Full text of DOI:10.1038/labinvest.3780408

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LABORATORY INVESTIGATION
Vol. 82, No. 2, p. 159, 2002
Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc.
Printed in U.S.A.
VASA Is a Specific Marker for Both Normal and
Malignant Human Germ Cells
Anne-Marie Zeeman, Hans Stoop, Marjan Boter, Ad J. M. Gillis, Diego H. Castrillon,
J. Wolter Oosterhuis, and Leendert H. J. Looijenga
Department of Pathology/Laboratory for Experimental Patho-Oncology (A-MZ, HS, MB, AJMG, JWO, LHJL),
University Hospital of Rotterdam/Daniel, Josephine Nefkens Institute, Erasmus University Rotterdam, The Nether-
lands; and Women’s and Perinatal Pathology Division, Department of Pathology (DHC), Brigham and Women’s
Hospital and Harvard Medical School, Boston, Massachusetts
SUMMARY: VASA is so far the only known gene in mammals whose expression is specific for the germ cell lineage. We
investigated the presence of VASA mRNA and protein in a series of germ cell tumors of different histologic subtypes and
anatomic location, as well as in nongerm cell tumors such as testicular lymphomas and Leydig cell tumors. We detected VASA
mRNA (by quantitative RT-PCR) and protein (by immunohistochemical staining) in normal spermatogenesis, seminoma (both
classic and spermatocytic), carcinoma in situ (the precursor of classic seminoma and nonseminoma), dysgerminoma, and
gonadoblastoma. VASA immunostaining was relatively weak in seminomas and dysgerminomas compared with spermatocytic
seminomas, despite similar mRNA levels, suggesting that VASA is regulated in part by post-transcriptional mechanisms. A higher
staining intensity compared with the invasive counterparts was observed in the precursor lesions (ie, carcinoma in situ and
gonadoblastoma). No VASA mRNA or protein was detectable in nonseminomatous germ cell tumors (such as embryonal
carcinoma, teratoma, and yolk sac tumor) and derived cell lines, or nongerm cell tumors such as lymphoma or Leydig cell tumor.
These results provide direct evidence that some germ cell tumors retain germ cell characteristics, whereas other tumors of germ
cell origin result from differentiation and loss of germ cell identity. Furthermore, these findings suggest that VASA is likely to serve
as a useful and highly specific biomarker for germ cell tumors, particularly classic and spermatocytic seminoma/dysgerminoma,
including their precursor stages. (Lab Invest 2002, 82:159–166).
he evolutionary highly conserved VASA genes
are specifically expressed in the germ cell lin-
of germ cells. The mouse homolog of VASA, known as
mvh (mouse Vasa homolog) was recently identified. Its
expression is also restricted to the germ cell lineage
(Tanaka et al, 2000). Although male knock-out mice for
mvh form primordial germ cells, they subsequently
develop testicular atrophy because of abnormal pro-
liferation and colonization of the primordial germ cells,
as well as death of zygotene spermatocytes, and are
infertile.
T
eage, both in vertebrates and invertebrates (for review,
see Raz, 2000). Members of this family have been
identified in C. elegans, Drosophila, Xenopus, ze-
brafish, chicken, rainbow trout, rat, and mouse. The
Drosophila vasa gene encodes a DEAD-box protein
with ATP-dependent RNA-helicase activity (Hay et al,
1
988; Lasko and Ashburner 1988). The helicase activ-
ity is required for the translation of at least two mRNAs
involved in germ cell migration and development, ie,
nanos (Forbes and Lehmann 1998; Gavis et al, 1996;
Kobayashi et al, 1996) and gurken (Gonzalez-Reyes et
al, 1995; Neuman-Silberberg and Schupbach 1993;
Roth et al, 1995; Styhler et al, 1998; Tomancak et al,
The human VASA gene was recently cloned. VASA
mRNA and protein are abundantly and specifically
expressed in germ cells in both sexes throughout
development. In contrast to mice, where the protein is
induced after the primordial germ cells reach the
genital ridge, immunohistochemistry demonstrated
that in humans, the VASA protein is already present in
migrating primordial germ cells. During normal sper-
matogenesis, VASA staining is relatively weak to in-
termediate in spermatogonia, strong in spermato-
cytes/spermatids, and absent in spermatozoa
1998). VASA is present in the developing oocyte and is
part of the germ plasm, a poorly understood ribonu-
cleoprotein complex crucial for germ cell determina-
tion. In Drosophila, vasa is essential for the formation
(
Castrillon et al, 2000). In the human adult testis, two
Received September 24, 2001.
This research was supported by the Dutch Cancer Society (KWF-DDHK
biologically and clinically distinct types of germ cell-
derived tumors are recognized. These are distin-
guished by epidemiologic characteristics, histology,
clinical behavior, and chromosomal constitution (Looi-
jenga and Oosterhuis, 1999 for review). The first
comprises the seminomas and nonseminomas, here
referred to as testicular germ cell tumors of adoles-
9
9-1968).
