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Endocrinology Vol. 139, No. 2 741-747
Copyright © 1998 by The Endocrine Society

ARTICLES

Regulation by Thyroid Hormone of the Expression of Basement Membrane Components in Rat Prepubertal Sertoli Cells1

Salvatore Ulisse, Nadia Rucci, Deamaria Piersanti, Eleonora Carosa, Filomena M. Graziano, Antonio Pavan, Piergiuseppe Ceddia, Mario Arizzi, Paola Muzi, Luisa Cironi, Lucio Gnessi, Massimino D’Armiento and Emmanuele A. Jannini

Department of Experimental Medicine (S.U., N.R., D.P., E.C., F.M.G., A.P., P.C., P.M., L.C., E.A.J.), University of L’Aquila, 67100 L’Aquila, Italy; and Departments of Medical Physiopathology (M.A., L.G.), and Experimental Medicine and Pathology (M.D’A.) University of Rome "La Sapienza," 00161 Rome, Italy

Address all correspondence and requests for reprints to: Prof. Massimino D’Armiento, Department of Experimental Medicine, Section of Endocrinology, University of L’Aquila, Coppito, Building 2, Room A2/54, 67100 L’Aquila, Italy. E-mail: darmiento{at}axscaq.aquila.infn.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study reports the modulation of basement membrane (BM) components, laminin, entactin, and type IV collagen, expression in prepubertal rat Sertoli cell by the thyroid hormone T3. Immunocytochemical studies of permeabilized Sertoli cells in culture showed that T3 treatment (10-7 M for 24 h) increased the number of cells staining positive for laminin and/or entactin (from 58 ± 5.3% to 86.4 ± 6.5%, P < 0.01). In contrast, a strong inhibition of type IV collagen immunopositivity was observed. Western blot analysis of Sertoli cell-conditioned media indicated that T3 treatment significantly (P < 0.01) increased the level of secreted entactin by 60–65% without affecting the levels of laminin A and B1/B2 chains. Moreover, thyroid hormone treatment of Sertoli cells significantly reduced type IV collagen secretion by 62% (P < 0.05). Slot blot analysis of poly-A RNA demonstrated a significant (P < 0.01) increase in the level of entactin messenger RNA (mRNA) by 140% (P < 0.01) and a 50% reduction of type IV collagen {alpha}1 chain mRNA after thyroid hormone treatment. No effect of the hormone was observed on the accumulation of the laminin B1 and B2 chain mRNAs in Sertoli cell cultures. These effects cannot be ascribed to changes in the degradation of BM components, because no effect of thyroid hormone was observed on plasminogen activators or metalloproteinase secretion by Sertoli cells.

These observations indicate the Sertoli cell as a source of entactin within the testis, demonstrate the ability of T3 to differentially regulate the expression of BM components, and can be regarded as a part of the integrated mechanism by which thyroid hormone affects testicular development and differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTHOUGH thyroid hormone is essential for growth and development of the majority of tissues in higher organisms, the adult rat testis has been classically regarded as unresponsive to iodothyronines (1). However, a growing number of reports suggest a regulatory role for thyroid hormone in testicular development and in gametogenesis (for review, see Refs. 2 and 3). The Sertoli cell is regarded as the main target for thyroid hormone action within the testis, because this cell possesses T3 nuclear binding activity and thyroid hormone receptor {alpha}1 messenger RNA (mRNA) expression (4, 5, 6). Moreover, thyroid hormone treatment of newborn rats hastens the Sertoli cell proliferation phase and the time taken for terminal Sertoli cell differentiation. As a consequence, an increase in the number of Sertoli and germ cells, together with an increase in the seminiferous cord and testicular size, has been observed (7, 8). The increased seminiferous cord diameter after T3 treatment, in vivo and in vitro, implies a remodeling of the tunica propria and, in particular, of basement membrane (BM) surrounding the seminiferous epithelium.

