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320
K. NISHINO
JOURNAL
ET
OF
AL.
EXPERIMENTAL ZOOLOGY 286:320–327 (2000)
Establishment of Fetal Gonad/Mesonephros
Coculture System Using EGFP Transgenic Mice
KOICHIRO NISHINO, MINORU KATO, KOU YOKOUCHI,
KEITARO YAMANOUCHI, KUNIHIKO NAITO, AND HIDEAKI TOJO*
Laboratory of Applied Genetics, Graduate School of Agricultural and Life
Sciences, University of Tokyo, Tokyo 113-8657, Japan
ABSTRACT
In developing mouse embryos, the Sertoli cells, Leydig cells, and seminiferous
cords are differentiated in the XY gonads. The migration of mesonephric cells into the gonads is
required during the developmental stage for seminiferous cord formation in the male gonads. In
previous experiments, an organ coculture system has been used to examine morphologically developing gonads. However, by the process used in this system for fixing and staining the gonad/
mesonephros complexes for examination, the kinetics of cell migration and the character of migrating cells cannot be observed. In the present study, we established an improved organ coculture
system, using transgenic mice ubiquitously expressing Enhanced Green Fluorescent Protein
(EGFP). In this system, time-dependent morphological changes in male-specific migration were
observable in the gonad/mesonephros complex. The cell migration occurred at around 20 hr of
coculture and began to spread at 25 hr with increases in the number of migrating cells occurring
at 45 hr of coculture. No degenerative changes were detected at the end of coculture. Our results
indicate that the present coculture system is very useful for investigating the mechanism of cell
migration, as well as the characteristics of the migrating cells, in developing gonads. J. Exp. Zool.
286:320–327, 2000. © 2000 Wiley-Liss, Inc.
In mice, the gonads of male embryos at 12.5
days post coitus (dpc) are dramatically differentiated and form cord-like structures containing pre-Sertoli cells and primordial germ cells.
In contrast, the histological structures of female
gonads are little changed at this time. Several
previous studies using the primary culture or
organ culture of male gonads have shown that
Sertoli cells, Leydig cells, and seminiferous
cords are differentiated in vitro (Magre and
Jost, ’84; Patsavoudi et al., ’85; Jost and Magre,
’88; Karl and Capel, ’98), and that the seminiferous cord formation requires cell migration
from the adjacent mesonephros. It has been reported that some of the stromal cells in male
gonads are of mesonephric origin (Buehr et al.,
’93). Merchant-Larios et al. (’93) demonstrated,
in a study using an organ coculture system at
the male gonad and the 3H-thymidine labeled
mesonephros, that Sertoli and Leydig cells are
differentiated without the mesonephros, while
endothelial and peritubular myoid-like cells migrate into the male gonad from the mesonephros. It was also shown that both male and
female mesonephric cells or even limb bud cells
migrate into the male gonad when they are
cocultured with male gonads (Buehr et al., ’93;
2000 WILEY-LISS, INC.
Merchant-Larios et al., ’93; Moreno-Mendoza et
al., ’95). Martineau et al. (’97) and Brennan et
al. (’98) verified, by using their own coculture
system of a transgenic mouse ubiquitously expressing β-galactosidase, that cell migration
from the mesonephros to the gonad is male-specific and that the migration is dependent on induction signals from the male gonad.
These organ coculture systems of a gonad
grafted on another individual mesonephros have
contributed to the morphological analysis of gonad development. In these previous experiments,
however, the gonad/mesonephros complexes were
fixed and stained for examination, which meant
that the kinetics of cell migration and the character of migrating cells, such as the differential
ability of cells or gene expression in the cells, could
not be examined. In the present study, we established an improved organ coculture system using
Grant sponsor: Ministry of Education, Science, Sports and Culture of Japan Grant-in-Aid for Scientific Research (A); Grant number: 07556063.
*Correspondence to: Hideaki Tojo, Laboratory of Applied Genetics,
Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. E-mail:
[email protected]
Received 16 February 1999; Accepted 2 June 1999
COCULTURE OF FETAL GONAD AND MESONEPHROS
transgenic mice ubiquitously expressing the enhanced green fluorescent protein (EGFP) under the
control of a CMV enhancer and β-actin promoter.
The green signal of EGFP can be readily detected
in a live organ under a fluorescent microscope without fixing and staining. The present coculture system enabled us to observe the time-dependent
morphological changes that occur in the male-specific migration of cells in the gonad/mesonephros
complex. The present coculture system showed a
two-phase migration of the mesonephros cells.
