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Biology of Reproduction 65, 1732-1742 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Somatostatin Inhibits Stem Cell Factor Messenger RNA Expression by Sertoli Cells and Stem Cell Factor-Induced DNA Synthesis in Isolated Seminiferous Tubules1

Isabelle Goddarda, Sylvian Bauerb, Alain Gougeona, Frédéric Lopezc, Nathalie Giannettid, Christiane Susinic, Mohamed Benahmeda, and Slavica Krantic2,a

a Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 407, Faculté de Médecine Lyon Sud, F-69921 Oullins, France b Neurosciences et Systèmes Sensoriels, Université Claude Bernard-Lyon 1, F-69622 Villeurbanne, France c Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 531, Institut Fédératif de Recherche 31, Centre Hospitalier Universitaire Rangueil, F-31403 Toulouse, France d Laboratoire de Neurosciences, Unité de Formation et de Recherche de Franche-Comté, F-25030 Besançon, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Immature porcine Sertoli cells have been reported to be targets for the regulatory peptide somatostatin (SRIF), which inhibits the basal and FSH-induced proliferation of Sertoli cells through a decrease of cAMP production. In the present study, we show that SRIF inhibits both basal and FSH-stimulated expression of the stem cell factor (SCF), a Sertoli cell-specific gene. The SRIF-mediated inhibition of forskolin-triggered, but not of 8-bromoadenosine-cAMP-triggered, SCF mRNA expression demonstrates the involvement of adenylyl cyclase in underlying peptide actions. Moreover, these effects require functional coupling of specific plasma membrane receptors to adenylyl cyclase via inhibitory G proteins, because pertussis toxin prevents SRIF-mediated inhibition of SCF mRNA expression. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assays suggest the involvement of sst2 receptors in SRIF actions on Sertoli cells. The biological relevance of these data is supported by an SRIF-mediated decrease in SCF-induced incorporation of [3H]thymidine in isolated seminiferous tubules. In situ hybridization and confocal microscopy show that, in seminiferous tubules only, spermatogonia display both c-kit and sst2 receptors. Taken together, these results suggest that SCF-stimulated DNA synthesis can be inhibited by SRIF in spermatogonia, but not in Sertoli and peritubular cells. Combined RT-PCR and immunohistochemical approaches point toward spermatogonia and Leydig cells as the source of testicular SRIF. These data argue in favor of paracrine/autocrine SRIF actions in testis.

cAMP, cytokines, gene regulation, Leydig cells, Sertoli cells, signal transduction, somatostatin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During spermatogenesis, the permanent mitotic renewal of spermatogonia pools allows maintenance of their stock in immature animals and continuous spermatogenesis during adulthood. Underlying mechanisms related to such a biological process remain poorly understood, and in particular, local regulators that are able to restrain or inhibit spermatogonial proliferation remain unknown [1].

In contrast, spermatogonia-related stimulatory factors have been studied in greater detail, and among them, the roles of stem cell factor (SCF) or Steel gene product are well known (for review, see [2]). Involvement of SCF in regulation of spermatogonial proliferation and survival is unequivocally demonstrated by a complete depletion of primordial germ cell lineage in mice bearing Steel gene mutation [3]. Deletions or mutations in White spotting (W) locus encoding the SCF receptor c-kit lead to a phenotype similar to that observed with Steel locus mutations (i.e., animals are sterile [4]). This corroborates the essential role of SCF/c-kit signaling in the size control of primordial germ cell and spermatogonia pools in fetal and adult gonads, respectively. The testicular SCF/c-kit signaling system displays a cell type-specific pattern of expression: The receptor is localized on spermatogonia and Leydig cells, but not on Sertoli cells, whereas the ligand is exclusively produced by Sertoli cells [2].

Testicular expression of SCF by Sertoli cells encompasses two biologically relevant forms. They are produced by alternative splicing of SCF mRNA, yielding either soluble (SCFs) or transmembrane (SCFm) forms. Their relative proportion is a function of the developmental stage: SCFm is predominantly expressed throughout fetal, pubertal, and adult life, whereas SCFs is the major form expressed during the perinatal period [5]. Involvement of exogenously added SCF in the regulation of survival and proliferation of spermatogonia has been explicitly demonstrated [6–8]. However, the precise biological role of SCFs remains poorly understood.

The hormonal mechanisms regulating alternative splicing of SCF mRNA also are not completely elucidated [2]. Nevertheless, growth hormone-releasing factor and FSH are both able to increase the overall SCF mRNA expression [9, 10]. The hormonal induction of SCF transcription is mimicked by drugs that trigger an increase in the intracellular cAMP content [9–12]. This raises the possibility for negative regulation of SCF mRNA expression by agonists that target Sertoli cells and decrease the intracellular cAMP concentration.

Our recently reported data on somatostatin (SRIF), a regulatory peptide with well-documented hormonal and modulatory roles [13], suggest that it may be a putative candidate for such an agonist. Indeed, SRIF14 inhibits basal and FSH-stimulated adenylyl cyclase activity in immature porcine Sertoli cell cultures [14]. Moreover, these inhibitory effects correlate with SRIF-dependent inhibition of basal and FSH-stimulated proliferation of Sertoli cells [14]. In the present study, we therefore focused on a possible regulation of spermatogonial proliferation by SRIF. The indirect SRIF actions on spermatogonial proliferation were addressed by analyzing spermatogonia-specific growth factor SCF expression by Sertoli cells in the presence and absence of SRIF. Direct SRIF actions were studied by measuring its impact on SCF-induced [3H]CH3-thymidine and 5-bromodeoxyuridine (BrdU) incorporation in seminiferous tubule explants. We also searched for cellular sources of intratesticular SRIF to corroborate its role as a local regulator. The relevant experiments were performed using perinatal porcine testis as a model, because at this developmental stage, Sertoli cells and spermatogonia are the only two cell types present in the seminiferous tubule and SCFs is the predominant form expressed, thus allowing us to examine its role specifically.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals

Dulbecco modified Eagle medium (DMEM)/Ham F-12 medium, Trizol reagent, Moloney murine leukemia virus (MMLV) reverse transcriptase, nitroblue tetrazolium chloride (NBT), and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (BCIP) were obtained from Life Technologies (Eragny, France). Taq Polymerase was from Promega (Charbonière, France). The [3H]CH3-thymidine (88.7 Ci/mmol) was from Dupont-NEN (Les Ulis, France). Complete, mini-EDTA-free protease inhibitor cocktail and collagenase dispase were purchased from Boehringer Mannheim (Meylan, France). Somatostatin SRIF14 was obtained from Peninsula Laboratories (San Carlos, CA). Recombinant human SCF was from Genzyme (Cambridge, MA). Porcine FSH (USDA-pFSH-B-1) was generously provided by the U.S. Department of Agriculture Animal Hormone Program, Beltsville Agricultural Research Center (Beltsville, MD). Digoxigenin-11-uridine-5'-trisphosphate (digoxigenin-UTP), proteinase K, EcoRI, and HindIII were obtained from Roche Diagnostics (Mannheim, Germany). If not otherwise stated, the products used in immunohistochemical studies were from Dako (Trappes, France). All other chemicals were purchased from Sigma (L'Isle d'Abeau, France).

