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Endocrinology, doi:10.1210/en.2003-0955
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Endocrinology Vol. 145, No. 3 1481-1489
Copyright © 2004 by The Endocrine Society

Evidence for Similar Expression of Protein C Inhibitor and the Urokinase-Type Plasminogen Activator System during Mouse Testis Development

Fanny Odet, Romain Guyot, Patrick Leduque and Brigitte Le Magueresse-Battistoni

Institut National de la Santé et de la Recherche Médicale, Unité 329, Hopital Debrousse, 69322 Lyon, 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 329, 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
 
Plasminogen activators (PAs) and their inhibitors (PAIs) are predicted to be involved in the restructuring events that characterize the testis throughout development. We here demonstrate that PAI-3 or protein C (PC) inhibitor (PCI) was expressed in a sexually dimorphic fashion during mouse gonad genesis, whereas PAI-1 and -2 exhibited no sex differences. PCI transcripts accumulated rapidly in the male gonad, from 12.5 d postcoitum onward. Western blot and immunohistochemistry analyses confirmed that male, but not female, fetal gonads produced PCI, and that Leydig cells are the site of PCI synthesis. The occurrence of testicular target proteases for PCI, i.e. PC and urokinase- and tissue-type PA, was further tracked using RT-PCR, plasminogen zymography, and/or immunohistochemistry. PC and tissue-type PA showed no variation between sexes. By contrast, urokinase-type PA and its receptor (uPAR; which dictates the site and extent of proteolysis) exhibited sex differences from 13.5–14.5 d postcoitum. At that time, uPAR expression was restricted to Leydig cells. At earlier ages, uPAR was uniformly and widely distributed in the gonads of both sexes. In adult testes, PCI and uPAR immunoreactivities were also present in Leydig cells. In addition, PCI, PC, and uPAR had a germinal origin. Collectively, these results support the hypothesis that PCI may contribute to proteolysis equilibrium within the testis by acting in tandem with urokinase in Leydig cells and with PC and/or urokinase in spermatogenic cells. It will be important to determine how this role is linked to the phenotype of sterility reported elsewhere in male mice with pci deleted.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TESTIS IS a highly dynamic organ not only in the fetal period but also during postnatal development and in adult life. It is composed of two main compartments: the interstitium with the steroidogenic Leydig cells, and the seminiferous tubules. The seminiferous tubules are surrounded by peritubular cells. They contain associations of Sertoli cells and germ cells at different developmental stages. Sertoli cells play key roles in spermatogenesis. They are target cells for FSH and testosterone, which are the hormones responsible for the initiation and maintenance of spermatogenesis. They form the tubule and provide structural and nutritional support for the developing germ cells (1, 2, 3, 4). Initially, the gonads emerge as an outgrowth and will develop either as a testis or an ovary depending on the presence of the Sry gene located on the Y chromosome (5, 6, 7). In response to Sry, Sertoli cells differentiate. They synthesize the anti-Mullerian hormone (AMH), also called the Mullerian inhibiting substance, and they aggregate to form the cords together with peritubular cells originating from the mesonephros. Subsequently, Leydig cells differentiate in the interstitial milieu and start producing testosterone (6, 7, 8, 9, 10, 11). At puberty, dynamic changes are associated with the transformation of cords into tubules and the initiation of spermatogenesis. In adult life, migration is the fate of germ cells, which move from the base to the apex of the tubule while differentiating further. Finally, spermatids are released from the apex of the seminiferous epithelium (specifically, Sertoli cells) into the tubular lumen, becoming sperm. A new wave of spermatogenesis is initiated.

This spatio-temporal and highly orchestrated process is expected to involve several proteases and their specific inhibitors, to finely tune dynamic changes while preserving tissue integrity. Previous reports have consistently suggested that proteases or their inhibitors of the serine-, cysteine-, or metallo-protease family could act specifically, either during testis development (12, 13, 14, 15) or at specific stages of spermatogenesis (3, 16, 17, 18, 19, 20). However, the fact that deletion of genes encoding several matrix-degrading enzymes did not alter testicular development in genetically modified mice (21, 22, 23) had impeded further studies.

