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Department of Physiology, University of Turku (T.E.-H., P.R.M., I.H.), and Biokemi och Farmaci, Åbo Akademi University (J.P.S.), FIN-20520 Turku, Finland; and Unità di Andrologia, Dipartimento di Fisiopatologia Clinica, Università di Firenze (M.L., E.B.), 50139 Firenze, Italy
Address all correspondence and requests for reprints to: Ilpo Huhtaniemi, M.D., Ph.D., Department of Physiology, University of Turku, Kiinanmyllynkatu 10, FIN-20520 Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi
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Abstract |
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9.3 nmol/liter) and a low affinity
(Kd, 284 nmol/liter) component, and the binding
displayed with specificity (P > dehydroepiandrosterone >
17-OHP). The binding was apparently different from that of the
classical nuclear PR in the following ways. 1) The P/glucocorticoid
antagonist RU 486 did not compete with [3H]P binding to
the mLTC-1 cells. 2) No expression of the classical PR messenger RNA
was detected, despite clear P binding to these cells, by Northern
hybridization or RT-PCR. 3) An antibody against the C-terminal end of
the classical PR (
c-262) revealed in mLTC-1 cells several molecular
size protein bands between 45–57 kDa on Western hybridization, whereas
these immunoreactive proteins were faintly recognized by another
antibody (-PR) directed toward the NH2-terminal region
of the classical PR. The sizes of the immunoreactive molecules were
relatively similar to those detected using the same antibodies in human
sperm lysates, but were at variance with the classical PR (120, 94, and
60 kDa), detected with these antibodies in human uterus. The
immunoreactive proteins bound peroxidase-labeled-P, which could be
displaced in the presence of a 10-fold excess of free P. 4) An
immediate increase in the intracellular free calcium level was observed
after P treatment in cultured mLTC-1 cells, whereas it also increased
the 45Ca2+ entry within 15 min in these cells.
5) Increasing doses of P (0.1–10 µmol/liter) demonstrated
significant inhibition of LH receptor messenger RNA levels in a
dose-dependent manner in mLTC-1 cells. In conclusion, a nonclassical PR
is expressed and functional in these cells, and it is clearly distinct
from the classical nuclear PR. It is apparent that recently reported
inhibitory effects of P on LH receptor gene expression and function are
mediated through this novel type PR in mouse Leydig cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of the two forms of PR, i.e. the nuclear and nonclassical forms, has been demonstrated in the ovary, particularly in granulosa cells (8, 9, 10, 11, 12, 13). In the ovary, the level of PR expression is under hormonal regulation throughout the menstrual cycle by steroids and LH (8, 13, 14, 15, 16). This R is suggested to play a key role in follicular development and maintenance of the corpus luteum (17, 18, 19). On the other hand, a recent study demonstrated the expression of an unusual PR in porcine granulosa cells, which mediates its actions through mobilization of Ca2+ from the endoplasmic reticulum through activation of phospholipase C (20). Adult female mice with disrupted classical PR displayed significant defects in reproductive functions, including anovulation and uterine hyperplasia and inflammation (21). In contrast, adult mutant male mice showed no impairment of fertility, indicating that, unlike in the female, nuclear PR is not essential for male reproduction. Studies of possible PR expression in the testis are scanty (22, 23), and there are no detailed studies on putative effects of PR on Leydig cell function. Only one report exists demonstrating P binding to the cytosol of rat Leydig cells (24).
Recently, we demonstrated the inhibitory effect of high P levels on LH receptor (LHR)expression and function in Leydig cells (25). The aim of the present study was to study further the mechanisms of the documented P actions on Leydig cells and to explore whether evidence could be obtained for the presence of classical or nonclassical PR to explain the functional responses detected. In this report we demonstrate the presence and function of a R in murine Leydig tumor cell line (mLTC-1) (26), which is distinct from the classical nuclear PR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ligand binding assays
The mLTC-1 cells were grown in HEPES-buffered Waymouth’s medium
(Life Technologies, Inc., Paisley, Scotland), as
previously described (27). For the binding experiments, 7 x
104 cells/well were plated on 24-well culture
dishes (Greiner, Frickenhausen, Germany), and 24 h later the
binding experiments were performed on the living cells. Cells were
incubated for 1 h at 37 C in serum-free Waymouth’s medium with
2.2 nmol/liter [1,2,6,7-3H]progesterone
([1,2,6,7-3H]P; 4.40 tetrabecquerels/mmol;
Amersham Pharmacia Biotech, Aylesbury, UK) in the presence
or absence of 0.1–10 µmol/liter of one of the following nonlabeled
steroids: P, cortisol, testosterone, estradiol, 17-hydroxy-P,
dehydroepiandrosterone (DHEA), pregnanediol
(5ß-pregnane-3, 20-diol), pregnanolone
(5ß-pregnen-3-ol-20-one; all from Sigma, St. Louis,
MO), or antiprogestin RU 486 (RU 38484, Roussel UCLAF, France).
