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Department of Cell Biology, Georgetown University Medical Center, Washington, D.C. 20007
Address all correspondence and requests for reprints to: Dr. Martine Culty, Department of Cell Biology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington D.C. 20007.
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and 
isoforms of
protein kinase (PK) C (PKC) and the phosphatidylinositol 3-kinase (PI
3-K). Because both the PKC and PI 3-K have been shown to play a role
in platelet-derived growth factor (PDGF)-induced cell proliferation, we
examined the effects of PDGF on gonocytes. For this, we developed a
method to obtain highly purified and viable gonocytes in culture. After
enzymatic digestion, differential adhesion, and two successive gradient
fractionations, the gonocyte suspension obtained was over 90% pure, as
assessed by light microscopy. The viability of cultured gonocytes
exceeded 90% after 48 h in the presence of 2.5% FBS used as a
survival factor. Immunodetection studies showed that isolated gonocytes
expressed PKC, PI 3-K, and the PDGF receptor. Treatment with 10
ng/ml PDGF induced a 4-fold increase of bromodeoxyuridine incorporation
into gonocytes (from 5% proliferative gonocytes under basal conditions
to 20% in the presence of PDGF). Because neonatal Sertoli cells
secrete high levels of the growth promoting steroid, 17ß-estradiol,
we also tested its effect and found that it induced gonocyte
proliferation at a level comparable with that of PDGF and that this
effect was blocked by the estrogen receptor antagonist, ICI 164384. The
combination of PDGF and estradiol, however, was not additive,
suggesting that their effects were mediated by common molecular
target(s). These results demonstrate that PDGF and estradiol activate
gonocyte proliferation in vitro, suggesting that they
may act as the physiological regulators of gonocyte development
in vivo. |
Introduction |
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Early studies have determined that gonocytes go through two phases of proliferative activity, one in the embryo and the other at neonatal days 3 and 4, at which point, gonocytes migrate from the center to the periphery of the seminiferous cords and further differentiate into type A spermatogonia (4, 5, 6). It was demonstrated in organ cultures of newborn rat testis that both the reentry into proliferation and the relocation of gonocytes occur in the absence of extratesticular influences, suggesting that intratesticular factors, probably produced by Sertoli cells, control these events (4). A few molecules already have been reported to affect gonocyte proliferation, such as thyroid hormone (7), thymulin (8), and the Müllerian inhibiting substance (9).
It has been reported recently that the fibroblast growth factor-2 (FGF2) exerts a proliferative effect and that leukemia inhibitory factor and ciliary neurotropic factor exert a survival effect on gonocytes cocultured with Sertoli cells (10, 11). However, the interpretation of these results is limited because the two cell populations (gonocytes and Sertoli cells) were mixed, making it difficult to identify the target cell on which these agents act.
To overcome the problems inherent to the use of Sertoli-gonocyte
cocultures, we decided to examine the regulation of purified gonocyte
proliferation. We first examined what type of signal transduction
molecules were expressed in neonatal gonocytes, as a way to determine
what agents may be involved in their proliferation, and found
that gonocytes expressed the PKC and the phosphatidylinositol
3-kinase (PI 3-K). The PKC belongs to a group of atypical PKCs that
are not activated by Ca++ and diacylglycerol (12) and has
been shown to be involved in cell mitosis (13, 14, 15). Similarly, the PI
3-K, which catalyses the phosphorylation in position 3 of the inositol
ring of phosphoinositides (16), is an active component of the mitotic
cascade of various cell types (17). Moreover, these two kinases have
been shown to be activated by platelet-derived growth factor (PDGF)
(15, 18). It has been suggested that Sertoli cells produce PDGF (19).
Neonatal rat Sertoli cells also are known to secrete high amounts of
17ß-estradiol (20), which is a growth promoting agent (21). Thus, we
decided to study the effect of PDGF and estradiol on the proliferation
of purified gonocytes. These experiments revealed that both PDGF
and estradiol stimulate gonocyte proliferation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Purification and culture of gonocytes
The method used here is adapted and modified from that of Van
Dissel-Emiliani et al. (22). After collection, testes from
40–50 pups (3 days old) were decapsulated, cut in small fragments, and
incubated with 0.7 mg/ml type IV collagenase (Sigma Chemical Co., St.
Louis, MO) and 0.2 mg/ml type III testicular hyaluronidase (Sigma) for
30 min in RPMI medium containing an antibiotic/antimycotic solution of
100 U/ml penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml fungizone
(Biofluids, Rockville, MD) at 37 C. After sedimentation, the first
digest, which contained no gonocytes, was discarded, and the fragments
were further incubated with 0.25% trypsin-1 mM
ethylenediaminetetraacetate (GIBCO-BRL, Gaithersburg, MD) and 0.15
mg/ml DNase I (Boerhinger Mannheim, Indianapolis, IN) for 30 min. The
digestion was then stopped by addition of FBS (GIBCO-BRL or Irvine
Scientific, Santa Ana, CA). The cell suspension was filtered through
47-µm nylon mesh (Small Parts Inc., Miami Lakes, FL) to discard large
cell aggregates and fragments. The cells were quantified using a
hemacytometer, where the gonocytes were easily identified by their
large size. Because no specific marker is available to characterize
gonocytes (10), their size and morphology were used as criteria to
quantify them during the whole purification procedure. The cell pellet,
which contained 1–2% gonocytes (1.5 ± 0.3 x
106 gonocytes vs. 86 ± 6 x
106 Sertoli and other small cells in average), was
resuspended in 240 ml RPMI containing 5% FBS, plated in 150 mm culture
plates (15–20 x 106 total cells per plate), and
cultured overnight in 5% CO2 atmosphere at 37 C. After
20 h culture, the floating cells (containing an average of
1.3 ± 0.1 x 106 gonocytes vs.