Protein C inhibitor (PCI) is unique in that male mice with pci deleted show abnormal spermatogenesis, and sterility ensues (24). PCI is a serine protease inhibitor (serpin) that opposes serine proteases, including protein C (PC) and the plasminogen activators (25). Interestingly, we demonstrate here that the expression of PCI is restricted to male mouse gonads from 12.5 d postcoitum (dpc) onward, and that Leydig cells are the source of PCI. The present comprehensive survey points out that the urokinase-type plasminogen activator (uPA) is a potential target protease for PCI. The serine protease uPA catalyzes plasmin formation from the zymogen plasminogen in the vicinity of the cells expressing the urokinase receptor (uPAR) (26), and we found that uPAR expression became restricted to Leydig cells during sexual differentiation. In adult testes, both Leydig cells and certain germ cells exhibited PCI and uPAR immunoreactivities. The present data are discussed in relation to the phenotype of sterility in male mice deleted for pci and the unexpected role of Leydig cells in controlling proteolysis activity within the testes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissues
Animals were purchased from Elevage Janvier (Le Genest, France). To determine the age of the CD-1 mouse embryos, the morning after vaginal plug formation was designated 0.5 dpc. Gonads with attached mesonephroi were dissected from embryos aged 11.5–14.5 dpc. At later fetal ages, only the gonads were dissected. Genotypic sex was determined by running PCR with primers to Sry for embryos of 11.5 and 12.5 dpc, and by morphological examination of the gonads at later embryonic ages (15). Postnatal testes were also collected at various ages. Other organs, such as seminal vesicles, adrenals, ovaries, kidneys, and livers, were dissected from adult mice. Once harvested, the various organs were immediately stored at -70 C and processed for RNA or protein analysis. Experiments were conducted with the approval of the local committee on animal care and in accord with the European guidelines.

RNA extraction, RT-PCR, and semiquantitative RT-PCR
The procedure for RNA extraction and RT-PCR has been described previously (15, 19). Specific primers were designed using Gene-Jockey sequence processor (Biosoft, Cambridge, UK), and the optimal temperature of annealing for each pair of primers was defined (Table 1Go). Negative controls contained water instead of cDNA. PCR with no RT reactions gave no band, eliminating the possibility of 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 Corp., Charbonnières, France) was loaded on each gel. PCR products were sequenced by Biofidal (Lyon, France). Conditions for reliable semiquantitative RT-PCR were optimized for each series of primers as described previously (15). Briefly, 0.5 µg RNA was reverse transcribed, and the resulting cDNA samples were normalized by PCR using hypoxanthine phosphoribosyl transferase (HPRT) with fetal gonads and 18S RNA when handling postnatal testes. The number of cycles for each gene was adjusted in test experiments to be within the linear range of detection. Linearity of the signal was obtained by using 30–60 ng RNA and running 22–26 cycles in the presence of [{alpha}-33P]deoxy-ATP (0.75 µCi; 2500 Ci/mmol; Amersham Pharmacia Biotech Europe GmbH, Orsay, France). The PCR products were separated on 8% polyacrylamide gel electrophoresis in 1x Tris-borate-EDTA buffer. Gels were transferred to filter paper, dried, and exposed to Kodak Biomax MR1 films (Sigma-Aldrich Corp., St. Louis, MO).


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  		TABLE 1. List and sequence of the designed specific primers for PCR studies

 
Protein extraction and analysis
Gonadal proteins were extracted in PBS containing 1% Nonidet P-40 and 5 mM EDTA, as previously described (15). For plasminogen zymography, proteins were electrophoresed at 4 C on 8% polyacrylamide gels 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 essentially as previously described (27). Plasminogen-degrading activity was visualized after incubation at 37 C for 24–48 h, and gels were scanned. SDS-PAGE (10%) and Western blotting were carried out as reported previously (15, 19). The antibodies used were a polyclonal antibody against recombinant mouse PCI raised in rabbit (dilution, 1:200; a gift from M. Geiger, Department of Vascular Biology and Thrombosis Research, University of Vienna, Vienna, Austria), and an antirabbit IgG (dilution, 1:2000) conjugated to peroxidase (Dako S.A., Trappes, France). 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).

