA binding protein Dnd1
Dnd1 is an RNA binding protein (RBP) capable of prohibiting the function of several
microRNAs (miRNAs, miR). Crucial processes in normal development as well as
cancer are regulated through miRNAs inhibiting the expression of specific mRNAs, a
mechanism conserved across metazoans. Translation and stability of specific target
mRNAs is disrupted by miRNAs associating with the 3‟ untranslated region (UTRs) of
mRNAs (Valencia-Sanchez et al., 2006; Ketting, 2007).
Most miRNAs are transcribed by the RNA polymerase II as long RNAs that are then
converted to ~22 nt mature miRNAs by the RNase III enzymes Drosha and Dicer.
They are subsequently assembled into ribonucleoprotein complexes and form a
multisubunit complex referred to as miRNA-induced silencing complex (miRISC)
(Kedde et al., 2007; Filipowicz et al., 2008).
Dnd1 can counteract the miRNA-mediated repression of mRNAs by binding to the
uridine-rich regions in the 3‟UTR of target transcripts and protecting them from
miRNAs. Dnd1 contains a conserved RNA binding domain (RBD), also known as
RNA recognition motif (RRM), that it requires to counter miRNA activity (Kedde et al.,
2007; Ketting, 2007; Kedde & Agami, 2008). Dnd1 does not bind directly to the
miRNA and just how it influences miRNAs remains to be uncovered. Two possible
explanations seem plausible: Dnd1 binding of mRNAs could either block miRNA
recognition of target structures or move mRNAs to a location not accessible to
miRNAs (Ketting, 2007).
2.3.6. Zebrafish dnd, miRNAs and target genes
In zebrafish miR-430 is responsible for clearing maternal transcripts from the
developing embryo and causes the repression of nanos1 and TDRD7 in somatic cells
(Mishima et al., 2006). Nanos1 and TRDR7 expression is enabled in germ cells only
as long as dnd is present, as demonstrated by reduced target gene expression after
inhibition of endogenous dnd (Kedde et al., 2007). Furthermore, a mutation in the
3‟UTR of miR-430 led to a no longer germ cell restricted, ubiquitous expression of the
18 Literature review
target gene transcripts (Mishima et al., 2006). The c-terminus of zebrafish DND
possesses a Mg2+-dependent ATPase activity, which is essential for the survival of
PGCs. Translation of DND mutant proteins lacking ATPase is accompanied by a
reduced expression of nanos1 and TDRD7, indicating that the ATPase of Dnd is
required for the protection of both target gene mRNAs (Liu & Collodi, 2010).
Functional DND translocates from the germ-cell nucleus to the perinuclear granules,
however, this phenomenon is not observed in proteins mutated in the RRM domain,
as these are restricted to the nucleus. This is in line with the hypothesis that DND
binds RNA in the nucleus and then transfers to the granules, where it protects the
RNA from miRNA-mediated inhibition (Slanchev et al., 2009).
2.3.7. Mammalian Dnd1, miRNAs and target genes
The human miR-93,-302,-372,-373 and-520 have been termed miRNA-373 family
and act as potential oncogenes in human germ cells (Voorhoeve et al., 2006; Kedde
& Agami, 2008). Rapid cellular proliferation as well as enhanced cellular migration,
invasion, and metastasis have been linked to the expression of the miRNA-373
family (Kedde & Agami, 2008).
Absence of p53 or alternatively the presence of miR-372/3 can lead to a neoplastic
transformation of cells (Voorhoeve et al., 2006). p53 is a tumor suppressor protein
that increases levels of p21 and blocks the cell cycle or induces apoptosis (Serrano
et al., 1997; Voorhoeve et al., 2006). High levels of p21 (Cip1) inhibit cyclin
dependent kinases (CDK) and causes cells to arrest in G1 phase. However, miR-
372/3 counteracts the p53-mediated CDK inhibition by inducing resistance to high
p21 levels (Voorhoeve et al., 2006).
Using human cell-culture based assays Kedde and coworkers found that Dnd1
antagonized the miRNA-mediated repression of three genes: LATS2, p27 (Kip1) and
Cx43 (Connexin43) (Kedde et al., 2007). Furthermore, an overexpression of Dnd1
was found to augment p27 and Lats2 expression in mouse cells (Cook et al., 2011).
Gene activity of the tumor suppressor LATS2, a serine threonine kinase, is directly
controlled by human miR-372/3. LATS2 augments p53-mediated apoptosis (Li et al.,
2003), and its inhibition might be responsible for the p21-resistance seen in miR-
372/3 expressing cells (Voorhoeve et al., 2006). The human miR-221 and miR-222
Literature review 19
are regulators of p27, a cell cycle inhibitor and tumor suppressor. In certain cancer
cell lines a high activity of miR-221/2 is required to maintain low p27 levels and
continue proliferation (le Sage et al., 2007). Moreover, independent of whether
zebrafish or human Dnd1 were transfected, p27 levels went up in HEK293 cells
expressing miR-221/2 (Kedde et al., 2007). Expression of Cx43 gap junction
channels is required for skeletal myoblast fusion in vitro. The gap junctions are
downregulated after the myoblast fusion by human miR-206 and miR-1, which inhibit
the expression of Cx43 protein during myoblast differentiation despite the presence
of mRNA (Anderson et al., 2006; Kedde et al., 2007).
Through comparisons of the 129 and 129-Chr19MOLF mouse strain, miRNA-107 was
identified as one of the genes involved in germ cell tumor development in 129-
Chr19MOLF mice (Zhu et al., 2007b). This led Kedde and colleagues to suggest a
connection between the GCT susceptibility gene miRNA-107 and the Dnd1 mutation
in the 129 strain (Kedde et al., 2007; Kedde & Agami, 2008).
2.3.8. Interactions of Dnd1 with Apobec
The RRM of Dnd1 exhibits a high homology to the RRM of ACF (APOBEC1
complementation factor) (Youngren et al., 2005; Matin, 2007). ACF is the essential
RNA-binding co-factor of the RNA-editing enzyme APOBEC1 (Zhu et al., 2007a).
This led to speculations whether Dnd1, much like ACF, might interact with APOBEC-
like proteins (Zhu et al., 2007a; Bhattacharya et al., 2008). The APOBEC family is a
group of cytidine deaminases that includes the DNA mutator and antiretroviral factor
APOBEC3 (Conticello, 2008), which is expressed in a variety of tissues including the
testis and also in EGCs in vitro. APOBEC3 binds mRNA to inhibit miRNA-mediated
repression and was found to interact with Dnd1 in mammalian cells and the
embryonic gonads of the mouse. The functional consequences remain unknown, but
together these two proteins might modulate miRNA function. A weak interaction
between Dnd1 and APOBEC1 is thought possible but has not been confirmed
(Bhattacharya et al., 2008).
20 Literature review
2.3.9. Role of Dnd1 in germ cell cycle and gene expression
A group of cell cycle inhibitors including p21, p27, Lats2, p53, pRB and Pten are
enriched in mouse cells by overexpressing Dnd1 (Cook et al., 2011). Both p27 and
p21 regulate the G1/G0 mitotic arrest in male germ cells via inhibition of CDKs
(Western et al., 2008). CDKs along with cyclins are the major control switches in the
cell cycle, causing the cell to move from G1 to S or G2 to M (Voorhoeve et al., 2006;
Chu et al., 2008). During the physiological transition from G1 to G0 at d14.5pc p21
and p27 are detectable in germ cells (Western et al., 2008); however, regardless of
strain background, neither is active in mutant Ter/Ter germ cells and these do not
enter G0 mitotic arrest by d17.5pc. Nevertheless, C57BL/6J Ter/Ter germ cells
committed to apoptosis arrest prior to M-phase and the cell cycle arrest is considered
the key event in preventing tumor formation (Cook et al., 2011).
Loss of Dnd1 also results in downregulation of differentiation genes (Mvh, nanos2,
nanos3) followed by an upregulation of meiotic markers (STRA8 and SCP3), which is
typical for females at this stage, and maintenance of pluripotency genes (Nanog,
Sox2, Oct4) (Cook et al., 2011). Under physiological circumstances the male-specific
Nanos2 gene inhibits meiosis by preventing Stra8 expression (Suzuki & Saga, 2008).
The genital ridge at d13.5pc in Bax and Dnd1 double mutants exhibits an
upregulation of the pluripotency marker Nanog and a downregulation of the
differentiation markers Mvh, Nanos2 and Nanos3 in comparison to Bax single
mutants (Cook et al., 2011). Mvh and E-cadherin both label male germ cells after
entry in the genital ridge while Nanog and Sox2 expression declines by the time germ
cells have entered mitotic arrest at d15.5pc (Durcova-Hills & Capel, 2008). In the
129/Sv Ter/Ter mice clusters of Mvh negative and E-cadherin, Sox2 and Nanog
positive cells are found in the gonad in lieu of individual germ cells. The C57BL/6J
Bax and Dnd1 double mutants at d17.5pc do express Mvh and E-cadherin and
downregulate Sox2 and Nanog (Cook et al., 2011).
Summarizing the data suggests that prevention of tumor formation requires
differentiation and cell cycle arrest, and Dnd1 regulates the exit from G1 phase of
mitosis to the quiescent G0 phase.
Literature review 21
2.4. Pluripotent germ cells and their in vivo origin
2.4.1. Primordial germ cell specification in the mouse
Cells destined for the germline depend on the repression of somatic pathways and
the induction of germ cell specific programs (Seydoux & Braun, 2006). In the mouse,
the transcriptional repressor Blimp1 (Prdm1) seems to be the critical factor initiating
germ cell specification (Vincent et al., 2005; Durcova-Hills et al., 2008). Blimp1 is first
upregulated at d6.25pc in about six epiblast cells that are then restricted to the germ
cell fate (Ohinata et al., 2005; Chuva de Sousa Lopes & Roelen, 2008). Further
Blimp1-positive PGC precursors emerge between d6.25-7.25pc by induction of
additional epiblast cells and division of the existing Blimp1 population (Chuva de
Sousa Lopes et al., 2007; Chuva de Sousa Lopes & Roelen, 2010). Specification is
not simply a cell-intrinsic process, but depends on extracellular Bmp-mediated
signaling from the extraembryonic ectoderm (Ying et al., 2001; McLaren, 2003). This
has been established through the transplantation of cells to the proximal epiblast that
gave rise to PGCs (Tam & Zhou, 1996) and a chimera experiment proving that Bmp4
is required in the extraembryonic tissue (Lawson et al., 1999). Furthermore, culture of
competent epiblast cells revealed that these acquired germ-cell properties and
expressed Blimp1 after addition of Bmp4 (Ohinata et al., 2009). Germ cells become
restricted to the germline at d7.25pc, meaning that, aside from apoptosis or necrosis,
no more cells can exit or enter the germline. The specification is characterized by
changes in gene expression, cell cycle length and morphology of PGCs (Chuva de
Sousa Lopes & Roelen, 2010).
2.4.2. Identification of murine PGCs
Histologically PGCs can be identified based on their large size, low nucleus to
cytoplasm ratio, clear nuclear borders and granular nuclear chromatin. Moreover,
germ cells can be distinguished from surrounding cells through distinctive patterns of
gene expression (Saiti & Lacham-Kaplan, 2007). Different markers of PGCs have
been extensively researched, however, no single germ cell-specific marker could be
identified that is continuously expressed. Instead, there is a variety of markers
22 Literature review
expressed in the different developmental stages (Chuva de Sousa Lopes & Roelen,
2010).
Blimp1 is the first marker of the PGC precursors at d6.25pc and is also expressed in
the 40 PGCs found at d7.25pc (Ohinata et al., 2005; Vincent et al., 2005; Chuva de
Sousa Lopes & Roelen, 2010). The PGCs continue to express Blimp1 until d13pc
and it is also expressed in various other tissues (Chang et al., 2002). Fragilis (Ifitm3)
appears to be expressed in response to Bmp4 signaling (Saitou et al., 2002). It is
detectable throughout the epiblast in d6.0pc embryos until the expression pattern
concentrates on about 150 cells including the PGCs by d7.2pc. Shortly afterwards, at
d8.5pc, Fragilis is downregulated (McLaren, 2003). Stella (Dppa3) is a PGC-specific
marker also detectable in the post natal oocyte, the zygote and the blastocyst (Saiti &
Lacham-Kaplan, 2007). Its expression commences at d7.2pc in cells showing high
Fragilis activity and continues till d15.5pc in males and d13.5pc in females. c-kit is
expressed in PGCs from their initial segregation at d7.5pc up to d13.5pc. Expression
of c-kit reappears in differentiated spermatogonia and spermatocytes until the round
spermatid stage where it is no longer detected (Prabhu et al., 2006; Saiti & Lacham-
Kaplan, 2007). Expression of the Nanos genes in the mouse is restricted to germ
cells. Nanos3 is expressed during PGC migration and expression is continued in the
genital ridge until it disappears prior to d15.5pc in the male and d13.5pc in the female
(Tres et al., 2004; Suzuki et al., 2007). The mouse vasa homologue (Mvh) gene
encodes an ATP-dependant RNA helicase of the DEADbox protein family and is also
known as DEAD box polypeptide 4 (DDX4). It is exclusively expressed in germ cells
after entry into the genital ridge, with its expression continuing in spermatogenic cells
and maturing oocyte stages of adult mice (Toyooka et al., 2000).
Primordial germ cells also maintain or reactivate the expression of typical markers of
pluripotency such as the cell surface markers alkaline phosphatase (AP) and SSEA1.
Other pluripotency markers expressed are the transcription factors required for the
pluripotency of the inner cell mass (ICM) of the blastocyst: Oct4, Nanog and Sox2
(De Felici et al., 2009; Chuva de Sousa Lopes & Roelen, 2010). In the embryo Oct4
becomes restricted to the germline at d8pc (McLaren, 2003). It persists throughout
male fetal development, and is maintained in prospermatogonia and undifferentiated
Literature review 23
spermatogonia. However, Oct4 expression is repressed in female germ cells at
meiotic prophase I and expression is resumed after birth during the growth phase of
the oocytes (Saiti & Lacham-Kaplan, 2007). Nanog is initially downregulated in the
epiblast and then expressed during germ cell proliferation from d8.0pc. It is lost
during meiotic entry in females and decreased during mitotic arrest in male PGCs
(Yamaguchi et al., 2005). Sox2 is progressively upregulated in PGCs prior to Stella
and is downregulated after sexual differentiation (Yabuta et al., 2006; Western,
2009).
2.4.3. Defining pluripotency
Pluripotency is defined as the potential of a cell to differentiate into cells of the three
germ layers and give rise to any fetal or adult cell type. Totipotent cells have the
additional ability to develop extraembryonic tissue and exist only temporarily from the
zygote to the 4-cell stage. Cells of the ICM of the blastocyst and the succeeding
epiblast cells are considered to be pluripotent embryonic cells (Zwaka & Thomson,
2005; De Miguel et al., 2010).
Immortal cell lines exhibiting pluripotent characteristic and capable of remaining in an
undifferentiated state can be generated in vitro by more than one way (Kerr et al.,
2006). These cells include embryonic stem cells (ESC) from the ICM, as well as
EGCs and ECC from the PGCs. Only recently, the first pluripotent cells have been
derived from the testis of both neonatal mice (Kanatsu-Shinohara et al., 2004) and
adult mice (Ko et al., 2009). Even somatic cells can be induced to pluripotency and
create induced pluripotent stem (iPS) cells by transduction with vectors encoding
factors such as Oct4, Sox2, Klf4 and c-myc (Chuva de Sousa Lopes & Roelen,
2010).
Pluripotency can be analyzed in vitro through the expression of the markers
mentioned in chapter 2.4.2 and the ability to differentiate and form embryoid bodies
(EB) under specific culture conditions (Keller, 1995). Developmental potential of cells
is tested in vivo by the ability to form teratocarcinomas after transplantation to ectopic
sites or by analyzing the fate of genetically marked cells after blastocyst injection
(Donovan, 1998; Kerr et al., 2006). Pluripotent cells are capable of integrating into
the blastocyst and produce chimeras, which are made of two genetically distinct
24 Literature review
populations of cells. The hallmark of true pluripotency is the ability of the injected cell
line to give rise to the gametes and to transmit through the germline (Kerr et al.,
2006). Cells in possession of said property can be used to generate genetically
modified animals. This is accomplished by targeting specific genes in the pluripotent
cells using homologous recombination, selecting a clone with the correct insertion
and injecting it into the blastocyst (Capecchi, 1989).
2.4.4. Differentiation potential of PGCs
Although PGCs express pluripotency associated genes, they are in fact differentiated
cells restricted in their developmental potency to becoming either oocyte or sperm.
However, the fusion of oocyte and sperm generates the totipotent fertilized oocyte
and marks the beginning of a complex new organism. Consequently, PGCs are
unipotent cells capable of giving rise to the totipotent germline. (McLaren, 1981;
Donovan, 1998; Kerr et al., 2006). This, along with the ability of PGCs to give rise to
pluripotent EGCs or ECCs, shows that PGCs retain a well-regulated capacity for
developmental pluripotency. Teratomas can be generated experimentally in vivo by
transplantation of pluripotent cells or PGCs to ectopic sites. PGCs from d11.5pc-
d13.5pc embryos generate ECCs after transplantation into the kidney or testis
capsule. The ability of genital ridges to give rise to teratomas declines with
developmental age to a point that no tumors developed from grafts of d14.5pc
gonads (Stevens, 1966). These results infer that the PGC potential changes after
colonization of the gonad anlagen (Stevens, 1967) and that mitotically proliferating
PGCs are responsible for teratoma development (Zhu et al., 2007a). However, unlike
pluripotent cells, PGCs do not differentiate into embryoid bodies and do not
contribute to either germline or soma after injection into a blastocyst (McLaren &
Durcova-Hills, 2001; Durcova-Hills et al., 2006; Kerr et al., 2006).
2.4.5. Embryonic stem cells
ESC are derived from the inner cell mass of the preimplantation blastocyst and were
first established from the 129/Sv mouse strain in 1981 by Evans and Kaufman using
feeder cells, LIF (leukemia inhibitory factor) (Evans & Kaufman, 1981). Mouse ESCs
have allowed the targeted manipulation of the mouse genome and have become the
Literature review 25
principal technology used for genetic engineering in the mouse, due to their relatively
high capacity to contribute to germline chimeras (Capecchi, 1989; Glaser et al., 2005;
Yu & Thomson, 2008). Human ESCs have been derived under different culture
conditions and expressing a set of markers different from the mouse (Thomson et al.,
1998). For instance, SSEA1 is expressed in mouse but not human ESCs, while
SSEA3 and SSEA4 are expressed in human but not mouse ESCs (De Miguel et al.,
2010).
All attempts to establish rat ESCs failed until recently, when the first germline-
competent rat ESC were generated by inhibiting the MEK and GSK-3 pathways (Li et
al., 2008). The same inhibitors used for rat ESCs have also enabled deriving mouse
ESCs from different strains (Ying et al., 2008; Nichols et al., 2009). The first report
about a knockout of p53 using rat ESC was given little later (Tong et al., 2010).
