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Biology of Reproduction 62, 1040-1046 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Regulation of Tissue Inhibitor of Metalloproteinases-1 in Rat Sertoli Cells: Induction by Germ Cell Residual Bodies, Interleukin-1{alpha}, and Second Messengers1

Line M. Grønning2,a, Jacob E. Wang3,c, Anne H. Reeb, Trine B. Haugenc, Kjetil Taskéna, and Kristin A. Taskéna

a Institute of Medical Biochemistry, b Departments of Tumor Biology and Oncology, Norwegian Radium Hospital, c Andrology Laboratory, Department of Gynecology and Obstetrics, National Hospital, University of Oslo, N-0317 Oslo, Norway


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the testis, FSH has been shown to induce the expression and secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) from Sertoli cells in vitro. This study was performed to elucidate further the cellular origin of testicular TIMP-1 and its expression by hormonal and paracrine factors. This is the first report on the expression of testicular TIMP-1 in vivo. TIMP-1 mRNA in whole testis was decreased after hypophysectomy and strongly increased by the injection of FSH-S17 to hypophysectomized rats. Primary cultures of both peritubular and Sertoli cells showed basal expression of TIMP-1 mRNA. In contrast, we were unable to detect TIMP-1 mRNA in Leydig cells, freshly isolated immature germ cells (primary spermatocytes and spermatids), or residual bodies. We further show that treatment of Sertoli cells with 8-(4-chlorophenyl)thio-cAMP (8-CPTcAMP) in combination with 12-O-tetradecanoylphorbol 13-acetate (TPA) or Ca2+ inducers (calcium ionophore A23187 or thapsigargin) had additive (TPA) and synergistic effects (Ca2+) on the level of TIMP-1 mRNA and secreted protein. We also show that both the level of TIMP-1 mRNA and secreted protein from Sertoli cells were strongly increased by residual bodies, as well as by the cytokine interleukin-1{alpha}. TIMP-1 was not up-regulated by either 8-CPTcAMP or interleukin-1{alpha} in peritubular cells. In contrast to the regulated secretory fraction of TIMP-1, we also detected constitutively expressed immunoreactive TIMP-1 in the nucleus of Sertoli cells, suggesting a role of nuclear TIMP-1 in these cells. In conclusion, our data show that secretion of TIMP-1 from Sertoli cells is highly regulated by hormonal and local processes in the testis, indicating that TIMP-1 is of physiological importance during both testicular development and spermatogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been shown to possess several biological functions, particularly the apparently contrasting activities of inhibiting matrix metalloproteinases (MMPs) in both physiological and pathological tissue remodeling and promoting growth factor effects [1]. The classical experimental studies revealed that TIMP-1 acts as a central inhibitor of tumor cell invasion [2–4], as well as showing erythroid-potentiating activity [5,6]. In the testis, FSH, the main regulator of Sertoli cell function [7,8], has been shown to induce the expression and secretion of TIMP-1 with MMP-inhibiting activity from Sertoli cells in vitro [9]. Moreover, Boujrad et al. [10] found that a TIMP-1/procathepsin L complex secreted from FSH-stimulated Sertoli cell cultures potently activated Leydig cell steroidogenesis.

In addition to endocrine regulation by FSH, local processes in the testis may regulate expression of testicular TIMP-1. Of particular interest in this respect is the phagocytosis of residual bodies (RB; cytoplasmic vesicles shed from spermatozoa at spermiation) by Sertoli cells, a process that for decades has been thought to constitute an important signal in the maintenance of spermatogenesis [11–14]. In keeping with this notion, it was recently demonstrated that plasminogen activator is induced in cocultures of Sertoli cells and RB, probably mediated by the autocrine action of secreted interleukin-1{alpha} (IL-1{alpha}) [15]. This may suggest that Sertoli cell phagocytosis of RB is involved in the regulation of testicular extracellular matrix (ECM) turnover. Testicular ECM needs constant remodeling because of the continuous migration of immature germ cells towards the lumen of the seminiferous tubules, a process that is believed to involve proteases and anti-proteases [16,17].