For Scatchard analysis, the binding experiments were performed in the presence of 86 µmol/liter aminoglutethimide (AMG; Sigma) to block the endogenous steroidogenesis and in the presence or absence of excess cold P (50 µmol/liter) with increasing concentrations of [3H]P (up to 30 nmol/liter). Cells were preincubated for 2 h with 86 µmol/liter AMG, and then the incubations for P binding were carried out in the presence or absence of 1 µmol/liter cortisol, to reduce nonspecific binding, as endogenous production of P was blocked by AMG. After a 1-h incubation with [3H]P, the media were removed, and cells were washed with PBS, pH 7.4 (Life Technologies, Inc.) and lysed in 1% SDS containing 0.3 N NaOH solution. The lysates were transferred into scintillation vials, and the radioactivity was measured in a ß-counter after addition of 1.5 ml scintillation liquid (Optiphase, Wallac, Inc. OY, Turku, Finland).
Isolation of RNA
The single step method (28) was used to isolate total RNA from
control and treated samples for Northern hybridization analysis. Cells
were stimulated in six-well culture dishes for 6 h in the presence
or absence of 50 µg/liter hCG (CR-121; 13,500 IU/mg by bioassay;
NICHHD, Bethesda, MD) and with or without 86 µmol/liter AMG or 10
µmol/liter of the selected steroids (see below). The tissues (testes,
uterus, and spleen) were snap-frozen in liquid N2
and stored at -70 C until isolation of RNA, and RT-PCR was carried out
using RNA of high purity (29).
RT-PCR and Southern hybridization
The oligonucleotide primers used were designed according to the
published complementary DNA (cDNA) sequence of the murine classical
nuclear PR (30). The sense primer, 5'-CTAAATGAGCAGAGG ATGAAGGAG-3',
corresponding to nucleotides 2320–2343, and the antisense primer,
5'-TGGGGCAACTGGGGCAGCAATAAC-3', corresponding to nucleotides
2725–2704, were used to amplify a 406-bp DNA fragment encoding the
conserved steroid-binding domain of the classical PR. The RT and PCR
reactions were performed sequentially in the same assay tube. Three
micrograms of total RNA were used for all samples except the uterus,
where 0.3 µg was used. RNA was reverse transcribed using the avian
myeloblastosis RT (Finnzymes Oy, Espoo, Finland) and the above
oligonucleotides as primers. The cDNAs generated were then amplified by
PCR using the same primers as those described above in a mixture
containing 1 nmol/liter of each primer, 200 µmol/liter deoxy-NTPs, 20
U ribonuclease inhibitor (RNasin, Promega Corp., Madison,
WI), 12.5 U avian myeloblastosis RT, and 2.5 U Taq DNA
polymerase. The reaction was started at 50 C for 10 min, followed by 3
min at 97 C, and then ran for 39 PCR cycles (97 C for 1 min, 55 C for 2
min, 72 C for 2 min, and final extension for 5 min at 72 C). An aliquot
of the PCR products was analyzed on gel electrophoresis, and molecular
sizes of the amplified products were determined by mol wt markers run
in parallel with RT-PCR products.