Antibodies

Polyclonal anti-sst2 antibody was generated in rabbits immunized with a peptide corresponding to amino acid residues 339–359 (peptide SC1) of rat sst2 SRIF receptor [15, 16]. Goat polyclonal c-kit antibody (sc-1494) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rat monoclonal BrdU antibody was obtained from Harlan Sera Laboratories (Loughborough, UK). Texas-red conjugated anti-rat immunoglobulin G (IgG) antibody and fluorescein (FITC)-conjugated horse anti-goat IgG antibody were from Vector Laboratories (Burlingame, CA). Alkaline phosphatase-conjugated antidigoxigenin antibody was purchased from Boehringer Mannheim.

Polyclonal anti-SRIF antibody 19608 was generously provided by Dr. J.A. Chayvialle (Centre Hospitalier Universitaire Edouard Herriot, Lyon, France). It was generated in rabbits immunized with SRIF14. Its specificity and the absence of cross-reactivity with other regulatory peptides have been documented previously [17].

Cell Isolation and Culture

Sertoli cells were isolated from testes of 3-wk-old pigs. At this perinatal age, pigs are routinely castrated to improve gain in body mass and taste of the meat. The isolation of Sertoli cells was performed by collagenase dispersion as previously described [18]. After decapsulation, testes were chopped and extensively washed in DMEM/Ham F-12 (1:1 [v/v]) medium. They were then treated with collagenase dispase (0.4 mg/ml, 90 min, 32°C). Collagenase was removed by centrifugation (200 x g, 10 min, 4°C). The resulting pellets were resuspended in the medium and allowed to sediment for 5 min. Sedimented seminiferous tubules were recovered, washed, and decanted three times. The seminiferous tubules were then incubated (room temperature, 20 min) in Ca2+/Mg2+-free PBS solution containing 1 M glycine, 2 mM EDTA, and 0.1 mg/ml of DNase I to remove the remaining Leydig cells.

After repeated washings by gravity sedimentation (three cycles), the seminiferous tubules were incubated in DMEM/Ham F-12 (1:1 [v/v]) medium supplemented with collagenase dispase (0.4 mg/ml), DNase I (0.1 mg/ml), and fetal calf serum (10%) for 30 min at 32°C. The supernatants containing peritubular myoid cells were discarded, and the tubule pellets were treated with collagenase dispase as described (0.4 mg/ml, 30 min, 32°C) until small clumps were obtained. Sedimented clumps contained highly enriched Sertoli cells and were devoid of Leydig and germ cells. Myoid cell contamination was determined as previously reported [19] and represented from 2% to 5% of cells at the end of the 5-day culture period.

Leydig cells were isolated by Percoll gradient centrifugation from the supernatants collected after the first enzymatic digestion. The content of Leydig cells in the preparations obtained exceeded 90% as determined histochemically by 3ß-hydroxysteroid dehydrogenase staining [18].

Sertoli and Leydig cells were cultured in DMEM/Ham F-12 (1:1 [v/v]) medium containing 1.2 mg/ml of sodium bicarbonate, 15 mM Hepes, 20 µg/ml of gentamicin, 5 µg/ml of transferrin, 10 µg/ml of {alpha}-tocopherol, 100 IU of penicillin, 0.05 mg/ml of streptomycin, and 2 µg/ml of insulin at 32°C in a humidified atmosphere of 5% CO2 and 95% air. Cultures were run for 5 days or more to restore FSH receptor function before starting the experiments [20, 21].

Seminiferous tubules were obtained as previously described [22]. The seminiferous tubules devoid of Leydig cells (see above) were incubated for 20 min at 32° C in DMEM/Ham F-12 (1:1 [v/v]) medium supplemented with 0.5 mg/ml of collagenase and 5.6 mM glucose and washed four times by gravity sedimentation. After centrifugation (200 x g, 10 min, 4°C), the tubules were resuspended (1:10 [v/v]) in the same medium as those used for Sertoli and Leydig cell culture, except that fetal calf serum and insulin were omitted.

Semiquantitative Reverse Transcription-Polymerase Chain Reaction of SCF mRNA Expression

Sertoli cells cultured in Petri dishes (60 mm x 15 mm; density, 5 x 106 cells/dish) were preincubated (1 h, 32°C) with 10 nM SRIF14. The FSH (0.25 µg/ml) was then added (or not), and cells were incubated at 32°C for the next 4 h. This stimulation period was determined during the preliminary experiments as the time necessary to double SCF mRNA expression in the presence of FSH (see Fig. 1). Hormonal stimulation was also mimicked by addition of forskolin (1 µM) or 8-bromoadenosine (8Br)-cAMP (1 mM). In parallel, the basal SCF mRNA expression was determined in Sertoli cell cultures left untreated (i.e., no drug addition) and incubated under identical (5 h, 32°C) conditions.



