Localization of urokinase- and tissue-type plasminogen activator mRNAs in rat testes

ELSEVIER

Molecular

and Cellular Endocrinology 105 (1994) 55-64

Localization of urokinase- and tissue-type plasminogen activator mFWAs in rat testes T.-L. PenttilS*, A. Kaipiab, J. Topparib, M. Parvinena, P. Malia Institute ofBiomedicine, ‘Department of Anatomy, bDepartment of Physiology, University of Turku, Kiinamyllynkatu IO, SF-20520 Turku. Finland

Received 2 March 1994; accepted 8 July 1994

Abstract

The expressions of urokinase (uPA) and tissue-type plasminogen activators @PA)in different stages of the rat seminiferous epithelial cycle were analyzed by in situ and Northern hybridizations combined with zymographic analysis. Irradiated rat testes were used to assess the cell localization. Both of the plasminogen activators were expressed in a strictly stage specific manner. Maximal expression of UPA mRNA was seen in Sertoli cells during stages VII-VIII of the cycle. The same expression in the basal compartment of the tubules was detected at 7 days post-irradiation (p-i), during a selective reduction of spermatogonia and preleptotene spermatocytes. Levels of tPA mRNA started to accumulate in Sertoli cells at stage VIII and were high during stages IX-XII and detectable during stages XIII-XIV. At 26 days p-i, reduction of pachytene spermatocytes, which are shown to be immunoreactive for tPA, did not have an effect on tPA mRNA expression. Catalytic activities of UPAand tPA changed concomitantly to their RNA levels in different stages of the cycle. However, at 7 days p-i, UPA activity was decreased at stages VII-VIII of the cycle suggesting that germ cell Sertoli cell interaction is important for UPAactivity. Keywords:

Rat, Testis,Sertoli cell, Plasminogenactivator

1. Introduction

Plasminogen activators (PAS) are serine proteases that convert latent plasminogen into plasmin which is able to degrade, directly or indirectly, most of the components of extracellular matrix and basement membrane (Dan@et al., 1985; Blasi et al., 1987; Saksela et al., 1988; He et al., 1989). Both plasminogen and PAS are synthesized in the seminiferous epithelium (Saksela and Vihko, 1986; Vihko et al., 1989). Spermatogenesis is a complex cell differentiation process, where different generations of germ cells and somatic Sertoli cells form associations with constant composition (Leblond and Clermont, 1952). In the rat, the 14 (I-XIV) different cell associations, also called stages of epithelial cycle, follow each other in a wave-like fashion along the seminiferous epithelium (Perey et al., 1961). During spermatogenesis several physiological events require tissue reconstruction. Plasminogen activators *Corresponding 7352.

author.

Tel. +358 21 633 7355. Fax +358 21 633

(PAS), urokinase (UPA) and tissue-type plasminogen activator (tPA), are thought to play a role in regulating extracellular proteolysis associated with gametogenesis (Beers, 1975; Lacroix et al., 1981; Fritz et al., 1993). One postulated role for PAS during spermatogenesis is the translocation of preleptotene spermatocytes from basal compartment into intermediate compartment of seminiferous epithelium. This is supported by high UPA mRNA expression and secretion of PA activity from seminiferous tubules during stages VII and VIII (Lacroix et al., 1981; Vihko et al., 1989). Also PA secretion in these stages requires the presence of preleptotene spermatocytes (Vihko et al., 1984). Immunohistochemical analysis shows UPA reactivity in Sertoli cells at stages VII-VIII, and tPA reactivity in pachytene and diakinetic spermatocytes (Vihko et al., 1988). However, in vitro studies suggest that tPA is also expressed in Sertoli cells. FSH increased tPA mRNA levels both in cultured seminiferous tubules and Sertoli cells (Vihko et al., 1989; Hettle et al., 1986). In order to gain further understanding of the role of plasminogen activators during spermatogenesis, we ana-

0167-8140/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0167-8140(94)03362-W

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lyzed the expression of tPA and uPA mRNAs by in situ hybridization in the rat testis. Irradiated rat testes model was used to study the possible role of preleptotene spermatocytes for uPA and pachytene spermatocytes for tPA mRNA expression and enzymatic activity.