2.4.6. Embryonic carcinoma cells
ECCs are the PGC-derived pluripotent stem cells of teratocarcinomas that can also
be kept in culture (Kahan & Ephrussi, 1970; Zwaka & Thomson, 2005). They were
the first pluripotent cells to be successfully maintained in vitro, thereby paving the
way for all others. They closely resemble EGCs and ESCs as well as their precursor
cells, the PGCs, in that they continue to express typical markers such as AP, Oct4
and Nanog (De Miguel et al., 2010). However, unlike ESC or EGC, the ECC lines are
usually genetically unstable and have an aneuploid chromosome set (Kerr et al.,
2006; De Miguel et al., 2010). Many human ECC lines and some murine ECC lines
display a limited developmental potency and are not capable of differentiation
(Andrews, 1998; Kristensen et al., 2008). Some of the mouse ECC lines are capable
of forming chimeras (Brinster, 1974), however, the majority is not, which has been
attributed to their karyotypic abnormalities (Nichols & Smith, 2009). In a couple of
cases the chimeras have morphological abnormalities and develop tumors (Rossant
& McBurney, 1982). Unlike other pluripotent cells in the embryo, the ECC remain
pluripotent in vivo. Therefore, they can be serially transplanted between adult mice
and always produce new teratomas in their transplantation site (Stevens, 1970).
26 Literature review
2.4.7. Features of rodent embryonic germ cell derivation and culture
PGCs have the ability to de-differentiate into pluripotent cells showing clonal growth
in vitro and are then termed EGCs (Durcova-Hills et al., 2006). In the mouse the
complete conversion of PGCs to EGCs takes about 10 days (Durcova-Hills et al.,
2006). The first EGC were derived from mouse PGCs on feeder cells with media
containing the cytokines bFGF, SCF and LIF (Matsui et al., 1992; Resnick et al.,
1992). Contribution of these cells to all somatic cell lineages and the germline in
chimeras confirmed that the cultured PGCs gave rise to truly pluripotent cells. Mouse
EGCs can be obtained from different embryonic stages: pre-migratory (8.0-8.5pc),
migratory (9.5pc) and post-migratory EGCs (11.5 and 12.5pc) (Matsui et al., 1992;
Resnick et al., 1992; Labosky et al., 1994; McLaren & Durcova-Hills, 2001). After
many failed attempts, culture of d13.5pc EGCs was achieved only recently, however,
it is not known whether these cells contribute to chimeras (Shim et al., 2008).
It was long thought that derivation of EGC depends on the presence of the above-
mentioned cytokines (Donovan, 1994; Donovan et al., 2001; De Miguel et al., 2010).
However, recently mouse EGCs were derived without SCF and bFGF using feeder
cells and the 2i-LIF media described in the following chapter 2.4.8. (Leitch et al.,
2010). A similar protocol also derived the first EGCs from the rat at d10pc, which is
the equivalent to d8.5pc in the mouse. The rat PGCs were collected in media
containing FCS and bFGF on SCF-producing feeder cells and on the third day of
culture the original media was exchanged for 2i-LIF (Leitch et al., 2010).
EGC and ESC from mouse and rat appear indistinguishable in most aspects such as
morphology and expression of specific markers (Shovlin et al., 2008; Leitch et al.,
2010). However, differences are found between the ICM-derived ESC and PGC-
derived EGC lines in their respective methylation status (De Miguel et al., 2010).
2.4.8. The 2i-LIF media enabling culture of EGC and ESC
As revealed by the name, the 2i-LIF media contains two inhibitors (PD0325901,
CHIR99021) blocking different cellular pathways and the cytokine LIF. Extrinsic
factors thought to induce differentiation or cell commitment such as serum and its
substitutes are not included in the medium (Buehr et al., 2008).
Literature review 27
Under physiological circumstances, the fibroblast growth factor FGF4 induces the
mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK)
MEK pathway and drives cells into committing to a cell lineage (Voigt & Serikawa,
2009). The inhibitor PD0325901 blocks the MEK pathway by suppressing the
activation of ERK1/ERK2 (Bain et al., 2007) and helps sustain pluripotent cells
(Buehr et al., 2008; Ying et al., 2008; Leitch et al., 2010). The Glycogen synthase
kinase-3 (GSK-3) is a negative modulator of various anabolic pathways and is part of
the Wnt-signaling cascade. Inhibition of GSK-3 by CHIR99021 increases cytoplasmic
and nuclear β–catenin, which in turn activates the Wnt pathway and enhances self-
renewal (Buehr et al., 2008; Bone et al., 2009). LIF is important for maintaining the
self renewal in culture via activation of STAT3 and induction of c-myc (Bone et al.,
2009).
2.4.9. Epigenetic signature of PGCs and EGCs
Unlike cells of the ICM the PGCs are highly specified cells and are subjected to an
array of epigenetic changes during development (Shovlin et al., 2008). The original
biparental pattern of imprinting of the zygote is erased, and paternal or maternal
imprints are established (Oosterhuis & Looijenga, 2005). PGCs do not self-renew for
longer periods of time but follow a series of differentiation events instead (De Miguel
et al., 2010). Between germ cell specification at d7.25pc and sex differentiation at
d12.5pc, the germ cells exchange histone modification, reactivate the silent X-
chromosome in females, remove parental imprints so that the parental genomes
become epigenetically equal and undergo DNA demethylation (Hajkova et al., 2002;
Shovlin et al., 2008; Chuva de Sousa Lopes & Roelen, 2010). Correct sex-specific
somatic imprints are reestablished after birth in females or in later embryonic
development in males (Yamazaki et al., 2003; Chuva de Sousa Lopes & Roelen,
2010; De Miguel et al., 2010).
Methylation status of PGCs is not necessarily reflected in EGC lines, as these have
undergone epigenetic reprogramming of their own (Durcova-Hills et al., 2001; Shovlin
et al., 2008). EGC lines of different developmental stage show a reduced methylation
of many imprinted genes in comparison to ESCs (Shovlin et al., 2008; De Miguel et
al., 2010). A few of the chimeras made with late mouse EGCs exhibit skeletal
28 Literature review
abnormalities, and this might be attributed to erasure of differential methylation in
imprinted genes. Epigenetic changes upon arrival in the genital ridge are also
thought to be responsible for the reduced efficiency seen in the derivation of EGCs
from later embryonic stages (Kerr et al., 2006; Durcova-Hills et al., 2008).
Furthermore, early EGC lines show heterogeneous DNA methylation patterns, in
contrast to the uniformity of methylation patterns seen in late EGC and ESC lines
(Shovlin et al., 2008).
Consequently, epigenetic programming displays differences between pluripotent
EGC or ESC lines and unipotent PGC lines. Clarifying the relationship between germ
cells and pluripotent cells could help shed some light on the nature of the pluripotent
state (Surani et al., 2008).
2.4.10. EGCs from different species
EGCs have been derived from a whole array of species; the mouse was simply the
first and rat the most recent. Mouse EGCs and ESCs have a similar propensity to
generate germline chimeras (McLaren & Durcova-Hills, 2001). After aggregation with
eight-cell embryos, the EGCs are shown to contribute to embryonic and to some
extent to extraembryonic lineages (Durcova-Hills et al., 2003). The EGCs from the rat
have led to genetically modified chimeras; however, these were not germline
competent (Leitch et al., 2010). Culture of porcine PGCs of the genital ridge
generated EGCs (Shim et al., 1997) and genetically modified porcine EGCs have
been able to contribute to chimeras shown to transmit through the germline
(Piedrahita et al., 1998). PGCs from chicken have been cultured and also genetically
modified while staying committed to the germline (van de Lavoir et al., 2006). The
culture conditions used to maintain chicken EGCs can also be used to obtain duck
EGCs (Guan et al., 2009). Furthermore, the successful culture of caprine EGCs
contributed to chimeric goats (Jia et al., 2008). Deriving EGCs from the PGCs in the
genital ridge of human embryos has also been possible. Reprogramming PGC to
EGCs is possible using embryos between 5-10 weeks of gestation in humans (Kerr
et al., 2006). These EGCs express the same markers as human ESCs with the
addition of SSEA1 (Shamblott et al., 1998). It has been suggested that the human
EGCs could be used as an ESC-alternative for cell and tissue replacement therapy
Literature review 29
(Shamblott et al., 2001), however, this would require improving culture conditions and
a better understanding of human EGCs (Aflatoonian & Moore, 2005).
2.4.11. From ESC to PGCs
ESC can develop into PGCs of various developmental stages in culture, either
spontaneously or by applying differentiating culture conditions such as retinoic acid
or Bmp4-producing cells (Toyooka et al., 2003; Geijsen et al., 2004; Ko & Schöler,
2006). Germ cell markers stella and fragilis are detectable in ESCs even before the
onset of differentiation, even though they are not expressed in the inner cell mass
and early epiblast cells in vivo. In vitro PGCs are identified based on the expression
of stella and fragilis coupled with Mvh (DDX4), a marker initially not expressed in the
studied ESCs. ESC-derived Mvh-positive cells can produce functional sperm in vitro
(Toyooka et al., 2003). Using a germ cell specific Oct4 promoter, ESCs were
differentiated into oogonia that enter meiosis (Hübner et al., 2003). However,
functionality of the putative oocytes and sperm has not been established yet (Ko &
Schöler, 2006). Thus, ESCs already express specific germ cell markers and it is
relatively simple to induce complete differentiation to PGCs. A possible explanation
given by Ko and Schöler is that a subpopulation of ESCs is already committed to the
germline (Ko & Schöler, 2006).
30 Goals and objectives
3. Goals and objectives
In the WKY/Ztm rat the spontaneous ter mutation initiated infertility as well as
congenital teratomas in the rat testes and ovaries. The primary aim of this project
was to gain a better understanding of tumorigenesis and the developmental potential
of germ cells in the WKY/Ztm-ter rat strain.
This strain is the only available rat model found among the small number of animal
models already existing for GCTs. The first objective of the project was to
characterize the genotype and phenotype of the ter rat. A strong genetic component
of susceptibility for GCTs is found in both humans and mice. This made it critical to
identify the mutation causing the neoplastic transformation in the ter rat. For one, this
enables direct comparisons between different species, and for another, this is
essential to help unravel the molecular pathways leading to tumor development. In
general teratocarcinomas of the ovary are much rarer than of the testis and this rat
model has the unique ability of causing both. Therefore, a special focus of the
phenotypic analysis was to identify gender differences and similarities. Another
central point was gathering data that would allow comparing the ter rat with the Ter
mouse.
PGCs can de-differentiate into pluripotent ECCs through tumorigenesis in vivo or
EGCs through specific culture conditions in vitro. EGCs could present a powerful tool
to determine the relevant mechanisms involved in the neoplastic transformation of
PGCs. Therefore, the second goal of the project was to establish a stable culture of
wild type and ter rat EGCs derived from late PGCs of the genital ridge.
Characteristics of these EGCs were examined in various pluripotency tests, and the
expression of specific germ cell and pluripotency markers was analyzed. The
capacity of PGCs to generate EGC lines was assessed along with the proliferation
rate of these cell lines, and compared between the two different genotypes to
determine the effects of ter. Furthermore, it was discerned whether or not the wild
type cell line has the ability to contribute to germline competent chimeras. This is the
final test of pluripotency and essential, should rat EGCs ever be used as a tool for
genetic manipulation in the rat.
Chapter 4 31
4. The ter mutation in the rat Dnd1 gene causes gonadal
teratomas and infertility in both genders
Nils-Holger Zschemisch1, Emily Northrup1, Regina Eisenblätter1, Silke Glage1, Dirk
Wedekind1, Edwin Cuppen2, Martina Dorsch1, Hans-Jürgen Hedrich1
Nils-Holger Zschemisch and Emily Northrup contributed equally to this work
1 Institute for Laboratory Animal Science, Hannover Medical School
Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
2 Hubrecht Institute, KNAW and University Medical Center Utrecht
Uppsalalaan 8, 3584CT Utrecht, the Netherlands
submitted
Acknowledgements:
We thank Isabell Wittur and Cindy Elfers for excellent laboratory assistance.
Funding for this project was provided by the Cluster of Excellence REBIRTH,
Grant number: EXC 62/1.
32 Chapter 4
4.1. Abstract
We detected the formation of congenital tumors in the ovaries and testes of
WKY/Ztm rats. Histological examination revealed derivatives from all three germ
layers, thereby identifying the tumors as teratomas. These teratomas are presumed
to originate from the embryonic precursors of germ cells and the underlying mutation
was referred to as ter. Linkage analysis of 58 (WKY-ter x SPRD-Cu3) F2 rats with 52
polymorphic microsatellite markers associated the ter mutation with RNO18 (LOD =
3.25). Fine mapping with 64 SNP markers polymorphic between WKY and SPRD
rats, narrowed the ter mutation down to RNO 18p11. Sequencing of candidate genes
detected a point mutation in exon 4 of the dead-end homolog 1 gene (Dnd1) that
introduced a premature stop codon and is assumed to cause a truncation of the
Dnd1 protein leading to a loss of function. Genotyping of the recessive ter mutation
revealed a complete penetrance of teratocarcinogenesis in homozygous ter rats of
both genders. Moreover, the loss of the c-terminus of the Dnd1 protein caused
infertility in male and female rats. Morphologically non-tumorous testes of
homozygous ter males were reduced in size and weight. Immunohistochemical and
histological staining linked the gonadal malformation with a lack of spermatogenesis.
Additionally, oocytes were completely absent in the primary follicles of homozygous
ter females, which indicates an ontogenetic loss of germ cells. Our WKY-Dnd1ter/Ztm
rat could serve as an animal model to investigate gonadal teratocarcinogenesis and
the molecular mechanisms of gamete development, as well as the differentiation
potential of primordial germ cells.
4.2. Introduction
Tumors classified as germ cell tumors (GCTs) are derived from cells belonging to the
germ lineage (Looijenga & Oosterhuis, 1999) and are a very heterogeneous group of
neoplasms with varying histopathological and clinical manifestations (Bahrami et al.,
2007). Testicular germ cell tumors (TGCT) are the most common malignant
neoplasms in young Caucasian men. The incidence of TGCTs ranges from 6-11 per
100,000 and has doubled during the past 50 years (Chieffi et al., 2009; Hotaling &
Chapter 4 33
Walsh, 2009). In young women, 20-25 % of all ovarian neoplasms are ovarian germ
cell tumors (OGCT). The majority of OGCTs are benign ovarian teratomas (dermoid
cysts), and only 5% are characterized as malignant OGCTs (Faulkner & Friedlander,
2000; Giambartolomei et al., 2009). Genetic factors play an important role in the
development of GCTs, although multiple loci and not one major mutation seem to be
involved. To date, the molecular principles underlying OGCT development remain
poorly understood. However, in the testes silencing of p53, microRNA-372/373
activity, as well as mutations in the c-KIT, KRAS-2, and BRAF genes can contribute
to the neoplastic transformation of germ cell precursors (Crockford et al., 2006;
Krausz & Looijenga, 2008). Various male reproductive disorders, including infertility
and TGCTs, are thought to result from similar disruptions during fetal development
and have been grouped together in the so-called testicular dysgenesis syndrome
(TDS) (Skakkebaek et al., 2001).
Teratocarcinomas are malignant GCTs consisting of derivatives from all three germ
layers and undifferentiated embryonic carcinoma cells (ECCs). The pluripotent ECCs
are the stem cells of GCTs that are capable of differentiating into the various tumor
tissues (Kleinsmith & Pierce, 1964) and originate from a neoplastic transformation of
germ cells during embryonic development (Stevens, 1967; Vilar et al., 2006). In
general, teratomas can be classified into prepubertal type I GCTs and the more
common postpubertal type II GCTs. Type I GCTs have a biparental partially erased
pattern of genomic imprinting suggesting that the precursor cells are earlier
primordial germ cells (PGCs) or gonocytes that are directly transformed into ECCs.
Genomic imprinting is erased in the aneuploid type II GCTs and probably involves
late PGCs/gonocytes that are transformed into precursor lesions (CIS/ITGCN) before
being transformed into ECCs (Oosterhuis & Looijenga, 2005; Kristensen et al., 2008;
van de Geijn et al., 2009).
Spontaneous formation of testicular teratomas was described in the 129/Sv mouse
strain in the early 1950ies (Stevens, 1959, 1973). TGCT development was detected
in 1% of male 129/Sv mice, while an additional mutation in a modifier gene referred
to as Ter increased the incidence of TGCT to 17 % in Ter/+ and to 94 % in Ter/Ter
males (Stevens, 1984; Noguchi & Noguchi, 1985; Asada et al., 1994).
34 Chapter 4
Teratocarcinogenesis in the male 129/Sv-Ter mice was associated with infertility in
male and decreased fertility in Ter/Ter female mice (Noguchi & Noguchi, 1985),
which has been attributed to the embryonic loss of PGCs (Rivers & Hamilton, 1986;
Sakurai et al., 1995). Linkage analysis by Asada and colleagues located the modifier
gene carrying the Ter mutation on mouse chromosome 18. The Ter mutation was
identified as a C to T substitution in the Dnd1 gene introducing a premature stop
codon and leading to functional inactivation (Asada et al., 1994; Youngren et al.,
2005).
Weidinger and co-workers showed that the downregulation of dnd in zebrafish
inhibited the embryonic migration of PGCs into the developing gonads and led to the
ontogenic loss of the PGCs (Weidinger et al., 2003). In Xenopus laevis embryos, the
ablation of dnd also blocked PGC migration and initiated the death of PGCs, thereby
showing the conserved functions of the dead end gene in anamnia (Horvay et al.,
2006).
Dnd1 is an evolutionary conserved RNA-binding protein (RBP) that competes with
microRNAs for the binding of target gene mRNAs, as shown in human and mouse
cells. Dnd1 inhibits the miRNA-mediated repression of the tumor suppressor genes
p27 and Lats2, the germ cell specific genes Nanos1 and Tdrd7 as well as
Connexin43 (Kedde et al., 2007; Ketting, 2007). Moreover, Dnd1 is postulated to
form complexes with APOBEC3, a multifunctional protein also involved in the
inhibition of miRNA-mediated mRNA repression (Bhattacharya et al., 2008).
In 2004 Länger and co-workers reported a spontaneous recessive mutation in
WKY/Ztm rats referred to as ter that initiated congenital teratomas in the rat testes
and ovaries (Länger et al., 2004). Here we report the identification of ter as a point
mutation in the rat Dnd1 gene, which leads to a premature stop codon. Unlike the
mouse, Dnd1 is essential for survival and inhibition of the neoplastic transformation
of germ cells in all rats of the WKY-Dnd1ter/Ztm strain. While the mouse Dnd1 was
characterized as a modifier gene that simply modulates teratoma formation in males,
our data suggests that rat Dnd1 acts as a tumor suppressor gene in both genders.
Altogether, the inactivation of Dnd induces the loss or reduction of fertility in
vertebrates and is accompanied by gonadal teratocarcinogenesis in rodents.
Chapter 4 35
4.3. Material and Methods
Animals
All animals were bred and maintained at the Institute for Laboratory Animal Science,
Hannover Medical School, Germany (subline code: Ztm: http://www.mh-
hannover.de/einrichtungen/tierlabor). The experiments were in accordance with the
German Animal Welfare Legislation (Tierschutzgesetz 2006).