Immunohistochemistry
The tissues were fixed at 4 C in 0.1 M phosphate buffer, pH 7.4, containing 4% formaldehyde plus 10% picric acid. They were fixed from overnight to 3 d depending on their size and embedded in Paraplast X-TRA (Labonord, Templemars, France). Serial 5-µm sections were cut, mounted onto gelatinized slides, and deparaffinized in toluene. Indirect immunoperoxidase staining was performed as previously described (28), with minor modifications (15). Briefly, the tissues were pretreated for 5 min with 1% lithium carbonate in 70% ethanol, and blocked for 15 min in 5% milk. Then the sections were sequentially incubated with primary antibody overnight at 4 C and antirabbit IgG-conjugated to peroxidase at a 1:500 dilution for 1 h at room temperature, and diaminobenzidine solution. The specificity of the staining was assessed as described previously (15, 28). Primary antibodies used in this study, include a rabbit anti-PCI antibody (also used in Western blot; diluted 1:100), a rabbit antisteroidogenic acute regulatory protein (anti-StAR) antibody (diluted 1:500; a gift from D. M. Stocco, Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX), a rabbit anti-uPAR (diluted 1:150; Santa Cruz Biotechnology, Santa Cruz, CA), a rabbit antimouse type 1 3ß-hydroxysteroid dehydrogenase (3ßHSD; diluted 1:1000; a gift from I. Mason, Reproductive and Developmental Sciences Division, Edinburgh, Scotland, UK), or a rabbit antisteroidogenic factor-1 (anti-SF-1; diluted 1:1000; a gift from K. Morohashi, Department of Developmental Biology, National Institute for Basic Biology, Myodaiji-cho Okasaki, Japan). Sections incubated in the absence of the primary antibody remained unstained.

Data analysis
All experiments were performed at least three times. The band densities obtained in the RT-PCR analyses were determined by scanning densitometric analysis (Alcatel TITN ANSWARE, Massy, France). Data are presented as the mean ± SEM (n = 3–5). The significance of the results was examined by ANOVA, followed by a linear regression analysis or t test for comparison between two groups. Differences are accepted as significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sex-dimorphic pattern of expression of PCI in fetal gonads
As a step toward understanding the involvement of the plasminogen-plasmin system in testis throughout development, male and female gonads were screened against the four defined inhibitors of plasminogen activators (PAIs) (25). The primers used for the RT-PCR analysis are listed in Table 1Go. Consistently (13), the PAI protease nexin-1 exhibited a 6.2-fold higher level in male than in female gonads (P < 0.002; n = 3). PAI-1 and -2 exhibited very weak signals. However, no sex difference could be evidenced in 18.5 dpc gonads (Fig. 1AGo) or at earlier ages (not shown). Interestingly, PCI (or PAI-3) expression was age dependent (P < 0.001) and restricted to male gonads (Fig. 1AGo) from 12.5 dpc onward (Fig. 1BGo), i.e. shortly after the peak of Sry expression at 11.5 dpc (6). In fact, PCI expression followed closely the pattern for StAR (Fig. 1Go, B and C) and was somewhat delayed relative to AMH (Fig. 1BGo). This finding is consistent with the expression of PCI in Leydig cells, which express StAR (29), rather than in Sertoli cells, which express AMH (8). By Western blot, a single protein was identified in male, but not female, gonads at 18.5 dpc (Fig. 2AGo) and in seminal vesicles rich in PCI (24). The calculated migrating size was consistent with the molecular mass of 46 kDa reported for PCI (24). Finally, we found that the PCI immunoreactivity was confined to interstitial cells positive for StAR, identifying the Leydig cell population. No PCI immunostaining was observed within the seminiferous cords in 18.5 dpc testes (Fig. 2BGo). Female gonads remained unstained (not shown).



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  		FIG. 1. Sex-dimorphic expression of PCI in fetal gonads. A, The expression of four serpins [PAI-1, PAI-2, PCI, and protease nexin-1 (PN-1)] was monitored in 18.5 dpc gonads using a semiquantitative RT-PCR procedure. The upper PCR bands correspond to the serpin bands, and the lower bands correspond to the HPRT signal. B, The ontogeny of PCI was monitored in fetal gonads from 11.5 dpc onward, and the profile was compared with the expression profile of AMH and StAR. HPRT was used as an internal control. C, Autoradiographs for PCI and StAR were scanned, and expression was normalized to the HPRT signal. Values are the mean ± SEM of three (StAR) and four (PCI) independent series of samples. m, Male gonad; f, female gonad; ND, not detectable.