Address reprint requests to: Dr. L. H. J. Looijenga, Department of Pathol-
ogy/Laboratory for Experimental Patho-Oncology, University Hospital Rot-
terdam/Daniel, Josephine Nefkens Institute, Erasmus University Rotter-
dam, Building Be, Room 430b, PO Box 1738, 3000 DR Rotterdam, The
Netherlands. E-mail: looijenga@leph.azr.nl
Laboratory Investigation • February 2002 • Volume 82 • Number 2 159

Zeeman et al
cents and adults (TGCT), and represent the most
common neoplasms in young Caucasian males
Results
Immunohistochemistry for VASA and GCAP/PLAP
(
1
Adami et al, 1994; Pottern et al, 1998; Swerdlow
993). Although it is most likely that the initiating event
To study the presence of VASA protein in different
human tumors, we performed immunohistochemistry
on formalin-fixed paraffin-embedded tissue sections
using previously described polyclonal antibodies
in the development of TGCT occurs during embryonic
development (Jørgensen et al, 1995; Møller, 1989, for
review), affecting a primordial germ cell, leading to
carcinoma in situ (CIS) (Skakkebæk 1972), also known
as intratubular germ cell neoplasm (ITGCN), the tu-
mors only become clinically manifest after puberty.
CIS cells, like primordial germ cells, are characterized
by a high glycogen content (Mostofi and Sesterhenn
(Castrillon et al, 2000). The tested samples include
gonadoblastoma (n ϭ 5), dysgerminoma (n ϭ 5),
normal testicular parenchyma (n ϭ 5) (containing
spermatogenesis), primary testicular lymphomas (n ϭ
3), Leydig cell tumors (n ϭ 3), testicular parenchyma
1
998), membranous staining for the stem cell factor
receptor c-KIT (Rajpert-De Meyts and Skakkebæk
994), and germ cell/placental alkaline phosphatase
GCAP/PLAP) (Roelofs et al, 1999, for review). CIS
containing different amounts of CIS-cells and Sertoli
only tubules (n ϭ 14), seminomas (n ϭ 13), nonsemi-
nomas (n ϭ 12), and spermatocytic seminomas (n ϭ
1
(
4). The results are summarized in Table 1. Represen-
cells can progress to seminoma, an invasive tumor in
which the malignant cells still show, like CIS cells,
morphologic and immunohistochemical characteris-
tics of primordial germ cells. CIS cells can also
progress to nonseminomas, tumors of germ cell origin
that have lost their primordial germ cell characteris-
tics, and recapitulate embryonic development (Looi-
jenga et al, 1997, for review). Nonseminomatous germ
cell tumors exhibit morphologic and immunohisto-
chemical evidence of differentiation into various so-
matic (teratoma) and extraembryonic lineages (yolk
sac tumor and choriocarcinoma). In addition, embry-
onal carcinoma can be found. Each of these histologic
subtypes can be found in a pure form or in combina-
tions with other types (Mostofi and Sesterhenn 1998).
Seminomas and nonseminomas can also be found in
the ovary and dysgenetic gonads (Oosterhuis et al,
tative examples are shown in Figure 1.
Relatively weak staining for VASA was found in
spermatogonia, whereas stronger staining was de-
tected in later developmental stages. Spermatozoa
were negative (Fig. 1A). These results are in accor-
dance with earlier data (Castrillon et al, 2000). CIS/
seminoma and gonadoblastoma/dysgerminoma cells
were identified by their positive staining for GCAP/
PLAP (see Fig. 1, B and C). Double immunohisto-
chemistry for GCAP/PLAP and VASA demonstrated
that all of these malignant cells are positive for VASA.
Although the overall intensity of the staining was in the
same order as found for spermatogonia, the preinva-
sive stage (ie, gonadoblastoma and CIS), showed a
more intense staining compared with their invasive
counterparts (ie, dysgerminoma and seminoma; see
1
993, for review). The ovarian counterpart of semi-
Table 1. Summary of the Results of
noma is known as dysgerminoma and is histologically
indistinguishable from seminoma. The CIS-like lesion
that arises in dysgenetic gonads is referred to as
gonadoblastoma (Scully 1970; Jørgensen et al, 1997,
for review).