Endocrinology

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Northern blots, the 1.3 kb BamHl fragment of ~I-19 cDNA clone (Arnheim, 1979) of mouse 28s rRNA was labelled by random priming (Promega Biotec, Madison WI) with [32P]dCTP (DuPont NEN, Boston, MA) and used as reference probe to verify the even loading and transfer of RNA,

2, Materials and methods 2.1. Experimental animals Adult Sprague-Dawley rats and NMRI mice were housed in constant temperature (20°C) and light-dark cycle (lights on 0400-2000 h). The animals had free access to food and water. 2.2. Irradiation of the testes Testes of ether-anaesthetized rats were locally irradiated by 3 Gy (calculated radiation dose in central target area, dose rate 2 Gy/min) of 4 MeV X-rays produced by a Clinac 41100 linear accelerator (Varian, Palo Alto, CA, USA) as described earlier (Kangasniemi et al., 1990). Focus skin distance was 1 m and field size 5 x 5 cm. Determination of the X-ray dose distribution was confirmed with TLD (Therm0 Luminescent Dosimeter, lithium fluoride TLD-pellet, Alnor, Turku, Finland) in water. Animals were killed at 7 and 26 days after irradiation when the following germ cells were reduced in number to values less than 25% of control: at 7 days, spermatogonia and preleptotene spermatocytes; at 26 days, pachytene spermatocytes (Dym and Clermont, 1970; Kangasniemi et al., 1990). 2.3. Preparation of probes The complementary RNA probes were synthesized in the presence of [35S]CTP or [32P]CTP (Amersham International, Buckinghamshire, UK) using following cDNA fragments as templates: tPA; a 798 bp PhiI-SpeI f%agment of the mouse tPA cDNA clone pTAM50 (Rickles et al., 1988) was subcloned into pGEM-1 lZf(+) vector (Promega Biotec, Madison, WI). The cloned plasmid containing the insert was linearized with EcoRI or Hi&III enzymes for the pr~uction of antisense and sense transcripts, respectively. For uPA, a 658 bp &I-Hi&III fragment of the mouse uPA cDNA clone pDB29 (Belin et al., 1985) was subcloned into Bluescript SK+ vector (Stratagene, La Jolla, CA). The plasmid was linearized with Hi&III or PstI enzymes prior to synthesis of antisense and sense RNAs, respectively. Each transcription reaction contained 1 ,ug of linearized DNA template and transcriptions were performed essentially as recommended by the manufacturer of the polymerases (Promega Biotec, Madison, WI). The length of the 35Slabelled transcripts was reduced with alkaline hydrolysis to less than 200 bp. For in situ hybridization, RNA probes transcribed from opposite strands of the same plasmid template, yielding sense and antisense transcripts, were adjusted to the same radioactivity concentration. For

