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From the * Department of Histology and Medical
Embryology, School of Medicine, "Sapienza" University of Rome,
Rome, Italy; and Department of Experimental
Medicine, Histology and Embryology Laboratory, School of Medicine, Second
University of Naples and National Institute of Biostructures and Biosystems
Interuniversitary Consortium—Unit of Sections of Naples, Naples,
Italy.
| Correspondence to: Michela Galdieri, Dip. Istologia ed Embriologia Medica, Via A. Scarpa 14, Roma 00161, Italy (e-mail: michela.galdieri{at}uniroma1.it). |
| Received for publication March 19, 2007; accepted for publication June 18, 2007. |
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Abstract |
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Key words: c-met, HGF, testosterone, caspase-3, apoptosis, testis
Leydig cells, the testosterone-producing cells of the mammalian testis, are differentiated cells that rarely proliferate in the adult (De Kretser and Kerr, 1994; Saez, 1994). During postnatal development of the testis, Leydig cells undergo a series of morphologic and functional transformations and act as proliferating precursors in prepuberal rats and as immature and mature adult Leydig cells in puberal rats (Ariyaratne and Mendis-Handagama, 2000; Haider, 2004). In both stages, the cells are able to produce testosterone but synthesize different levels of the hormone (Haider, 2004). Leydig cell proliferation and endocrine functions are regulated by several hormones, including interleukins (Svechnikov et al, 2001; Walch and Morris, 2002), transforming growth factor ß (Khan et al, 1992; Dickson et al, 2002), insulin-like factors (Khan et al, 1992; Ge and Hardy, 1997), and ghrelin (Barreiro et al, 2004). It is also known that Leydig cells enter the programmed cell death pathway in particular situations such as in response to ethylene dimethanesulfonate (EDS) (Kerr et al, 1985; Morris et al, 1997; Kim et al, 2000) or glucocorticoids (Gao et al, 2002), and the regulation of apoptosis could play an important role in the maintenance of the proper number of Leydig cells. We recently demonstrated that in the embryonic testes HGF regulates testosterone production in fetal Leydig cells (Ricci et al, 2006). Interestingly, HGF increases the amount of testosterone secreted in the culture medium of in vitro cultures of testes isolated from 18.5-day embryos but does not modulate the amount of testosterone secreted by testes isolated from 15.5-day embryos (Ricci et al, 2006).
In this article, we report that c-Met is expressed by rat Leydig cells isolated from puberal rats, c-Met protein is present on the cells, and the receptor is functionally active. We demonstrate that HGF modifies several metabolic activities of these cells, including their steroidogenetic activity.
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Materials and Methods |
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Leydig Cell Isolation and Culture
Leydig cells were isolated as previously reported
(Morris et al, 1997) with
slight modifications. Briefly, decapsulated testes were incubated with minimum
essential medium (MEM; Invitrogen srl, Milan, Italy) containing 0.25 mg/mL
collagenase or trypsin (0.18%) at 32°C in a shaking water bath (90
cycles/min) for 15 minutes. After dissociation, the enzyme was diluted with
culture medium, and the seminiferous tubules were removed by gravity
sedimentation (4 minutes). Tubules were washed again to detach the
interstitia, and the two supernatants were collected and centrifuged at 300
x g for 10 minutes at room temperature. The pellet was
resuspended in MEM containing 0.1% bovine serum albumin (BSA) and 0.01% DNase,
and the cell suspension was loaded on a discontinuous Percoll gradient
(20%–86% Percoll) and centrifuged at 800 x g for 20
minutes at 18°C. After centrifugation, fractions at 1.056 and 1.068 g/mL
were collected, washed with buffer, and counted. Isolated Leydig cells were
resuspended in MEM culture medium containing 15 mM HEPES, nonessential amino
acids, 5 µg/mL gentamicin, 100 U/mL penicillin, and 100 µg/mL
streptomycin. Cells were seeded on Falcon culture plates (Becton, Dickinson
and Co, Lincoln Park, NJ) at a concentration of 0.5 to 0.7 x 106
cells/mL of medium. Viability of Leydig cells was assessed by the trypan blue
dye exclusion method. Briefly, isolated Leydig cells were mixed with an equal
volume of 0.4% trypan blue (Flow Laboratories, Irvine, United Kingdom),
incubated for 5 minutes at 37°C, and examined under a microscope. After 24
hours of incubation, Leydig cells were almost totally viable. To assess the
effects of HGF in vitro, cells were cultured for 26 hours at 32°C in a
humidified 5% CO2–95% air atmosphere in the presence of the
growth factor (100 U/mL–30 ng/mL) for the last 24 hours of culture.
