Molecular Human Reproduction, Vol. 5, No. 10, 934-940,
October 1999
© 1999 European Society of Human Reproduction and Embryology
Regulation of testis function |
Factors of the plasminogen activator system in human testis, as demonstrated by in-situ hybridization and immunohistochemistry
1 Department of Urology and 2 Research Laboratory of the Department of Obstetrics and Gynecology, Lund University, University Hospital, Lund and 3 Health Sciences, Malmö University, Malmö, Sweden
Abstract
A growing body of evidence suggests that the plasminogen activator (PA) system is crucially involved in reproductive physiology in both sexes. Certain factors of the PA system have been found to be present in organs of the male reproductive tract in various species, and the presence of several of the factors was recently demonstrated in monkey testis. The present morphological study was therefore designed to investigate the occurrence and distribution of the tissue and urokinase plasminogen activators (t-PA and u-PA), the u-PA receptor (u-PAR) and the plasminogen activator inhibitors (PAI-1 and PAI-2) in normal human testis. Intraoperative specimens from seven patients undergoing orchidectomy or testicular biopsy were studied using in-situ hybridization (ISH) and immunohistochemistry (IHC). All five factors studied could be detected with both the ISH and IHC procedures. The ISH signals were localized to sites in the testicular tubules in a stage-specific manner. There was good correlation between results obtained with the two methods, though in IHC the tubules were stained more uniformly. The findings provide support for the involvement of the PA system in human male reproductive physiology.
plasminogen activator/plasminogen activator inhibitor/testis/u-PAR
Introduction
The plasminogen activator (PA) system is involved in a diversity of physiological and pathological processes such as thrombolysis, inflammation, angiogenesis and neoplastic growth and invasion (see reviews: Saskela and Rifkin, 1988; Vassalli et al., 1991; Carmeliet, 1995). Five specific factors of the PA system are known: the activators, t-PA (tissue type activator) and u-PA, (urokinase type activator) whose activities are regulated by two plasminogen activator inhibitors, type 1 (PAI-1) and type 2 (PAI-2), and the u-PA-specific receptor, u-PAR.
Several observations have suggested that the PA system may also be involved in mammalian fertilization in both sexes. In the male, the presence of t-PA and u-PA has been demonstrated in seminal plasma (Åstedt et al., 1979
), and at high levels (Maier et al., 1991) and factors of the PA system have also been localized to spermatozoa in different species (Huarte et al., 1987; Smokovitis et al., 1987; Manske et al., 1994). Several animal studies have shown both t-PA and u-PA to be present in cells from the seminiferous tubules (Lacroix et al., 1977, 1981; Vihko et al., 1984), and secretion of these activators seems to be dependent on the stage of the spermatogenic cycle in the seminiferous epithelium. In the rat, t-PA was immunohistochemically localized to spermatogenic cells in stages VII–XIII, whereas u-PA was seen in Sertoli cells in stages VII and VIII (Vihko et al., 1988). However, using the in-situ hybridization (ISH) technique, Penttilä and co-workers were able to demonstrate the expression of both t-PA and u-PA in rat Sertoli cells (Penttilä et al., 1994). Liu and co-workers have studied this system in monkey (Liu et al., 1995a, 1996; Zhang et al., 1997a) and in a recent study, ISH showed that t-PA, u-PA, u-PAR and PAI-1 were expressed in the rhesus monkey testis (Zhang et al., 1997b).
The purpose of our morphological study was to elucidate the occurrence and distribution of the five specific factors of the PA system, t-PA, u-PA, u-PAR, PAI-1 and PAI-2, in normal human testicular tissue, at the RNA and protein levels, using ISH and IHC techniques.
Material and methods
Tissue specimens
Tissue specimens were obtained at surgery from seven men, in four of whom orchidectomy was performed on the following indications: prostatic cancer (two patients, aged 75 and 76 years), chronic testicular pain (one patient, aged 31 years) and contralateral testis lymphoma (one patient, aged 74 years). The remaining three patients underwent testicular biopsy because of obstructive azoospermic infertility (two patients, aged 31 and 32 years) or because of contralateral testicular cancer (one patient, aged 42 years). None of the patients had had hormonal, cytostatic or radiation therapy pre-operatively, and all specimens were of normal histopathological appearance.
