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Endocrinology Vol. 141, No. 10 3679-3686
Copyright © 2000 by The Endocrine Society

ARTICLES

Transforming Growth Factor-ß2 Mediates Mesenchymal-Epithelial Interactions of Testicular Somatic Cells1,2

Lutz Konrad, Martin Albrecht, Heiner Renneberg and Gerhard Aumüller

Department of Anatomy and Cell Biology, Philipps University, D-35033 Marburg, Germany

Address all correspondence and requests for reprints to: Dr. L. Konrad, Department of Anatomy and Cell Biology, Robert Koch Strasse 6, D-35033 Marburg, Germany. E-mail: konrad{at}mailer.uni-marburg.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transforming growth factor-ß2 (TGFß2) is an important mediator of growth and differentiation. We here describe for the first time the complete sequence of the TGFß2 complementary DNA derived from peritubular myoid cells of the rat testis. The size of the rat TGFß2 complementary DNA was 1245 bp, and the deduced protein sequence contained 414 amino acids. Sequence comparison with the human and mouse amino acid sequences demonstrated 96.4% and 97.9% sequence identities, respectively. To elucidate the functional role of TGFß2 in testicular somatic cells, we studied its secretion in vitro in monocultures and cocultures of mesenchymal peritubular and epithelial Sertoli cells. The highest amounts of TGFß2 protein were secreted in the cocultures and by peritubular cells, whereas Sertoli cells secreted only minor amounts. Stimulation experiments with FSH revealed a reduced secretion of TGFß2 in cocultures, probably mediated by a paracrine interaction of the FSH-responsive Sertoli cells. In contrast, TGFß2 secretion by peritubular cells was increased after stimulation with glucocorticoids and after addition of recombinant TGFß2, indicating an autoregulation of TGFß2. Furthermore, application of recombinant TGFß2 to cocultures resulted in an enhanced aggregation and cell clustering of Sertoli cells, pointing to an important role of TGFß2 in the paracrine interaction of peritubular and Sertoli cells of the developing rat testis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MAMMALS, three isoforms of transforming growth factor-ß (TGFß1, -ß2, and -ß3) have been identified. All three isoforms are generally disulfide-linked 25-kDa homodimers and are synthesized as precursors with large prosegments, which are probably cleaved from the C-terminal mature monomer by dibasic processing endoproteases (1). Mature TGFß2 is a homodimer of two 112-amino acids monomers, containing nine cysteine residues, with eight cysteines forming intrachain disulfide bonds, and one cysteine being involved in forming an interchain disulfide bond linking the two monomers (2, 3). Newly synthesized TGFß is released in a latent form that cannot interact with TGFß receptors. This is due to noncovalent association with the so-called latency-associated peptide representing the TGFß propeptide homodimer (4). After an activation step, most often resulting from proteolytic cleavage of latency-associated peptide by plasmin, the biologically active TGFß is able to bind to the TGFß receptors.

TGFß action is transduced by two transmembrane serine/threonine kinases (receptor types I and II) that are essential for intracellular signaling. Furthermore, TGFß receptor type III (betaglycan) potentiates TGFß binding to type I and type II receptors, of which TGFß2 particularly has only low affinity for the type II receptor (5).

The TGFßs are secreted by many cell types and exert three major activities: 1) proliferation inhibition of most epithelial cells, except for some mesenchymal cells; 2) immunosuppressive effects, mainly due to their antimitogenic action; and 3) stimulation of extracellular matrix production (for review, see Refs. 6 and 7).

In the testis, TGFßs modulate paracrine/autocrine actions (8, 9). All three mammalian TGFß isoforms are expressed and secreted by Sertoli cells (SC) and peritubular cells (PC) of the rat (10). Bioactive TGFß1 is secreted by SC and Leydig cells of the pig (11). Furthermore, the messenger RNA (mRNA) of the three receptor types (I–III) is found in somatic and germ cells of the rat testis (12).

TGFß2 is the predominant hormone-dependent isoform in the rat testis (10). Suppression of TGFß2 expression, as occurs after FSH stimulation, correlates with the onset of puberty and the induction of spermatogenesis. Based on immunohistochemical studies, Teerds and Dorrington (13) demonstrated that a TGFß2 antibody intensively labeled the elongated spermatids in the adult rat testis. This study was substantiated by the observations of Olaso et al. (14), who investigated immunolocalization in the fetal and neonatal rat testis. In the fetus, TGFß2 staining of SC appeared on day 13.5, that of Leydig cells on day 16.5, and that in germ cells on fetal day 20.5. No immunolabeling was observed in the mesenchymal PC.