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  		FIG. 1. SRIF14 inhibits SCFs mRNA expression in immature Sertoli cells. SRIF14 (10 nM) inhibits basal, FSH-stimulated (0.25 µg/ml), and forskolin-stimulated (FK; 1 µM) SCFs mRNA expression, but not that induced by 8Br-cAMP (cAMP; 1 mM). The upper panel shows the representative autoradiograms from one experiment. The lower panel shows the histograms representing the mean ± SEM of the ODs measured on corresponding autoradiograms for each determination performed in duplicate in three independent experiments. The ODs were normalized taking as a reference 100% of the values obtained for the ratio SCFs:ß-actin in basal conditions, as described in Materials and Methods. These reference values were 0.377, 0.256, and 0.353 for the three experiments performed. aP < 0.05, bP < 0.01 for SCFs mRNA expression in the presence of SRIF compared to that measured in its absence and given by the adjacent left-side histogram. The letter symbol is omitted for the last histogram bar, because SRIF had no significant effect on cAMP-induced SCF mRNA expression. *P < 0.05, **P < 0.01 for comparisons between basal and stimulated (by either FSH, FK, or cAMP) SCFs mRNA expression

In some experiments, Sertoli cells were treated with pertussis toxin (30 ng/ml, 24 h). The toxin was washed-out before beginning the 5-h test period (1 h of preincubation with SRIF14 + 4 h of stimulation with FSH).

At the end of the different treatments, cells were washed with PBS solution. Total RNAs were then extracted with Trizol reagent according to the manufacturer's instructions.

For reverse transcription-polymerase chain reaction (RT-PCR), 3 µg of total RNA were reverse-transcribed in 10 µl of reaction mixture containing 200 µM dNTPs, 10 U/ml of MMLV reverse transcriptase, 0.01 M 1,4-dithiothreitol, and 5 µM random hexamer primers in 20 mM Tris-HCl buffer for 60 min at 37°C, followed by heating at 100°C for 5 min.

Two microliters of the RT reaction mixture from each sample were amplified with either SCF or ß-actin primers (1 µM), [{alpha}33P]dATP (0.75 µCi), Taq polymerase (0.01 U/µl), dNTPs (100 µM), MgCl2 (1.5 mM), and buffer (50 mM Tris-HCl) in a final volume of 20 µl. The primers used to study SCF mRNA expression (Table 1) amplified transcripts corresponding to both SCFm and SCFs [5]. The primer sequences [5] and conditions used to study ß-actin expression are given in Table 1.


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  		TABLE 1. Primers and conditions used in PCR reactions

The number of PCR cycles was determined during preliminary experiments by performing from 20 to 35 and 15 to 30 amplification reactions for SCF and ß-actin, respectively. Amplification for SCF and ß-actin was linear up to 30 and 25 cycles, respectively (data not shown). To ensure the amplification products to be analyzed were obtained during the linear phase of the reaction, the number of PCR cycles was fixed at 25 and 20 cycles for SCF and ß-actin, respectively. The identity of the amplified fragments was checked by manual sequencing (data not shown). For negative controls, cDNA was replaced by water.

The PCR products were resolved by 8% polyacrylamide gel electrophoresis. Products of amplification were visualized by using Kodak X-Omat S films (Eastman Kodak, Rochester, NY). The intensities of the autoradiographic bands were estimated by densitometric scanning using the Intelligent quantifier (Omni Media scanner XRS; Bioimage, Bedford, MA). The results correspond to the optical densities (ODs) of autoradiograms relative to three different RT-PCRs performed with RNAs extracted from Sertoli cells from three independent cultures. The ODs corresponding to SCFs and ß-actin mRNA expression in each individual sample were expressed in arbitrary units as a ratio of SCFs over ß-actin densitometric intensities. The SCFs:ß-actin ratios corresponding to the duplicate determination for each individual sample were then normalized, taking as a reference (100%) value the SCFs:ß-actin ratio obtained under basal conditions. The latter was chosen as a reference because it was reproducible from one experiment to another. Described normalization was performed to allow comparison of the results obtained in different rounds of RT-PCR on Sertoli cell mRNAs obtained from different cultures.

RT-PCR of SRIF and sst2 Receptor mRNA Expression

Total RNA extracted with Trizol reagent from Sertoli cells after 5 days of culture was used for RT-PCR as described above. To ascertain that cDNA was not contaminated by genomic DNA, each RNA sample was also incubated in the absence of MMLV reverse transcriptase.

For the analysis of SRIF mRNA expression, one-tenth (2 µl) of the synthesized first-strand cDNA was added to PCR buffer (100 mM Tris) containing 200 µM dNTP, 1.5 mM MgCl2, and 1.25 U Taq DNA polymerase in a total volume of 50 µl. The sequences of the chosen SRIF primers and the PCR conditions are given in Table 1. After 40 PCR cycles, the amplified products were visualized on 2% agarose gel with ethidium bromide staining.

Expression of sst2 receptor mRNA was assessed as summarized in Table 1 and detailed elsewhere [14]. Amplification was performed by reiterating 40 PCR cycles, after which the amplification products were separated by electrophoresis on 2% agarose gel and stained with ethidium bromide.

Western Blot Analysis of sst2 Receptor

At the end of the 5-day culture period, Sertoli cells were washed twice in PBS solution and then scraped off Petri dishes (100 mm x 20 mm; density, 20 x 106 cells/dish) in 50 mM Tris-HCl buffer (pH 7.5) containing 140 mM NaCl, 1 mM EDTA, 0.1 mg/ml of soybean trypsin inhibitor, 0.1 mM phenylmethylsulfonyl fluoride in the presence of 1.5% 3-[(3-cholamidopropyl)dimethylammoniol]-1-propane-sulfonate (CHAPS), and 0.5 mM sodium orthovanadate. The mixture was incubated for 30 min at 4°C and then centrifuged at 13 000 x g for 20 min. The aliquots of soluble proteins contained in the cleared supernatant were frozen and kept at -20° C until used.

For Western blot analysis, solubilized proteins (100 µg) were resolved through 7.5% SDS-polyacrylamide gels and transferred to a nitrocellulose membrane (Hybond-C extra; Amersham, Les Ulis, France). The membranes were saturated with 5% dried milk in PBS solution containing 0.05% Tween-20 (PBST; pH 7.6) and then immunoblotted overnight (4°C) with either preimmune serum, anti-sst2 antibody, or anti-sst2 antibody preadsorbed with the immunogenic peptide, each at a 1:500 (w/w) dilution. After washing three times with PBST at room temperature, the detection of bound antisera was performed with horse radish peroxidase-labeled goat-anti-rabbit IgG at a 1:2000 (w/w) dilution. The membranes were then washed and the immune complexes visualized using CovaLight chemiluminescence detection system (CovalAb, Lyon, France).

