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Endocrinology, doi:10.1210/en.2006-0484
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Endocrinology Vol. 147, No. 9 4374-4383
Copyright © 2006 by The Endocrine Society

The Mouse Testis Is the Source of Various Serine Proteases and Serine Proteinase Inhibitors (SERPINs): Serine Proteases and SERPINs Identified in Leydig Cells Are under Gonadotropin Regulation

Fanny Odet, Adélie Verot and Brigitte Le Magueresse-Battistoni

Institut National de la Santé et de la Recherche Médicale, Unité 418, Institut National de Recherche et de Sécurité Unité Mixte de Recherche 1245, and Université Lyon 1, Hopital Debrousse, 69322 Lyon cedex 05, France

Address all correspondence and requests for reprints to: Dr. B. Le Magueresse-Battistoni, Institut National de la Santé et de la Recherche Médicale Unité 418/Institut National de la Recherche Agronomique, Unité Mixte de Recherche 1245, University of Lyon Claude Bernard, Hopital Debrousse, 29 rue soeur Bouvier, 69322 Lyon cedex 05, France. E-mail: lemagueresse{at}lyon.inserm.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The occurrence of various serine proteinases and serine proteinases inhibitors (SERPINs) was investigated by RT-PCR in whole testes of 1-, 3-, and 8-wk-old mice in crude and enriched germ cell fractions, mouse Leydig tumor cells (mLTC-1), and primary cultures of 3- and 8-wk-old enriched fractions of Leydig cells and 3-wk-old Sertoli cells. New members were identified in the testis protease repertoire. Within the Leydig repertoire, a PCR product was found for plasminogen activators urokinase plasminogen activator (uPA) and tissue plasminogen activator (8-wk-old cells), matriptase-2 (mLTC-1), kallikrein-21, SERPINA5, SERPINB2 (primary cultures), and serine peptidase inhibitor Kunitz type 2 (SPINT2). The gonadotropin regulation was explored by semiquantitative RT-PCR, using steroidogenic acute regulatory protein (StAR) as a positive control. Matriptase-2, kallikrein-21, SPINT2, and SERPINA5 were down-regulated, whereas uPA and its receptor were up-regulated by human chorionic gonadotropin (hCG) via cAMP in the mLTC-1 cells. Positive effects were observed transiently after 1–8 h of hCG exposure, and negative effects, first evidenced after 6 h, lasted 48 h. The hCG-induced effects were confirmed in primary cultures. In addition, SERPINB2 was augmented by hCG in primary cultures. Addition of either trypsin or protease inhibitors did not alter the hCG-induced surge of StAR. Because hCG regulated proteases and SERPINs (whereas testosterone did not), it could alter the proteolytic balance of Leydig cells and consequently the metabolism of extracellular matrix components. Therefore, even though a direct interplay between the early hCG-induced surge of uPA and StAR is unlikely, our data together with the literature suggest that extracellular matrix proteins alter Leydig cell steroidogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A NUMBER OF important processes that regulate the activity and fate of many proteins are strictly dependent on proteolytic processing events. The serine proteinase family is one of the oldest characterized and largest families of proteolytic genes (227 members in the mouse) (1, 2), which has well-characterized roles in diverse cellular activities including blood coagulation, platelet activation, fibrinolysis and thrombolysis, extracellular matrix (ECM) remodeling, and cancer invasion (3). The serine proteinases can be further subdivided into 16 families, among them the plasminogen activators, transmembrane serine proteinases, and kallikreins. Serine proteinase activity is regulated by serine proteinase inhibitors (SERPINs). The SERPINs belong to an expanding superfamily of structurally similar but functionally diverse proteins, and most of them share a substrate suicide mechanism irreversibly inactivating the SERPINs (4, 5, 6).

The testis is a reproductive organ that serves two decisive functions, the production of spermatozoa taking place in the seminiferous tubules and the synthesis and secretion of testosterone by the Leydig cells located in the interstitium. Spermatogenesis is hormonally regulated, pituitary gonadotropins positively regulating Leydig and Sertoli cell secretions through LH and FSH, respectively. Testosterone, produced as a result of LH action on Leydig cells, acts via androgen receptors localized on Sertoli, peritubular, and Leydig cells. Sertoli cells play several key roles in spermatogenesis. They are targets for FSH and testosterone, these hormones being responsible for the initiation and maintenance of spermatogenesis. Moreover, together with the peritubular cells, they also form the cytoarchitectural scaffolding of the tubule, providing structural and nutritional support for the developing germinal cells (7, 8, 9, 10).