In the study presented here we sequenced the complete rat TGFß2 complementary DNA (cDNA) and investigated TGFß2 secretion by PC, SC, and cocultures in vitro. Cocultures of immature epithelial Sertoli cells and mesenchyme-derived peritubular cells represented a model system of embryonic tubulogenesis in the rat testis (reviewed in Ref. 15). Furthermore, we analyzed the modulation of TGFß2 secretion by FSH, glucocorticoids, and recombinant TGFß2 of somatic testicular cells. Our conclusions provide strong evidence that TGFß2 is an important mediator of mesenchymal-epithelial interactions in rat testicular somatic cells during testicular development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Primary cultures of Sertoli cells and peritubular cells
Peritubular and Sertoli cells were prepared from 19-day-old Wistar rats as previously described (16, 17). SC were plated and maintained in 75-cm2 flasks (1 x 107 cells/flask) in serum-free medium A [RPMI 1640 (Life Technologies, Inc., Eggenstein, Germany) supplemented with 22.5 mM HEPES (Life Technologies, Inc.), 4.3 mM L-glutamine (Sigma, Deisenhofen, Germany), 100 µg/ml streptomycin sulfate, and 100 U/ml penicillin G (both from Biochrom, Berlin, Germany)]. To remove germ cells and PC from the SC-enriched fraction, cells were hypotonically shocked with 20 mM Tris-HCl (pH 7.5) for exactly 2.30 min and washed twice with PBS (with Ca2+/Mg2+; Life Technologies, Inc.).

The PC fraction was resuspended in medium A containing 10% FCS (Linaris, Bettingen, Germany), and cells were washed after 24 h with PBS (with Ca2+/Mg2+, Life Technologies, Inc.). After 2 and 4 days, respectively, the PC were briefly trypsinized (0.05% trypsin/0.02% EDTA, Life Technologies, Inc.). Seven-day-old PC were used in the experiments. Both PC and SC, were incubated at 32 C in a humidified incubator with 5% CO2. To assay for TGFß2 secretion as a function of time, PC were seeded in six-well plates (6.0 x 105 cells/well) in duplicate and after some hours topped with freshly prepared SC (2.0 x 105 cells/well). For monocultures, 1.5 x 106 PC and 4.5 x 106 SC, respectively, were seeded in six-well plates. Cocultures and PC were cultivated in medium B (medium A with 1% FCS), except for the SC, which were kept without FCS.

The purity of primary PC was checked immunohistochemically with an smooth muscle {alpha}-isoactin antibody (18) and an enzyme-linked immunosorbent assay (ELISA) for fibronectin (19) modified according to Hoeben et al. (20). The purity of primary SC was evaluated by staining with oil red O (21) and the absence of smooth muscle {alpha}-isoactin immunoreactivity. Only 99% pure SC and PC, respectively, were used for the experiments described.

RNA isolation and RT
Total RNA was prepared with TRIzol (Life Technologies, Inc.) according to the manufacturer’s protocol. Total RNA was treated with ribonuclease-free deoxyribonuclease I (Roche, Mannheim, Germany), and RT was carried out for 90 min at 37 C in a final volume of 20 µl containing 1 x RT buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2), 5 mM dithiothreitol, 0.5 M each of deoxy-NTP, 2.5 pmol oligo(deoxythymidine)12–18, 40 U ribonuclease inhibitor (Promega Corp., Madison, WI), and 200 U Moloney murine leukemia virus (Promega Corp.).

Oligonucleotide primers and plasmids
Oligonucleotide primers were obtained from Biosource Technologies, Inc. (Ratingen, Germany). The primer pairs for sequencing the complete rat TGFß2 cDNA are shown in Table 1Go. The identity of the PCR products obtained was confirmed by sequencing after subcloning of the PCR products in the pCRII vector (Invitrogen, Groningen, The Netherlands).