Husbandry
The WKY/Dnd1ter/Ztm (further abbreviated as WKY-ter) colony is maintained as a
segregating inbred strain by mating littermates or parents known to carry the
mutation. Microbiological status was monitored according to FELASA
recommendations (Nicklas et al., 2002) and the WKY/Ztm rats were positive for
parvovirus and apathogenic protozoa. Rats were kept in groups of three animals
under a 14:10 light-dark cycle and 55 5% humidity. They received an autoclaved
commercial pelleted diet (Altromin 1314) (protein 22%, fat 5%, raw fiber 4.5%, ash
7%, utilizing energy 3.1 kcal/g) and water ad libitum. The commercial softwood
granulate bedding was sterilized (Lignocel, Altromin; Lage, Germany).
Linkage analysis
We created a F1 generation by mating heterozygous male WKY-ter rats with female
SPRD-Cu3 rats to identify teratocarcinoma susceptibility loci. SPRD-Cu3 was
chosen, because it is easily distinguished from the WKY-ter strain by microsatellites.
The F1 generation was used to generate a [WKY-ter x SPRD-Cu3] F2 generation
(n=58). All offspring was monitored daily for tumors by inspection and palpation of the
scrotum and the abdominal cavity.
DNA samples
Genomic DNA was extracted from the tissues using the NucleoSpin™ Tissue kit
(Macherey-Nagel, Dueren, Germany) according to the manufacturer‟s instructions.
36 Chapter 4
Microsatellite analyses
All oligonucleotides used in this study were synthesized by MWG Biotech AG
(Ebersberg, Germany). The microsatellite markers were selected using rat genome
maps published by the Whitehead Institute, the Rat Genome Database and Ratmap.
All primers were tested against a panel of the progenitor strains and the F1
generation to determine the polymorphic nature of the microsatellite markers.
Approximately 52 microsatellite markers proved to be polymorphic between WKY-ter
and SPRD-Cu3 and were therefore used in a genome wide screen of the F2
progeny. A complete list of all used microsatellites can be requested from the
authors.
The PCR reaction was performed according to the manufacturer‟s instructions
(Peqlab Biotechnologie, Erlangen, Germany) using 100 ng DNA template per well in
a 96-well plate (MultiRigid Ultra Plates™, Roth, Karlsruhe, Germany) and amplified in
a PTC-200 thermocycler (Biozym, Hess. Oldendorf, Germany). The amplification
consisted of the following steps: 4 min at 94 °C; 35 cycles: 15 s at 94 °C, 1 min at
55°C, 2 min at 72 °C; 7 min final extension at 72°C. PCR products were analyzed by
electrophoresis in 3 % Nusieve™ agarose gels (Biozym, Hess. Oldendorf, Germany).
Gels were stained using Gelstar™ (Cambrex, Apen, Germany) and documented by
UV light illumination at 312 nm.
Sequencing
Resequencing of all four exons was managed using LIMSTILL, LIMS for Induced
Mutations by Sequencing and TILLing (Victor Guryev, E.C., unpublished). This web-
based publicly accessible information system (http://limstill.niob.knaw.nl) was used to
generate the Dnd1 project and visualize the gene structure based on the Ensembl file
ENSRNOG00000016894. The primer design application within LIMSTILL is Primer3-
based, and parameters are set to design primers with an optimal melting temperature
of 58°C.
PCR was done using a touchdown thermocycling program (92C for 60 s; 12 cycles
of 92C for 20 s, 65C for 20 s with a decrement of 0.4C per cycle, 72C for 30 s;
followed by 20 cycles of 92C for 20 s, 58C for 20 s and 72C for 30 s and 72C for
Chapter 4 37
180 s; GeneAmp9700, Applied Biosystems). PCR reaction mixes contained 5 µl
genomic DNA, 0.2 µM forward primer and 0.2 µM reverse primer, 200 µM of each
dNTP, 25mM Tricine, 7.0% Glycerol (w/v), 1.6% DMSO (w/v), 2 mM MgCl2, 85 mM
Ammonium acetate pH 8.7 and 0.2 U Taq Polymerase in a total volume of 10 µl.
PCR products were diluted with 20 µl H2O, and 1 µl was used as template for the
sequencing reactions. Sequencing reactions, containing 0.25 µl BigDYE (v1.1;
Applied Biosystems, Foster City, CA), 3.75 µl 2.5x dilution buffer (Applied
Biosystems) and 0.4 µM gene specific primer in a total volume of 10 µl, were
performed using cycling conditions as recommended by the manufacturer.
Sequencing products were purified by ethanol precipitation in the presence of 40 mM
sodium-acetate and analyzed on a 96-capillary 3730XL DNA analyzer (Applied
Biosystems). Sequences were analyzed for polymorphisms using polyphred software
(Nickerson et al., 1997). Primers for PCR amplification and sequencing were
designed using the Ensembl genome database (http://www.ensembl.org) and a
customized interface to Primer3.
Genotyping
For the identification of the ter allele PCRs were performed with the primer pair
(terFor2: 5‟-GTCTGGTCTTAAGTGCTTGG-„3; terRev2: 5‟-TCACTGCTTCACCACAG
AAC-3‟) amplifying a 560 bp sequence in a total volume of 25 µl containing 12.5 µl
HotStarTaqMatermix Kit (Qiagen, Hilden, Germany), 10 pmol of each primer and 1 µl
template DNA. After initial 15 min at 95°C followed by 35 cycles (95°C for 30 s, 54°C
for 30 s, 72°C for 30 s), the PCR products were digested adding 0,5 µl KpnI-HF, 3 µl
NEB4 buffer and 0.3 µl BSA (100x) amplification (NEB, Ipswich, MA). Cleavage
fragments from PCR products of wild type Dnd1 (180 bp and 380 bp) and unchanged
560 bp ter allele products were separated by electrophoresis in a 1.5 % agarose gel
in 1x TBE buffer.
Tumors and tissues
Ten males and ten females from WKY as well as from WKY-Dnd1ter with +/+, ter/+
and ter/ter genotype were sacrificed at 3, 6 and 9 weeks of age by cervical
dislocation after CO2 anesthesia. Testes and ovaries were prepared, inspected,
38 Chapter 4
weighed and fixed for histology. In addition, GCTs were harvested from 46 male and
38 female Dnd1-deficient rats at the pre-final stage of cancer, which were
characterized by a significant increase of body weight accompanied by abdominal
swelling, inactivity, reduced food intake and piloerection/rough coat. Tumors were
excised, examined macroscopically, weighed and prepared for histology.
Histology and Immunohistochemistry
Tissues were fixed in 4% formaldehyde solution or in Bouin‟s solution (Sigma-Aldrich,
St.Louis, MO) for 3-5 days. Paraffin embedded tissues were cut in 3 µm slices,
transferred to SuperFrost slides (Menzel, Braunschweig, Germany) and stained with
hematoxylin-eosin following standard procedures.
For immunohistochemical staining, slides from formaldehyde-fixed, paraffin-
embedded tissues were heated in 10 mM citric buffer (pH 6) for 5 min at 125°C in a
pressure cooker for antigen retrieval. Samples were stained with a 1:100 dilution
(stock 200µg IgG/ml PBS) of an anti c-kit antibody (sc-168; Santa Cruz
Biotechnology Inc, Santa Cruz, CA) for 1 hour. For secondary antibody reaction and
HRP staining, the Zytochem Plus HRP Polymer System (Zytomed Systems, Berlin,
Germany) was used according to the manufacturer‟s instructions.
Statistical analyses
Linkage analyses were performed with the JoinMap V 2.0 program (Agricultural
Research Department, Wageningen, Netherlands). The LOD scores of the
teratocarcinoma susceptibility region were calculated using the R/qtl program
provided by Dr. K. Browman (Department of Biostatistics, Johns Hopkins University,
Baltimore, MD) (Xu & Atchley, 1996; Broman et al., 2003). E/M algorithms estimated
susceptibility regions in a binary model using the teratocarcinoma of the animals as a
trait.
A permutation test was performed based on our current genotypic and phenotypic
data to calculate an individual threshold value for significance (LOD score > 2.3)
independent from the theoretical model of Lander and Kruglyak (Churchill & Doerge,
1994; Lander & Kruglyak, 1995). Tumor data analysis and Kaplan-Meyer survival
analysis were performed and statistically verified using One-way Anova and
Chapter 4 39
Bonferroni Multiple Comparison Test by the PRISM 5 for Mac OSX Software
(GraphPad Software Inc., La Jolla, CA).
4.4. Results
We traced the ter mutation in the WKY/Ztm rat strain to a point mutation in the Dnd1
gene and observed teratocarcinomas and infertility in all homozygous rats of both
genders.
Teratocarcinomas in the WKY strain
We established a coisogenic segregating inbred WKY strain that carries a mutation
referred to as ter leading to germ cell tumors (GCT) in both testes and ovaries (Fig.
4.1A). Histological examination of these tumors revealed different tissues that
originated from all three germ layers, thereby characterizing the GCTs as teratomas.
Besides mesodermal tissues such as cartilage, skeletal and cardiac muscle we also
identified glandular structures arising from the endodermal germ layer. Furthermore,
immature neuronal tissue, neural tube-like formations and squamous epithelia
originating from the ectoderm were present in teratomas of both ovaries and testes
(Fig. 4.1B). Tumor development in the ovaries was often accompanied by ascites
and cystic alterations in the early stages of tumorigenesis (Fig. 4.1C, left).
Identification of the ter mutation
Linkage analysis of 58 (WKY-ter x SPRD-Cu3) F2 rats using 52 polymorphic
microsatellite markers indicated an association of RNO18 (D18Rat61, LOD = 3.25)
with tumor development (Fig. 4.2A). A panel of 64 SNP markers, which are
polymorphic between WKY and SPRD-Cu3 was selected. Genomic loci were
amplified by PCR from 58 F2 animals of the WKY-ter x SPRD-Cu3 cross and
genotyped by dideoxy re-sequencing. Strong linkage to a single locus in the middle
of chromosome 18 was observed (data not shown). Based on synteny with the
recently cloned mouse Ter mutation within MMU18qB2, the homologous rat Dnd1
gene in RNO18p11 was re-sequenced (Fig. 4.2B).
40 Chapter 4
A G to A mutation was identified in exon 4 at position 1975, which introduces a
premature stop codon thought to result in a 62 amino acid truncation at the c-
terminus of the Dnd1 protein (Fig. 4.2C) and most likely a complete loss-of-function
of the encoded protein. The ter point mutation also disrupts the recognition site of the
restriction endonuclease KpnI. A 560 bp PCR amplification product of wild type Dnd1
was cleaved by KpnI into a 380 bp and a 180 bp fragment, while no cleavage was
detected in homozygous ter/ter animals, which confirms the absence of the KpnI
restriction site (Fig. 4.3A).
Using this genotyping approach we were able to distinguish between rats carrying
only one ter allele (ter/+), and animals carrying two ter-alleles (ter/ter), and wild type
(+/+) animals. The ter/+ animals showed normal fertility in both genders and were
used to establish the coisogenic WKY-Dnd1ter strain. Litters from breeding
heterozygous rats were of the same size (7-10 pups) as litters from wild type matings
of WKY-Dnd1ter or WKY. Offspring from ter/+ parents showed normal Mendelian
ratios with 22.8% +/+, 53.7 % ter/+ and 24.5% ter/ter genotypes.
Age dependent GCT development
Teratomas in WKY-Dnd1ter became clinically apparent during adolescence. OGCT in
female ter/ter rats were detected between 21 and 66 days after birth with a median
survival of 34 days, and only 7.8 % of the ter/ter females survived day 43 (Fig. 4.3B).
Of the male ter/ter rats 51% had to be sacrificed by day 58 because of
tumorigenesis, and the rapid increase of tumor progression led to the sacrifice of an
additional 39% of ter/ter males by day 70. However, 10% of the ter/ter males
exhibited a delayed TGCT formation and survived up to 196 days, before exhibiting
visual teratomas. Altogether, the median survival was 58 days in males.
In contrast to the 100% tumor incidence in ter/ter rats, tumorigenesis was entirely
absent in the gonads of heterozygous animals. Furthermore, there was no
spontaneous teratocarcinogenesis detectable in wild type rats from WKY-Dnd1ter or
WKY strains (Fig. 4.3B).
Tumor size and localization were examined in male and female rats at three, six and
nine weeks of age to determine tumor progression. The mean TGCT weight
increases in ter/ter male rats from 2.5 g (SD±2.32) at 3 weeks to 3.8 g (SD±2.96) and
Chapter 4 41
4.7 g (SD±3.76) at 6 and 9 weeks (Fig. 4.3B). At 3 and 6 weeks of age, 40% of the
homozygous ter rats had unilateral TGCTs, while both testes were affected in 20%
and in 40% both testes were macroscopically degenerated without signs of tumor
formation. At 9 weeks of age bilateral teratomas increased to 68%, while 18% had
unilateral tumors and 14% degenerated testes.
At 3 weeks of age the OGCTs in female ter/ter rats were smaller (0.54 g±0.38) than
the TGCTs. Rapid growth of the OGCTs was detected from 3 to 6 and 9 weeks of
age up to mean weights of 4.68 g (SD±4.58) and 5.11g (SD±4.86), with large
variations in tumor size (Fig. 4.3C). Only 4 females survived up to 9 weeks of age
and 3 of these females had bilateral OGCTs. Only 1 of the surviving 4 females at 9
weeks of age exhibited unilateral teratocarcinogenesis, however, the non-tumorous
ovary was cystic (Fig. 4.1C). This was the only case of a female ter/ter rat developing
a teratoma in one ovary and not in both. Ovary degeneration in ter/ter females
without tumor formation was never found due to an early onset of tumor
development. The rapid tumor progression was correlated to the fast drop of survival
between 3 and 6 weeks of age compared to male ter/ter rats (Fig. 4.3C).
Testicular malformation in ter/ter males
We defined the degenerated testis as lower in weight and size than the wild type
testis (Fig. 4.1C, right). The mean weight of wild type testes increased from 0.14 g
(SD±0.02) at 3 weeks to 0.83 g (SD±0.07) at 6 weeks and to 1.14 g (SD±0.07) at 9
weeks in WKY males, reflecting sexual maturation. In wild type WKY-Dnd1ter males
the mean testes weight was 0.09 g (SD±0.02) at 3 weeks, 0.65 g (SD±0.05) at 6
weeks and 1.05g (SD±0.14) at 9 weeks of age. There was no significant difference to
heterozygous WKY-Dnd1ter males with 0.1 g (SD±0.03), 0.7 g (SD±0.04) and 1.12 g
(SD±0.14) of testes weight at 3, 6 and 9 weeks of age. In ter/ter males the
malformed, non-tumorous testes were reduced in size with 0.06 g (SD±0.01), 0.32 g
(SD±0.13) and 0.81 g (SD±0.26) from 3 to 9 weeks of age (Fig. 4.4A).
Histological evaluation revealed normal testicular development and maturation in wild
type WKY-Dnd1ter rats from 3 to 9 weeks. In ter/ter testes the failure of germ cell
development was demonstrated by HE staining, which showed a reduced diameter of
the tubuli seminiferi, a loss of germ cells and a lack of spermatogenesis at 3 and 6
42 Chapter 4
weeks of age. At 9 weeks of age, the degenerated ter/ter testes showed an onset of
neoplastic transformation in the seminiferous tubules (Fig. 4.4B).
To confirm the loss of the sperm precursor cells we performed immunohistological
staining with an anti c-kit antibody as a specific marker for stem and germ cells. As
expected, at 3 weeks of age wild type testes showed widely undifferentiated germ
cell precursors in the seminiferous tubules, while at 9 weeks of age the complete
differentiation process from spermatogonia to sperm could be observed. However, in
the testes of both 3- and 6-week-old ter/ter rats only a few c-kit positive cells were
detected in the seminiferous tubule epithelium, and this suggests that primordial
germ cells were lost during embryonic development. Remaining germ cells often
appeared to be aggregated to foci of neoplastic cells (Fig. 4.5).
Ovarian malformation in ter/ter females
At 3 weeks of age, we found no significant differences in ovary size between females
from the WKY or WKY-Dnd1ter strain (0.005-0.01 g). Three weeks later the ovaries
grew to 0.032 g (SD±0.009) in WKY while the ovaries remained significantly smaller
in WKY-Dnd1ter wild type (+/+) with 0.02 g (SD±0.006) and 0.015 g (SD±0.007) in
heterozygous (ter/+) females. At 9 weeks of age the ovaries from WKY/Ztm females
grew to 0.058 g (SD±0.011), in WKY-Dnd1ter/Ztm wild type (+/+) to 0.034 g
(SD±0.007), and to 0.04 g (SD±0.007) in heterozygous (ter/+) females confirming
sexual maturation (Fig. 4.6A). All ter/ter females had developed tumors at 6 and 9
weeks of age (Fig. 4.3C). Histological examination of early OGCTs in female ter/ter
rats revealed that the non-neoplastic primary follicles lack oocytes, indicating the loss
of primordial germ cells (Fig. 4.6B).
4.5. Discussion
Teratocarcinogenesis in the rat: a novel animal model for GCTs
Teratomas are rare tumors in rats. Only a very few cases of spontaneous
teratocarcinogenesis in adrenal gland, kidney, central nervous system or abdomen
have been reported (Schardein & Fitzgerald, 1977; Ninomiya, 1983; Itoh et al., 1985).
Chapter 4 43
Aside from the extra gonadal teratomas, the formation of testicular GCT has been
described in testes of a SPRD IGS rat (Sawaki et al., 2000). Hereditary teratomas
have also been found in testes and ovaries of the Tera strain that was developed
from the Csk:Wistar-Imamichi rat (Miwa et al., 1987); however, the underlying
mutation was never identified. We detected a congenital, recessive point mutation
referred to as ter in the rat Dnd1 gene on chromosome 18p11 that induces
teratocarcinogenesis and infertility in all animals of both genders. The mutant rat
strain was denominated WKY-Dnd1ter/Ztm and it is a valuable new animal model for
research on germ cell development, TDS, TGCTs and OGCTs.
The 129/Sv-Ter mouse is most likely a model for prepubertal type I TGCTs, whereas
there are no animal models available for type II TGCTs (Looijenga & Oosterhuis,
1999; Oosterhuis & Looijenga, 2005). It remains to be established whether the
teratomas of the ter rat show a closer resemblance to type I or type II GCT. In
analogy to the mouse, and because the majority of rat teratomas occurred before or
during puberty, it seems more plausible that the ter rat is a model for type I GCTs.
Species and gender differences in Dnd1-related teratocarcinogenesis
In the WKY-Dnd1ter/Ztm rat, we identified a point mutation at position 1975 of the
Dnd1 gene that substituted G for A and introduced a premature stop codon, which is
presumed to cause a truncation of 62 amino acids of the Dnd1 protein at the c-
terminus. A similar point mutation has been found in the mouse Dnd1 gene with a C
to T exchange in exon 2 on chromosome 18 that also generates a premature stop
codon thought to result in a c-terminal truncation of the Dnd1 protein (Noguchi and
Noguchi, 1985; Asada et al, 1996; Youngren et al, 2005). A rescue of Dnd1 in mutant
Ter mice showed that the loss of functional Dnd1 is responsible for the phenotype
observed in the mouse. Dominant negative effects were excluded in the mouse, as
tumors and germ cell deficient testes no longer expressed regular or truncated Dnd1
(Youngren et al., 2005). The close similarities found between Dnd1 mutant mouse
and rat in the mutation and the phenotype make it highly probable that a functional
inactivation of Dnd1 also causes germ cell loss and teratomas in the ter rat.