The cDNA fragments generated from RT-PCR were resolved in 1.2% agarose gel and transferred onto nylon membranes (Hybond, Amersham Pharmacia Biotech, Arlington Heights, IL). The membranes were prehybridized for 4 h in 5 x SSP (0.18 mol/liter NaCl, 0.01 mol/liter sodium phosphate, and 1 mmol/liter EDTA, pH 7.7), 5 x Denhardt’s solution [0.02% BSA, 0.02% (wt/vol) Ficoll, and 0.2% (wt/vol) polyvinylpyrrolidone], 0.5% SDS, and heat-denatured sonicated calf thymus DNA (20 mg/liter). The hybridizations were performed overnight at 42 C using a 32P end-labeled nested oligonucleotide probe, 5'-TCGACAGCTTGCATGATCTTG-3', corresponding to bases 2601–2621 of the classical PR cDNA. The membranes were washed twice for 10 min each time with 2 x SSPE-0.1% SDS at room temperature and then exposed to Kodak x-ray films (Kodak XAR-5, Eastman Kodak Co., Rochester, NY) for 4 days at -50 C. The molecular sizes of the RT-PCR amplicons were determined by comparison with molecular size markers.
Assessment of the RT-PCR products and Northern hybridization
The PR fragment produced by RT-PCR from adult mouse uterus was
eluted from the agarose gel, and its identity was confirmed with the
EcoRI restriction endonuclease digestion, which provided
100- and 306-bp fragments. The 406 PCR fragment was then directly
cloned into a plasmid vector (T-vector, Promega Corp.),
and its identity corresponded to the classical mouse PR sequence (30).
T7 polymerase was used to generate a 32P-labeled
antisense complementary RNA (cRNA) probe using the cloned cDNA as
template. Prehybridization and hybridization were carried out as
previously described (31), and the membranes were washed in decreasing
concentrations of SSC containing 0.1% SDS at 64 C and treated for 5
min at room temperature with 2 x SSC (1 x SSC = 0.15
mol/liter NaCl and 15 mmol/liter sodium citrate, pH 7.0) in the
presence of ribonuclease-A (2 mg/liter), followed by two washes in
0.1 x SSC and 0.1% SDS at 64 C. The membranes were exposed to
Kodak x-ray films for 5 days at -50 C before and after
the ribonuclease treatment.
Western and ligand blot analysis
The mLTC-1 cells from six-well culture plates were scraped off
into PBS under ice, washed, and lysed in 50 µl buffer [20 mmol/liter
Tris (pH 7.4), 150 mmol/liter NaCl, 0.25% Nonidet P-40, 1 mmol/liter
Na3VO4, and 1 mmol/liter
PMSF]. The proteins were measured in the cell lysates using a
Bio-Rad Laboratories, Inc., kit (Hercules, CA), and 50
µg protein were diluted in an equal volume of 2 x Laemmli’s
reducing sample buffer. The samples were vortexed, incubated at 95 C
for 5 min, and then loaded onto 10% polyacrylamide-bisacrylamide
midigels. After SDS-PAGE, the proteins were transferred onto
nitrocellulose membranes (Sigma). The lysates of human
sperm from healthy donors were used as controls. The transferred
nitrocellulose membranes were blocked for 3 h at room temperature
in TTBS (0.1% Tween-20, 20 mmol/liter Tris, and 150 mmol/liter NaCl)
containing 5% BSA. Thereafter, the membranes were washed repeatedly in
TTBS and incubated for 2 h in 2% BSA-TTBS containing a 1:400
dilution of the c-262 PR antibody directed against the C-terminal
region of the classical PR (32). Another antibody (-PR, at a 1:100
dilution) was also used, which recognizes the N-terminal region of the
classical PR. After washing, the membranes were incubated with goat
antimouse IgG-POD (1:5000 in 2% BSA-TTBS). The membranes were then
washed several times in TTBS, and immunoreactive proteins were
visualized by the enhanced chemiluminescence system (ECL,
Amersham Pharmacia Biotech). Alternatively, for the ligand
blot analysis, the nitrocellulose membranes were incubated for 30 min
in 3% Nonidet P-40/0.1% BSA, then for 2 h in 0.3% BSA/0.1%
Tween-20/PBS, followed by 10 min in 0.1% Tween-20/PBS, and finally
overnight in 0.3% BSA/0.1% Tween-20/PBS with or without a 10-fold
concentration of P containing 1 µmol/liter peroxidase-conjugated P
(P-POD; Sigma). Peroxidase was conjugated to P by
carboxymethyl oxime at position 3 [progesterone
3-(O-carboxymethyl)oxime-6-amino caproic acid-POD type IV].
After several washes in 0.1% Tween-20/PBS, the protein bands binding P
were visualized by ECL Western blotting kit.
