Effect of Hypoxia on the Release of Testosterone and Vascular Endothelial
Growth Factor in Mouse TM3 Leydig cells
1
Wei-Ju Huang, 1Paulus S. Wang
1
Department of Physiology, Schools of Life Science and Medicine, National
Yang-Ming University, Taipei 11221, Taiwan, Republic of China
Short Title: Hypoxia & VEGF, T secretion
Correspondence:
Keywords: hypoxia, mouse TM3 Leydig cells, VEGF, testosterone, HIF-1
Abbreviations:
ERK 1/2, extracellular signal-regulated kinases 1 and 2; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; hCG, human chorionic gonadotropin;
HIF-1, hypoxia-inducible factor-1; LH, luteinizing hormone; MAPK, mitogen
activated protein kinase; StAR protein, steroidogenic acute regulatory protein; VEGF,
vascular endothelial growth factor
Abstracts
Previous studies indicated that intermittent hypoxia resulted in an enhancement
of plasma testosterone, increased response of Leydig cells to human chorionic
gonadotropin (hCG), and angiogenesis in rat testes. Hypoxia has been shown to
stimulate the expression of vascular endothelial growth factor (VEGF), which is a
major mediator for angiogenesis and vasculogenesis. During hypoxia, VEGF
promotes the angiogenesis in the testis. However, the effect of VEGF on the
steroidogenesis of testosterone in Leydig cells is not clear. A mouse TM3 Leydig cell
line has been used as a research model. Under hypoxic condition, the Leydig cells
were incubated in an incubator chamber (95 % N2, 5 % CO2) for 1~24 hours. The
cultured media were collected and assayed for testosterone by radioimmunoassay
(RIA) and for VEGF by enzyme immunoassay (EIA). The cytosolic and nuclear
proteins were extracted and intracellular protein expression was determined by
Western blot. MTT test was used for detecting the proliferation of Leydig cells. The
present data showed that the proliferation of Leydig cells was enhanced significantly
under hypoxia condition. During hypoxia, administration of hCG or VEGF could
stimulate proliferation of Leydig cells, but the stimulatory effect was abolished by the
administration of anti-VEGF antibody. The basal release of VEGF was increased, and
the response of VEGF production to hCG was also enhanced in hypoxic condition.
Furthermore, induced expression of hypoxia inducible factor-1 α (HIF-1α) in Leydig
cells resulted in an increase of VEGF release. Hypoxia failed to cause an increase of
testosterone release in Leydig cells. Expression of phospho-extracellular
signal-regulated kinase 1 and 2 (P-ERK1/2) was enhanced in response to hypoxia or
hCG treatment. PD98059 (an inhibitor of MEK) inhibited the hCG or
hypoxia-induced VEGF release and diminished the hCG-stimulated testosterone
release. Meanwhile, the interaction between VEGF and testosterone were also
investigated. Testosterone did not affect VEGF release in Leydig cells, but higher
dose of VEGF could stimulate testosterone release in a dose dependent manner.
Administration of anti-VEGF antibody abolished the stimulatory effect of VEGF on
testosterone release. Expression of P-ERK1/2 was enhanced after treatment with
VEGF. PD98059 inhibited VEGF-induced P-ERK1/2 expression. These data
demonstrated that the enhancement of testosterone release during hypoxia was
resulted from proliferation of Leydig cells by an increase of VEGF generation.
Apparently, VEGF plays an important role in regulating steroidogenesis of
testosterone in Leydig cells during hypoxia.
Introduction
Oxygen is essential for life in human and other mammals. Insufficient oxygen,
hypoxia, in tissue or cell has profound physiological and pathological responses.
Cellular hypoxia causes an induction of hypoxia-response genes, relate to the
angiogenesis, oxygen transport and metabolism and so on (Adrian et al., 2001).
Vascular endothelial growth factor (VEGF), one of the genes induced by hypoxia, is a
key regulator for angiogenesis and vascular formation in vascular endothelial cells.
