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Is Constitutively Expressed in Murine Leydig Cells and Regulates 3β-Hydroxysteroid Dehydrogenase Type 1 Promoter Activity
From the Departments of * Urology and
Endocrinology, University of Virginia Health
System, Charlottesville, Virginia; The Centre
for Research in Biology of Reproduction, Department of Obstetrics and
Gynecology, Laval University, Quebec City, Canada; and the Departments of
Pediatrics and || Cell
Biology, University of Virginia Health System, Charlottesville,
Virginia.
| Correspondence to: Jeffrey J. Lysiak, Department of Urology, University of Virginia, Charlottesville, VA 22908 (e-mail: jl6n{at}virginia.edu). |
| Received for publication July 2, 2008; accepted for publication October 7, 2008. |
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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(HIF-1) is a transcription factor
that plays an essential role in oxygen homeostasis. HIF-1 is
constitutively made in cells; however, it is ubiquitinated and degraded under
normoxic conditions. Hypoxia prevents the ubiquitination of HIF-1,
resulting in stabilization of the protein and activation of target genes.
Because of its vascular arrangement and the high metabolic demand of
spermatogenesis, the testis has been described previously as functioning on
the brink of hypoxia; thus, we have hypothesized that HIF-1 is
constitutively expressed and stabilized in the testis, where it could play a
role in testicular homeostasis. Western blot analysis using nuclear proteins
from liver, kidney, and testis revealed the presence of HIF-1 only in
the testis. Immunohistochemistry confirmed this result and revealed that
HIF-1 was specifically located in interstitial Leydig cells.
Electromobility shift assays employing nuclear extracts from the TM3 Leydig
cell line revealed that these cells express HIF-1 that is capable of
binding DNA under normoxic conditions. Furthermore, we found that protein
levels can be increased further when the TM3 cells are cultured under hypoxic
conditions. Finally, transient transfections of TM3 Leydig cells revealed that
the promoter of the mouse 3β-hydroxysteroid dehydrogenase type 1 (Hsd3b1)
gene, which encodes a key enzyme in testosterone production, is a potential
target of HIF-1. In conclusion, HIF-1 is constitutively present
in the Leydig cells of the murine testis, where it potentially regulates
Hsd3b1 transcription, and thus male reproductive function.
Key words: Reproductive tract, steroidogenesis, testis
HIFs are heterodimeric proteins composed of and β subunits.
Three oxygen-regulated subunits have been described thus far:
HIF-1, HIF-2, and HIF-3
(Jiang et al, 1996a;
Maxwell and Salnikow, 2004).
HIF-1 is constitutively made in cells; however, under normoxic
conditions it is constantly degraded. In the presence of oxygen, 2 critical
prolines are hydroxylated which ultimately target HIF-1 for proteasomal
degradation (Maxwell et al,
1999; Jaakkola et al,
2001; Maxwell et al,
2001). Under hypoxic conditions, however, this degradation does
not occur, and HIF-1 accumulates in the cell, translocates into the
nucleus, and binds to its partner, HIF-1β. HIF-1β is also known as
aryl-hydrocarbon-receptor nuclear translocator and is constitutively
expressed. Both HIF-1 and HIF-1β are members of the basic
helix-loop-helix family of transcription factors, and the complex of the two
then binds to hypoxia response elements (HREs) in the promoter region of
specific hypoxia-regulated genes and recruits transcriptional coactivators
(Wenger and Katschinski,
2005). In the presence of oxygen, an asparagine residue in the
C-terminal transactivation domain of HIF-1 is hydroxylated by HIF
asparaginyl hydroxylase, termed factor-inhibiting HIF (FIH), and this
posttranslational modification essentially blocks the recruitment of
transcriptional coactivators. Thus, HIF activity is tightly regulated (Lando
et al,
2002a,b;
Wenger and Katschinski,
2005).
Marti et al (2002) first
reported HIF-1 in the murine testis and noted that spermatocytes,
spermatids, and spermatozoa express the mouse HIF-11.1 isoform.
Recently, this same group speculated that the low partial pressure of oxygen
(PO2) in the lumen of the seminiferous tubules is a stimulant for
HIF-1 and that HIF-1 may play a role in spermatogenesis
(Wenger and Katschinski,
2005). Wenger and colleagues have also reported a human
testis-specific HIF-1 isoform that does not bind DNA and have suggested
it may function as a dominant-negative form of HIF-1
(Depping et al, 2004).