2.4. Histology and in situ hybridization Rat testes and mouse ovaries, oviducts and vasa deferentia were fixed in 10% buffered formalin at room temperature for 24 h, dehydrated in ethanol, cleared in xylene and embedded in paraffin. Sections (5 pm) were placed on microscope slides treated with Denhardt’s solution (Denhardt, 1966) and acetylated as described by Brahic and Haase (1978). In situ hybridization was performed according to the Current Protocols for Molecular Biology (Zeller and Rogers, 1991) with some modifications (for detailed information, see Kaipia et al., 1992). Autoradiography was performed by dipping the slides into Kodak NTB-3 emulsion, exposed for 9 to 65 days in desiccant-containing boxes at 4”C, developed in Kodak D-19 developer, and counterstained with haematoxylin. 2.5. ~icrodi~~ectio~ of semi~~ero~s tubules Rats were killed by C02-asphyxiation and the testes were decapsulated. Seminiferous tubule segments were isolated in phosphate-buffered saline (pH 7.4) under a stereomicroscope by transillumination-assisted microdissection (Parvinen and Vanha-Perttula, 1972). For accurate analysis of the stage-specificity, 10 pools (50 2-mm segments in each) of seminiferous tubule segments from stages I, II-III, IV-V VI, VIIab, VIIcd, VIII, IX-XI, XII and XIII-XIV were dissected within 3 h of death. In addition, stage-specificity was confirmed with rapid fourstage dissection, because Ny and co-workers (1988) have shown that the 3’ untranslated region of rat tPA mRNA contains the sequence AUUUA which may confer the instability to mRNA transcripts (Caput et al., 1986). For four-stage Northern and zymography analysis, 20 S-mm seminiferous tubule segments were dissected from stages II-VI, VII-VIII, IX-XII and XIII-I from control animals, from animals immediately after irradiation, at 7 and 26 days p-i. 2.6. RNA extraction and Northern blot hybridization Total RNA was extracted by the acid guanidinium thiocyanateJphenol/chloroform extraction method (Chomzynski and Sacchi, 1987). The quality and amount of RNA was determined by measuring optical densities at 260 and 280 nm by UV spectrophotometry (Beckman Instruments Inc. Fullerton, CA). The optical density ratios of absorbance at 260 nm to that at 280 nm were between I .7 and 1.9. RNA samples (IOyg of total RNA) were fractioned on 1% agarose gel in the presence of formaldehyde. The gel was stained with ethidium bromide to verify the even loading of RNA. RNA was transferred

T.-L. Penttikiet al. I Molecularand CellularEndocrinologyIO5 (1994) 5544

onto a nylon membrane (GeneScreen, E.I. du Pont de Nemours et Co., Boston, MA) (Thomas, 1980). After baking for 2 h at 80°C, filters were prehybridized in 50% formamide, 3 X SSC, 5X Denhardt’s solution (1 mg/ml Ficoll, 1 mg/ml polyvinylpyrrolide and 1 mg/ml BSA), 1% SDS and 10% dextran sulfate containing lOOpg/ml heat-denaturated calf thymus DNA and lOO~g/ml yeast tRNA at 65OC for 6-16 h. Hybridization was performed at the same temperature for 16-24 h by adding 32P-labelled tPA or UPA RNA probe (5 @ml). The filters were washed twice for 15 min each with 2X SSC at room temperature, followed by two washes of 45 min in 0.2~ SSC/O.l% SDS at 65OC and by two washes of 30 min in 0.1 x SSC at room temperature. When 32P-labelled PI- 19 cDNA probe was used, the prehybridizations, hybridizations and washes were performed essentially as recommended by the manufacturer of the hybridization membrane. Filters were exposed to Kodak XARJ film at -80°C between intensifying screens. 2.7. Zymography To study the tissue-associated PAS, the pools of seminiferous tubules (20 times 5 mm in each) dissected from defined stages were sonicated for 5 s on ice in 10 ~01s. of 0.1 M Tris-HCl (pH 8.0) containing 0.1% Triton X-100 and 100 III/ml aprotinin. The homogenates were centrifuged at 2500 X g for 15 min at 4OC, and the supernatants were transferred into new tubes. The protein concentrations of the extracts were determined by the Bradford Protein Assay Kit (Bio-Rad, Richmond, CA, USA). To study the secreted PAS, the pools of staged seminiferous tubule segments (20 X 5 mm) were cultured as triplicates for 24 h in 500~1 Ham’s F12 Dulbecco’s modified Eagle’s medium (1: 1; Flow Laboratories, McLean, VA, USA) supplemented with HEPES (15 mM; Sigma, St Louis, MO, USA), sodium bicarbonate (1.25 g/l), gentamycin sulphate (10 mg/l; Sigma), G-penicillin (60 mg/l; Sigma) and aprotinin (100 IU/ml; Sigma) at 34°C in humidified 5% CO2 atmosphere. The tissue-homogenates and culture media were analyzed by fibrin- and caseinagarose zymography as previously described (GranelliPiperno and Reich, 1978). After incubation at 37°C for 20-72 h, the fibrin-agarose indicator gels were stained with 0.1% amido black and photographed under transillumination. Casein-agarose indicator gels were not stained and photographs were taken using epi-illumination. Three separate experiments were analyzed by zymography and repeated at least twice. 3. Results 3.1. Localization of tPA and uPA mRNA in normal rat testes by in situ hybridization In normal adult rat testis, tPA mRNA was expressed in a highly stage-specific manner. The expression of tPA mRNA was first detectable in Sertoli cells just prior to