[3H]CH3-Thymidine Assay in Isolated Seminiferous Tubules

One day after beginning the culture, the isolated seminiferous tubules (cultured in 24-well plates in a final volume of 0.5 ml) were washed twice in the medium, and cells were treated (or not) for 24 h with either SRIF14 (10 nM) alone, SCF (20 ng/ml) alone, or both. All experiments were done in the presence of the Complete protease inhibitor cocktail containing antipain, bestatin, chymostatin, leupeptin, pepstatin, phosphoramidon, pefabloc, and aprotinin. The [3H]CH3-thymidine pulse (1 µCi/well) was performed during the last 5 h of culture according to the previously established conditions [14]. The plates were extensively washed, and the radioactivity was quantified by liquid scintillation ß-counting. The protease inhibitor cocktail used during the culture had no cytotoxic effect, because its addition did not modify the [3H]CH3-thymidine incorporation in the absence of drugs (data not shown).

The results of the assays are expressed as cpm/106 cells. The total cell number present in the seminiferous tubule fragments at the end of the experiment was determined on duplicate wells that were processed identically to those used for 3H-thymidine assays, except that the label was not added. Standard 10x trypsin-EDTA solution was diluted to 1x in the culture medium, added at the rate of 1 ml/well, and incubated for 15 min at 32°C. Trypsin action was arrested by the addition of 0.1 ml of bovine serum albumin (final concentration, 10%). Cells were mechanically dispersed by vigorous pipetting and counted under the optic microscope using a Malassez counting chamber.

In the control experiments, Sertoli cells (cultured in 24-well plates at a final volume of 0.5 ml with 0.5 x 106 cells/well) were washed twice in the medium and treated for 24 h with either SCF (20 ng/ml) or FSH (0.25 µg/ml). The [3H]CH3-thymidine pulse was performed as described for seminiferous tubule fragments.

Indirect Fluorescent Assay of BrdU Incorporation in Seminiferous Tubule Explants

Isolated seminiferous tubules were grown on the glass coverslips (inserted at the bottom of the 60 mm x 15 mm Petri dishes) in the same conditions as described above for [3H]CH3-thymidine assays. The adherent explants obtained after 24 h of culture were washed and further incubated with 30 µM BrdU for 24 h in the absence or presence of SCF (20 ng/ml), which was added either alone or in combination with SRIF14 (10 nM). After several rinses in PBS, tubule explants were fixed in 4% paraformaldehyde in PBS for 15 min, rinsed, and stored in PBS at 4°C.

For BrdU immunocytochemistry, if not otherwise stated, all reactions were performed at room temperature. Tubular explants were first treated with a 0.5% solution of Triton X-100 in PBS (30 min) followed by a treatment with a pepsin solution (0.006 mg/ml) in 0.1 M HCl (2 min). After several washings in PBS, DNA was denaturated by incubation in ice-cold 0.1 M HCl (20 min), followed by incubation in 2 M HCl (1 h, 37°C). The HCl was then neutralized with 0.1 M sodium tetraborate (pH 8.5, 10 min). After rinsing, cells were incubated in PBS containing 0.125% BSA, 0.05% Triton X-100, and 2.5% normal goat serum for 30 min. They were then incubated overnight at 4°C with the anti-BrdU antibody (1:100 [w/w] dilution) in the same blocking buffer. After washing, preparations were further incubated (2 h) in Texas red-conjugated anti-rat IgG antibody diluted to 1:100 (w/w) in PBS containing 2% normal swine serum. After washing, coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and observed under the fluorescent microscope.

For double immunohistochemistry experiments, after the last washing, tubular explants were incubated in the second blocking buffer (PBS containing 0.125% BSA, 0.05% Triton X-100, and 2.5% normal swine serum) for 1 h at room temperature and then overnight at 4°C in the same blocking buffer containing a 1:500 (w/w) dilution of anti-c-kit antibody. Tubular explants were then extensively washed in PBS. They were further incubated (2 h) with the FITC-conjugated secondary mouse anti-goat IgG antibody diluted to 1:200 (w/w) in PBS containing 2% swine serum. After few PBS rinses, explants were mounted with Vectashield and observed under the laser scanning confocal microscope (Zeiss LSM-10, Jena, Germany).

In Situ Hybridization

The mouse SSTR2 probe was a generous gift of Dr G. Bell (Howard Hughes Medical Institute, University of Chicago, Chicago, IL). This probe is a 458-base pair (bp) BstEII-XbaI fragment of the mouse sst2 cDNA encoding amino acids 254–369, the stop codon, and 107 nucleotides of the 3'-flanking/untranslated region. The probe was subcloned into the plasmid vector pGEM-3Z. The latter was linearized with EcoRI and transcribed with the SP6/T7 Transcription Kit (Roche Diagnostics, Mannheim, Germany) using SP6 RNA polymerase and digoxigenin-UTP to obtain the antisense probes. Sense probes were prepared from HindIII-linearized plasmid DNA using T7 RNA polymerase and digoxigenin-UTP.

To perform in situ hybridization, 5-µm testicular sections from three immature donor pigs were dewaxed, treated with 4 µg/ml of proteinase K (37°C, 15 min), and hybridized overnight at 55°C with 1.5 µg of antisense probe per milliliter of hybridization buffer containing 50% formamide, 10% dextran sulfate, 4x standard saline citrate (SSC), 1x Denhardt solution, 250 µg/ml of salmon DNA, and 250 µg/ml of yeast tRNA. To control for nonspecific hybridization, additional sections were incubated in parallel with the equivalent concentration of sense probe. Sections were then washed twice in 1x and once in 0.5x SSC buffer (30 min) at room temperature. The hybridized digoxigenin-labeled probe was detected with alkaline phosphatase-conjugated antidigoxigenin antibody (1:200 [w/w], 90 min, room temperature) and visualized with NBT/BCIP chromogen.

Immunohistochemical Localization of SRIF and c-kit

Testes of 3-wk-old pigs (n = 3) were fixed in 4% paraformaldehyde in PBS (pH 7.4) and embedded in paraffin. Sections (thickness, 5 µm) were mounted on positively charged glass slides (SuperFrost* Plus; Menzel-Glaser, Freiburg, Germany) and thereafter deparaffinized, hydrated, and treated for 20 min at 95°C in citric buffer (pH 6). After washing in PBST, sections were preincubated for 10 min at 37°C in peroxidase blocking reagent (Envision+ kit; DAKO), washed in PBST, and incubated overnight at 4°C with anti-SRIF antibody (1:1000 [w/w] dilution). After two washings with PBST, the sections were incubated (30 min, 37°C) with goat-anti-rabbit IgG attached to a peroxidase-conjugated polymer backbone (Envision+ kit; DAKO). At the end of the incubation period, the unbound secondary antibody was washed, and sections were incubated for 10 min at room temperature with 3-amino-9-ethylcarbazole to reveal a red color at the site of peroxidase activity. After an extensive wash, the nuclei were counterstained with Mayer hematoxylin.