Despite the similarities in the rodent Dnd1 mutations, differences between the
species mouse and rat were apparent in tumor incidence and severity of fertility
44 Chapter 4
disorders. In the mouse, Dnd1 has been characterized as a modifier gene amplifying
the incidence of spontaneous teratocarcinogenesis in heterozygous and homozygous
Ter males. Other modifier genes (KitlSl-J, Trp53nul, Ay, M19, M19-A2, M19-C2) interact
with mouse Dnd1 and induce a 2-3 fold increase of TGCT incidence in heterozygous
Ter/+ males (Lam et al, 2007). Unlike the co-dominant Ter mutation in the mouse, the
recessive ter mutation in rat Dnd1 induced gonadal teratoma formation in 100% of
the homozygous animals, while heterozygous and wild type animals completely
lacked teratocarcinogenesis. Therefore, we postulate that Dnd1 is an essential factor
involved in teratocarcinogenesis and acts as a tumor suppressor gene and not as a
tumor modifier in germ cells of the WKY/Ztm rat strain.
Furthermore, the malignant transformation of rat germ cells initiates not only
testicular, but also ovarian teratocarcinogenesis. OGCTs developed in all
homozygous Dnd1-deficient female rats, whereas no tumor development has been
observed in female mice. Cook and colleagues demonstrated that the testicular
environment is a crucial factor in GCT development of mice, by showing that female
Dnd1Ter/Ter germ cells neoplastically transform in the testes (Cook et al., 2009). The
somatic cells of the testes secrete paracrine factors such as SCF, retinoic acid,
FGF2, LIF, EGF and GDFN as well as androgenic hormones, and these might play a
role in providing a tumor-promoting environment in the mouse (Krentz et al., 2009).
The gender in the WKY-Dnd1ter/Ztm rats had no effect on tumor incidence, which
was 100%, and only modified the tumor progression in homozygous animals.
Surprisingly, on the average tumors became clinically apparent earlier in females
where both ovaries were affected, while tumors developed slower in males with
about half of the tumors being unilateral. This could indicate that, in the rat, the
surroundings provided by the ovary might contain tumor-promoting factors or,
alternatively, the testis might include inhibitory factors.
Dnd1 ablation causes PGC loss and infertility
In response to the repression or ablation of dnd in Xenopus laevis and zebrafish
embryos, PGCs failed to migrate into the developing gonads (Weidinger et al., 2003;
Horvay et al., 2006). A recent publication showed that the c-terminus of the zebrafish
Dnd protein possesses an ATPase domain, which is essential for PGC formation and
Chapter 4 45
survival (Liu & Collodi, 2010). The presumed c-terminal truncation of Dnd1 in the
mouse and rat was also linked to the ontogenetic loss of germ cells; however, it
remains to be established which parts of the c-terminus are crucial for a functioning
Dnd1 protein in rodents and whether an ATPase domain is involved.
Loss of PGCs started on embryonic day 8.5 post coitum in Dnd1 deficient mice of
both genders (Sakurai et al., 1995). Apoptosis is at least in part responsible for the
germ cell loss, as additional inactivation of the pro-apoptotic Bax gene in Dnd1Ter/Ter
mice rescued up to 50% of the PGCs in both genders (Cook et al, 2009). Deficit of
the PGCs caused abnormally small testes and male sterility in homozygous 129/Sv-
Ter mice, while in the homozygous females a few germ cells survived and even
matured to oocytes in ovaries of reduced size (Noguchi and Noguchi, 1985).
In contrast to the mouse, gametogenesis was absent in both degenerated testes and
in non-neoplastic ovarian follicles of ter/ter rats, and this may be the result of a
complete ontogenic loss of normal PGCs. The WKY-Dnd1ter/Ztm rat strain exhibited
infertility, small degenerated testis and TGCTs in males. The older the male ter/ter
rats were, the lower the rate of degenerated testes and the higher the rate of bilateral
tumors. Hence, it is highly likely that the infertile and degenerated testes exhibited by
ter/ter males are the predecessors of cancer. Every one of the ter females was
infertile and all but one developed bilateral OGCTs without ever exhibiting any cases
of degenerated ovaries, and this might be due to the early onset of cancer (under 66
days of age) in females compared to males (under 196 days of age).
Conclusion
The WKY-Dnd1ter/Ztm rat provides a promising new model to study Dnd1-dependent
GCT development in gonads of both genders and investigate the molecular
mechanisms of GCT development in testes and ovaries. This model offers the
opportunity to establish new therapeutic and diagnostic approaches for GCTs in men
and women.
46 Chapter 4
4.6. Figures
Figure 4.1
Chapter 4 47
Fig. 4.1: Teratomas in 6 week old ter/ter rats of the strain WKY-Dnd1ter/Ztm: (A) Bilateral
GCTs in female rat (black arrows: ovarian GCTs) and unilateral GCT in male rat (white
arrow: non-neoplastic testes; red arrow: left TGCT. (B) Upper left: ectodermal tissues in
OGCT of a 5-week-old female; neural tube formation (black star) and squamous epithelium
(black arrow). Upper right: mesodermal and endodermal tissues in an OGCT of a 9-week-old
female; cartilage (white star), glandular structures (white arrow) and skeletal muscle (black
arrow). Lower left: heart muscle-like tissue from a contractile ovarian teratoma. Lower right:
immature neuronal tissue from a TGCT of a 6-week-old male. (C) Rat gonads from 3 weeks
old siblings. Left: non-tumorous ter/ter testes are significantly smaller than wild type. Right:
normal wild type ovaries compared to an OGCT and an abnormal, cystic ovary (confirmed by
histological section and HE staining).
Figure 4.2
48 Chapter 4
Fig. 4.2: Identification of the ter mutation in the rat Dnd1 gene: (A) Genome-wide mapping of
teratoma susceptibility loci of the WKY-ter rat. 58 (WKY-ter x SPRD-Cu3) F2 animals were
typed using genomic DNA and the complete panel of polymorphic microsatellite markers as
described in Material and Methods. The incidence of teratomas of the F2 population was
15%. The LOD scores of the teratoma susceptibility region were calculated using the R/qtl
program. A permutation test assumed a LOD score > 2.3 to be associated with the teratoma
development. (B) Resequencing of all 4 exons of Dnd1 was managed using LIMSTILL, LIMS
for Induced Mutations by Sequencing and TILLing (Victor Guryev, E.C., unpublished). This
web-based publicly accessible information system (http://limstill.niob.knaw.nl) was used to
generate the Dnd1 project and visualize the gene structure based on Ensembl file
ENSRNOG00000016894. (C) The 2625 bp coding sequence of the Dnd1 gene is separated
in 4 exons. The RNA Recognition Motif (RRM) essential for nucleic acid binding of Dnd1 was
located in exon 3. The ter point mutation was identified as G to A substitution in exon 4 at
position 1975 introducing a premature stop codon.
Chapter 4 49
Figure 4.3
Fig. 4.3: (A) The ter mutation disrupts a KpnI restriction site used for genotyping of the ter
allele performing PCR amplification and restriction digest. KpnI digest (black arrows) cleaved
the PCR product of 560bp into 380bp and 180bp fragments, N negative control. (B) Kaplan-
Meyer survival analysis of male and female WKY-Dnd1ter/Ztm rats carrying heterozygous and
homozygous ter alleles compared to wild type animals. (C) Sizes of TGCTs and OGCTs after
3, 6 and 9 weeks of age in homozygous WKY-Dnd1ter/Ztm rats.
50 Chapter 4
Figure 4.4
Chapter 4 51
Fig. 4.4: non-tumorous testes. (A) Box plot analysis of the size of non-tumorous testes in
WKY/Ztm and WKY-Dnd1ter/Ztm with wild type (+/+), heterozygous (ter/+) and homozygous
(ter/ter) genotype at 3, 6 and 9 weeks of age. ter/ter testes are degenerated and significantly
smaller than +/+ and ter/+ testes (B) HE staining of paraffin sections from wild type and
homozygous ter/ter testes of WKY-Dnd1ter/Ztm rats from 3 to 9 weeks of age (: p <
0,001).
Figure 4.5
Fig. 4.5: Immunohistochemical anti c-kit staining of paraffin section from wild type and
homozygous ter/ter testes of WKY-Dnd1ter/Ztm rats at 3 and 6 weeks of age (black arrows:
clusters of surviving c-kit positive germ cell).
52 Chapter 4
Figure 4.6
Fig. 4.6: non-tumorous ovaries. (A) Box plot analysis of non-tumorous ovaries from
WKY/Ztm and WKY-Dnd1ter/Ztm with wild type (+/+), heterozygous (ter/+) and homozygous
(ter/ter) genotype (: p < 0.01, : p < 0.001). (B) HE staining of paraffin section from
wild type (+/+, upper panel) and homozygous ovaries (ter/ter, lower panel) of WKY-
Dnd1ter/Ztm rats at 6 weeks of age. While in the wild type ovaries different stages of follicle
maturation (upper panel, left) and primary follicles with a central oocyte (upper panel, right)
were evident, in the ter/ter ovaries only a very few follicle-like structures without germ cells
(lower panel, right, black arrows) remained.
53
54 Chapter 5
5. Loss of Dnd1 facilitates the cultivation of genital ridge-
derived rat embryonic germ cells
Emily Northrup1, Regina Eisenblätter1, Silke Glage1, Cornelia Rudolph2, Martina
Dorsch1, Brigitte Schlegelberger2, Hans-Jürgen Hedrich1, Nils-Holger Zschemisch1
1 Institute for Laboratory Animal Science
Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
2 Institute of Cell and Molecular Pathology
Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
submitted
Acknowledgements:
We thank Isabell Wittur and Cindy Elfers for excellent laboratory assistance.
Funding for this project was provided by the Cluster of Excellence REBIRTH,
Grant number: EXC 62/1.
Chapter 5 55
5.1. Abstract
Pluripotent cells referred to as embryonic germ cells (EGCs) can be derived from the
embryonic precursors of the mature gametes: the primordial germ cells (PGCs). A
homozygous mutation (ter) leading to the ablation of the dead-end homolog 1 gene
(Dnd1) in the rat causes gonadal teratocarcinogenesis and sterility due to neoplastic
transformation and loss of PGCs. We mated heterozygous ter/+ WKY-Dnd1ter/Ztm
rats and were able to cultivate the first genital ridge-derived EGCs of the rat embryo
at day 14.5 post coitum (d pc). Genotyping revealed that ten EGC lines were Dnd1
deficient, while only one wild type cell line had survived in culture. This suggests that
the inactivation of the putative tumor suppressor gene Dnd1 facilitates the
immortalization of late EGCs in vitro. Injection of the wild type EGCs into blastocysts
resulted in the first germ-line competent chimeras. These new pluripotent rat EGCs
offer an innovative approach for studies on germ cell development and germ cell
tumor development as well as a new tool for genetic manipulations in rats.
5.2. Introduction
The gametes of adult vertebrates, oocytes and sperm, develop from their embryonic
precursors, the primordial germ cells (PGCs), and are responsible for the
transmission of genetic information from one generation to the next. During
ontogenesis, the PGCs migrate through various tissues before finally reaching the
genital ridge. PGCs themselves are highly specialized cells restricted in their
developmental potency (McLaren, 1981). Pluripotent cells referred to as embryonic
germ cells (EGCs), can be obtained from PGCs in vitro. Culture of PGCs leads to this
not well-understood process of epigenetic reprogramming, which transforms the
unipotent PGCs to pluripotent EGCs (McLaren, 2003). The EGCs proliferate
indefinitely and, unlike PGCs (McLaren & Durcova-Hills, 2001), can result in germ-
line competent chimeras upon blastocyst injection (Labosky et al., 1994; Stewart et
al., 1994). EGCs have been made from PGC in the mouse before and during
migration, as well as after entry into the genital ridge (Durcova-Hills et al., 2001;
McLaren & Durcova-Hills, 2001). Only recently, the first EGC were obtained from pre-
56 Chapter 5
migratory PGCs of the rat. Culture conditions similar to those for rat ESC, using the
two inhibitors (2i) and LIF on feeder cells, could procure these rat EGCs (Leitch et al.,
2010).
Loss of Dnd1 can lead to the formation of germ cell tumors and infertility. Dnd1 is an
RNA-binding protein that competes with miRNAs for the binding of specific target
gene mRNAs. In doing so, Dnd1 inhibits the miRNA-mediated repression and
enables the expression of certain target genes even though miRNAs are present.
The persistence of mRNAs from the tumor suppressor genes Lats2 and p27 as well
as the genes Nanos1, Tdrd7 and Connexin43 in germ cells depends on expression
of Dnd1 (Kedde et al., 2007; Ketting, 2007). Dnd1 has also been shown to interact
with the multifunctional protein APOBEC3 (Bhattacharya et al., 2008). The Ter
mutation in the 129/Sv mouse strain was traced back to a point mutation leading to a
premature stop codon in Dnd1 (Asada et al., 1994; Youngren et al., 2005). This
mutation causes decreased fertility in Ter/Ter females and infertility in Ter/Ter males
and increases the teratoma incidence in the testis to 17% in Ter/+ and 94% in
Ter/Ter males, compared to the 1% in wild type 129/Sv (Stevens, 1984; Noguchi &
Noguchi, 1985). Loss of PGCs during ontogenesis was first detectable in Ter/Ter
mouse embryos of both genders at day 8.5 post coitum (d8.5pc) (Sakurai et al.,
1995).
A spontaneous mutation with a recessive mode of inheritance leading to teratomas in
ovaries and testis also occurred in the WKY/Ztm rat strain and was named ter
(Länger et al., 2004). This ter mutation was identified as a point mutation in exon 4 of
the Dnd1 gene that introduces a premature stop codon. Resulting loss or truncation
of the Dnd1 protein in the homozygous ter/ter rats leads to infertility due to the
ontogenetic death of germ cells, and initiates gonadal teratoma formation in all
animals through neoplastic transformation of surviving germ cells. The onset of
teratocarcinogenesis and the teratoma progression were influenced by gender. While
tumors were detected between 21 and 66 days of age in female ter/ter rats with a
mean survival of 34 days, the tumorigenesis was delayed in Dnd1-deficient males
where 61% survived longer than day 70 and 10% longer than day 196 with a mean
Chapter 5 57
survival of 58 days. Furthermore, both ovaries were affected in the females whereas
50% of the males had unilateral tumors in the testis (chapter 4).
Thus, Dnd1 fulfils different roles in PGC migration, development and survival,
depending on the species. So far the molecular mechanisms involved in the rat
remain entirely unknown. We cultured rat embryonic germ cells (EGC) in vitro derived
from embryonic PGCs of the genital ridge as a tool to investigate the mechanisms of
gender-dependent germ cell differentiation and germ cell tumor development.
5.3. Material and Methods
Animals
All animals were bred and maintained at the Institute for Laboratory Animal Science,
Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany
(subline code: Ztm: http://www.mh-hannover.de/einrichtungen/tierlabor). The
experiments were in accordance with the German Animal Welfare Legislation
(Tierschutzgesetz 2006), approved by the local Institutional Animal Care and
Research Advisory Committee and permitted by the Animal Welfare Service of the
Lower Saxony State office for Consumer Protection and Food Safety (Az.10/0226;
Az.10/0209).
Strain origin
The rat strain WKY-Dnd1ter/Ztm traces back to a spontaneous mutation that occurred
in the Institute for Laboratory Animal Science, Hannover Medical School in the inbred
generation F73 of the WKY/Ztm rat strain(Länger et al., 2004). The ter mutation leads
to the development of teratomas and infertility with an incidence of 100% in
homozygous animals, while no effects are seen in heterozygous animals. WKY-
Dnd1ter/Ztm colony is maintained as a segregating, coisogenic inbred strain.
Husbandry
Animals were maintained under standardized conditions in the following environment:
temperature of 22 ± 2°C, relative humidity of approximately 55%, and artificial light
58 Chapter 5
for 14h. The commercial softwood granulate bedding was sterilized (Lignocel,
Altromin; Lage, Germany). They received an autoclaved commercial pelleted diet
(Altromin 1314) (protein 22%, fat 5%, raw fiber 4.5%, ash 7%, utilizing energy 3.1
kcal/g) and water ad libitum.
The rats were kept under conventional housing conditions, as pairs or in sibling
groups. Mice were housed under specified pathogen free (SPF) conditions,
separated by gender and in individually ventilated cages according to ETS123.
Microbiological status was monitored according to FELASA recommendations
(Nicklas et al., 2002). The mice were free of the listed microorganisms. The DA.1M
rats were positive for parvovirus, helicobacter hepaticus and apathogenic protozoa
and the WKY/Ztm rats were positive for parvovirus and apathogenic protozoa.
EGC Derivation
WKY-Dnd1ter/Ztm rats with a ter/+ genotype were mated and the pregnant females
were sacrificed at day 14.5 post coitum. Embryos were collected and EGCs were
isolated as previously described by Cooke and colleagues. with minor modifications
(Cooke et al., 1993): The genital ridge containing the PGC was dissected free from
the embryo without the mesonephros and a single-cell suspension was obtained by
mechanical disruption and digestion with accutase.
EGC Culture
EGC were maintained on -irradiated TRF-O3 cells as feeder in N2B27 media
supplemented with 2i-LIF (Buehr et al., 2008; Li et al., 2008). 2i-LIF includes the
MEK-inhibitor PD032591 (1µM; Axon, Groningen, The Netherlands), the GSK-3
inhibitor CHIR99021 (3µM; Axon) and rat LIF (1000 U/ml; Millipore, Billerica, MA).
The TRF-O3 cells stem from a teratoma that developed in the ovaries of a female
ter/ter WKY-Dnd1ter/Ztm rat and are maintained in DMEM media with FCS. Between
7-10 days of primary culture all visible EGC colonies were picked and expanded. The
EGC colonies were identified based on their morphology and the fact that they were
easy to detach from the feeder cells. To maintain the cells, medium was changed
every other day and cells were split with accutase every 3-4 days. Cells were
Chapter 5 59
cryopreserved in 2i-LIF medium supplemented with 10% DMSO and survived
multiple freeze-thaw cycles.
Immunocytofluorescence and Alkaline Phosphatase Staining
Immunostaining was performed by fixation of the cells in 4% paraformaldehyde for
2h. Cells were then made permeable using 0.05% Triton-X-100 (Sigma) together and
1% SDS in PBS for 10min. Primary antibodies were: rabbit IgG anti Oct-4 (Millipore,
1:50), rabbit IgG anti DDX4/MVH (Abcam, 1:50), rat IgM anti SSEA3 (Millipore,
1:100), goat IgG anti Nanog (Everest, 1:100), mouse IgM anti SSEA1 (Santa Cruz,
1:100). The following Alexa Fluor secondary antibodies (Invitrogen, Carlsbad, CA)
were used: donkey anti-rabbit IgG (1:800), donkey anti-goat IgG (1:800), goat anti-rat
IgM (1:400), goat anti-mouse IgM (1:800).
