Advances in Bioscience and Biotechnology, 2010, 1, 444-452 ABB
doi:10.4236/abb.2010.15058 Published Online December 2010 (http://www.SciRP.org/journal/abb/).
Published Online December 2010 in SciRes. http://www.scirp.org/journal/ABB
Ubiquitous expression of Sry induces embryonic lethality
related to suppression of Tie2/Tek expression
Kazuhisa Yoshida1, Masanori Ito1,2, Kou Yokouchi1,3, Kiyoshi Kano1, Kunihiko Naito1, Hideaki Tojo1,4
1Laboratory of Applied Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo-ku,
Tokyo, Japan;
2Department of Pharmacology, School of Medicine, Faculty of Medicine, Toho University, Omori-Nishi, Otaku, Tokyo, Japan;
3Shirakawa Institute of Animal Genetics, Odakura, Nishigo, Fukushima, Japan;
4Yamazaki College of Animal Health Technology, Minamioosawa, Hachioji, Tokyo, Japan.
Received 21 October 2010; revised 29 October 2010; accepted 29 October 2010.
ABSTRACT
Sry (sex-determining region on the Y chromosome) is
a mammalian sex-determining gene on the Y chro-
mosome. In mice, the transient expression of Sry in
supporting cell precursor cells between 10.5 and 12.5
days post-coitus (dpc) triggers the differentiation of
Sertoli cells from granulosa cells. The importance of
the strict regulation of Sry expression remains un-
known. Thus, we attempted to produce a Sry ubiqui-
tous-expressing transgenic (Tg) mouse in which for-
eign Sry is driven by the CAG (cytomegalovirus im-
mediate-early enhancer, chicken beta-actin promoter,
and the fusion intron of chicken beta-actin and rabbit
beta-globin)-Sry gene for ubiquitous expressing Sry. A
low rate (2/127) of Tg pups was observed, whereas
the rate of early-stage transgenic embryos before
birth was 19.2% (5/26). The Sry ubiquitous-express-
ing embryos showed abnormal development. The
results suggest that ubiquitous expression of Sry ex-
erts a negative effect on embryonic development. One
of the two adult Tg mice showed low levels of Sry ex-
pression. The other Tg mouse showed high Sry
transgene expression, but was mosaic for the trans-
gene. Developmental analysis of transgenic F1 em-
bryos produced from the mosaic Tg mouse revealed
that ubiquitous expression of Sry had a lethal effect
on embryonic development around 12.5 dpc. The
histological data indicated that ubiquitous expression
of Sry induced abnormal cardiovascular development,
resulting in embryonic death. Enhanced expression of
Sry suppressed endogenous Tie2/Tek (tyrosine kinase
with Ig and EGF homology domains 2/tunica interna
endothelial cell kinase) expression in Sry-transfected
primary cultured cells from wild type embryonic
hearts. The results indicate that the tissue-specific
and stage-specific expression of Sry is essential for
normal embryogenesis.
Keywords: Sry; Ti e2/Tek; Transgenic Mice
1. INTRODUCTION
Sry (Sex determining region on the Y chromosome) is a
transcription factor with a DNA-binding domain referred
to as the high mobility group (HMG), which triggers a
gene expression cascade required for initiating male sex
differentiation in the bipotential indifferent gonads of
mammals [1]. Mouse Sry is expressed for a brief period
between 10.5 and 12.5 days post-coitus (dpc) in the
supporting cells of undifferentiated gonads that differen-
tiate into Sertoli cells instead of granulosa cells [2-5].
It is well documented that Sry is a trigger and decisive
gene for mammalian sex determination. Sry expression
induces down- or up-regulation of the expression of
various genes linked to the sex-determination cascade
and subsequent testicular development. A large number
of factors driving gonadal differentiation are encoded by
autosomal genes. Testicular development after Sry ex-
pression has been shown to be regulated by various
genes such as SfI/Ad4bp (steroidogenic factor 1/Adrenal
4 binding protein) [6], Wt1 (Wilms’ tumor suppressor 1)
[7,8], Amh/Mis (Anti-Mulerian hormone/Mulerian-in-
hibiting substance) [9], and Sox9 (Sry-related high-mo-
bility group box 9) [10]. In addition, the regions respon-
sible for stage-specific and tissue-specific regulation of
mouse Sry expression have also been investigated
[11-14]. However, the gene responsible for gonadal dif-
ferentiation, i.e., the direct target of Sry, remains to be
identified.