33 ± 3 x 106 other cells, corresponding to
4–10% gonocytes) were collected, centrifuged, and resuspended in 3 ml
serum-free RPMI. This suspension was then applied on a 2–4% BSA
(Sigma) STAPUT gradient (23). After 2 h sedimentation by gravity,
30 fractions of 2 ml were collected, and the cells were counted in a
hemacytometer. The fractions containing the most gonocytes (as judged
by their morphology and large size) were pooled and centrifuged. The
cell pellet (0.6 ± 0.1 x 106 gonocytes
vs. 1.2 ± 0.2 x 106 small cells in
average) was resuspended in 1 ml RPMI containing 0.1% BSA and applied
on a 10-ml density gradient made of 5–27% Nycodenz (GIBCO-BRL). After
centrifugation, two major bands of cells were visible in the gradient,
which was collected in 5 fractions. This additional step allowed the
removal of most of the small cells still present (which were mostly
dead cells, as determined by trypan blue exclusion). The final cell
suspension (in average 0.48 ± 0.02 x 106
gonocytes with a purity of 92 ± 5%), was diluted at 20,000
cells/ml and was kept in culture in RPMI supplemented with 2.5% FBS
and the antibiotic/antimycotic solution for 1–2 days at 37 C.
Electron microscopy
Gonocytes were fixed after centrifugation in a solution of 1%
paraformaldehyde, 2% glutharaldehyde in PBS for 15 min and washed
several times with PBS. The cells were then embedded in Epon-araldite
and further processed, as we described previously (24).
Immunocytochemistry
Day 19 fetuses (1 day before birth) and newborn male rats (day
zero to 7) were killed and their testis dissected and fixed in 3.5%
formaldehyde. After several washes in PBS, the tissues were embedded in
Polyester wax (Gallard-Schlesinger Chemical Manufacturing Corp., Carle
Place, NY) as described in Ref. 25, with minor modifications. Briefly,
the testes were dehydrated through a graded series of ethanol,
incubated for 1 h at 38 C in 50% Polyester wax (vol/vol in
ethanol), then for 1 h in 90% Polyester wax, and finally embedded
in 90% Polyester wax and kept at 4 C. Sections of 5 µm were cut with
a microtome at 4 C, collected on egg albumin-coated slides, allowed to
dry, and kept at 4 C until staining. For this, the sections were first
dewaxed through a graded series of ethanol solutions, washed with PBS
and water, and then permeabilized by a microwave heating process as
described in Ref. 26. The slides were immersed in 0.01 M
citrate buffer, pH 6.0, and heated in a microwave for 30 min at high
power (800W), allowed to cool slowly for 2 h at room temperature,
and after a few PBS washes, the immunostainings were carried out. The
sections were incubated with the primary antibodies diluted (1:50 to
1:250) in PBS containing 10% calf serum (PBS/CS) overnight at 4 C.
These antibodies were: mAb anti-PKA (Transduction Laboratories,
Lexington, KT); mAb anti-PKC isoforms 
, ß, 
, 
, 
, 
, and
(Transduction Labs); polyclonal anti PKC (GIBCO-BRL); mAb
antiphosphotyrosine residue (PY20; Transduction Labs), polyclonal
antiphospholipase C (Upstate Biotech Inc., Lake Placid, NY),
polyclonal antiphospholipase A2 (Upstate Biotech Inc., Lake
Placid, NY); mAb anti-PI 3-K (Transduction Labs); and polyclonal
anti-PDGF type A/B receptor (UBI). After a few washes in PBS, the
sections were incubated for 1 h with a secondary antibody
(antirabbit or antimouse, depending on the host of the first Ab)
coupled to peroxidase (Transduction Labs) diluted at 1:1000 in PBS/CS,
followed by 1 h incubation with the substrate mixture for
peroxidase (0.03% H2O2 + 0.2 mg/ml
3-amino-9-ethylcarbazole in 0.05 M Na acetate, pH 5.0;
Sigma). As a negative control, the first antibody was omitted in the
treatment of some slides, whereas a neutralizing peptide was added with
the primary antibody in the case of the staining for the PKC.
Moreover, some primary antibodies that did not detect any signal in
gonocytes, but were positively staining other cell types in the
sections, provided a good indication of the background staining for
gonocytes. At the end of the reaction, the sections were counterstained
with hematoxylin (Sigma), the slides further coated with Crystal-mount
(Biomeda Corp., Foster City, CA) and dried at 80 C for 10 min, and
coverslips were added using Permount (Fisher Scientific, Columbia, MD).
