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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 11 5521-5528
Copyright © 2003 by The Endocrine Society

Characterization of Endozepines in the Human Testicular Tissue: Effect of Triakontatetraneuropeptide on Testosterone Secretion

Celine Duparc, Herve Lefebvre, Marie-Christine Tonon, Hubert Vaudry and Jean-Marc Kuhn

European Institute for Peptide Research (Institut Fédératif de Recherche Multidisciplinaires sur les Peptides 23) (C.D., H.L., M.-C.T., H.V., J.-M.K.), Laboratory of Cellular and Molecular Neuroendocrinology, Institut National de la Santé et de la Recherche Medicale, Unité 413, Unité Affiliée Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan, France; and Department of Endocrinology and Metabolic Diseases (H.L., J.-M.K.), Centre Hospitalier Universitaire of Rouen, 76031 Rouen, France

Address all correspondence and requests for reprints to: Dr. Hubert Vaudry, Laboratory of Cellular and Molecular Neuroendocrinology, Institut Fédératif de Recherche Multidisciplinaires sur les Peptides 23, Institut National de la Santé et de la Recherche Medicale, Unité 413, Unité Affiliée Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont-Saint-Aignan Cedex, France. E-mail: hubert.vaudry{at}univ-rouen.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have shown that endozepines, i.e. endogenous ligands of benzodiazepine (BZD) receptors, stimulate steroidogenesis in adrenocortical and Leydig cells. In the present report, we have investigated the presence and action of endozepines in the human testis. Immunohistochemical labeling revealed the occurrence of endozepine-like immunoreactivity in Leydig, Sertoli, and germ cells. HPLC analysis combined with a specific RIA resolved two immunoreactive peaks that coeluted with synthetic octadecaneuropeptide (ODN) and triakontatetraneuropeptide (TTN). RT-PCR amplification showed that the mRNA encoding the endozepine precursor diazepam-binding inhibitor is expressed in the human testis. The action of endozepines on testosterone production was studied in vitro using perifused human testicular fragments. Administration of TTN provoked a dose-dependent increase in testosterone secretion, whereas ODN had no effect. The stimulatory action of TTN on testosterone production was totally blocked by flunitrazepam, a peripheral-type BZD receptor antagonist/central-type BZD receptor (CBR) agonist. Conversely, the CBR agonist clonazepam and the CBR antagonist flumazenil did not affect testosterone secretion. Collectively, these results suggest that, in the human testicular tissue, TTN may exert an intracrine and/or paracrine control of steroidogenesis through activation of a peripheral-type BZD receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TERM ENDOZEPINES designates a family of regulatory peptides that have been originally isolated from the rat brain on the basis of their ability to competitively displace benzodiazepines (BZD) from their binding sites (1). All endozepines characterized so far derive from an 86-amino acid polypeptide named diazepam-binding inhibitor (DBI) that generates, through proteolytic cleavage, several biologically active peptides including the triakontatetraneuropeptide DBI[17–50] (TTN) and the octadecaneuropeptide DBI[33–50](ODN) (2, 3).

The DBI gene is widely expressed in the central nervous system and in various peripheral tissues. Notably, DBI and its processing products are particularly abundant in steroidogenic organs, i.e. in the adrenal cortex, testis, and ovary (4, 5, 6, 7, 8, 9, 10, 11), suggesting that endozepines could be involved in the regulation of steroid hormone secretion. Consistent with this hypothesis, it has been shown that endozepines stimulate steroid biosynthesis in mitochondria isolated from Y-1 adrenocortical, MA-10, and R2C Leydig cell lines (12, 13), and from rat Leydig cells (14). It has also been recently reported that TTN stimulates corticosterone and aldosterone secretion from frog adrenocortical cells (15).