VEGF was a homodimeric glycopepetide, had been characterized as a potential
growth factor in angiogenesis of endothelial cells (Gospodarowicz et al., 1989). This
growth factor was also called a vascular permeability factor (VPF), because of a 5,000
times permeability than histamine. It was been studied that, five human VEGF
isoforms (VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206) have been
characterized (Houck et al., 1991). VEGF121, VEGF145, and VEGF165 were considered
as secreted VEGF isoforms, induced proliferation of endothelial cells mediated by
VEGF receptor. Different type of VEGF receptor also been identified. VEGFR-1
(Flt-1) is thought to be a negative role in angiogenesis (Gille et al., 2000), but
VEGFR-2 (Flk-1/KDR) is the main mediator of the mitogenic and angiogenenic
effects of VEGF (Ferrara, 1999).
It is well documented that, several transcription factors were involved in the
response to hypoxia. For instance, Ap-1, NF-κB and HIF-1 have a potential role in
induction of hypoxia-response genes (Faller et al., 1999). Hypoxia-inducible factor-1
(HIF-1) is a heterodimer that composed of the α and β subunit, also known as
hypoxic response factor and aryl hydrocarbon receptor nuclear translocator (ARNT),
respectively. In the hypoxic conditions, the transcriptional activity of HIF-1 is
elevated and the expressions of hypoxia-response genes were also activated by HIF-1
through hypoxia-response elements (HREs) of genes, such as VEGF and
erythropoietin (Epo). Previous studies have shown that, in hypoxic cells the stability
of HIF-1α was regulated by Von Hippel-Lindau (VHL) protein mediated pathway
(Adrian et al., 2001), and the activity might also be enhanced by p42/p44 MAPK
mediated pathway (Edurne et al., 2000).
Luteinzing hormone (LH) and follicle-stimulating hormone (FSH), released from
anterior pituitary, were well known major regulators of testicular functions in male.
Apart from these endocrine effectors, autocrine and paracrine control of Leydig cells
has been studied in stimulatory or inhibitory role in steroidogenesis. Lejeune et al.
showed that certain growth factors possessed positive (IGF-1, inhibin, activin) or
negative (TGF-β, TGF-α/EGF, bFGF) regulation of LH/hCG receptor number and
mRNA and steroidogenic enzyme mRNA and activites, to altered the responsiveness
to LH in the immature porcine Leydig cells (Lejeune et al., 1996).
Our studies have previously shown that intermittent hypoxia (12 % O2, 88 % N2)
of male rats for 7~14 days caused an increase of plasma testosterone levels.
Angiogenesis and vasodilation were observed in rat testicular tissues after intermittent
hypoxia. It is plausible to postulate that the increase of plasma testosterone levels may
result from certain angiogeneis-related growth factors, which were triggered by
hypoxia. Therefore, in present study, using mouse Leydig cells, we investigated the
effects of hypoxia on (a) cell proliferation, (b) release of testosterone and VEGF, and
(c) basic cellular regulatory pathway involved.
Materials and Methods
Cell culture
TM3 Leydig cells, a non-tumorogenic cell line derived from mouse testis, were
obtained from the Culture Collection and Research Center (Food Industry Research
and Development Institute, Taiwan, Republic of China). This cell line responds to
luteinzing hormone (LH) by increasing testosterone production and secretion, through
mechanisms similar to those encountered in freshly isolated cells. Cells were cultured
in 1:1 mixture of Ham-12 and Dulbecco’s MEM (Sigma, St. Louis, MO, USA),
containing 15 mM HEPES, 0.12 % NaHCO3 and supplemented with 0.45 % glucose,
5 % horse serum, 2.5 % fetal calf serum (Kibbutz Beit, Haemek, Israel) and 100
IU/ml potassium penicillin G + 100 μg/ml streptomycin sulfate (Sigma, St. Louis,
MO, USA), in 75 cm2 flasks (Falcon, Franklin Lakes, NJ, USA). Cells were cultured
at 37℃ in humidified atmosphere of normoxic conditions (95 % Air and 5 % CO2) or
placed in a modular incubator chamber (Billups-Rothenberg, NY, USA), flushed with
hypoxic gas (95 % N2, and 5 % CO2).