Previously, an XX-sex-reversal mouse line carrying
the Sry transgene driven by a weak basal Hsp70.3 pro-

K. Yoshida et al. / Advances in Bioscience and Biotechnology 1 (2010) 444-452 445
moter (Hsp-Sry) was established. Comparison of Hsp-
Sry/XY gonads with wild-type/XY and Hsp-Sry/XX go-
nads has suggested that Sry mRNA expression alone is
not likely to provide positional or timing information
needed for male-specific Sox9 activation in developing
gonads [15]. The ability of Sry to induce testis develop-
ment is limited to approximately 11.0-11.25 dpc, a time
window of only 6 hours after the normal onset of Sry
expression in XY gonads [16]. It is generally expected
that the tightly regulated spatiotemporal expression profile
of Sry during embryogenesis is crucial for assuring the
normal development not only of gonads, but also of other
fetal organs; however, the precise biological significance
of limited Sry expression has yet to be clarified.
It is well established that the phenotype of transgenic
(Tg) mice exhibiting ubiquitous expression of a gene of
interest can provide evidence for speculations regarding
natural biological functions [17-19]. Thus, to evaluate
the role(s) played by Sry in development, we utilized
transgenic mice. For our specific purposes, we con-
structed a CAG (cytomegalovirus immediate-early en-
hancer, chicken beta-actin promoter and fusion intron of
chicken beta-actin and rabbit beta-globin)-Sry fusion
gene construct that induces strong ubiquitous expression
of a gene of interest [20], and we then attempted to gen-
erate the corresponding Tg mice (Figure 1). Here, we
describe the developmental effects of ubiquitous Sry
expression in these Tg mice.
2. MATERIALS AND METHODS
2.1. Animals
The following strains of mice were purchased from a
commercial animal breeder (Sankyo Labo-Service Cor-
poration, Inc., Tokyo, Japan): B6C3F1 (C57BL/6Nx
C3H/HeN), C57BL/6J, and ICR. The mice were kept in
an environment with regulated temperature (22-25℃),
humidity (40-50%), and illumination cycles (14-h light,
10-h dark), and were provided with food and water ad
libitum. The experiments were conducted according to
guidelines for the care and use of laboratory animals at
the College of Agriculture, the University of Tokyo.
2.2. Tg Mouse Generation
Tg mice were generated by microinjecting DNA into the
pronuclei of zygotes collected from the oviducts of su-
perovulated B6C3F1 females that were mated with
B6C3F1 males. All methods for generating the Tg mice
used here have been described in the protocol reported
by Hogan et al. [21]. The construction of pCX-Sry has
been described previously [22]. The SalI/BamHI DNA
fragment containing the CAG-Sry fusion gene was ex-
cised from pCX-Sry and separated by electrophoresis
Figure 1. Schematic representation of gonadal sex determina-
tion and expression levels of endogenous Sry and CAG-Sry
transgene. In mice, gonads are formed around 10.5 days
post-coitus (dpc). Sry (Sex-determining region on the Y chro-
mosome) triggers the testis developmental pathway in the bi-
potential indifferent gonads. Sry is expressed in the mouse
gonads during a narrow window of development, between 10.5
and 12.5 dpc. In this study we constructed a CAG (cytomega-
lovirus immediate-early enhancer, chicken beta-actin promoter
and fusion intron of chicken beta-actin and rabbit beta-glo-
bin)-Sry fusion gene construct that induces strong ubiquitous
expression of Sry and generate the transgenic (Tg) mice.