At the molecular level, endozepines appear to interact with the two types of BZD receptors: ODN predominantly modulates central-type BZD receptors (CBR) associated with the GABAA-receptor complex (16), whereas TTN is a selective ligand of peripheral-type (mitochondrial) BZD receptors (PBRs) (3, 17, 18). Several lines of evidence indicate that PBR mediate the intracrine and/or autocrine/paracrine actions of endozepines on steroidogenesis 1) PBRs are extremely abundant in steroidogenic cells (19, 20, 21) where they are predominantly located to the outer mitochondrial membrane (22); 2) high-affinity PBR agonists stimulate steroid formation both in vitro (23, 24, 25, 26, 27) and in vivo (28, 29); and 3) targeted disruption of the PBR gene in R2C rat Leydig tumor cells suppresses steroid biosynthesis (30). In addition to their PBR-mediated steroidogenic effects, endozepines may also regulate steroid production through activation of a membrane receptor positively coupled to adenylyl cyclase and calcium influx, as recently shown in frog adrenocortical cells (31).

In man, only few studies have been conducted to examine a possible action of endozepines on endocrine testicular function. Three clinical case reports have described BZD-induced decrease in plasma free testosterone index associated with an increase in plasma LH levels. These observations suggest that endozepines could play a significant role in the regulation of testosterone production from the human testis (32, 33, 34). However, this hypothesis has not yet been tested by in vitro studies.

The aim of the present study was to characterize endozepines in the human testicular tissue using immunohistochemical and biochemical approaches. In addition, we have investigated the effect of TTN, ODN, and BZD receptor ligands on testosterone secretion by human testicular fragments.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissues

Human testicular tissue was obtained from patients aged 72.5 ± 10.6 yr undergoing pulpectomy for metastatic prostate carcinoma. None of them was on GnRH agonist and/or antiandrogen therapy before the study. The protocol of collection of the tissues and the experimental procedure had been approved by the regional ethical committee.

Test substances, reagents, and antibodies

Synthetic ODN and TTN (human sequences) were purchased from Neosystem (Strasbourg, France). Clonazepam (Rivotril), flunitrazepam (Narcozep), and flumazenil (Anexate) were obtained from Roche Laboratories (Neuilly-sur-Seine, France). Human chorionic gonadotropin (hCG, 5000 IU) was from Organon (St. Denis, France). Ro5–4864 was from Fluka Chimie (Mulhouse, France). PK11195 and testosterone (4-androsten-17ß-ol-3-one) were from Sigma (St. Louis, MO). [1,2,6,7,3H]Testosterone was from Amersham International (Les Ulis, France).

The ODN antibodies used for immunohistochemical studies and RIA were raised by immunizing rabbits with synthetic human ODN covalently conjugated to BSA with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide-HCl (Sigma) as previously described (7). Polyclonal rabbit antibodies to cytochrome P450c17 was kindly provided by Drs. V. Luu The and G. Pelletier (Laval University Medical Center, Quebec, Canada). Fluorescein isothiocyanate-conjugated goat antirabbit {gamma}-globulin was from Nordic Immunology Laboratories (Tillburg, The Netherlands).

Immunohistochemical procedure

Testicular tissue obtained at surgery was immediately fixed in formalin and embedded in paraffin. Sections were deparaffinized in cyclohexane, rehydrated in a descending alcohol series, and washed in 0.1 M (pH 7.3) phosphate buffer (PB). Tissue sections were incubated overnight at 4 C in a humid atmosphere with the rabbit antiserum against human ODN (diluted 1:200). The antiserum was diluted in PB containing 1% BSA and 0.3% Triton X-100. The sections were rinsed four times (10 min) in PB and incubated for 2 h at room temperature with fluorescein isothiocyanate-conjugated goat antirabbit {gamma}-globulin at the working dilution of 1:100. Finally, the slices were rinsed in PB, mounted with PB/glycerol (1:1), coverslipped, and observed with an UV microscope (Olympus, Tokyo, Japan). To verify the specificity of the immunoreaction, adjacent sections were incubated with the ODN antiserum (diluted 1:200) preabsorbed with synthetic human ODN (10-6 M).

Consecutive sections were labeled with the antiserum against ODN (diluted 1:2000) or the antiserum against cytochrome P450c17 (diluted 1:2000), and the immunoreactivities were detected with the peroxidase-based EnVision+ kit (K4010; Dako, Trappes, France).