In an attempt to elucidate the role of proteinases and proteinase inhibitors in testicular physiology, we examined the presence of various serine proteinases and SERPINs in the whole testes of mice as well as isolated testicular cells. For this purpose, we used total RNA recovered from whole testes of 1-, 3-, and 8-wk-old mice, from crude and enriched germ cell fractions, primary cultures of 3-wk-old Sertoli cells, 3- and 8-wk-old enriched preparations of Leydig cells, and mouse Leydig tumor cells (mLTC-1). Because Leydig cells were found to express several proteinases and SERPINs, we determined whether they were under gonadotropin regulation. We also examined the influence of testosterone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, tissues, and cell preparations
CD-1 mice were purchased from Elevage Janvier (Le Genest, France). Testes were collected from 1-, 3-, and 8-wk-old mice. Enriched preparations of pachytene spermatocytes and early spermatids (75–80% enrichment) were obtained after centrifugal elutriation of a crude germ cell preparation recovered from adult mice testes by trypsination (11). Once harvested, testes or cells were immediately stored at –70 C and processed for RNA analysis. Experiments were conducted according to the Guide for the Care and Use of Laboratory Animals.

Leydig-enriched fractions and Sertoli cells were isolated from 3-wk-old mice testes and cultured in Ham’s F12-DMEM (Life Technologies, Grand Island, NY) at 32 C in a humidified atmosphere of 5% CO2 as previously described (12), except that a hyaluronidase treatment (0.1%, 20 min, 32 C) was introduced after the second collagenase digestion to reduce peritubular cell contamination in the Sertoli cell fraction (13). Leydig cells were recovered in the supernatant of the first collagenase digestion, and the resulting suspension was hypotonic treated to eliminate the contaminating germ cells, as described recently (14). After several washes, the cell suspension was plated in 12-well plates (50,000 cells/well). Enrichment of the cell suspension was higher than 80% as judged by 3ß-hydroxysteroid dehydrogenase immunostaining (not shown). At the end of the enzymic procedure, the Sertoli cell suspension was washed with fresh culture medium, and cells were seeded in 6-well plates at a ratio of 1 x 106 viable cells/well in Ham’s F12-DMEM. They were cultured for 2 d. At that time, the purity of the cultures was higher than 90%, and contamination was mainly due to residual germ cells (an average of 5–7%) (not shown).

Leydig-enriched fractions were also isolated from 8-wk-old mice testes and cultured in Ham’s F12-DMEM at 32 C in a 5% CO2 incubator as described for the 3-wk-old testes. They were recovered in the supernatant of the first collagenase digestion, and the resulting suspension was harvestly washed, filtered through a 45-mm nylon mesh and hypotonic treated. After several washes, the cell suspension was plated in 12-well plates (50,000 cells/well). Enrichment of the cell suspension examined on the next day was higher than 70% as judged by 3ß-hydroxysteroid dehydrogenase immunostaining (not shown).

Cell line cultures
The immortalized Leydig cell mLTC-1 line was kindly provided by D. M. Stocco (Texas Tech, Lubbock, TX). The mLTC-1 cells were cultivated at 37 C in a 5% CO2 incubator. Culture medium was a RPMI 1640 medium (Sigma-Aldrich Corp., St. Louis, MO) supplemented with 10% fetal calf serum until cells reached subconfluency (70%), as described (15). Serum was then omitted, cells were rinsed abundantly, and fresh medium was replaced.

Cell treatment
In the relevant experiments, testosterone (0.1 µM), human chorionic gonadotropin (hCG; from 1 to 100 ng/ml; Organon, Puteaux, France), bu2cAMP (1 mM), actinomycin D (5 µg/ml), cycloheximide (50 µg/ml), trypsin (from 1 to 1000 ng/ml), or proteases inhibitors (a cocktail of aprotinin and leupeptin at 100 µM each) (all from Sigma) were added to the cultures at the doses and for the time periods indicated.

RNA extraction, RT-PCR, and semiquantitative RT-PCR
Procedures for RNA extraction and RT-PCR have been described elsewhere (16). Specific primers were designed using the Gene-Jockey sequence processor (Biosoft, Cambridge, Cambridgeshire, UK), and the optimal temperature of annealing was defined for each couple of primers (Table 1Go). Negative controls contained water instead of cDNA. PCR with no reverse transcription gave no product, eliminating the possibility of a genomic DNA contamination in the RNA preparations. Amplified cDNAs were visualized in a 1.5% agarose gel stained with ethidium bromide. A DNA ladder (Promega, Charbonnières, France) was loaded on each gel, and 18S was used to ensure equal loading. PCR products were sequenced by Biofidal (Lyon, France). Conditions for reliable semiquantitative RT-PCR were optimized for each series of primers in the presence of {alpha}-33P dATP (0.75 µCi; 2500 Ci/mmol; Amersham Pharmacia Biotech Europe GmbH, Orsay, France), as described elsewhere (17, 18). The PCR products were separated on 8% polyacrylamide gel electrophoresis in 1x Tris borate EDTA (TBE) buffer. Gels were transferred to filter paper, dried, and exposed to Kodak biomax MR1 films (Sigma). The band densities were determined by scanning densitometric analysis (Scion image ß, 4.03; Scion Corp.). Densitometry data were normalized using hypoxanthine phosphoribosyl transferase (HPRT).