5-I

sperm release in stage VIII of the seminiferous epithelial cycle. Strong hybridization signal was found during stages IX-XII followed by a decrease in signal intensity in stages XIII-XIV (Fig. 1). In these stages, the majority of silver grains overlayed the basal parts of the Sertoli cells. A proportion of silver grains at lower abundance were also observed around some germ cells and residual bodies. Interestingly, in late stage XIV seminiferous tubule sections, a hybridization signal was found around spermatocytes undergoing second meiotic division (Fig. 1F). No specific hybridization signal was seen in stages IVII. The expression pattern of uPA mRNA was distinctly different from that of tPA mRNA. The highest grain densities after in situ hybridization with uPA RNA probe were seen over basal compartments of Sertoli cells in stages VII-VIII of the seminiferous epithelial cycle (Fig. 2). In other stages, the grain density did not differ from the background. Controls of specificity were performed by hybridizing tissue sections of rat testis and mouse oviduct and vas deferens on the same slide. Previously, it was shown that mouse oviduct contains predominantly tPA mRNA while vas deferens has more uPA mRNA (Huarte et al., 1987; Sappino et al., 1989). We obtained similar results by using RNA probes for tPA and uPA mRNA, whereas in tissue sections hybridized with tPA or uPA RNA sense probes, no specific signals were seen (data not shown). 3.2. Stage-specific pattern of tPA and uPA mRNA expression in the rat seminiferous epithelium by Northern analyses To determine the specificity of RNA probes used and to confirm the stage-specific pattern of tPA and uPA mRNA expression in the seminiferous epithelium, total RNA was extracted from staged seminiferous tubules and analysed by Northern hybridization. Total RNA was prepared from ten or four different pools of seminiferous tubules. Northern analyses revealed a 2.8 kb band of tPA mRNA (Figs. 3 and 4), and stages IX-I of the cycle contained more tPA mRNA than other stages. Also a faint smaller band was occasionally detected. Hybridization of uPA mRNA showed a single 2.5 kb mRNA species (Figs. 3 and 4). The steady-state levels of UPA mRNA were most abundant in stages VIIVIII of the epithelial cycle. 3.3. Stage-specific catalytic activity of tPA and uPA Zymography of tissue extracts of seminiferous tubules in different stages of the epithelial cycle showed the highest tPA activity during stages IX-XII, while uPA activity was mainly detected during stages VII-VIII (Fig. 4). Only uPA activity was found in media from cultured seminiferous tubules at defined stages of the epithelial cycle by zymography. The secreted uPA activity was also detected during stages VII-VIII of the cycle (data not shown).

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T.-L. Penttiki et al. I Moleculur und Cellular Endocrinology 105 (1994) 55-64

Fig. 1. Bright (A, C, D, E and B) and a dark field (A’, C’, D’, E’ and F) photomicrographs of tPA mRNA expression in adult rat testis. A strong hybridization signal is seen in stages IX-XI. High magnifications of stage IX-X (C, C’), XI (D, D’), XIII (E, E’) and XIV (F, F’) tubules show grain accumulation over Sertoli cell cytoplasm (arrow, Sertoh cell nucleus). In stages IX-X a proportion of silver grains is also seen around some germ cells or residual bodies (C, C’) and in stage XIV (F, F’) around spematocytes in second meiotic divisions (M II, second meiotic division; 9, 10, I1 and 13, different steps of spermiogenesis). In situ hybridization with tPA sense RNA (B and B’): no specific labelling is observed. Slides were exposed for 13 days. Two different experiments with identical results were performed. Magnifications: A, A’, B and B’ 90x; C, C’, D, D’, E, E’, F and F’ 260x