Evaluation of Isolated Leydig Cell Purity
Purity of the Leydig cell preparation was routinely assessed on the basis
of positive staining of cells for the enzyme 3ß-hydroxy steroid
dehydrogenase (HSD) (Payne et al,
1980). Briefly, an aliquot of Leydig cell fraction was added to a
tube containing 0.5 mL of ß-NAD (9 mg/mL; Sigma-Aldrich, St Louis, Mo),
0.2 mL of dehydroepiandrosterone (1 mg/mL in methanol), and 0.25 mL of
nitroblue tetrazolium (2 mg/mL in phosphate buffer [pH 7.4]; Sigma-Aldrich).
The reaction mixture was allowed to stand for 1 hour at 37°C. The
percentage of positively (dark blue) stained cells was examined under the
microscope. The cell purity was consistently higher than 90%. The purity of
our cell populations was also evaluated by immunocytochemistry for cytochrome
P450 side-chain cleavage (P450scc). The cells were paraformaldehyde fixed,
washed extensively with phosphate-buffered saline (PBS) supplemented with 1%
BSA and 0.2% Triton X-100, and incubated for 30 minutes in PBS containing 10%
goat serum. Cells were then incubated with a polyclonal antibody against
P450scc (AB1294; 1:200 dilution; Chemicon International, Temecula, Calif) for
16 hours at 4°C. At the end of the incubation period, the cells were
washed extensively with PBS and incubated for 45 minutes at room temperature
with a fluorescein isothiocyanate–conjugated goat anti-rabbit antiserum
(Sigma-Aldrich). Cells were rinsed again with PBS and mounted in buffered
glycerol (pH 9). As a negative control, the primary antibody was omitted and
substituted with rabbit IgG.
Organ Culture
Fragments of approximately 1 mm3 were isolated from 8 testes of
30- to 32-day-old rats and placed on steel grids previously coated with 2%
agar. Grids were then placed in organ culture dishes (Becton, Dickinson and
Co) with 0.8 mL of medium necessary to wet the grid. The chemically defined
medium is used in Leydig cell isolation and culture section. HGF (150 U/mL;
Sigma-Aldrich) was added to the culture medium when indicated. Samples were
cultured for 24 hours at 32°C in a humidified atmosphere of 5%
CO2 in air. After culture, the samples were washed twice in PBS,
fixed overnight in Bouin fixative, dehydrated, embedded in paraffin, sectioned
at a thickness of 7 µm, and used in the in situ terminal deoxynucleotidyl
transferase biotin-dUTP nick end labeling (TUNEL) assay. At least three
experiments in triplicate were performed, and the number of TUNEL-positive
interstitial cells/500 transverse tubules was evaluated. The morphology of the
samples appeared well preserved in all of the experiments.
Immunocytochemistry
Leydig cells prepared as indicated above were fixed in methanol according
to the antibody manufacturer's recommendation for 10 minutes at
–20°C, treated with 5% BSA (Sigma-Aldrich) for 30 minutes at room
temperature to minimize nonspecific binding, and then exposed to a polyclonal
antibody against the carboxy terminus of c-Met (sc-162; 1:50 dilution; Santa
Cruz Biotechnology, Santa Cruz, Calif) for 16 hours at 4°C. At the end of
the incubation period, the cells were washed extensively with PBS and
incubated for 45 minutes at room temperature with a fluorescein
isothiocyanate–conjugated goat anti-rabbit antiserum (Sigma-Aldrich).
The cells were rinsed again with PBS and mounted in buffered glycerol (pH 9).
As a negative control, the primary antibody was omitted and substituted with
rabbit IgG or 10-fold excess by weight of a blocking peptide (sc-162P; Santa
Cruz Biotechnology). Samples were analyzed using a Zeiss Axioplan fluorescence
microscope (Oberkochen, Germany).
RNA Isolation and Reverse Transcription Polymerase Chain Reaction Analysis
Total RNA was extracted from cultured cells using a Perfect RNA Eukaryotic
Mini kit (Eppendorf, Hamburg, Germany). The purity of isolated mRNA was
evaluated spectrophotometrically using the A260/280 ratio. To
reduce contamination by genomic DNA, total RNA was treated with
ribonuclease-free DNase I for 15 minutes as recommended by the manufacturer
(Invitrogen srl). Samples of total RNA (200 ng) were reverse transcribed with
reverse transcriptase (Invitrogen srl) in the presence of oligo(dT) primers
(Invitrogen srl) for 50 minutes at 37°C, and the reaction was terminated
by heating for 15 minutes at 70°C. Polymerase chain reaction (PCR) was
performed using HotMaster Taq DNA polymerase (Eppendorf) and the following
primers: c-met sense 5'-AATGTGTCAGGAGGTGTTTGG-3' and
antisense 5'-GAATAATCGGGAGGGTAGGAAG-3', S-16 sense
5'-TCCAAGGGTCCGCTGCAGTC-3' and antisense
5'-CGTTCACCTTGATGAGCCCAT-3'. The amplification program for
c-met consisted of one denaturing cycle at 94°C for 5 minutes,
followed by the following steps: 35 cycles of amplification defined by
denaturation at 94°Cfor 45 seconds, annealing at 57°C for 45 seconds,
and extension at 72°C for 1 minute. The final incubation was performed at
72°C for 5 minutes. The amplification program for S-16 was
similar with the exceptions of the number of cycles of amplification (30) and
annealing temperature (60°C). Negative controls contained water instead of
cDNA. PCR with no reverse transcriptase produced no product, eliminating the
possibility of genomic DNA contamination in the RNA preparations. PCR products
were separated by 2% agarose gel electrophoresis, visualized by ethidium
bromide staining, and quantitated by computer analysis. Reverse transcription
PCR (RT-PCR) analysis was conducted with primers for S-16 to be sure
that equal amounts of cDNA were used. A DNA ladder was included in the gels to
determine the sizes of the PCR products.