This study was performed to further examine the cellular origin of testicular TIMP-1. The in vivo effect of FSH on TIMP-1 expression in testis was studied in hypophysectomized rats. Furthermore, we studied the regulation of TIMP-1 by both hormonal and paracrine factors in primary cultures of rat Sertoli cells via perturbation of several second messenger pathways (protein kinases A [PKA] and C [PKC], Ca2+ signaling) and by culturing Sertoli cells in the presence of RB or IL-1{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Cell Cultures

Primary cultures of rat Sertoli cells were made from testes of 19- to 45-day-old Sprague Dawley rats (Møllegaard Breeding Center Ltd., Copenhagen, Denmark, and B&K Universal AS, Nittedal, Norway) according to the method of Dorrington et al. [18] with some modifications [19]. The cells were plated in 10-cm culture dishes (Nunc, Copenhagen, Denmark) for RNA and protein analysis and cultured in minimum essential medium Eagle (MEM; 212090-022, Gibco BRL, Grand Island, NY) with addition of streptomycin (100 mg/L), penicillin (105 IU/L), fungizone (0.25 mg/L), L-glutamine (2 mM), and fetal calf serum (10%; 11099-117, Gibco BRL) at 32°C in a humidified atmosphere with 5% CO2. After three days, the cells were incubated further in a serum-free modified MEM. Leydig cell tumor H-540, peritubular cells, and different populations of germinal cells were prepared as previously described [19]. The Sta-Put Technique was used to prepare different fractions of germ cells [19]. Pachytene spermatocytes (90–95% purity) and round spermatids (85–90% purity) were isolated from 32-day-old rat testes, in which no elongating spermatids are yet present. From 44-day-old rats, pachytene spermatocytes (75–80% purity, contaminated with round spermatids), round spermatids (65–70% purity, contaminated with elongating spermatids), and elongating spermatids (45–50% purity, contaminated with round spermatids) were isolated. RB was prepared from 55-day-old rats by centrifugal elutriation [20] as previously described [14]. The fraction enriched in RB contained a mixture of RB and cytoplasts from elongating spermatids and less than 5% cells [14].

Stimulation of Sertoli Cells

After two days of culture in serum-free medium, the medium was changed, and incubation was continued in the presence or absence of FSH (1 µg/ml ovine FSH-S17; NIH, Bethesda, MD), 8-(4-chlorophenyl)thio-cAMP (8-CPTcAMP) (10-4 M; C-3912, Sigma Chemical Co., St. Louis, MO), Ca2+-ionophore A23187 (5 x 10-7 M; C-5149, Sigma), thapsigargin (10-5 M; T-9033, Sigma), with 12-O-tetradecanoylphorbol 13-acetate (TPA; 10-7 M; P-8139, Sigma), RB (5 x 105/ml) [14], or recombinant IL-1{alpha} (5 ng/ml; murine IL-1{alpha}, Genzyme, MA; rat IL-1{alpha}, R&D Systems, Minneapolis, MN).

Animals

Immature Sprague Dawley rats of the same weight (65 g) were selected at Day 20 and hypophysectomized by Møllegaard Breeding Center Ltd. (Copenhagen, Denmark). More than 80% of the animals appeared completely hypophysectomized as judged by testes size and body weight (65 ± 5 g) at Day 29. Animals received s.c. injections of 250 µg FSH-S17 in 0.9% saline with 0.1% BSA [21].

