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INSERM U. 407, Communications Cellulaires en Biologie de la Reproduction (C.M., I.G., V.B., M.B.), and INSERM U. 189 (C.R., F.G.), Faculté de Médecine Lyon Sud, 69921 Oullins Cedex, France; URA INRA-CNRS 1291, PRMD, Nouzilly, France (F.D.)
Address all correspondence and requests for reprints to: Dr. C. Mauduit, Institut National de la Santé et de la Recherche Médicale U 407, Communications Cellulaires en Biologie de la Reproduction, Faculté de Médecine Lyon Sud, BP 12, 69921 Oullins Cedex, France. E-mail mauduit{at}lsgrisnl.univ-lyon1.fr
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Abstract |
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Introduction |
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Specifically, in the reproductive systems, LIF is an important cytokine in early pregnancy. Indeed, female LIF knock-out mice are infertile because of a defect in the process of embryonic implantation (5, 7, 8, 9). Cultured granulosa cells from mature follicle, but not from immature follicle, exhibit an increase in LIF production after ßhCG (ß-human CG) (10), suggesting that LIF might be involved in ovulation and final oocyte development (11). In the testis, LIF has been shown to promote primordial germ cell proliferation (12, 13, 14) and to enhance survival of gonocytes (15) and Sertoli cells (16). In the present paper, we report on a novel biological activity of this cytokine in the testis, the inhibition of Leydig cell steroidogenesis. Indeed, by using cultured porcine Leydig cells as a model, we: 1) show that LIF is a potent inhibitor of steroid hormone formation; and 2) further localize the site(s) of action of the cytokine to the transport of cholesterol substrate into mitochondria.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 6ß,
9-trihydroxy-labd-14-en-11-one), cholera toxin, insulin,
transferrin, vitamin E, HEPES
(4-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic
acid]) and deoxyribonuclease type I were purchased from
Sigma (St. Louis, MO). Oligonucleotide primers were
obtained from Genset (Paris, France), and Taq polymerase was
purchased from Promega Corp. (Lyon, France). LIF R rabbit
polyclonal antibody (sc-659) and LIF goat polyclonal antibody (sc-1339)
were purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). [3H]PK11195 was obtained from
NEN Life Science Products (Boston, MA). Horseradish
peroxidase-labeled goat or rabbit antibodies, and Covalight reagent,
were purchased from Covalab (Lyon, France).
Leydig cell preparation and culture
Isolated Leydig cells were prepared from immature porcine testes
(2–3 weeks old) by collagenase treatment as previously reported
(17). Briefly, decapsulated testes were minced and washed
in DMEM/Ham’s F-12 medium (1:1). After collagenase dissociation (0.5
mg/ml, 90 min at 32 C), cells were washed by centrifugation (200
x g for 10 min). The pellet was then resuspended and
submitted to two successive sedimentations of 5 and 15 min. The crude
interstitial cells were recovered from the supernatants, and Leydig
cells were prepared from this fraction by Percoll gradient
centrifugation. The purity of Leydig cells was more than 90%, as
determined by histochemical 3ß-hydroxysteroid dehydrogenase staining
(18). Leydig cells were plated in Falcon (Los Angeles, CA)
24-multiwell plates (0.5 x 106 cells/dish)
and cultured at 32 C in a humidified atmosphere of 5% CO2–95% air in
DMEM/Ham’s F-12 medium (1:1) containing sodium bicarbonate (1.2
mg/ml), 15 mM HEPES, and gentamicin (20 µg/ml).
This medium was supplemented with insulin (2 µg/ml), transferrin (5
µg/ml), and vitamin E (10 µg/ml). At the end of the experiments,
the culture medium was collected and stored at -20 C until assayed for
steroid hormone content.
Leydig cell steroidogenic activity
Cultured porcine Leydig cell steroidogenic activity was mainly
evaluated through the secretion of testosterone. The main
characteristic of this culture system was that the secretion of this
hormone in response to LH/hCG remains high and stable for several days
and particularly between day 2 and day 6 of culture. Because, in
cultured porcine, Leydig cells’ accumulation of unconjugated steroids
is close to linear only during the first 4 h (19
and our unpublished data), the steroidogenic capacity of these cells
was tested, after a 3-h stimulation with hCG, on day 6 of culture.