Testicular sections used for immunohistochemical localization of c-kit were processed identically, except that the primary antibody dilution used was 1:200 (w/w).

Data Analysis

If not otherwise specified, data are presented as the mean ± SEM of triplicate determinations performed in at least three independent experiments carried out with different cell preparations or cell cultures. Statistical significance of the differences observed between experimental groups was determined by one-way ANOVA using the InStat (Graph Pad Software, San Diego, CA) computer program. Post-hoc comparisons between treatment group means were made with the Bonferroni test for multiple comparisons. Differences were accepted as being significant if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
SRIF Inhibits SCF mRNA Expression

After 5 days of culture, perinatal porcine Sertoli cells express predominantly SCFs, whereas SCFm was barely detectable (Fig. 1).

Exogenous addition of SRIF14 to the Sertoli cell cultures inhibited basal SCF mRNA expression in a dose-dependent manner, with a plateau for SRIF concentrations between 10 nM and 1 µM (Fig. 2A).



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  		FIG. 2. Pertussis toxin pretreatment (24 h, 30 ng/ml) prevents SRIF14 inhibition of both basal (A) and FSH-stimulated (B; 0.25 µg/ml) SCFs mRNA expression in immature Sertoli cells. Data are the mean ± SEM obtained in three independent experiments, each performed in duplicate. Empty symbols represent the results obtained in control cultures (in the absence of toxin); filled symbols correspond to the data obtained in pertussis toxin-pretreated cultures. The ODs were normalized taking as a reference 100% of the values obtained for the ratio SCFs:ß-actin in basal conditions for cultures performed in the absence of the toxin. These reference values were 0.377, 0.256, and 0.353 for the three experiments performed. bP < 0.01 for the differences between the percentage of SCFs mRNA expression in the presence versus that in the absence of SRIF14

We next studied the SRIF actions in FSH-stimulated conditions. To do that, we first quantified the hormone effects on SCF mRNA expression. When added at the rate of 0.25 µg/ml [14], FSH doubled SCF mRNA expression as measured over the 4-h stimulation period (Fig. 1). Addition of SRIF14 in the concentration range from 1 pM to 1 µM inhibited the FSH-stimulated SCF expression in a dose-dependent manner (Fig. 2B). This inhibition was maximal for SRIF14 concentrations from 10 nM to 1 µM.

To study the underlying mechanisms, SRIF14 concentration was fixed at 10 nM (corresponding to the 10-fold affinity constant Kd reported for all known SRIF receptors [13]). In these conditions, SRIF14 inhibition of basal and FSH-stimulated SCF mRNA ranged from 30% to 50% and from 50% to 70%, respectively (Fig. 1). To assess the involvement of adenylyl cyclase inhibition in the observed SRIF actions, we used pharmacological tools such as forskolin and 8Br-cAMP. At the concentrations tested, forskolin (1 µM) increased SCF mRNA expression to a similar extent as FSH (approximately twofold over basal), whereas 8Br-cAMP (1 mM) triggered an almost threefold increase (Fig. 1). The SRIF14 (10 nM) significantly (P < 0.01) inhibited forskolin-induced, but not 8Br-cAMP-induced, SCF mRNA expression (Fig. 1), thus demonstrating unequivocally that the mechanism of the observed SRIF actions implied adenylyl cyclase inhibition.

Pertussis Toxin Prevents SRIF14-Mediated Inhibition of SCF mRNA Expression

Involvement of plasma membrane SRIF-specific receptors in the inhibitory actions of SRIF14 on SCF mRNA expression was further addressed functionally through assessment of their coupling to inhibitory G (Gi) proteins. This was done by treating Sertoli cells for 24 h with 30 ng/ml of pertussis toxin [14].

In the absence of SRIF14, the basal SCF mRNA expression measured between the toxin-treated and the control cells was similar (Fig. 2A). This allowed us to discard a possible cytotoxic effect of pertussis toxin per se and, consequently, to compare the SRIF actions on SCF mRNA in the presence and absence of the toxin. As expected, pertussis toxin blocked SRIF14-mediated inhibition of SCF mRNA expression, whereas in control cells, such inhibition was observed (Fig. 2A).

In contrast, the toxin treatment significantly (P < 0.01) decreased the effect of FSH added alone on SCF mRNA level compared to the control (i.e., not toxin-treated) FSH-stimulated cells (Fig. 2B). The latter data made the interpretation of pertussis toxin effects on SRIF14-mediated inhibition of FSH-induced SCF mRNA expression inconclusive.

sst2 Receptor Involvement in Observed SRIF Actions

The sst2 receptor mRNA has been recently identified as the sole SRIF receptor transcript expressed by Sertoli cells [14]. To unequivocally demonstrate the presence of this plasma membrane receptor on immature porcine Sertoli cells, RT-PCR and Western blot assays of sst2 receptor were performed in parallel.

According to our previous study [14], the analysis of PCR products amplified with primers specific for sst2 demonstrated that the 415-bp fragment was present in reactions using reverse cDNA transcribed from Sertoli cells (Fig. 3A, lane 1). A similar fragment was also amplified from genomic DNA (Fig. 3A, lane 3) used as a positive control. The negative controls corresponding to PCR reactions in which either mRNA (Fig. 3A, lane 2) or water (Fig. 3A, lane 4) were used confirmed the absence of any template or reactive contaminations.