Laminin, entactin/nidogen, and type IV collagen are major structural components of BMs, including that of the seminiferous tubules. BMs have been shown to play a role in the differentiation of a variety of cell types and to promote cell attachment and motility (9, 10). In the testis, BM components have been reported to modulate Sertoli cell functions, survival, and responsiveness to FSH (11). Both Sertoli cells and peritubular myoid cells contribute to the synthesis of BM components (12). Studies have demonstrated that Sertoli cells synthesize laminin and type IV collagen (11, 12), whereas peritubular myoid cells produce type I and IV collagen and fibronectin (11, 12).

The site of entactin production within the testis is still uncertain. Entactin is necessary for BM formation, epithelial morphogenesis, and laminin/type IV collagen interaction (13, 14, 15).

Alterations in specific BM component expression are likely to influence structure and function of the BM deposited. BM turnover results from the balance between the levels of component synthesis and degradation. Two of the major groups of enzymes implicated in BM degradation are the family of matrix metalloproteinases (MMPs) and the enzymes of the plasmin secreting system. The MMPs family is comprised of the collagenases, stromelysins, and gelatinases (16), the activities of which are regulated by specific tissue inhibitors of MMPs (TIMPs) (17). Sertoli cells have been shown to secrete plasminogen activators (PAs), gelatinolytic metalloproteinases, and TIMPs (18, 19).

In the present study, we have investigated the role of thyroid hormone on BM metabolism by examining the effect of T3 on the expression of laminin, entactin, type IV collagen, and extracellular matrix-degrading enzymes by prepubertal rat Sertoli cells in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
MEM, trypsin (type I), collagenase (type II), hyaluronidase (type 1-S), T3, soybean trypsin inhibitor, EDTA, gelatin, bovine casein, human plasminogen, antirabbit IgG biotin-conjugated, extravidin peroxidase-conjugated, and 3'-3' diaminobenzidine were purchased from Sigma Chemical Co. (St. Louis, MO). Nonessential amino acids and antibiotics were purchased from Flow Laboratories (Irvine, UK). Antimouse EHS (Engelbreth-Holm-Swarm mouse tumor) laminin (20) and type IV collagen (21) polyclonal antibodies, standard EHS mouse type IV collagen preparation, EHS laminin, and entactin-free laminin preparations were purchased from Collaborative Research (Bedford, MA). The mouse nidogen complementary DNA (cDNA) and specific antimouse nidogen antibody were kindly provided by Prof. M. L. Chu (Thomas Jefferson University, Philadelphia, PA) (14) and Dr. R. Timpl (Max-Planck Institut für Biochemie, Martinsried, FRG). The cDNA probes for human B1 and B2 laminin chains and human {alpha}1 type IV collagen chain were obtained from the American Type Culture Collection (ATCC, Rockville, MD) (22, 23, 24). Nylon membranes (Nytran) were from Schleicher and Schuell Inc. (Keene, NH) and the fast-track kits from Invitrogen (Leek, The Netherlands). RNA molecular weight marker II was from Boehringer Mannheim (Monza, Milan, Italy). The Bradford protein assay kit, electrophoresis and Western blotting reagents, and molecular weight markers were purchased from Bio-Rad Laboratories (Richmond, CA). The ECL detection system was obtained from Amersham (Little Chalfont, UK). Microconcentrators (Centricon 10 and 100) were from Amicon (Beverly, MA) and plastic Lab-Tek chamber slides from Nunc Inc. (Naperville, IL). FCS was purchased from Mascia Brunelli (Milan, Italy).

Sertoli cell cultures
Wistar rats were reared in our Institute facilities, and all the experimental protocols were approved by the local ethical committee. Purified Sertoli cells were prepared from 4- to 5-day-old Wistar rats, as described (25), and plated in 1% charcoal-stripped FCS-MEM. In preliminary experiments, the purity of Sertoli cell preparations was assessed after 2 days of culture by staining for peritubular cells with alkaline phosphatase (26). In these preparations, Sertoli cells accounted for approximately 90% of the cell population, as judged by phase-contrast examination, with the major contaminant being germ and peritubular cells. For immunocytochemical studies, Sertoli cell preparations were seeded on plastic Lab-Tek chamber slides at low cell density. The following day, the cells were washed three times with serum-free MEM and incubated for 24 h at 32 C in 5% CO2, in the absence or presence of T3 (10-7 M) (27). For all other experiments, Sertoli cells were cultured on 6-well plates and treated as described above. At the end of the culture period, the supernatants were collected and concentrated 20-fold in Centricon 100 concentrators. The effect of T3 on extracellular matrix degrading enzymes was determined using Sertoli cells cultured on 96-well Costar plates and incubated for 24 or 48 h, with or without T3.