Effect of hypoxia on basal or hCG-stimulated cell proliferation in mouse TM3 Leydig
cells
Mouse TM3 Leydig cells (2,000 cells 200 μl-1) were preincubated for 48 hours in
96-well plates and then incubated for 1~16 hours with or without hCG at 1 IU/ml in
normoxic or hypoxic condition. To test the role of hypoxia on VEGF-stimulated cell
proliferation in TM3 Leydig cells, the cells were incubated with VEGF (5~20 ng/ml)
in the hypoxic conditions. Moreover, to determine the proliferated effect in Leydig
cells through VEGF receptor, the cells were incubated with hCG at 1 IU/ml or VEGF
at 5 ng/ml in the presence or absence of anti-VEGF antibody at 0.1 μg/ml. At the end
of incubation, the cells were performed to MTT assay for cell proliferation
assessment.
Effectr of hypoxia on basal and hCG-stimulated VEGF and testosterone secretion in
mouse TM3 Leydig cells
After preincubation for 48 hours in 12-well plates, the cells (105 cells ml-1) were
incubated for 1~16 hours with or without hCG at 1 IU/ml in normoxic or hypoxic
condition. At the end of incubation, the cultured medium was collected and stored at
-20℃ until analyzed for VEGF by ELISA and testodterone for ELISA. Furthermore,
the cells (106 cells 10 ml-1) were seeded in 10 cm dishes (Falcon, Franklin Lakes, NJ,
USA), and then incubated in normoxic or hypoxic condition in presence or absence of
hCG at 1 IU/ml. After the treatments, the cells were collected, and the nuclear and
cytoplasmic extracts were determined for protein expression by Western blot.
Role of ERK1/2 on VEGF and testosterone release in mouse TM3 Leydig cells
Mouse TM3 Leydig cells (106 cells 10 ml-1) were seeded in 10 cm dishes, and
then incubated with or without hCG at 1 IU/ml for 1~16 hours in normoxic or
hypoxic condition. At the end of incubation, the cells were extracted for cytoplasmic
protein to determine ERK1/2 expression by Western blot. Furthermore, the cells (105
cells ml-1) were seeded in 12-well plates, and then incubated with or without hCG at 1
IU/ml plus 50 μM PD98059 (an inhibitor of MEK) or not in normoxic or hypoxic
condition. The medium was collected and stored at -20℃ until analyzed for VEGF by
ELISA and for tstosterone by RIA.
Effect of VEGF on testosterone release and ERK1/2 expression in mouse TM3 Leydig
cells
To ascertain the dose-dependent effect of VEGF, the cells (105 cells ml-1) were
seeded in 12-well plates, and then incubated with VEGF (5~20 ng/ml) or not. The
medium was collected and stored at -20℃ until analyzed for tstosterone by RIA.
Furthermore, the cells (106 cells 10 ml-1) were seeded in 10 cm dishes, and then
incubated with or without hCG at 1 IU/ml, VEGF at 20 ng/ml in the presence or
absence of PD98059 at 50 μM. At the end of incubation, the cells were extracted for
cytoplasmic protein to determine ERK1/2 expression by Western blot.
Antibodies and reagents
Human chorionic gonadotropin (hCG) and mouse vascular endothelial growth
factor (mVEGF) were purchased from Sigma Co. (Sigma, St. Louis, MO, USA).
PD98059 was purchased from TOCRIS Cooksoon Co.. The first antibody,
anti-HIF-1α (1:500 dilution, Cayman Chemicals) and anti-HIF-1β (1:2,000 dilution,
Novus Biologicals) were used for Western blot. Other first antibody, anti-VEGF
(1:200 dilution), anti-P-ERK1/2 (1:1,000 dilution), anti-β-actin (1:8,000 dilution) and
anti-GAPDH (1:500 dilution) all were purchased from Santa Cruz Co.. The
horseradish peroxidase-conjugated IgG, goat anti-rabbit IgG (1: 6000 dilution) and
goat anti-mouse IgG (1: 8000 dilution) were purchased from ICN Pharmaceuticals,
Inc. (Aurora, Ohio, U.S.A.).