T.-L, Penttilii et al. I Molecular and Cellular Endocrinology 105 (1994) X5-64

Fig. 2. Bright (A, B) and a dark field (A’, B’) photomicrographs of UPA mRNA expression in adult rat testis. The specific hybridization signal is seen over Sertoli celis (arrow) at stages VII-VIII of the epithelial cycle. In situ hyb~dization with uPA sense RNA (C, c’, D and D’). No specific Iabelling is observed. Slides were exposed for 9 days. Two different experiments with identicai results were performed. Magni~cations: A, A’, C and C 90x; B, B’, D and D’ 250 x

T.-L. Penttilii et ul. I Molecular und Cellular Endocrinology 105 (1994) 55-64

Sertoli cells (Fig. 68). However, zymographic analyses showed that at 7 days p-i, both tissue-associated (Fig. 6A, a) and secreted (Fig. 6A, b) uPA activity decreased in

A 1

2

3

4

(28s tPA>

(28s uPA> Fig. 3. Northern analysis of tPA (A) and uPA (B) mRNA in rat seminiferous tubules at defined stages of the cycle. The highest UPA mRNA leves arc found in stages VII-VIII while stages IX-XIV contain more tPA mRNA than stages I-VIII. Total RNA (1Opg) was isolated from pooled seminiferous tubule segments from stages I (lane 1), II-III (lane 2). IV-V (lane 3). VI (lane 4) V&h (lane 5). V&d (lane 6) VIII (lane 7) IX-XI (lane 8). XII (lane 9) and XIII-XIV (lane 10). As controls, total RNA (lO@g) from mouse oviduct (lane 11) and mouse vas deferens (lane 12) were analyzed. The same filter was hybridized to uPA and tPA RNA probes. Migration of ribosomal RNAs is indicated. The corresponding ethidium bromide staining of the blot is shown to verify the even loading and transfer of RNA. Similar results were obtained in five separate experiments.

3.4. Expression of tPA mRNA and enzymatic activity in irradiated rat testes Previously, tPA immunoreactivity was localized in pachytene spermatocytes in the rat seminiferous epithelium (Vihko et al., 1988). At 26 days p-i, when pachytene spermatocytes were depleted, the expression pattern of tPA mRNA was similar to controls as judged by in situ hybridization (Fig. 5B) and Northern analyses (data not shown). Zymography of tissue extracts of seminiferous tubules from stages IX-XII of the epithelial cycle showed no significant differences in tPA activity at 26 days p-i compared to controls (Fig. 5A). 3.5. Expression of uPA mRNA and enzymatic activity in irradiated rat testes Detachment of preleptotene spermatocytes from the basement membrane at the onset of meiosis has been proposed to require plasmin mediated proteolysis (Vihko et al., 1984). The absence of spermatogonia and preleptotene spermatocytes in irradiated testes at 7 days p-i did not influence the expression pattern of uPA mRNA in

B

tPA)

(65

UPA)

(43

4. mRNA levels stages of the seminiferous

at defined in normal

adult

or tissue-extracts at stages II-VI (lanes l), VII-VIII (lanes 2). IX-XII (lanes and XIII-I 4) were analysed by Northern hybridization To verify of RNA. 19 (28s rRNA) cDNA by SDS-PAGE by the fibrin-overlay A representative of three similar experiments shown.