Immunocytochemical analysis
Sertoli cells, cultured on chamber slides, were fixed with acetone-methanol 1:1 at -20 C for 5 min, rinsed in PBS, and [after inactivation of endogenous peroxidase (0.5% H2O2)] incubated with the primary antilaminin (1:200) or antitype IV collagen (1:200) antibodies overnight at 4 C in a moist chamber. After rinsing in PBS, the secondary antibody (antirabbit IgG biotin-conjugated, diluted 1:20) was applied for 1 h at room temperature. After rinsing in PBS, the extravidin peroxidase-conjugate was applied at room temperature for 30 min; and after washing, immunoreactive sites were visualized by incubation in 3'-3' diaminobenzidine, 25 mg/100 ml PBS, with 60 µl H2O2, 36 volumes. Sections were lightly counterstained with hematoxylin and mounted in permanent medium. Microscopy was performed using a Leitz Dialux microscope with photographic equipment. The percentage of cells that stained positive for laminin and/or entactin or type IV collagen was assessed by counting the total number and the number of positive-stained cells in 10 fields (x40 magnification) for each culture, in 3 independent experiments. Negative controls were perfomed by the omission of the first antibody on the permeabilized cells; and in these conditions, no immunoreactivity was observed.

Western blot analysis
To study the secretion of BM components, aliquots of 20-fold concentrated Sertoli cell supernatants, from an equal number of cells, were separated on 5% or 4–15% gradient SDS-PAGE, under reducing conditions (28), and proteins transferred electrophoretically to nitrocellulose membrane. Nonspecific binding sites were blocked with 10% nonfat dry milk in Tris-buffered saline-Tween 20 (0.3%) (20 mM Tris, 137 mM NaCl, pH 7.6) for 2 h at room temperature and blots incubated overnight with antilaminin antibody (1:400) or antitype IV collagen antibody (1:4000) or antinidogen antibody (1:600) diluted in blocking solution containing 5% nonfat dry milk. After washing in Tris-buffered saline-Tween 20, nitrocellulose membranes were incubated for 1 h with goat antirabbit-horseradish peroxidase-conjugated IgG (Bio-Rad, Hercules, CA), diluted 1:3000 in blocking solution, and immunoreactivity assessed by chemiluminescence reaction using the ECL Western blotting detection system. Immunopositive bands were quantified by scanning densitometry, using Molecular Analyst PC software for the Bio-Rad model 670 scanning densitometer.

Substrate gel electrophoresis (zymograms)
SDS-PAGE zymograms (containing either 0.1% gelatin, 0.1% casein, or 0.1% casein) plus 12 µg/ml plasminogen were prepared as described (29). Supernatants were compared according to cell number. Approximately 2 µg protein were examined in unconcentrated samples and 20 µg in those subject to concentration. After electrophoresis, gels were rinsed in 50 mM Tris-HCl (pH 7.4) containing 2% Triton X-100, followed by 50 mM Tris-HCl (pH 7.4), and incubated overnight for gelatinase and PAs or 72 h for caseinases, in a buffer containing 50 mM Tris-HCl (pH 7.4), 0.2 M NaCl, 5 mM CaCl2, and 1% Triton-X100 at 37 C. PA zymograms were also incubated in the presence of 15 mM EDTA, to inhibit metalloproteinases. Enzyme activity was detected after staining with 0.1% Coomassie blue in a mixture of acetic acid:methanol:water (1:3:6) and destaining in the same mixture without dye.