Testosterone Evaluation
Leydig cells were cultured for 24 hours in the presence of different doses
of HGF (25–300 U/mL), and the culture medium was tested for testosterone
levels by radioimmunoassay. The Access immunoassay system (Beckman Coulter
Inc, Fullerton, Calif) was used for the testosterone determination
(Wilson and Foster, 1992).
TUNEL Assay
Leydig cells were cultured for 24 hours in the presence of different doses
of HGF (25–300 U/mL). Apoptotic cells were detected by the TUNEL method
using the ApopTag Peroxidase kit (Q-BIOgene, Irvine, Calif). As positive
controls, Leydig cells or testicular sections were treated with DNase I. We
omitted the terminal deoxynucleotidyl transferase enzyme in the reaction
mixture for negative controls. The samples were counterstained with hemalum
and analyzed using a Zeiss Axioscope microscope. Four experiments in duplicate
were performed, and the number of TUNEL-positive cells/3000 cells was
evaluated.
Protein Extraction and Western Blot Analysis
Freshly isolated Leydig cells were lysed with ice-cold PBS (pH 7.4)
containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease
inhibitor cocktail (1:100 dilution; Sigma-Aldrich). Lysates were sonicated,
and the protein content was determined by s bicinchoninic acid protein assay
(Pierce Biotechnology, Rockford, Ill). Equal amounts of protein (40 µg)
were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes.
After the membranes were blocked, they were incubated for 16 hours at 4°C
with 0.2 mg/mL rabbit polyclonal anti-human caspase-3 antibody that recognizes
full-length caspase-3 and proteolytic fragments (Upstate Biotechnology, Lake
Placid, NY) and then incubated for 60 minutes at room temperature with a
horseradish peroxidase–conjugated secondary antibody (1:7000 dilution;
Amersham Biosciences UK Ltd, Buckinghamshire, United Kingdom). Peroxidase
activity was visualized with the SuperSignal West Pico Trial kit (Pierce
Biotechnology) according to the manufacturer's instructions. The caspase-3
protein content was determined densitometrically. The nitrocellulose membranes
were also probed with an anti-tubulin monoclonal antibody (Sigma-Aldrich).
Statistical Analysis
All experimental data were expressed as 
± SE of at least 3 separate experiments. Statistical analysis was
performed by Student's t-test. Differences were considered
significant at P < .05. Analysis of variance followed by Duncan's
test for multigroup comparison was also employed to evaluate the significance
of differences.
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Results |
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HGF Effects on Leydig Cells
c-met Expression—
To evaluate the effect of HGF on c-met mRNA expression levels,
purified cell preparations of Leydig cells were cultured for 24 hours in
control medium or in medium supplemented with HGF (100 U/mL) and total RNA was
extracted. RT-PCR was performed, and RNA was extracted from the livers of the
same rats used for Leydig cell isolation to be used as positive controls. We
found that c-met mRNA levels were not changed after incubation with
HGF (Figure 3B).
Testosterone Production— The potential functional role of HGF signaling in the control of testicular function was explored. To this end, Leydig cells were cultured for 24 hours and the culture media of cells cultured in medium alone or supplemented with HGF were used to evaluate the amount of testosterone secreted. The media were collected and testosterone levels determined by radioimmunoassay; the relative amounts of testosterone secreted in the 7 different experiments are reported in Figure 4A. The results indicated that the amounts of testosterone secreted by the cells cultured in the presence of HGF (100 U/mL) are significantly higher with respect to the control samples. We also present the dose-response curve of testosterone produced in the presence of different doses of HGF (25–300 units/mL) (Figure 4B). The amount of testosterone produced was significantly higher at doses of HGF ranging from 50 to 300 U/mL, whereas the dose of 25 U/mL did not produce a significant increase in hormone production.