The sections were examined with an Olympus microscope and photographs
were taken.
For the immunostaining of cultured cells, the gonocytes were first collected by centrifugation, resuspended in 200 µl fresh medium, then transferred onto microscopic slides by centrifugation on a Cytospin 2 centrifuge (Shandon, Pittsburgh, PA) for 5 min at 1000 rpm and allowed to dry overnight at 4 C. The cells were then fixed in 70% ethanol for 15 min and processed directly (no microwave treatment) for staining as described above. Some slides were treated directly with the secondary antibodies to determine the background staining.
In vitro study of gonocyte proliferation
The proliferative response of gonocytes was followed by
determination of 5-Bromo-2'-deoxyuridine (BrdU) incorporation according
to the manufacturer’s recommendations (cell proliferation kit from
Zymed Laboratories Inc., South San Francisco, CA). For this, the cells
were incubated with a mixture of 30 µg/ml BrdU and 3 µg/ml
5-fluoro-2'deoxyuridine, together with the agents to be tested, for
20 h at 37 C. The agents used were: 0.1–100 ng/ml PDGF (type BB,
human recombinant), 0.1–10 µM 17ß-estradiol, 2.5 ng/ml
epidermal growth factor (EGF), 1 ng/ml nerve growth factor (NGF), 25
ng/ml aFGF, and 10 ng/ml bFGF (all purchased from Sigma); 100
µM ICI 164384 (gift from Dr. Alan Wakeling, Zeneca Corp.,
Macclesfield, UK), in the presence of 2.5% FBS. The cells were
cultured in the presence of FBS because preliminary experiments had
shown that it improved the viability and responsiveness of the cells
without changing significantly the basal level of proliferation. At the
end of the incubation, the cells were collected and applied on slides
by Cytospin as described above. After fixation in 70% ethanol for 15
min, the cells were stained using the biotinylated anti-BrdU antibody,
streptavidin-peroxidase, and 3,3'-diaminobenzidine tetrahydrochloride
substrate mixture provided in the kit, following the manufacturer’s
protocol. Pictures were taken of each slide (usually 4) to visualize
all the cells present in each sample. The number of positively stained
gonocytes was determined and expressed as a percent of the total number
of gonocytes (an average of 800 cells were scored for each sample). The
results represent means ± SEM of two to six
individual experiments, in which each condition was tested in
duplicate. Statistical analyses were performed by ANOVA followed by the
Student-Newman-Keuls and the Bonferroni multiple comparisons tests
using the Instat 2 (v2.04) package from Graphpad Inc. (San Diego,
CA).
Some proliferation experiments also were carried out on gonocytes isolated from 1- or 2-day-old rats using the same procedures described above. After purification, part of these cells was put back in culture on Sertoli cell monolayers from the same experiment grown in Supercell-8 well-tissue culture chambers (Fisher Scientific; Pittsburgh, PA), while others were kept as isolated cell suspensions. The gonocytes in coculture were incubated with or without 2.5% FBS, while 10 ng/ml PDGF or 1 µM estradiol were added together with FBS on the isolated gonocytes. In both cases, BrdU was added for 20 h with the other agents. At the end of the incubations, the gonocytes in coculture were fixed in the chambers with ethanol, which were then transformed into microscopic slides by removal of their walls. The isolated gonocytes were collected on slides by cytospin as described before. Gonocyte proliferation was then examined as described above.
Immunoblot analysis
Isolated gonocytes were collected by centrifugation, washed
twice with PBS, and solubilized in Laemmli buffer (27). After
electrophoretic separation by SDS-PAGE on a 10% acrylamide gel
(reagents and equipment from Bio-Rad Laboratories, Melville, NY), the
proteins were transferred onto nitrocellulose membranes (Immobilon-NC,
Millipore, Bedford, MA) at 0.9 amperes for 30 min using a Trans-Blot
Cell (Idea, Corvalis, OR). Nonspecific adsorption of the antibodies was
prevented by incubating the sheets in 5% milk. The blots were then
treated for immunodetection of the PKC, PKC, PI 3-K, and PDGF
receptor using the same antibodies and peroxidase reaction used for the
immunocytochemistry, except that these reagents were diluted (1:250 to
1:1000 for the primary antibodies; 1:2000 for the secondary antibodies)
in PBS/CS containing 0.02% Tween 20, and that a chemiluminescent
peroxidase substrate (Western Blot Chemiluminescence kit from
Dupont-NEN, Boston, MA) was used. Some blots were treated with
nonspecific IgG, instead of the first antibody, to determine the
background staining. A neutralizing peptide for the PKC antibody
also was used as another negative control, whereas the identity of the
bands recognized by the antibodies was compared with that obtained for
positive control cell extracts.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ß,
, , , , , ), the phospholipases C and
A2, the PI 3-K, and tyrosine kinase activity (identified by
detection of phosphotyrosine residues (P-tyr) on proteins). As shown in
Fig. 1
, A–E, only 3 of the molecules studied were
expressed strongly by the gonocytes: the PKC, PKC, and the PI
3-K. The phospholipase A2 was detectable in some gonocytes.