Extraction and analysis of RNA
The acid phenol guanidinium isothiocyanate method of Chomczynsky and Sacchi (30) was used to prepare total RNA from cultured Sertoli cells, treated with or without T3 (10-7 M for 16 h). The purity and integrity of the RNA preparations were checked spectroscopically and by gel electrophoresis before carrying out the analytical procedure. Poly-A RNA was prepared using the commercial kit Fast-Track. For Northern analysis, RNA was resolved on 1% agarose gel, containing formaldehyde, and blotted onto nylon membranes. Slot blot analysis was performed by blotting mRNA from control and T3-treated Sertoli cells onto nylon membranes using a slot blot apparatus (Hybri-slot manifold from BRL, Life Technology, Gaithersburg, MD), as described (31). After transfer, RNA was cross-linked to membrane by UV exposure. Immobilized RNA was then hybridized with 32P-labeled entactin, {alpha}1 type IV collagen chain, B1 and B2 laminin chain cDNAs in hybridization buffer (5x Denhardt’s, 50 mM phosphate, 0.1% SDS, 6x saline sodium citrate, and 200 µg/ml of salmon sperm DNA) for 14–16 h at 65 C. Filters were then washed twice with 2x SSC and 0.1% SDS, twice with 0.5 x SSC and 0.1% SDS, and twice with 0.1 x SSC and 0.1% SDS at 65 C for 15 min each wash. Filters were then exposed to X-OMAT Kodak film at -70 C for 24–48 h. Stripped filters were reprobed with ß-actin cDNA to normalize the amount of RNA loaded in each lane. Autoradiographs were quantified using a scanner densitometer (GS670, Bio-Rad). mRNA sizes were estimated according to the migration of RNA molecular weight marker II.

Statistical analysis
Results are expressed as the mean ± SE of at least three experiments, and values were statistically compared using the Student’s t test. Results were determined to be significantly different if P values were lower than 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Figure 1Go shows the effect of T3 treatment of Sertoli cell cultures from 5-day-old rats on laminin and/or entactin and type IV collagen expression. In basal conditions, laminin and/or entactin immunopositivity was present in 58 ± 5.3% of the cells. T3 treatment (10-7 M for 24 h) induced a significant increase (P < 0.01) in the number of cells staining positive for laminin and/or entactin to 86.4 ± 6.5%. In contrast, in the same cell cultures, type IV collagen immunostaining, very intense in all untreated cells, was dramatically reduced after T3 treatment (Fig. 1Go).



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  		Figure 1. Effect of thyroid hormone treatment on intracellular laminin/entactin and type IV collagen accumulation in prepubertal rat Sertoli cells in culture. Sertoli cells from 5-day-old rats were cultured at low-density on plastic Lab-Tek chamber slides. After 24 h, the cells were incubated for an additional 24 h in serum-free MEM in the absence or presence of T3 (10-7 M). At the end of the hormonal treatment, cells were processed for immunocytochemical analysis, as described in the Materials and Methods section. Data presented are representative of three independent experiments (125x magnification).

 
In Western blots of concentrated Sertoli cell supernatants, antilaminin polyclonal antibody recognized the laminin A and B1/B2 chains, together with a major 100-KDa protein, which may correspond to degraded entactin also present in the standard laminin preparation (Fig. 2AGo) (14). Densitometric analysis of different blots from 5 independent experiments indicated that T3 treatment of cultured Sertoli cells did not affect laminin A and B1/B2 chain expression (Fig. 2BGo), whereas a significant (P < 0.01) increase (65.1 ± 14.6%) in entactin immunoreactivity was observed (Fig. 2BGo). To better characterize this effect, Western blots were repeated using a specific antimouse entactin antibody. As shown in Fig. 2CGo, this antibody recognizes the 150- to 160-kDa entactin present in standard laminin preparation, together with degraded entactin fragments. A very similar immunoreactive profile was observed in Sertoli cell supernatants, whereas no cross-reactivity of the antibody was found with standard entactin-free laminin preparation. Densitometric analysis of different blots (n = 3) confirms the increase (59 ± 4.2%) in entactin immunoreactivity after T3 treatment (P < 0.01; Fig. 2DGo). The effect of thyroid hormone treatment on secreted type IV collagen levels is shown in Fig. 3Go. Two major immunoreactive bands, of approximately 170- and 180-kDa (type IV collagen {alpha}1 and {alpha}2 chains, Fig. 3AGo), which comigrated with EHS collagen standard, were present in concentrated Sertoli cell supernatants. Densitometric analysis of three independent experiments, reported in Fig. 3BGo, revealed that thyroid hormone treatment significantly (P < 0.05) reduced secreted levels of type IV collagen by 62.3 ± 16%.