 


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  		FIG. 2. PCI expression in 18.5 dpc gonads. A, A Western blot analysis using an anti-PCI antibody indicated that PCI was only produced in male gonads (50 µg proteins loaded/lane). Seminal vesicles (SV) were used as a positive tissue (20 µg proteins loaded). Three experiments were performed, and a representative blot is shown. B, Immunohistochemical (IHC) localization of PCI and StAR in 18.5 dpc male gonads using serial sections. PCI immunoreactivity was confined to cells positive for StAR, identifying the Leydig cell population (arrowheads). m, Male gonad; f, female gonad; I, interstitium; SC, seminiferous cords. Bar, 70 µm.

 
Localization of PCI within StAR-expressing tissues
Inasmuch as the patterns of expression of PCI and StAR could be superposed, that adult ovaries have been found to express StAR (29, 30) and to contain transcripts for PCI (Fig. 3AGo) (24), whereas fetal ovaries are devoid of both StAR (29) and PCI (this study), we examined whether other StAR-expressing cells could produce PCI. Within adult ovaries, the corpus luteum, which stained for StAR as expected (29), was weakly labeled for PCI (Fig. 3BGo). By contrast, thecal cells that express StAR (29) did not immunostain for PCI (not shown). Adrenals had a PCI signal through RT-PCR (Fig. 3AGo) and strong PCI immunostaining was confined to the medulla, which stained weakly for StAR as shown previously (31). By contrast, the cortex, which is extremely rich in StAR (29), was devoid of PCI immunostaining (Fig. 3BGo), underlining that PCI and StAR are not under the same transcriptional machinery.



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  		FIG. 3. PCI and StAR are not systemically coexpressed. A, PCI and StAR expression was examined by a classical (not semiquantitative) RT-PCR in adult tissues using 32 cycles of amplification. Amplified cDNAs were visualized in a gel stained with ethidium bromide. HPRT was used as an internal control, and a DNA ladder was included in the gel. B, Immunohistochemical localization of PCI and StAR in ovary and adrenal of adult mice using serial sections. The arrowheads point to positive areas, and the asterisk indicates the absence of PCI labeling in a StAR-positive area. CL, Corpus luteum; Cx, cortex; Md, medulla. Bar, 150 µm.

 
Analysis of PAs in fetal testes
PAs are target proteases for PCI (25). PAs are of two types, the tissue type (tPA) and the urokinase type (uPA), and the two PAs are produced within the testis (16). PAs catalyze the conversion of plasminogen (produced in testis) (16) into plasmin, and plasmin is a powerful matrix-degrading enzyme (16, 21). RT-PCR analysis demonstrated that tPA RNA levels decreased significantly (P < 0.001) as a function of age. However, no significant variations were observed between male and female samples throughout the period examined (Fig. 4AGo). With regard to uPA, we made three major observations (Fig. 4BGo). Firstly, at the two earliest ages investigated (11.5 and 12.5 dpc), uPA RNA levels in male gonads were not significantly different compared with uPA RNA levels in female gonads. Secondly, uPA RNA levels in female gonads decreased continuously and significantly (P < 0.001) as a function of age, and at 12.5, 13.5, 14.5, and 17.5 dpc, uPA RNA levels corresponded to 79%, 41%, 34%, and 28%, respectively, of the levels observed at 11.5 dpc. Thirdly, uPA RNA levels in males decreased by only 1.36-fold (P < 0.01) between 11.5 and 17.5 dpc. As a consequence, a sex-dimorphic pattern (P < 0.01) could be seen at 14.5 and 17.5 dpc.



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  		FIG. 4. Analysis of tPA (A) and uPA (B) gene expression in male and female gonads from 11.5 until 17.5 dpc using a semiquantitative RT-PCR procedure. HPRT was used as a control. A representative autoradiograph is shown for tPA and uPA in the left half of A and B, respectively. Autoradiographs for tPA (A) and uPA (B) were scanned, and expression was normalized to the HPRT signal. Values are the mean ± SEM of four or five independent series of samples. a, P < 0.01 (compared with female gonads of the same age).

 
To determine whether the higher uPA RNA levels detected in males compared with female gonads corresponded to an increase in urokinase activity, a zymographic analysis was performed. Kidney samples were used because they have long been recognized as a major source of PAs (32). As expected from the RT-PCR study, fetal gonads exhibited the two types of plasminogen activators. tPA and uPA migrated at approximately 75 and 45 kDa, respectively (Fig. 5AGo), and tPA was the minor form synthesized. Given that tPA was weakly detectable, it was difficult to visualize the age-dependent decrease in tPA that might be expected from the RT-PCR analysis. By contrast, the uPA was present at equal and very high levels in male and female undifferentiated gonads. Thereafter, the uPA signal decreased with the advancing age of the gonad donor. Such a decrease, however, was less intense in males compared with females. As a consequence, a sex-dimorphic profile was observed at approximately 13.5 dpc (Fig. 5AGo).