AP staining was performed with the Stem Tag™ alkaline phosphatase staining kit
(Cell Biolabs, San Diego, CA) according to the manufacturer‟s instructions.
Genotype and Gender Determination
DNA from rat tissue or EGCs was extracted using TENS buffer (50mM Tris-HCl,
100mM EDTA, 100mM NaCl, 1% SDS and 0,27 mg/ml proteinase K) followed by
isopropanol precipitation. For the identification of the ter allele, PCR and restriction
digest were performed as described (chapter 4). Cleavage fragments from PCR
products of wild type Dnd1 (180 bp and 380 bp) and unchanged 560 bp ter allele
products were separated by electrophoresis in a 1.5 % agarose gel in 1x TBE buffer.
The gender of the EGCs was determined by PCR amplification of the Y-
chromosome-specific Sry gene using the HotStarTaqMastermix Kit (Qiagen, Hilden,
Germany) with the primers 5‟-CTGAAGACCCTACACAGAGA-3‟ and 5‟-
GCTTTTCTGGTTCTTGGAGG-3‟ at an annealing temperature of 52°C. The 210 bp
product in males was identified by electrophoresis in a 2% agarose gel in 1 x TBE
buffer.
Cloning
Dnd1 from the wild type EGC line and one ter/ter EGC line were cloned into the
vector pSCA-amp/kan using the StrataClone PCR cloning kit (Agilent Technologies,
60 Chapter 5
Waldbronn, Germany) according to the manufacturer‟s recommendations. For
cloning, 0.5µl cDNA were amplified using Phusion® Hot Start High-Fidelity DNA
Polymerase (NEB, Ipswich, MA). An A-overhang was ensured by adding 1µl of Taq
Polymerase (Qiagen, Hilden, Germany) to the samples and incubating for an
additional 10 min at 72°C after amplification. The forward primer 5‟-
CCCGGGCATGCAGTCCAAACGGGAGTG- 3‟ was used with the reverse primer 5‟-
CCCGGGGGCCGGCCCTGCTTCACCACAGAACC-3‟ for wild type Dnd1, while the
5‟-CCCGGGGGCCGGCCGAAGCGATGCCAGCCGGC-3‟ reverse primer was used
for the mutated Dnd1. The additional XmaI and FseI restriction sites were added to
the forward and the reverse primers for further cloning efforts. The primers were
synthesized by Eurofins MWG Operon (Ebersberg, Germany). Sequencing of Dnd1
cDNA was performed by Seqlab (Göttingen, Germany) using the primers T3 and T7.
Cytogenetic Analysis
Metaphase chromosomes were prepared by treating the cells with colcemid at a final
concentration of 0.035µg/ml for 5.5 hours, incubated in 0.075M KCl for 20 min at
37°C and fixed in a freshly prepared mixture of methanol:acetic acid (3:1) at room
temperature. Cell suspension was dropped onto glass slides in a climate chamber
(Polymer, Kassel, Germany) at 22°C and 48% humidity and stained with Giemsa.
Images were acquired using a Zeiss Axio Imager.M1 microscope equipped with the
BandView EXPO Software (ASI; Applied Spectral Imaging, Ltd., Migdal HaEmek,
Israel). Altogether, 20-25 metaphases per case were selected for chromosome
counting.
Cell Proliferation Assay
Cell proliferation reagent WST-1 (Roche, Mannheim, Germany) was used according
to the manufacturer‟s instructions. 1x104 cells were seeded onto a 96-well plate and
after 48h of culture 10µl of the cell proliferation reagent WST-1 were added per well
followed by incubation for 3.5h.
Chapter 5 61
Embryonic Stem Cells (ESC)
ESCs were derived from the inner cell mass of +/+ WKY-Dnd1ter/Ztm rat embryos
d4.5 pc as described previously and maintained under the same culture conditions as
the EGCs (Buehr et al., 2008; Li et al., 2008).
RNA Extraction and Reverse Transcription
RNA was extracted from cell culture and tissue using the RNeasy Mini Kit (Qiagen)
according to the manufacturer‟s instructions. cDNA was synthesized from 1µg of
RNA using the Omniscript Reverse Transcription Kit (Qiagen, Hilden, Germany) with
Oligo(dt)18 primers (Fermentas, St. Leon Rot, Deutschland) following the suppliers
recommendations.
Gene Expression Analysis
Expression analysis was performed with the HotStarTaqMastermix Kit (Qiagen)
according to the supplier‟s instructions using 0.1µl of cDNA.
The primers for c-Myc, Klf4, Nanog, Oct4, Stella, Rex1, Sox2, Blimp1, DDX4/MVH
and GAPDH have been described previously (Komiya & Tanigawa, 1995;
Zschemisch et al., 2006; Buehr et al., 2008). Other primers used were: Nanos3
forward 5‟-CTTCTGTCTACTGCTACACC-3‟ and reverse 5‟-
CTTCCTGCCACTTTTGGA-3‟; Fragilis forward 5‟-AGAACTCTCCATCCTCTACC-3‟
and reverse 5‟-CAGAGTAGGCATAGGCAATG-3‟; c-kit forward 5‟-
TCAAGGAAGGTTTCCGAA-3‟ and reverse 5‟-AGGAGAGGCTGTGTGGAAGA-3‟.
Teratoma Induction
EGC were harvested and resuspended in PBS to form a slightly clumpy suspension.
Cells were drawn into a 1ml syringe, kept at 4°C and warmed to room temperature
prior to injection. Teratomas were then induced by subcutaneous injection of the
5x106 EGCs in 150µl of PBS cells into the ventro-lateral region of NOD.CB17-
Prkdcscid/J mice. Teratomas were removed when they had reached a maximal
diameter of 1-1.5cm or weight loss of over 20% was evident in the recipients.
Formalin-fixed and paraffin-embedded teratomas were cut into 4µm slices before
62 Chapter 5
staining with HE. Microphotographs were taken using a Zeiss AxioCam MRc camera
and analyzed histologically for structures of all three germ layers.
Blastocyst Injection and Embryo Transfer
Blastocyst injection was performed as described with some modifications (Hogan et
al., 1994). DA.1M blastocysts were injected at d5pc after mating. 10 embryos were
transferred to each uterine horn of a pseudopregnant rat (Hedrich, 1990). Chimeric
rats were identified by coat color. Germline transmission was tested by mating of
chimeras with WKY/Ztm rats.
5.4. Results
Derivation of embryonic germ cells
Our goal was to culture and characterize pluripotent rat EGCs from the WKY-
Dnd1ter/Ztm rat strain. We isolated PGCs from embryos at day 14.5 post coitum after
breeding of Dnd1ter/+ rats. The genital ridges were dissected from the embryo,
mechanically disrupted and digested with accutase (Fig. 5.1A). Resulting cells were
seeded in 2i medium containing recombinant rat LIF on radiation-inactivated rat
tumor fibroblasts as feeder cells. After 7-10 days of culture clonal growth of colonies,
consisting of small and spherical cells referred to as embryonic germ cells (EGC),
was observed along with differentiated cells. To establish the EGC lines, the colonies
from 60 embryos were picked and expanded leading to 11 different cell lines. All cells
lines were propagated in an undifferentiated state for more than 40 passages by
dissociation with the enzyme accutase (Fig. 5.1B). Morphology did not differ
significantly between passages and is also comparable to that of the rat EGCs from
d10pc depicted in the publication by Leitch et al. (2010).
Characterization of the embryonic germ cell lines
Due to the fact that heterozygous ter/+ WKY-Dnd1ter/Ztm rats were mated, it was
essential to determine the genotype of the cell lines as well as the gender. The
results for the 11 EGC lines showed that 3 male and 7 female cell lines were Dnd1
Chapter 5 63
deficient while only one, the female cell line denominated rEGC2, carried the
homozygous wild type Dnd1 (Fig. 5.2A). These genotyping results were verified by
cloning Dnd1 from the +/+ cell line and a ter/ter cell line into the vector pSCA-
amp/kan and subsequent sequencing. The point mutation in which a G-base was
substituted for a T-base could be confirmed in the ter/ter cell line, whereas no
abnormalities were seen in Dnd1 of the +/+ cell line (data not shown). In accordance
with the mating of heterozygous rats, 25% +/+, 50% ter/+ and 25% ter/ter cell lines
would be expected if Dnd1 played no role in EGC survival. However, there were no
ter/+ and only one +/+ cell line with the rest of the cell lines being ter/ter.
Furthermore, no +/+ EGCs could be established from the mating of +/+ WKY-
Dnd1ter/Ztm, as all surviving cells ended up differentiating in culture. These results
suggest that loss of Dnd1 facilitates EGC survival in vitro. Aside from the Sry-PCR,
the gender of the EGCs could be determined through the morphology of cell
colonies. While the female EGCs produced mostly round colonies with smooth
edges, male EGC were prone to a more irregular growth with an angular shape of the
colonies (Fig. 5.2B).
Cytogenetic analysis revealed that four of the eight analyzed rEGC lines (rEGC 1, 2,
4, 8) retained euploid chromosome complements with 42 chromosomes in 67-80% of
the analyzed metaphase at passage 10. A slight increase of numerical aberrations
was detectable in these cell lines over the next passages, with only 60-70% of the
metaphases being euploid at passage 20. The other four cell lines (rEGC 3, 5, 6, 7)
showed mostly aneuploid chromosome complements and included tetraploid and
complex aberrant metaphases at passage 10 and 20. The wild type rEGC2 line was
among the cell lines with a euploid chromosome complement and was also analyzed
at passage 30 showing numerical aberrations in less than 40% of the metaphase
spreads (Fig. 5.3A). Since there were gender-differences in tumor progression in
vivo, we decided to evaluate the proliferation rate of the different EGC lines. A WST-
1 assay was performed with cell lines rEGC1-8 and the inactivated TRF-O3 feeder
cells as control. Proliferation rate varied between the cell lines; however, this seemed
to be independent of gender, genotype or chromosome number. The genetically
instable cell lines showed both the two highest (rEGC5: absorbance 1.655 SD±0.080;
64 Chapter 5
rEGC7: absorbance 1.684 SD±0.169) and the lowest (rEGC3: absorbance 1.316
SD±0.084) proliferation rates (Fig. 5.3B).
Proof of Pluripotency
EGCs, much like ESC, are pluripotent cells and as such express specific markers.
Pluripotency of EGCs could be demonstrated by strong activity of alkaline
phosphatase (AP) in various passages, as late as passage number 25 (Fig. 5.2B).
Furthermore, immunostainings demonstrated the expression of the pluripotency
markers Nanog, Oct4, SSEA1 and SSEA3 in all of the 8 examined cell lines. EGCs
were characterized as PGC-derived by detection of DDX4/MHV in
immunofluorescent stainings (Fig. 5.4). DDX4/MVH, also known as RVLG (rat vasa-
like gene), is the vasa-homologue in the rat and used as a specific marker for germ
cells (Komiya & Tanigawa, 1995; Toyooka et al., 2000). Pluripotency and germ cell
associated markers expressed in the ICM or in PGCs can differ from those
expressed in their in vitro counterparts ESCs and EGCs. Therefore, expression
analysis was performed by RT-PCR with cDNA from EGCs, ESCs and tissue
containing PGCs. The pluripotency-associated genes Nanog, Oct4, Sox2, Rex1, c-
kit, c-myc and Stella were detected in the ESCs and EGCs as well as in the genital
ridge containing the PGCs. The levels of Nanog were higher in the ESCs than in the
EGCs and very low in the genital ridge. There was a strong signal for Klf4 in the
feeder cells; therefore it cannot be excluded that the signal in the ESCs and EGCs
could be due to a slight contamination with feeder cells. Nanos3, which is expressed
during hind gut migration of the PGCs and essential for PGC survival in the mouse
(Tsuda et al., 2003), was found in the genital ridge and a very weak expression in
EGCs and ESCs. The results for Blimp1 mirror those found previously in mouse and
rat at an earlier embryonic stage (Durcova-Hills et al., 2008; Leitch et al., 2010):
Blimp1 could only be identified in tissue from the embryo and was no longer present
in the EGC lines. DDX4/MVH was expressed in the genital ridge and in the EGC and
unexpectedly also in the ESCs. When comparing the genital ridges it was noted that
the DDX4/MVH and Sox2 expression was lower in ter/ter than in +/+ embryos. To put
it briefly, the RT-PCR showed that the EGCs expressed all of the expected
pluripotency and germ cell markers (Fig. 5.5). To allow the use of EGCs as a tool for
Chapter 5 65
further research it was also vital to show their pluripotency through the ability to
develop into the different tissue types. The differentiation potential was demonstrated
through subcutaneous injection of rat EGCs into immunodeficient NOD.CB17-
Prkdcscid/J mice, which resulted in tumor formation. Histological analysis revealed
derivatives from all three embryonic germ layers thereby showing the classical
characteristics of teratomas. Cartilage, neuronal tissue, skeletal muscle tissue,
mucosal epithelium and squamous epithelium could be found among the identified
tissues. Genotype and gender of the cell line played no role in the ability to induce
teratomas as these were obtained from every single injected cell line (Fig. 5.6A).
True pluripotency of cells is only given when cells are capable of integrating into the
developing embryo and are transmitted through the germline. Wild type rEGC2 cells
from different passages injected into blastocysts were capable of integrating into the
embryo. Chimeras were identified through coat coloring. The EGCs stem from an
albino rat and the injected blastocysts from the agouti colored rat strain DA.1M.
Blastocysts were injected with cells from passage 5 before being transferred to a
pseudopregnant female. Of the 5 pups born in the litter, 1 female and 3 males were
chimeric. The chimeras showed a normal development and demonstrated no defects
or malfunctions up to 8 months of age. Furthermore, the female chimera was capable
of transmitting through the germline with 36% (14 of 38 pups) of the offspring
carrying the cell line genome (Fig. 5.6B).
5.5. Discussion
We were able to culture EGCs from post-migratory PGCs of the genital ridge using
d14.5 embryos from the rat strain WKY-Dnd1ter/Ztm. The loss of Dnd1 clearly helped
render the isolation of EGC lines possible. Propagated EGCs were pluripotent and
half of the isolated cell lines retained a stable chromosome number. The wild type
cell line, rEGC2, contributed to the first germline competent chimera made with rat
EGCs.
EGCs have been derived from the genital ridge of other species such as mice, pigs,
chicken and goats (Matsui et al., 1992; Resnick et al., 1992; Shim et al., 1997; Jia et
66 Chapter 5
al., 2008). However, the only rat EGC derived so far are early pre-migratory EGCs
from Sprague Dawley rat embryos at d10pc (Leitch et al., 2010). Here we isolated
late EGCs from the genital ridge. Our protocol for EGC-derivation differs from that of
Leitch et al. (2010) in the embryonic stage (d14.5pc compared to d10pc), in the
feeder cell line (TRF-O3 instead of stem cell producing Sl4-m220 and MEF) and
additionally our EGC colonies emerged earlier in culture (at passage zero and not
passage one).
Despite the fact that we cultured wild type as well as ter/ter PGCs, with the exception
of +/+ rEGC2, only the latter could be propagated in an undifferentiated state. The
mutation of the putative tumor suppressor gene Dnd1 supports or simply accelerates
the immortalization of cultured PGCs from d14.5pc to EGCs. This reveals that the
fate of the PGCs is influenced as early as d14.5pc by the loss of Dnd1. Something
similar can be seen in mice where the tumor suppressor phosphatase and tensin
homologue protein (PTEN) was knocked out, as the PGC-restricted PTEN knockouts
resulted in a high incidence of testicular tumors and the mutant mPGCs formed
EGCs more efficiently than wild type PGCs (Kimura et al., 2003). Moreover,
hyperactivation of the serine/threonine kinase AKT and its suppression of p53
activation also augmented the efficiency of EGC establishment (Kimura et al., 2008).
Dnd1 is known to protect certain mRNAs, including those of tumor suppressor genes
such as p27 and Lats2, from repression by miRNAs (Kedde et al., 2007). The
immortalization of PGCs could be facilitated through the inactivation of tumor
suppressor genes, a well-known process in other (tumor) cell lines. An example
would be the inactivation of p53, a tumor suppressor gene, which enhances
spontaneous cellular immortalization and initiates the tumorigenic transformation of
cells in vivo while playing a key role in the immortalization of murine embryonic
fibroblasts (MEFs) in vitro (vom Brocke et al., 2006; Fridman & Tainsky, 2008).
Gonadal teratocarcinomas originate from PGCs and consist of differentiated tissues
from all three germ layers along with undifferentiated, pluripotent cells (Stevens,
1967). In ter/ter rats all teratomas were restricted to the gonads, meaning that all
tumor precursor cells reach the genital ridge. The growth rate of the teratomas was
gender-specific. First gender-dependent differences such as varying gene expression
Chapter 5 67
profiles can be seen after sex differentiation which starts at d12.5pc in the mouse
embryo (McLaren, 2003; Sabour et al., 2010). The embryonic stage of a mouse at
d12.5pc is comparable to d14pc in the rat (Hill, 2010). These facts made late rat
EGCs from the genital ridge our preferred tool for further research on teratoma
development in connection with Dnd1.
Although seven of the cell lines stem from ter/ter WKY-Dnd1ter/Ztm the number of
chromosomes remained remarkably stable in three of them. In fact, the chromosomal
stability was comparable to that found in rEGC2 or ESC (unpublished data) of +/+
animals from the same rat strain.
The pluripotency of our EGCs could be shown by AP-staining, immunostainings, RT-
PCR, and teratoma development after injection of EGCs into immunodeficient mice.
High alkaline phosphatase activity is characteristic for all pluripotent cells.
Immunocytology detected further markers of pluripotency (Oct4, SSEA1, SSEA3 and
Nanog) along with the germ cell maker vasa (DDX4/MVH). Derivatives of all three
germ layers were identified in the tumors that developed in the NOD.CB17-Prkdcscid/J
mice and confirmed the pluripotent potential of the cells.
Comparative expression analysis by RT-PCR for different pluripotency and germ cell
markers in EGC and/or ESC lines have been performed before in humans, mouse
and rat (Toyooka et al., 2000; Clark et al., 2004; Buehr et al., 2008; Conrad et al.,
2008; Durcova-Hills et al., 2008; Leitch et al., 2010). In the following we compare our
results to those of others. Most markers (Nanog, Oct4, Sox2, Rex1, c-kit, c-myc,
Stella) were expressed in EGCs, ESCs and the genital ridges of rats, which is in
accordance with the cited literature (Buehr et al., 2008; Durcova-Hills et al., 2008;
Leitch et al., 2010). Unexpectedly, the PCR amplification of germ cell markers
showed that all ESC and EGC lines expressed DDX4/MVH. In the mouse,
DDX4/MVH is a specific marker from the late migration to the post-meiotic stage only
expressed in germ cells after entry into the genital ridge. It is not expressed in ESC,
EGCs from migrating PGCs or in PGCs before entering the genital ridge (Toyooka et
al., 2000). The results for undifferentiated human ESCs are varied: Clarke et al.
(Clark et al., 2004) found no expression of VASA while Conrad et al.(Conrad et al.,
2008) detected high levels of VASA in their ESCs. This difference could therefore be
68 Chapter 5
either species-specific or a result of different culture conditions, as it is known that
ESCs can differentiate to germ cells in vitro (Toyooka et al., 2003; Clark et al., 2004).