HIF-1 has also been reported in the rat testis and has been shown to
increase with periods of testicular ischemia, although the cell type
expressing HIF-1 was not determined
(Powell et al, 2002).
Interestingly, the von Hippel-Lindau (VHL) protein is necessary for the
process of HIF-1 degradation, and mice with an inactive VHL
gene have small testes, oligospermia, and are infertile
(Ma et al, 2003).
Setchell and Waites (1964) speculated that the testis operates on the verge of hypoxia. They suggested this based on the observations that 1) oxygen consumption in the testis is high because of the demands of spermatogenesis, 2) the testis has little capacity to increase blood flow, and 3) the PO2 in the testis is relatively low. Studies from our lab employing an isolated oximeter have also described a low testicular PO2 averaging 12.5 mmHg in the rat testis, and the PO2 values oscillated in synchrony with simultaneously recorded vasomotion (Lysiak et al, 2000). The values reported in that study were in line with the results of Free et al (1976), who reported a PO2 of 15.2 mmHg in the interstitium of the rat testis. Similarly, a PO2 of 11.6 mmHg was described by Cross and Silver (1962) in the rabbit testis (Free et al, 1976), and in both those latter studies the testis values were approximately half of that found in reference tissues. Thus, data across species confirm Setchell and Waites's (1964) speculation that the microenvironment of the mammalian testis is relatively hypoxic.
The facts that the testis has a relatively low PO2, that
HIF-1 has been localized to the testis, and that disruption of the
HIF-1 degradation pathway results in infertility suggest an important
role for HIF-1 in normal testicular function; however, the exact role
of HIF-1 in the mammalian testis has yet to be determined. The current
study investigates the presence, localization, activity, and potential targets
of HIF-1 in the murine testes as well as its regulation under
restricted oxygen conditions.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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antibodies C-19 and Y-15 were purchased from Santa
Cruz Biotechnology Inc (Santa Cruz, California), anti–HIF-1 from
BD Transduction Laboratories (San Jose, California), and
anti–HIF-1 from Novus Biochemical (Littleton, Colorado).
Anti-HIF-1β antibodies were purchased from Novus Biochemical, Santa Cruz
Biotechnology Inc, and BD Transduction Laboratories. The
3β-hydroxysteroid dehydrogenase (3βHSD) antibody was from Santa Cruz
Biotechnology. HIF-11.1– and HIF-11.2–specific
primers (Marti et al, 2002)
were purchased from Invitrogen (Carlsbad, California).
Nuclear Extract Preparation
Testes from adult and from postnatal days 10, 15, and 21 C57BL/6 mice were
removed, decapsulated, and homogenized, and nuclear extracts were collected as
described below. TM3 Leydig cells were dissociated with trypsin and
centrifuged at 1200 x g, and the resultant cell pellets were
suspended in 4 volumes of Buffer A (10 mM Tris-HCl, pH 7.5; 1.5 mM
MgCl2; and 10 mM KCl) and incubated on ice for 10 minutes. The
cells were then homogenized, and the nuclei were pelleted and resuspended in
0.42 mM KCl; 20 mM Tris-HCl, pH 7.5; 1.5 mM MgCl2; 20% glycerol; 2
mM dithiothreitol [DTT]; 0.4 mM phenylmethyl sulfonylfluoride; 1 mM
Na3VO4, 2 mg/mL leupeptin; 2 mg/mL pepstatin A; and 2
mg/mL aprotinin. This mixture containing the nuclear proteins then was
incubated for 30 minutes, centrifuged, and dialyzed with 1 change of 20 mM
Tris-HCl, pH 7.5; 0.1 mM KCl; 0.2 mM EDTA; and 20% glycerol for 4 hours at
4°C.
Western Blot Analysis
The protein concentration in the nuclear extracts was determined using the
BCA kit (Pierce, Rockford, Illinois), and 50 µg of protein per lane was
subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
The gel contents were electrotransferred to nitrocellulose membranes (BioRad,
Hercules, California), blocked with 5% nonfat dried milk, 0.1% Tween-20 in
PBS, and incubated with anti–HIF-1 antibody (Santa Cruz
Biotechnology) overnight at 4°C. Blots then were washed and incubated with
peroxidase-conjugated secondary antibody, and immunocomplexes were detected
with enhanced chemiluminescence (SuperSignal West Pico; Pierce).