For the determination of steroidogenic enzyme activities, cultured
Leydig cells were incubated with a hydroxylated cholesterol derivative
(22R hydroxycholesterol), which can readily diffuse across the
mitochondrial membranes and the aqueous space between membranes and can
be used as a substrate for the mitochondrial cholesterol side chain
cleavage enzyme activity (cytochrome P450scc) (20). The
other steroidogenic enzyme activities were determined by incubation
with different steroid substrates: pregnenolone, DHEA, and
androstenedione. Porcine Leydig cells use the following pathway for
testosterone biosynthesis: cholesterol 
pregnenolone 17
hydroxypregnenolone DHEA 
4 androstenedione
testosterone. To assess whether LIF affects this steroidogenic pathway,
cultured Leydig cells (in the absence or presence of LIF) were
incubated with different steroid substrates i.e.
22Rcholesterol, pregnenolone, DHEA, and 4
androstenedione, which are used for testosterone synthesis.
Testosterone levels were measured in the culture medium by using a
previously reported specific RIA (21).
RNA extraction
Total RNA was extracted from porcine Leydig cells with TRIzol
reagent, a monophasic solution of phenol and guanidine isothiocyanate.
This reagent is an improvement to the single-step RNA isolation
developed by Chomczynski and Sacchi (22). The amount of
RNA was estimated, by spectrophotometry, at 260 nm.
RT-PCR analysis
Single-stranded complementary DNAs (cDNAs) were obtained from RT
of 1.5 µg total RNA using random hexanucleotides as primers (5
µM), in the presence of deoxynucleotides (200
µM), dithiothreitol (10 mM), and M-MLV (10
U/µl), for 1 h at 37 C. cDNAs (1 µl RT mixture) were amplified
by PCR with Taq polymerase (0.01 U/µl), deoxynucleotides
(50 µM), 0.75 µCi
[-33P]deoxy-ATP, and specific primers (1
µM). The mixture was first heated at 94 C for 5
min and then X cycles of 94 C for 30 sec, Tm (melting
temperature) for 30 sec, 72 C for 30 sec, then 72 C for 5 min (see
Table 1
). PCR products were analyzed on
an 8% polyacrylamide gel. Dried gels were exposed to Biomax MR-1 films
(Eastman Kodak Co., Rochester, NY) for 1–2 days at
room temperature. Intensity of bands was estimated by
densitometric scanning using the BioImage scanner (BioImage, Cheshire,
UK). The data were expressed by steroidogenic acute regulatory protein
(StAR)/ßactin or peripheral benzodiazepine receptor (PBR)/ßactin
messenger RNA (mRNA) ratio.
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Northern blotting analysis
About 15 µg total RNAs [denatured 15 min at 65 C in the
presence of formaldehyde (2.2 M), formamide (12.5
M), 1x 3(N-morpholino)propanesulfonic acid] were
electrophoresed on 1.2% agarose/2.2 M formaldehyde gels.
After migration in 0.02 M 3(N-morpholino)propanesulfonic
acid running buffer, RNAs were transferred onto a nylon membrane in
10x SSC (1.5 M NaCl, 0.15 M sodium citrate)
and fixed at 80 C for 2 h. The plasmid containing the StAR cDNA
(kindly given by Dr. D. M. Stocco, Texas Tech University Health
Sciences Center, Lubbock, TX) was linearized with SalI. The
StAR DIG-labeled riboprobe was obtained by in vitro
transcription using the Sp6 enzyme according to the manufacturer’s
recommendations. The nylon membranes were prehybridized for 1 h at
68 C, and the filters were then hybridized with StAR RNA DIG-labeled
probe (100 ng/ml) overnight at 68 C in 50% formamide, 5x SSC, 2%
blocking reagent, 0.02% SDS, and 0.1% N-lauryl sarcosin. Afterwards,
membranes were washed twice in 2x SSC, 0.1% SDS (5 min, at room
temperature), followed by 15 min at 68 C in 0.1x SSC, 0.1% SDS. The
filters were then equilibrated for 1 min in buffer 1 [maleic acid (100
mM), NaCl (150 mM), pH
7.5] and blocked for 30 min in buffer 1 containing 1% blocking
reagent. The antibody (anti-DIG AP) was diluted (1:10,000) in blocking
solution and incubated for 30 min with the filters. The membranes were
washed twice in buffer 1 (15 min, at room temperature) and equilibrated
in detection buffer [Tris (100 mM), NaCl (100
mM), pH 9.5]. Detection was performed with CPD
Star chemiluminescent substrate solution at a 1:100 dilution in
detection buffer (5 min, at room temperature). Filters were
autoradiographed for 15 min. After dehybridation [formamide (80%),
Tris-HCl (pH 8, 50 mM), SDS (1% for 2 x 30 min
at 75 C)], the filters were hybridized with an 18S RNA DIG-labeled
probe (20 ng/ml) as described above. Intensities of autoradiographic
bands were estimated by densitometric scanning using the BioImage
scanner. The data were expressed as StAR/18S mRNA ratios.