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A

Fig. 5. Stage-specific tPA mRNA expression and enzymatic activity at 26 days post-irradiation(p-i). (A) Characterizationof plasminogen activatorsby casein-overlay method in tissueextracts (10,ugilane) from seminiferous tubules at stages IX-XII dissected from three control animals (lanes 1, 2, 3), two animals immediately after irradiation(lanes 7, g) and three animals at 26 days p-i (lanes 4, 5, 6). (B) Detection of tPA mRNA expression in rat testis at 26 days p-i by in situ hyb~di~tion. A tPA mRNA signal can be seen in Sertoli cells during stages IX-X {a is a bright-field p~tomic~graph and a’ is a corresponding dark-field photomicrograph).High magnifications of stage IX (b, b’) and XIII (c, c’) tubules show silver grains over Sertoli cells (9, step 9 spermatid; 13, step 13 spermatid).Two parallelexperiments gave identical results. Magnifications:a and a’ 90X; b, b’, c and c’ 260x.

stages VII-VIII of the seminiferous epithelial cycle when compared to controls. 4. Discussion Sertoli cells function in a 12.9-day cycle (Clermont and Harvey, 1965; Parvinen, 1982) that is divided into 14 (I-XIV) stages characterized by the presence of distinct spermatogenic cell associations (Leblond and Clermont,

1952). By in situ hybridization, we demonstrate that both tPA and UPA mRNAs are localized in Sertoli cells in different stages of the epithelial cycle suggesting differential regulation and function of these enzymes in the seminiferous epithelium. The localization of tPA mRNA in Sertoli cells correlates well with previous studies in which cultured Sertoli cells and seminiferous tubule segments were capable of producing tPA upon FSH-stimulation (Hettle et al., 1986; Vihko et al., 1989; Nargolwalla et al.,

5

6

bi23

4

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Fig. 6. Stage-specific uPA activity (panel A) and uPA mRNA expression (panel B) in rat testis at 7 days post-irradiation. (Aa) Tissue associated uPA in seminiferous tubules at stages VII-VIII dissected from two control animals (lanes al and a2,), two animals at 7 days p-i (lanes a3 and a4) and two animals immediately after irradiation (lanes a5 and a@. (Ab) uPA secretion by seminiferous tubules at stages VII-VIII after 24 h culture. Lanes bl-2 and b3-4 are duplicate cultures from two different control animals. Lanes b5-6 and lanes b7-8 arc duplicate cultures from two different animals at 7 days p-i. Casein-overlay zymography was used to detect uPA activities (IOO~g protein or IOOpl culture media/lane). At 7 days p-i, both tissueassociated and secreted uPA activities am decreased as compared to controls. (B) Detection of uPA mRNA expression in rat testis at 7 days p-i by in situ hyb~dization. The figure shows grain accumulation over Sertoli celh (arrow) in stage VII (a and b, bright-field photomic~graphs; a’ and b’, corresponding dark-field photomicrographs). Two parallel experiments gave identical results. Magnifications: a and a’ 90x ; b and b’ 260x.