Immunofluorescence
Testes from adult C57BL/6 mice were harvested, fixed in Bouin solution, and
sectioned. Sections then were deparaffinized, rehydrated, and incubated in 10%
goat serum in phosphate-buffered saline (PBS). Sections were incubated
overnight at 4°C with anti–HIF-1 antibody (Santa Cruz
Biotechnology) or anti-3βHSD (Santa Cruz Biotechnology), followed by
incubation with a fluorescent secondary antibody. Sections incubated in the
absence of primary antibody served as negative controls. Sections were washed
and were mounted with Vectashield (Vector Laboratories Inc, Burlingame,
California) containing 4',6'-diamidino-2-phenylindole, and images
were captured.
Immunohistochemistry
C57BL/6 mice were killed at postnatal days 10, 15, 21, and 28, and the
testes were harvested. Testis sections were deparaffinized, rehydrated,
incubated with 10% H2O2 methanol to block endogenous
peroxidases, and incubated in 10% goat serum in PBS. Sections then were
incubated overnight at 4°C with anti–HIF-1 antibody, followed
by incubation with biotinylated secondary antibody. The immunocomplexes were
visualized with avidin-biotin-peroxidase complex (Elite ABC Kit; Vector
Laboratories) and diaminobenzidene as the chromagen. Incubation without
primary antibody served as a specificity control.
Testicular Ischemia
This work was conducted in accordance with the Guiding Principals of
the Care and Use of Research Animals promulgated by the Animal Care and
Use Committee at the University of Virginia. Adult male C57BL/6 mice were
anesthetized with an intraperitoneal injection of 0.01 mg/g sodium
pentobarbital, and the testis was subjected to ischemia as described by Lysiak
et al (2001). Briefly, the
testis was exteriorized, the gubernaculum was divided, and the testis was
freed from the epididymotesticular membrane. The testis was rotated 720°
for 1, 2, or 4 hours, during which time it remained in the abdomen with a
closed incision. This procedure has been demonstrated many times to obstruct
testicular blood flow and induce testicular ischemia
(Lysiak et al, 2007), and was
documented to do so intraoperatively in these experiments by use of a
laser-Doppler flowmeter and flow probe (ALF-21; Transonic Systems Inc, Ithaca,
New York). The flow probe is capable of monitoring microvascular perfusion in
a tissue volume of approximately 1 mm3. The laser-Doppler flow
probe was carefully positioned at the testicular surface to avoid local
pressure effects and to monitor flow over microvascular fields only. Three
30-second measurements of blood flow were recorded and averaged (data not
shown). Following induction of ischemia, the incision was reopened, and the
testis was removed and snap frozen in liquid nitrogen. Sham-operated animals
were treated identically, except that upon completion of the rotation maneuver
the testis was immediately counterrotated.
Cell Line
The Leydig cell line, TM3, was obtained from the Cell Culture Core facility
at the University of Virginia and maintained under conventional culture
conditions in Dulbecco modified Eagle medium supplemented with 10% fetal
bovine serum, and 50 mg/L of gentamycin and streptomycin sulfate at 34°C
and 5% CO2.
Hypoxic Treatment of Cells
TM3 Leydig cells maintained in standard culture conditions are considered
to be normoxic. To induce hypoxia cells were plated in 6-well plates at a
concentration of 104 cells/well in complete media and placed in a
hypoxic chamber housed in a culture incubator. Hypoxia was induced by purging
the chamber with nitrogen to an approximately 5% O2 atmosphere, and
cells were incubated under these conditions for 24 hours.
Electromobility Shift Assay
Nuclear extracts were obtained from TM3 cells incubated in normoxic or
hypoxic conditions or treated with either vehicle or 250 µM
CoCl2 for 24 hours. Ten-microgram aliquots of nuclear extracts were
preincubated in 10 mM Tris, 50 mM KCl, 50 mM NaCl, 1 mM MgCl2, 1 mM
EDTA, 5 mM DTT, and 5% glycerol for 5 minutes at 4°C. The
32P-labeled, double-stranded oligonucleotides used as probes were
as follows: Hsd3b1 HRE element (shown in bold) at –1012 bp: sense,
5'-CTG TGG GCC CAC GTT ACT TGA TTC-3'; and antisense,
5'-GAA TCA AGT AAC GTG GGC CCA CAG-3'. For the competition
experiments, a double-stranded oligonucleotide containing a mutation in the
HRE (CACGT to CAATT) was used. For supershift
experiments, 2–4 µg of a commercially available
anti–HIF-1 antiserum was also added to the binding reaction. The
mixture was incubated on ice for 15 minutes, loaded onto a 4% nondenaturing
polyacrylamide gel, and electroporesis was performed in 0.5 x
tris-borate-EDTA at 4°C. The gel was dried and autoradiography
performed.