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  		FIG. 5. A, Plasminogen zymographic analysis of male (m) and female (f) gonads from 11.5 until 17.5 dpc (40 µg proteins loaded/lane). Kidney extracts were used for identification of the size of the lytic bands of uPA and tPA (45 and 75 kDa, respectively). In that case, plasminogen-degrading activity was visualized after a 24-h incubation at 37 C instead of a 48-h incubation with gonad samples. Three different experiments were performed with three independent series of gonadal proteins, and a representative zymogram is shown. B, Analysis of uPAR gene expression using a semiquantitative RT-PCR procedure in male and female gonads from 11.5 to 17.5 dpc. HPRT was used as a control. A representative autoradiograph is shown in the left half of B. Autoradiographs were scanned, and expression was normalized to the HPRT signal. Values are the mean ± SEM of four or five independent series of samples. a, P < 0.05; b, P < 0.01 (compared with female gonads of the same age).

 
Expression of uPAR in fetal gonads
Proteolysis mediated by uPA requires the presence of a receptor, i.e. uPAR. The uPAR is a glycosylphosphatidylinositol anchor membrane protein that localizes the generation of plasmin to the close environment of the cell expressing it (26). RT-PCR analysis demonstrated that the uPAR levels in female gonads decreased significantly (P < 0.001) as a function of age, and at 17.5 dpc, they represented 27% of the levels detected at 11.5 dpc (Fig. 5BGo). By contrast, in male gonads uPAR RNA levels increased significantly (P < 0.001) as a function of age from 12.5 dpc onward. They were 2-fold higher (P < 0.001) at 17.5 vs. 12.5 dpc. Given that the uPAR RNA levels were of the same order in male and female gonads of 11.5 dpc, a sex-dimorphic pattern was evident from 13.5 dpc onward, and at 17.5 dpc, uPAR RNA levels were 5.7-fold (P < 0.01) higher in males compared with the females of the same age (Fig. 5BGo).

Localization of uPAR immunoreactivity was further examined in fetal gonads. The earliest age investigated was 11.5 dpc when male and female gonads are morphologically identical. At that time, we observed a high uPA activity coupled to high levels of uPA and uPAR through RT-PCR (Figs. 4Go and 5Go). We found that uPAR immunostained the whole gonadal territory (the coelomic epithelium and the mesonephros) in a strong and uniform fashion (Fig. 6Go). SF-1 was used as a marker of the coelomic epithelium (the genital ridge) and adrenal territories (33). Progressively, uPAR staining became specifically restricted to male gonads within the cells located in the interstitium and positive for the 3ßHSD type I isoform, i.e. the Leydig cells (34). The first uPAR-positive Leydig cells were seen at 13.5 dpc. At 17.5 dpc, uPAR staining was exclusively restricted to Leydig cells (Fig. 7Go). A uniform and low staining persisted in female gonads from 13.5 dpc onward (Fig. 7Go; not shown for older ages).



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  		FIG. 6. Immunohistochemical localization of uPAR in 11.5 dpc male and female gonads. The boundary between the coelomic epithelium (CE), which immunostained for SF-1 (see arrowheads), and the mesonephros (M) is indicated by a dotted line. The adrenal (Ad) territory also immunostained for SF-1. Immunoreactivity for uPAR was widely distributed within 11.5 dpc gonads of both sexes. Bar, 90 µm.

 


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  		FIG. 7. Immunohistochemical localization of uPAR in fetal gonads during sexual differentiation. The testis comprises the interstitium (I) containing Leydig cells positive for 3ßHSD and the seminiferous cords (SC). First uPAR-positive Leydig cells were seen at 13.5 dpc. They were more numerous at 15.5 and 17.5 dpc. Serial sections were used, and the arrowheads point to Leydig cells. In female gonads, no organization was evident at 13.5 dpc, and the whole gonadal territory exhibited immunopositive staining for uPAR. Bar: for 13.5 dpc gonads, 200 µm; for 15.5 and 17.5 dpc gonads, 100 µm.