Cell proliferation assessment
We
used
the
modified
colorimetric
3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay to
quantified cell proliferation (Chung et al., 1999). Living cells reduced the yellow
MTT to blue formazen, which was soluble in dimethyl sulfoxide (Wako, Osaka,
Japan). In the culture medium, the intensity of blue staining was proportional to the
number of cell alive at analysis. Described briefly, cells were incubated in 96-well
microplates (Falcon, Franklin Lakes, NJ, USA) for 24 hours. Cells were plated at
2,000 cells per 200 μl per well with medium supplemented with 7.5 % serum. The
culture medium was removed and replaced by serum free medium. Incubated for 24
hours, the culture medium was replaced again by serum free medium containing
various drugs. After the treatments, the medium was removed and replaced by 50 μl 1
mg/ml MTT solution (Sigma, St. Louis, MO, USA). After a further 4-hour incubation
period, the MTT solution was removed and replaced by 50 μl dimethyl sulfoxide and
plates were shaken for 3 min. The optical density of each well was determined using
microplate reader (Dynatech Laboratories, Chantilly, VG, USA) at a wavelength of
570 nm with a reference wavelength of 630 nm.
VEGF ELISA
Leydig cells were incubated under normoxic or hypoxic conditions for a period.
At the end of incubation, the supernatant was collected, and VEGF levels were
determined using Quantikine VEGF ELISA kit (R&D Systems, Minneapolis, MN)
following the manufacturer’s instructions. This kit specifically measures rodent
VEGF164 and VEGF120 variants, and the limit of detection is 3 pg/ml. VEGF
concentrations were normalized relative to cellular protein concentrations (Braford
protein asssay).
Testosterone RIA
The concentrations of testosterone in medium were determined by RIA as
described previously (Wang et al., 1994; Tsai et al., 1996). The anti-testosterone
serum no. W8. was used, the sensitivity of testosterone RIA was 2 pg per assay tube.
The intra- and interassay coefficients of variance (CV) were 4.1 % (n = 6) and 4.7 %
(n = 10), respectively.
Nuclear and cytoplasmic protein extraction
Nuclear and cytoplasmic protein extracts were prepared from Leydig cells using
following method. Briefly, cells were scraped from cultured dishes, and harvested by
centrifugation at 8,000 rpm at 4℃ for 3 min. Cell pellets were resuspended in 100 μl
hypotonic buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.1 % PMSF, 0.1 %
aprotinin) in iced-bath for 5min. Followed by adding of 12.5 μl lysis buffer (10 mM
HEPES, 10 mM KCl, 1.5 mM MgCl2, 2.5 % NP-40), and the nuclei pelleted by
centrifugation at 3,800 rpm at 4℃ for 10 min. Collected the supernatants as the
cytoplasmic fraction, and the nuclei pellets were resuspended in 50 μl of buffer C (20
mM HEPES, 0.45 M NaCl, 1 mM EDTA) while mixing for 20 min at 4℃ followed
by centrifugation at 14,000 rpm for 10 min at 4℃. The supernatants were stored at
-70℃ until analysis.
Western blot
Protein concentrations were determined by the Braford protein assay using BSA
as the standared. Thirty microgram aliquots of nuclear and cytoplasmic proteins were
separated by SDS-PAGE using 7.5~15 % polyacrylamide gels. Proteins were
electroporetically transferred to polyvinylidene difluoride (PVDF) membranes (NEN
Life Science Products, Inc., Boston, MA, USA) or nitrocellulose (NC) membrane
(Schleoicher & Schuell, Inc., Keene, NH, USA) using a Trans-Blot SD semi-dry
transfer cell (170-3940, Bio-Rad, Hercules, CA, USA). The membrane were washed
in TBS-T buffer (0.242 % Tris-base, 0.8 % NaCl, 0.1 % Tween-20) and blocked in
TBS-T containing 5 % nonfat dry milk for 2 hours at room temperature with gentle
agitation. Then the membranes were incubated with first antibodies in TBS-T buffer
containing 0.05 % BSA more then 16 hours at 4℃. Followed by incubated for 1 hour
with horseradish peroxidase-conjugated secondary antibodies in 5 % nonfat dry milk
of TBS-T buffer. The membranes were washed three times with TBS-T buffer, and
then the blots were developed by enhanced chemiluminescence using ECL kit (ECL,
Western blotting detection reagents, Amersham International, UK) and exposed to
X-ray film. Using the computerized image analytic densitometry (Personal
Densitometer, Molecular Dynamics, Sunyale, CA, USA) performed quantification of
chemiluminescence signal data.