Transfections and Luciferase Reporter Assay
All transfections were done using the Ca(PO4)2
precipitation method. The day prior to transfection, TM3 cells were plated at
a density of 5 x 104 cells per well. Culture media was
changed between 12 and 16 hours after transfection, and the cells were
harvested the following morning. Cells were lysed by adding 50 µL lysis
buffer (100 mM Tris-HCl, pH 7.9; 0.5% Igepal [Sigma]; and 5 mM DTT) directly
to the culture wells. An aliquot of the lysate was assayed for luciferase
activity using an EG&G Berthold LB 9507 luminometer and luciferine (BD
Pharmingen, San Diego, California) as substrate. In experiments with
CoCl2 stimulation, cells were treated with 250 µM
CoCl2 for 24 hours prior to harvesting. Several DNA preparations of
the plasmids were used to ensure reproducibility of the results. In all
experiments, the total amount of DNA was kept constant at 1.5 µg/well using
Sp64 (Promega, Madison, Wisconsin) as carrier DNA. Data are reported as the
average of at least 3 experiments, each done in duplicate.
Plasmids
The mouse Hsd3b1 promoter fragment (–1375 to +17 bp) was isolated by
polymerase chain reaction (PCR) from mouse genomic DNA using the following
oligonucleotide primers: a forward primer containing an XhoI cloning
site (underlined), 5'-CCG CTC GAG TCC CAT CCT CAC AAA TGC
CTC-3, and a reverse primer containing a KpnI cloning site
(underlined), 5'-GGG GTA CCT CAG CCC TCA GAT CAG
GAC-3'. The deletion at –100 bp was generated by PCR using the
–1375-bp plasmid as template and the same reverse primer described above
along with the forward primer (XhoI cloning site underlined),
5'-CCG CTC GAG CAA TCA CTG GGA AGG ACA G-3'. The
–1375-bp Hsd3b1 reporter containing a mutation of the HRE at –1012
bp was obtained by site-directed mutagenesis using the QuikChange XL
mutagenesis kit (Stratagene, La Jolla, California) and the following pair of
oligos (the mutation is underlined): sense, 5'-GAG CCC GTG TGG GCC
CAA TTT ACT TGA TTC TGT GTG-3'; and antisense, 5'-CAC
ACA GAA TCA AGT AAA TTG GGC CCA CAG GGG CTC-3'. The rat
–1013-bp Nur77 (NGFI-B) promoter sequence was amplified by PCR from rat
genomic DNA using the following primers: forward (includes a BamHI
site shown underlined), 5'-CGG GAT CCG CTA CTA CCT AGC TTA
GTG ACC-3'; reverse (contains a KpnI site shown underlined),
5'-CTG GTA CCG CGT GCG CTC TGC AAT CCT TC-3'. Deletion
of the Nur77 promoter to –65 bp was obtained by PCR using the
–1013-bp Nur77 promoter as template, along with the same reverse primer
described above and the following forward primer containing a BamHI
(underlined) cloning site, 5'-CGG GAT CCA TGC GTC ACG GAG CGC
TTA AGA G-3'. All of the amplified promoter fragments were cloned into
the corresponding sites of a modified pXP1-luciferase reporter plasmid and
confirmed by sequencing. The HIF-1 expression vector was kindly
provided by Dr Darren Richards (Centre de Recherche de l'Hotel-Dieu de Quebec,
Quebec City, Canada).
Statistic Analysis
All statistic evaluations were either by ANOVA followed by Tukey's range
test or Student's t test (P < .05) after evaluation of
each data set by Chauvenet's criterion for homogeneity.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Is Constitutively Expressed in the Testis in proteins from nuclear extracts of
the testes as well as from the liver and kidney revealed that HIF-1 was
only detected in the testis (Figure
1). HIF-1 was detected as 2 bands at approximately 120 and
125 kd in the nuclear proteins from the testis, possibly representing the 1.1
and 1.2 isoforms of HIF-1.