Immunohistochemistry for VASA on Germ Cell Tumors of
Different Histology and Anatomical Localization, as well
as Nongerm Cell Tumors and Normal
Testicular Parenchyma
The second category of testicular tumors derived
from the germ cell lineage are the spermatocytic
seminomas. These tumors develop usually at a rela-
tively advanced age, rarely metastasize, and are as-
sumed to be derived from a later developmental stage
of spermatogenesis (spermatocytes) than the precur-
sor cells of TGCT (Burke and Mostofi, 1993; Eble,
a
Histology
Cases tested
19
Staining
Testicular parenchyma
Leydig cells
Sertoli cells
Ϫ
Ϫ
Ϫ
ϩϩ
ϩϩϩ
ϩϩϩ
Ϫ
Ϫ
Ϫ
ϩϩ
ϩ
ϩϩ
ϩ
Stromal cells
Spermatogonia
Spermatocytes
Spermatids
1
1
1
994; Muller et al, 1987; Romanenko and Persidskii,
983; Rosai et al, 1969; Stoop et al, 2001; Talerman,
974, 1980; Talerman et al, 1984). Their different
Spermatozoa
Leydig cell tumor
Testicular lymphoma
Gonadoblastoma
Dysgerminoma
Carcinoma in situ
Seminoma
3
3
5
pathogenesis from TGCT is also illustrated by the lack
of glycogen (Mostofi and Sesterhenn, 1998), c-KIT,
and GCAP/PLAP (Dekker et al, 1992), their positive
staining for SSX, SCP1, and XPA (Stoop et al, 2001),
as well as their unique chromosomal constitution
5
14
13
12
4
(
Looijenga et al, 1994; Rosenberg et al, 1998).
b
Nonseminoma
Ϫ
ϩϩϩ
We investigated whether tumors derived from the
Spermatocytic seminoma
germ cell lineage (ie, gonadoblastoma/CIS, dysgermi-
noma/seminomas, nonseminomas, and spermato-
cytic seminomas) can be identified specifically on the
basis of the presence of VASA mRNA and protein.
a
Staining intensity: Ϫ, negative; ϩ, weak; ϩϩ, intermediate; ϩϩϩ,
strong.
b
The nonseminomas included multiple independent elements of embryonal
carcinoma, teratoma, and yolk sac tumor.
1
60 Laboratory Investigation • February 2002 • Volume 82 • Number 2

VASA and Human Germ Cell Tumors
Figure 1.
Immunohistochemical detection of VASA (blue) and GCAP/PLAP (red) in several testicular tissues (original magnification, ϫ200). A, VASA staining is clearly visible
in normal testis, with a weak intensity in spermatogonia (Spgo), a strong intensity in spermatocytes till spermatids (Spcy/Spti), and no staining in spermatozoa (Spzo)
and Sertoli cells (S); B, Testicular parenchyma without carcinoma in situ (CIS) cells are negative for GCAP/PLAP. However, CIS cells are positive for GCAP/PLAP as
well as VASA. Spermatogonia of the normal tubules (arrowhead in upper right) show VASA staining intensity similar to CIS tubules. C, Classic seminomas are overall
weakly VASA positive and show strong GCAP/PLAP staining. D, Teratoma and E, embryonal carcinoma are negative for VASA, although some positive cells for
GCAP/PLAP are identified in the latter. F, Spermatocytic seminomas are strongly positive for VASA and negative for GCAP/PLAP. Red blood cells appear orange.
Fig. 1, B and C). A similar staining intensity was
observed with single immunohistochemical labeling
which demonstrates that the presence of GCAP/PLAP
is not coupled to the presence of VASA. This is also
demonstrated by the lack of GCAP/PLAP in spermato-
cytic seminomas, whereas these tumors were strongly
positive for VASA (see Fig. 1F). The intensity of stain-
ing in spermatocytic seminomas appears to corre-
spond to the pattern in normal spermatogenesis,
(
data not shown). None of the nonseminomas showed
staining for VASA (see Fig. 1, D and E). As described
previously (Roelofs et al, 1999), some of the embryo-
nal carcinoma cells still contain GCAP/PLAP. How-
ever, these cells were negative for VASA (see Fig. 1E),
Laboratory Investigation • February 2002 • Volume 82 • Number 2 161

Zeeman et al
Figure 2.
Relative VASA transcript levels in several testicular tissues as determined by semiquantitative RT-PCR. A, Representative autoradiograms of semiquantitative RT-PCR
on several tissues (Lymph.: lymphocytes; Test. paren.: normal testicular parenchyma; Class. sem.: classic seminoma; Nonsem.: nonseminoma; Sperm. sem.:
spermatocytic seminoma). The number of amplification cycles are depicted above the autoradiograms. Lymphocytes and nonseminomas show a lower transcript level
of VASA compared with classic seminoma and spermatocytic seminomas. B, Summary of the results of the semiquantitative RT-PCR. Increasing amounts of CIS-cells
(
from 15% to more than 80% of the seminiferous tubules) as indicated in the graph, correlate with decreasing VASA/HPRT ratios. Classic seminomas (Class. sem.)
do not show significant differences in VASA mRNA levels compared with normal testicular parenchyma (Test. paren.), nor do spermatocytic seminomas (Sp. ϩsem.).
Nonseminomas of all histologic subtypes (EC: embryonal carcinoma, YS: yolk sac tumor, TE: teratoma,) show a reduced transcript level of VASA. The results of the
individual samples and the mean and standard deviation of the histologic groups are indicated.
where staining is strongest in spermatocytes (see Fig.
A).
ing gene with a relatively constant level of expression.