Intracellular free Ca2+ measurements
To test the P actions on intracellular free
Ca2+
([Ca2+]i) concentrations,
mLTC-1 cells were plated on glass coverslips at a density of 3 x
104 cells/cm2. Cells were
loaded with 2 µmol/liter fura-2/AM (Sigma) at 37 C for
30 min in serum-free culture medium. The cells were incubated further
for 10 min with serum-free medium to wash off the unincorporated
fura-2/AM. The coverslips were taken into assay buffer (137 mmol/liter
NaCl, 5 mmol/liter KCl, 0.44 mmol/liter
KH2PO4, 4.2 mmol/liter
NaHCO3, 1.2 mmol/liter
MgCl2, 20 mmol/liter HEPES, 1 mmol/liter
CaCl2, and 10 mmol/liter glucose, pH 7.0) on ice
until [Ca2+]i
measurements were performed using the Hitachi F-2000
double wavelength fluorometer (Hitachi, San Jose, CA).
Calibration for the measurements was performed using 0.2% (wt/vol)
digitonin (Sigma) and 3 µmol/liter EGTA. The
concentration of P (1 µmol/liter) was used for
([Ca2+]i) determination.
Alternatively, mLTC-1 cells were trypsinized, centrifuged, and loaded
with fura-2/AM at 37 C for 30 min, followed by
([Ca2+]i) measurement in
the suspension, as described above.
The immediate action of P on the Ca2+ entry was studied in mLTC-1 cells plated on 24-well dishes at a density of 6 x 104 cells/well. Twenty-four hours later, cells were washed once with Ca2+-free medium (Life Technologies, Inc.), and 500 µl Ca2+-free medium containing 5 µCi/ml 45Ca2+ isotope (Ca45, NEN Life Science Products, Boston, MA) were added to each well in the presence or absence of varied doses of P (0.1–10 µmol/liter). The mLTC-1 cells were incubated for 15 min at 37 C, washed, and lysed in 500 µl 1% SDS containing 0.3 N NaOH. The lysates were counted in a ß-spectrometer (Rack ß, Wallac, Inc., Turku, Finland) after adding 1 ml scintillation liquid.
Steroid regulation of LHR messenger RNA (mRNA) expression
The mLTC-1 cells (7 x 105/well in
six-well plates) were cultured for 6 h in the presence or absence
of 10 µmol/liter P, cortisol, testosterone, estradiol,
17-hydroxyprogesterone (17-OHP), DHEA, pregnanediol,
pregnanolone, or the antiprogestin RU 486. In additional experiments,
cells were also treated for 48 h with increasing concentrations of
P (0.1–10 µmol/liter). Fifteen to 20 µg total RNA were analyzed by
Northern hybridization using an antisense
32P-labeled cRNA probe corresponding to bases
441–849 of extracellular domain of the rat LHR cDNA (33). The
hybridized membranes were exposed to x-ray films (Kodak)
for 5 days at -70 C. The intensities of the specific mRNA species were
quantified using the Tina software (Raytest, Straubenhardt, Germany)
and normalized according to the intensities of the 18S ribosomal RNA
bands in the gels stained with ethidium bromide.
[125I]Iodo-hCG binding
Radioiodination of hCG (CR-121, NIDDK) was carried out with
Na[125I]iodide (IMS 300, Amersham Pharmacia Biotech), using a solid phase lactoperoxidase method
(34). The specific activity of the labeled hormone was 30 µCi/µg,
and hCG binding studies were conducted under optimized conditions, as
described previously (25). Briefly, mLTC-1 cells were treated for
48 h in the presence or absence of 10 µmol/liter of one of the
steroids, and 3 x 105 cells were incubated
with [125I]iodo-hCG
(105 cpm/incubation) in the absence (total) or
presence (nonspecific) of 50 IU unlabeled hCG (Pregnyl,
Organon, Oss, The Netherlands). The reaction was
terminated after overnight incubation in 3 ml ice-cold Dulbecco’s-PBS
containing 0.1% BSA. After centrifugation, supernatant was discarded
by careful aspiration, and pellet was counted in a 
-spectrometer
(1260 Multigamma II, Wallac, Inc.).