HPLC

Characterization of endozepines in human testicular extracts was performed by reversed phase HPLC analysis coupled to RIA. Testicular fragments obtained at surgery were frozen on dry ice and stored at -80 C. The tissues were immersed in boiling acetic acid (0.5 M) and maintained for 10 min in a water bath at 56 C to inactivate proteolytic enzymes. The tissues were chilled on ice and homogenized with a glass Potter. The homogenates were centrifuged (4800 x g, 4 C, 30 min) and the supernatants were prepurified on a Sep-Pak C18 cartridge (Waters Associates, Milford, MA) equilibrated with 50% acetonitrile. The cartridge was rinsed with 0.12% trifluoroacetic acid (TFA) and the peptide fraction was eluted with 50% acetonitrile. Samples were evaporated in a Speed-Vac concentrator (Savant, Hicksville, NY), and the dried extracts were stored at room temperature until analysis.

The testicular extracts were resuspended in 500 µl of 0.12% TFA and centrifuged 10 min at 13,000 x g. Supernatants were analyzed on a 0.46 x 25-cm Vydac 218TP54 C18 column (Touzart et Matignon, Courtaboeuf, France) equilibrated with a solution of 14% acetonitrile/0.12% TFA at a flow rate of 1 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 56% over 60 min. One-minute fractions were collected and evaporated in a speed-Vac concentrator. All fractions were kept dry until RIA determination. Synthetic human ODN and TTN, used as reference peptides, were chromatographed in the same conditions.

Concentrations of ODN-like material were measured in each fraction using a double-antibody RIA method as previously described (35). Briefly, human (Tyr0)-ODN (1 µg) was iodinated with 0.5 mCi Na125I (ISM30, Amersham International) using 20 µg chloramine-T in 20 µl 0.5 M phosphate buffer (pH 7.4). The labeling of the peptide was completed within 15 sec and the reaction was stopped by adding 20 µl of a sodium metabisulfite solution (3 mg/ml). The iodination mixture was then applied to a Sep-Pak C18 cartridge and eluted with a step gradient of acetonitrile (10–54%) in 0.1% aqueous TFA. Radioiodinated (Tyr0)-ODN was eluted at 26% acetonitrile. The radioactive peptide was kept frozen for 6 wk without noticeable loss of immunoreactivity. The RIA was performed using 0.02 M veronal buffer (pH 8.6) containing 0.4% BSA. The final dilution of the human ODN antiserum was 1:10,000 and the total amount of tracer was 6,000 cpm per tube. After a 2-d incubation, the antibody-bound ODN fraction was immunoprecipitated by addition of 200 µl of goat antirabbit {gamma}-globulin (1:30) and 200 µl of normal rabbit serum (1:100). The incubation was carried on for another 2 d. After centrifugation, the supernatant was removed and the precipitate containing the bound fraction was counted on a {gamma} counter (LKB Wallac, Rockville, MI). Standard curves were set up with synthetic human ODN at concentrations ranging from 2–5000 pg/tube. The sensitivity threshold of the assay was 2 pg/tube. The ODN antibodies exhibited a 24% cross-reactivity with human TTN.

RNA extraction and RT-PCR

Testicular tissue explants obtained at surgery were immediately frozen on dry ice. Total RNAs from three different testicular samples were extracted according to the method of Chomczynski and Sacchi (36) using Tri Reagent (Sigma). The concentration of total RNA was determined by measuring the OD at 260 nm (UV-1605, Shimadzu, Kyoto, Japan). Samples (10 µg mRNA each) were treated by ribonuclease-free deoxyribonuclease I (Pharmacia Biotech, Orsay, France) to remove potential contamining DNA. Total RNA from each human testicular tissue sample was converted into single-stranded cDNA using Superscript II (Life Technologies, Eragny, France) with oligo (deoxythymidine)12–18 primer (500 µg/ml) and amplified by PCR with the primers 5'-TGG CCA CTA CAA ACA AGC AA-3' and 5'-TGG CAC AGT AAC CAA ATC CA-3' corresponding, respectively, to bases 138–157 and 321–340 of the human DBI cDNA. Two other primers (5'-TGC TGA GTA YGT CGT GGA GTC-3' and 5'-TTG GTG GTG CAG GAK GCA TTG C-3'), corresponding respectively to bases 297–317 and 467–488 of the glyceraldehyde-3-phosphate dehydrogenase sequence (GenBank accession no. M17701), were used for semiquantitation of reverse-transcribed mRNAs. RT(-) experiments were also carried out with the same RNA samples to check for possible amplification of genomic DNA.