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in the adult testis revealed distinct
localization to the interstitial Leydig cells
(Figure 2A). Faint staining of
the germ cells in the seminiferous tubules was detected but poorly evident in
the images. The Leydig cell localization was confirmed in serial sections
stained for 3βHSD, a common marker for Leydig cells
(Figure 2B).
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HIF-1 in day 10 postnatal testes was immunolocalized to interstitial
cells. Cells within the seminiferous tubules were largely negative
(Figure 3). By postnatal day 15
the interstitial cells were still prominently stained, with occasional faint
staining of cells within the seminiferous tubules. At postnatal day 21 the
interstitial cells remained positively stained, and selected germ cells within
the seminiferous tubules were displaying HIF-1 immunoreactivity. In
testis sections from the adult, interstitial cells continued to be
HIF-1 immunoreactive. Germ cells within the seminiferous tubules also
were stained (Figure 3).
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in the testis demonstrated
prominent labeling in interstitial cells, the TM3 murine Leydig cell line was
obtained and employed in subsequent studies. To determine whether TM3 cells
express HIF-1 and to determine whether the expressed HIF-1
actively binds DNA, electromobility shift assays (EMSAs) were performed with
nuclear proteins isolated from TM3 cells. When cultured under normoxic
conditions (20% O2), 2 protein bands were detected that bound to
the P32-labeled probe (inducible nitric oxide synthase promoter
with HRE; Figure 4). The
addition of nonlabeled probe actively competed the binding of
higher–molecular weight protein, suggesting it was HIF-1
(Figure 4). To determine
whether hypoxia influenced HIF-1 levels in the TM3 Leydig cells, the
cells were placed under hypoxic conditions (5% O2) for 24 hours
prior to collection of nuclear proteins. As observed in
Figure 4, an increase in the
upper band occurred when cells were placed in a hypoxic environment. The
addition of nonlabeled probe again actively competed for binding.
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To confirm that the upper band on the EMSAs was indeed HIF-1, a
panel of HIF-1 and HIF-1β antibodies was incubated with the
proteins to determine whether any of the antibodies induced a supershift in
the binding of the radiolabeled probe. Two of the four anti–HIF-1
antibodies induced a supershift, as did all of the anti–HIF-1β
antibodies (Figure 4). The fact
that 2 anti–HIF-1 antibodies did not cause a supershift may be
due to the location of their binding epitopes on HIF-1 or their
affinities; thus, these data suggest that TM3 Leydig cells express
HIF-1 and that HIF-1 can actively interact with its partner
HIF-1β and subsequently bind to DNA containing an HRE.
Testicular Ischemia Did Not Increase HIF-1 Levels
To determine whether brief periods of testicular ischemia led to an
increase in HIF-1 levels, testes were subjected to periods of ischemia
prior to being harvested. A period of ischemia up to 4 hours did not further
increase the level of HIF-1 relative to that constitutively expressed
in the sham-operated murine testis (Figure
5).
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Regulates Hsd3b1 Transcription in Leydig Cells in Leydig cells,
transient transfection assays were performed in TM3 cells using a panel of
promoters of genes known to be expressed in Leydig cells. These included the
promoters of P450c17 (Cyp17), platelet-derived growth factor receptor alpha,
steroidogenic acute regulatory protein, P450 side-chain cleavage, insulinlike
factor 3, and mouse 3β-hydroxysteroid dehydrogenase type 1 (Hsd3b1). Of
all of the promoters tested, only the mouse Hsd3b1 promoter was activated when
TM3 cells were transfected with HIF-1
(Figure 6A) or when treated
with cobalt chloride (CoCl2) to stabilize endogenous HIF-1
levels (Figure 6B). Combination
of HIF-1 and CoCl2 led to a further enhancement of Hsd3b1
promoter activity (Figure 6B).
In this way, the mouse Hsd3b1 promoter behaves similarly to the rat Nur77
promoter used here as a positive control
(Figure 6), because Nur77 is a
known target for HIF-1 in renal cells
(Choi et al, 2004;
Yoo et al, 2006). Sequence
analysis of the mouse Hsd3b1 promoter revealed the presence of a consensus HRE
(RCGTG) located at position –1012 bp. A deletion construct to –100
bp that removes this element was no longer activated by HIF-1 and/or
CoCl2 treatment (Figure
6B). Similarly, HIF-1/CoCl2-mediated activation
of the –1375-bp Hsd3b1 construct was significantly blunted when a point
mutation was introduced in the HRE at –1012 bp
(Figure 6C).