through 1% agarose gel; the fragment was then purified
by CsCl ultra-centrifugation. The purified DNA frag-
ment was dissolved in a solution containing 10 mM
Tris-HCl (pH 7.4) and 0.25 mM EDTA (pH 7.4) and was
used for pronuclear microinjection. To identify Tg foun-
der animals, genomic DNA was isolated from the tip of
the tail, and the genomic DNA was screened by poly-
merase chain reaction (PCR) amplification using the
following primers: 5’-CTC-TGC-TAA-CCA-TGT-TCA-
TGC-CTT-3’ and 5’-CCA-CTG-CAG-AAG-GTT-GTA-
CAG-TTT-3’, which span the CAG promoter and Sry
coding region (Figures 2(a) and (b)). The PCR amplify-
cations were carried out using the following parameters:
35 cycles of 30 s at 94℃, 30 s at 58℃, and 1 min at
72℃. Sex chromosome karyotypes (XX or XY) were
determined by PCR using the primers 5’-GCT-CGT-
TAA-TTT-CTC-ACG-TTA-GTC-C-3’ and 5’-ACA-CTT-
TAG-CCC-TCC-GAT-GAG-GCT-GA-3’, which span the
Sry promoter and Sry coding region (Figures 2(c) and
(d)). The PCR amplifications were carried out using the
following parameters: 35 cycles of 30 s at 94℃, 30 s at
58℃, and 1 min at 72℃.
2.3. Preparation of Primary Cultured Cells and
Transfection of Plasmids
Primary cultured cells were prepared from the hearts of
12.0-dpc embryos according to methods described in our
previous paper [12,14]. pCX-Sry and pCAGGS (mock)
were transfected using Effectene Transfection Reagent
(Qiagen, Valencia, CA) according to the manufacturer’s

K. Yoshida et al. / Advances in Bioscience and Biotechnology 1 (2010) 444-452 446
Figure 2. CAG-Sry construct and genotyping. (a) A schematic representation of the CAG-Sry transgene construct used in the micro-
injection. The arrows indicate the location of primers used to identify the transgenic pups or embryos. (b) PCR analysis with genomic
DNA to identify Tg mice. (c) A schematic representation of the endogeneous Sry gene. The arrows show the primer positions used to
identify presence of Y chromosome. (d) PCR analysis with genomic DNA to identify presence of Y chromosome. Embryos were
collected from pregnant foster mothers and their developmental level was analyzed. Non-transgenic embryo at 17.5 dpc (e) and Tg
embryo at 17.5 dpc (f) were shown. Embryonic death is observed in CAG-Sry Tg embryo. Tg, transgenic; No, Non-transgenic; P,
positive control; N, Negative control; XX, XX karyotype; XY, XY karyotype; M, size marker.
instructions, and the transfected cells were cultured at
37℃ under 5% CO2 for 3 days.
2.4. Reverse Transcription Polymerase Chain
Reaction (RT-PCR)
Transcripts of the Sry transgene and endogenous Tie2/
Tek (tyrosine kinase with Ig and EGF homology domains
2/tunica interna endothelial cell kinase) in the embryonic
tissues, and Sry-transfected primary cultured cells from
the hearts of 12.0-dpc embryos were deter- mined as
previously described [12,14,22]. PCR was done using
the appropriate primer sets for the target gene: for Sry,
forward primer 5’-AAG-CGC-CCC-ATG- AAT-GCA-
TTT-ATG-GT-3’ and reverse primer 5’-ACA-CTT-TAG-
CCC-TCC-GAT-GAG-GCT-GA-3’; for Tie2/Tek, 5’-
TAC-ATA-GGA-GGA-AA C-CTG-TTC-ACC-3’ and 5’-
GGA-GGT-AAG-ACT-CGG-TTG-ACA-GTG-3’; for gly-
ceraldehyde-3-phosphate dehydrogenase (G3PDH), 5’-
TGA-AGG-TCG-GTG-TCA-ACG-GAT-TTG-GC-3’ and
5’-CAT-GTA-GGC-CAT-GAG-GTC-CAC-CAC-3’.
2.5. Histology
For the staging of embryos, midday for the vaginal plug
was considered to be 0.5 dpc. Part of a limb was re-
moved from the embryos for DNA extraction and geno-
typing. For the histological analysis, embryos were fixed
in 4% paraformaldehyde. After fixation, the embryos
were processed for paraffin embedding as previously

K. Yoshida et al. / Advances in Bioscience and Biotechnology 1 (2010) 444-452 447
described [5]. The embryos prepared in this manner were
then sectioned at 6 μm, and the sections were used for
hematoxylin and eosin staining.