To exclude DNA contamination, the VASA primers
were designed to span several introns (see “Materials
and Methods”). Representative examples are shown
in Figure 2A, and the results are summarized in Figure
2B. Lymphocytes were used as negative control, and
normal testicular parenchyma (containing spermato-
genesis) as positive control. As expected, lympho-
cytes showed extremely low levels of VASA expres-
sion, even after 24 amplification cycles, whereas a
relatively high level (0.6 times the level of HPRT) was
1
Quantification of VASA Transcript Levels
To study whether the different levels of VASA protein
in the different types of germ cell-derived tumors are
due to transcriptional or post-transcriptional regula-
tion, we determined the VASA mRNA levels using
semiquantitative RT-PCR. The VASA-transcript levels
were compared with the level of HPRT, a housekeep-
1
62 Laboratory Investigation • February 2002 • Volume 82 • Number 2

VASA and Human Germ Cell Tumors
detected in testicular parenchyma with spermatogen-
esis. The level of VASA expression was inversely
proportional to the percentage of CIS-containing sem-
iniferous tubules; in samples with more than 80%
positive tubules, the VASA/HPRT ratio was reduced
by a factor of two. It is important to note that with
increasing percentage of CIS-containing seminiferous
tubules, the percentage of seminiferous tubules with
normal spermatogenesis is reduced.
Classic seminoma samples showed VASA expres-
sion levels in the same range as in samples of normal
testicular parenchyma (mean VASA/HPRT ratio, 0.56;
standard deviation, 0.16). In contrast, a significantly
lower level of expression (p Ͻ 0.00013, Student’s t
test) was found in all histologic types of nonsemino-
mas (mean ratio, 0.13; standard deviation, 0.012). In
fact, the level of expression was reduced by a factor of
tion and partial meiosis (Looijenga et al, 1994; Rosen-
berg et al, 1998; Stoop et al, 2001).
The germ cell origin of CIS/classic seminoma and
spermatocytic seminomas is supported by our find-
ings on the specific expression of VASA mRNA and
protein in these tumors. This also is true for gonado-
blastoma/dysgerminoma, which are the CIS and clas-
sic seminoma counterparts in dysgenetic gonads and
ovary. Although classic seminomas and spermato-
cytic seminomas showed a comparable level of VASA
mRNA, about 0.6 times the level of HPRT, more
intense immunohistochemical staining was found in
spermatocytic seminomas than in classic seminomas.
This indicates that post-transcriptional mechanisms
regulate VASA protein levels. Indeed, it is known that
in Drosophila oocytes, vasa is subjected to post-
translational modification (Ghabrial and Schupbach
1999). The intensity of staining in the spermatocytic
seminomas was comparable to that found in more
differentiated germ cells during spermatogenesis (ie,
from spermatocytes till spermatids; Castrillon et al,
2000, and this paper). This supports the model that
spermatocytic seminoma cells are spermatocyte-like
or are capable of developing to this stage of matura-
tion. In addition, the lower staining intensity observed
in CIS/seminoma–and gonadoblastoma/dysgermi-
noma cells–supports the notion that these tumor cells
originate from an earlier germ cell precursor, and are
less capable of undergoing maturation in the germ cell
lineage. In contrast, all nonseminomas, independent
of the histology, as well as cell lines derived from
them, have lost VASA expression, at both the mRNA
and protein level. This is therefore due to transcrip-
tional down-regulation on differentiation and loss of
the germ cell phenotype. The mechanisms involved
are unknown, and regulators of VASA expression
remain to be identified.
4.7 compared with normal testis. In addition, no or
extremely low levels of VASA expression were ob-
served in four nonseminoma-derived cell lines (data
not shown). These expression levels likely account for
the difference in protein level as detected by immuno-
histochemistry in nonseminomas. Although spermato-
cytic seminomas showed a stronger staining intensity
for VASA than classic seminoma (see above), only a
slightly higher level of expression was found (mean
VASA/HPRT ratio, 0.69; standard deviation, 0.037)
(
see Fig. 2, A and B).
Discussion
In the human adult testis, on the basis of biologic and
clinical data, two categories of germ cell-derived tu-
mors are distinguished: (1) seminomas and nonsemi-
nomas, here referred to as TGCT, and (2) spermato-
cytic seminomas (Looijenga and Oosterhuis, 1999, for
review). The TGCT originate from CIS (Skakkebæk,
1
972), also referred to as intratubular germ cell neo-
It is of interest that the actual protein level of mvh
(the mouse homolog of VASA) in primordial germ cells
is regulated by interaction with gonadal somatic cells
(Toyooka et al, 2000). In this context, the lower stain-
ing intensity in the dysgerminomas/classic semino-
mas compared with their preinvasive stage (ie, go-
nadoblastoma/CIS) is of interest. The composition of
the intratubular microenvironment of the precursor
cells is remarkably different from that of the invasive
tumors; in contrast to the invasive tumor cells, both
gonadoblastoma and CIS cells are still interacting with
their physiologic supportive cells (granulosa/Sertoli
cells). Thus, differences in cell-cell interactions may
account for differing protein levels we observed, sim-
ilar to observations made in the mouse. It is, however,
from our results, not possible to draw any conclusion
about possible differences in levels of transcription of
VASA between preinvasive and invasive cells. The
reason is that in parenchyma with 80% CIS, only a few
of the cells are CIS cells (Mosselman et al, 1996). The
reduced level of VASA transcript in CIS containing
parenchyma is most likely explained by the replace-
ment of spermatogenesis by CIS.