The localization of P-tyr-proteins in gonocytes was difficult to
ascertain, because it gave a strong signal at the lateral and apical
borders of the Sertoli cells that are tightly apposed to the gonocyte
plasma membranes. All of the other molecules were not expressed or
their expression was below detection levels in gonocytes (see Table 1), although some were clearly present in other cell
types, validating the antisera reactivity and the immunostaining
procedure used. For example, PKA was expressed by the Sertoli cells,
whereas the isoform of PKC was present in the peritubular and
Leydig cells. The immunolocalization of most proteins was similar
across the time-window studied. However, the cellular expression of the
PKC was exclusively nuclear in the gonocytes at the day before
birth, whereas it was mostly cytosolic at days 3 and 4 (Fig. 1, A and
B). These results, together with the information available on the
functions of PKC and PI 3-K, suggest that these enzymes could
participate in the proliferative response of the gonocytes.
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by phase contrast and electron
microscopy, and corresponds to what has been previously reported for
gonocytes in vivo (6). After 24 h in culture, 91% of
the gonocytes were alive in RPMI medium, as showed by trypan blue dye
exclusion. The addition of 2.5% FBS raised the survival to 97% and,
after 48 h, 87% gonocytes were still alive. Numerous experiments
were performed to substitute FBS with a chemically defined medium
supplement such as a mixture of
Insulin-Transferin-Selenium-BSA-linoletic acid (Collab. Biomed. Prod.,
MA) to culture the cells, but without success because the viability of
the gonocytes in the presence of these supplements was not as good as
that in the presence of low amounts of FBS (data not shown). Therefore,
the culture medium was supplemented subsequently with 2.5% FBS as a
survival factor. Also, in some preliminary experiments, some culture
plates were precoated with extracellular matrix components (laminin,
fibronectin, collagen, Matrigel) or poly-L-lysine to
promote gonocyte adherence, but most of the cells remained floating or
poorly attached to the substratum (data not shown). Thus, gonocytes
were cultured as cell suspensions that had to be collected at the end
of the incubations and applied onto microscopic slides by
centrifugation on a Cytospin device as described above.
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and PKC, PI 3-K, and
P-tyr proteins in purified gonocytesPKC and PI 3-K in gonocytes from the
testis sections pinpointed PDGF as a potential proliferative agent, we
then examined the expression of the PDGF receptor and that of PKC,
PKC, PI 3-K, and P-tyr proteins in purified gonocytes kept in
culture for 24 h. As shown in Fig. 3, the PDGF
receptor was present in some, but not all, gonocytes, whereas both
PKC and PI 3-K were localized in the cytosol of most gonocytes.
Subsequent immunolocalization of the PDGF receptor on testis sections
from day-zero-to-7 pups further revealed that it was expressed in
different cell types as a function of the development stage; whereas
PDGF receptor was localized in the gonocytes and Sertoli cells during
the first 5 days after birth, it was mostly expressed in the Leydig and
myoid cells at day 7 (Fig. 1
, G and H). The presence of P-tyr proteins
was evident, suggesting that at least part of the staining observed
earlier in the testis sections was localized at the surface of the
gonocytes. The PKC was expressed in a few cells only and at low
levels (not shown).
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). Proteins of molecular size
identical to that reported in the literature for these proteins were
observed. In the cases of the PKC from brain tissue and the PI 3-K,
the antibodies revealed one additional band of lower molecular weight
than that of the enzyme, which may represent a breakdown product
(especially for the PKC, because addition of the neutralizing
peptide displaced that band too) or crossreactivity of the antibody
with another molecule. These data further confirmed the results
obtained by immunocytochemistry.
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, A and B). We found that
2.5% FBS alone slightly improved the basal proliferation found in
serum-free medium, probably by providing the necessary elements for
survival and responsiveness of the cells in vitro. As shown
in Figs. 5C and 6, the addition of PDGF stimulated
gonocyte proliferation in a dose-dependent and biphasic manner,
inducing a 4-fold increase at a maximal dose of 10 ng/ml. The addition
of estradiol gave a similar biphasic response, with a maximal increase
for a dose of 1 µM (Figs. 5C and 7). This
effect was abolished by the addition of the antiestrogen, ICI 164384
(Fig. 5C). As shown in Fig. 5C, the simultaneous addition of PDGF and
estradiol had only a slight, insignificant, additive effect. When other
growth factors were used at concentrations reported to be optimal in
the literature (10, 28, 29), we found that EGF and NGF had no
significant effect on gonocyte proliferation. Both a- and bFGF induced
a slight, but not significant, increase in proliferation similar to
that produced by low concentrations of PDGF.
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) or estradiol (not
shown). However, when some of these purified cells were put back in
culture on the Sertoli cell monolayers and further incubated with BrdU
in the presence or absence of 2.5% FBS for 20 h, we found
different results; the gonocytes were easily identified by their large
size and round shape as compared with the small elongated Sertoli cells
(Fig. 8B). Even in the absence of FBS, a few gonocytes were clearly
proliferative, as shown by their incorporation of BrdU (data not
shown), and in the presence of FBS, a large proportion of gonocytes
were proliferating (Fig. 8C).