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  		Table 1. Primer pairs used to sequence rat TGF-ß2 gene

 
PCR
PCR amplification was carried out in a volume of 50 µl with the following final concentrations: 2 µl first strand cDNA, 1 x PCR buffer [16 mM (NH4)2SO4, 50 mM Tris-HCl, and 0.01% Tween-20, pH 8.8], 0.2 mM of each deoxy-NTP, and 100 pmol forward and reverse primers, respectively, were used as standard conditions. PanScript DNA Polymerase (1.25 U; PanSystems, Nürnberg, Germany) was used for each reaction. Negative controls were performed without the primers. The reaction mixture was covered with 30 µl mineral oil (Sigma) and run on a Hybaid Omnigene thermocycler (MWG Biotech, Ebersberg, Germany). Amplification was carried out for 35 cycles. After an initial heating to 94 C for 4 min, each cycle consisted of denaturing at 94 C for 45 sec, annealing at 57 C or annealing at 55 C (Table 1Go) for 45 sec, and elongation at 72 C for 90 sec, except for the last extension, which lasted 5 min.

TGFß2 ELISA
TGFß2 concentrations were quantitated using a TGFß2-specifc ELISA kit (Promega Corp.), according to the manufacturer’s instruction. Briefly, total TGFß2 was determined by acid activation of the samples with HCl at pH 2.0–3.0 for 15 min and neutralization with NaOH to approximately pH 7.6. Bioactive TGFß2 was determined without acid activation. TGFß2 levels were normalized to 1 x 106 cells. Cells were trypsinized and counted after staining with trypan blue in a hemocytometer.

Stimulation experiments
Dexamethasone (Dex) and FSH from porcine pituitary were obtained from Sigma, and recombinant TGFß2 was purchased from R and D Systems (Wiesbaden, Germany). In control incubations, stimulating agents were omitted. For the TGFß2 ELISA, aliquots from the medium were taken from the wells, and benzamidine (0.1 mM) and phenylmethylsulfonylfluoride (25 µM) were added.

Dexamethasone
PC (2 x 104/well; 24-well plates) were cultured for 2 days in medium A supplemented with 10% FCS. After an overnight incubation with medium A, cells were stimulated for 5 days with Dex (1.0 µM, 10 nM, and 0.1 nM) in medium B.

FSH
For monocultures, 6 x 105 PC and SC, respectively, were seeded in 24-well plates, whereas for cocultures, 6 x 105 PC were topped after 4–6 h with freshly prepared SC (1.5 x 105 cells) in medium B. After 5 days, cells were stimulated for 4 days with 50 ng/ml FSH in medium B.

Autoinduction and effects of TGFß2
PC (9 x 105) and 3 x 105 SC were seeded in 12-well plates and incubated for 5 days in medium B. Stimulation experiments were also performed with monocultures of PC (1.2 x 106 cells/well). Medium was replaced, and cells were stimulated with 20 ng/ml recombinant human TGFß2. After 2 days, the cells were intensively washed several times with PBS, and medium B was added. After 3 days, the number of SC aggregates was counted by three different individuals. During this time, aliquots were taken for the TGFß2 ELISA. The use of 1% FCS is necessary for SC aggregation in cocultures. However, no detectable amount of latent or active TGFß2 could be measured in 1% FCS alone (data not shown).

Statistical methods
Results were obtained from at least three independently performed experiments (n = 3). Variables were summarized as the mean ± SEM. Data were analyzed using two-way ANOVA followed by Dunnett’s multiple comparisons test (22). Differences between two means at P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sequence analysis of the rat TGFß2 cDNA
The sequence of the complete rat TGFß2 cDNA was determined using PCR-based techniques. The initial experiments were based on two partial rat TGFß2 sequences published by McKinnon et al. (23) (EMBL accession no. X71904) and Nishida et al. (24) (accession no. M96643). The homology comparison of these partial sequences with the human TGFß2 gene (25) (accession no. M19154) and the mouse cDNA sequence (26) (accession no. X57413) led to the hypothesis that the rat cDNA sequence M96643 could contain some noncoding nucleotides that are not present in the coding regions of the human and mouse genes (Fig. 1AGo). Based on this assumption, we deduced two primer pairs, 5-B2M/3-B2M and 5-B2E/3-B2E (Table 1Go), the sequences of which were taken from the rat sequences X71904 and M96643, respectively. The sequence of the primer 3-B2E comprising the stop codon was derived from the mouse sequence X57413.