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  		FIG. 3. Expression of sst2 SRIF receptor mRNA (A) and protein (B) in immature Sertoli cells. A) Amplification products obtained in PCR reaction with sst2-specific primers. Templates used in the reaction were cDNA (lane 1), mRNA (lane 2), genomic DNA (lane 3), and water (lane 4). The size (in bp) of DNA fragments is given in lane 5. B) Western blot analysis of sst2 receptor immunoreactivity in CHAPS-solubilized proteins (100 µg/lane) separated by 7.5% SDS-PAGE. After the electrotransfer of proteins, the corresponding nitrocellulose membrane was divided into three parts. Two parts were immunoblotted with either polyclonal anti-sst2 antibody alone (lane 3) or in the presence of the immunogenic peptide (lane 2). The third part was incubated in parallel with the preimmune serum (lane 4). Molecular mass markers (in kDa) are shown in lane 1. The arrow indicates the amplification fragment corresponding to sst2 mRNA (A) and to sst2-immunoreactive protein (B)

Immunoblotting of protein extracts prepared from the same batch of Sertoli cells with the anti-sst2 antibody revealed an immunoreactive band with an apparent molecular mass of 110 kDa (Fig. 3B, lane 3). In the presence of 12 nM immunogenic peptide SC1, immunoreactivity of the 110-kDa protein was completely inhibited. Parallel immunoblotting with the preimmune serum revealed the absence of any detectable immunostaining (Fig. 3B, lane 4).

Functional Relevance of SRIF Actions on SCF mRNA Expression

To correlate the SRIF actions on SCF expression by Sertoli cells with its possible actions on spermatogonial proliferation, we next studied the [3H]CH3-thymidine incorporation in seminiferous tubule explants. Comparison by global ANOVA between [3H]CH3-thymidine incorporation in the absence of any agonist versus in the presence of either SCF alone, SRIF alone, or both indicated significant (F = 10.28, P < 0.01) differences in proliferation rates.

More precisely, addition of 20 ng/ml of SCF for 24 h increased by approximately 60% to 70% the incorporation of [3H]CH3-thymidine in seminiferous tubules (Fig. 4A), but it had no effect on Sertoli cells (Fig. 4B), thus demonstrating the spermatogonial specificity of SCF actions. Immunohistochemistry performed on the tissue sections of donor testis showed that, as expected, SCF receptor in seminiferous tubules is exclusively expressed by spermatogonia; Sertoli and peritubular cells do not display this receptor (Fig. 5G). Peritubular cells that rapidly proliferate could not, by increasing thymidine incorporation background, lead to a misestimation of the specific SCF-induced [3H]CH3-thymidine incorporation. Indeed, the morphological examination of the explants indicated that their presence remained minor during the culture period (Fig. 5, A–C). Increased DNA synthesis in the seminiferous tubule explants treated with SCF (20 ng/ml, 24 h) was also observed in situ by monitoring the BrdU incorporation (Fig. 5, D and E). However, this increase could not be quantified, because multiple cellular layers in the explants prevented an accurate cell counting. Laser confocal scanning microscopy of the c-kit/BrdU double-labeled spermatogonia confirmed that they were actively synthesizing DNA when stimulated by SCF in similar experimental conditions (Fig. 5, H and I).



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  		FIG. 4. SRIF14 inhibits SCF-induced proliferation in isolated seminiferous tubules. Experimental values correspond to the mean ± SEM of [3H]CH3-thymidine incorporation obtained in four independent experiments, each performed in triplicate, for seminiferous tubule explants (A) and homologous control Sertoli cell culture (B). Data are expressed as a percentage of the basal rate of [3H]CH3-thymidine incorporation obtained in the absence of agonists. Basal values measured in the four different experiments were 1844, 1923, 1916, and 1778 cpm/106 cells for tubule fragments and 1197, 1175, 1328, and 1414 cpm/106 cells for Sertoli cell preparations. aP < 0.05, bP < 0.01, cP < 0.001



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  		FIG. 5. SCF specifically targets spermatogonia in seminiferous tubule explants. Morphological aspect of the seminiferous tubule fragments after 24 h (A and B) and 48 h (C) of culture shows a minor peritubular cell contamination at the end of the culture period. Indirect fluorescence of BrdU incorporation observed in the absence of agonists (D), after the treatment with 20 ng/ml of SCF over 24 h added either alone (E) or in combination with 10 nM SRIF14 (F) is also shown. In seminiferous tubules visualized on porcine testis sections by hematoxylin staining, only spermatogonia display c-kit, whereas Sertoli and peritubular cells are devoid of this receptor (G). Laser scanning confocal micrographs of a seminiferous tubule explant (H) and spermatogonia (I) incubated with SCF (20 ng/ml, 24 h) and double-stained with anti-c-kit (1:500 [w/w] dilution) and anti-BrdU (1:100 [w/w] dilution) antibodies are also shown. Bar = 40 µm (A and B) and 20 µm (all other micrographs except I, in which the whole micrograph width equals 20 µm)

Treatment of seminiferous tubule explants for 24 h with 20 ng/ml of SCF added simultaneously with SRIF14 (10 nM) led to a significant (P < 0.05) decrease in [3H]CH3-thymidine incorporation compared to that measured in the presence of SCF alone (Fig. 4A). In contrast, SRIF14 alone did not significantly modify the basal rate of [3H]CH3-thymidine incorporation (Fig. 4A). The SRIF-dependent decrease of SCF-induced DNA synthesis was also observed by indirect fluorescence of the BrdU incorporation (Fig. 5F).

To assess whether spermatogonia can be directly targeted by SRIF, we attempted to localize the sst2 receptor in situ by using the polyclonal antibody employed in Western blot assays of Sertoli cell extracts. However, our attempts remained inconclusive even when different types of tissue sections (frozen and formalin-fixed, paraffin-embedded), antibody dilutions (up to 1:100 [w/w]), and detection methods (peroxidase staining, indirect fluorescence) were tested (data not shown). These technical limitations were circumvented by the in situ hybridization analysis of sst2 receptor expression. The assays indicated that spermatogonia express sst2 receptor transcripts in a heterogeneous manner (Fig. 6, A and C). The absence of any labeling with sense probe demonstrates the specificity of this expression (Fig. 6B). Moreover, the in situ hybridization approach allowed us to further confirm the expression of sst2 receptor mRNA in Sertoli cells, a phenomenon already documented by RT-PCR (Fig. 3A) and Western blot analysis (Fig. 3B). In addition, no sst2 mRNA was seen in peritubular cells (Fig. 6, A and C).