Radioligand binding assays for PBR
Leydig cell cultures were washed with medium, scraped from the
dishes, and collected by centrifugation at 180 x g for
10 min. PK 11195 was used as a ligand for PBR.
[3H]PK11195 binding studies on 20 µg of
proteins from cell suspensions in Tris-buffered saline (TBS) were
performed at 0 C in a final incubation vol of 0.25 ml, using 0.9
nM of the radiolabeled ligand. Nonspecific
binding was determined in the presence of
10-5
M unlabeled ligand. After 30 min incubation, the
assays were stopped by filtration through GF/C filters
(Whatman, Maidstone, UK) pretreated with 10
µM unlabeled ligand and washed with 15 ml
ice-cold PBS. Radioactivity trapped on the filters was determined by
liquid scintillation counting. Total binding was approximately 10% of
the total free radioligand included in the assay, and specific binding
was 90% of the total binding.
Western blot analysis
StAR protein contents were identified in isolated mitochondria;
PBR, LIF, and LIF R protein contents were identified in whole
Leydig cells. For isolation of crude mitochondria, the harvested Leydig
cells were pelleted by centrifugation at 200 x g for
10 min. The pelleted cells were resuspended in ice-cold buffer A,
consisting of 275 mM sucrose, 10
mM Tris-HCl (pH 7.4), and 1
mM EDTA (0.5 ml of buffer/75 x
106 cells) and homogenized with a Teflon
homogenizer. Cell debris and nuclei were removed from homogenates by
centrifugation at 960 x g for 15 min. The pellet was
resuspended in buffer A, homogenized, and centrifuged under the same
conditions. The supernatants were pooled and centrifuged at 8,600
x g for 15 min to yield the mitochondrial pellet. To purify
mitochondria, the crude mitochondrial pellet was suspended in isolation
buffer and centrifuged at 960 x g for 3 min. The
resultant supernatant was centrifuged twice at 8,600 x
g for 15 min.
Proteins from whole Leydig cells (30 µg) or from mitochondria (80 µg) were resolved on 5% (LIF R), 10% (LIF), or 12% (StAR, PBR) SDS/polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes using 25 mM Tris, 185 mM glycine (pH8.3) containing 20% methanol. The transfer was performed at a constant voltage of 100 V for 1 h. After transfer, the membranes were incubated in blocking buffer (TBS buffer containing 5% blocking reagent) for 1 h at room temperature. The membranes were rinsed three times with TBS and incubated with primary antibody (1/100, 1/250, 1/500, 1/1000 dilution in TBS containing 2% blocking reagent for LIF R, LIF, StAR, and PBR, respectively) overnight at +4 C. The membranes were rinsed with TBS/Tween 0.1% (3 x 10 min) and then incubated with horseradish peroxidase-labeled goat or rabbit antibodies (1/2000 degree). Bound antibodies were detected by chemiluminescence using a Covalab kit and Biomax MR-1 films (Eastman Kodak Co.).
Immunohistochemistry
Large White boars obtained from INRA (Nouzilly, France)
were electrocuted and immediately bled. Testes from 15-,
21-days-postpartum (perinatal), 3.5-months (prepubertal), and
6.5-months (adult) animals were removed and fixed overnight in 4%
paraformaldehyde in PBS and postfixed in aqueous Bouin for 6 h at
room temperature. Paraffin sections (5 µM) of
Bouin-fixed testis were mounted on glass slides. The sections were
deparaffinized and rehydrated. The UltraVision Detection System (Lab
Vision Corp., Fremont, CA) was used as recommended by the
manufacturer. Briefly, endogenous peroxidases were blocked with 3%
H2O2 for 15 min. The
sections were incubated for 5 min with a protein-blocking solution to
minimize nonspecific binding. The primary antibody was diluted (LIF,
1:100; LIF R, 1:250) in antibody diluant (DAKO Corp.,
Trappes, France) and incubated with the sections for 2 h at room
temperature. After washing and incubation with the biotinylated
secondary antibody, a peroxidases streptavidin complex was applied.