PCR-based procedures were performed in a final volume of 50 µl containing first-strand cDNA (1 µl), 1.5 U Taq polymerase (Promega, Charbonnières, France), PCR buffer (Life Technologies), 1.5 mM MgCl2, dimethylsulfoxide (5%), 0.2 mM deoxynucleotide triphosphates and 20 pmol of each primer. The PCRs were performed for 30 cycles (94 C, 10 sec; 58 C, 10 sec; 72 C, 60 sec). The PCR products were analyzed on a 1.5% agarose gel, blotted on a nylon membrane, and hybridized with [32P]-ATP-labeled internal oligonucleotide (5'-GAC TTC CAA GGA AGA TGC CA-3') corresponding to bases 243–262 of the DBI cDNA. PCR products were subcloned into pGEM-T (Promega) and sequenced using the Thermosequenase kit (Amersham, Orsay, France) on a Li-Cor 4200L DNA sequencer (ScienceTec, Les Ulis, France) using fluorescent T7 and T3 primers (MWG-Biotech, Courtaboeuf, France).

Perifusion experiments

The effect of test substances on testosterone secretion by human testicular tissue was studied using a perifusion system technique derived from that previously described for frog and human adrenal tissues (37, 38). Briefly, human testicular explants were immediately immersed into Krebs-Ringer buffer (NaCl 120 mM, MgSO4 0.67 mM, KCl 5 mM, KH2PO4 1.2 mM, CaCl2 2.6 mM, NaHCO4 22.5 mM, glucose 10 mM, BSA 1 g/liter (KRB), chilled at 4 C, and rapidly transported to the laboratory. The testicular tissue was carefully dissected and diced into small pieces (1–2 mm3). Testicular fragments were rinsed three times with fresh KRB and layered between several beds of Bio-Gel P2 (Bio-Rad, Marnes-la-Coquette, France) into perifusion chambers. Each chamber consisted of a polystyrene column (id, 10 mm) delimited by two Teflon pestles. The testicular tissue was perifused with KRB, continuously gassed with a 95% O2-5% CO2 mixture, at constant flow rate (320 µl/min), temperature (35 C) and pH (7.4). The test substances, dissolved in gassed KRB, were administered at the same flow rate as KRB alone. Effluent fractions were collected at 5-min intervals and kept at -20 C until assay.

Pilot experiments showed that, in basal conditions, the rate of testosterone secretion from perifused human testicular tissue gradually declined during the first 2 h and then stabilized during the following 7 h (data not shown). Thus, test substances were administered after a 2-h stabilization period.

Testosterone RIA

Testosterone concentrations were determined by RIA, in 100-µl samples of effluent perifusate. The testosterone antiserum (Sigma; catalog no. T4276) significantly cross-reacted with 5{alpha}-dihydrotestosterone (23%) but exhibited weak cross-reactions with 5ß-dihydrotestosterone and 11ß-hydroxytestosterone (2.0 and 2.8%, respectively). Cross-reactivity with all other steroids tested was lower than 2%. The working range of the RIA was 150-5000 pg/ml. The sensitivity of the assay was 5 pg/tube.

Statistical analysis

Each perifusion profile was established as the mean profile of testosterone release (±SEM) calculated over at least three independent experiments. The levels of testosterone release were expressed as percentages of the basal values calculated as the mean of eight samples (40 min) immediately preceding the administration of test substances.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunohistochemical localization of endozepines

Labeling of human testicular sections with the antihuman ODN antiserum revealed the presence of ODN-like immunoreactivity within seminiferous tubules. The immunoreaction was located in the cytoplasm of Sertoli cells as well as in germ cells (Fig. 1Go, A and B). Preincubation of the ODN antiserum with synthetic human ODN (10-6 M) resulted in a complete loss of the immunoreaction (Fig. 1Go, C and D). ODN-like immunoreactivity was also detected in Leydig cells (Fig. 1Go, B and E) that could be identified by labeling of consecutive sections with an antiserum against cytochrome P450c17 (Fig. 1FGo).