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  		Figure 2. Effect of thyroid hormone on prepubertal Sertoli cell laminin and entactin secretion. Prepubertal rat Sertoli cells were cultured on 6-well plates; and after 24 h, the medium was changed and cells incubated for further 24 h in the absence (B) or in presence of T3 (10-7 M). At the end of the hormonal treatment, supernatants were concentrated 20-fold in Centricon 100 and processed for Western blotting, as described in the Materials and Methods section. Densitometric values represent the mean ± SE of five and three different experiments, respectively, for laminin and entactin. St. Lam, Standard laminin; *, P < 0.01.

 


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  		Figure 3. Thyroid hormone effect on prepubertal Sertoli cell type IV collagen secretion. Experimental conditions are as in Fig. 2Go. Densitometric values represent the mean ± SE of three different experiments. St. Coll., Standard type IV collagen; *, P < 0.05.

 
To further characterize this effect, the expression of BM component mRNAs was studied. Northern analysis of RNA extracted from cultured Sertoli cells showed the presence of mRNA for laminin B1 and B2 chains, collagen {alpha}1 chain, and entactin of the expected size (Fig. 4AGo). Quantification of T3 effect was performed by slot blot analysis with total or poly-A RNA from control and T3 (10-7 M for 24 h)-treated cells. Densitometric analysis of autoradiographs from three different experiments demonstrated that T3 increases the levels of entactin mRNA by 140 ± 28%, whereas it decreases that of type IV collagen {alpha}1 chain by 54 ± 6.4% (Fig. 4BGo). However, no effect by T3 was observed on the expression of laminin B1 and B2 chain mRNAs.



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  		Figure 4. Thyroid hormone effect on laminin, entactin, and type IV collagen RNA in prepubertal Sertoli cells. A, Northern blots showing the expression of the different BM component mRNAs of the expected size in rat Sertoli cell cultures; B, densitometric analysis of slot blots, showing the effects of T3 treatment (10-7 M, 24 h) on the accumulation of BM component mRNAs. Northern and slot blot for laminin were performed on total RNA (20 µg), whereas those for entactin and type IV collagen were on poly-A RNA (2–3 µg). Densitometric results were normalized with the values obtained by ß-actin rehybridization. Values represent the mean ± SE of three separate experiments. *, P < 0.01.

 
Because BM turnover involves a balance between synthetic and degradation phases, we analyzed the effect of T3 on the activity of gelatinolytic and caseinolytic MMPs and PAs secreted by Sertoli cells (Fig. 5Go). It can be observed that T3 treatment did not modify gelatinase (panel A), caseinase (panel B), or PA (panel C) activity secreted by prepubertal rat Sertoli cells in vitro.



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  		Figure 5. Lack of effect of thyroid hormone on secreted gelatinase (A), PAs (B), or caseinase (C) activity by prepubertal Sertoli cells in culture. Sertoli cells from 5-day-old animals were treated for 48 h in the absence or presence of T3 (10-7 M). At the end of the hormonal treatment, cell supernatants were concentrated 20-fold in Centricon 10 and aliquots used in zymograms, as described in the Materials and Methods section.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent studies, from our own and other laboratories, have demonstrated that the administration of T3 to neonatal rats accelerates the proliferation of Sertoli cells and shortens the time taken to reach terminal differentiation (7, 8), whereas resumption of thyroid hormone secretion after transient prepubertal hypothyroidism causes increased adult testis size (32), sperm production (33), and Sertoli cell number (34, 35). A major consequence of these effects is the augmentation of testicular size, histologically characterized by increased Sertoli and germ cell number. The effects on seminiferous cord size should imply a remodeling of the tunica propria and, in particular, of the BM surrounding the seminiferous epithelium. Our findings provide evidence in cultured prepubertal Sertoli cells of a differential effect of thyroid hormone on the expression of the major components of the BM, laminin, entactin/nidogen, and type IV collagen.

Immunocytochemistry studies showed, in basal conditions, a faint laminin and/or entactin immunopositivity localized in the perinuclear region and confined to about 50% of the cells. In contrast, type IV collagen immunopositivity was very intense and present in nearly all the cells.