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Discussion |
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Considering that nothing was known about the signals acting on
gonocytes and the signal transduction molecules present in these cells,
and taking advantage of the availability of antibodies allowing the
detection of most of the molecules involved in signal transduction
pathways, it seemed that an initial screening of the gonocytes with
such tools could give us indications of the type of regulatory
mechanisms that are present and that may control gonocyte
proliferation. Thus, we examined the expression of known signal
transduction molecules in gonocytes from 3-day-old rat testis sections.
These experiments revealed the presence in gonocytes of the and
PKC and the PI 3-K. Both the and PKC are newly described
atypical isoforms of PKC that share high sequence homology. Although
nothing is yet known about the function of the PKC (30), a novel
protein, the [Lambda]-interacting protein, recently has been shown to
activate it (but not the PKC) (31), and it was proposed that this
activation may be involved in cell proliferation. The PKC has been
more widely studied, and its presence in gonocytes seemed very
interesting because this kinase has been shown to be involved in cell
mitosis; indeed, PKC was found to be associated with the mitotic
apparatus of cells (13). Another recent study described how the direct
interaction of PKC with the Ras GTPase resulted in the activation of
the serine/threonine kinase MEK (MAP kinase kinase) and its substrate
the MAPK (mitogen-activated PK) (14, 15), two kinases that belong to
the transduction cascade of growth factors and trigger the
transcription of genes associated with mitosis (32). There are only a
few agents known to activate PKC: the ceramides, generated in cells
stimulated by TNF and interleukin-1 during inflammation (14); the
phosphatidylinositol 3,4,5-triphosphate, product of the PI 3-K (33),
which we also found in gonocytes; and the Ras GTPase (15). Both the PI
3-K and Ras are involved in cell proliferation and are activated by
PDGF (18). The findings that the product of the PI 3-K and Ras were
potent activators of the PKC suggest a functional link between these
enzymes (33, 15). Such a relationship is suggested also by the data of
Moscat et al., who found that a specific PI 3-K inhibitor
abolished the activation of the PKC by PDGF (15).
Another intriguing finding of our study is that the levels of
expression of PKC were different in the in vivo and
in vitro models used, because it seemed to be downregulated
in vitro, whereas the expression of PKC under these
conditions was not changed. It may be that the expression of the PKC
isoform requires a constant signal from the Sertoli cells, whereas the
maintenance of the isoform, once activated, is independent of the
Sertoli cells.
The presence of proteins with phosphorylated tyrosine residues in gonocytes was consistent also with the involvement of a growth factor, such as PDGF, because its receptor is a tyrosine kinase, which, upon activation, phosphorylates itself and other proteins (34).
Altogether, these initial results pinpointed PDGF as a potential candidate for the activation of gonocyte proliferation. Another potential regulator studied here was the 17ß-estradiol, which had been shown to be secreted transiently at high levels by immature rat Sertoli cells during the same time-frame as that of gonocyte proliferation, although nothing is known about its function in this system (20). The interaction of the estrogen and its receptor have been shown to have a growth promoting function in certain tissues such as breast and uterus (21, 35).
Cell suspensions containing more than 90% pure and viable gonocytes were obtained by using a modified version of the existing methods. The identity of the gonocytes during the purification process was assessed by their size and morphology, as it has been done previously by other authors (4, 5, 6, 7, 8, 9, 10, 11) studying these cells. When the cells are in suspension, the difference in size between the gonocytes and the other cell types is obvious, the gonocytes appearing as very large round cells (twice as big as any other cell type) with big nuclei. These criteria are still the only ones available because no specific marker for gonocytes from new born rats have been yet identified. In addition, we have confirmed the identity of the purified gonocytes by transmission electron microscopy analysis. The identity of the small percentage (10%) of small-size, trypan blue-positive cells remaining at the end of the purification procedure is not clear. However, the observations that they are trypan blue positive and nonadherent excludes the possibility of these cells being live Sertoli cells. The gonocytes could be maintained in culture for at least 2 days without major effect on their viability in the presence of low amounts of FBS. High amounts of FBS have been reported to have a negative effect on early embryonic gonocytes, but not in gonocytes from 16-day-old embryos (36), whereas FBS has been shown to exert a positive effect on gonocyte proliferation in organ culture, probably through the Müllerian inhibiting substance present in it (9, 37, 38). Here, we found that low amounts of FBS actually improved the survival and responsiveness of newborn gonocytes.