The fact that we did not find DDX4/MVH expression in the distal part of the embryo at
d10.5pc (supplementary data), which contains the PGCs, leads us to lean towards
the latter. The visibly reduced expression of DDX4/MVH and Sox2 expression found
in ter/ter genital ridges when compared to +/+ genital ridges could be explained
through an early loss of PGCs in this tissue. This would be analogous to the
observations made in the Ter mouse where fewer PGCs survive and enter the genital
ridge of the Ter/Ter than of the +/+ embryo (Sakurai et al., 1994). Furthermore, a
slight expression of Klf4 was seen in the embryo at d10.5 pc, whereas Klf4 was not
detected in PGCs at a comparable embryonic stage in the mouse (Durcova-Hills et
al., 2008) or in the posterior fragment of a rat at d10pc (Leitch et al., 2010).
The +/+ EGC line was capable of chimera contribution and also germline
transmission. This is the first time that germline competence could be shown for
EGCs from the rat, thereby proving that the conversion of once unipotent PGCs to
truly pluripotent EGCs is possible in the rat. Leitch and colleagues (2010) showed
that genetically manipulated EGCs can result in chimeric rats; however, the offspring
did not carry the transgene. The fact that germline transmission could not be shown
in those rat EGCs could be either due to the genetic manipulation or maybe only the
increased number of passages. EGCs have even been used as an alternative to
ESC for the successful germline-competent genetic manipulation in other species
such as pig (Piedrahita et al., 1998) or chicken (van de Lavoir et al., 2006) and could
present an alternative tool to ESCs for gene targeting in the rat.
Conclusion
Here we cultured the first pluripotent and germline competent EGCs from late rat
embryos. The genital ridge-derived rat EGCs offer an innovative approach for studies
on germ cell differentiation and germ cell tumor formation in both genders.
Furthermore, our data indicates that the loss of the putative tumor suppressor gene
Dnd1 enhances the cultivation of late rat EGCs and supports EGC immortalization.
The culture of EGCs gives us a new opportunity to research the interactions between
Dnd1 and its target genes.
Chapter 5 69
5.6. Figures
Figure 5.1
Fig. 5.1: (A) Embryo at d14.5pc and the extracted genital ridges (white arrow) attached to
the mesonephros. The genital ridges contain the PGCs and are cultured. (B) Genital ridge-
derived EGC colonies at passage 0 (P0) and passage 10 (P10)
70 Chapter 5
Figure 5.2
Fig. 5.2: Genotype and gender. (A) Genotyping: Dnd1 was amplified by PCR followed by a
restriction digest with Kpn1. The wild type allele was cut, whereas the ter allele lost the KpnI
recognition site and remains uncleaved (rEGC2: +/+; rEGC1, 3-11: ter/ter). The gender
detected by PCR amplification of the Y chromosome specific Sry gene (rEGC 4, 7, 8: male;
rEGC1-3, 5, 6, 9-11: female). (B) Alkaline phosphatase (AP) staining. Morphological
differences visible between the AP positive colonies of a male and a female EGC line.
Chapter 5 71
Figure 5.3
Fig. 5.3: Differences in-between EGC lines. (A) Metaphase spreads of rEGCs. Euploid
chromsosome complement of rEGC2 showing 42 chromosomes in passage 10 and 20.
EGC3 with structural and numerical aberrations in passage 10. Aneuploid chromosome
complement of rEGC7 with a reunion figure (arrow) in passage 20. (B) WST-1 assay to
determine the proliferation rates of rEGC1-8 and inactivated TRF-03 as control.
72 Chapter 5
Figure 5.4
Fig. 5.4: Staining of rat EGC for germ cell and pluripotency markers. The
immunocytofluorescence was positive for SSEA3, SSEA1, Nanog and Oct4, as well as the
germ cell marker DDX4/MVH.
Chapter 5 73
Figure 5.5
Fig. 5.5: Expression analysis: RT-PCR of pluripotency markers using cDNAs from late rat
EGC lines (rEGC1-8), rat ESC from WKY-Dnd1ter/Ztm, and the genital ridges of d14.5pc
embryos containing PGCs. Controls: TRF-O3: feeder cells cultured in 2i medium. C: negative
control.
74 Chapter 5
Figure 5.6
Fig. 5.6: Differentiation potential of EGCs (A-D) Injection of EGCs into immunodeficient
NOD.CB17-Prkdcscid/J mice led to formation of teratomas with derivatives of all three germ
layers. (A) Mucosal epithelium (representing Endoderm). (B) Neuronal tissue (representing
Ectoderm). (C) Cartilage (representing Mesoderm). (D) Skeletal muscle tissue (representing
Mesoderm). (E-F) Chimeras and germline transmission: (E) Chimeric siblings. (F) Chimeric
mother with albino germline pup and littermates.
Chapter 5 75
5.7. Supplementary data
Recently, we mated heterozygous ter/+ rats from the WKY-Dnd1ter/Ztm and cultivated
pre-migratory PGCs from rat embryos at d10.5pc (Suppl. Fig. 1). PGCs were
collected as described by Leitch and colleagues (2010). The culture conditions used
were identical to those described for the embryos d14.5pc, aside from the fact that
the EGC colonies were detached from the feeder between 9-12 days of culture and
not 7-10 days. Altogether PGCs from 21 different embryos were cultured and each
embryo gave rise to EGCs. Genotyping of the EGC lines revealed that ter/ter, wild
type and heterozygous cell lines from both genders had survived in vitro (Suppl. Fig.
2). This shows that inactivation of Dnd1 does not play a role in deriving pluripotent
cells from early PGC in vitro. Pluripotency was depicted in all cell lines by staining for
alkaline phosphatase (Suppl. Fig. 1B). Expression analysis was performed by RT-
PCR with four randomly chosen wild type and ter/ter cell lines from d10.5pc. The
posterior fragment of the embryo, which contains the PGCs, was used as a control.
The pluripotency and germ cell markers Nanog, Oct4, Sox2, Klf4, Rex1, c-kit and
Stella were detected in the EGCs and the embryo. Expression of DDX4/MVH was
only exhibited by EGCs, and not by the PGCs in the embryo d10.5pc (Suppl. Fig. 3).
76 Chapter 5
Supplementary Figures
Supplementary Figure 5.1
Suppl. Fig. 5.1: (A) Embryo at d10.5pc. The pre-migratory PGCs are located in the distal
end of the embryo. (B) Early WKY-EGC colonies stained with AP.
Supplementary Figure 5.2
Suppl. Fig. 5.2: Genotyping and gender determination of early WKY-EGC lines from 21
embryos. 8 EGC lines are male and 13 are female. 9 EGC lines are ter/+, 4 are +/+, and 8
are ter/ter.
Chapter 5 77
Supplementary Figure 5.3
Suppl. Fig. 5.3: Expression analysis: RT-PCR of pluripotency markers using cDNAs from
four early WKY-EGC lines (21, 22, 25, 37), posterior fragment of d10.5pc embryos containing
pre-migratory PGCs (dE), and ESCs from the WKY-Dnd1ter/Ztm rat strain; C: negative
control.
78
Discussion and Conclusions 79
6. Discussion and Conclusions
Incidence of infertility and TGCTs are on the rise and the key to understanding these
diseases lies in discerning the factors and genetics regulating germ cell
differentiation to mature gametes and de-differentiation to pluripotent cells (Huyghe
et al., 2003; Hotaling & Walsh, 2009). A new phase in the intensive research on
genetic and molecular mechanisms involved in germ cell differentiation was
introduced by generating germ cells from embryonic stem cells and the isolation of
pluripotent spermatogonial stem cells from germ cells as a therapeutic tool
(Mardanpour et al., 2008; Guan et al., 2009; Ko et al., 2009). The goal of this study
was to characterize and establish two models that can be used for further research
on germ cell development. In general, this is the WKY-Dnd1ter/Ztm rat strain and, in
particular, the cultured EGCs of said strain. Dnd1 is a relevant gene in germ cell
development of the rat and this is elucidated in the manuscripts containing the two
parts of this project:
Chapter 4: The ter mutation in the rat Dnd1 gene causes gonadal teratomas and
infertility in both genders
Chapter 5: Loss of Dnd1 facilitates the cultivation of genital ridge-derived rat
embryonic germ cells
This chapter summarizes the principal results of the two aforementioned chapters
and provides a combined discussion based on the literature review (chapter 2) while
giving an outlook on the future perspectives for germ cell and teratoma research.
6.1. The WKY-Dnd1ter/Ztm rat as a model for germ cell tumors
Linkage analysis, fine mapping and subsequent sequencing of candidate genes
traced the WKY/Ztm-ter rat to a point mutation in exon 4 of the Dnd1 gene on
RNO18. Accordingly, the strain was denominated WKY-Dnd1ter/Ztm, and it is a
valuable animal model for research on GCTs in both genders (chapter 4). Studies on
80 Discussion and Conclusions
germ cell development have confirmed a central role of Dnd1 in the regulation of
PGC migration, survival and tumor suppression in different vertebrates (Ketting,
2007).
6.1.1. Highly conserved functions of Dnd1 in germ cell development
The Dnd1 mutations on Chr 18 in mice (Youngren et al., 2005) and rats occurred
spontaneously with a single base change introducing a premature stop codon, which
probably causes a c-terminal truncation of the Dnd1 protein. The manifestation of
these highly related genetic modifications in impairment of gametogenesis along with
neoplastic transformation of PGCs to ECC emphasizes a central and conserved
function of Dnd1, in particular of the c-terminal end of the protein. The c-terminus of
the zebrafish Dnd protein possesses an ATPase domain, which is essential for PGC
formation and survival (Liu & Collodi, 2010); however, it remains to be established
which parts of the c-terminus are crucial for a functioning Dnd1 protein in rodents and
whether an ATPase domain is involved. Downregulation of dnd in the germ plasm of
zebrafish and African clawed frog inhibits PGC migration and results in their
disappearance (Weidinger et al., 2003; Horvay et al., 2006). Mammalian PGCs lack
migration disorders, while tumorigenesis is not observed in anamnia, thereby
illustrating the varying functions of Dnd1. The ontogenetic death of PGCs in response
to the functional inactivation of the dead-end gene, however, is a common
phenomenon in all species, including the rat. Consequently, dead end is a highly
conserved and essential gene regulating the survival of PGCs in both anamnia and
amniota.
6.1.2. Gonadal tumorigenesis and infertility in rodents
Genotyping of WKY-Dnd1ter/Ztm rats revealed that the homozygous Dnd1 mutation
resulted in complete penetrance with 100% of the ter/ter animals of either gender
developing teratomas, whereas no teratomas were found in their wild type or
heterozygous counterparts. This deviates from the phenotype observed in the
129/Sv-Ter mouse model for GCTs. In general mice of the 129/Sv strain develop
spontaneous teratomas on a regular basis without a mutation in Dnd1, which is
something unheard of in rats of the WKY strain. Loss of functional Dnd1 in male
Discussion and Conclusions 81
129/Sv mice is responsible for increasing the TGCT incidence from 1% in wild type to
17% in heterozygous and 94% in homozygous males. At the same time, none of the
mutant females develop any teratomas (Noguchi & Noguchi, 1985). Consequently,
the Dnd1 mutation followed a recessive mode of inheritance in male and female rats,
which is distinct from the co-dominant Ter-allele involved in teratocarcinogenesis of
male mice.
Rats with a homozygous mutation in the Dnd1 gene were infertile due to a loss of
germ cells, while the heterozygous animals possessed normal fertility and litter sizes.
Similarly, all male Ter/Ter mice are also sterile and Ter/+ mice do not show any signs
of germ cell deficiency. The fertility of female Ter/Ter mice is only reduced as some
germ cells are capable of survival and progress to mature oocytes (Noguchi &
Noguchi, 1985; Sakurai et al., 1995).
Teratoma development in the rat testis was either bilateral or unilateral. The
underlying defects initiated proliferation of neoplastic cells in one or both testicles
while the non-tumorous testicle showed a complete depletion of germ cells and loss
of the typical structure of the tubuli seminiferi without GCT. Every one of the ter
females was infertile and developed OGCTs without ever exhibiting any cases of
degenerated ovaries. This could be related to the relatively short time period of about
42 days (at 21-66 days of age) during which tumors were observed in all females, not
being long enough to allow complete germ cell deterioration in the entire ovary prior
to neoplastic proliferation. The fact that non-neoplastic primary follicles found in early
OGCTs did lack oocytes supported this presumption. In comparison, tumor
progression varied strongly between male individuals, and tumors were diagnosed
over a time period of 182 days (at 14-196 days of age). The wide range of time
probably allowed one testis to develop teratomas, while the other degenerated.
6.1.3. Implications of the ter rat for human TDS and OGCTs
Human disorders of germ cell development leading to the assorted symptoms of TDS
are common and are believed to stem from ontogenetic disruptions (Skakkebaek et
al., 2001; Walsh et al., 2009). The WKY-Dnd1ter/Ztm rat strain is a model for TDS as
the Dnd1 mutation could result in infertility, small degenerated testis and TGCTs in
males. The older the male ter/ter rats were, the lower the rate of degenerated testes
82 Discussion and Conclusions
and the higher the rate of bilateral tumors. Hence, it is highly likely that the infertile
and degenerated testes exhibited by ter/ter males are the predecessors of cancer.
This supported the view of Burns and colleagues (2010) that infertile men are at an
increased risk of developing testicular cancer.
In contrast to the Ter mouse, the WKY-Dnd1ter/Ztm rat was also a model for OGCTs.
It has been proposed that TGCTs and OGCTs share a common pathogenic
mechanism as, for the most part, both ovaries and testis are capable of developing
identical types of GCTs (Faulkner & Friedlander, 2000). Furthermore, clusters of
OGCTs and TGCTs have been discovered in one and the same family
(Giambartolomei et al., 2009) and chromosomal abnormalities of HSA 12p are found
in GCTs of either gender in humans (Faulkner & Friedlander, 2000). The phenotype
of the WKY-Dnd1ter/Ztm supported the idea of a common pathogenesis, as a shared
mutation in a genetic locus resulted in GCTs with equal frequency in both genders. It
is probable that closely related or even identical mechanisms were responsible for
the neoplastic transformation of germ cells in both genders of the WKY-Dnd1ter/Ztm
rat. This might also be the case in GCTs of some men and women.
6.1.4. Gender and environment
Gender and environmental influences affect the frequency of germ cell tumors in
humans (Skakkebaek et al., 2007; Greene et al., 2010), and this is to some extent
reflected in the gender dissimilarities observed in the rodent models. Deviating
factors present in the gonad as well as intrinsic factors of the germ cells can both be
held responsible for the gender differences observed in mammalian
teratocarcinogenesis. In humans, factors secreted by the male sertoli cells, as well as
estrogen and follicle-stimulating hormone (FSH), have long been strongly believed to
play a role in TGCT development (Sharpe, 2006). Furthermore, estrogen-like
compounds and endocrine disrupters are both discussed as possible causes for the
rising incidence of the TDS (McGlynn et al., 2008). A hormonal aetiology has also
been proposed for human OGCTs (Walker et al., 1988). The surroundings provided
by the gonad seem to be required in teratocarcinogenesis of both Dnd1–mutant
mouse (Matin, 2007) and rat, since neither species developed teratomas in ectopic
sites. This is substantiated by the fact that the restriction of teratomas to male Ter
Discussion and Conclusions 83
mice is linked to the testicular environment, wherein female germ cells are as
capable as male germ cells of generating neoplastic clusters (Cook et al., 2009).
Differences between male and female rats existed, but were limited to tumor
progression. On the average tumors were diagnosed earlier in female than in male
rats, which is obviously the opposite of testicular surroundings enabling teratoma
growth in the mouse. Nevertheless, regardless of whether in the mouse or in the rat,
gender-specific features alter the course of germ cell development towards
teratocarcinogenesis in Dnd1-mutants. Which factors are responsible for inhibiting or
promoting tumorigenesis in the Dnd1 rodent models remains to be determined, but
the WKY-Dnd1ter/Ztm rat shows us that female factors have to be taken into account
as much as those of males. Using both the mouse and the rat as models for GCT will
help in identifying the different paracrine, autocrine or hormonal signals involved in
tumorigenesis. This could help comprehend the pathogenesis of human GCTs and
identify the source of the growing incidence.
6.1.5. Genetic background
The genetic component in susceptibility to TGCTs is as strong as it is complex. This
is applicable for humans with their high familial index of TGCTs and despite all efforts
no known susceptibility genes (Heaney & Nadeau, 2008), and is also true for the Ter
mouse and the ter rat. In the Ter/Ter mouse tumors only develop in the 129/Sv strain
and modifier genes (SlJ, Trp53null, Ay, CSS M19, M19-A2 and M19-C2) modulate the
teratoma incidence by interacting with the mutated Dnd1 (Lam et al., 2007).
Replacing the X-chromosome from the 129/Sv-ter mouse through that of the
C57BL/6 mouse was enough to shift the Ter/Ter phenotype from teratoma formation
to infertility (Hammond et al., 2007). In the rat backcrossing of the ter mutation to the
DA strain also yielded a change in phenotype, which proved that genetic factors
other than Dnd1 are associated with tumorigenesis in the rat (results not shown).
6.1.6. DND1 in humans
The role of DND1 in humans remains uncertain, but screening of over 500 men with
type II TGCTs made it fairly obvious that a DND1 mutation is an unlikely cause in the
majority of cases (Linger et al., 2008; Sijmons et al., 2010). Nevertheless, it is still
84 Discussion and Conclusions
conceivable that DND1 plays a role in teratocarcinogenesis through mechanisms
more complicated than those investigated in the two previous studies. This could
include the presence of duplications and large deletions in the gene (Sijmons et al.,
2010), as well as imprinting mechanisms, miRNA suppression or epigenetic
silencing. Testisin, MGMT, SCGB3A1, RASSF1A, HIC1 and PRSS21 are examples
for putative tumor suppressor genes that are inactivated through imprinting or
epigenetic silencing in TGCTs (Honorio et al., 2003; Kempkensteffen et al., 2006;
Lind et al., 2007). Furthermore, elucidating the pathways of Dnd1 in the mouse and
rat could help identify new processes and modifier genes that are involved in human
GCT development since the pathways of DND1 and GCTs do cross. For instance,
DND1 inhibits the miR-373 mediated LATS2 repression (Kedde et al., 2007), and the
same miR-373 is believed to act as oncogene in TGCTs (Voorhoeve et al., 2006).
Additionally, increasing the knowledge on DND1 is of importance since it could play a
role in other cancers such as the tongue squamous cell carcinoma associated with a
DND1 downregulation (Liu et al., 2010). Information on the role of DND1 in
prepubertal GCTs is lacking. The 129/Sv-ter mouse lacks CIS or characteristic
karyotypic abnormalities, and teratomas are detected soon after birth, which makes it
a model for human prepubertal type I TGCTs. Hence, it is quite possible that human
DND1 mutations could be responsible for prepubertal TGCT (Zhu et al., 2007a;
Anderson et al., 2009). The new-found role of Dnd1 in rat OGCTs signifies that the
involvement of DND1 in human OGCTs should be evaluated in future studies. Dead-
end is a highly conserved protein associated with reduced fertility and infertility
throughout different species, strains and genders. Therefore, it is feasible that
disruption of DND1 could cause infertility or reduced fertility in adult human beings.