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  		TABLE 1. Nomenclature and selected information on the serine proteinases and SERPINs hereby studied (1 )

 
Protein extraction and analysis
Gonadal proteins were extracted in PBS containing 1% Nonidet P-40 and 5 mM EDTA, as previously described (16). For plasminogen zymography, 10-fold concentrated proteins were electrophoresed at 4 C on 10% PAGE in the absence of any reducing agent. After electrophoresis, sodium dodecyl sulfate was removed from the gel by exchange in Triton X-100 (two washes, 30 min each, at room temperature in 2.5% Triton X-100, followed by three washes, 30 min each, in distilled water). The gel was subsequently placed on a casein-agar-plasminogen underlay as previously described (18, 19). Plasminogen-degrading activity was visualized after incubation at 37 C for 12 h, and gels were scanned. SDS-PAGE (10%) and Western blotting were carried out as described elsewhere (16, 17, 18), using the concentrated culture media also assayed in plasminogen zymography. A rabbit polyclonal antibody was used against recombinant mouse protein C inhibitor (PCI or SERPINA5; dilution 1:1000; a gift from M. Geiger, Department of Vascular Biology and Thrombosis Research, University of Vienna, Vienna, Austria) and was detected by an antirabbit IgG (dilution 1:10,000) conjugated to peroxydase (Jackson ImmunoResearch, Baltimore, MD). Precision protein standards (Bio-Rad Laboratories, Hercules, CA) were loaded for estimation of the molecular masses of the bands, which were revealed using an ECL+ chemiluminescent detection system (Amersham Pharmacia Biotech).

Data analysis
The densitometric data are given as the mean ± SEM (n = 3–4), and experiments were repeated at least three times (twice for the 8-wk-old preparations of Leydig cells). The significance of the results was determined by ANOVA followed by a Mann-Whitney U test. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RT-PCR screening of serine proteinases and SERPINs
Selected information and nomenclature for the seven serine proteinases and six SERPINs studied are summarized in Table 1Go, and the list and sequences of the designed specific primers for PCR studies are described in Table 2Go. Total RNA was recovered from 1-, 3-, or 8-wk-old testes from total germ cells or enriched fractions in pachytene spermatocytes or early spermatids; the mLTC-1; Leydig-enriched fractions recovered from 3- and 8-wk-old mice testes; and 3-wk-old Sertoli cells, which had been cultured for 2 d in basal conditions. A PCR product of the right size and sequence (not shown) was detected for each of the serine proteinases and SERPINs (Fig. 1Go). Plasminogen activators (PAs) were among the most abundant serine proteases within the testis at the three ages investigated, and tissue plasminogen activator (tPA) was more abundant than urokinase plasminogen activator (uPA). SERPINA5, serine peptidase inhibitor Kunitz type 1 (SPINT1), and SPINT2 were the most abundant SERPINs within the testis. Germ cells had the smallest repertoire with hepatocyte growth factor activator (HGFA), activated protein C (PC), SERPINA5, SPINT1, and the longest form of SPINT2. The weak bands seen for tPA and SERPINE2 in the nonelutriated fraction probably resulted from a somatic contamination because they were not detected in the enriched fractions (Fig. 1Go). These data extend previous results showing activated PC (aPC) in mouse germ cells (24) and SPINT2 in human germ cells (27).


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  		TABLE 2. List and sequence of the designed specific primers for PCR studies

 

Figure 1
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  		FIG. 1. RT-PCR analysis of various serine proteinases and SERPINs in 1-, 3-, 8-wk-old mice testes; germ cells either from total germinal cells (total) or fractions enriched in pachytene spermatocytes (scytes) or early spermatids (stids); mLTC-1 cells; Leydig-enriched fractions recovered from 3- and 8-wk-old mice testes; or Sertoli cells recovered from 3-wk-old mice testes (SC 3-wk), which had been cultured for 2 d in basal conditions. The set of primers used is listed in Table 2Go. RT-PCR studies were also conducted using primers directed against 18S to ensure that equal amounts of material were used. A DNA ladder was included in each gel for accurate determination of the size of the PCR product (not shown). Mat.-2, Matriptase-2; kall.-21, kallikrein-21.

 
Primary cultures of Leydig-enriched fractions as well as mLTC-1 cells exhibited a PCR product for six of the 13 serine proteases and SERPINs investigated, including the two plasminogen activators, matriptase-2, kallikrein-21, SERPINB2, SERPINA5, and SPINT2. No signals were seen with primers designed against activated protein C, HGFA, or hepsin (Fig. 1Go). Interestingly, the PCR band for matriptase-2 was detected in mLTC-1 but not Leydig-enriched fractions, Sertoli cells, or germ cells. Thus, the origin of the weak band in whole testes is yet unknown. Inversely, a PCR band for SERPINB2 was found in Leydig-enriched fractions but not mLTC-1 cells. Finally, a PCR band for tPA was found in 8-wk-old enriched fractions of Leydig cells (Fig. 1Go). All these data on Leydig cells are original except for tPA (28) and kallikrein-21 (22). Sertoli cells exhibited stronger PCR products corresponding to uPA and tPA than Leydig cells. A weak PCR product corresponding to hepsin was also found in Sertoli cells. Regarding the SERPINs, Sertoli cells exhibited a PCR product for each of them. In addition, PCR products were stronger in Sertoli cells than Leydig cells, with the exception of SERPINB2 (Fig. 1Go).