Our results demonstrated that PDGF is able to activate the proliferation of isolated gonocytes in a dose-dependent manner. In contrast, neither EGF nor NGF had such an effect. The effects of EGF and NGF (which induced a 30–60% increase over the basal level) were not found to be significant using 2 different statistical tests, although both EGF and NGF were used at concentrations reported to be optimal on testicular cells (28) and neurons (29), respectively. Both a- and bFGF seemed to have a small effect (inducing, respectively, 70 and 100% increases over the basal), which required higher concentrations than those used for PDGF. While this study was in progress, a report was published showing that FGF2 (bFGF) had a survival effect on Sertoli cells and gonocytes in coculture and that it seemed to stimulate gonocyte proliferation by 2-fold (10). However, it could not be excluded that the proliferative effect observed was the result of a primary effect of FGF2 on the Sertoli cells, inducing them to secrete factors that would, in turn, activate gonocyte proliferation. In this regard, immature rat Sertoli cells have been shown to express the FGF receptor type 1, which binds bFGF (39), and bFGF has been shown by other investigators (40) to be mitogenic for these cells. Our present finding that bFGF is able to induce a small (2-fold) increase in purified gonocyte proliferation, in the same range as that found on gonocytes-Sertoli cell cocultures (10), confirms that at least part of its effect observed on cocultures can be attributed to a direct effect on the gonocytes.
The present study showed that 17ß-estradiol also has a dose-dependent mitogenic effect on gonocytes, comparable with that triggered by PDGF. The inhibition of this effect by the estrogen antagonist, ICI 164384, suggested that the effect observed was mediated by the binding of the estrogen to its receptor, because this antiestrogen has been found to bind in the same region of the estrogen receptor as estradiol and to block the dimerization of the receptor and its subsequent binding to DNA (41). Moreover, the fact that the effects of PDGF and estradiol were not additive or synergistic suggest that they may have common target molecule(s) in their pathways. In this regard, a recent study suggested that 17ß-estradiol was able to modulate the PDGF (type BB)-dependent uterine artery growth by both estrogen receptor and nonreceptor-mediated mechanisms (42), whereas another report described the activation of the estrogen receptor by the MAP kinase pathway (43).
The proliferative effects observed were dependent on the developmental stage of the gonocytes because we found that gonocytes obtained from 1 or 2-day-old pups were viable, but unresponsive, to these agents (data not shown). The fact that the gonocytes obtained from 2-day-old rats were maintained for 1 night in coculture with Sertoli cells before their purification, and therefore were 3 days old when isolated from the Sertoli cells but still did not have the ability to proliferate, suggests that an irreversible change occurs in the gonocytes isolated from 3-day-old rats that allows them to be responsive to proliferative agents in the absence of Sertoli cells. Moreover, we found that the 2-day-old gonocytes could proliferate when kept in coculture with Sertoli cells for 1 more day, suggesting that the Sertoli cells themselves were changing during this time, probably acquiring the ability to secrete factors responsible for the induced changes in gonocyte responsiveness. These results were not surprising because Orth et al. had previously found that gonocyte proliferation occurred in the narrow time-frame of days 3 and 4 (5).
The facts that Sertoli cells have been shown to express the PDGF mRNA (19) and secrete estradiol in the neonatal period (20) suggest that they may constitute the source of these molecules in vivo. Thus, our next objective will be to determine whether the proliferative effects of PDGF and estradiol observed in vitro correspond to existing regulatory systems in vivo.
Our results validate the use of purified gonocytes as a model to study their proliferation and provide the means to study how gonocytes can progress from the stage of quiescent primordial germ cells to the stage of differentiation into spermatogonial stem cells. In addition, this model system also may provide the tool to understand the cellular and molecular mechanisms involved in pathological situations such as infertility and testicular tumorigenesis.
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Acknowledgments |
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Footnotes |
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Received August 7, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 K. L. Porter, G. Shetty, G. A. Shuttlesworth, C. C. Y. Weng, I. Huhtaniemi, P. Pakarinen, and M. L. Meistrich Estrogen Enhances Recovery From Radiation-Induced Spermatogonial Arrest in Rat Testes J Androl, July 1, 2009; 30(4): 440 - 451. [Abstract] [Full Text] [PDF]
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Y. Han, H. L. Feng, J. I. Sandlow, and C. J. Haines Comparing Expression of Progesterone and Estrogen Receptors in Testicular Tissue From Men With Obstructive and Nonobstructive Azoospermia J Androl, March 1, 2009; 30(2): 127 - 133. [Abstract] [Full Text] [PDF]