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EMSA was used next to test whether HIF-1 can bind to the
–1012-bp HRE. In unstimulated TM3 cells, a weak band
(Figure 7, lane 2) was
observed. This binding was strongly enhanced in nuclear extracts from
CoCl2-treated TM3 cells (Figure
7, lane 6). Furthermore, this band was displaced/supershifted by
the addition of an anti–HIF-1 antiserum
(Figure 7, lanes 4 and 12).
Binding of HIF-1 to this HRE was found to be specific because it was
competed by increasing doses of unlabeled oligonucleotides
(Figure 7, lanes 7 and 8) but
not by oligonucleotides harboring a mutation in the HRE
(Figure 7, lanes 9 and 10).
Together, these data indicate that endogenous HIF-1 can specifically
bind to the novel HRE element at –1012 bp and activate the mouse Hsd3b1
promoter activity.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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in the rodent testis,
although no exact role for HIF-1 was described. HIF has been described
as the "master regulator" of the cell's adaptive response to low
oxygen levels. It regulates the expression of gene products involved in
cellular metabolism, glucose transport, angiogenesis, erythropoiesis, pH
regulation, apoptosis, and cell proliferation
(Jiang et al, 1996b;
Iyer et al, 1998;
Maxwell, 2005). The results of
the present investigation confirm the results of Marti et al
(2002) with regard to the
presence of HIF-1 in developing germ cells; further, they demonstrate
that HIF-1 is made constitutively by murine Leydig cells from early
postnatal life into adulthood (Figures
2 and
3) and can regulate the
transcription of the mouse Hsd3b1 gene (Figures
6 and
7), which encodes a critical
enzyme in testosterone production.
In the study by Marti et al
(2002), in situ hybridization
was performed to identify which HIF-1 isoform is expressed in the
testis. Their results show that the 1.1 isoform is expressed in germ cells of
the seminiferous tubules and exhibited stage specificity, whereas the 1.2
isoform showed a ubiquitous expression pattern in cells at the periphery of
the seminiferous tubules. The resolution of those authors' images does not
allow determination of whether the peripheral 1.2 isoform hybridization was
intratubular or peritubular, and they do not discuss the possibility that it
was the interstitial cells of the testis that were positive for
HIF-11.2.
In the human, a novel testis-specific isoform of HIF-1 has been
described (Depping et al,
2004). Based on the predicted coding region, this testis-specific
isoform is thought to lack DNA-binding capabilities, and thus may function as
a dominant-negative regulator of normal HIF-1
(Depping et al, 2004). To
determine whether the HIF-1 in Leydig cells binds to DNA, and therefore
is most likely functional, or does not bind to DNA, and therefore may function
as a dominant-negative regulator, EMSAs were employed. The EMSA results
unequivocally show that Leydig cell HIF-1 interacts with its partner
HIF-1β and binds to DNA containing an HRE
(Figure 4). Thus, HIF-1
constitutively expressed in the relatively hypoxic environment of the testis
is capable of binding DNA and may be functional.
When testes were rendered ischemic by testicular torsion, no increase in
intratesticular HIF-1 was detected
(Figure 5), but when the TM3
Leydig cells were cultured in a hypoxic environment, HIF-1 levels were
dramatically increased compared with when the cells were cultured under normal
culture conditions (20% O2;
Figure 4). It is possible that
a Leydig cell–specific response to hypoxia in the intact testis was
undetectable against the background of constitutive HIF-1 production in
the relatively hypoxic in vivo environment, or it is possible that the 4-hour
ischemic period was not of sufficient duration.
Mice deficient in HIF-1 die in utero between days 8 and 11
postcoitum (pc) from neural tube defects, cardiovascular malformations, and
increased cell death in the cephalic mesenchyme
(Iyer et al, 1998), but a
phenotype in the testis has not be described. Nevertheless, specifically
interfering with the degradation pathway of HIF-1 by selectively
knocking out VHL in the testes resulted in mice with defects in
spermatogenesis, suggesting a functional role for HIF-1
(Ma et al, 2003). Also,
knockout of HIF-2, a closely related family member, renders the animal
azoospermic, along with other anomalies
(Scortegagna et al, 2003).