6.2. Derivation of pluripotent EGCs from the WKY-Dnd1ter/Ztm rat
PGCs of various species can be reprogrammed into pluripotent EGCs through the
application of specific culture conditions. We were able to culture the first pluripotent
EGCs from post-migratory PGCs of the genital ridge using the rat strain WKY-
Dnd1ter/Ztm. Mating of heterozygous WKY-Dnd1ter/Ztm rats and culture of PGCs from
60 embryos resulted in clonal proliferation of ten Dnd1-deficient ter/ter EGC lines of
Discussion and Conclusions 85
both genders, while only one female wild type EGC line and no heterozygous cell
lines survived without differentiating in vitro. Therefore, Dnd1 played a role in
reprogramming cultured PGCs from d14.5pc to EGCs (chapter 5). EGCs were also
cultured from early rat embryos at d10.5pc, however, no Dnd1-dependent effects
were observed (chapter 5, supplementary data).
6.2.1. EGCs as a new approach to study rat Dnd1
The new rat EGCs can be used as a tool to determine the relevant mechanisms
involved in the neoplastic transformation of ter/ter PGCs. Previously, mouse EGCs
were employed to help clarify the interactions of Dnd1 with the APOBEC family
(Bhattacharya et al., 2008). Use of PGCs in research is restricted by their low number
and limited survival in the embryo, while their in vitro counterparts have the
advantage of being immortal and growing indefinitely. PGCs are the precursor cells
of both EGCs and ECCs and in the mouse these two pluripotent cell lines express
the same markers and differ only in their genetic stability. The tight link between
EGCs and ECCs was ascertained by the fact that mutations in Pten or p53 trigger
teratomas while also enhancing EGC derivation (Kimura et al., 2003; Kimura et al.,
2008). The advantage of culturing EGCs instead of ECCs as a tool to research Dnd1-
related effects is that the former enables comparisons between the wild type and
ter/ter genotype. Additionally, the EGCs have had less time than the instable ECCs
to accumulate genetic aberrations on top of the Dnd1 mutation. In general, ECCs are
aneuploid, while EGCs are usually euploid and maintain a normal set of
chromosomes (De Miguel et al., 2010). Almost half of the tested ter/ter EGC lines
and the +/+ EGC line used here remained euploid in culture for multiple passages.
In this project we cultured EGCs from d14.5pc WKY-Dnd1ter/Ztm embryos, despite
the fact that reprogramming of PGCs is more difficult and less efficient in late
embryonic stages (McLaren, 2003; Kerr et al., 2006). Both the neoplastic
transformation and sexual differentiation of PGCs starts at d12.5pc in the mouse,
which is the equivalent embryonic stage to the rat at d14.5pc. Furthermore, all tumor
precursor cells reach the genital ridge in Dnd1 mutants, as seen by the fact that no
ectopic GCTs developed. Altogether, the genital ridge-derived EGCs were more
likely than their earlier counterparts to display differences between wild type and
86 Discussion and Conclusions
ter/ter EGCs; making them more prone to successfully identify the differences
causing ter/ter PGCs to develop into teratomas and +/+ to develop into gametes in
the adult animal. This hypothesis proved to be true as seen by the influence of
mutated Dnd1 on the efficiency of deriving pluripotent cells from late, and only late,
PGCs. Early EGCs from d10.5pc were also cultured and Dnd1 did not exert such
strong effects in primary culture. In fact, no significant differences were seen between
embryos of different genotypes in their aptitude to generate EGCs. Teratoma
progression in the rat was gender-specific, and the late EGCs were more likely to
display germ cell intrinsic differences between the sexes. However, the growth rate
did not differ significantly between female and male ter/ter EGC lines. Consequently,
the differences in tumor progression of the rat are either induced by the somatic
surroundings of the germ cell or possibly the result of sexual differentiation of
PGCs/ECCs in later stages.
6.2.2. The culture of EGCs
Though EGCs of mouse and other species have been successfully cultured for some
time now, the rat proved to be more elusive, and rat EGC culture was achieved only
recently. The only rat EGCs to date stem from pre-migratory PGCs of Sprague
Dawley (SD) rat embryos at d10pc (early SD-EGC) (Leitch et al., 2010). Slightly
modified culture conditions were used to generate our WKY-Dnd1ter/Ztm EGCs from
d14.5pc embryo (late WKY-EGC). The specification of PGCs in the embryo is
accompanied by an upregulation of pluripotency markers such as Nanog, Oct4 and
Sox2. Nevertheless, PGCs differ significantly from EGCs as they are unipotent cells
restricted in their developmental potency to the gametes and incapable of integrating
into the blastocyst (McLaren & Durcova-Hills, 2001; Durcova-Hills et al., 2008). The
complete conversion of mouse PGCs to EGCs takes about 10 days, even though
some large EGC-like colonies can be observed after 6 days in culture (Durcova-Hills
et al., 2006). Similar to this, most of our rat EGC colonies from the genital ridge of
WKY-Dnd1ter/Ztm appeared between 7-10 days. This differs from findings of Leitch
and colleagues, who passaged their cells after 14 days of primary culture and
described the emergence of the first rat EGC colonies 7-15d after passaging, thereby
adding up to a total of 21-29 days. It is possible that loss of Dnd1 accelerated the
Discussion and Conclusions 87
formation of ter/ter EGC colonies, and that further +/+ EGC colonies might have
formed during a longer period of culture. However, that does not explain the
presence of the one EGC line from a d14.5pc +/+ embryo and the fact that d10.5pc
+/+ colonies also formed without having to be passaged first. Another reason might
be the culture conditions applied, which in the case of the early SD-EGCs where
based on the culture of mouse EGCs. The cells were cultured in media containing
FCS and bFGF and were plated on SCF-producing feeder cells. The media was
changed on day 3 and replaced with serum free N2B27 media supplemented with 2i-
LIF (Leitch et al., 2010). In contrast, our late WKY-EGCs were cultured using the
same conditions applied to rat ESCs. They were cultured directly in 2i-LIF media and
did not come into contact with either serum, or bFGF and SCF for that matter. Serum
induces differentiation or death in ESCs of the rat (Li et al., 2008), and a similar effect
might be inhibiting or prolonging the derivation of pluripotent early SD-EGCs from
PGCs.
To this date the culture conditions for rat EGCs have not been optimized, and almost
certainly do not achieve the utmost efficiency in deriving EGCs. Even though we
showed that 2i-LIF media is sufficient to generate EGCs, it remains to be determined
whether bFGF or SCF might enhance derivation or growth. In the mouse this is the
case, as neither SCF nor bFGF are required when using the 2i-LIF media, yet bFGF
still augments the number of EGCs developing from PGCs. As feeder cells are
eliminated during initial exposure to the inhibitors and removed in subsequent
changes of media, the number of media changes during primary culture might play
an important role in EGC derivation. Replacing media every other day as described
for both the early SD and our late WKY-EGCs might be excessive and unwarranted.
A greater sensitivity of rat EGCs than mouse EGCs to feeder cell elimination was
proposed by Leitch and colleagues (2010) to be one cause for the species
differences between mouse and rat in EGC derivation.
Prior to 2i-LIF media, long-term culturing of any pluripotent cells of the rat and many
mouse strains was not possible. Until now, human EGCs have been cultured in
media containing bFGF, LIF and forscolin (Shamblott et al., 1998). However, these
culture conditions are in need of improvement, as these human EGCs appear to
88 Discussion and Conclusions
have a limited long-term proliferative potential and do not contribute to teratomas (Yu
& Thomson, 2008). Therefore it would be interesting to see whether applying 2i-LIF
media might be a superior method for culturing human EGCs. This might not only
hold true for humans, but might also open new doors for deriving EGCs from other
species.
6.2.3. Molecular networks of pluripotency in EGCs
Comparisons of pluripotency and germ cell markers demonstrate almost identical
expression patterns between mouse or rat PGCs and EGCs (De Miguel et al., 2010).
Yet there are minor differences, and Blimp1 is the key gene expressed in PGCs but
not in EGCs or ECCs (Ancelin et al., 2006; Durcova-Hills et al., 2008; Leitch et al.,
2010). This result could be confirmed for all of the established genital ridge-derived
EGC lines. It is hypothesized that Blimp1 is crucial for repressing PGCs from
acquiring a genuinely pluripotent phenotype. High levels of the Blimp1 target genes
Dhx38 (after 8 days of culture), c-myc (after 7 days of culture) and Klf4 (after 3 days
of culture) are expressed in EGC while neither one is transcribed in the mouse PGCs
at d8.5pc (Durcova-Hills et al., 2008). Nevertheless, gene activity of Klf4 and c-myc
was found in the genital ridges of the WKY-Dnd1ter/Ztm rat. In analogy to the mouse
(Durcova-Hills et al., 2008) the c-myc expression probably results from the
surrounding somatic cells of the gonad, however, the source of the Klf4 expression
remains unknown. In general, the expression of homologs of the vasa gene (VASA,
Mvh, DDX4/MVH) is restricted to germ cells. The Mvh protein is only detectable in
mouse PGCs after entry in the genital ridge at d11.5 pc, and it is lost after 3 days of
culturing in EGCs (Durcova-Hills et al., 2006). The situation observed in our culture is
different, as the expression of the rat DDX4/MVH was retained by the rat EGCs and
even acquired by rat ESCs. This could result from differences between the two
species mouse and rat or be a direct result of our culture conditions. We found no
expression of DDX4/MVH in the early embryo containing the PGCs and suspect that
our culture conditions are responsible for its expression. In the mouse Mvh-activity
can be induced in EGCs, but not in ESCs, through co-culture with gonadal somatic
cells (Toyooka et al., 2000) and the differentiation of human or mouse ESC to vasa-
positive PGCs can occur spontaneously or in specialized culture conditions (Ko &
Discussion and Conclusions 89
Schöler, 2006). Consequently, the teratoma fibroblasts (TRF-O3) used as feeder
cells may produce factors inducing ESCs and EGCs to express DDX4/MVH. To this
date, no other study has investigated the expression of vasa in pluripotent cells in 2i-
LIF media. Hence another possibility is that 2i-LIF media induces pathways activating
DDX4MVH expression. However, whatever the reason, the gene activity of
DDX4/MVH does not compromise the pluripotency of said EGCs or ESCs.
6.2.4. Germline competence of rat EGCs
Pluripotent cells have the extraordinary ability to give rise to all cells of the body and
this includes those of the germline. The pluripotency of our +/+ and ter/ter EGC lines
could be shown by alkaline phosphatase staining, immunostainings and RT-PCR for
pluripotency markers, as well as teratoma development after injection of EGCs into
immunodeficient mice. Furthermore, the +/+ EGC line produced chimeric offspring
after injection into the blastocyst. Mating of a female chimeric animal with an albino
rat resulted in 40% albino germline pups, thus proving that the EGC line contributed
to germ cells. This is the first time that germline competence could be shown for rat
EGCs and proved that the 2i-LIF conditions suffice to generate truly rat pluripotent
EGCs. Leitch and colleagues show that their genetically manipulated EGCs can
result in chimeric rats; however, these lack the ability of producing germline offspring.
The fact that germline transmission is not seen in those chimeras made with early
SD-EGCs could result from minor difference such as culture conditions, the genetic
manipulation or the higher passage number of the injected EGCs.
Germline transmission opens new possibilities for EGCs of the rat as they could
present an alternative tool to ESCs for gene targeting. EGCs have even been used
for the successful genetic manipulations in other species such as pig or chicken
(Piedrahita et al., 1998; van de Lavoir et al., 2006). The capacity of mouse ESC and
EGC in generating chimeras is identical (McLaren & Durcova-Hills, 2001). The
frequency of chimeras after injection of the early rat EGCs is higher than that
previously published for ESCs. This led Leitch and co-workers to speculate on
whether they might present a better tool than ESCs for genetic manipulations in the
rat (Leitch et al., 2010). Our late WKY-EGC line was highly capable of generating
chimeras, which would be in agreement with this. Further data is necessary to prove
90 Discussion and Conclusions
or refute this hypothesis, as factors such as rat strain, passage number and
karyotype of the specific cell line all influence the ability to contribute to chimeras.
6.3. Dnd1-related molecular mechanisms at work in germ cells
Nothing was known about the functions of Dnd1 in the rat prior to this study. A
spontaneous mutation causing teratomas gave rise to this project and ended up
demonstrating the importance of Dnd1 in germ cell development. Loss of Dnd1
enhanced the ability of germ cell precursors to form pluripotent germ cells in vivo
(ECC) and in vitro (EGC), as described in the previous two chapters 4 and 5.
Although the effects of the Dnd1 mutation in the rat are described here, the involved
molecular pathways remain in the dark for now. Nevertheless, the newfound
knowledge on Dnd1 in the rat in addition to the molecular data gained in other
species provides ample room for speculations and conclusions.
6.3.1. Interactions of Dnd1 and miRNAs
Dnd1 interacts with miRNAs of mice, zebrafish and humans and permits the
expression of specific target genes. The extent to which Dnd1 modifies the activity of
what genes remains to be established (Kedde & Agami, 2008). In zebrafish Dnd
modulates Nanos1 and Trdr7 expression through miR-430. Human cell culture
showed that DND1 allows the transcription of Lats2 through miR-372/373 inhibition,
Cx43 through miR-1/206 inhibition and p27 through miR221/222 inhibition (Kedde et
al., 2007). In the mouse Dnd1 is thought to allow upregulation of cell cycle inhibitors
in mitotically arresting germ cells even though inhibiting miRNAs such as the miR-17-
92 and the miR 290-295 are present (Hayashi et al., 2008; Cook et al., 2011). Due to
the ter-mutant rat and the culture of its EGCs, it becomes possible to elucidate the
role of Dnd1 with hither to unspecified target genes and miRNAs.
6.3.2. Effects of Dnd1 on EGC derivation and culture
The zebrafish miR-430 family, the mouse miR-290 cluster (miR-291-3p, miR-294,
and miR-295), and the human miR-371/2/3 and miR-302 clusters are homologous to
one another and highly expressed in the early embryo (Wang & Blelloch, 2009). In
Discussion and Conclusions 91
humans, miR-372 maintains p21 at a low level and is probably in part responsible for
the accelerated G1/S transition of dividing ESCs (Qi et al., 2009). The mouse miR-
290 cluster is known to be highly expressed in PGCs and pluripotent cells, including
in EGCs. Its presence has been associated with the expression of pluripotency
markers and maintaing a pluripotent state (Hayashi et al., 2008; Zovoilis et al., 2008).
Similar to human miR-372, miR-290 controls cell cycle progression by allowing a
rapid G1/S transition in ESC through suppression of multiple inhibitors such as p21,
p27, pRB and Lats2 (Wang & Blelloch, 2009). Incidentally, p21, p27 and Lats2 are
the cell cycle inhibitors linked to Dnd1 expression, and absence of Dnd1 causes loss
of p21 and p27 in the mouse embryo during mitotic arrest (Cook et al., 2011).
Therefore, the enhanced EGC derivation of the rat in absence of Dnd1 could possibly
be related to a miRNA-mediated absence of specific cell cycle inhibitors. However, it
was deemed improbable to be the main cause after a proliferation assay was
performed with the +/+ and ter/ter cell lines and showed no significant Dnd1-
dependent differences in growth rates. The results of the proliferation assay indicated
that Dnd1 affects the process of immortalization of PGCs rather than their in vitro
growth rate as EGCs.
The precise pathways causing PGCs to transform into pluripotent EGCs or ECCs are
unknown, but similar mechanisms seem to be involved. Hyperactivation of Akt
signaling in PGCs increases the efficiency with which EGCs are established. The
tumor suppressor Pten is a negative regulator of the PI3K dependent Akt activation
(Kimura et al., 2003). Distinct mutations in Pten or p53 induce gonadal teratomas in
mice while also augmenting the number of EGCs derived from PGCs of the genital
ridge in the mouse (Kimura et al., 2003; Kimura et al., 2008). The same was also true
for the Dnd1 mutation of the rat, where the efficiency of deriving late WKY-EGCs was
increased in the mutant animals sure to develop teratomas. Effects of the Ter
mutation on EGC-derivation in the mouse are likely, but unknown. In view of the
similar effects on PGCs, it is conceivable that Pten, p53 and Dnd1 could be involved
in similar pathways; a notion which is supported by the fact that mouse Dnd1 is
capable of binding transcripts of the negative cell cycle regulators p53 and Pten
(Cook et al., 2011). Furthermore, the Dnd1 and the p53 mutation both require similar
92 Discussion and Conclusions
factors for the induction of GCTs in vivo, as teratomas primarily develop in the 129/Sv
strain and not on a mixed C57BL/6 x 129/Sv genetic background (Donehower et al.,
1995).
6.3.3. Onset of germ cell loss and transformation
Dnd1 is upregulated in PGCs after specification at d7.25pc and the first effects in the
Ter-mutant mice are seen shortly afterwards when germ cell deficiency starts at
d8.0pc. At what point germ cells are lost in the rat remains to be determined,
however, a situation similar to that of the mouse is not unlikely. Germ cells are
completely depleted in immature males at three weeks. An embryonic onset of germ
cell loss is insinuated by the lower expression of germ cell-specific markers, such as
DDX4/MVH and Sox2, in ter/ter than in +/+ rat genital ridges.
The morphological changes found in PGCs of ter/ter mice in the genital ridge
pinpoints the beginning of teratocarcinogenesis in Ter/Ter mice to d12.5pc (Matin,
2007). Intrinsic changes of ter/ter rat PGCs were exhibited at a similar embryonic
stage: the loss of Dnd1 was found to enhance derivation of pluripotent EGCs at
d14.5pc. The ter mutation played no substantial role in generating pluripotent cells as
such, since deriving EGCs from embryos at d10.5pc showed no significant Dnd1-
related effects (chapter 5, supplementary data). These results reveal that the loss of
Dnd1 had influenced the fate of the PGCs by d14.5pc, and the development leading
to teratocarcinomas instead of gametes commenced.
6.3.4. Dnd1 in mitosis and meiosis
In the mouse, tumor development depends on male factors present in the genital
ridge, and the initiation at d12.5pc (Matin, 2007) coincides with the time healthy male
germ cells start entering G0 mitotic arrest (Western et al., 2008). Ter/Ter germ cells
fail to enter G0, which has led to the suggestion that Dnd1 regulates mitotic arrest in
male germ cells (Cook et al., 2011). The contrasting situation in the rat, with
teratocarcinogenesis in both sexes, expands the role previously conferred to Dnd1.
In the rat it cannot be reduced to the inability of germ cells to enter mitotic arrest in
males, as this does not offer an explanation for teratocarcinogenesis in females. The
high level of the Dnd1 target gene p27 found in mitotically arresting germ cells
Discussion and Conclusions 93
between d13.5pc and 15.5pc is not exhibited by male mutant mice or by female germ
cells between d12.5-14.5pc (Western et al., 2008). Should the male rat mirror the
situation in the male mouse, this would signify that Dnd1 target genes and
tumorigenesis differs between female and male rats. An alternative explanation could
be that the downregulation of specific cell cycle inhibitors might be a secondary effect
instead of the cause of tumor development.