Statistic analysis
All data were expressed as mean ± SEM. Treatment means were tested for
homogeneity using the analysis of variance (ANOVA), and the differences between
the specific means were tested for the significance by Duncan's multiple range test
(Steel & Torrie, 1960). The level of significance chosen was P < 0.05.
Results
Stimulatory effect of cell proliferation on basal or hCG-treatment under hypoxic
conditions
To study the effect of hypoxia on TM3 cell proliferation, cells were exposed to
normoxic or hypoxic conditions for 1~16 hr. Proliferation status were assessed by
MTT assay. As shown in Fig. 1, proliferation of TM3 cells was significantly increased
by hCG-treatment for 16 hr in normoxic and hypoxic conditions. As expected,
hypoxia caused a greater induction in TM3 cell proliferation than normoxia observed
in 16 hr. Under hypoxic conditions, VEGF and hCG had more significant stimulatory
effect on cell proliferation than normoxic conditions in a dose dependent manner (Fig.
2). To examine whether the VEGF receptor was involved in the induction of cell
proliferation, we incubated the TM3 cell with anti-VEGF antibody. A similar
inhibitory effect on cell proliferation was observed when the anti-VEGF antibody was
added prior to stimulation of the cells with hCG, VEGF and hypoxic treatment (Fig.
3).
Stimulation of VEGF release and expression by hCG in hypoxic conditions
The effects of hypoxia on VEGF release by TM3 cells are shown in Fig. 4. After
treating with hCG, the levels of medium VEGF were enhanced significantly at 4 hr in
normoxia and at 2 hr in hypoxia. As stimulatory effect on TM3 cell proliferation,
hypoxia also caused a significant elevation of VEGF release in basal status and
hCG-treatment after 16 hr. Furthermore; to investigate whether the expression of
VEGF was affected by hCG and hypoxia, the cells were incubated in normoxic or
hypoxic conditions in the presence or absence of hCG. The data demonstrated that
hypoxia didn’t cause an observably differential staining of VEGF in Western blots,
but the hCG caused after treatment for 16 hr (Fig. 5). Previous studies had shown that
HIF-1 was involved in several physiological responses to hypoxia. The transcriptional
activity of HIF-1 mediated the specific genes transcription, such as the genes for
glycolytic enzymes, erythropoietin, and VEGF (Adrian, 2001). As expected, HIF-1α
expression was observed under hypoxic conditions, but the expression was barely
detectable under normoxic conditions (Fig. 5). None of the treatments caused any
change in the staining of HIF-1β, as previous knows that HIF-1β was consistent
expression in the cells (Wang et al., 1995).
Activation of ERK 1/2 by hCG and hypoxia in TM3 cells
It had been shown that, in mouse Leydig cells, VEGF secretion could been
mediated by an MEK 1/2-ERK 1/2 dependent pathway (Ravinder et al., 2003). Thus,
we incubated the TM3 cells with the MEK inhibitor, PD98059, blocked the
phosphorylation of ERK 1/2. Consequently, a significant altitude of enhancement in
VEGF release in hypoxic conditions was abolished by PD98059 (Fig. 4). Takashi et al.
had shown that hCG provokes a 2- to 3-fold increase in the levels of phosphorylated
ERK 1/2 in the MA-10 cells transiently transfected with an vector coding the wilid
type hLHR (Takashi et al., 2002). Therefore, we tested the role of ERK 1/2 involved
in testosterone production of TM3 cells. As expected, whatever in normoxic or in
hypoxic conditions, PD98059 could inhibit the release of testosterone in
hCG-stimulated cells (Fig. 6). Moreover, Minet et al. demonstrated that in human
microvascular endothelial cells-1 (HMEC-1), ERK 1/2 were activated during hypoxia,
and ERK 1 was needed for hypoxia-induced HIF-1 transcriptional activity (Minet et
al., 2000). To verify the whether the expression of ERK 1/2 was affected by hypoxia
in TM3 cells, we treated the cells with hCG in normoxic and hypoxic conditions. The
results revealed that the expression of phospho-ERK 1/2 (P-ERK 1/2) were activated
by hCG and hypoxia for 1 and 16 hr (Fig. 7). The levels of P-ERK 1/2 expression
were judged by the staining of GAPDH, which consistent expression in the cells, as
an internal control.