Statistics
All results presented are from two to four independent
experiments (unless otherwise specified). The data were analyzed by
one-way ANOVA, followed by Duncan’s new multiple range test, and
P < 0.05 was considered statistically significant. The
results shown in the figures represent the mean ±
SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
revealed that cultured mLTC-1 cells bound [3H]P
with a high and a low affinity component, the former having a
Kd of 9.3 ± 4.6 nmol/liter and the latter
having a Kd of 284 ± 12.8 nmol/liter, in
the presence of 1 µmol/liter cortisol, to exclude the confounding
effect of glucocorticoid receptors. Determinations of the affinities of
P in the absence of cortisol or binding carried out in ice provided
similar results (data not shown). In ligand displacement assays, the
[3H]P binding was displaced by increasing
concentrations of P, 17-OHP, and DHEA in the incubation
medium. Other steroids studied, including the antiprogestin RU 486,
cortisol, and testosterone, failed to displace P binding (Fig. 1, B and
C). On the other hand, a 2-h incubation with AMG increased the specific
P binding of mLTC-1 cells (Fig. 1D).
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). However,
the cDNA fragment from the uterine RNA was the only one that was
confirmed by restriction endonuclease digestion using EcoRI
to be cleaved into the expected 100- and 300-bp fragments (data not
shown). In accordance, Southern hybridization using a
32P end-labeled nested oligonucleotide probe
revealed strong positive hybridization to the uterine sample and weak
signal to the testis, but no hybridization was found to the spleen or
mLTC-1 cell lysates. The RT-PCR products of testis and mLTC-1 cells
were subcloned into the T-vector (Promega Corp.), and
their sequencing revealed homology to phospholipase A2 (result not
shown). In contrast, restriction endonuclease digestion and sequencing
of the uterine amplicons revealed identity with the classical murine PR
(30), and the cDNA was later used as a template for PR cRNA in Northern
hybridization (see below).
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).
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c-262) known to recognize the C-terminal region of the classical
nuclear PR. This antibody is known to recognize all species of PR,
including the cell membrane PR in human sperm (35).
In sperm lysates, we observed several immunoreactive bands between 45
and 66 kDa, and proteins with 54 and 66 kDa sizes gave the strongest
signals. We also tested another antibody (-PR), which is directed
against the N-terminal domain of the classical PR. This antibody has
recently been shown to recognize a 66-kDa protein in human sperm
lysates (35). However, we observed that the c-262 antibody detected
in mLTC-1 cell lysates similar immunoreactive protein bands between 45
and 57 kDa, whereas the -PR antibody faintly recognized proteins of
the same sizes (Fig. 4A). In addition, both
antibodies identified two additional bands of similar intensities at 35
kDa, which were absent in sperm lysates and may be nonspecific.
Importantly, using either antibody, the mLTC-1 cells did not clearly
show the 66-kDa band, but had similar immunoreactive proteins at 45–57
kDa (Fig. 4A). Ligand blot analysis was then performed using P-POD as
probe to reveal which proteins in the lysates can specifically bind to
P. In both sperm and mLTC-1 cell lysates, P-POD bound largely to the
same proteins revealed by the c-262 and -PR antibodies, and most
of the bands were displaced with a 10-fold excess of free P, except for
a 50-kDa band in the sperm lysates (Fig. 4B).
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). In addition, 0.1–3 µmol/liter P
increased 45Ca2+ entry into
mLTC-1 cells after a 15-min incubation. The
45Ca2+ entry returned to
normal levels at the high (10 µmol/liter) concentrations of P (Fig. 5B).
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). In addition, DHEA and 17-OHP
displayed moderate, but significant, inhibition of the LHR mRNA signal.
The effect was confined to these steroids, as no effects were found
after 6-h treatment with the other steroids tested (Fig. 6), including
the antiprogestin RU 486. The effects of the selected steroids on
[125I]iodo-hCG binding to the mLTC-1 cells were
qualitatively similar after 48-h treatment at 10 µmol/liter
concentrations of the steroids (Fig. 7).
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show dose-dependent inhibition of LHR
mRNA content with increasing P concentrations. These results further
confirmed the previous data regarding involvement of P in the
down-regulation of LHR mRNA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results of Western blot demonstrated several PR-like immunoreactive
proteins between 45–57 kDa in the mLTC-1 cell lysates. These bands
were especially observed when an antibody raised against the
steroid-binding domain of the classical nuclear PR (c-262) was used.