RNA Extraction and Northern Analysis

Whole testes were homogenized in guanidine isothiocyanate and centrifuged at 500 x g for 5 min [21]. Total RNA from cell cultures and tissue specimens was extracted by the guanidine isothiocyanate/CsCl method as previously described [19,22]. Northern blot analysis was performed using 20 µg total RNA that was denatured in 50% (v:v) formamide and 6% (v:v) formaldehyde and subjected to electrophoresis in a 1.5% (w:v) agarose gel containing 6.7% formaldehyde. Ethidium bromide staining of the gel verified equal loading in each lane, and RNA was blotted onto Biotrans membranes by capillary blotting technique (ICN Biomedicals, Costa Mesa, CA). Human cDNA probes for TIMP-1 (0.7-kilobase [kb] EcoRI fragment; British Biotech, Oxford, UK), and ribosomal protein L27 (American Type Culture Collection, Rockville, MD) were labeled with [{alpha}-32P]dCTP using the megaprime DNA labeling system (RPN 1607, Amersham, Arlington Heights, IL) to a specific activity of 0.5–1.0 x 109 cpm/µg. Hybridization was performed with 50% formamide at 42°C according to the manufacturer (ICN). After hybridization, the filters were washed four times in a solution containing double-strength standard saline citrate (300 mM NaCl and 30 mM sodium citrate, pH 7.0) with 0.1% SDS at 25°C for 5 min and twice in 0.7-strength standard saline citrate with 0.1% SDS at 50°C for 30 min. Northern blots were subjected to autoradiography using Amersham Hyperfilm MP. The signal intensities of suitably exposed films were estimated by the use of PHOTO-CAPT software, Version 99.01 (SAVEEN, Malmø, Sweden).

Preparation of Nuclear Extracts

Sertoli cells (2 culture dishes [10 cm; ~12 x 106 cells/dish]) were scraped in Hanks' Balanced Salt Solution containing 0.1% fatty acid-free BSA, harvested by centrifugation at 320 x g at 4°C for 5 min, and washed in cold PBS. Cell pellets were resuspended in 450 µl hypotonic buffer (10 mM Tris pH 7.6, 10 mM NaCl, 3 mM MgCl2), 50 µl 5% NP-40 lysis buffer (N-3516, Sigma) was added, and the nuclei were pelleted by centrifugation at 130 x g at 4°C for 5 min. Nuclei were resuspended in 1 ml hypotonic buffer and centrifuged at 130 x g at 4°C for 5 min. Pellets were resuspended in 100 µl of a buffer containing 5 mM Hepes pH 7.9, 26% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM PMSF. Then 1/10 volume of 4 M NaCl were added, and the samples were incubated on a roller for 30 min at 4°C and centrifuged at 30 000 x g for 20 min at 4°C. The supernatants (nuclear extracts) were stored at -70°C until analysis.

Subcellular Fractionation of Sertoli Cells

Sertoli cells (10 culture dishes [10 cm; ~12 x 106 cells/dish]) were washed in cold PBS and then scraped into 3.0 ml isotonic sucrose buffer containing 250 mM sucrose, 20 mM Tris-HCl pH 7.8, 1 mM EGTA, 10 mM MgCl2, 10 mM ß-mercaptoethanol, 50 mM NaF, and Complete (a trademark name) protease inhibitor mix (1 tablet/10 ml; 1836170, Boehringer Mannheim GmbH, Mannheim, Germany), benzamidine (0.0078 g/ml; B-6506, Sigma), Calpain inhibitor II (42 µM; 1086103, Boehringer Mannheim), and PMSF (0.5 mM; 837091, Boehringer Mannheim). Cell suspensions were lysed by 2 x 10 strokes Dounce homogenization followed by centrifugation for 1 h at 200 000 x g at 4°C. The supernatants were stored at -70°C until analysis. The pellets were washed twice in 1 ml isotonic sucrose buffer and resuspended in 750 µl isotonic sucrose buffer containing 1% Triton X-100 (T-9284, Sigma), extracted for 30 min on a roller at 4°C, and centrifuged at 14 000 x g at 4°C for 15 min. The supernatants (1% Triton) were stored at -70°C until analysis. The Triton X-100-insoluble material was resuspended in 600 µl radioimmunoprecipitation assay (RIPA) buffer containing 150 mM NaCl, 1% Nonidet-P40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 8.0, Complete proteinase inhibitor mix (1 tablet/10 ml), and PMSF (0.5 mM); extracted for 30 min on a roller at 4°C; and centrifuged at 14 000 x g for 15 min at 4°C. The supernatants (RIPA) were stored at -70°C until analysis. The pellet was resuspended in Laemmli buffer.