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 S. Basciani, G. De Luca, S. Dolci, M. Brama, M. Arizzi, S. Mariani, G. Rosano, G. Spera, and L. Gnessi Platelet-Derived Growth Factor Receptor {beta}-Subtype Regulates Proliferation and Migration of Gonocytes Endocrinology, December 1, 2008; 149(12): 6226 - 6235. [Abstract] [Full Text] [PDF]
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A. Bouskine, M. Nebout, B. Mograbi, F. Brucker-Davis, C. Roger, and P. Fenichel Estrogens Promote Human Testicular Germ Cell Cancer through a Membrane-Mediated Activation of Extracellular Regulated Kinase and Protein Kinase A Endocrinology, February 1, 2008; 149(2): 565 - 573. [Abstract] [Full Text] [PDF]
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C. Miura, T. Higashino, and T. Miura A Progestin and an Estrogen Regulate Early Stages of Oogenesis in Fish Biol Reprod, November 1, 2007; 77(5): 822 - 828. [Abstract] [Full Text] [PDF]
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 G. Delbes, C. Duquenne, J. Szenker, J. Taccoen, R. Habert, and C. Levacher Developmental Changes in Testicular Sensitivity to Estrogens throughout Fetal and Neonatal Life Toxicol. Sci., September 1, 2007; 99(1): 234 - 243. [Abstract] [Full Text] [PDF]
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 K. Boukari, M. L. Ciampi, A. Guiochon-Mantel, J. Young, M. Lombes, and G. Meduri Human fetal testis: source of estrogen and target of estrogen action Hum. Reprod., July 1, 2007; 22(7): 1885 - 1892. [Abstract] [Full Text] [PDF]
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Y. Wang and M. Culty Identification and Distribution of a Novel Platelet-Derived Growth Factor Receptor {beta} Variant: Effect of Retinoic Acid and Involvement in Cell Differentiation Endocrinology, May 1, 2007; 148(5): 2233 - 2250. [Abstract] [Full Text] [PDF]
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 G. Delbes, C. Levacher, and R. Habert Estrogen effects on fetal and neonatal testicular development. Reproduction, October 1, 2006; 132(4): 527 - 538. [Abstract] [Full Text] [PDF]
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V. Stabile, M. Russo, and P. Chieffi 17{beta}-Estradiol induces Akt-1 through estrogen receptor-{beta} in the frog (Rana esculenta) male germ cells. Reproduction, September 1, 2006; 132(3): 477 - 484. [Abstract] [Full Text] [PDF]
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M. D. Anway and M. K. Skinner Epigenetic Transgenerational Actions of Endocrine Disruptors Endocrinology, June 1, 2006; 147(6): s43 - s49. [Abstract] [Full Text] [PDF]
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 C Roger, S Lambard, A Bouskine, B Mograbi, D Chevallier, M Nebout, G Pointis, S Carreau, and P Fenichel Estrogen-induced growth inhibition of human seminoma cells expressing estrogen receptor {beta} and aromatase J. Mol. Endocrinol., August 1, 2005; 35(1): 191 - 199. [Abstract] [Full Text] [PDF]
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J. J. Lysiak, H. J. Bang, Q. A. T. Nguyen, and T. T. Turner Activation of the Nuclear Factor Kappa B Pathway Following Ischemia-Reperfusion of the Murine Testis J Androl, January 1, 2005; 26(1): 129 - 135. [Abstract] [Full Text] [PDF]
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Y. Wang, R. Thuillier, and M. Culty Prenatal Estrogen Exposure Differentially Affects Estrogen Receptor-Associated Proteins in Rat Testis Gonocytes Biol Reprod, November 1, 2004; 71(5): 1652 - 1664. [Abstract] [Full Text] [PDF]
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G. Delbes, C. Levacher, C. Pairault, C. Racine, C. Duquenne, A. Krust, and R. Habert Estrogen Receptor {beta}-Mediated Inhibition of Male Germ Cell Line Development in Mice by Endogenous Estrogens during Perinatal Life Endocrinology, July 1, 2004; 145(7): 3395 - 3403. [Abstract] [Full Text] [PDF]
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B. T. Akingbemi, C. M. Sottas, A. I. Koulova, G. R. Klinefelter, and M. P. Hardy Inhibition of Testicular Steroidogenesis by the Xenoestrogen Bisphenol A Is Associated with Reduced Pituitary Luteinizing Hormone Secretion and Decreased Steroidogenic Enzyme Gene Expression in Rat Leydig Cells Endocrinology, February 1, 2004; 145(2): 592 - 603. [Abstract] [Full Text] [PDF]
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S. Basciani, S. Mariani, M. Arizzi, M. Brama, A. Ricci, C. Betsholtz, C. Bondjers, G. Ricci, A. Catizone, M. Galdieri, et al. Expression of Platelet-Derived Growth Factor (PDGF) in the Epididymis and Analysis of the Epididymal Development in PDGF-A, PDGF-B, and PDGF Receptor {beta} Deficient Mice Biol Reprod, January 1, 2004; 70(1): 168 - 177. [Abstract] [Full Text] [PDF]
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T. Miura, T. Ohta, C. I. Miura, and K. Yamauchi Complementary Deoxyribonucleic Acid Cloning of Spermatogonial Stem Cell Renewal Factor Endocrinology, December 1, 2003; 144(12): 5504 - 5510. [Abstract] [Full Text] [PDF]
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S. Migrenne, C. Racine, F. Guillou, and R. Habert Pituitary Hormones Inhibit the Function and Differentiation of Fetal Sertoli Cells Endocrinology, June 1, 2003; 144(6): 2617 - 2622. [Abstract] [Full Text] [PDF]