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  		FIG. 1. Immunohistochemical localization of endozepines in the human testis. A, Microphotograph of a testicular section labeled with ODN antibodies showing immunoreactive cells within seminiferous tubules (ST) and interstitial tissue (I). B, Higher magnification showing ODN-immunoreactive germ cells (G, solid arrows), Sertoli cells (S, open arrows), and Leydig cells (L, arrowheads). C and D, Adjacent control sections showing complete loss of immunoreaction after preabsorption of the ODN antibodies with 10-6 M synthetic human ODN. E and F, Comparative distribution of endozepines (E) and cytochrome P450c17 (F) on consecutive sections of human testis. Scale bars, A and C, 100 µm; B and D–F, 50 µm.

 
Biochemical characterization of endozepines

Serial dilutions of testicular extracts produced displacement curves that were parallel to those obtained with synthetic human ODN and TTN (Fig. 2AGo). The concentration of endozepines in crude testicular extracts (n = 5) was 1.14 ± 0.19 ng/g wet tissue.



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  		FIG. 2. Characterization of ODN-immunoreactive material in human testicular extracts. A, Semilogarithmic curves comparing competitive inhibition of antibody-bound 125I-labeled ODN by synthetic human ODN ({blacktriangleup}-{blacktriangleup}) and TTN (•-•), and by three different human testicular extracts ({blacktriangleup}-{blacktriangleup}, {blacksquare}-{blacksquare}, {blacktriangledown}-{blacktriangledown}). B, Reversed phase HPLC analysis of endozepine-like immunoreactivity in human testicular extracts. Representative profile obtained with an extract containing predominantly TTN. Arrows, Retention times of synthetic human ODN and TTN. Dashed line, Concentration of acetonitrile in the eluting solvent.

 
HPLC analysis of testicular extracts resolved two major peaks of ODN-like immunoreactivity. The first peak (retention time 23 min) coeluted with synthetic human ODN, whereas the second peak (retention time 30 min) coeluted with synthetic human TTN (Fig. 2BGo). The actual amounts of ODN and TTN contained in testicular extracts were determined by using standard curves made with ODN and TTN. Of five samples tested, four predominantly contained TTN (Fig. 2BGo), whereas one contained mainly ODN.

RT-PCR amplification of DBI mRNA

The presence of DBI mRNA in human testicular tissue was investigated by RT-PCR analysis. A cDNA band of the expected size (203 bp) was detected in the reverse transcribed products from three different testicular samples (Fig. 3Go). The cDNA bands were excised, ligated into PGEM-T, and sequenced. All sequences corresponded to the published sequence of the human DBI cDNA (GenBank accession no. NM_020548).



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  		FIG. 3. RT-PCR analysis of DBI mRNA in human testicular tissue. Specific primers for DBI and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to amplify DNA fragments of 203 and 192 bp, respectively, from three different testicular extracts. PCR products were electrophoresed on an agarose gel and stained with ethidium bromide (upper and lower photographs). The gel was then blotted and hybridized with a 32P-labeled internal DBI probe (central photographs). The control lane (C) contained no DNA.

 
Effect of endozepines on testosterone secretion from perifused human testicular tissue fragments

The effect of graded doses of synthetic TTN on testosterone production by perifused testicular fragments is shown in Fig. 4AGo. A dose-dependent increase in testosterone secretion was observed for concentrations of TTN ranging from 10-8 to 10-5 M (pEC50 = 6.58 ± 0.72). A significant response occurred within 20 min and the maximum effect was achieved 30–40 min after the beginning of TTN administration. Maximum stimulation (+252%) was obtained at a concentration of 10-6 M. In contrast, ODN (10-7 to 10-5 M) did not significantly affect testosterone secretion (Fig. 4AGo). As a comparison, the effect of hCG on perifused testicular fragments is shown in Fig. 4BGo. Graded concentrations of hCG (10-8 to 10-6 M) induced a dose-dependent stimulation of testosterone secretion (pEC50 = 6.26 ± 0.09). For each concentration of hCG, the lag period of the response was 20 min and the maximum effect on steroid output was observed 40–50 min after the onset of hCG administration. The efficacy (+120%) obtained at a concentration of 10-6 M, was significantly lower than that of TTN (P < 0.05, using unpaired t test with Welch’s correction).