plasia (ITGCN), the premalignant counterpart of pri-
mordial germ cells. Although classic seminoma cells
are morphologically and immunohistochemically
highly similar to CIS, nonseminomas such as tera-
toma, yolk sac tumor, and choriocarcinoma reflect
differentiation to other lineages with concomitant loss
of their germ cell-like characteristics (Mostofi and
Sesterhenn 1998, for review). In contrast, spermato-
cytic seminomas still exhibit similarities to cells be-
longing to the germ cell lineage (Burke and Mostofi,
1
993; Cummings et al, 1994; Eble, 1994). They have a
different etiology and clinical behavior from TGCT
Burke and Mostofi, 1993; Dekker et al, 1992; Looi-
jenga et al, 1994; Rosenberg et al, 1998; Stoop et al,
001). Available data suggest that spermatocytic sem-
(
2
inomas originate from a later developmental stage of
the germ cell than TGCT, most likely spermatocytes
(
Burke and Mostofi, 1993; Dekker et al, 1992; Eble,
1
1
994; Muller et al, 1987; Romanenko and Persidskii,
983; Rosai et al, 1969; Talerman, 1974, 1980; Taler-
man et al, 1984; ). However, we recently found indi-
cations that the cell of origin of spermatocytic semi-
noma might be at an earlier stage of development than
spermatocytes. This cell is capable of further matura-
We have shown that VASA is an informative marker
for normal germ cells and germ cell tumors that retain
Laboratory Investigation • February 2002 • Volume 82 • Number 2 163

Zeeman et al
germ cell features; ie, classic seminoma/dysgermi-
noma and spermatocytic seminomas. Currently, im-
munohistochemical staining for GCAP/PLAP is fre-
quently used to confirm the histopathologic diagnosis
of classic seminoma/dysgerminoma, either in metas-
tases or in cases in which the primary tumor exhibits
histologic overlap with other tumor types (ie, lympho-
ma). Although it is a fairly sensitive marker for classic
seminoma/dysgerminoma, GCAP/PLAP is not ex-
pressed in spermatocytic seminoma (Dekker et al,
mas, 2 yolk sac tumors, and 2 teratomas), and the 4
spermatocytic seminomas (see above) were analyzed
for VASA transcript levels using semiquantitative
RT-PCR.
Immunohistochemistry
Tissue sections 3 ␮m thick of formalin-fixed paraffin-
embedded samples were mounted on APES (3-
aminopropyl-triethoxysilane)-coated glass slides and
used for simultaneous incubation with antibodies di-
rected against the VASA protein and GCAP/PLAP.
Slides were pretreated for antigen-retrieval (Shi et al,
1991), using 10 mM citrate buffer, pH 7.0. VASA
immunohistochemical stainings were performed over-
night at 4° C using a rabbit polyclonal antibody against
the human VASA protein, as previously described
(Castrillon et al, 2000), at 1:1500 dilution. Visualization
was performed by incubation with the second anti-
body biotinylated swine-anti-rabbit (DAKO A/S,
Glostrup, Denmark; cat. no. E0431) at 1:200 dilution
followed by incubation with avidin-biotin-alkaline
phosphatase complex (DAKO A/S; cat. no. K0391)
using Fast-blue BB (Sigma, St. Louis, Missouri) as
chromogen. For immunohistochemical staining of
GCAP/PLAP, a monoclonal antibody (Novocastra,
Newcastle, United Kingdom; NCL-PLAP-8A9) was
used at 1:10 dilution. The primary antibody was de-
tected using goat-anti-mouse-HRP (DAKO; cat. no.
P0447) at 1:100 and mouse-anti-peroxidase-PO (Sig-
ma; cat. no. P3039) 1:100 dilution with AEC (ϭ
3-amino-9-ethyl-carbazole) as chromogen. The stain-
ing results were found to be sensitive to fixation. This
was demonstrated by an overall weaker staining in-
tensity in the center of the tissue block than at the
edges of all samples. Processing tissue as thin tissue
slices, allowing optimal fixation, avoided this weaker
staining in the center of the block.
1992). The diagnostic usefulness of GCAP/PLAP is
further limited by lack of specificity, because it is
frequently expressed in nongerm cell tumors such as
ovarian carcinoma (in as many as half of ovarian
papillary serous carcinomas) (Nakopoulou et al, 1995)
and other neoplasms, particularly those of gastroin-
testinal origin (Hamilton-Dutoit et al, 1990). In addition,
GCAP/PLAP is expressed in several normal tissues
(
Hamilton-Dutoit et al, 1990). In contrast, the highly
specific expression of VASA in normal germ cells and
in specific subtypes of germ cell tumors (our study)
suggests that VASA is likely to be a useful diagnostic
marker with greater specificity than GCAP/PLAP.