3,3'-Diaminobenzidine was used as a peroxidases chromogen.
Sections were briefly counterstained with Harris hematoxylin and
mounted in mounting medium. The same antibodies were used for
immunohistochemistry and Western blot analyses. Negatives controls were
performed by using saturated antibodies with corresponding immunogenes.
The saturation was performed by preincubating (6 h at 4 C) the usual
antibody dilution with a 5-fold concentration of the immunogene
peptide.
Data analysis
All experimental data are presented as the mean ±
SD of triplicate determinations of steroid production by
three replicate cultures within each treatment group. Triplicates were
handled as three independent values. All experiments reported here were
repeated at least three times with independent cell preparations. A
representative experiment of each series is presented. Statistical
significance between groups was determined by Student’s t
test using the StatWorks (Hyden and Son Ltd, London, UK) package on a
Macintosh computer. Differences are accepted as significant at
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The maximal and half-maximal
inhibitory effects were observed, respectively, with 10 ng/ml and 2.5
ng/ml LIF (Fig. 1A). Such an inhibitory effect of LIF was
time-dependent and was maximal (P < 0.01) after
48 h with LIF (25 ng/ml) (Fig. 1B). Finally, as shown in Fig. 1C, the inhibitory action of LIF (25 ng/ml, 72 h) was observed on
testosterone synthesis stimulated with different gonadotropin
concentrations (0.01–9 ng/ml). LIF affected the maximal steroidogenic
capacity of Leydig cells but not the ED50 of hCG
(0.1 ng/ml) required to stimulate testosterone synthesis.
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Indeed, the removal of the cytokine from Leydig cell culture medium
resulted in a gradual and progressive recovery of most of the
hCG-stimulated testosterone levels. The inhibition of hCG-induced
androgen production decreased gradually from 57% of inhibition at the
moment after the removal of LIF (day 0) to only 14% of inhibition
72 h after the removal of the cytokine (Fig. 2). Furthermore, the
number of Leydig cells cultured in the presence or absence of LIF (25
ng/ml, 72 h) was not affected (data not shown).
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). These findings suggest that
LIF interferes with cAMP production and/or action. The significant
(P < 0.002) and dramatic decline (about 60% decrease)
in testosterone biosynthesis, similarly observed in LIF-treated Leydig
cells stimulated with 8-bromo-cAMP (3 mM, 3
h) (Fig. 3B), indicates that the cytokine affects Leydig cell
steroidogenesis at a biochemical step (or steps) located beyond cAMP
formation.
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5 pregnenolone, DHEA, and 4
androstenedione. As shown in Fig. 3C
, in the presence of these
substrates, the inhibitory effect of LIF on testosterone formation was
no longer observed. These observations indicate that the potent
inhibitory effect of the cytokine on the gonadotropin action occurs at
a step(or steps) related to cholesterol transport/availability in the
mitochondria rather than to cholesterol conversion into steroid
hormones. Specifically, the disappearance of the inhibitory action of
the cytokine on testosterone formation in the presence of
22R-hydroxycholesterol suggests that LIF may exert its inhibitory
action on cholesterol substrate transport. Because two proteins (StAR
and PBR) have been mainly involved in the cholesterol transport, we
further examine the effect of LIF on their expression.
LIF inhibited, in a dose-dependent manner, StAR mRNA levels, as shown
by the RT-PCR approach. The maximal inhibitory effect
(P < 0.03) of LIF was observed with 6.6 ng/ml (Fig. 4A). The inhibitory action of LIF on StAR
expression was also time-dependent, with a maximal effect observed at
48 h (Fig. 4B). The inhibitory effect of LIF on StAR was confirmed
by identification and quantification of StAR mRNA through Northern
blotting analysis (mRNA size, 4.4 kb) (Fig. 4C) and identification of
StAR protein (30 kDa) through Western blotting (Fig. 5B). In contrast, PBR mRNA levels were
not modified by LIF at the different doses and times tested (Fig. 6, A and B). Similarly, PBR binding (Fig. 6C) and PBR (18 kDa) protein content (Fig. 5A) were not affected by LIF
at the different doses (0.07–20 ng/ml, 72 h) tested.