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  		FIG. 4. Effect of increasing concentrations of endozepines and hCG on testosterone secretion by perifused testicular fragments. A, After a 120-min equilibration period, graded doses of TTN (10-8 to 10-5 M; filled circles) or ODN (10-7 to 10-5 M; open circles) were administered for 30 min. B, Graded doses of hCG (10-8 to 10-6 M) were administered for 20 min. The data represent the means (±SEM) of at least three independent perifusion experiments. Each point is the mean testosterone production (expressed as a percentage of spontaneous steroid output) of two consecutive fractions collected over 5 min. The mean basal levels of testosterone secretion in these experiments were (A) 1.54 ± 0.13 and (B) 1.55 ± 0.13 ng/min·g testicular tissue.

 
Pharmacological characterization of the human testicular endozepine receptor

In an attempt to determine the pharmacological profile of the receptor involved in the stimulatory effect of TTN on testicular steroidogenesis, we have investigated the action of various synthetic BZD receptor ligands on testosterone production from perifused testicular fragments. Administration of the PBR antagonist/CBR agonist flunitrazepam, at a concentration of 10-6 M, totally abrogated the stimulatory action of TTN on testosterone production (Fig. 5AGo). It was also noticed that graded concentrations of flunitrazepam (10-8 to 10-5 M) induced a dose-dependent decrease in spontaneous testosterone secretion (Fig. 5BGo). The lag period of the response was 20 min and the maximum effect on testosterone output (-45% of basal level) was achieved 100 min after the beginning of flunitrazepam administration (Fig. 5BGo). The inhibitory effect of flunitrazepam was dose-related for concentrations ranging from 10-8 to 10-5 M (pIC50 = 7.08 ± 0.05; Fig. 5CGo). The maximum response (-50% of basal level) was observed at a concentration of 5 x 10-7 M. In addition, flunitrazepam (10-5 M) totally inhibited the stimulatory effect of hCG (10-6 M) on testosterone secretion (Fig. 6AGo). Administration of graded concentrations of flunitrazepam (10-9 to 10-5 M) to the tissues induced a dose-dependent inhibition of hCG-stimulated testosterone secretion (pIC50 = 6.81 ± 0.26; Fig. 6BGo). In contrast, basal testosterone production was not affected by administration of the selective PBR agonists Ro5–4864 (10-5 M; Fig. 7Go) or PK11195 (10-5 M; data not shown).



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  		FIG. 5. Effect of the peripheral-type benzodiazepine receptor antagonist/central-type benzodiazepine receptor agonist flunitrazepam on testosterone secretion by perifused testicular fragments. A, Effect of TTN alone or during prolonged infusion of flunitrazepam. After a 120-min equilibration period, a pulse of TTN (10-6 M) was administered alone for 20 min (control conditions; left) or during prolonged infusion of flunitrazepam (10-6 M; right). B, Typical perifusion profiles showing the effect of three concentrations of flunitrazepam (10-8, 10-7 and 10-6 M) on testosterone secretion. After a 120-min equilibration period, fragments were perifused for 110 min with flunitrazepam. C, Semilogarithmic plot showing the effect of graded concentrations of flunitrazepam (from 10-8 to 10-5 M). All experimental values were calculated from data similar to those presented in panel B. The mean testosterone concentration in two consecutive fractions collected at the nadir during flunitrazepam administration (amplitude of inhibition) was compared with the mean testosterone level observed just before the infusion of flunitrazepam. The mean basal levels of testosterone secretion in these experiments were (A) 1.42 ± 0.23, (B) 1.90 ± 0.13, and (C) 1.66 ± 0.24 ng/min·g testicular tissue. See legend to Fig. 4Go for other designations.