MATERIALS AND METHODS
EGFP-transgenic mice
Mice of BDF1 (C57BL/DBA F1) and ICR strains
were purchased from a dealer (Japan SLC, Shizuoka, Japan), and were kept under the regulated
temperature (22–25°C), humidity (40–60%), and
illumination cycles (14 hr light, and 10 hr dark)
throughout the experiments. The pCX-EGFP plasmid containing CMV enhancer, chicken β-actin
promoter, and 733 bp cDNA encoding EGFP was
a generous gift from Dr. M. Okabe, Research Institute for Microbial Diseases, Osaka University,
Osaka, Japan (Okabe et al., ’97). Briefly, the 3.2kbp insert fragment of the EGFP gene was prepared by digesting pCX-EGFP plasmid with Sal I
and BamHI (Takara, Otsu, Japan). The DNA fragments were purified by the cesium chloride ultracentrifugation method, and approximately 4.0 µg/
ml of the linear fragments were used for microinjection into the pronuclei of fertilized eggs. The 7to 8-week-old F1 females (C57BL/DBA) were superovulated by an injection of 7.5 IU of pregnant
mare’s serum gonadotropin (PMSG; Teikokuzoki,
Tokyo, Japan), followed 48 hr later by the injection of 7.5 IU of hCG. Fertilized eggs were collected from the oviducts of the females that had
been mated with males of the same strain the day
after mating. Microinjection was performed through
the use of an inverted microscope equipped with a
micromanipulator (Narishige, Tokyo, Japan). Approximately 2 pl of DNA solution were injected into
the pronuclei of collected eggs and the microinjected
embryos were cultured for 4 days in M16 medium
(Sigma, St. Louis, MO) until they reached the
morula or blastocyst stage. The EGFP-positive embryos were then transferred to the uteri of the
pseudo-pregnant female ICR strain female mice
(>10 weeks old). The transgenic mice were identified by both Southern blotting and PCR analyses
using DNA extracted from the tail tips of 4-weekold pups. The expression of EGFP was determined
321
by northern blotting and by observing the fluorescence on the entire body of transgenic mice using an excitation light. The several transgenic
lines ubiquitously expressing EGFP were used for
the present experiments.
Organ cultures
The 12.5-dpc mouse embryos were collected from
the pregnant females (C57BL/DBA F1) that had
been mated with the transgenic males. To determine whether the collected embryos expressed
the transgene, the embryos were examined for
the expression of EGFP under a fluorescent stereomicroscope (Leica MZFLIII/CLS150, Leica
AG, Heerbrugg, Switzerland) using an excitation light. The whole gonad and mesonephros were dissected from all embryos, and the
gonad and mesonephros were then separated
using a 27-gauge needle in ice-cold PBS under
a stereomicroscope (Martineau et al., ’97). Gonads separated from non-transgenic littermates
and mesonephroi from EGFP transgenic pups
were placed onto ice-cold F12/DME medium
(Gibco BRL, Grand Island, NY) containing 1.2
g/liter of NaHCO3, 10% fetal bovine serum, 105
units/liter of penicillin, 100 mg/liter of streptomycin and 50 µl of gentamicin. Gonad and mesonephros were placed in a groove on a 1.5% agar plate
(Bacto Agar: Difco, Detroit, MI) in 35 mm culture
dishes containing the culture medium without serum. Excess medium was removed from the groove
to promote adhesion of the gonad to the mesonephros. One hour after the start of the coculture,
gonad/mesonephros complexes were added to 2 ml
of the culture medium and incubated for 45 hr
under a humidified atmosphere of 5% CO2 in air
at 37°C. The culture media were exchanged at 2–
4 hr, 20 hr, and 40 hr of coculture.
Cell migrations from the mesonephros expressing EGFP into the gonads of non-transgenic embryos were directly observed every 5 hr without
fixation under the fluorescent stereomicroscope.
Photographs were taken under UV light with a
Leica GFP2 filter.
Hematoxylin-eosin (HE) staining
Gonad/mesonephros complexes cocultured for
45 hr were fixed with 4% paraformaldehyde in
PBS for 2 hr. After dehydration through an
ethanol series, the samples were embedded in
paraffin, sectioned at 5.0 µm, and stained with
Mayer’s hematoxylin and eosin solution. The
preparation was examined under a light-inverted microscope.
322
K. NISHINO ET AL.
RESULTS
Transgenic mice ubiquitously
expressing EGFP
In the transgenic mice ubiquitously expressing
EGFP, green signals of EGFP were observed in
the heart, liver, kidney, testis, and muscle upon
irradiation by the excitation light (Fig. 1). Deep
green fluorescence was also observed under a fluorescent stereomicroscope in the whole-mount
specimens of gonads and mesonephroi collected
from the 12.5-dpc transgenic fetuses (Fig. 2).