After collection the specimens were immediately fixed in Bouin's fluid. After rinsing, dehydration and paraffin embedding, the specimens were cut into 5 µm sections and mounted on slides (Super Frost/Plus; Menzel-gläser Menzel, Germany).
cRNA probes
The cRNA probes for ISH were prepared from cDNA fragments, cloned in plasmid vectors and linearized as previously described. Human t-PA cDNA was cloned into the EcoRI site of pGEM-3, nucleotides 70–803, (Pennica et al., 1983). Templates for generating antisense cRNA probes were prepared by linearizing t-PA with one round of BglII digestion. Sense control cRNA probes were prepared from a 472 bp cDNA fragment in pGEM-3. Templates for generating sense control cRNA probes were prepared by linearizing t-PA with HindIII digestion.
Human u-PA cDNA was cloned into the PstI site of pBluescript SK8, nucleotides 1–1340 (Verde et al., 1984). Templates for generating antisense cRNA probes were prepared by linearizing u-PA cDNA with XbaI digestion. Sense control cRNA probes were prepared by analogue linearization with SalI.
cDNA for human u-PAR was cloned into the BamHI site of pBluescript KS(+), nucleotides 497–1081 (Roldan et al., 1990). Templates for generating antisense cRNA were prepared by linearizing u-PAR with EcoRI digestion. Sense control cRNA probes were prepared analogues with XbaI.
Human PAI-1 cDNA, nucleotides 1–2876, was cloned into the EcoRI site of pGEM-1 (Ny et al., 1986). Templates for generating antisense cRNA probes were prepared by linearizing PAI-1 with BglII digestion, and sense control cRNA probes were prepared with SalI.
cDNA for human PAI-2 was cloned into the EcoRI site of pGEM-4, nucleotides 1–1880 (Ny et al., 1989). Templates for generating antisense cRNA probes were prepared by linearizing PAI-2 with NheI digestion and sense control cRNA probes were prepared with SalI.
The cRNA probes were labelled with 20 µmol/l [35S]UTP (Du Pont; Dreich, Germany, 800.0 Ci/mmol). The following RNA polymerases were used: sense T3 and antisense T7 for u-PA, sense T3 and antisense T7 for u-PAR, sense T7 and antisense T7 fore t-PA, sense T7 and antisense SP6 for PAI-1, and sense SP6 and antisense T7 for PAI-2. The probes were reduced by limited hydrolysis at pH 10.2.
In-situ hybridization
Prior to hybridization, tissue sections were pretreated essentially as previously described (Young, 1990). Briefly, after deparaffinization, the slides were acetylated in 0.1 mol/l triethanolamine–HCl (pH 8.0), 0.25% acetic anhydride for 10 min. After rinsing, dehydration and drying, the hybridization procedure was performed (Cox et al., 1984; Whitefield et al., 1990). The labelled cRNA probes were denatured at 65°C for 5 min and placed on ice for 5 min. The final hybridization buffer, with 2x106 c.p.m. denatured probe per 80 µl, consisted of 20 mmol/l Tris–HCl (pH 7.4), 1 mmol/l EDTA (pH 8.0), 300 mmol/l NaCl, 50 % formamide, 10% dextran sulphate, 1xDenhardt's, 25 mg/ml yeast tRNA, 100 µg/ml salmon sperm DNA, 250 µg/ml total yeast RNA (fraction XI; Sigma, St. Louis, USA), 100 mmol/l dithiothreitol (DTT), 0.1% sodium thiosulphate, and 0.1% sodium dodecyl sulphate (SDS). Hybridization buffer (70 µl/15 cm2) was applied to sections on each slide and covered with untreated glass coverslips (Kebo, Lund, Germany). The slides were then incubated at 55°C in chambers humidified with 2xSSC/50% formamide for 24 h. After hybridization, the slides were washed, immersed in RNase A, desalted, dehydrated and dried (Simmons et al., 1989). The slides were then coated with nuclear track emulsion (NBT-3, Kodak, Stockholm, Sweden) undiluted. Following exposure for 4 weeks at 4°C, the slides were developed in D-19 developer (Kodak), fixed and counterstained with 1% toluidine blue. The sections were finally analysed using an Olympus-BX 60 microscope with brightfield and darkfield illumination.
Antibodies
The following previously described or commercial monoclonal antibodies (mAb) were used: t-PA mAb (TechnoClone, Vienna, Austria, No. 21023; Wojta et al., 1987), u-PA mAb (American Diagnostica Inc., Greenwich, USA No. 3689; Sier et al., 1991), u-PAR mAb (American Diagnostica Inc. No. 3936; Del Vecchio et al., 1993), PAI-1 mAb (a gift from Peter Andreasen, Aarhus, Denmark, Christensen et al., 1996), and PAI-2 mAb (from our laboratory; Åstedt et al., 1985).