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  		FIG. 6. In situ hybridization of sst2 receptor mRNA. Hybridized anti-sense sst2 probe was revealed by alkaline phosphatase-conjugated antidigoxigenin (A and C). The specificity of hybridization signal was controlled by a parallel incubation of testicular sections with sense probe (B). Note the intense intratubular labeling of Sertoli cell cytoplasm (A and C). Note also the heterogeneity of Leydig cell and spermatogonia labeling. Strongly labeled cells are indicated by thick arrows, whereas nonlabeled cells are indicated by thin arrows (A and C). Lc, Leydig cell; Sc, Sertoli cell; Sg, spermatogonia. Bar = 40 µm (A and B) and 10 µm (C)

Cellular Origin of Intratesticular SRIF

The possible testicular expression of SRIF was studied by immunohistochemistry on tissue sections from 21-day-old pigs. Positive immunostaining with the anti-SRIF antibody was observed in both intratubular and interstitial compartments (Fig. 7A). This immunoreactivity could be attributed to spermatogonia and Leydig cells. However, both cell types immunostained heterogeneously, with varying (from low to strong) intensities of labeling (Fig. 7, A and B). The observed immunostaining was specific, because it was abolished in the presence of exogenously added 125 nM SRIF14 (Fig. 7C). Testicular sections processed without incubation with the anti-SRIF antibody displayed no immunoreactivity, thus excluding any artifactual labeling (Fig. 7D).



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  		FIG. 7. SRIF-like immunoreactivity on formalin-fixed, paraffin-embedded testis sections. Tissue sections were incubated with 19608 anti-SRIF antibody (1:1000 [w/w] dilution) alone (A) or in the presence of exogenously added SRIF14 (C). Negative control consisted in a staining procedure from which anti-SRIF antibody was omitted (D). Whereas in the seminiferous tubules Sertoli cells do not immunostain in the presence of the SRIF antibody, some, but not all, spermatogonia are strongly immunostained (A and B). Note the staining heterogeneity of Leydig cells and the strong immunostaining of the interstitial space (A and B). is, Interstitial space; Lc, Leydig cell; Sc, Sertoli cell; Sg, spermatogonia. Bars = 20 µm (A, C, and D) and 10 µm (B)

To distinguish between testicular and systemic origin of the detected SRIF, we searched for its mRNAs in the two immunoreactive compartments. The RT-PCR showed that a single product of the expected size (211 bp) was amplified with primers specific for SRIF when cDNA corresponding to mRNA isolated from either Leydig cells or seminiferous tubules was used as a template (Fig. 8, lanes 3 and 5, respectively). The reactions (i.e., negative controls) in which mRNA was used as a template (Fig. 8, lanes 4 and 6) and in which the template was replaced by water (Fig. 8, lane 2) further confirmed the absence of any template or reactive contamination.



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  		FIG. 8. SRIF mRNA expression in Leydig cells (lanes 3 and 4) and seminiferous tubules (lanes 5 and 6) of immature porcine testis. Lanes 3 and 5 correspond to the products of amplification in which cDNA was used as template; lanes 4 and 6 correspond to the reactions performed with RNA. Negative control consisted in replacing templates by water (lane 2). Lane 1 shows the DNA ladder, indicating the size (in bp) of the amplified fragments


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This work provides evidence for the inhibitory role of SRIF in the control of spermatogonial proliferation. In perinatal porcine testis, SRIF might exert its actions both directly on spermatogonia by preventing SCF-induced proliferation and indirectly by inhibiting SCF mRNA expression by Sertoli cells. The indirect mechanism of regulation concerns SRIF effects on SCF mRNA expression not only in basal but also in FSH-stimulated conditions. The latter further corroborates the recently proposed role for SRIF as a negative regulator of FSH actions in immature Sertoli cells [14]. To our knowledge, SRIF is the first known candidate for such a role. Indeed, another putative regulator, transforming growth factor-ß, although able to inhibit SCF actions on hematopoietic stem cells [23, 24] and various testicular functions [25, 26], has been reported to be inefficient in regulating testicular SCF gene expression [10].

The culture conditions used here were validated by the predominant SCFs mRNA expression observed in perinatal porcine Sertoli cells, which is in agreement with that reported in perinatal murine Sertoli cells [5, 27]. Similarly, in semiquantitative RT-PCR, 8Br-cAMP, forskolin, and FSH all triggered an increase in SCF mRNA expression of the same order of magnitude as that previously observed in rodent Sertoli cells [9, 10, 28]. The experimental conditions of the proliferation assays permitted the measurement of a similar SCF-induced [29] and FSH-induced [20, 21] increase of [3H]CH3-thymidine incorporation, as previously reported in immature rodent spermatogonia and Sertoli cells, respectively.

Our data show that SRIF14 inhibits both basal and FSH-stimulated SCF mRNA expression in Sertoli cells. The SRIF-induced inhibition of forskolin-induced, but not of 8Br-cAMP-induced, SCF mRNA expression demonstrates explicitly the block of cAMP production as a mechanism for SRIF14 actions. This effect involves the interaction of SRIF-specific, Gi protein-linked receptors with adenylyl cyclase. Accordingly, basal inhibition of SCF mRNA expression is prevented by pertussis toxin-mediated inactivation of Gi/Go proteins and subsequent blockage of SRIF transduction at the level of receptor coupling to adenylyl cyclase [14].

By contrast, the implication of specific membrane receptors in SRIF14 actions on FSH-stimulated SCF mRNA expression cannot be studied using pertussis toxin insofar as, in the absence of SRIF14, the toxin blocked FSH-induced SCF expression. Similar observations regarding pertussis toxin inhibition of FSH actions in Sertoli cells have been previously reported [14].

The present RT-PCR, Western blot, and in situ hybridization data further corroborate our previous conclusions concerning sst2-selectivity of adenylyl cyclase inhibition in Sertoli cells [14]. Based on Western blot analysis, the apparent molecular mass of sst2 receptor is approximately 110 kDa. In agreement with this finding, the molecular mass of the sst2-immunoreactive proteins identified previously range from 60 to 110 kDa, depending on the cell and tissue types studied [30–34]. Nevertheless, our efforts to visualize Sertoli-specific sst2 receptor labeling by using the same anti-sst2 antibody remained inconclusive. The absence of testicular labeling is presumably due to the low expression of sst2 protein, because the antibody used clearly labeled sst2 receptor on formalin-fixed, paraffin-embedded brain sections (data not shown).