Immunoblotting

Media (12 ml) collected from Sertoli cells were concentrated by ultrafiltration in VIVASPIN 4-ml concentrator columns with cut-off at 10 000 Mr (VS0403, Vivascience Limited, Binbrook Hill, UK). Protein samples were diluted in SDS sample buffer and denatured for 5 min at 100°C before being loaded on a one-dimensional SDS-polyacrylamide gel (4.5% stacking gel, 12% separating gel). Concentrated Sertoli cell media and 40 µg of total protein from various Sertoli cell fractions were loaded in each lane, subjected to electrophoresis, and subsequently transferred to polyvinyl difluoride membranes (Millipore, Bedford, MA) by electroblotting. The membranes were blocked in a solution containing 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20, and 5% milk, and incubated with goat polyclonal antibody against TIMP-1 (1 µg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in blocking solution. Membranes were washed in a solution containing 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween-20. Immunoreactive proteins were visualized by ECL (RPN 2106, Amersham) using a horseradish peroxidase-conjugated secondary antibody (1:20 000) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TIMP-1 Was Expressed in Sertoli and Peritubular Cells and Induced by FSH in Whole Testes of Hypophysectomized Rats

Northern blot analysis of various testicular cell types showed that TIMP-1 (0.9 kb) was present in Sertoli cells and peritubular cells, and not in germ cells or Leydig cells (Fig. 1A). The mRNA expression level of TIMP-1 was low and almost undetectable in Sertoli cells from 19-day-old rats, with increasing levels in Sertoli cells from 35- to 45-day-old rats. To study whether TIMP-1 mRNA is induced by FSH in vivo, groups of hypophysectomized rats (n = 3) either received injections of FSH-S17 (250 ng) or were left untreated, and the animals were killed 6 h later. Figure 1B shows Northern blot analysis of TIMP-1 levels in total RNA from testes of a representative hypophysectomized rat (lane 1), a hypophysectomized rat treated with 250 ng FSH-S17 (lane 2), and a control rat (lane 3). Treatment with FSH strongly induced TIMP-1 mRNA above the levels in both hypophysectomized and control rats. Furthermore, TIMP-1 levels in control rats were significantly higher than in hypophysectomized rats.



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  		FIG. 1. Cell-specific expression and hormonal regulation of TIMP-1 mRNA in rat testis. A) Northern blot showing TIMP-1 mRNA. Lanes 1–3, Sertoli cells (SC) from 19-, 35-, and 45-day old rats; lane 4, peritubular cells (PT) from 19-day-old rats; lane 5, Leydig cell tumor (LT); lanes 6–9, pachytene spermatocytes (PS) and round spermatids (RST) from 32- and 44-day old rats; lane 10, elongated spermatids (ES) from 44-day-old rats. Results shown are representative of three independent experiments. B) Rats were hypophysectomized at Day 20, and completely hypophysectomized animals (Hypox) were randomized into two groups at Day 29, given injections of FSH (250 µg ovine FSH-S17) or left untreated, and killed 6 h later. Total RNA from whole testes of untreated rats (-FSH, lane 1), rats treated with FSH (+FSH, lane 2), and control rats was prepared and examined by Northern blot analysis using cDNA for human TIMP-1 and the ribosomal factor L27 (control). Representative data from one of three individual animals in each group are shown