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  		Figure 1. A, Putative 3'-end of the rat TGFß2 gene being composed of the nucleotide sequence as given by Nishida et al. (24 ) (accession no. M96643) and the partial 3'-end of the mouse TGFß2 gene (26 ) (accession no. X57413). The nucleotides belonging to the coding region are given in capital letters, whereas the presumptive noncoding nucleotides are given in shaded and lowercase letters; the nucleotides from the mouse sequence are italicized. The stop codon is shown in bold letters, and the primer sequences are underlined. B, RT-PCR performed with PC and primer pairs 5-B2M/3-B2M (left panel) and 5-B2E/3-B2E (right panel). c, control; M, marker.

 
The initial RT-PCRs were performed with total RNA from PC isolated from 20-day-old rat testes and revealed two amplicons of 280 bp (5-B2M/3-B2M) and 234 bp (5-B2E/3-B2E; Fig. 1BGo). Sequence analysis of the PCR fragments demonstrated 1) the additional nucleotides in M96643 were noncoding, 2) the presence of the 3'-end of the rat TGFß2 gene, and 3) the 100% identity of the partial sequence X71904 with the newly obtained sequence of the testis-specific TGFß2 cDNA.

The 5'-end of the rat TGFß2 cDNA was obtained with the primer pairs 5-B23N/3-B23 and 5-B20/3-B20 (Table 1Go), whose sequences were deduced from highly conserved regions of the mouse sequence X57413, except for 3-B23, which was derived from our new rat sequence. After sequencing the respective amplicons, we obtained the complete rat TGFß2 cDNA sequence and the deduced amino acid sequence (Fig. 2Go). Sequence comparison revealed that the rat TGFß2 shared high amino acid identity with human (96.4%) and mouse (97.9%) TGFß2. A second rat-specific TGFß2 cDNA sequence has recently become available from the EMBL database (accession no. AF135598). However, the sequence differed in only three positions from the one presented here [positions (99) Y instead of C (282), P instead of L, and (315) D instead of H].



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  		Figure 2. Nucleotide and deduced amino acid sequences of rat TGFß2. Nucleotides are numbered on the left beginning with the start codon, and amino acids are numbered on the right. The start and stop codons are double underlined. The secretory signal sequence is underlined ({triangledown} indicates the cleavage site). The PEST motif is given in bold capital letters.

 
Using computer-assisted analysis (PC/Gene), we identified in the N-terminal region an expected secretory signal sequence (27) (Fig. 2Go), because TGFßs are secreted molecules. At amino acid positions 50–74, a half-life determining PEST (Pro-Glu-Ser-Thr) region (28) was identified (Fig. 2Go). No RGD (Arg-Gly-Asp) motif was found, which is a recognition sequence for interactions with extracellular matrix molecules and integrins; in contrast, TGFß1 and TGFß3 were both reported to contain one RGD (Arg-Gly-Asp) sequence.

TGFß2 secretion by peritubular cells, SC, and cocultures
The ability of PC, SC, and cocultures to secrete TGFß2 was determined by a highly sensitive ELISA. During 1 week, PC and cocultures secreted increasing amounts of TGFß2, whereas SC secretion was only marginal (Fig. 3Go, A and B). After 7 days of culture, a plateau phase in secretion was reached. In the cocultures, the total amount of TGFß2 on the first 2 days was similar to that of PC, but starting with day 3, it was subsequently reduced (Fig. 3AGo). The reduced TGFß2 secretion of PC in cocultures was due to the reduced number of PC present in the coculture relative to the monoculture of PC (the ratio of PC to SC being 3:1 in the coculture). Secretion of active TGFß2 was highest in the cocultures (Fig. 3BGo) and demonstrated a small peak on day 2 comparable to the peak of the total amount of TGFß2 (Fig. 3AGo). This peak possibly precedes the formation of SC aggregates, which appeared after 3 days of culture. After 6 days, secretion of active TGFß2 by PC and cocultures reached a plateau (Fig. 3BGo).



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  		Figure 3. The secretion of total (A) and active (B) TGFß2 protein by PC, SC, and cocultures as a function of time. The concentrations of TGFß2 were quantified by ELISAs. Each data point represents the mean ± SEM from three independently performed experiments (n = 3). d, Day.

 
Modulation of TGFß2 secretion
Secretion of total TGFß2 in cocultures and SC was reduced when the cells were treated with 50 ng FSH (Fig. 4AGo). Similar effects were also observed with 25 and 100 ng/ml, respectively (data not shown). The reduction was highest and significant (P < 0.05) on days 2 and 3, with approximately 25% in the cocultures and approximately 40% for SC.