Addition of hCG altered the expression of proteinases and SERPINs in the mLTC-1 cells
Leydig mLTC-1 cells were cultured to 80% confluence, serum starved, and then treated with hCG at 1, 10, or 100 ng/ml for 6 h. Cells were then scraped, total RNA was extracted, and RT-PCR experiments performed. Primers used included those specific for uPA, kallikrein-21, matriptase-2, SPINT2, SERPINA5, HPRT as a house keeping gene, and the steroidogenic acute regulatory protein (StAR) to ensure that Leydig cells responded to hCG under our experimental conditions. Addition of hCG resulted in a significant enhancement of StAR at the three doses, and 100 ng/ml induced a 2.5-fold increase (P < 0.05). Interestingly, hCG positively regulated the expression levels of uPA and down-regulated the expression levels of matriptase-2, kallikrein-21, SERPINA5, and SPINT2 (Fig. 2Go). These effects depended on the doses of hCG, and a significant (P < 0.05) effect was obtained with the dose of 100 ng/ml. Indeed, uPA levels increased 4.2-fold, whereas levels of kallikrein-21, matriptase-2, SPINT2, and SERPINA5 were halved in the hCG-treated cultures, compared with the untreated cultures.


Figure 2
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  		FIG. 2. Regulation of StAR, uPA, kallikrein-21, matriptase-2, SPINT2, and SERPINA5 in the mLTC-1 cells by hCG. Cells were cultured for 6 h in the absence or presence of three different doses of hCG (1, 10, and 100 ng/ml, respectively). Three experiments were performed and a representative autoradiograph is shown. HPRT was used as a control in the RT-PCR studies.

 
Gonadotropins positively regulate the expression of uPA, its receptor (uPAR), and the uPA enzymatic activity via cAMP
The addition of hCG at a dose of 100 ng/ml for 2 h, which significantly increased (2.5-fold increase; P < 0.05) the expression of StAR, also significantly enhanced (a 5.2-fold increase; P < 0.05) the expression of uPA (Fig. 3Go). The hCG-induced expression of uPA was maintained after 8 h (a 2-fold increase; P < 0.05), but it was no longer observed after 24 h of stimulation. The time-course effects of hCG were mirrored by the addition of bu2cAMP 1 mM, indicating the involvement of the protein kinase A (Fig. 3AGo). We next examined the expression levels of the uPAR because uPAR dictates the site and extent of proteolysis of uPA (3). Addition of hCG or bu2cAMP was found to exert a significant but transient stimulatory effect on the expression of uPAR. The effect was observed after 2 h (a 3- and 2.9-fold increase, respectively; P < 0.05) but not after 8 or 24 h of stimulation (Fig. 3AGo).


Figure 3
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  		FIG. 3. Effects of hCG and bu2cAMP on the expression levels of StAR, uPA, uPAR (RT-PCR), and uPA biological activity (plasminogen zymography) in mLTC-1 cells. A, mLTC-1 cells were stimulated for 2, 8, or 24 h in the presence of hCG 100 ng/ml or bu2cAMP 1 mM. C, Control, i.e. untreated cells. B, mLTC-1 cells were pretreated for 30 min with actinomycin D (Act) or cycloheximide (CHX) and stimulated with hCG for 6 h. Representative autoradiographs are shown in A and B. Autoradiographs were scanned and the expression was normalized to the HPRT signal. Values are the mean ± SEM of n = 3; *, P < 0.05, compared with untreated dishes. C, Zymographic analysis of the culture media of mLTC-1 cells stimulated for 24 h in the presence or absence of 100 ng/ml hCG or bu2cAMP 1 mM. A representative zymogram of four experiments performed is shown. The uPA lytic band is noted on the left. Cont, Control.

 
The role of transcription and ongoing protein synthesis in the hCG-induced uPA mRNA levels was evaluated. For this purpose, mLTC-1 cells were pretreated for 30 min with actinomycin-D or cycloheximide and stimulated with hCG 100 ng/ml for 6 h. The results indicate that the presence of the inhibitor of either RNA or protein synthesis abolishes the hCG-induced positive regulation of uPA and StAR (Fig. 3BGo).

The biological activity of uPA was examined by zymography in culture media from mLTC-1 treated for 24 h with hCG or bu2cAMP. We observed a strong enhancement of the uPA lytic band in the stimulated cells. No tPA lytic band was detectable (Fig. 3CGo).