VEGF increase testosterone release in TM3 cells
It has been reported that some steroids, androgens, estrogens or gestagens are
able to induce VEGF production in a variety of steroid-dependent cells. Johanna et al.
had demonstrated that both estrogen and androgen stimulate the expression of VEGF
by increasing gene expression and mRNA stability (Ruohola et al., 1999). Thus, the
interaction between VEGF and testosterone were also investigated. Our results
indicated that, the levels of VEGF were not altered in testosterone treatment at
10-12~10-6 M (data not shown). In a contrary, VEGF produced significant
concentration dependent increase in testosterone production in TM3 cells (Fig. 8).
Furthermore, administration of anti-VEGF antibody, to block the signaling through
VEGF receptor, resulted in an inhibitory effect of testosterone release (Fig. 8). To
further verify the MAPK signaling pathway associated with this stimulatory effect by
VEGF on testosterone release, the expression of ERK 1/2 was also detected. Our
results were shown in Fig. 9, the VEGF induced clear elevation of ERK 1/2
expression that was suppressed by MEK inhibitor, PD98059.
Discussion
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140
130
∗
Normoxia
Vehicle (n= 6)
hCG (1 IU/ ml, n= 6)
120
110
100
90
170
160
Hypoxia
Vehicle (n= 6)
hCG (1 IU/ ml, n= 6)
+
150
∗++
0
140
++
Value of Cell Proliferation Percentage to Control Cells at Hour 0 (%)
Figures
130
120
110
100
90
0
1 2
4
8
16
Incubation Time after Challenge (hr)
Fig. 1.
∗
∗∗
Normoxia (n= 4)
Hypoxia (n= 4)
∗
Value of Proliferation Percentage
to Untreated Group (%)
160
140
120
100
=
0
VEGF (ng/ ml)
hCG (IU/ ml)
Fig. 2.
−
−
5
−
10
−
20
−
−
1
Vehicle (n= 5)
Anti-VEGF Antibody (0.1 μg/ ml, n= 5)
160
140
−
+
−
80
=
0
Hypoxia (16 hr)
hCG (1 IU/ ml)
VEGF (5 ng/ ml)
Fig. 3.
+
−
−
∗
100
∗
120
∗
Value of Cell Proliferation Percentage
to Untreated Group (%)
180
−
−
−
−
−
+
10
∗
∗∗
∗∗
6
Vehicle (n= 6)
hCG (1 IU/ ml, n= 6)
PD98059 (50 μM, n= 6)
PD98059 + hCG (n= 6)
4
2
0
Vehicle (n= 6)
hCG (1 IU /ml, n= 6)
PD98059 (50 μM, n= 6)
PD98059 + hCG (n= 6)
12
++
++
14
∗∗ ++
16
Hypoxia
++
18
∗∗++
10
6
4
2
∗∗ ++
8
∗∗
Release of Vascular Endothelial Growth Factor
(pg/ μg protein)
Normoxia
8
0
1
2
4
8
16
Incubation Time after Challenge (hr)
Fig. 4.
hCG (1 IU/ ml)
Normoxia
−
+
Hypoxia
−
+
25 KDa
β-Actin
45 KDa
HIF-1α
120 KDa
HIF-1β
92 KDa
VEGF / β-Actin
VEGF
4
3
Normoxia (n= 1)
Normoxia + hCG (1 IU/ ml, n= 1)
Hypoxia (n= 1)
Hypoxia + hCG (1 IU/ ml, n= 1)
2
1
HIF-1α / HIF-1β
0
4
Normoxia (n= 1)
Normoxia + hCG (1 IU/ ml, n= 1)
Hypoxia (n= 1)
Hypoxia + hCG (1 IU/ ml, n= 1)
3
2
1
0
Treatment
Fig. 5.