 


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  		FIG. 6. Effect of flunitrazepam on hCG-induced testosterone secretion by perifused testicular fragments. A, Typical perifusion profile showing the effect of flunitrazepam (10-5 M) on the steroidogenic response to hCG (10-6 M). After a 120-min equilibration period, a pulse of hCG was administered alone for 20 min (control conditions; left) or during prolonged infusion of flunitrazepam 10-5 M; right). B, Semilogarithmic plot showing the effect of graded concentrations of flunitrazepam (from 10-9 to 10-5 M) on hCG-stimulated testosterone production. All experimental values were calculated from data similar to those presented in Fig. 6AGo. The results are expressed as a percentage of the maximum response induced by hCG in the absence of flunitrazepam. The mean basal levels of testosterone secretion in these experiments were (A) 2.04 ± 0.11 and (B) 1.45 ± 0.06 ng/min·g testicular tissue. See legend to Fig. 4Go for other designations.

 


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  		FIG. 7. Effect of the peripheral-type benzodiazepine receptor agonist Ro 5-4864 on testosterone secretion by perifused testicular fragments. After a 120-min equilibration period, 10-6 M Ro 5-4864 was infused for 120 min. The mean basal level of testosterone secretion in these experiments was 2.32 ± 0.30 ng/min·g testicular tissue. See legend to Fig. 4Go for other designations.

 
We have also examined the effect of CBR agonists and antagonists on testicular steroidogenesis. Administration of the CBR agonist clonazepam did not influence spontaneous testosterone secretion. Similarly, the specific CBR antagonist, flumazenil, had no effect on testosterone production.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As far as we know, this is the first report describing the occurrence and effect of endozepines in the human testis. Immunohistochemical labeling with specific antibodies against human ODN revealed the presence of ODN-like immunoreactivity in seminiferous tubules and Leydig cells. In seminiferous tubules, endozepine-like immunoreactivity is located in the cytoplasm of Sertoli cells as well as in germinal cells. These observations are consistent with previous data showing that, in the rat testis, DBI-like immunoreactivity is found in the cytoplasm of Sertoli cells, Leydig cells (6, 14, 39) and late-differentiated germ cells (11).

Although the antibodies used in the present study have already been used for labeling endozepine-containing cells in the brain and peripheral endocrine glands (10, 40), a degree of caution is warranted in interpreting results of immunohistochemical studies. In particular, it has been shown that, in rat, germ cells contain significant amounts of a DBI-related peptide called endozepine-like peptide, which may cross-react with the endozepine antibodies (41). However, several lines of evidence indicate that the immunoreactivity visualized in the human testis actually corresponds to authentic DBI. 1) Preabsorption of the antibodies with synthetic human ODN totally abolished the immunolabeling, showing the specificity of the immunoreaction. 2) The dilution curves obtained with testicular extracts were strictly parallel to those generated with synthetic ODN and TTN. 3) HPLC analysis of human testicular homogenates revealed two major peaks of immunoreactive material that exhibited the same retention time as synthetic human ODN and TTN. Previous reports have shown that HPLC analysis of rat testicular extracts resolves only one major endozepine-immunoreactive peak that elutes later than ODN and may thus correspond to TTN (6), whereas in MA-10 Leydig cells four immunoreactive compounds were detected, three of which coeluted with ODN, TTN, and DBI (12). 4) Finally, RT-PCR analysis and nucleotide sequencing of the amplified product confirmed that the DBI gene is actually expressed in the human testis.

The occurrence of endozepines in the human testis raises the possibility that DBI and its derived processing products may act locally to modulate steroidogenesis. The present study shows that TTN, but not ODN, is capable of stimulating testosterone secretion by human testicular tissue with a potency and an efficacy similar to those observed in MA-10 Leydig cells (12). In rodent Leydig cells, it has been found that both TTN and ODN stimulate steroid secretion (12, 14), whereas, in the frog adrenal gland, in very much the same way as in the human testis, TTN but not ODN stimulates corticosteroid secretion (15). Comparison of the steroidogenic responses induced by TTN and hCG shows that the two factors are approximately equipotent but that TTN is significantly more efficient than hCG to stimulate testosterone secretion. This observation supports the view that the endozepine TTN may play an important role in the control of the secretory activity of human Leydig cells.