Gonadotropins negatively regulate the expression of matriptase-2, kallikrein-21, SERPINA5, and SPINT2 via cAMP
Matriptase-2, kallikrein-21, SERPINA5, and SPINT2 were decreased in hCG-treated cells after a kinetics different from that described for uPA and uPAR (Fig. 4AGo), and no effects were observed after 2 h of stimulation. However, in cells treated for 8 h with hCG, matriptase-2, kallikrein-21, SERPINA5, and SPINT2 were significantly decreased (P < 0.05), and their levels represented 33, 40, 36, and 50% of their respective control. After 24 h of hCG exposure, the inhibition was stronger than that after 8 h (except for SPINT2), with levels of matriptase-2, kallikrein-21, and SERPINA5 decreased to baseline levels. The kinetics of the effects induced by hCG were mimicked by the addition of bu2cAMP, indicating the involvement of the protein kinase A (Fig. 4Go).


Figure 4
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  		FIG. 4. Effects of hCG and bu2cAMP on the expression levels of matriptase-2, kallikrein-21, SERPINA5, and SPINT2 assessed by RT-PCR in mLTC-1 cells. A, mLTC-1 cells were stimulated for 2, 8, or 24 h with hCG 100 ng/ml or bu2cAMP 1 mM. C, Control cells, i.e. untreated cells. Representative autoradiographs are shown. mLTC-1 cells were cultured with (B) (+ hCG) or without (C) hCG 100 ng/ml for 16 h. Cells were washed and fresh hCG-free medium was added for 24 h. In A and B, autoradiographs were scanned and the expression was normalized to the HPRT signal (the same as in Fig. 3Go for A). Values are the mean ± SEM of n = 3. *, P < 0.05, compared with untreated dishes in A and the time-matched controls in B. C, Western blot analysis of culture media of mLTC-1 cells cultured for 24 h with or without (C) 100 ng/ml hCG or bu2cAMP 1 mM (same culture media as in Fig. 3CGo). Thirty micrograms of proteins were loaded per lane. Seminal vesicles (SV) and testis from adult mice (testis) were used as a positive control. The arrow points to the 46-kDa migrating band corresponding to SERPINA5. A representative gel of the three experiments performed is presented.

 
To determine the reversibility of the hCG-induced inhibition, mLTC-1 cells were treated for 16 h with hCG 100 ng/ml, after which cells were washed and fresh culture medium was added. After 24 h, cells were scraped and total RNA was extracted for RT-PCR studies. A densitometry analysis indicated a full recovery of the expression levels of matriptase-2 and kallikrein-21, whereas the expression levels of SERPINA5 and SPINT2 had only partially recovered (Fig. 4BGo). After 48 h, half and full recovery of the expression levels of SERPINA5 and SPINT2 were observed, respectively (not shown).

By Western blotting, a single protein, comigrating at 46 kDa with proteins extracted from seminal vesicles rich in PCI (24), was detected in testis extracts as well as the culture media of mLTC-1. Addition of hCG 100 ng/ml or bu2cAMP 1 mM for 24 h resulted in a strong decrease in the 46-kDa migrating band corresponding to PCI (Fig. 4CGo).

Testosterone did not influence the expression of proteases and SERPINs in the mLTC-1 cells
We also investigated a possible influence of testosterone 0.1 µM added for 2, 8, or 24 h. Androgen receptors were first evidenced by RT-PCR (not shown). No effects of testosterone were observed on uPA, matriptase-2, kallikrein-21, SERPINA5, or SPINT2 (not shown).

Gonadotropins regulate serine proteases and SERPINs expressed in primary cultures
Because mLTC-1 cells were tumor cells, we wished to examine whether hCG and testosterone could regulate serine proteases and SERPINs expressed in normal Leydig cells. For this purpose, enriched fractions of Leydig cells were isolated from 3- and 8-wk-old mouse testes, cultured for 2 d, and stimulated with hCG 100 ng/ml or testosterone 0.1 µM for 2, 24, or 48 h (Fig. 5Go). Androgen receptors were first evidenced by RT-PCR (not shown). The serine proteases and SERPINs examined include uPA, kallikrein-21, SERPINA5, SPINT2, and SERPINB2. tPA was also studied using the 8-wk-old preparations, but levels in the culture were too weak to be quantified (not shown). The data presented indicate that hCG significantly enhanced uPA and SERPINB2 and that kallikrein-21, SERPINA5, and SPINT2 were down-regulated by hCG in both types of primary cultures, i.e. from the 3- and 8-wk-old preparations (Fig. 5Go). After 2 h of stimulation, fold increases of uPA were 2.5 and 4.9 (P < 0.05), respectively for the 3- and 8-wk-old preparations in primary cultures. No effects of hCG were observed in long-term cultures (24 or 48 h) (Fig. 5Go A). The addition of hCG significantly increased SERPINB2 levels after 24 h of stimulation (a 4.1-fold increase; P < 0.05), using the 8-wk-old preparations. The fold increase was 1.6 (P < 0.05) after 48 h of stimulation using the 3-wk-old preparations. No significant effects were observed when the cells were treated for 2 h with hCG (Fig. 5AGo). The effects induced by hCG in primary cultures from the 8-wk-old preparations were mimicked by bu2cAMP (not shown). Primary cultures from the 3-wk-old preparations were not examined. The expression levels of kallikrein-21, SERPINA5, and SPINT2 were not modified by a 2-h exposure with hCG or testosterone (not shown). However, their levels were significantly (P < 0.05) halved in primary cultures treated for 24 or 48 h with hCG (Fig. 5BGo) or bu2cAMP (not shown). Testosterone had no effect (Fig. 5BGo).