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, a–d). In the seminiferous tubules,
LIF was detected exclusively in Sertoli cells at 3.5 and 6.5 months
(Fig. 7, a–d). The presence of LIF mRNA (Fig. 8C) and LIF protein (Fig. 8C) were
evidenced in isolated and purified Leydig and Sertoli cells from
21-day-old porcine testes.
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, a–d). Moreover, the
LIF R was detected in isolated and purified Leydig and Sertoli cells
from 21 days, in terms of mRNA (Fig. 8C) and protein (Fig. 8B).
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and immunohistochemistry (data not shown) approaches. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The inhibitory effect of LIF on Leydig cell steroidogenesis was not exerted in a context of a toxic or deleterious action of the cytokine on the testicular steroidogenic cells. Indeed, LIF was without significant effect on Leydig cell number, and its inhibitory effect was reversible. The reversibility of LIF action was shown by the capacity of Leydig cells to recover most of their steroidogenic activity in response to gonado-tropin stimulation after removal of the cytokine. This indicates that, in the experimental conditions used in this study, LIF has a specific (noncytotoxic) regulatory action on Leydig cell steroidogenic activity. Furthermore, the inhibitory effect of LIF on the gonadotropin-stimulated testicular testosterone production may result from a direct interaction between the cytokine and the steroidogenic cells. Such an observation is supported by the identification of LIF R in postnatal porcine Leydig cells, in terms of mRNA identified through RT-PCR approach and protein identified through immunohistochemical and Western blotting analyses.
Considering the biochemical mechanisms and the molecules involved in the inhibitory effect of the cytokine on the gonadotropin-induced androgen production, the major site(s) of action of the cytokine seems to be located at a post-cAMP level. The similar dramatic decrease in hormonal production (in LIF-pretreated Leydig cells) observed after an acute stimulation with LH/hCG or with cAMP formation stimulators (e.g. cholera toxin, PGs, forskolin) indicates that the inhibitory action of LIF might be related to cAMP formation and/or action. The fact that testosterone synthesis was similarly and dramatically reduced in LIF-treated Leydig cells stimulated either with the gonadotropin or with 8-bromo-cAMP suggests a predominant action of the cytokine at a site(s) located beyond cAMP generation. Furthermore, incubation of Leydig cells with 22R-hydroxycholesterol (a cholesterol derivative that can readily diffuse across the mitochondrial membranes and the aqueous space between membranes to be delivered to P450scc) reversed most of the inhibitory effect of LIF. These observations suggest that LIF antagonizes the gonadotropin hormonal action predominantly by decreasing cholesterol substrate transport and/or availability for cytochrome P450scc activity in the inner mitochondria.
In steroidogenic tissues, and particularly in Leydig cells, cholesterol transport can be thought of as occurring in two separate processes. The first part of this process is the mobilization of cholesterol from cellular stores to the outer mitochondrial membrane, whereas the second part consists of the transfer of cholesterol from the outer to the inner mitochondrial membrane (for review, see 23, 24). It is now accepted that the rate-limiting step regulated by LH/hCG is the delivery of cholesterol to P450scc (for review, see 24). Although all the mechanisms involved in the cholesterol delivery to P450scc are not completely understood, recent years have brought enlightenment and two strong candidates: StAR and PBR. Different arguments suggest the involvement of StAR in such a process. Indeed, the StAR protein is expressed in steroidogenic tissues and is up-regulated by trophic hormones, and particularly LH/hCG, in Leydig cells (25). StAR protein has been shown to be associated with the mitochondria (26). A strong evidence for the role of StAR in cholesterol transfer was the finding that mutations in the StAR gene result in a disease named congenital lipoid adrenal hyperplasia, in which an almost-complete blockade of steroid hormone synthesis was observed (27). The generation of StAR knock-out mice showed a phenotype identical to that of the human disease (28). These data strongly support the involvement of StAR in cholesterol delivery to P450scc. Although, the mechanisms of action of StAR are not entirely known, mutation studies showed that the C-terminal region of StAR supports the cholesterol transfer function of the protein (29). Moreover, StAR is synthesized as a 37-kDa precursor and has to be phosphorylated to produce its full activity (30). Concerning PBR, although present in all tissues examined, it was found to be particularly high in steroid producing tissues (for a review, see 31). In these tissues, PBR is primarily localized in the outer mitochondrial membrane and preferentially located in the outer/inner membrane contact sites involved in the cholesterol delivery to P450scc (32). It has been shown that mitochondrial ligand binding to PBR is sensitive to hormone treatment (33). Moreover, targeted disruption of the PBR gene in Leydig cells resulted in the arrest of cholesterol transport into mitochondria and steroid formation, whereas transfection of the mutant cells with PBR cDNA rescued steroidogenesis (34). These data suggest that PBR is involved in cholesterol transfer to the mitochondrial inner membrane, and molecular modeling of PBR suggests that it might function as a channel for cholesterol (35). Recently, a consensual hypothesis suggested that PBR could interact with StAR to promote cholesterol transfer (24).