Effects of FSH and Second Messengers on TIMP-1 mRNA in Sertoli Cells

Figure 2A shows a representative Northern blot of TIMP-1 mRNA from untreated Sertoli cells and Sertoli cells treated with FSH-S17 (1 µg/ml). FSH strongly induced TIMP-1 mRNA from low basal levels to maximal levels after 6 h of stimulation, with sustained induction for up to 12 h. The level of TIMP-1 mRNA was back to basal levels after 24 h of stimulation (data not shown). Treatment with 8-CPTcAMP (10-4 M) induced TIMP-1 mRNA after 4 h, with maximum levels at 6–12 h of stimulation and a subsequent decline towards basal levels at 24 h (Fig. 2B). TIMP-1 mRNA was constitutively expressed in peritubular cells and was not induced by 8-CPTcAMP (data not shown). We next examined the effect of combined treatment with TPA and 8-CPTcAMP or the cAMP analogue and Ca2+ inducers (ionophore A23187 or the inhibitor of the Ca2+-ATPase of the endoplasmatic reticulum, thapsigargin) on the expression of TIMP-1 mRNA in Sertoli cells. As seen from Figure 3A, combined treatment with TPA (10-7 M) and 8-CPTcAMP (10-4 M) had an additive effect on TIMP-1 mRNA compared to treatment with TPA (4-fold) or the cAMP analogue alone. Furthermore, combined treatment with 8-CPTcAMP and thapsigargin (10-5 M) or Ca2+-ionophore A23187 (5 x 10-7 M) had a synergistic effect on TIMP-1 mRNA compared to treatment with either agent alone (Fig. 3B). Treatment with TPA alone resulted in an approximately 4-fold induction of TIMP-1 mRNA (Fig. 3A), whereas only minor effects of thapsigargin or A23187 were observed (Fig. 3B).



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  		FIG. 2. Time-dependent regulation of TIMP-1 mRNA by FSH and cAMP in Sertoli cell primary cultures. A) Northern blot showing mRNAs for TIMP-1 and the ribosomal factor L27 (control). RNA from Sertoli cells (19-day-old rats) incubated in the absence (-; basal at same level at 1.5–12 h) or presence of ovine FSH-S17 (1 µg/ml) for 1.5 to 12 h. B) RNA from Sertoli cells incubated in the absence (-) or presence (+) of 8-CPTcAMP (10-4 M) for up to 24 h. Representative data from one each of two (A) and three (B) independent experiments are shown



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  		FIG. 3. Effect of TPA or calcium in combination with 8-CPTcAMP on the expression of TIMP-1 mRNA. A) Northern blot showing mRNAs for TIMP-1 and the ribosomal factor L27 (control). RNA from Sertoli cells (19-day-old rats) incubated in the absence or presence of TPA (10-7 M) alone or in combination with 8-CPTcAMP (10-4 M). B) RNA from Sertoli cells incubated in the absence (-) or presence (+) of thapsigargin (10-5 M) or calcium ionophore A23187 (5 x 10-7 M) alone or in combination with 8-CPTcAMP (10-4 M) for 12 h. Densitometric scanning results are shown above the autoradiograms. The experiments were repeated two times with similar results

Effects of Second Messengers on Expression of TIMP-1 Protein in Sertoli Cells

We investigated the level of immunoreactive TIMP-1 in concentrated media collected from Sertoli cell cultures (Fig. 4A) and in subcellular fractions of Sertoli cells (Fig. 4B). The identity of Sertoli cell TIMP-1 was determined by its molecular weight, which is reported to be 28 kDa in its glycosylated form [1,9]. However, the molecular mass can range from 28 to 34 kDa depending on the degree of glycosylation [1]. As shown in Figure 4A, there were no detectable levels of secreted TIMP-1 protein in media from unstimulated Sertoli cells after 6 and 12 h in culture. After 24 h, low basal levels were detected. 8-CPTcAMP induced secreted TIMP-1 protein after 12 h of stimulation, with further induction after 24 h. The observation that cAMP-induced TIMP-1 mRNA was decreasing at 24 h whereas the TIMP-1 protein level increased was probably due to a time lag caused by posttranslational processing and secretion. Combined treatment with TPA and 8-CPTcAMP for 24 h strongly induced TIMP-1 protein levels. Treatment with TPA alone induced TIMP-1 protein slightly more strongly than treatment with 8-CPTcAMP according to what we observed at the mRNA level. Examination of subcellular compartments of Sertoli cells revealed that TIMP-1 protein was constitutively expressed in the nucleus of Sertoli cells regardless of stimuli (8-CPTcAMP, TPA/8-CPTcAMP, TPA, IL-1{alpha}) or the time period studied (2–48 h) (Fig. 4B and data not shown). Furthermore, TIMP-1 protein was detected in the 1% Triton X-100 extractable fraction prepared from 8-CPTcAMP-treated Sertoli cells (Fig. 4B).