T.-L. Penttilii et al. I Molecular and Cellular Endocrinology 105 (1994) 55-64

1990). Pituitary follicle stimulating hormone (FSH) is one of the main hormones supporting spermatogenesis, and the Sertoli cells are generally considered to be the main targets of FSH in the seminiferous epithelium. However, our results may seem difficult to reconcile with the immunohistochemical analysis, in which the tPA immunoreactivity was detected in pachytene and diakinetic spermatocytes at the same stages of the epithelial cycle at which we detected the tPA mRNA in Sertoli cells (Vihko et al., 1988). In our study, the absence of pachytene and diakinetic spermatocytes, however, had no effect either in the localization and stage-specific pattern of tPA mRNA expression or in the activity of tPA enzyme in rat seminiferous epithelium. Several explanations could account for this apparent discrepancy. One possibility is that tPA is produced in Sertoli cells and the protein is rapidly taken up by germ cells. In other model systems it has been found that some cells are capable of producing specific cellular receptor for tPA (Hajjar et al., 1987; Bizik et al., 1990; Bassel-Duby et al., 1992; Nguyen et al., 1992). However, our results do not rule out the possibility that tPA mRNA may also be present in small amounts in germ cells. The accumulation of tPA mRNA in growing mouse oocytes and its translation and subsequent degradation during meiotic maturation has been reported (Huarte et al., 1985; Huarte et al., 1987; Strickland et al., 1988; Huarte et al., 1992). The finding that uPA mRNA is expressed in Sertoli cells during stages VII-VIII is consistent with the previous observation that uPA immunoreactivity is localized in these stages in the same cells (Vihko et al., 1988). Zymographic analyses showed that of the two known plasminogen activators, only uPA was detectable in media from cultured seminiferous tubules. This finding supports the view that PA secreted by rat seminiferous tubules is mostly urokinase-type PA while most of the tPA remains associated with the cells. Both secreted and tissueassociated uPA activity were highest during stages VIIVIII which is in good agreement with the expression pattern of uPA mRNA. Our results suggest that uPA enzymatic activity is regulated by spermatogenic cells. At 7 days p-i, the amount of uPA mRNA was similar to controls while uPA activity was decreased during stages VII-VIII. The decreased uPA activity in stages VII-VIII of the seminiferous epithelial cycle was concomitant to the loss of spermatogonia and preleptotene spermatocytes. It has been shown that uPA activity is enhanced when it is bound to its receptor (Quax et al., 1991). One explanation for decreased uPA activity at 7 days p-i, is that the cells that have uPA receptor on their surface were depleted. Whether spermatogonia and preleptotene spermatocytes play a role in the regulation of uPA mRNA translation or in the activation of pro-uPA, cannot be assessed on the basis of the present data. Also the role of specific inhibi-

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tor in the regulation PAS during spermatogenesis remains to be explored. The expression of uPA and tPA mRNAs in Sertoli cells during different stages of the seminiferous epithelial cycle suggest a different regulatory function for plasminogen activators during spermatogenesis. Although the experiments reported here do not further define the cellular function of PAS during spermatogenesis, they support the view that plasmin mediated proteolysis is involved in the movement of preleptotene spermatocytes from basal to adluminal compartment of the seminiferous epithelium at the onset of meiosis (Vihko et al., 1984). Recently it was found that cell surface-associated uPA can activate, not only indirectly but also directly the latent type IV collagenase which is capable of degrading the major basement membrane protein type IV collagen (Keski-Oja et al., 1992). Ossowski et al. (1991) have shown that uPA produced by one cell can, by a paracrine interaction, modulate the invasive potential of a uPA-receptorexpressing tumor cell. The expression of uPA receptor is generally required for the function of uPA (Stoppelli et al., 1985; Vassalli et al., 1985; Roldan et al., 1990). Whether this is also the case in the seminiferous epithelium remains to be determined. Although it is possible that both tPA and uPA are involved in cell migration processes, this function has been most commonly assigned to uPA (Pepper et al., 1987; Hearing et al., 1988; Ossowski et al., 1991), whereas tPA is considered as a key enzyme in thrombolysis (Collen, 1980). However, several findings suggest an additional function for tPA as a post-translational processor of polypeptides (Virji et al., 1980; Granelli-Piperno and Reich, 1983; Kristensen et al., 1985; Liu et al., 1986). Both meiotic oocytes (Liu et al., 1986; Huarte et al., 1987) and spermatocytes (Vihko et al., 1988) have been found to contain tPA, but further studies are needed to define the role of this enzyme during meiosis. The stage-specific localization of tPA and uPA mRNA to Sertoli cells and the cellular regulation of uPA activity in rat testis strongly support the view of paracrine interaction between Sertoli cells and spermatogenic cells and enables the focusing of future studies of PA action in specific events during spermatogenesis. Acknowledgements The excellent technical assistance of Ms. Leena Simola and Mr. Lam-Henrik Wikgren is acknowledged. Dr. Dominique Belin is acknowledged for donating the urokinase cDNA clone. The tissue-type plasminogen activator cDNA clone was a gift from Dr. Sidney Strickland. This study was supported by grants from the Academy of Finland project 1071023 (M.P.) and 1071076 (J.T.), the Sigrid JusClius Foundation, the Paul0 Foundation and the Farmos Research and Science Foundation.

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