In the present study, we showed that LIF was an inhibitor of StAR
expression at the levels of mRNA and protein. In contrast, LIF did not
modulate PBR mRNA expression, PBR binding, and PBR protein contents.
Thus, the inhibitory action of LIF on LH/hCG-induced testosterone
synthesis might be mainly attributable to a decrease in StAR
expression. In addition, we showed in our porcine Leydig cell model
that, in contrast with the murine model (33), hCG did not
enhance PBR expression. These discrepancies could be attributed to
interspecies specificity but could not be accounted for by an absence
of PBR regulation in porcine Leydig cells, because we recently
demonstrated that PBR expression is increased after TNF treatment in
porcine-cultured Leydig cells (36). It is of interest to
note that different recent reports have indicated that StAR expression
is targeted by a large number of molecules that affect
gonadotropin-induced testosterone formation in Leydig cells. Different
cytokines, including transforming growth factor ß (our unpublished
data), IL-1ß (37), tumor necrosis factor-
(38), and interferon
(39), have been
reported to exert their inhibitory action on LH/hCG-induced
testosterone production through a decrease in StAR expression.
Moreover, injection of LPS, which is a potent inductor of cytokine
secretion, decreases StAR expression in Leydig cells
(40). Other signaling molecules, such as insulin-like
growth factor I (41), CRH (42),
T3 (43), GH (44), and
retinoic acid (45), enhanced LH/hCG-induced testosterone
formation by increasing StAR expression. Finally, more recently,
pesticides [such as lindane (46) and dimethoate
(47)] have been shown to inhibit Leydig cell
steroidogenesis through a decrease in StAR expression.
LIF and IL-6 family-related cytokines bind to specific cell surface receptors that are coupled to a common gp 130 signal transducing receptor component in diverse cell types and induce the homodimerization of the gp 130 protein with the IL-6 receptor chain (in the case of IL-6) or heterodimerization of gp 130 and the LIF R component (in the case of LIF, ciliary neurotrophic factor, cardiotrophin, and oncostatin M). Upon binding to these receptors, the cytokine triggers a rapid intracellular tyrosine phosphorylation of the gp 130 protein, Janus kinase (JAK) 1, JAK 2, and tyrosine kinase 2. These phosphorylations activate the signal transducer and activator of transcription (STAT-3/STAT-1) signal transduction (for review, see 48, 49). Although in the present report we have shown that LIF R is expressed in testicular Leydig cells, the transducing systems used by LIF to regulate StAR expression remain to be identified. Considering the mechanisms involved in the negative effect of LIF on StAR mRNA levels, it might be attributable to a decrease in the transcriptional activity and/or mRNA stability. LIF may affect StAR gene expression through interactions with transcriptional factors that might bind to the StAR gene promoter. In this context, LIF may antagonize cAMP production and/or cAMP action (the present data) and therefore the induction of proteins that regulate StAR gene transcription such as SF-1 (steroidogenic factor-1), previously shown to control StAR gene expression (50, 51, 52).