In conclusion, our results on the expression of VASA
and the presence of the corresponding protein con-
firm the germ cell lineage origin of gonadoblastoma/
CIS, dysgerminoma/classic seminoma, and sper-
matocytic seminoma. The data support the model that
TGCT are derived from an early germ cell, and that
spermatocytic seminomas mimic the developmental
stage of spermatocytes. The lack of VASA, both
mRNA and protein in nonseminomas and derived cell
lines is in accordance with differentiation toward em-
bryonal and extraembryonic tissue and concomitant
loss of their germ cell–specific characteristics.
Materials and Methods
Collection of Tissue Samples
Semiquantitative RT-PCR
Freshly obtained tumor samples were collected in
collaboration with urologists and pathologists in the
southwestern part of The Netherlands. Immediately
after removal, portions of the tumor were snap-frozen
or fixed overnight in 10% buffered formalin and em-
bedded in paraffin. Standard procedures were fol-
lowed for tumor diagnosis, according to WHO classi-
fication (Mostofi and Sesterhenn 1998).
A total of 2 dysgerminomas, 2 gonadoblastomas, 3
mixed gonadoblastoma/dysgerminoma, 5 normal tes-
tes (containing spermatogenesis), 3 Leydig cell tu-
mors, 3 primary testicular lymphomas, 14 samples
containing CIS and/or Sertoli-only tubules adjacent to
tumor or normal testicular parenchyma, 13 semino-
mas, 12 nonseminomas, and 4 spermatocytic semi-
nomas (from 3 patients; one patient had a bilateral
tumor) were analyzed for the presence of the VASA
protein. In addition, one peripheral blood lymphocyte
sample, normal testes (n ϭ 2), a series of samples
containing 15% CIS (n ϭ 2), 50% CIS (n ϭ 1), 65% CIS
Total RNA was isolated from fresh frozen tissue sec-
tions. Adjacent sections were stained with hematoxy-
lin and eosin as histologic control. Standard proce-
dures were used for RNA isolation with TRIzol reagent
(InVitrogen, Carlsbad, California) from five 30-␮m-
thick sections per tumor. Cell-line RNA was isolated
similarly. First-strand cDNA was synthesized from 4
␮g total RNA with SuperScript-II reverse transcriptase
following the manufacturer’s instructions (InVitrogen).
Relative quantification of the VASA-transcript levels
was performed by duplex PCR with VASA specific
primers (primer sequences: 5' AAG AGA GGC TAT
CGA GAT GGA 3' and 5' CGT TCA CTT CCA CTG
CCA CTT CTG 3') compared with HPRT (primer se-
quences: 5' CGT GGG GTC CTT TTC ACC AGC AAG
3' and 5' AAT TAT GGA CAG GAC TGA ACG TC 3';
Jolly et al, 1983), resulting in amplification products of
238 bp and 387 bp, respectively. The expression level
of HPRT is constant in most tissues, including the
testis, and can therefore serve as an internal RT-PCR
standard (Pannetier et al, 1993).
(
n ϭ 1), and 80% CIS (n ϭ 2) tubules, 5 classic
seminomas, 6 nonseminomas (2 embryonal carcino-
1
64 Laboratory Investigation • February 2002 • Volume 82 • Number 2

VASA and Human Germ Cell Tumors
PCR reactions were performed in a Peltier Thermal
Cycler 200 machine (MJ Research, Watertown, Mas-
sachusetts) under the following conditions: 3 minutes
at 94° C, then 30 seconds at 94° C, 30 seconds at
extensive sequence similarity to ATP-dependent helicases.
Cell 55:577–87.
Jolly DJ, Okayama H, Berg P, Esty AC, Filpula D, Bohlen P,
Johnson GG, Shively JE, Hunkapillar T, and Friedmann T
(1983). Isolation and characterization of a full-length express-
ible cDNA for human hypoxanthine phosphoribosyl trans-
ferase. Proc Natl Acad Sci USA 80:477–81.
5
6° C, and 30 seconds at 72° C (26 cycles), 2 minutes
at 72° C (final extension step) in 60 ␮l reaction volume
1 ␮l cDNA (the equivalent of 250 ng RNA), 50 mM KCl,
(
1
0
0 mM Tris (pH 9.0), 0.1% Triton X-100, 1.5 mM MgCl ,
.2 ␮M dGTP, dTTP, dCTP, and dATP, 0.25 ␮l ␣ P-
2
Jørgensen N, Muller J, Jaubert F, Clausen OP, and Skakke-
baek NE (1997). Heterogeneity of gonadoblastoma germ
cells: Similarities with immature germ cells, spermatogonia
and testicular carcinoma in situ cells. Histopathology 30:177–
32
dATP (0.0925 MBq), 0.2 ␮M of each primer, 0.5 U
TaqPolymerase (QIAGEN, Hilden, Germany). After 20,
2
86.