In the present experiment, the sex of the 12.5dpc embryos was identified by the presence or absence of testis cords, and male or female gonads
that were separated from the nontransgenic littermates were attached to the mesonephroi of the
nonsexed EGFP transgenic pups. Only the mesonephros of transgenic embryos expressed green
fluorescence under a fluorescent stereomicroscope,
but the gonads of non-transgenic embryos did not
express fluorescence (Fig. 3).
Observation of male-specific cell migration
into gonads
To determine whether the mesonephros cells
migrated into the gonads, organ complexes cultured for 35 hr were directly observed under a
fluorescent stereomicroscope using an excitation
light. We found that mesonephric cells migrated
into the male gonad (Fig. 4A), whereas no cell migration of such cells into the female gonad was
observed (Fig. 4B). This finding of male-specific
migration corresponds with that in previous studies, and confirms that the present system using
EGFP transgenic mice enabled observation of the
cell migration in the gonad/mesonephros complex.
To survey temporal morphological changes of the
male-specific migration in detail, the kinetics of
the cell migration were examined every 5 hr starting at 15 hr of coculture (Fig. 5). Figure 5A–F
shows a visual field representing typical migration kinetics. No sign of the cell migration from
the mesonephros into the gonad was observed at
15 hr of coculture (Fig. 5A). Cell migration was
first detected as a penetration of green signals
into the gonads at around 20 hr of coculture (Fig.
5B), and at 25 hr of coculture (Fig. 5C), the green
signals elongated and became clear. Although the
elongated green signal had not spread at 30 hr, a
deep green circle appeared in the gonad along with
the green signal (Fig. 5D), and the number of deep
green circles on the line had increased at 35 hr
(Fig. 5E). At 40–45 hr of coculture, the green
circles began to accumulate at the top of the green
signal (Fig. 5F).
Histological analysis
The section of 45-hr cocultured male gonad/mesonephros complex revealed cord-like structures
comparable to the 12.5-dpc normal male gonads.
The histological examination of cocultured gonads
did not indicate degenerative changes during the
in vitro culture (Fig. 6).
DISCUSSION
The purpose of the present study was to establish an experimental coculture system that did
not require fixing and staining, by which the cell
migration from mesonephroi to gonads could be
observed. For this purpose, we generated transgenic mice in which ubiquitous expression of
EGFP, visible under excitation fluorescence, was
used as a signal. The transgenic mice produced
in the present study expressed EGFP strongly in
the adult heart, testis, and muscle, though diffusely in the liver and kidney. This difference in
the expression patterns of EGFP might be characteristic of the chicken β-actin promoter activity. Our results are in agreement with those
reported previously (Okabe et al., ’97); the strong
and uniform expression of EGFP in early-stage
embryos they reported were also shown in the
mesonephroi and gonads of 11.5–12.5-dpc embryos
in the present study.
Cell migrations from the mesonephroi into the
gonads of 11.5-dpc mouse embryos have been reported by Buehr et al. (’93), Merchant-Larios et
al. (’93), and Martineau et al. (’97). Martineau et
al (’97) showed that this cell migration was malespecific. The present study, in which a clear malespecific cell migration was also observed, confirms
their results. These previous reports did not clarify
the point at which cell migration occurred in their
respective coculture systems. Our results show
that migration is initiated at around 20 hr of
coculture. The green signal of EGFP was elongated between 20 and 25 hr of culture, and the
signal did not spread until the end of culture (45
hr after the start of coculture). The present findings suggest that the migration process consists
of two phases, the first of which, evidenced by the
elongation of the very thin green line, indicates
migration from the mesonephros into gonad. The
second phase, during which deep green circles appear on the green line, show an increase in number of migratory cells throughout the culture
period and a peak accumulation at the top of the
COCULTURE OF FETAL GONAD AND MESONEPHROS
Fig. 1. Adult tissues of EGFP transgenic (Tg) mice. Left
panels indicate the light field and right panels indicate the
dark field under fluorescence. Green signals were observed
323
in all of the adult tissues. A and B, heart; C and D, liver; E
and F, kidney; G and H, testis; I and J, muscle.
324
K. NISHINO ET AL.
Fig. 2. The 12.5-dpc fetuses collected from pregnant nonTg females mated with EGFP Tg males (A). Strong green
fluorescence is observed in the whole mount of Tg fetuses (B,
right), but not expressed in the non-Tg littermate (B, left).