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  		Figure 4. Effects of FSH (A) on the secretion of TGFß2 in cocultures (Co) and Sertoli cells and of TGFß2 (B) on the secretion of TGFß2 by PC and in cocultures. Cells were treated for 4 days with FSH (50 ng/ml); during this time aliquots were taken for the TGFß2 ELISA. In contrast, cells were only treated for 3 days with TGFß2 (20 ng/ml) in medium A supplemented with 1% FCS. After a medium change to medium A without TGFß2, aliquots were taken for the TGFß2 ELISA. The controls (ctrl) in both experiments were performed without the agents. Each data point represents the mean ± SEM of at least four independently performed experiments (n = 4). Statistically significant differences are indicated (*, P < 0.05; **, P < 0.01).

 
Treatment of PC and cocultures with recombinant TGFß2 caused a dramatic rise in TGFß2 secretion (Fig. 4BGo). The increase was more pronounced in the PC, with 4.83-, 2.53-, and 2.54-fold increases on days 1–3, compared with the cocultures, with 2.3-, 1.6-, and 1.44-fold increases on days 1–3, respectively. This effect was not caused by remnants of recombinant TGFß2 used for the stimulation, because 4 h (day 0, Fig. 4BGo) after the medium change the TGFß2 levels were really low compared with those on day 1. In both experiments the differences between treated cultures and controls were statistically significant (P < 0.01 for the PC and P < 0.05 for the cocultures).

The effect of Dex on TGFß2 secretion by PC on days 2 and 3 is shown in Fig. 5Go. Cells were treated for 5 days followed by a TGFß2 ELISA to determine alterations in secretion. TGFß2 secretion by PC was dose dependently increased after 24 h and lasted for 5 days. The maximal level of secretion was already reached on day 2, with only a slight increase thereafter. A concentration of 1.0 µM dexamethasone stimulated TGFß2 secretion significantly differently (P < 0.05) from the control value by 53% and 59% on days 2 and 3, respectively. The up-regulation in TGFß2 secretion was not caused by proliferative effects, as dexamethasone has been shown to be antimitogenic on peritubular cells (29).



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  		Figure 5. Effects of different concentrations of Dex (1.0 µM to 0.1 nM) on the secretion of TGFß2 by PC on days 2 and day 3 as quantified by an ELISA. The enhanced secretion was concentration dependent, with a minimal effective concentration of 1.0 µM on both days. *, Statistically significant difference (P < 0.05). Each experiment was independently repeated three times (n = 3) in duplicate, with each value given as the mean ± SEM.

 
Functional role of TGFß2 in the coculture
When immature epithelial SC are seeded on top of immature mesenchymal PC, SC aggregate and form tubule-like structures that resemble testicular seminiferous cords of the differentiating testis. In the cocultures, Sertoli cells migrated into cell colonies and formed cell clusters (Fig. 6Go, A and B). The aggregation of the SC, after administration of 20 ng/ml recombinant TGFß2, was significantly increased 2-fold compared with that in the untreated control (Fig. 6CGo). The monocultures of PC and SC did not show any enhanced cell clustering after stimulation with TGFß2 (data not shown). Our observations indicate that TGFß2 has a significant effect in promoting Sertoli cell aggregation and the formation of cell clusters in coculture in the presence of peritubular cells.



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  		Figure 6. Effect of TGFß2 (20 ng/ml) on the formation of SC aggregates in cocultures (B) compared with an untreated control (A) on day 3. The number of aggregates was significantly enhanced 2-fold after stimulation (C). **, Statistically significant difference (P < 0.01). Each data point represents the mean ± SEM of five independently experiments (n = 5) performed in triplicate. Magnification, A and B, x100.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Sequence analysis of the TGFß2 cDNA from the rat revealed for the first time the complete nucleotide sequence of 1245 bp coding for 414 amino acids. Comparison of the amino acid sequence showed identity to the human and mouse sequences of 96.4% and 97.9%, respectively. Sequence identities of TGFß2 among various species demonstrated 82% in the pro region and 95% in the mature region (30). All 112 amino acids of the mature protein (amino acids 313–414) that are involved in the three-dimensional structure of TGFß2, as deduced from x-ray diffraction studies of the human sequence (2, 3), are also highly conserved in the rat TGFß2 sequence. Only three amino acids in the mature rat amino acid sequence are different from those in human TGFß2.