This antibody is able to detect the classical PR in breast cancer cells
(32), human uterus (36), and insect cells transfected with the A form
of PR (35); however, the sizes of those reactive bands are 120, 94, and
60 kDa (35, 36). The mouse uterus PR species are also similar in size,
ranging between 66–115 kDa (30). The c-262 has been shown to
prevent P-mediated calcium influx into human sperm when added to the
incubation medium (36). Using this antibody we were able to show that
mLTC-1 cells only expressed shorter forms of PR that were close in size
to those observed in human sperm lysates (36).
The immunoreactive P-binding proteins in sperm and mLTC-1 cell lysates
were also faintly detected by another antibody (-PR) that is
directed toward the N-terminal domain of the classical nuclear PR. They
apparently represented minimal cross-reactivity and suggested the
specificity toward the C-terminal region. These findings together with
the fact that no expression of the nuclear PR mRNA was observed in
mLTC-1 cells indicated that these cells may express an unusual form of
PR. Moreover, the pattern of steroid binding suggested different ligand
specificity for the R. Interestingly, a recent study (37) demonstrated
that P binds to the specific proteins purified from porcine liver with
two binding affinities (Kd, 11 and 286
nmol/liter). The Scatchard analyses of P binding in the mLTC-1 cells
appear to have two different affinity components at similar ranges of
Kd values, i.e. 9.3 ± 4.6 and
284 ± 12.8 nmol/liter. In ligand blot assays, only some bands
completely disappeared in the presence of a 10-fold excess of cold P,
whereas others were only decreased in intensity. Although the antibody
used did not clearly recognize the 60-kDa band in the mLTC-1 cell
lysate, a protein of similar size was revealed using P-POD as the
detection system. It is suggested that some forms of PR in mLTC-1 cells
are similar, yet not identical, to the sperm membrane PR, which may be
due to differences in the structure and function of the two cell types,
as spermatozoa are terminally differentiated cells whereas mLTC-1 cells
are immortalized Leydig tumor cells. Subsequently, Western and ligand
blot analyses also confirmed the absence of classical nuclear PR in
mLTC-1 cells. As the gene of the nonclassical sperm PR is not yet
known, we are currently unable to study whether the apparent
differences in the sperm and mLTC-1 cell PR-representing molecules are
products of different genes or due to alternative splicing or
posttranslational modification of the same gene.
Similar to its reported actions in granulosa cells (20) and human sperm (3), P also induced a rapid increase in [Ca2+]i in mLTC-1 cells. Nonspecific interaction of P in increasing [Ca2+]i can be ruled out, as the ethanol concentration (0.01%) used for P dilution was without effect (data not shown). Ca2+ is a known regulator of many enzymes, including protein kinase C (PKC), where it is known to participate in its translocation to the cytoplasmic leaflet of the cell membrane (38), to be activated there by diacylglycerol. The activated PKC, in turn, serves as an interacting signal in the regulation of gene expression. In accordance, the induction of PKC is known to down-regulate LHR expression and function in Leydig tumor cells (39). The classical pathway for the nuclear PR in the regulation of LHR expression is unlikely, as the murine LHR promoter does not contain a complete P response element within appropriate distance from the transcription initiation site (40). The P-mediated increase in [Ca2+]i was observed in mLTC-1 cells cultured on coverslips, but not in trypsinized cells. This may point out that the observed P effect is mediated through a plasma membrane PR, which is disturbed after trypsinization of these cells or that other signaling pathways important for Ca2+ immobilization/influx are disturbed after this procedure. In the ovary, PR is down-regulated by elevated concentrations of P or its metabolites (41). The decrease in P binding and action upon Ca2+ entry, when measured in cells plated for 48 h, might be due to down-regulation by the accumulated high basal production of P or its metabolites.