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  		FIG. 4. Time-dependent effect of 8-CPTcAMP and TPA on TIMP-1 secretion and subcellular distribution in Sertoli cells. A) Total protein samples from media concentrated 16x were examined by immunoblotting using anti-TIMP-1 antibody. B) Immunoblot of various subcellular Sertoli cell fractions (200 000 x g; 1% Triton, RIPA, and nucleus extracts). Sertoli cells were incubated in the absence (-) or presence (+) of 8-CPTcAMP (10-4 M) alone or in combination with TPA (10-7 M) for up to 24 h. Horizontal lines indicate mobility of Rainbow molecular weight markers (RPN 756, Amersham)

Effects of RB and IL-1{alpha} on TIMP-1 mRNA and Protein Levels in Sertoli Cells

We next examined whether coculture of Sertoli cells and RB would induce TIMP-1 mRNA and protein (Fig. 5). Indeed, as seen from Figure 5A, after 2 h of stimulation with RB (5 x 105/ml), TIMP-1 mRNA was strongly induced, with sustained levels up to 28 h of stimulation and then a further increase up to 48 h. This increase in the level of TIMP-1 mRNA was not due to TIMP-1 mRNA released from RB since TIMP-1 mRNA was not detected in RB (data not shown). RB weakly induced immunoreactive TIMP-1 secreted from Sertoli cells after 12 h of stimulation, with maximal induction after 48 h (Fig. 5B). We have previously shown that the expression of IL-1{alpha} mRNA is stimulated in Sertoli cells cocultured with RB [14]. For this reason, we investigated whether IL-1{alpha} was able to induce TIMP-1 mRNA and protein in Sertoli cells (Fig. 6). Recombinant murine IL-1{alpha} (5 ng/ml) strongly increased the level of TIMP-1 mRNA after 2 h of stimulation, and levels remained high at observations up to 7 h (Fig. 6A). IL-1{alpha} did not induce TIMP-1 mRNA in peritubular cells (data not shown). Immunoreactive TIMP-1 secreted from Sertoli cells was weakly increased in Sertoli cells stimulated with recombinant rat IL-1{alpha} (5 ng/ml) for 6 h (Fig. 6B). After 24 h of treatment, IL-1{alpha} strongly increased secreted TIMP-1 protein. The basal level of secreted TIMP-1 increased during time in culture.



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  		FIG. 5. Effect of RB on the expression of TIMP-1 mRNA and secreted protein in Sertoli cell primary cultures. A) Northern blot showing mRNA for TIMP-1 and the ribosomal factor L27 (control). RNA from Sertoli cells (19-day-old rats) was incubated in the absence (-) or presence (+) of RB (5 x 105/ml) for up to 48 h. B) Total protein samples from media concentrated 5x were examined by immunoblotting using anti-TIMP-1 antibody. Horizontal lines indicate mobility of Rainbow molecular weight markers (RPN 756, Amersham)