2.6. Statistical Analysis
The results are expressed as mean  SEM. The signifi-
cance of differences between groups was determined by
Student’s t-test.
3. RESULTS
3.1. Low Production Rate of Sry Transgenic
Mice
The construct containing Sry under the control of the
CAG promoter (CAG-Sry) was used for DNA microin-
jection to produce Tg mice (Figure 2(a)). The CAG
promoter was selected because it is a ubiquitously and
strongly expressed promoter. In analyses of DNA pups
born, the percentage of Tg animals was 1.5% (2/127).
Two CAG-Sry Tg lines (female and male) were gener-
ated. The female karyotype was XX, and that of the male
was XY. Then, in the next experiment, pregnant mice
that had undergone a transfer of CAG-Sry microinjected
eggs were sacrificed between 11.5 dpc and 17.5 dpc, and
the percentage of Tg embryo was investigated. The per-
centage of Tg embryo taking into consideration both
embryonic stages, was approximately 19.2% (5/26). Tg
embryos sacrificed at 17.5 dpc showed histological ab-
normalities (Figure 2(f)), suggesting that the survival
rate of Tg embryos was reduced by the ubiquitous ex-
pression of Sry.
3.2. Lethal Effect of Sry-Ubiquitous Expression
during Embryonic Development
Although the transgene was detected in F1 mice in the
case of female CAG-Sry Tg mouse, low transcripts from
the transgene were detected by RT-PCR, and female-
to-male sex reversal did not occur with the Sry transgene
(data not shown). The male Tg mouse was characterized
as XY and fertile. No newborn pups resulting from
breeding with the male Tg mouse were transgenic. This
result suggested that ubiquitous and strong expression of
Sry yields embryonic lethality; the mosaic integration of
the transgene in the founder Tg mouse could explain
why the founder Tg mouse was able to live to adulthood.
To further investigate this lack of generation of trans-
genic offspring, we performed a series analysis of litters
at embryonic stages. Tg embryos showing high levels of
Sry transgene expression were detected at a rate of 7%
(12/175). The rate of occurrence of Tg embryos indi-
cated the mosaic integration of the transgene. Next, we
attempted to determine the stage of embryonic develop-
ment at which the ubiquitous-expression of Sry could
exert a negative impact. At 11.5 dpc, control and
CAG-Sry Tg embryos displayed no morphological dif-
ferences (Figures 3(a) and (b)). In contrast, edema and
congestion were observed in CAG-Sry Tg embryos at
12.5 dpc (Figure 3(d)). The CAG-Sry Tg embryos were
dead by 13.5 dpc (Figure 3(f)). Therefore, the present
results indicate that the ubiquitous expression of Sry has
a lethal effect on embryonic development at approxi-
mately 12.5 dpc. Histological analysis of the genital
ridge of CAG-Sry transgenic embryos (XY) at 12.5 dpc
showed no testis cord formation (Figure 4(g)), and an
enlargement of the diameter of the atrium was also ob-
served (Figure 4(h)). The layer of smooth muscle cells
of Tg embryos was thin, compared to that of wild-type
embryos, in the dorsal aorta region (Figure 4(d) and (i)).
Moreover, the endothelial cells of Tg embryos were ab-
normally round (Figure 4(j)).
3.3. Suppression of Tie2/Tek Expression by Sry
Expression
We also examined the expression levels of Tie2/Tek in
Sry-transgenic tissues, because Ti e2/Tek has been re-
ported to be involved in cardiovascular development. In
wild-type embryos, Tie2/Tek expression levels were
highest in the hearts, compared with those of the other
two tissue types examined, i.e., brains and gonads, at
12.0 dpc (Figure 5(a)). As regards the rates of expres-
sion observed in these three tissues, Ti e2/Tek expression
levels of Sry-transgenic tissues were lower than that of
wild-type tissues (Figure 5(a)). Thus, in these embry-
onic tissues, Tie2/Tek expression was suppressed by the
ubiquitous expression of foreign Sry. This suppression of
Tie2/Tek expression by Sry expression was also seen in
Sry-transfected primary cultured cells from wild-type
embryonic hearts (Figure 5(c)). The present findings sug-
gest that ubiquitous Sry expression exerts a negative
effect on cardiovascular development via changes in the
expression of other genes, which ultimately results in
embryonic lethality. The Tg male became unable to im-
pregnate any females, which rendered it impossible to
conduct further analyses of embryos from the Tg male.