Immunohistochemistry
Biotinylated rabbit anti-mouse IgG, avidin–biotin complex– horseradish peroxidase (ABC), normal mouse IgG1 and IgG2 mAb, and mouse IgG1 anti-Aspergillus niger mAb were obtained from Dako A/S (Glostrup, Denmark).
The ABC procedure, according to the manufacturer's (Dako A/S) instructions, was performed with some modifications. After deparaffinization, the slides were microwave treated at full effect (800 W) for two min and then at 30% effect for 10 min. The slides were allowed to cool for 20 min before being rinsed in water. The primary monoclonal antibodies were used at concentrations of 10–20 µg/ml. To detect biotinylated horseradish peroxidase, we used diaminobenzidine as a chromophore.
For negative controls two different methods were used: the primary antibody being replaced either by a non-immune normal mouse IgG mAb or by an unrelated monoclonal anti-Aspergillus niger mAb.
Results
In-situ hybridization
For all factors of the plasminogen activator system studied, t-PA, u-PA, u-PAR, PAI-1 and PAI-2, ISH signals were obtained, the strongest signals being obtained for t-PA, u-PA and PAI-1. In the case of t-PA, u-PA, u-PAR and PAI-1, the ISH signals were localized to sites within the seminiferous epithelium, in a stage-specific manner. All specimens tested with sense probes as controls were negative.
For t-PA, a strong ISH signal was obtained in all specimens, localized in a typical stage-specific manner to certain areas of the seminiferous epithelium (Figure 1). Comparison of a consecutive IHC section showed the ISH ignals to be associated with specific cells in the seminiferous tubule (Figure 2). A similar signal pattern was seen for PAI-1, and comparison of consecutive sections showed that the PAI-1 and t-PA signals were often associated with the same cells in the same area in the testicular tubules (Figure 3).
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For u-PA, a signal pattern similar to that for t-PA was seen in some specimens (Figure 4a
), but the u-PA signal was not detected in the same areas as the t-PA and PAI-1 signals, as shown by comparison of consecutive sections. The u-PAR signal was generally less constant but in some sections it could be detected in the testicular tubules (Figure 4b). In the case of PAI-2, the ISH signals were more widespread, being seen in the testicular tubules and in interstitial tissue as well, and not organized in typical spots, as were those of the other factors (Figure 4c). The signals were not consistently detected in all specimens.
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Immunohistochemistry
With the ABC procedure we were able to obtain immunostaining for all five plasminogen activator system factors in all specimens examined and consistently confirmed by negative controls. In contrast to the typical pattern seen in the ISH procedure, immunostaining for t-PA, u-PA, u-PAR and PAI-1 was more uniformly distributed in the testicular tubules (Figures 2B and 5
). The PAI-2 staining was weaker but clearly visible as compared with negative control (Figure 5E). Even if the IHC staining was clear, it was often somewhat low in saturation and intensity, leading to difficulty in distinguishing cytoplasm and extracellular fluids. However, the nucleolar and the paranucleolar regions of the cells were often not stained.
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Discussion
Involvement of the PA system in male reproductive physiology has been proposed on the basis of findings in several studies. The chief source of support for this interpretation is the presence of PA system components in reproductive organs (Kester, 1971; Huarte et al., 1987), at high concentrations in seminal plasma (Mac Gregor ***et al., 1987; Van Dreden et al., 1988; Maier et al., 1991) and their presence in spermatozoa (Smokovitis et al., 1992; Manske et al., 1994). Even clinically, correlation has been found to exist between infertility/pathological semen samples and certain factors of the PA system (Maier et al., 1991; Lison et al., 1993). In basic research, however, the most important studies have focused on the PA system components and the testis in various species. Using cell culture and IHC techniques, the presence of t-PA and u-PA in seminiferous tubules has been demonstrated in the rat (Lacroix et al., 1981; Vihko et al., 1988) and in the mouse (Liu et al., 1995b). In an IHC study in humans, t-PA and u-PA immunostaining were found both in Sertoli cells and in spermatogenic cells in different stages (Balboni et al., 1991). Recently, Zhang and colleagues, using ISH with digoxigenin-labelled cRNA probes, showed mRNA for t-PA, u-PA, u-PAR and PAI-1 to be present in rhesus monkey testis (Zhang et al., 1997b). Stage-specific variation in the expression of t-PA, u-PA and u-PAR was seen within the seminiferous tubules, t-PA and u-PA being expressed in Sertoli cells and u-PAR and PAI-1 in spermatogenic cells. We designed our study to determine the occurrence and distribution of all known specific factors of the plasminogen activator system, t-PA, u-PA, u-PAR, PAI-1 and PAI-2, at both the RNA and protein levels in normal human testis.