 
Immunolocalization of uPAR and PCI in postnatal testes
Inasmuch as male mice deleted for pci are sterile secondarily to abnormal spermatogenesis (24), we wanted to determine whether PCI and the urokinase system may work in tandem in postnatal testes. During normal testicular development, two separate populations of Leydig cells arise in a sequential manner. The fetal population is essential for masculinization of the fetus. The adult population differentiates after birth, and testosterone secretion by this population acts to induce male behavior, secondary sexual characteristics, and fertility (34, 35, 36). The 3ßHSD type I isoform is expressed by the two populations of Leydig cells (34), and we used it to examine PCI and uPAR in postnatal testes.

Around 2 wk of age, the two populations of Leydig cells coexist in the interstitium (34, 35). The cords are now tubes with a lumen in the center. Sertoli cells have ceased dividing and create the testis barrier by establishing tight junctional complexes (1). Spermatogenesis has progressed, and primary spermatocytes are present in tubules. We observed some uPAR and PCI immunoreactivities within Leydig cells. No staining could be evidenced within the seminiferous epithelium (Fig. 8Go). Around 8 wk of age, testes are adult, spermatogenesis is cyclical, and 12 stages (I–XII) have been defined depending on the specific germ cell associations found in the tubular cross-sections (37). PCI and uPAR immunoreactivities were found in Leydig cells, but also within the seminiferous epithelium (Fig. 9Go). Interestingly, a distinct pattern of staining was observed with uPAR and PCI depending on the stage of the seminiferous epithelium. Immunostaining for PCI was restricted to the early spermatids within the acrosomal region, from stages V–VI (not shown) to stages VII and VIII (Fig. 9Go). Both cytoplasm and flagellum of the elongated spermatids at stage VII were also immunoreactive. A very faint signal persisted in spermatids at stages IX–X (Fig. 9Go). Spermatogonia did not show any staining for PCI throughout the entire period of development, nor did Sertoli cells. The situation was somewhat different for uPAR, in that its staining was restricted to stages VII–VIII of the seminiferous epithelium (Fig. 9Go). Precisely, we found staining at the level of the spermatocytes migrating toward the apical compartment as well as in the elongated spermatids that are released into the tubular lumen. Sertoli cells and spermatogonia were never labeled regardless of the stage (Fig. 9Go).



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  		FIG. 8. Immunohistochemical localization of PCI and uPAR in testes at 16 postnatal d. At that age, a lumen (Lu) is visible in the center of the cord termed tubule; spermatogenesis has progressed, and primary spermatocytes are present in tubules. In the interstitium (I), Leydig cells (arrowheads) that stained for 3ßHSD (A and D) are immunoreactive for PCI (B) and uPAR (E). Serial sections were used in A–C and in D–F. No staining was present within the seminiferous epithelium. Sections incubated in the absence of the primary antibodies remained unstained (C and F). Bar, 120 µm.

 


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  		FIG. 9. Immunohistochemical localization of PCI and uPAR in testes at 56 postnatal d. At that age, testes are adult, spermatogenesis is cyclical, and 12 stages have been defined (indicated in Roman numerals). Leydig cells immunostained for PCI and uPAR independently of the stage (short arrows). PCI and uPAR immunoreactivities were also found within the seminiferous epithelium. However, in that case staining was stage dependent (arrowheads). PCI immunoreactivity was detected in early spermatids within the acrosomal region (shown for stages VII and VIII). Spermatids at stages IX–X were weakly stained. Elongated spermatids were also labeled for PCI at stage VII. Immunoreactivity for uPAR was restricted to stages VII–VIII, at the level of the migrating spermatocytes and the elongated spermatids. Bar, 90 µm.

 
Analysis of PC in testis
Active PC is the active enzyme of the anticoagulant PC pathway and a preferential substrate for PCI (25). In addition, PC is produced in testis (38). We thus investigated PC ontogeny using a semiquantitative RT-PCR procedure. During fetal life, PC levels were hardly detectable and were roughly constant between male and female gonads (Fig. 10AGo). The signal disappeared in the first days of life, and from 21 d onward levels increased dramatically, coinciding with spermatids populating massively the tubules (Fig. 10BGo). These data extend the findings of a previous in situ study identifying spermatogenic cells (spermatids) as the major source of PC mRNA in the testis and excluding interstitial cells (38).