Between d12.5pc and 14.5pc, Dnd1 is downregulated in XX and upregulated in XY
germ cells. Similar to the rostro-caudal wave of meiotic entry, the progressive
downregulation of Dnd1 ensues from anterior to posterior in the female gonad
(Youngren et al., 2005). Whether rat (or mouse) Dnd1 played a major role during
meiosis is unknown, however, it was deemed improbable. After all, Dnd1 is
downregulated in the mouse during meiosis (Youngren et al., 2005), and the rat
ter/ter germ cells are on the road to pluripotency before meiotic entry as seen by their
ability to generate EGCs at d14.5pc. Furthermore, the absence of Dnd1 in the mouse
actually results in an upregulation of meiotic markers in male germ cells (Cook et al.,
2011). It is more likely that Dnd1 is required for the suppression of
teratocarcinogenesis before the meiotic entry of germ cells. This, together with the
fact that female rats were affected by teratomas, also makes improbable the idea
harbored by Youngren and colleagues (2005), that tumor resistance or susceptibility
might be conferred through the gender-dependent patterns of Dnd1 activity. To what
extent differences and similarities exist in the tumor development of male and female
ter/ter rat needs to be established and might provide new insights into
teratocarcinogenesis.
6.4. Final conclusion and future perspectives
Two models capable of giving new information in the area of rat germ cell
development are described in this study: the WKY-Dnd1ter/Ztm rat and the EGCs
derived from it.
Both models clearly emphasize the importance of Dnd1 in germ cell development.
Loss of Dnd1 is responsible for an increased tendency of germ cells to form
94 Discussion and Conclusions
pluripotent cells in vivo and in vitro. Furthermore, its absence results in infertility due
to the inability of non-neoplastic germ cells to survive. The other principal
achievement of this study is the establishment of the first germline competent EGC
line of the rat. This proves that the conversion of previously unipotent PGCs to truly
pluripotent EGCs is possible in the rat by applying the described culture conditions.
Further research in the WKY-Dnd1ter/Ztm rat strain includes continuing the analysis of
mRNA expression and protein translation of both the truncated and wild type Dnd1.
Moreover, a project is in progress to generate a rescue with an inducible expression
of wild type Dnd1 in the ter/ter rat. The revision of the ter/ter phenotype would be the
final proof that the ter mutation leads to the loss of a functional Dnd1. Additionally,
the inducible Dnd1-expression would allow investigating the role of Dnd1 during
different developmental stages and in prospective target locations other than the
gonad, such as the heart.
In the mouse the Ter phenotype differs between strains. Backcrossing of the mutated
rat Dnd1 onto another strain, in this case the DA rat, has resulted in a modulation of
the phenotype (results not shown). The next step is the comparison of different
strains with varying phenotypes to help identify other aspects involved in
teratocarcinogenesis. Backcrossings of the mutated Dnd1 to different rat strains such
as LEW and BN remain to be established. Having the ter mutation in different rat
strains could help identify modifier genes regulating the ter phenotype and define the
precise functions of Dnd1 in the rat.
At what point in time the teratocarcinogenesis or the germ cell loss commences
remains unknown and this could potentially be clarified through immunohistological
staining of germ cells or ECC in the embryo and early gonads. Ascertaining the
absence or presence of ECCs in the degenerated testes or a unilateral orchiectomy
in ter/ter males, followed by long term tumor check-ups, could help identify whether
the non-tumorous testis found in some male rats stays degenerated and does not
form teratomas. Identifying genomic imprinting and chromosomal abnormalities in the
ECCs would help identify the embryonic stage in which teratoma precursors develop
Discussion and Conclusions 95
and allow more precise comparisons between the rat animal model and the different
types of TGCTs in humans.
The environment allowing growth of TGCTs includes specific autocrine, paracrine
growth factors and hormones. The effects of different factors on the efficiency of
EGC formation and growth could be tested in vitro. As TGCT and EGC derivation are
linked, this could help identify the factors responsible for tumor growth and the
gender dependent differences visible in the WKY-Dnd1ter/Ztm rat. The expression
levels of Dnd1 and its target genes need to be identified in the EGCs and compared
between +/+ and ter/ter cell lines. Microarrays could be another method with which to
identify differences between +/+ and ter/ter cell lines. Also of interest would be the
determination whether culture of earlier EGCs or ESCs exhibit any Dnd1-dependent
effects. Preliminary data from early WKY-EGCs (d10.5pc) showed no significant
differences in EGC derivation between +/+, ter/ter and ter/+ animals; however,
growth rate and genetic stability remain to be evaluated. Additionally, the ability of
early +/+ EGCs to contribute to germline chimeras needs to be assessed.
A whole new field of opportunities has been opened in rat research through the
breakthrough in culturing pluripotent and germline competent cells from the rat. The
capacity of pluripotent cells to generate knockouts or to replace tissue has been long
studied in the mouse; which pluripotent rat cells are the most resourceful remains to
be determined. For now, a comparison of multiple ESC and EGC lines, cultured and
injected using the same conditions and rat strains, would be required to find out
which has the higher potential of resulting in germline chimeras.
The basic functions of Dnd1 in the rat have now been identified. However, much
more remains to be accomplished, particularly in terms of germ cell development and
pluripotency. This project might simply mark the beginning of significant future
research.
96 Summary
7. Summary
Emily Northrup
“Dnd1: From Germ Cells to Teratomas in the Rat”
Human germ cell tumors (GCT) can be observed in all age groups, ranging from
infancy to adulthood, and are frequently diagnosed at a young age.
Teratocarcinomas are GCTs consisting of derivatives from all three germ layers and
undifferentiated embryonic carcinoma cells (ECCs). A disruption during embryonic
development transforms unipotent primordial germ cells (PGC)/gonocytes into
pluripotent ECCs. Other reproductive problems including infertility have been
associated with testicular GCTs. For unknown reasons the incidence of male GCTs
and infertility are on the rise in humans, making it increasingly important to elucidate
the mechanisms triggering the disease.
The key to understanding GCTs and infertility lies in discerning the factors and
genetics regulating germ cell differentiation to mature gametes or de-differentiation to
pluripotent cells. The ter mutation in the WKY/Ztm rat strain causes infertility along
with ovarian and testicular teratomas. Genetics and phenotype of the WKY/Ztm-ter
rat were investigated in the first part of this thesis. Linkage analysis, fine mapping
and sequencing of candidate genes traced the ter mutation of the rat to a point
mutation introducing a premature stop codon in the dead-end homolog 1 gene
(Dnd1). Genotyping revealed a recessive mode of inheritance with complete
penetrance of teratocarcinogenesis and infertility in homozygous ter rats of both
genders, and no teratomas in their wild type or heterozygous counterparts. This
differs strongly from the co-dominant Ter mutation in the Dnd1 gene of the 129/Sv
mouse, which induces testicular teratoma and infertility but never ovarian teratomas.
Gonadal teratocarcinogenesis indicates that Dnd1 acts as a tumor suppressor gene
in mouse and rat. Teratoma progression in the rat was gender dependant and on the
average tumors developed earlier in females than males. Morphologically non-
tumorous testes of homozygous ter rats were reduced in size and weight.
Immunohistochemical along with histological stainings associated the gonadal
Summary 97
malformation with the absence of spermatogenesis. Additionally, oocytes were
lacking in non-neoplastic follicles of homozygous ter females, thereby showing that
Dnd1 is required for survival of non-neoplastic rat germ cells. The advantage of the
ter rat over the Ter mouse model is the ability of the former to allow studies on
ovarian GCTs and enable comparative studies on GCT development between
genders. In general, the WKY-Dnd1ter/Ztm rat is a novel animal model for further
studies on gonadal teratocarcinogenesis and the molecular mechanisms involved in
germ cell differentiation or de-differentiation. A special focus lies on the tumor
precursor cells, the PGCs, and identifying the factors inducing or accelerating their
neoplastic transformation to ECCs.
Although the developmental potency of PGCs is restricted to the germ lineage, ECCs
are not the only pluripotent cells derived from PGCs. Through in vitro culture,
unipotent PGCs can be converted into pluripotent embryonic germ cells (EGCs).
EGCs from the WKY-Dnd1ter/Ztm rat present an innovative approach allowing in vitro
studies on Dnd1 in germ cell differentiation and tumorigenesis. The successful
culture of EGCs from the genital ridge of the WKY-Dnd1ter/Ztm rat was established in
the second part of this thesis. Heterozygous ter/+ rats were mated and the PGCs
from embryos at day 14.5 post coitum (d pc) were cultivated in media containing
inhibitors of differentiating pathways (2i-LIF). The generated cell lines were
genotyped, and almost all were found to stem from Dnd1-deficient ter/ter embryos.
EGCs were also cultured from early rat embryos at d10.5pc, however, no Dnd1-
dependent effects were observed. Taken together, these facts demonstrate that the
inactivation of Dnd1 facilitates the immortalization of late, but not of early, PGC in
vitro. This reveals that the loss of Dnd1 has altered the fate of PGCs by d14.5pc, and
the development resulting in teratocarcinomas instead of gametes has commenced.
The EGC lines could be maintained in a pluripotent state as demonstrated by
expression of pluripotency markers and teratoma formation in immunodeficient mice.
Injection of the wild type cell line into blastocysts resulted in germline competent
chimeras, making this the first time that germline competence could be shown for a
rat EGC line.
98 Summary
In conclusion, this thesis demonstrates the importance of Dnd1 in rat germ cell
development, with its loss resulting in de-differentiation of germ cells to pluripotent
cells in vivo and in vitro. A further achievement is establishing the culture of genital
ridge-derived rat EGCs. Both, the characterized WKY-Dnd1ter/Ztm rat strain and the
established culture of EGCs from the genital ridge, open new possibilities for
research in conjunction with the Dnd1 gene and the mechanisms involved in germ
cell development.
Zusammenfassung 99
8. Zusammenfassung
Emily Northrup
„Dnd1: Von Keimzellen zu Teratomen in der Ratte“
Humane Keimzelltumoren (GCT) kommen vom Kleinkind zum Erwachsenen in allen
Altersgruppen vor und werden häufig in einem frühen Lebensabschnitt diagnostiziert.
Teratokarzinome, eine Form von GCT, weisen die Besonderheit auf, Gewebe aller
drei Keimblätter zu bilden und undifferenzierte Embryonale Karzinomzellen (ECC) zu
enthalten. Pluripotente ECC entstehen durch neoplastische Transformation aus
unipotenten Primordialen Keimzellen (PGC) bzw Gonozyten. Weitere
Reproduktionsstörungen, wie z.B Infertilität, wurden bereits mit GCT der Hoden in
Verbindung gebracht. Aus noch unbekannten Gründen nimmt die Inzidenz von
männlichen GCT und Infertilität zu, wodurch die Identifizierung der
krankheitsauslösenden Mechanismen zunehmend an Bedeutung gewinnt.
Um GCT und Infertilität zu ergründen, ist es erforderlich die Faktoren und
genetischen Hintergründe zu identifizieren, welche für die Differenzierung von
Keimzellen zu reifen Gameten oder De-Differenzierung zu pluripotenten Zellen
verantwortlich sind. Die ter Mutation in dem WKY/Ztm Rattenstamm verursacht
Infertilität sowie Keimzelltumoren von Ovar und Hoden. Genetik und Phänotyp der
WKY/Ztm-ter Ratte wurden in dem ersten Teil dieser Dissertation untersucht. Die ter
Mutation konnte anhand von Kopplungsanalysen, Feinkartierung und
Sequenzanalysen von Kandidatengenen auf eine Punktmutation zurückgeführt
werden, welche ein vorzeitiges Stopcodon in dem dead-end homolog 1 Gen (Dnd1)
verursacht. Genotypisierungen zeigten, dass der genetische Defekt rezessiv vererbt
wird mit kompletter Penetranz von Teratokarzinogenese und Infertilität in
homozygoten ter Ratten beider Geschlechter, wobei keine Teratome in wildtypischen
oder heterozygoten Tieren vorkommen. Dies unterscheidet sich stark von der
codominanten Ter Mutation in dem Dnd1 Gen der 129/Sv Maus, welche Infertilität
und Hodenteratome, jedoch niemals ovariale Teratome, zur Folge hat. Die
Teratokarzinogenese legt nahe, dass es sich bei Dnd1 um ein Tumorsuppressorgen
100 Zusammenfassung
handelt. Die Tumorprogression in der Ratte war zudem geschlechtsspezifisch
unterschiedlich. Im Durchschnitt entwickelten sich die Teratome früher in weiblichen
Ratten als in männlichen. Die Tatsache, dass morphologisch nicht tumoröse Hoden
von homozygoten ter Ratten kleiner und leichter waren, konnte anhand von
histologischen und immunhistochemischen Nachweisverfahren mit fehlender
Spermatogenese in Zusammenhang gebracht werden. Da außerdem die nicht-
neoplastischen Follikel von homozygoten ter Weibchen frei von Oogonien waren,
liegt der Schluss nahe, dass Dnd1 als Survivalfaktor die Erhaltung von Keimzellen in
der Ratte sichert. Der Vorteil der Ter Ratte gegenüber der ter Maus als Tiermodell
liegt darin, dass diese auch Studien an Ovarteratomen ermöglicht, sowie einen
Vergleich zwischen den Geschlechtern erlaubt. Generell ist die WKY-Dnd1ter/Ztm
Ratte als neues Tiermodell anzusehen, insbesondere für weitere Studien zur
gonadalen Teratokarzinogenese und zur Identifikation von molekularen
Mechanismen, die im Rahmen von Keimzelldifferenzierung und De-Differenzierung
eine Rolle spielen. Von zentraler Bedeutung sind hierbei die Tumorvorläuferzellen,
die PGCs, und das Erkennen von Faktoren, die die neoplastische Transformation der
PGCs zu ECCs induzieren oder beschleunigen.
Ungeachtet der Tatsache, dass sich das Entwicklungspotential von PGCs auf die
Keimbahn beschränkt, sind ECC nicht die einzigen pluripotenten Zellen, die von PGC
abstammen. Durch in vitro Kultur können unipotente PGC zu pluripotenten
embryonalen Keimzellen (EGC) reprogrammiert werden. EGC von der WKY-
Dnd1ter/Ztm Ratte können als innovativer Ansatz dienen, um in vitro Studien zur Rolle
von Dnd1 in Keimzelldifferenzierung und Tumorigenese zu ermöglichen. Die
erfolgreiche Kultur von EGCs aus der Keimleiste der WKY-Dnd1ter/Ztm Ratte wurde
im zweiten Teil dieser Dissertation etabliert. Heterozygote ter/+ Ratten wurden
verpaart und die PGCs von Embryonen an Tag 14.5 post coitum (d pc) wurden in
Medium kultiviert, das Inhibitoren enthielt, welche eine Ausdifferenzierung verhindern
(2i-LIF). Die generierten Zelllinien wurden genotypisiert. Bis auf eine Zelllinie
stammten alle von Dnd1-defizienten Embryonen ab. Dnd1 hatte dagegen keinen
Einfluss auf die Kultivierung von prä-migratorischen EGC aus einem früheren
Embryonalstadium. Dies bedeutet, dass die Inaktivierung von Dnd1 die
Zusammenfassung 101
Immortalisierung von späten, nicht jedoch von frühen PGC in vitro beeinflusst hat.
Somit hat der Verlust von Dnd1 in PGC an d14.5pc vermutlich bereits die
Entwicklungsrichtung in der Gametendifferenzierung zu Teratokarzinomen geändert.
Die EGC-Linien konnten in Kultur Ihren pluripotenten Status aufrechterhalten, was
anhand der Expression von Pluripotenzmarkern und der Bildung von Teratomen in
immundefizienten Mäusen gezeigt wurde. Keimbahngängige Chimäre konnten durch
Injektion der Wildtyp Zelllinie in Blastozysten generiert werden. Somit wurde
erstmalig Keimbahngängigkeit für eine Ratten EGC-Linie gezeigt.
Zusammenfassend konnte durch diese Arbeit die Bedeutung von Dnd1 in der
KeimzelIentwicklung der Ratte aufgezeigt sowie veranschaulicht werden, dass der
Verlust die De-Differenzierung von Keimzellen zu pluripotenten Zellen in vivo und in
vitro zur Folge hat. Eine weitere Errungenschaft ist die Etablierung der Kultur von
Keimzellen aus der Keimleiste der Ratte. Sowohl der charakterisierte WKY-
Dnd1ter/Ztm Rattenstamm als auch die etablierte Kultur von EGCs aus der Keimleiste
schafften neue Möglichkeiten für die weitere Erforschung des Dnd1 Gens und der
Mechanismen, die an der Keimzellentwicklung beteiligt sind.
102 References
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Acknowledgements
The completion of this thesis would not have been possible without the support and
guidance of many people.
First, I would like to express my gratitude to my thesis supervisor, Prof. Hedrich, for
giving me the opportunity and the freedom to develop my experiments in his
laboratory. I am especially thankful for his discovery that Christmas, his numerous
contributions, the scientific advice, and his full support.
I am grateful to Dr. Martina Dorsch for her helpfulness, her suggestions and her
encouragements, as well as the constructive criticism throughout the last years.
A special and sincere thank you to Dr. Nils-Holger Zschemisch, with all his
enthusiasm, optimism and inspirations. His patience, sound advice and good
teaching were essential for this thesis, from beginning to end.
Furthermore, I thank my other supervisors Prof. Haas, Prof. Martin and Prof. Greiser-
Wilke († 2010) for their interest and guidance throughout this work. I am grateful to
Prof. Greiser-Wilke for the encouragement and advice at the beginning of my studies.
I owe my deepest gratitude to the staff of the Institute for Laboratory Animal Science,
without whom I could not have accomplished this thesis. A special thank you goes to
Matthias Meyer and Elena Wiebe for their technical support, as well as to Dr. Dirk
Wedekind for helpful suggestion in many situations. Furthermore, I would like to
thank Isabell Wittur, Cindy Elfers and especially Regina Eisenblätter, who contributed
to this project with all their knowledge, their patience, their time and their assistance
in varying circumstances.
I am indebted to Dr. Cornelia Rudolph for instructions on cytogenetic analysis,
evaluation of the metaphase spreads and valuable discussions.
Acknowledgements 119
I would like to thank all my friends for the emotional support, comradery,
entertainment, and caring they provided. Silke Glage, Lydia Janus and Gwen
Büchler: your help was indispensible, the regular food intake was essential, and it
would have been a lonely lab without you.
For many hours of happiness I thank Wilma Feuerstein, who made sure I got out of
the lab and exercised on a daily basis.
My special thanks to Tobias Tiedtke for his love, his encouragement, and for being
by my side in good and bad times. He has the common sense that I tend to lack and
the ability to make me laugh in any given situation.
I am grateful to my siblings Jenica and Lukas, who make life so much more fun, and
though both are far away, they are always there when needed.
Lastly, and most importantly, I wish to thank my parents, Sibylle Northrup and Scott
Northrup. They bore me, raised me, supported me, and taught me with unconditional
love. They are responsible for everything I achieved and are the only parents I know
that are continuously encouraging their children to work less, spend more and live life
to its fullest!