∗∗
15
Vehicle (n= 5)
hCG (1 IU/ ml, n= 5)
PD98059 (50 μm, n= 5)
PD98059 + hCG (n= 5)
∗
20
Normoxia
10
5
0
20
15
Vehicle (n= 5)
hCG (1 IU/ ml, n= 5)
PD98059 (50 μm, n= 5)
PD98059 + hCG (n= 5)
∗∗
Hypoxia
∗
Release of Testosterone (pg/ μg protein)
25
10
5
0
1
16
Incubation Time after Challenge (hr)
Fig. 6.
1 hr
hCG (1 IU/ ml)
Normoxia
−
+
16 hr
Hypoxia
−
+
Normoxia
−
+
Hypoxia
−
+
44 KDa
42 KDa
GAPDH
37 KDa
P-ERK 1/2 / GAPDH
P-ERK 1/2
12
10
8
Normoxia (n= 3)
Normoxia + hCG (1 IU/ ml, n= 3)
Hypoxia (n= 3)
Hypoxia + hCG (1 IU/ ml, n= 3)
6
4
2
0
1
16
Incibation Time after Challenge (hr)
Fig. 7.
∗∗
Vehicle (n= 5)
Anti-VEGF Antibody (0.1 μg/ ml, n= 5)
20
15
∗
Release of Testosterone
(pg/ μg protein)
25
+
10
5
0
0
5
10
VEGF (ng/ ml)
Fig. 8.
20
hCG (1 IU/ ml)
VEGF (20 ng/ ml)
PD98059 (50 μm)
−
−
−
−
−
+
+
−
−
+
−
+
−
+
+
P-ERK 1/2
44 KDa
42 KDa
GAPDH
37 KDa
P-ERK 1/2 / GAPDH
2.0
Vehicle (n= 3)
PD98059 (50 μm, n= 3)
1.5
1.0
0.5
0.0
Treatment
Fig. 9.
−
+
−
Figure legends
Figure 1.
Effect of hypoxia on basal and hCG-stimulated cell proliferation in TM3 Leydig cells.
* P < 0.05 compared to vehicle group. + P < 0.05, ++ P < 0.01 compared to normoxic
vehicle group at indicated time.
Figure 2.
Effect of VEGF and hCG on cell proliferation of TM3 Leydig cells after tretament for
16 hr. ** P < 0.01 compared to corresponding vehicle group.
Figure 3.
Effect of anti-VEGF antibody on hCG-, VEGF- or hypoxia-stimulated cell
proliferation in TM3 Leydig cells after treatment for 16 hr. * P < 0.05 compared to
corresponding vehicle group.
Figure 4.
Effect of PD98059 (a MEK inhibitor) on hCG-stimulated VEGF release in TM3
Leydig cells. * P < 0.05, ** P < 0.01 compared to vehicle group at indicated time. ++
P < 0.01 compared to normoxic vehicle group at indycated time..
Figure 5.
Effect of hypoxia on HIF-1α, HIF-1β and VEGF expression in TM3 Leydig cells
after treatment for 16 hr.
Figure 6.
Effect of PD98059 (a MEK inhibitor) on hCG-stimulated testosterone release in TM3
Leydig cells after treatment for 16 hr. * P < 0.05, ** P < 0.01 compared to normoxic
group.
Figure 7.
Effect of hypoxia on basal and hCG-stimulated phospho-ERK 1/2 (P-ERK 1/2)
expression in TM3 Leydig cells.
Figure 8.
Effect of anti-VEGF antibody on VEGF-stimulated testosterone release in TM3
Leydig cells after treatment for 16 hr. * P < 0.5, ** P < 0.01 compared to
corresponding vehicle group. + P < 0.05 compared to vehicle group.
Figure 9.
Effect of PD98059 (a MEK inhibitor) on basal and hCG-, VEGF-stimulated
phospho-ERK 1/2 (P-ERK 1/2) expression in TM3 Leydig cells.