1, 22, 23, and 24 cycles of amplification, 10 ␮l
samples were taken for analysis, to which 2 ␮l loading
buffer (type 2; Sambrook et al, 1989) was added. Four
microliters of each sample was loaded on a 4% native
polyacrylamide gel and run for 2 1/2 hours (400 V, 13
mA, 8 W), and then the gel was dried under vacuum.
Radioactive signals were quantified with the Storm
Jørgensen N, Rajpert-De Meyts E, Graem N, Müller J, Giwer-
cman A, and Skakkebæk NE (1995). Expression of immuno-
histochemical markers for testicular carcinoma in situ by
normal fetal germ cells. Lab Invest 72:223–231.
Kobayashi S, Yamada M, Asaoka M, and Kitamura T (1996).
Essential role of the posterior morphogen nanos for germline
development in Drosophila. Nature 380:708–11.
820 Phosphor Imager System (Molecular Dynamics,
Sunnyvale, California), using Image-quant software.
Lasko PF and Ashburner M (1988). The product of the
Drosophila gene Vasa is very similar to eukaryotic initiation
factor-4A. Nature 335:611–7.
References
Adami HO, Bergström R, Möhner M, Zatonski W, Storm H,
Ekbom A, Tretli S, Teppo L, Ziegler H, Rahu M, Gurevicius R,
and Stengrevics A (1994). Testicular cancer in nine northern
European countries. Int J Cancer 59:33–38.
Looijenga LHJ, Gillis AJM, Van Gurp RJHLM, Verkerk AJMH,
and Oosterhuis JW (1997). X inactivation in human testicular
tumors: XIST expression and androgen receptor methylation
status. Am J Pathol 151:581–590.
Looijenga LHJ, Olie RA, Van der Gaag I, van Sluijs FJ,
Matoska J, Ploem-Zaaijer J, Knepfle C, and Oosterhuis JW
Burke AP and Mostofi FK (1993). Spermatocytic seminoma:
A clinicopathologic study of 79 cases. J Urol Pathol 1:21–32.
(1994). Seminomas of the canine testis; counterpart of sper-
Castrillon DH, Quade BJ, Wang TY, Quigley C, and Crum CP
matocytic seminoma of men? Lab Invest 71:490–496.
(2000). The human VASA gene is specifically expressed in the
Looijenga LHJ and Oosterhuis JW (1999). Pathogenesis of
testicular germ cell tumors. Rev of Reproduction 4:90–100.
germ cell lineage. Proc Natl Acad Sci USA 97:9585–9590.
Cummings OW, Ulbright TM, Eble JN, and Roth LM (1994).
Spermatocytic seminoma: An immunohistochemical study.
Hum Pathol 25:54–59.
Møller H (1989). Decreased testicular cancer risk in men born
in wartime. J Natl Cancer Inst 81:1668–9.
Mosselman S, Looijenga LHJ, Gillis AJM, Van Rooijen MA,
Kraft HJ, Van Zoelen EJJ, and Oosterhuis JW (1996). Aber-
rant platelet-derived growth factor ␣-receptor transcript as a
diagnostic marker for early human germ cell tumors of the
adult testis. Proc Natl Acad Sci USA 93:2884–2888.
Dekker I, Rozeboom T, Delemarre J, Dam A, and Oosterhuis
JW (1992). Placental-like alkaline phosphatase and DNA flow
cytometry in spermatocytic seminoma. Cancer 69:993–996.
Eble JN (1994). Spermatocytic seminoma. Hum Pathol 25:
1
035–1042.
Mostofi FK and Sesterhenn IA (1998). Histological typing of
testis tumours. Berlin: Springer.
Forbes A and Lehmann R (1998). Nanos and Pumilio have
critical roles in the development and function of Drosophila
germline stem cells. Development 125:679–90.
Muller J, Skakkebaek NE, and Parkinson MC (1987). The
spermatocytic seminoma: Views on pathogenesis. Int J An-
drol 10:147–56.
Gavis ER, Curtis D, and Lehmann R (1996). Identification of
cis-acting sequences that control nanos RNA localization.
Dev Biol 176:36–50.
Nakopoulou L, Janinis J, Giannopoulou I, Lazaris AC,
Koureas A, and Zacharoulis D (1995). Immunohistochemical
expression of p53 protein and proliferating cell nuclear anti-
gen in hepatocellular carcinoma. Pathol Res Pract 191:1208–
Ghabrial A and Schupbach T (1999). Activation of a meiotic
checkpoint regulates translation of Gurken during Drosophila
oogenesis. Nat Cell Biol 1:354–7.
13.
Neuman-Silberberg FS and Schupbach T (1993). The Dro-
sophila dorsoventral patterning gene gurken produces a
dorsally localized RNA and encodes a TGF alpha-like protein.
Cell 75:165–74.
Gonzalez-Reyes A, Elliott H and St Johnston D (1995).
Polarization of both major body axes in Drosophila by
gurken-torpedo signalling. Nature 375:654–8.
Hamilton-Dutoit SJ, Lou H, and Pallesen G (1990). The
expression of placental alkaline phosphatase (PLAP) and
PLAP-like enzymes in normal and neoplastic human tissues:
An immunohistological survey using monoclonal antibodies.