Figure 5
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  		FIG. 5. Effects of hCG and testosterone on primary cultures of Leydig cells isolated from 3- and 8-wk-old testes. Leydig cells were cultured for 2, 24, or 48 h with or without hCG 100 ng/ml or testosterone 0.1 µM, as indicated. RT-PCR studies were performed using uPA and SERPINB2 (A) and kallikrein-21, SPINT2, and SERPINA5 (B). In A, the 3-wk-old Leydig cells were stimulated for 2 and 48 h, and the 8-wk-old Leydig cells were stimulated for 2 and 24 h; in B, the 3-wk-old Leydig cells were stimulated for 48 h, and the 8-wk-old Leydig cells were stimulated for 24 h. Representative autoradiographs are shown for serine proteases and SERPINs but not HPRT. Autoradiographs were scanned and expression was normalized to the HPRT signal. The values correspond to the mean ± SEM of n = 4 (3 wk old Leydig cells) and n = 3 (8 wk old Leydig cells). *, P < 0.05, compared with untreated cells of the time-matched control.

 
Effects of the exogenous addition of protease inhibitors or of trypsin
We explored the hypothesis that the enhancement of uPA and StAR may be interdependent events. For this purpose, we carefully examined the kinetics of the hCG-induced stimulatory effects during the first hours of treatment to determine which of uPA or StAR was first induced by hCG. Our data show that the hCG-induced enhancement of StAR and uPA followed closely the same pattern of expression up to 4 h of stimulation, after which uPA levels decreased, whereas StAR levels were maintained at a significantly higher level than in the controls, in mLTC-1 cells (Fig. 6AGo). We also examined SERPINB2 expression levels in the hCG-stimulated mLTC-1 cells. We observed the appearance of a PCR product in the hCG-treated mLTC-1 cells, whereas no PCR product was detected in untreated cells. However, the signal was too weak to be quantified (not shown). The expression levels of StAR were also significantly increased in primary cultures after a 2-h treatment with hCG (Fig. 6BGo).


Figure 6
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  		FIG. 6. Kinetics of the early effects of hCG on StAR and uPA in the mLTC-1 cells (A). Cells were stimulated with hCG 100 ng/ml for 15 min, 30 min, 60 min, 2 h, 4 h, and 8 h. Autoradiographs of the PCR products were scanned, the expression was normalized to the HPRT signal, and levels of uPA and StAR at the beginning of the experiment were arbitrarily fixed to 1. The values are the mean ± SEM of n = 3; *, P < 0.05. B and C, Effects of a cocktail of protease inhibitors and trypsin on the hCG- or bu2cAMP-induced StAR expression in mLTC-1 cells (B and C) and primary cultures of Leydig cells prepared from 3- and 8-wk-old testes (B). Cells were pretreated with a cocktail of inhibitors or trypsin (100 or 1000 ng/ml) and stimulated for 2 h with hCG 100 ng/ml (B) or bu2cAMP 1 mM (C). Autoradiographs of the PCR products were scanned and expression was normalized to the HPRT signal. The values are the mean ± SEM of n = 4. *, P < 0.05, compared with untreated cells of the time-matched control. In C, the number of PCR cycles was lowered to 19 instead of 22 for the bu2cAMP samples to avoid saturation of the signal. This explains why the intensity of the PCR bands between the treated and control samples are of the same amplitude.

 
We then explored whether the hCG-induced early peak of StAR could be mimicked by the addition of serine proteases. If so, the addition of serine protease inhibitors would decrease the hCG-induced increase of StAR. Cells were therefore pretreated with protease inhibitors or trypsin and then exposed to hCG for 2 h and the expression of StAR was examined. The addition of serine protease inhibitors (in both normal and tumor cells) or trypsin 1, 100, or 1000 ng/ml (performed in the mLTC-1 cells) had no effect on the hCG- or bu2cAMP-induced StAR (Fig. 6Go, B and C). Higher doses of trypsin resulted in cell detachment, and thus, their effect on StAR could not be examined (not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrate that the mouse testis is the source of several serine proteinases and inhibitors and that not only Sertoli cells and germ cells but also Leydig cells (enriched fractions from 3- or 8-wk-old mice testes or mLTC-1 cells) contribute to the protease repertoire. In addition, serine proteases and inhibitors detected in Leydig cells were regulated by hCG in both normal and tumor cells. The physiological significance of the data is discussed, taking into account the predicted role of proteinases and inhibitors in degrading the ECM components as well as data from the literature showing that ECM proteins alter Leydig cell steroidogenesis.