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  		FIG. 10. Analysis of PC gene expression in fetal gonads at 12.5, 14.5, and 18.5 dpc (A) and during testicular development (B) using a semiquantitative RT-PCR procedure. HPRT was used as an internal control in A, and 18S was used as an internal control in B. A representative autoradiograph is shown in the upper part of B. Autoradiographs were scanned, and expression was normalized to the 18S signal. Values are the mean ± SEM of three or four independent series of samples. m, Male; f, female; ND, not detectable; dpn, days postnatal.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we demonstrated that PCI was produced by Leydig cells and spermatids. We illustrated that PC and uPA were differentially expressed within the testis, and uPAR had its expression restricted to Leydig cells and certain germ cells at stages VII–VIII of the seminiferous epithelium. The present data will be discussed in relation to the sex-dimorphic pattern of expression of PCI, the occurrence and localization of PCI and its target proteases in fetal and adult testes, and the described phenotype of sterility in male pci-/- mice.

In a first series of experiments we pointed out that PCI had a sex-dimorphic pattern of expression. The profile closely paralleled the expression pattern described for StAR (Ref. 29 and this study), and Leydig cells appeared as the unique site of expression in the fetal testis. This raised the possibility that some of the transcription factors driving StAR expression (30) also regulate PCI transcription in Leydig cells. Definitive answers await molecular analysis. This study also highlighted that the interstitial compartment may participate in the regulation of proteolytic equilibrium during fetal life. Indeed, testicular architecture in two distinct compartments relies on Sry-inducing events of cell migration from the coelomic epithelium (Sertoli cells), the mesonephros (peritubular cells), or both (Leydig cells) (7, 9, 10, 11), and the three inhibitors of proteases reported to date with a sex-dimorphic pattern of expression (12, 13, 15) were products of the seminiferous cords.

Based on the profile of expression of the target proteases for PCI, PCI would rather oppose uPA than tPA or PC in fetal testes. In addition, the observation that the levels of both uPAR (promoting uPA activity) and PCI (opposing a protease activity) were increasing with the advancing age of the testis donor, whereas in the meantime uPA zymographic activity (reflecting the net activity between the protease and its inhibitor) did not increase further supports the hypothesis that uPA is a target for PCI in fetal testes. We also demonstrated that Leydig cells that express uPA (39) and PCI (this study) were the site of plasmin generation in fetal testes. In fact, high and homogeneous staining for uPAR was present in undifferentiated gonads, consistent with the RT-PCR data and the zymographic analysis. During testis organogenesis, differentiating Leydig cells were found to retain the capacity to express this receptor and thus to develop uPA activity at their cell membrane. The other cell types did not. These observations raise the importance of examining uPAR and PCI expression in fetal testes of genetically engineered mice in which Leydig cell differentiation is profoundly impaired (10, 11). Also PCI should be monitored in uPAR-deficient mice (21).

A thorough examination of the PCI immunoreactivity throughout testis development pointed out that the adult population of Leydig cells as well as the spermatids were a source of PCI, extending a previous result identifying PCI in the acrosomal cap of epididymal spermatozoa in humans (40). Interestingly, PCI was found to be under androgen control in rats even though testosterone itself did not affect PCI gene transcription (shown in seminal vesicles) (41). In addition, male, but not female, mice deleted for pci are sterile. Precisely, testis extracts of pci-/- mice had higher amidolytic activity compared with wild-type or heterozygous animals (24). Inasmuch as this amidolytic activity could be quenched by exogenous recombinant PCI or amiloride (an inhibitor of the urokinase activity), these researchers concluded that increased or unopposed proteolytic activity of the urokinase type might be responsible for premature release and degeneration of developing germ cells in the lumen of the tubules. Furthermore, testosterone inhibits the synthesis of PAs by Sertoli cells (16), and its withdrawal promotes premature detachment of early spermatids in stages VII–VIII in the rat (42). Collectively, these data prompted us to examine the occurrence of target proteases for PCI in adult testes.

As depicted for PCI, Leydig cells, and germ cells were two different sources for uPAR in adult testes, extending previous data identifying that spermatogenic cells and spermatozoa could display uPA-binding activity (43). Precisely, uPAR immunostaining was restricted to germ cells at stages VII–VIII of the seminiferous epithelium. Interestingly, uPA immunoreactivity was also exclusively detected at these stages in rat Sertoli cells (44). In the rat as in mouse testis, these stages cover the important remodeling events of translocation of the spermatocytes through the testis barrier and of release of the elongated spermatids into the lumen, followed by phagocytosis of the shed cytoplasts (residual bodies) by Sertoli cells (1). Whether germ cells bearing uPAR facilitate their movement (spermatocytes) and their release into the lumen (elongated spermatids) by localizing plasmin generation at their cell membrane is an attractive hypothesis. In vitro studies documenting that spermatocytes may induce Sertoli cell uPA activity (20) (Le Magueresse-Battistoni, B., unpublished observations) support this hypothesis. In that situation, however, inasmuch as PCI is not expressed in either spermatocytes or Sertoli cells, an alternate serpin would have to oppose excessive uPA activity. PAI-1 produced by Sertoli cells (17) appears as a likely candidate.