APMIS 98:797–811.
Oosterhuis JW, Looijenga LHJ, Geurts van Kessel A, and De
Jong B (1993). A cytogenetic classification of germ cell
tumors, and its biological relevance. Eur Urol 23:6–8.
Pannetier C, Delassus S, Darche S, Saucier C, and Kourilsky
P (1993). Quantitative titration of nucleic acids by enzymatic
amplification reactions run to saturation. Nucleic Acids Res
21:577–83.
Hay B, Jan LY, and Jan YN (1988). A protein component of
Drosophila polar granules is encoded by Vasa and has
Laboratory Investigation • February 2002 • Volume 82 • Number 2 165

Zeeman et al
Pottern ML, Morris Brwn L, and Devesa SS (1998). Epidemi-
ology and pathogenesis of testicular cancer. In: Ernsthoff
MS, editor. Testicular and penile cancer. Oxford: Blackwell
Science, 2–10.
Skakkebæk NE (1972). Possible carcinoma-in-situ of the
testis. Lancet 516–517.
Stoop H, Van Gurp RHJ, LM, De Krijger R, Geurts van Kessel
A, Koberle B, Oosterhuis JW, and Looijenga LHJ (2001).
Reactivity of germ cell maturation stage-specific markers in
spermatocytic seminoma: Diagnostic and etiological implica-
tions. Lab Invest 81:919–928.
Rajpert-De Meyts E, and Skakkebæk NE (1994). Expression
of the c-kit protein product in carcinoma-in-situ and invasive
testicular germ cell tumours. Int J Androl 17:85–92.
Raz E (2000). The function and regulation of Vasa-like genes
in germ-cell development. Genome Biol: 1–6.
Styhler S, Nakamura A, Swan A, Suter B, and Lasko P (1998).
Vasa is required for Gurken accumulation in the oocyte, and
is involved in oocyte differentiation and germline cyst devel-
opment. Development 125:1569–78.
Roelofs H, Manes T, Millan JL, Oosterhuis JW, and Looijenga
LHJ (1999). Heterogeneity in alkaline phosphatase isozyme
expression in human testicular germ cell tumors: An en-
zyme-/immunohistochemical and molecular analysis.
J Pathol 189:236–244.
Swerdlow AJ (1993). The epidemiology of testicular cancer.
Eur Urol 23:35–38.
Talerman A (1974). Spermatocytic seminoma. J Urol 112:
Romanenko AM and Persidskii YV (1983). Ultrastructure and
histogenesis of spermatocytic seminoma. Voprosi Onkologii
212–6.
Talerman A (1980). Spermatocytic seminoma. Cancer 45:
19:61–66.
2169–2176.
Rosai J, Khodadoust K, and Silber I (1969). Spermatocytic
seminoma. Cancer 24:103–116.
Talerman A, Fu YS, and Okagaki T (1984). Spermatocytic
seminoma: Ultrastructural and microspectrophotometric ob-
servations. Lab Invest 51(3):343–349.
Rosenberg C, Mostert MC, Bakker Schut T, Van de Pol M,
Van Echten-Arends J, De Jong B, Raap T, Tanke H, Ooster-
huis JW, and Looijenga LHJ (1998). Chromosomal constitu-
tion of human spermatocytic seminomas: Comparative
genomic hybridization supported by conventional and inter-
phase cytogenetics. Genes Chromosom Cancer 23:286–
Tanaka SS, Toyooka Y, Akasu R, Katoh-Fukui Y, Nakahara Y,
Suzuki R, Yokoyama M, and Noce T (2000). The mouse
homolog of Drosophila Vasa is required for the development
of male germ cells. Genes Dev 14:841–53.
2
91.
Roth S, Neuman-Silberberg FS, Barcelo G, and Schupbach T
1995). Cornichon and the EGF receptor signaling process
Tomancak P, Guichet A, Zavorszky P, and Ephrussi A (1998).
Oocyte polarity depends on regulation of gurken by Vasa.
Development 125:1723–32.
(
are necessary for both anterior-posterior and dorsal-ventral
pattern formation in Drosophila. Cell 81:967–78.
Toyooka Y, Tsunekawa N, Takahashi Y, Matsui Y, Satoh M,
and Noce T (2000). Expression and intracellular localization
of mouse Vasa-homologue protein during germ cell develop-
ment. Mech Dev 93:139–49.
Sambrook J, Frisch EF, and Maniatis T (1989). Molecular
cloning: A laboratory manual. New York: Cold Spring Harbor
Laboratory Press.
Scully RE (1970). Gonadoblastoma: A review of 74 cases.
Cancer 25:1340–1356.
Shi SR, Key ME, and Kalra KL (1991). Antigen retrieval in
formalin-fixed, paraffin-embedded tissues: An enhancement
method for immunohistochemical staining based on micro-
wave oven heating of tissue sections. J Histochem Cytochem
39:741–8.
1
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