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  		FIG. 6. Effects of IL-1{alpha} on the expression of TIMP-1 mRNA and secreted protein in Sertoli cell primary cultures. A) Northern blot showing mRNA for TIMP-1 and the ribosomal factor L27 (control). RNA from Sertoli cells (19-day-old rats) were incubated in the absence (-) or presence (+) of murine recombinant IL-1{alpha} (5 ng/ml) for up to 7 h. B) Total protein samples from media concentrated 30x were examined by immunoblotting using anti-TIMP-1 antibody. Sertoli cells were incubated with rat recombinant IL-1{alpha} (5 ng/ml) for 6- and 24 h. Horizontal lines indicate mobility of rainbow molecular weight markers (RPN 756, Amersham)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies in vitro have shown that stimulation of Sertoli cells with FSH induces the expression and secretion of TIMP-1 [9,10]. To our knowledge, no other cell types in the testis have been shown to express TIMP-1. In the study reported here, we observed basal expression of TIMP-1 mRNA both in Sertoli cells and in peritubular cells. In whole testis, FSH induced the expression of TIMP-1 mRNA in vivo by injection of FSH-S17 to hypophysectomized rats. This is the first report on the expression of testicular TIMP-1 in vivo. The significantly higher testicular TIMP-1 mRNA level in normal rats compared to untreated hypophysectomized rats is most likely due to physiological FSH stimulation. Since Sertoli cells are the primary targets for FSH in the testis and TIMP-1 mRNA was not induced by cAMP in peritubular cells, this in vivo regulation most likely reflects induction of Sertoli cell TIMP-1 mRNA. In line with this notion, we were not able to detect TIMP-1 mRNA in freshly isolated germ cells (primary spermatocytes and spermatids), or in RB. However, we cannot rule out the possibility that FSH through stimulation of Sertoli cells indirectly regulates the level of TIMP-1 mRNA in other testicular cells.

Besides acting through the cAMP-signaling pathway, FSH may influence intracellular levels of Ca2+ [23,24]. The Ca2+ level in Sertoli cells is also regulated by adenosine through P2-purinergic receptors, endothelin-1, insulin-like growth factor-I, and several other hormones [25–28]. Furthermore, germinal cells have been shown to induce both PKC and Ca2+ signaling in Sertoli cells [29]. We show a strong induction of TIMP-1 after combined treatments with 8-CPTcAMP and TPA or 8-CPTcAMP and Ca2+, implicating cooperation between FSH and local paracrine factors in the testis in the induction of TIMP-1 in Sertoli cells. The additive effect of 8-CPTcAMP and TPA on TIMP-1 expression and secretion from Sertoli cells shown in this study is consistent with the hypothesis put forward by Ulisse et al. [9] that the PKA and PKC signaling pathways may converge at the transcription factor level through activation of CREB and AP-1 proteins (fos/jun), respectively. Treatment of Sertoli cells with Ca2+ and 8-CPTcAMP had a synergistic effect on TIMP-1 expression, indicating that Ca2+ signaling pathways also cooperate with cAMP to regulate TIMP-1.

In addition to hormonal regulation by FSH, we show that RB and the cytokine IL-1{alpha} induce expression and secretion of TIMP-1 from Sertoli cells. Sertoli cell phagocytosis of RB has been suggested to play a role in the maintenance of spermatogenesis, possibly mediated by IL-1{alpha} [14,15]. Although the function of IL-1{alpha} within the testis is unknown, IL-1{alpha} has been implicated in the regulation of ECM by fibroblasts [30–32]. Phagocytosis of RB induces the secretion of IL-1{alpha} from Sertoli cells [13,33], which is further regulated by an autocrine loop in these cells [14]. In addition, IL-1{alpha} may also be produced by immature germ cells [34]. In the present study, both RB and IL-1{alpha} induced TIMP-1 mRNA after only 2 h of stimulation. This is in contrast to the slow kinetics of FSH/cAMP-mediated induction of TIMP-1 mRNA, suggesting that different mechanisms are involved in the regulation of TIMP-1. Complete RB-phagocytosis by Sertoli cells occurs first after 24 h of coculture with RB [35], suggesting that the early RB-mediated induction of TIMP-1 mRNA is a result of binding of RB to Sertoli cells, probably via specific adhesion molecules [36]. After 32 h of coculture with RB, the level of TIMP-1 increased to very high levels. This strong second wave of TIMP-1 induction may be due to release of specific RNAs and proteins after complete RB phagocytosis or due to newly synthesized factors in the Sertoli cells, including IL-1{alpha}. We further suggest that the higher basal expression level of TIMP-1 mRNA observed in Sertoli cells from prepubertal compared to infantile rats was due to influence of germ cells present in much higher numbers in testes from prepubertal (35- and 45-day-old) rats [37].