The situation is probably more complex at the spermatid level. Round spermatids at stages V–VIII express PCI, but not uPAR, contrasting with elongated spermatids at stages VII–VIII that express both. Considering that elongated spermatids and their shed cytoplasts can induce Sertoli cell uPA activity in a coculture system (18), it might be proposed that the balance PCI/urokinase favors PCI in round spermatids and urokinase activity in elongated spermatids at stages VII–VIII. This would permit the detachment of elongated spermatids (which occurs in the early stage VIII) while preventing premature release of the early one. Whether this situation is prevailing in mice deleted for pci (24) needs further investigation. Also, the role played by Leydig cells that produce PCI, uPAR, and testosterone remains unknown.

With regard to PC, it has yet to be demonstrated that the protein is indeed produced by spermatids. Notwithstanding, the PC pathway contributes to the maintenance of tubular fluidity in various human body fluids where PC is secreted (38). Spermatids regulation of seminiferous tubular fluidity produced by Sertoli cells would be consistent with the concept of mutual assistance between these two cell types, as documented previously (1, 2, 3, 4, 19, 20).

Despite their proteolytic activities, it has to be kept in mind that PCI and urokinase display other multiple functions. The urokinase system can promote cell adhesion or detachment, and uPA, by binding to its receptor, can behave as a growth and/or chemotactic factor, independently of its proteolytic activity (26). Also, PCI has recently been identified as a binding protein for retinoic acid (45), and Leydig cells are targets for retinoids (36). These findings may explain why mice deleted for either uPA or uPAR (14) do not exhibit the phenotype of sterility observed in male mice deleted for pci (24).

In conclusion, PCI and uPAR are markers of both Leydig cells and some specific germ cells, extending previous reports documenting the presence of PCI and uPAR on human spermatozoa and emphasizing their role in sperm-oocyte interactions (24, 40, 43). Our current interest is to examine how testes of pci-/- mice develop throughout the fetal as well as the postnatal period, when the testis barrier first develops, and to determine the cascade of events leading to spermatogenesis failure in these animals. Such developmental studies might assist in unraveling the role of PCI as a serpin as well as be instrumental in elucidating the role and the relevance of PCI in vivo.


   Acknowledgments
 
We are indebted to Margarethe Geiger (University of Vienna, Vienna, Austria), Douglas M. Stocco (Texas University, Lubbock, TX), Ian Mason (University of Edinburgh, Edinburgh, Scotland, UK), and Ken-Ichirou Morohashi (National Institute for Basic Biology, Okazaki, Japan) for providing us with the anti-PCI, anti-StAR, anti-3ßHSD, and anti-SF-1 antibodies, respectively. We are grateful to Maguelone Forest (Hopital Debrousse, Lyon, France) for her careful reading the manuscript, and to Hervé Le Jeune for his help with the statistical analysis.


   Footnotes
 
This work was supported by Institut National de la Santé et de la Recherche Médicale and Ministère de l’Aménagement du Territoire et de l’Environnement (MATE AC014G, to B.L.M.B.), Ministère de la Recherche et de la Technologie (to F.O.), and Organon (Faro, France; to R.G.).

Abbreviations: AMH, Anti-Mullerian hormone; dpc, day postcoitum; HPRT, hypoxanthine phosphoribosyl transferase; 3ßHSD, 3ß-hydroxysteroid dehydrogenase; PA, plasminogen activator; PAI, plasminogen activator inhibitor; PC, protein C; PCI, protein C inhibitor; serpin, serine protease inhibitor; SF-1, steroidogenic factor-1; StAR, steroidogenic acute regulatory protein; uPA, urokinase-type plasminogen activator; uPAR, urokinase-type plasminogen activator receptor; tPA, tissue-type plasminogen activator.

Received July 29, 2003.

Accepted for publication November 21, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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