In agreement with findings in previous animal studies, we found a manner of stage-specific expression within the testicular tubules of the factors studied. However, we could not determine the exact spermatogenic stage, or specific cell, in which factors were expressed, though, unlike that of animals, the human seminiferous epithelium represents a mosaic of several stages of spermatogenesis in each cross-section (Clermont, 1963). In addition, with our radioactive ISH method, using sections counterstained with Toluidine Blue, it is not histologically possible to determine the specific spermatogenic cycle stage of the area, or the specific cell, where the ISH signals were localized. Furthermore, although the nuclei of human Sertoli cells rest on the basement membrane, the cytoplasm reaches up to the lumen of the tubule, and thus, comparing ISH and IHC results in consecutive sections, it was not possible to be certain about the cellular origin of the ISH signals obtained. However, previous animal studies using the ISH technique have shown t-PA and u-PA to be predominantly expressed in Sertoli cells both in the rat (Penttilä et al., 1994) and in the monkey (Zhang et al., 1997b). In conflict with this, Vihko and co-workers, using the IHC procedure, demonstrated t-PA to be specific to spermatogenic cells in the rat testis (Vihko et al., 1988).
The pattern of IHC staining differed from that of the ISH signals, IHC staining being more uniformly distributed in all the testicular tubules in a non-stage-specific manner. This IHC staining pattern seems to be the same as that obtained by Balboni and co-workers for t-PA and u-PA in human testis, using polyclonal antibodies (Balboni et al., 1991). This apparent discrepancy between the results of the ISH and IHC procedures can be explained by the fact that the ISH signal is intracellular whereas the IHC staining pattern may indicate an extracellular appearance of the proteins in the PA system. The low intensity of IHC staining in the nucleolar and paranucleolar cell regions also indicates that IHC staining could be extracellular. However, it was not possible with the IHC technique to distinguish between cytoplasm and extracellular fluid. This could be due to problems in fixation or microwave treatment of the specimens. As proposed in a previous study, an alternative explanation for the difference between the ISH and IHC results could be a paracrine interaction between Sertoli cells and spermatogenic cells (Penttilä et al., 1994).
The physiological role of the PA system in human testis is still speculative. It has been proposed that the PA system is involved in the restructuring of the seminiferous epithelium, germ cell migration from basal compartments, and the release of mature spermatozoa into the lumen of the tubule (Russel, 1980; Vihko et al., 1988). Our study provides the morphological evidence for an important role of the PA system in human testis and nothing in our results is inconsistent with any of these above-mentioned theories. In addition, the association of the PA factors with the mature spermatozoa leaving the testicular tubule may be important for their further maturation in the male genital tract and their fertilization capacity once in contact with female reproductive tract and the oocyte.
To prevent the negative effects of the cascade of enzymatic activities induced by u-PA and t-PA, the occurrence of PAI may be important in the physiological processes within the seminiferous epithelium as well as on the mature spermatozoa. PAI-1 has been demonstrated in rat and monkey testis (Manske et al., 1994; Zhang et al., 1997b) but we were also able to show PAI-2 in human testis. However, further studies will have to elucidate if both PAI-1 and PAI-2 are physiologically significant in human testis.
To sum up, using both ISH and IHC techniques, we were able to demonstrate for the first time the presence of all specific factors of the plasminogen activation system (t-PA, u-PA, u-PAR, PAI-1 and PAI-2) in normal human testis specimens. In good agreement with findings in previous studies, t-PA, u-PA, u-PAR and PAI-1 were expressed in a stage-specific manner localized to specific cells in the seminiferous epithelium. Overall IHC staining was more uniformly distributed in all types of cells. These findings provide support for the putative involvement of the plasminogen activator system as a crucial feature of human male reproductive physiology. The results also suggest the need for further investigation of the PA system in male accessory genital glands and its possible association with human spermatozoa.
Acknowledgments
We wish to thank Katina Intoftsi Bengtsson and Gunilla Martinsson for their excellent technical assistance. The work was supported by grants from the Swedish Medical Research Council (grant no. 04523), the Medical Faculty, University of Lund, and the Crafoord Foundation, Sweden.
Notes
4 To whom correspondence should be addressed 
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Submitted on March 12, 1999; accepted on July 14, 1999.
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