The fetal gonad and mesonephros (C) also strongly express
green fluorescence (D).
Fig. 3. The grafted male gonad of a 12.5-dpc non-Tg littermate on mesonephros from the Tg embryo (A). Under an
excitation light, only the mesonephros, not the gonad, expresses green fluorescence (B).
COCULTURE OF FETAL GONAD AND MESONEPHROS
325
Fig. 4. The male or female gonad/mesonephros at 35 hr
of coculture. The mesonephros cells migrate into the male
gonad (A), whereas no cell migration is observed into the female gonad (B).
initial green signal between 25 hr and 45 hr of
culture. Although the cells corresponding to the
initial green signal and to the green circle were
not identified in the present study, it is thought
that several types might migrate during the
coculture. The relationship between cell migration
and components of the extracellular matrix (ECM)
has been well investigated in several tissues. The
construction and disintegration of ECM by proteolytic enzymes and their inhibitors are important
for the formation, maintenance, and remodeling of
the tissues. Testicular cord formation, Sertoli cell
differentiation and migration, and germ cell development are also regulated by ECM (Hadley et
Fig. 5. The time-dependent morphological changes in
male-specific migration in a gonad/mesonephros complex. A–
F are a higher magnification of the same area. No sign of cell
migration into the gonad was detected at 15 hr of coculture
(A). Cell migration was first detected as a penetration of green
signals into the gonads at around 20 hr of coculture (B), and
the green signals elongated and became clear at 25 hr of
coculture (C). At 30 hr, although the range of the elongated
green signals had not spread, a deep green circle appeared
in the gonad along with each green signal (D), and at 35 hr,
the number of green circles increased in a line on each green
signal (E). The green circles accumulated at the top of each
green signal at 45 hr of coculture (F). The arrows indicate an
elongated green. Arrowheads indicate deep green circles.
326
K. NISHINO ET AL.
Fig. 6. The paraffin section of a 45 hr-cocultured male
gonad/mesonephros complex stained with hematoxylin and
eosin. Cord-like structures found in the section did not show
any degenerative changes during in vitro culture.
al., ’85; Tung and Fritz, ’86). Therefore, one possible explanation is that cells initially migrated,
then stromal cells probably determine the migration site and produce specific ECM proteins.
Several proteinases such as matrix metalloproteinases (MMPs) and plasminogen activators are expected to play a key role in testicular
development (Lacroix et al., ’77; Marzowski et al.,
’85; Vihko et al., ’87; Sang et al., ’90a,b; Ailenberg et al., ’91). Similarly, tissue inhibitors of
metalloproteinases (TIMPs) have also been reported to be an important factor in the testicular
developmental processes (Ailenberg et al., ’91;
Ulisse et al., ’94; Grima et al., ’96). Therefore,
MMPs and TIMPs may be involved in the initial
invasion of mesonephros cells. Other types of stromal cells might migrate along with the initial
ECM guide. Further studies, however, are necessary to elucidate the mechanism of cell migration from mesonephroi to the gonads.
No degenerative changes were observed in the
gonad/mesonephros complexes cocultured up to 45
hr in the present system. In addition, cord-like
structures comparable to the 12.5-dpc normal
male gonads were observed in an organ complex,
showing that EGFP does not have deteriorative
effects on tissue development and indicating that
the present coculture system was subphysiological.
It has been reported that the migrated cells from
the mesonephros are located outside the testicular cord and differentiate to myoid cells surrounding seminiferous tubules or endothelial cells in the
vasculature, but not to Leydig cells, since they do
not express steroidogenic enzymes. The differentiation capabilities of the migrated cells, however,
were not fully examined in our investigation. In
our preliminary experiment, we were able to find
migrated cells expressing EGFP that were conclusively derived from the mesonephros in the primary culture; only gonads were collected from the
gonad/mesonephros complexes cocultured for 45
hr and digested with trypsin for the subsequent
primary cell culture. This culture system may be
very useful for investigating the differentiation
and gene-expression abilities of migrated cells expressing EGFP.
In conclusion, we have successfully established
a coculture system, without fixing and staining,
that uses transgenic mice ubiquitously expressing EGFP in which the cell migration from the
mesonephroi to the gonads can be observed. This
system is subphysiological and can be used for
subsequent primary culture containing migrated
cells. We are currently investigating the mechanisms of cell migration and the characteristics of
the migrating cells using this coculture system.
ACKNOWLEDGMENT
We thank Dr. M. Okabe, Research Institute for
Microbial Diseases, Osaka University, Japan, for
providing the EGFP-expression vector (pCXEGFP) used in this study.
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