It has been shown in various cell models that ODN is a preferential ligand for CBR, whereas TTN is a selective ligand for PBR (2, 3, 17). The fact that TTN, but not ODN, was able to stimulate testosterone production from the human testis strongly suggested that the stimulatory action of endozepines on testicular steroidogenesis could be accounted for by activation of PBR. Pharmacological characterization of the receptors mediating the effect of TTN confirmed the involvement of PBR in the mechanism of action of the peptide. Thus, flunitrazepam, a PBR antagonist with CBR agonistic properties, totally abolished TTN-induced steroidogenesis. Consistent with this observation, it has previously been reported that, in MA-10 and R2C Leydig tumor cells, flunitrazepam induces a concentration-dependent inhibition of DBI-evoked steroidogenesis (13, 14, 42, 43). The lack of effect of Ro5–4864 and PK11195, which has also been observed in R2C Leydig cells (13), can be likely ascribed to the weak affinity of the PBR expressed in the human testis for these compounds. In agreement with this hypothesis, it has been shown that the affinity of PBR for BZD receptor ligands exhibits marked differences depending on the mammalian species (44, 45, 46) and tissues examined (47, 48). The implication of PBR in endozepine-induced testicular steroidogenesis is further supported by the fact that neither the CBR agonist clonazepam nor the CBR antagonist flumazenil had any effect on testosterone production, indicating that the stimulatory effect of TTN on testosterone secretion is not mediated through CBR.

Interestingly, flunitrazepam induced a concentration-dependent inhibition of both spontaneous and hCG-evoked testosterone secretion, similar to that observed in MA-10 Leydig cells (42), suggesting the existence of an endogenous tone exerted by native endozepines in the human testis. Consistent with this notion, there is now accumulating evidence that, in the rat testis, PBR play an important role in basal and LH/hCG-stimulated steroidogenesis by mediating the entry of cholesterol into the mitochondria (26, 49). The involvement of endozepines in testicular steroidogenesis in humans is also supported by clinical observations that have shown a reduction in the plasma free testosterone index associated with an increase in plasma LH level in patients treated with BZD (32, 33, 34). Thus, endozepines, acting as inverse agonists on PBR (3, 50), stimulate, whereas BZD (agonists) inhibit testosterone production in human.

In conclusion, the present study has shown that the endozepines ODN and TTN are located in germ cells, Sertoli cells, and Leydig cells in the human testis. Our data have also revealed that TTN stimulates the secretion of testosterone through activation of PBR. Collectively, these results suggest that locally produced endozepines may act as autocrine and/or paracrine factors to modulate steroidogenesis in the human testicular tissue.


   Acknowledgments
 
We thank the urologists, Drs. J. M. Cleret, P. Grise, D. Pavard, C. Pfister, L. Roubach, O. Rousseau, and L. Sibert for providing testicular tissue. The authors also acknowledge Dr. V. Contesse for statistical analysis and D. Degrave, A. M. Heldé, D. Cartier, F. Pereira, and M. Guervin for skillful technical assistance.


   Footnotes
 
This work was supported by the Centre Hospitalier Universitaire of Rouen, Institut National de la Santé et de la Recherche Medicale, Unité 413, Institut Fédératif de Recherche Multidisciplinaires sur les Peptides 23, and the Conseil Régional de Haute-Normandie. C.D. was the recipient of a fellowship from the Société d’Andrologie de Langue française.

Abbreviations: BZD, Benzodiazepine; CBR, central-type BZD receptor; DBI, diazepam-binding inhibitor; hCG, human chorionic gonadotropin; KRB, Krebs-Ringer buffer; ODN, octadecaneuropeptide; PB, phosphate buffer; PBR, peripheral-type BZD receptor; TFA, trifluoroacetic acid; TTN, triakontatetraneuropeptide.

Received May 1, 2003.

Accepted August 11, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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