The affinity of the mLTC-1 cell PR for its ligand may be different in cytosolic extract and whole cell preparation. The P actions on Leydig cells require high, i.e. micromolar, concentrations of this steroid, also indicating that mouse Leydig tumor cells express a special form of PR. This is logical in view of the fact that PR in Leydig cells needs elevated ligand concentrations to reach the activation threshold. The P concentration in the testis in basal in vivo conditions is far higher than that in the blood, and it reaches up to 0.3–3 µmol/liter in testes when adult rats are treated with a high (600 IU/kg) down-regulating dose of hCG (42). Therefore, despite the apparently high levels of P that are needed to elicit an effect on Leydig cells, this regulatory system can well be physiological, acting at the site of P synthesis, where very high hormone levels are attained. In this respect, the nonclassical PR in mLTC-1 cells is relatively similar to the receptor characterized in human sperm, as in both cases, P acutely increases the Ca2+ flux. Noteworthy, nonclassical binding sites for P have been demonstrated in bovine follicular and luteal membranes, whether they have been suggested to be involved in autocrine/paracrine regulation of follicular function (43, 44). We recently showed that P regulates LHR function in primary cultures of adult mouse Leydig cells (25). In addition, involvement of P inhibits LHR mRNA levels in mLTC-1 cells in a dose-response manner. Thus, our present results may not only be confined to a specific tumor cell, but also to normal Leydig cells. We also investigated the effects of selected steroids in the regulation of LHR function in mLTC-1 cells by quantitative [125I]iodo-hCG binding studies, and the pattern of effects was similar to their actions on LHR mRNA in primary adult Leydig cells (El-Hefnawy, T., unpublished observations).
Although cortisol displayed an inhibitory effect on LHR mRNA and binding [in agreement with our recent report (25)], the competition assay for P binding to mLTC-1 cells showed that cortisol does not compete with P for binding to the same R. As the glucocorticoid receptor is expressed in Leydig cells (45, 46, 47), the cortisol effects on cultured Leydig cells are apparently mediated through its own receptor and/or by an pathway independent from that of P action. Another difference between the two steroids is that corticosterone affects testicular function in an endocrine fashion originating from the adrenal gland, whereas the effect of P on Leydig cells seems to be of an autocrine nature.
In conclusion, our study sheds more light on direct P effects on Leydig tumor cells. The P binding detected in Leydig cells is different from the classical nuclear PR, but probably structurally and functionally related to the membrane variant of PR that has recently been characterized in human sperm. The signaling pathway of this R involves increased [Ca2+]i levels, through which P may regulate LHR gene regulation in Leydig cells, possibly through PKC and other Ca2+-dependent pathways. Further studies of the physiological significance will clarify the novel P actions as well as other signaling pathways involved in Leydig cells.
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Acknowledgments |
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Footnotes |
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Received March 1, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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m AC, Gustafsson JÅ, Pelto-Huikko M 1993 Localization of the glucocorticoid receptor in testis and accessory
sexual organs of male rat. Mol Cell Endocrinol 95:115–120[CrossRef][Medline]
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 C. Wang, Y.-G. Cui, X.-H. Wang, Y. Jia, A. Sinha Hikim, Y.-H. Lue, J.-S. Tong, L.-X. Qian, J.-H. Sha, Z.-M. Zhou, et al. Transient Scrotal Hyperthermia and Levonorgestrel Enhance Testosterone-Induced Spermatogenesis Suppression in Men through Increased Germ Cell Apoptosis J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 3292 - 3304. [Abstract] [Full Text] [PDF]
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 J. Cheng, S. C. Watkins, and W. H. Walker Testosterone Activates Mitogen-Activated Protein Kinase via Src Kinase and the Epidermal Growth Factor Receptor in Sertoli Cells Endocrinology, May 1, 2007; 148(5): 2066 - 2074. [Abstract] [Full Text] [PDF]
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G. Groyer, B. Eychenne, C. Girard, K. Rajkowski, M. Schumacher, and F. Cadepond Expression and Functional State of the Corticosteroid Receptors and 11{beta}-Hydroxysteroid Dehydrogenase Type 2 in Schwann Cells Endocrinology, September 1, 2006; 147(9): 4339 - 4350. [Abstract] [Full Text] [PDF]