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The effect of HGF on testosterone production was also studied by culturing small explants of testicular tissue prepared as described above (Figure 4C). The explants cultured for 24 hours in medium supplemented with HGF produced significantly higher amounts of testosterone with respect to the control explants.
Apoptosis— Four separate experiments were performed to investigate the role of HGF on Leydig cell apoptosis. Leydig cells were cultured for 24 hours in medium alone or medium supplemented with HGF (100 U/mL). In Figure 5A, the morphologic appearances of the control (C) and HGF-treated Leydig cells (HGF) are shown. Leydig cells undergoing apoptosis are evident for the brown, morphologically abnormal nuclei (arrows). The number of apoptotic cells was not high; however, by counting the apoptotic cells under both culture conditions, we detected an antiapoptotic effect of HGF. As shown in Figure 5B, the number of apoptotic cells was significantly lower when Leydig cells were cultured in the presence of HGF. Different doses of HGF (25–300 units/mL) were also used to evaluate the antiapoptotic effect of HGF (Figure 5C), and the results obtained demonstrate that HGF significantly reduces the number of apoptotic cells starting from the dose of 50 U/mL. Apoptosis of Leydig cells was also evaluated by culturing small explants of testicular tissue. Testicular samples were incubated for 24 hours in fresh medium or medium containing HGF (150 U/mL), and Figure 6A shows the morphology of the cultured tissues. After culture, the number of apoptotic cells present in the interstitial tissue, defined by 500 transverse seminiferous tubules, was evaluated in both samples. As shown in Figure 6B, the number of apoptotic cells is strongly reduced in the presence of the growth factor.
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Caspase-3 Expression— The expression of caspase-3 mRNA in Leydig cells cultured in control medium and in medium supplemented with HGF (100 U/mL) was evaluated by RT-PCR (Figure 7A), and the results indicated that gene expression is not modified by the HGF treatment. On the contrary, Western blot analysis of the proteins extracted from control and HGF-treated Leydig cells (Figure 7B) showed that HGF significantly reduced the amount of the 17-kd active fragments as shown in Figure 7C. A statistically significant variation in the inactive form of the enzyme was not detected as reported in Figure 7D, which summarizes the amount of the inactive and active form of caspase-3 obtained in control and HGF-treated cells.
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Discussion |
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Considering the relevance of the apoptotic process in the regulation of the number of Leydig cells, we then evaluated the role of HGF in this process and our experiments clearly demonstrated that HGF prevents apoptosis of cultured Leydig cells. Culturing explants of testicular tissue, we also found that HGF strongly reduces the number of apoptotic interstitial cells. We cannot ascribe the effect exclusively to Leydig cells; however, the finding that testosterone is highly secreted under this culture condition allows us to conclude that, besides other interstitial cells, Leydig cells are protected against apoptosis by HGF. Caspases are enzymes that play a critical role in the execution of apoptosis in a number of different cell types (Villa et al, 1997). Most of them are synthesized as inactive proenzymes that are processed to active forms in cells undergoing apoptosis (Nuñez et al, 1998). Among the caspases, caspase-3, one of the effector caspases, appears to be a key protease in the apoptotic pathway (Porter and Janicke, 1999): activated caspase-3 targets DNA fragmentation factor, which is integrally involved in degrading DNA (Liu et al, 1997; Nagata, 1997). It has been reported that caspase-3 is associated with testicular germ cell apoptosis (Kim et al, 2001) and with Leydig cell apoptosis induced by EDS (Kim et al, 2000). Therefore, we studied the effect of HGF on caspase-3 gene expression, and we found that treatment with the growth factor does not modify the expression levels of caspase-3 mRNA. On the contrary, HGF appears to affect the activation of the enzyme because we found by Western blot that the amount of the active fragment of the enzyme was significantly reduced when Leydig cells were cultured in the presence of HGF. We reported that in the rat testis peritubular myoid cells secrete HGF (Catizone et al, 1999, 2001, 2005); therefore, myoid cells could be the source of the factor. However, it is unknown now which of the cells of the interstitial compartment secrete HGF. Further studies are necessary to clarify this point.
In conclusion, we demonstrated that rat Leydig cells express the receptor of HGF, c-Met, and that the receptor is functionally active because HGF influences the steroidogenic pathway and significantly increases the amount of testosterone secreted by Leydig cells. Moreover, HGF decreases the number of Leydig cells undergoing apoptosis, and the antiapoptotic effect of HGF is mediated by caspase-3 activity because amounts of the active fragment of the enzyme are decreased in Leydig cells cultured in the presence of HGF. On the contrary, treatment with the growth factor does not modify the expression levels of c-met and caspase-3 mRNA. All of these data indicate that HGF regulates the functional activities of Leydig cells, the steroidogenetic cells of the interstitial compartment of the testis.
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Footnotes |
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