TIMP-1 is able to inhibit several members of the MMP family, and TIMP-1 secreted from Sertoli cells has MMP-inhibiting activity as shown by reverse zymography [9]. Different types of MMPs, in particular MMP-2 (gelatinase A) and MMP-9 (gelatinase B), are produced in the testis by both Sertoli cells and peritubular cells, where MMP-2 is up-regulated by FSH and cAMP [38–41]. Recently, a new MMP, MMP-23, was cloned and found to be predominantly expressed in ovary, testis, and prostate, suggesting that it may play a specialized role in reproductive processes [42]. The authors further show that TIMP-1 was able to inhibit the proteolytic activity of recombinant MMP-23. Whether TIMP-1 is also able to inhibit testicular MMP-23 in vivo remains to be elucidated.

FSH stimulates proliferation of immature Sertoli cells, and at 15 days of age in the rat, the Sertoli cells cease to divide. At this stage, FSH seems to be required for the final maturation of Sertoli cells, where it appears to be essential for the formation of tight junctions and for stimulation of the first wave of spermatogenesis [8]. In the adult rat, the action of FSH appears to vary in the different stages of the cycle of the seminiferous epithelium because of variations in FSH-receptor numbers [43]. The FSH- and RB-inducible secretory fraction of TIMP-1 is therefore likely to be involved in ECM remodeling through inhibition of MMP activity during both testicular development and spermatogenesis. In addition to reports of TIMP-1 as an MMP inhibitor, Boujrad et al. [10] have reported a novel function of TIMP-1. They showed that a TIMP-1/procathepsin L complex secreted from FSH-stimulated Sertoli cells potently activated Leydig cell steroidogenesis in vitro. Procathepsin L is secreted by seminiferous tubules during the period immediately before spermiation [44], and medium from cultured seminiferous tubules at this stage shows the greatest stimulatory effect on testosterone production by Leydig cells [45]. Thus, the testicular TIMP-1/procathepsin L complex secreted in adult rats is possibly under the control of specific germ cell types. Testosterone, hormonally controlled by LH, is the main regulator of spermatogenesis in adults. Since TIMP-1 is induced by contact activation and Sertoli cell phagocytosis of RB, and is a co-inducer of testosterone production, we postulate that TIMP-1 is a mediator by which RB contribute to initiation of new spermatogenesis.

In conclusion, our data show that secretion of TIMP-1 from Sertoli cells is highly regulated by both hormonal and paracrine factors/processes including IL-1{alpha} and phagocytosis of RB. This indicates that TIMP-1 is of physiological importance during both testicular development and spermatogenesis.


   ACKNOWLEDGMENTS
 
We greatly appreciate the skilful technical assistance of Gladys Josefsen and Guri Opsahl.


   FOOTNOTES
 
First decision: 28 October 1999.

1 This work was supported by the Norwegian Research Council, the Norwegian Cancer Society, Novo Nordisk Foundation Committee, and Anders Jahres Foundation for the Promotion of Science. Back

2 Correspondence: Line M. Grønning, Institute of Medical Biochemistry, University of Oslo, P.O. Box 1112, Blindern, N-0317 Oslo, Norway. FAX: 47 22851497; l.m.gronning{at}basalmed.uio.no Back

3 Current address: Jacob E. Wang, Institute of Surgical Research, National Hospital, University of Oslo, N-0027 Oslo, Norway. Back

Accepted: November 12, 1999.

Received: October 5, 1999.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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