Vascular endothelial cell growth factor, erythropoietin, phosphoglycerate
kinase-1, and heme oxygenase-1 are all well-studied HIF-regulated genes;
however, we chose to determine whether Leydig cell HIF-1 influenced the
expression of several genes known to be expressed by Leydig cells. To address
this, luciferase reporter assays were performed with promoters of several
Leydig cell–specific genes. Increasing Leydig cell levels of
HIF-1 either by transfection with wild-type HIF-1 or by
stabilization with CoCl2 significantly increased mouse
Hsd3b1 promoter activity (Figure
6). HSD3B1 is the enzyme that converts pregnenolone to
progesterone in the pathway to testosterone in Leydig cells; thus, Leydig cell
HIF-1 may significantly contribute to testosterone production in vivo
and have downstream effects on secondary sex characteristics and
spermatogenesis.
In the mouse and most other mammals, including humans, there are 2
populations of Leydig cells, the fetal and the adult
(Habert et al, 2001). The fetal
population of Leydig cells arises in the developing testis at 12.5 days pc in
the mouse. These fetal Leydig cells produce androgens, including testosterone,
that are essential for the masculinization of the fetus
(Jost, 1970), but unlike their
adult counterparts, fetal Leydig cells produce androgens in the absence of
luteinizing hormone (O'Shaughnessy et al,
1998). The stimulus for testosterone production in these cells
remains largely unexplored. Fetal Leydig cells regress during late fetal/early
postnatal life and are virtually absent from the adult testis. For those few
that persist, they no longer support steroidogenesis and never enter mitosis
or transform into adult-type Leydig cells. The adult population of Leydig
cells originates from undifferentiated fibroblast-like cells in the testicular
interstitium 4 days after birth in the mouse
(Vergouwen et al, 1991). At
puberty these Leydig cells begin producing testosterone and are responsible
for the male secondary sex characteristics, male behavior, and male fertility.
At present we do not know whether both Leydig cell populations express
HIF-1; however, it is interesting to speculate that the hypoxic
environment of the testis during development and in adulthood stabilizes
HIF-1 and contributors to testosterone production. Indeed, previous
studies have demonstrated a link between oxygen tensions and Leydig cell
steroidogenesis (Quinn and Payne,
1984a,b;
Georgiou et al, 1987). Thus
far, the role of HIF in the developing testis has not been examined, but this
is an area of future investigation in our laboratory.
The constitutive stabilization of HIF-1 by Leydig cells under
putative normoxic conditions in vivo is unlike other tissues in their normoxic
conditions. This may be due to: 1) The usual condition of the testis is
relatively hypoxic. Our finding of the constitutive presence of HIF-1
in the testis provides a molecular confirmation of the physiologic facts
reported long ago (Setchell and Waites,
1964; Free et al,
1976; Lysiak et al,
2000). 2) High intracellular levels of reactive oxygen species
(ROS) in Leydig cells due to active steroidogenesis
(Chen et al, 2006;
Hanukoglu, 2006) may
contribute to stabilized HIF-1. Besides functioning as key enzymes in
steroidogenesis, mitochondrial P450-type enzymes can function as a futile
NADPH oxidase and leak electrons, thus producing superoxide and ROS
(Hanukoglu, 2006). 3) ROS
produced from testicular macrophages
(Hales, 2002;
Allen et al, 2004) may also
contribute to a relative hypoxic microenvironment, leading to HIF-1
stabilization in Leydig cells. Because in the last 2 cases ROS is known to
decrease steroidogenesis in Leydig cells
(Hales, 2002;
Allen et al, 2004;
Murugesan et al, 2005;
Hanukoglu, 2006), a balance
between ROS and HIF-1 may need to exist in Leydig cells to ensure
optimal steroid production. As an aside, these results also raise the question
of the appropriate oxygenation of Leydig cell cultures, because their
"normoxic" condition is actually relatively hypoxic. It may well
be that culturing Leydig cells under conventional, 
20% O2
conditions actually exposes them to a relatively hyperoxic environment that
can alter experimental results.
Finally, the involvement of HIF-1 in regulating Leydig cell mouse
Hsd3b1 and, likely, testosterone production indicates a novel role
for HIF-1 in testis function. That role may well have an influence on a
variety of androgen-related processes, including spermatogenesis, and
disruption of HIF-1 signaling may even result in male infertility, a
possibility that will be addressed by our laboratory.
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Acknowledgments |
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