Regarding the functional significance of SRIF actions, our data point to the capacity of SRIF14 to decrease SCF-stimulated proliferation in isolated seminiferous tubules back to the basal level. It should be stressed that it is still impossible to maintain viable, purified spermatogonial stem cells in a primary culture devoid of fetal calf serum [35, 36]. These technical limitations can be overcome by the use of seminiferous tubule explants, in which Sertoli cells provide the necessary growth factors for spermatogonia. Hence, this model allows the assessment of mitogenic actions of exogenous SCF by exclusively targeting spermatogonial proliferation. Consistently, the observed failure of SCF to trigger the increase of [3H]CH3-thymidine incorporation by Sertoli cells isolated from the same batch of "SCF-responsive" seminiferous tubules confirms biochemically the lack of SCF receptors on Sertoli cells reported in the present as well as previous studies [37, 38].

The molecular mechanisms by which SRIF modulates the cell cycle of spermatogonia could not be assessed with the model used here. Indeed, the relevant studies require in situ quantification of the number of cytologically identified, dividing spermatogonia. This is not possible in the seminiferous tubule explant model, in which cells form multilayers, thus rendering their accurate count impossible. However, it is worth noting that SRIF might inhibit either SCF-induced cell-cycle progression [6] or block the antiapoptotic effects of SCF [7, 8, 39]. In accordance with these mechanisms, sst2 SRIF receptor has been involved both in G1 cell-cycle arrest [40] and in triggering apoptosis [41]. Future studies using rodent models for in vivo injection of BrdU and subsequent double immunostaining of testicular tissue sections are now required to elucidate these points.

In the seminiferous tubule, SRIF acts at two different levels. It inhibits SCF expression by Sertoli cells, and it apparently blocks SCF action on spermatogonial proliferation. However, a direct causality between SRIF14 inhibition of FSH-stimulated SCF mRNA expression by Sertoli cells and spermatogonial proliferation cannot be unequivocally established in the seminiferous tubule explants. Indeed, gonadotropin affects Sertoli cell proliferation directly [14, 20] and spermatogonial proliferation indirectly through Sertoli cell-derived factors. Many of these factors are induced in Sertoli cells on FSH stimulation as, for example, SCF ([9], present study), basic fibroblast growth factor (bFGF) [42], inhibin [43], and interleukin 6 [44]. Among them, SCF ([29], present study) and bFGF [45, 46] are known to increase spermatogonial proliferation, whereas inhibin [47] and interleukin 6 [48] inhibit it. Consequently, precise evaluation of the suggested SRIF actions on SCF-induced spermatogonial proliferation after stimulation by FSH must await development of more suitable models.

Finally, SRIF immunoreactivity of both spermatogonia and Leydig cells on testicular sections, combined with the presence of SRIF-specific mRNA in corresponding testicular compartments, demonstrates the local production of this peptide. These data, combined with the expression of sst2 SRIF receptor transcripts revealed by in situ hybridization on Sertoli cells, point to the paracrine role of SRIF in the regulation of Sertoli cell function. Furthermore, autocrine actions of SRIF on Sertoli cells can be excluded. In addition, sst2 transcripts in spermatogonia and SRIF capacity to inhibit exogenously added SCF-induced spermatogonial proliferation imply that these cells can also be directly targeted by SRIF, thus suggesting the autocrine actions of SRIF on spermatogonia.

In conclusion, the reported antiproliferative actions of SRIF in immature testis might be of particular biological importance, especially in the light of their functional impact on adult fertility. This impact can now be assessed in vivo by analyzing the consequences of perinatal treatment with stable SRIF analogues specific for sst2 receptor subtype [49] on adult male fertility.


   ACKNOWLEDGMENTS
 
We are indebted to Drs. Graeme Bell and A.F. Parlow for the generous gifts of sst2 probe and FSH, respectively. We are grateful to Dr. Jean-Alain Chayvialle for providing us with anti-SRIF antibody and to Professor Jean-Claude Bernengo (Centre Commun de Quantimetrie, Université C. Bernard, Lyon, France) for help in performing laser scanning confocal microscopy. We would also like to thank Dr. Patrick Mehlen for critical reading of the manuscript and helpful suggestions, Professor Paul Dubois for advice during the initiation of immunohistochemistry, Dr. Cecile Violet for her precious help during setting-up of SRIF RT-PCR assays, and Mrs. Glynis Thoiron for the English revision.


   FOOTNOTES
 
First decision: 31 January 2001.

1 Supported by INSERM (Unité 407), ARC grant 7244 (S.K.) and Ligue contre le Cancer, Comité de la Savoie (S.K.). Back

2 Correspondence and current address: Slavica Krantic, Interactions Cellulaires Neuroendocriniennes, UMR 6544 CNRS-Université de la Méditerranée, Faculté de Médicine Secteur Nord, Boulevard Pierre Dramard, 13916 Marseille, Cedex 20, France. FAX: 33 4 91 69 89 20; krantic.s{at}jean-roche.univ-mrs.fr Back

Accepted: July 24, 2001.

Received: November 20, 2000.


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J. Fombonne, C. Charrier, I. Goddard, E. Moyse, and S. Krantic
Leptin-Mediated Decrease of Cyclin A2 and Increase of Cyclin D1 Expression: Relevance for the Control of Prepubertal Rat Leydig Cell Division and Differentiation
Endocrinology, May 1, 2007; 148(5): 2126 - 2137.
[Abstract] [Full Text] [PDF]

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M. Duran-Prado, C. Bucharles, B. J. Gonzalez, R. Vazquez-Martinez, A. J. Martinez-Fuentes, S. Garcia-Navarro, S. J. Rhodes, H. Vaudry, M. M. Malagon, and J. P. Castano
Porcine Somatostatin Receptor 2 Displays Typical Pharmacological sst2 Features but Unique Dynamics of Homodimerization and Internalization
Endocrinology, January 1, 2007; 148(1): 411 - 421.
[Abstract] [Full Text] [PDF]

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A. Yoneda, M. Kashima, S. Yoshida, K. Terada, S. Nakagawa, A. Sakamoto, K. Hayakawa, K. Suzuki, J. Ueda, and T. Watanabe
Molecular cloning, testicular postnatal expression, and oocyte-activating potential of porcine phospholipase C{zeta}.
Reproduction, September 1, 2006; 132(3): 393 - 401.
[Abstract] [F