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M. J. Walton, R. A. L. Bayne, I. Wallace, D. T. Baird, and R. A. Anderson Direct Effect of Progestogen on Gene Expression in the Testis during Gonadotropin Withdrawal and Early Suppression of Spermatogenesis J. Clin. Endocrinol. Metab., July 1, 2006; 91(7): 2526 - 2533. [Abstract] [Full Text] [PDF]
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 H. Wang, L. Zhou, A. Gupta, R. R. Vethanayagam, Y. Zhang, J. D. Unadkat, and Q. Mao Regulation of BCRP/ABCG2 expression by progesterone and 17beta-estradiol in human placental BeWo cells Am J Physiol Endocrinol Metab, May 1, 2006; 290(5): E798 - E807. [Abstract] [Full Text] [PDF]
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 B.M. Brady, J.K. Amory, A. Perheentupa, M. Zitzmann, C.J. Hay, D. Apter, R.A. Anderson, W.J. Bremner, P. Pollanen, E. Nieschlag, et al. A multicentre study investigating subcutaneous etonogestrel implants with injectable testosterone decanoate as a potential long-acting male contraceptive Hum. Reprod., January 1, 2006; 21(1): 285 - 294. [Abstract] [Full Text] [PDF]
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L. P. Ly, P. Y. Liu, and D. J. Handelsman Rates of suppression and recovery of human sperm output in testosterone-based hormonal contraceptive regimens Hum. Reprod., June 1, 2005; 20(6): 1733 - 1740. [Abstract] [Full Text] [PDF]
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 H. Youn, Y. Koo, I. Ji, and T. H. Ji An Upstream Initiator-Like Element Suppresses Transcription of the Rat Luteinizing Hormone Receptor Gene Mol. Endocrinol., May 1, 2005; 19(5): 1318 - 1328. [Abstract] [Full Text] [PDF]
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 S. Goldman, A. Weiss, I. Almalah, and E. Shalev Progesterone receptor expression in human decidua and fetal membranes before and after contractions: possible mechanism for functional progesterone withdrawal Mol. Hum. Reprod., April 1, 2005; 11(4): 269 - 277. [Abstract] [Full Text] [PDF]
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B.M. Brady, M. Walton, N. Hollow, A.T. Kicman, D.T. Baird, and R.A. Anderson Depot testosterone with etonogestrel implants result in induction of azoospermia in all men for long-term contraception Hum. Reprod., November 1, 2004; 19(11): 2658 - 2667. [Abstract] [Full Text] [PDF]
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G. Shetty, C. C. Y. Weng, O. U. Bolden-Tiller, I. Huhtaniemi, D. J. Handelsman, and M. L. Meistrich Effects of Medroxyprogesterone and Estradiol on the Recovery of Spermatogenesis in Irradiated Rats Endocrinology, October 1, 2004; 145(10): 4461 - 4469. [Abstract] [Full Text] [PDF]
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 C. Fix, C. Jordan, P. Cano, and W. H. Walker Testosterone activates mitogen-activated protein kinase and the cAMP response element binding protein transcription factor in Sertoli cells PNAS, July 27, 2004; 101(30): 10919 - 10924. [Abstract] [Full Text] [PDF]
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J. J. Peluso, A. Pappalardo, G. Fernandez, and C. A. Wu Involvement of an Unnamed Protein, RDA288, in the Mechanism through which Progesterone Mediates Its Antiapoptotic Action in Spontaneously Immortalized Granulosa Cells Endocrinology, June 1, 2004; 145(6): 3014 - 3022. [Abstract] [Full Text] [PDF]
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H. Schwarzenbach, G. Chakrabarti, H. J. Paust, and A. K. Mukhopadhyay Gonadotropin-Mediated Regulation of the Murine VEGF Expression in MA-10 Leydig Cells J Androl, January 1, 2004; 25(1): 128 - 139. [Abstract] [Full Text] [PDF]
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 R. M. LOSEL, E. FALKENSTEIN, M. FEURING, A. SCHULTZ, H.-C. TILLMANN, K. ROSSOL-HASEROTH, and M. WEHLING Nongenomic Steroid Action: Controversies, Questions, and Answers Physiol Rev, July 1, 2003; 83(3): 965 - 1016. [Abstract] [Full Text] [PDF]
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 H. Schwarzenbach, P. R. Manna, D. M. Stocco, G. Chakrabarti, and A. K. Mukhopadhyay Stimulatory Effect of Progesterone on the Expression of Steroidogenic Acute Regulatory Protein in MA-10 Leydig Cells Biol Reprod, March 1, 2003; 68(3): 1054 - 1063. [Abstract] [Full Text] [PDF]
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 R. A. Anderson and D. T. Baird Male Contraception Endocr. Rev., December 1, 2002; 23(6): 735 - 762. [Abstract] [Full Text] [PDF]
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J.J. Peluso, G. Fernandez, A. Pappalardo, and B.A. White Characterization of a Putative Membrane Receptor for Progesterone in Rat Granulosa Cells Biol Reprod, July 1, 2001; 65(1): 94 - 101. [Abstract] [Full Text] [PDF]
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