In a first series of experiments using a RT-PCR procedure, we show the presence of mRNAs encoding several proteinases and inhibitors in the mouse testis of various ages, enriched germ cell fractions, 3-wk-old mouse Sertoli cells, Leydig-enriched fractions from 3- and 8-wk-old mice testes, and the mLTC-1 Leydig cells. The purpose of using these different fractions was to evidence potential cross-contaminations because enriched Leydig fractions but not pure Leydig cells could be isolated and mLTC-1 cells are tumor cells. In addition, because of the differential proportion of somatic and germinal cells during testicular development, an age-developmental decrease of a PCR product suggests a somatic origin of the corresponding transcript, whereas an age-developmental enhancement of a PCR product suggests a germ cell origin. New members were identified in the mouse testis protease repertoire including HGFA in germ cells, hepsin in Sertoli cells, SPINT1 in Sertoli cells, and SPINT2 in the different fractions examined. Because all these molecules converge on the HGF signaling pathway and considering the importance of HGF in testicular physiology (29), our data support the hypothesis that in addition to the standard transcriptional level, the regulation of this pathway could involve a proteolytic level of regulation. This expression screening also demonstrates that Leydig cells exhibit a PCR product corresponding to six of the 13 proteases and inhibitors investigated. In addition to SERPINA5 (18), kallikrein-21 (22) and tPA weakly present in adult Leydig cells (28); the somatic form of SPINT2 was identified in Leydig cells. Interestingly, SERPINB2 was found in primary cultures of Leydig cells but not the mLTC-1 cells, and matriptase-2 was found in the mLTC-1 cells but not the primary cultures.

In the second part of this study, we show that hCG regulates the expression of serine proteinases and SERPINs identified in the mLTC-1 cells and the primary cultures. Furthermore, a positive regulation was highlighted for uPA (which depended on ongoing RNA and protein synthesis; studied in the mLTC-1 cells) and SERPINB2 (shown in normal cells), whereas SPINT2, kallikrein-21, SERPINA5, and matriptase-2 (shown in tumor cells) were negatively regulated by hCG. The hCG concentrations effective in regulating proteinases and SERPINs were identical with those required to stimulate the expression of StAR in the mLTC-1 cells (15). The striking difference was the kinetics of the hCG-induced effects. Indeed, we essentially observed an early and transient positive effect or a delayed (not evidenced before 6 to 8 h of stimulation) negative effect, which lasted 48 h. Interestingly, the hCG-induced enhancement of uPA paralleled the hCG-induced StAR during the first 4 h, after which uPA but not StAR levels decreased. Given that SERPINB2 may oppose PA activity (3, 4, 5, 6, 30), the hCG-induced enhancement of SERPINB2 observed in primary cultures might contribute to restrict excessive proteolysis of uPA. Therefore, the presence of matriptase-2 (not found in primary cultures) together with the very low level of SERPINB2 detected on hCG stimulation could account for the specific characteristics of the mLTC-1 cells. For instance, if the role of SERPINB2 is to oppose uPA within the Leydig cells, the lack of inhibition of uPA by SERPINB2 in mLTC-1 cells could explain a high uPA activity at a time (24 h of hCG stimulation) when uPA and uPAR mRNA levels return to their basal levels. Future experiments are required to determine the precise kinetics of the hCG-induced enhancement of SERPINB2 in normal Leydig cells and test whether SERPINB2 could degrade the protein uPA and its receptor in the primary Leydig cell cultures, keeping in mind that uPA and SERPINB2 actually perform many additional and unrelated functions (30).

We also explored the hypothesis that the hCG-induced enhancement of StAR and of uPA may be interdependent events. For this purpose, we examined the effect of a pretreatment with either serine protease inhibitors or trypsin on the expression of StAR in basal and hCG-treated cultures. We did not observe any effect. Because hCG can induce testosterone formation as early as 30 min in normal Leydig cells (9, 31, 32, 33), we also examined whether androgens could stimulate uPA. We did not observe any effect of testosterone on the proteases or SERPINs analyzed in the mLTC-1 cells or the Leydig cell primary cultures. We could not explain why kallikrein-21 was unresponsive to testosterone in our hands, whereas a positive effect had previously been reported (22). In addition, using the same batch of testosterone and mouse Sertoli cells from 3-wk-old mice testes, we observed that testosterone could induce the expression of SERPINA5 (Odet, F., A. Verot, and B. Le Magueresse-Battistoni, unpublished data), extending previous findings (10, 14). Collectively, our data could be interpreted as a lack of a direct interaction between the hCG-induced surge in StAR and uPA.