In vitro treatment of Sertoli cells with thyroid hormone induced a significant increase in the number of cells expressing laminin and/or entactin, whereas type IV collagen expression was greatly reduced. These results were mirrored in Western blot analysis of the secreted BM components. Indeed, thyroid hormone treatment did not affect laminin A and B1/B2 chain expression, whereas a significant increase of entactin/nidogen immunopositivity was observed. These observations were confirmed by Northern blot experiments showing a significant stimulation of entactin mRNA level after T3 treatment of prepubertal Sertoli cells.

Our observations point to Sertoli cells as a source of entactin in rat testis, as demonstrated at both protein and mRNA levels. In fact, in in vitro experiments, a direct effect of thyroid hormone on contaminant peritubular (rather than Sertoli) cells is unlikely because our Sertoli cell preparations contain a very small amount of peritubular cells (1–2%), which may account for the presence of entactin in Western blots. Furthermore, thyroid hormone modulates specific Sertoli and Leydig cell functions, including glucose uptake (27), aromatase activity (36), insulin growth factor I (37), androgen binding protein (6, 38), inhibin expression (39), androgen and estrogen receptor content (40), and steroidogenic function (41, 42, 43). Thus, entactin secretion by prepubertal Sertoli cells can be regarded as an additional specific function of these cells that is regulated by thyroid hormone. It is possible also that this effect may be amplified in vivo. T3 could interact with its own receptors in Sertoli cells, leading to the production of some paracrine factors that may, in turn, stimulate neighboring peritubular cells to produce entactin.

All together, these observations demonstrate, at both protein and mRNA levels, the capability of T3 to differentially modulate, in the same cell system, the expression of BM components. In fact, laminin was not affected by hormone treatment, whereas entactin and type IV collagen expression were, respectively, stimulated and inhibited. This could be of physiological relevance, considering the essential role of entactin in establishing the network of collagen IV and laminin required to form stable BM structure (15).

BM turnover results from the balance between synthetic and degradative phases. Enzymes of the plasminogen-activating system and MMPs have been implicated in BM degradation. Although it is known that thyroid hormone regulates the expression of MMPs in mammals and low vertebrates (44, 45, 46), the results of this study demonstrate that it did not modulate MMPs or PA activity secreted by Sertoli cells that are under FSH control (47, 48). However, peritubular myoid cells have been reported to act in concert with Sertoli cells in the production and deposition of extracellular matrix components in vitro (12). For this reason, further studies are currently underway, in vivo and/or in organ culture, to evaluate the effect of thyroid hormone on BM components and on matrix-degrading enzyme expression.

Even though thyroid hormone has been shown to modulate the expression of extracellular matrix components (such as fibronectin and collagen) in both rat and human tissues (49, 50), this is the first report demonstrating the ability of T3 to differentially regulate BM components in Sertoli cell cultures. This could be relevant during testis development and differentiation. Indeed, BM components have been shown to promote Sertoli-Sertoli tight junction and blood testis barrier formation. In addition, they alter responsiveness to FSH and are important for Sertoli and germ cell survival (11). Furthermore, in vitro studies have demonstrated that prepubertal Sertoli cells, cultured on BM substrate, showed an increased secretion of specific proteins and differentiate into a phenotype close to that observed in vivo (11, 51, 52). Thus, the ability of T3 to regulate the expression of BM components may be regarded as a part of the integrated mechanism by which thyroid hormone affects development and differentiation of rat testis and may contribute to the continuous remodeling of the architecture of the prepubertal seminiferous epithelium.


   Acknowledgments
 
The authors are grateful to Drs. M. L. Chu (Thomas Jefferson University) and R. Timpl (Max-Planck Institut für Biochemie) for the mouse entactin cDNA and the antimouse entactin. Our compliments and gratitude to P. Minelli and D. Di Gregorio for their secretarial work and to Dr. R. Caruso for adapting her English expertise to our needs.


   Footnotes
 
1 This work was supported by Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST) and Consiglio Nazionale delle Ricerche (CNR) grants. Back

Received July 2, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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