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J. Lassurguere, G. Livera, R. Habert, and B. Jegou Time- and Dose-Related Effects of Estradiol and Diethylstilbestrol on the Morphology and Function of the Fetal Rat Testis in Culture Toxicol. Sci., May 1, 2003; 73(1): 160 - 169. [Abstract] [Full Text] [PDF]
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S. Hasthorpe Clonogenic Culture of Normal Spermatogonia: In Vitro Regulation of Postnatal Germ Cell Proliferation Biol Reprod, April 1, 2003; 68(4): 1354 - 1360. [Abstract] [Full Text] [PDF]
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R. Thuillier, Y. Wang, and M. Culty Prenatal Exposure to Estrogenic Compounds Alters the Expression Pattern of Platelet-Derived Growth Factor Receptors {alpha} and {beta} in Neonatal Rat Testis: Identification of Gonocytes as Targets of Estrogen Exposure Biol Reprod, March 1, 2003; 68(3): 867 - 880. [Abstract] [Full Text] [PDF]
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 T. L. Gaskell, L. L. L. Robinson, N. P. Groome, R. A. Anderson, and P. T. K. Saunders Differential Expression of Two Estrogen Receptor {beta} Isoforms in the Human Fetal Testis during the Second Trimester of Pregnancy J. Clin. Endocrinol. Metab., January 1, 2003; 88(1): 424 - 432. [Abstract] [Full Text] [PDF]
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 K. E. Orwig, B.-Y. Ryu, M. R. Avarbock, and R. L. Brinster Male germ-line stem cell potential is predicted by morphology of cells in neonatal rat testes PNAS, September 3, 2002; 99(18): 11706 - 11711. [Abstract] [Full Text] [PDF]
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S. Basciani, S. Mariani, M. Arizzi, S. Ulisse, N. Rucci, E. A. Jannini, C. D. Rocca, A. Manicone, C. Carani, G. Spera, et al. Expression of Platelet-Derived Growth Factor-A (PDGF-A), PDGF-B, and PDGF Receptor-{alpha} and -{beta} during Human Testicular Development and Disease J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2310 - 2319. [Abstract] [Full Text] [PDF]
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A. M. M. van Pelt, H. L. Roepers-Gajadien, I. S. Gademan, L. B. Creemers, D. G. de Rooij, and F. M. F. van Dissel-Emiliani Establishment of Cell Lines with Rat Spermatogonial Stem Cell Characteristics Endocrinology, May 1, 2002; 143(5): 1845 - 1850. [Abstract] [Full Text] [PDF]
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M. A. Amer, T. Miura, C. Miura, and K. Yamauchi Involvement of Sex Steroid Hormones in the Early Stages of Spermatogenesis in Japanese Huchen (Hucho perryi ) Biol Reprod, October 1, 2001; 65(4): 1057 - 1066. [Abstract] [Full Text] [PDF]
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 L. O'Donnell, K. M. Robertson, M. E. Jones, and E. R. Simpson Estrogen and Spermatogenesis Endocr. Rev., June 1, 2001; 22(3): 289 - 318. [Abstract] [Full Text] [PDF]
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 S. Makinen, S. Makela, Z. Weihua, M. Warner, B. Rosenlund, S. Salmi, O. Hovatta, and J.-A. Gustafsson Localization of oestrogen receptors alpha and beta in human testis Mol. Hum. Reprod., June 1, 2001; 7(6): 497 - 503. [Abstract] [Full Text] [PDF]
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F. J. P. Ebling, A. N. Brooks, A. S. Cronin, H. Ford, and J. B. Kerr Estrogenic Induction of Spermatogenesis in the Hypogonadal Mouse Endocrinology, August 1, 2000; 141(8): 2861 - 2869. [Abstract] [Full Text] [PDF]
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B. Boulogne, C. Levacher, P. Durand, and R. Habert Retinoic Acid Receptors and Retinoid X Receptors in the Rat Testis During Fetal and Postnatal Development: Immunolocalization and Implication in the Control of the Number of Gonocytes Biol Reprod, December 1, 1999; 61(6): 1548 - 1557. [Abstract] [Full Text]
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P. Parma, E. Pailhoux, and C. Cotinot Reverse Transcription-Polymerase Chain Reaction Analysis of Genes Involved in Gonadal Differentiation in Pigs Biol Reprod, September 1, 1999; 61(3): 741 - 748. [Abstract] [Full Text]
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G. Cobellis, R. Pierantoni, S. Minucci, R. Pernas-Alonso, R. Meccariello, and S. Fasano c-fos Activity in Rana esculenta Testis: Seasonal and Estradiol-Induced Changes Endocrinology, July 1, 1999; 140(7): 3238 - 3244. [Abstract] [Full Text]
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A. M. M. van Pelt, D. G. de Rooij, B. van der Burg, P. T. van der Saag, J.-A. Gustafsson, and G. G. J. M. Kuiper Ontogeny of Estrogen Receptor-{beta} Expression in Rat Testis Endocrinology, January 1, 1999; 140(1): 478 - 483. [Abstract] [Full Text]
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R. Olaso, C. Pairault, B. Boulogne, P. Durand, and R. Habert Transforming Growth Factor {beta}1 and {beta}2 Reduce the Number of Gonocytes by Increasing Apoptosis Endocrinology, February 1, 1998; 139(2): 733 - 740. [Abstract] [Full Text] [PDF]
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 L.-X. Feng, N. Ravindranath, and M. Dym Stem Cell Factor/c-kit Up-regulates Cyclin D3 and Promotes Cell Cycle Progression via the Phosphoinositide 3-Kinase/p70 S6 Kinase Pathway in Spermatogonia J. Biol. Chem., August 11, 2000; 275(33): 25572 - 25576. [Abstract] [Full Text] [PDF]
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