4. DISCUSSION
In this study, we used a Tg approach to characterize the
biological function of Sry. We generated Tg mice that
ubiquitously expressed Sry as a means of elucidating the
biological functions of this transcription factor. The effi-
ciency of transgenesis was remarkably low (1.5%) com-
pared with the efficiency of our usual transgenic ex-
speriments [23-26]. Furthermore, no newborn pups gen-
erated by breeding of the CAG-Sry Tg founder (mosaic
for the transgene) carried the CAG-Sry transgene. Time-
series analyses of F1 CAG-Sry Tg embryos clearly re-

K. Yoshida et al. / Advances in Bioscience and Biotechnology 1 (2010) 444-452 448
(a) (b)
(c) (d)
(e) (f)
Figure 3. Comparison of CAG-Sry transgenic embryos to
wild-type embryos at different stages. Gross morphology of
wild-type (a, c, e) and CAG-Sry Tg (b, d. f) embryos at 11.5
dpc (a, b), 12.5 dpc (c, d) and 13.5 dpc (e, f). Arrow in d shows
congestion. Arrowhead in D shows edema. CAG-Sry Tg em-
bryos appear normal up to 11.5 dpc. At 12.5 dpc, the Tg em-
bryos begin to appear deformed and are dead by 13.5 dpc.
vealed the negative effects of the ubiquitous expression
of Sry on cardiovascular development; these negative
effects were apparent as early as the 12.5 dpc, indica-
ting that neither the spermatogenesis of germ cells, nor
embryonic development, may be influenced by the ubiq-
uitous expression of Sry prior to 11.5 dpc. The results
also suggest that the precisely regulated expression of
Sry in gonads appears to be essential for normal em-
bryogenesis as well as for sex differentiation. Foreign
mouse Sry has been shown to induce XX sex reversal [1].
The ability of Sry to induce testis development is limited
to approximately 11.0-11.25 dpc [16]. Interestingly, al-
though the gene-regulation system of goat SRY differs
from that of the mouse Sry gene [14], transgenic mice
with goat SRY showed XX sex reversal [27]. The previ-
ous results suggest that tissue-specific and stage-specific
Sry expression might not necessarily be required for tes-
tis differentiation. Indeed, Sry, when under the control of
the Hsp70.3 promoter (which induces weak yet broad
expression), induced XX sex reversal [15]. In this study,
it remained unclear whether or not the CAG-Sry trans-
gene could induce testis development, because gonadal
development had already stopped prior to testis cord
formation (Figure 4(g)).
Knockout-mouse disrupted genes related to cardio-
vascular development (e.g., fetal liver kinase-1, fms-like
tyrosine kinase-1, vascular endothelial growth factor,
Tie2/Tek, and angiopoietin-1) have been associated with
embryonic lethality at 8.5-12.5 dpc [28-32]. Ubiquitous
Sry expression might influence the expression of these
genes after 11.5 dpc. A Ti e2/Tek promoter region analy-
sis suggested that the Octamer-binding protein-1 (Oct-1)
co-factor complex mediates the expression of Tie2/Tek
[33,34]. There are 10 SOX (Sry-related High Mobility
Group box) binding motifs, AACAA(T/A), within 5 kb
of the 5’-flanking region of mouse Tie2/Tek . It is prob-
able that the SOX-Oct complex regulates Tie2/Tek expres-
sion. Interestingly, Tie2/Tek expression was found to be
downregulated in the heart of CAG-Sry Tg embryos at 12
dpc, and was also downregulated by the transfection of
pCX-Sry into primary cultured cells prepared from em-
bryonic mouse hearts (Figure 5). The SOX transcription
factor family contains 20 (human and mouse) members,
which have been classified into 8 groups [35]. SoxF genes
(Sox7, Sox17, and Sox18) are expressed in endothelial
cells and are required for vascularization [36-42]. As the
morphology of endothelial cells was found to be malig-
nant in CAG-Sry Tg embryos (Figure 3(j)), the
CAG-Sry transgene might interrupt the function of SoxF
genes in endothelial cells by competition with SoxF genes,
thereby inducing abnormal development of the cardio-