However, considering that plasmin generated by uPA is highly active in degrading ECM components and initiating a proteolytic cascade through activation of other proteases (3, 34), indirect interactions are likely. A similar situation has been described in the ovary in which plasmin action takes place early after the LH peak to ensure follicular rupture and ovulation (35, 36). In addition, ECM serves as a reservoir for a variety of biologically active molecules, which may interfere with Leydig cell steroidogenesis ability (31, 33, 37) when released and/or activated by proteases (3, 34). Thus, it could be suggested that uPA is involved in the metabolism of ECM components in the interstitial tissue surrounding the Leydig cells in the testis. In this context, it should be mentioned that adhesion, shape, proliferation, and gene expression of mouse Leydig cells are influenced by ECM in vitro (38), that rat Leydig cells are able to synthesize ECM proteins in vitro (39), and that fibronectin and type IV collagen induce the down-regulation of steroidogenic response to gonadotropins in adult rat Leydig cells (40, 41). It would be of interest to determine whether hCG modulates the nature of the ECM components synthesized in vitro by Leydig cells.

The second set of serine proteases and SERPINs expressed in Leydig cells, i.e. matriptase-2 (in tumor cells), kallikrein-21, SERPINA5, and SPINT2, was negatively regulated by hCG. Taking into account that serine proteinases exhibit degrading activity against ECM (3, 34), whereas inhibitors oppose this activity, our data suggest that, in contrast to its early-induced effects, the hCG-induced long-term effects may stabilize the ECM proteins in the interstitial area surrounding the Leydig cells in the mouse testis. However, the situation is far from clear regarding SERPINA5. Indeed, we previously reported that SERPINA5 is expressed in Leydig cells throughout development in which it could oppose a uPA activity (18). The present findings on the kinetics of the hCG-induced effects on uPA and SERPINA5 do not support our previous hypothesis, and SERPINB2 is a better candidate for opposing uPA activity in Leydig cells. SERPINA5 is unique in that serpina5–/– male mice are sterile because of increased or unopposed proteolytic activity, which might be responsible for premature release and degeneration of developing germ cells in the lumen of the tubules (42). The recent findings that SERPINA5 is also a Sertoli cell product and that its expression is positively controlled by testosterone (Refs. 10 and 14 and our unpublished data) may explain the phenotype of the transgenic mice. In this context, the relationship between the hCG down-regulation of SERPINA5 and the phenotype of the serpina5–/– male mice is still unclear. Future studies will be useful to clarify this point.

In conclusion, we demonstrate that gonadotropins substantially modify the proteolytic balance in Leydig cells and probably the metabolism of ECM proteins. Therefore, even though a direct interplay between the early hCG-induced surge of uPA and StAR is unlikely, our data should be considered together with the described influence of ECM proteins on Leydig cell steroidogenesis. Future studies will aim at clarifying whether the molecules studied here interact or display independent proteolytic roles within Leydig cells. It would also be of interest to determine whether common transcription factors drive their expression in Leydig cells.


   Acknowledgments
 
We are indebted to Douglas M. Stocco (Texas University, Lubbock, TX), and Margarethe Geiger (Department of Vascular Biology and Thrombosis Research, University of Vienna, Vienna, Austria) for providing us with the MLTC-1 cells and the anti-PCI antibody, respectively. Thierry Blachère and Annick Lefèvre are thanked for providing us with the enriched fractions of germ cells from mouse testes. We are grateful to Maguelone G. Forest (Hopital Debrousse, Lyon, France) and Sophie Rousseaux (Institut National de la Santé et de la Recherche Médicale U309, La Tronche, France) for their contribution in careful reading of the manuscript.


   Footnotes
 
This work was supported by Institut National de la Santé et de la Recherche Médicale, Institut National de la Recherche Agronomique, and the University of Lyon Claude Bernard. F.O. is funded by the Ministère de la Recherche et de la Technologie and the Fondation pour la Recherche Médicale. A.V. is funded by Organon (Azko, Nobel).

Author disclosure summary: F.O., A.V., B.L.M.-B. have nothing to declare.

First Published Online June 1, 2006

Abbreviations: aPC, Activated protein C; ECM, extracellular matrix; hCG, human chorionic gonadotropin; HGFA, hepatocyte growth factor activator; HPRT, hypoxanthine phosphoribosyl transferase; mLTC, mouse Leydig tumor cell; PA, plasminogen activator; PC, protein C; PCI, protein C inhibitor; SERPIN, serine proteinase inhibitor; SPINT1 or 2, serine peptidase inhibitor Kunitz type 1 or 2; StAR, steroidogenic acute regulatory protein; tPA, tissue plasminogen activator; uPA, urokinase plasminogen activator; uPAR, uPA receptor.

Received April 13, 2006.

Accepted for publication May 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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