Whether the in vitro data presented here reflect a potential physiological or pathological role of LIF in the male gonad function required further studies. To answer this question, we have performed, as a first step, the identification of LIF and LIF R proteins during porcine testis development at three critical periods: neonatal, prepubertal, and adult periods. LIF and LIF R proteins were identified by using immunohistochemical and Western blot analyses. During testicular development, immunoreactive LIF and LIF R were identified in Leydig and Sertoli cells but not in the other testicular cell types, except for the presence of LIF R in spermatogonia. By using Western blotting analysis, the presence of LIF and LIF R proteins was confirmed in porcine Leydig and Sertoli cells as early as 21 days post partum. To our knowledge, this is the first report on the in situ localization of LIF and LIF R proteins in the different testicular cell types during the postnatal development. Furthermore, in these testicular cells, we confirmed the expression of the genes through the presence of the LIF and LIF R mRNA by using the RT-PCR approach. LIF was detected in porcine Sertoli cells at 21 days post partum by RT-PCR and Western blot analyses but not by immunohistochemistry. These discrepancies could be attributable to a lower sensitivity of the immunohistochemical approach. Studies from other laboratories have also been performed to identify LIF and LIF R mRNA through RT-PCR and LIF bioactivity in the postnatal testicular cells (16, 53). These authors first isolated the different testicular cell types and then performed LIF and LIF R detection on each cell population (16, 53). Concerning LIF mRNA, it has been detected in Leydig cells, Sertoli cells, spermatogonia (16, 53), peritubular myoid cells, and macrophages (53) in rat and mouse models (16, 53). By using a bioassay, LIF activity was detected in peritubular myoid cells and Leydig cells under basal conditions (53). Concerning LIF R mRNA, it has been detected in Leydig cells, Sertoli cells, and pre- and postmeiotic germ cells (16). In our study, we also detected LIF R in germ cells but only in spermatogonia. These discrepancies could be attributable to a difference in sensitivity between the techniques used (immunohistochemistry vs. RT-PCR) or could be attributed to interspecies variations (porcine vs. murine models). Indeed, Jenab et al. (16) detected the LIF R mRNA in murine germ cells but not in rat germ cells. The LIF R detected in porcine testes was the membrane-bound form, because the primers used amplified only this form and because soluble LIF R was not detected in the testis (54). The gp130 protein, which is a LIF R partner for signal tranduction, was detected in rat Leydig, Sertoli, and germ cells (16), suggesting that LIF could directly interact with Leydig cells.
The presence of LIF and its receptor in both the neonatal and adult Leydig cells suggests that the inhibitory action of LIF on testosterone synthesis may occur during the testicular development in the fetal and adult Leydig cell populations. In this context, it will be of interest to determine how testicular LIF and LIF R expressions are modulated in physiological or pathological conditions. Specifically, the role of the gonadotropins and other local testicular signaling molecules affecting LIF and its receptors are currently being investigated in our laboratory.
In summary, by using cultured purified porcine Leydig cells as a model, this study has evidenced a novel role of LIF. The cytokine antagonizes the gonadotropin steroidogenic action at post-cAMP level(s) and, more specifically, at the level of the expression of StAR, a protein involved in cholesterol transport/availability into mitochondria.
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Acknowledgments |
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Footnotes |
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Received September 18, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 R.-S. Ge, Q. Dong, C. M. Sottas, H. Chen, B. R. Zirkin, and M. P. Hardy Gene Expression in Rat Leydig Cells During Development from the Progenitor to Adult Stage: A Cluster Analysis Biol Reprod, June 1, 2005; 72(6): 1405 - 1415. [Abstract] [Full Text] [PDF]
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I. Dorval-Coiffec, J.-G. Delcros, H. Hakovirta, J. Toppari, B. Jegou, and C. Piquet-Pellorce Identification of the Leukemia Inhibitory Factor Cell Targets Within the Rat Testis Biol Reprod, March 1, 2005; 72(3): 602 - 611. [Abstract] [Full Text] [PDF]
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 A Bozec, F Chuzel, S Chater, C Paulin, R Bars, M Benahmed, and C Mauduit The mitochondrial-dependent pathway is chronically affected in testicular germ cell death in adult rats exposed in utero to anti-androgens J. Endocrinol., October 1, 2004; 183(1): 79 - 90. [Abstract] [Full Text] [PDF]
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 A. Omezzine, S. Chater, C. Mauduit, A. Florin, E. Tabone, F. Chuzel, R. Bars, and M. Benahmed Long-Term Apoptotic Cell Death Process with Increased Expression and Activation of Caspase-3 and -6 in Adult Rat Germ Cells Exposed in Utero to Flutamide Endocrinology, February 1, 2003; 144(2): 648 - 661. [Abstract] [Full Text] [PDF]
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) or
presence (
) of LIF (25 ng/ml), and then simulated with hCG (3 ng/ml,
3 h) (B); for 72 h, in the absence (
) or presence (•) of
LIF (25 ng/ml), and then stimulated with increasing doses of hCG
(0.01–9 ng/ml, 3 h) (C). The results represent the mean ±
SD of three separate determinations in three different
dishes.
4 androstenedione). Leydig cells were cultured in
the absence (