vascular system; this was the case with a Sox18 mutant
(Ra, RaJ, Raop and Ragl), which acted as a dominant
negative [43-45]. It has been reported that Tie2/Tek
knockout mice exhibit embryonic lethality accompanied

K. Yoshida et al. / Advances in Bioscience and Biotechnology 1 (2010) 444-452 449
Figure 4. Histological analysis of CAG-Sry transgenic embryos and wild-type littermates. Hematoxylin and eosin staining of whole
embryos (a, f), gonad regions (b, g), heart regions (c, h) and dorsal aortic regions (d, e, i, j) of wild-type XY embryos (a to e) and Tg
XY embryos (f to j) from breeding of wild-type female mouse and male CAG-Sry Tg mouse are shown. Tg embryos show the accu-
mulation of blood cells in the cardinal veins (f, arrow). In the sections of genital ridge region, the tubule structure (arrow) is observed
in gonad region of wild-type XY embryo (b). There is no tubule structure in gonad region of Tg XY embryo (g). Arrow shows bleed-
ing in abdominal cavity. Sections of heart region of wild-type (c) and Tg (h) embryos show enlarged atrium in Tg embryo (arrow).
Thin layer of muscle cells of aortic region (indicated by two arrows) is observed in section of Tg embryo (i) compared with that of
wild-type embryo (d). Normal endothelial cells of aorta show flat morphology (arrows in e) and abnormal round shape of endothelial
cells are observed in sections of Tg embryo (arrows in j). bar, 50 μm.
by abnormal cardiovascular development [31]. In the
present study, it was revealed that Tie2/Tek expression
was suppressed by enhanced Sry expression in both the
Sry-transgenic heart and Sry-transfected primary cul-
tured heart cells. As hypothesis of mutant Sox18 by
Downes and Koopman [45], Sry proteins might act as
dominant negative form by disruptive interaction with
co-factor(s) of Sox18 (Figure 5).
In conclusion, we demonstrated that the tissue-speci-
fic and stage-specific expression of Sry is essential for
normal embryogenesis, and in particular for cardiovas-
cular development.

K. Yoshida et al. / Advances in Bioscience and Biotechnology 1 (2010) 444-452 450
Figure 5. Effects of Sry-transgene on endogenous Ti e2/Tek expression. (a) Ti e2/Tek expression levels
relative to those of G3PDH in the CAG-Sry transgenic tissues at 12.0 dpc. Expression levels of
Tie2/Tek in the Sry transgenic tissues were reduced (n = 1). (b) Determination of expression of Sry
transgene in the primary cultured cells collected from embryonic hearts at 12.0 dpc. pCX-Sry:
Sry-transfected cells, Mock: mock-transfected cells, G3PDH: a house-keeping gene used as a refer-
ence, RT-: no reverse transcription. (c) Tie2/Tek expression levels relative to those of G3PDH.
Tie2/Tek expression levels were reduced in the Sry-transfected primary cultured cells. *: P < 0.05,
Vertical bars indicate the means  SEM (n = 3). (d-f) The hypothesized effects of ubiquitous expres-
sion of Sry on the function of Sox18. Sox18 binds to the upstream regulatory sequence of a Ti e2/Tek
gene. The functional trans-activation domain is shown as opening that complement in shape its re-
spective interacting co-factor (d). When affected by mutation, the domain is depicted as
non-complementary opening (e). In the case of CAG-Sry Tg mice, Sox18 is replaced by Sry proteins
which act as if mutant Sox18 (f). Successful interaction of Sox18 domain with its co-factor is indi-
cated by an arrow, while disrupted interaction is indicated by a crossed arrow.

K. Yoshida et al. / Advances in Bioscience and Biotechnology 1 (2010) 444-452 451
5. ACKNOWLEDGEMENTS
We would like to thank Dr. Toshiyasu Matsui (The University of Tokyo)
for expert advice on histology. This work was supported in part by a
Grant-in-Aid for Scientific Research from the Ministry of Education,
Science, Sports and Culture of Japan (No.16380197).
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