Previous examination of TGFß2 secretion of testicular cells by Western blot analysis (10) has been extended in the present study using the more sensitive and quantitative ELISA system and to cocultures of PC and SC. Secretion of TGFß2 was found to be highest in immature peritubular cells and cocultures, whereas SC secrete only very low amounts of the protein.

In an attempt to elucidate the modulation of TGFß2 production in testicular cells, we analyzed the influences of FSH, Dex, and TGFß2 on TGFß2 production of PC and SC alone and in cocultures. All three factors are known to alter TGFß2 levels and to exert effects on the testis.

The new finding of the present study that Dex caused a concentration-dependent increase in TGFß2 secretion by PC corroborates earlier results showing antimitogenic effects in the immortalized and immature peritubular cell line RTC-8T12 (29). Additionally, fibronectin secretion was abrogated. Similar, but even higher, concentrations of dexamethasone (10 µM to 1.0 nM) were used to stimulate the secretion of {alpha}2-macroglobulin of Sertoli cells (31). In view of the inducibility of TGFß2 expression by glucocorticoids and the similar effects exerted by both agents, it was speculated that glucocorticoids indirectly mediate their antiproliferative effects by induction of TGFß2 (32). In fact, at least four glucocorticoid response elements (5'-AGAACA) originally identified in the human TGFß1 promoter (33) could be also found in the human TGFß2 promoter (EMBL accession no. M87843; Konrad, L., unpublished data).

The present data not only confirm the previously observed down-regulation of TGFß2 production in SC by FSH (10, 34), but show for the first time a comparable down-regulation in cocultures after 48 h. As the contribution of SC to TGFß2 secretion is only marginal, we hypothesize that the reduced TGFß2 secretion of the FSH receptor-deficient PC in cocultures is mediated in a paracrine manner by the FSH-responsive SC. However, the kind of interaction of both cell types or paracrine factor(s) that mediates this down-regulation has to be determined in future studies. Also in other cells, FSH apparently down-regulates TGFß2 expression, e.g. in granulosa cells (35), possibly mediated by the cAMP response element/activating transcription factor-like element, which has been shown to confer cAMP responsiveness to a wide variety of genes, including FSH, and was found in the human TGFß2 promoter (36, 37, 38). In a recent publication (39) using testis organ cultures from pubertal day 0 no effects of FSH (25–50 ng/ml) on TGFß2 mRNA expression were observed, thus pointing to different effects of FSH on TGFß2 in in vitro systems and organ cultures.

The data summarized in Fig. 4BGo suggest a dramatic autoinduction of TGFß2 secretion after TGFß2 stimulation in peritubular cells and cocultures. This up-regulation was not due to some adherence of TGFß2 to the cells, because there was a clear difference in the up-regulation of TGFß2 secretion of PC compared with that in the cocultures. Treatment of fibroblasts with TGFß2 resulted in increased mRNA expression of TGFß1, TGFß2, and TGFß3, with the autoinduction of TGFß2 occurring rapidly after 1–3 h (40). In addition, autoinduction of TGFß1 mRNA and protein synthesis by TGFß1 was reported to be mediated by the AP-1 element (41, 42). Indeed, one AP-1 element was identified in the human TGFß2 promoter (37), indicating the possibility of a similar autoregulation mechanism for TGFß2.

Aggregation of SC and formation of cord-like structures have been observed by several researchers (43, 44, 45, 46, 47, 48) in coculture, where immature epithelial SC were seeded on top of immature mesenchymal PC. This has been regarded as an important and significant example of mesenchymal-epithelial interaction during testicular development.

In our coculture experiments, SC aggregation or tubule formation in vitro was 2-fold elevated after stimulation with TGFß2, whereas the monocultures remained unaltered. Based on this observation, we suppose that TGFß2 is an important paracrine mediator of the mesenchymal-epithelial interaction in developing rat testicular cells. Although the function of TGFß2 in the mesenchymal-epithelial interaction is new for the testis, it was described in other systems. TGFß2 mRNA was also reported to be mainly expressed in the mesenchymal components of tissues of mouse embryos from 10.5 days postcoitum to 3 days postpartum (49). These tissues included bone, cartilage, tendon, gut, blood vessels, skin, and fetal placenta. Therefore, the researchers concluded that the high levels of TGFß2 expression in the mesenchymal cells raise the possibility that some tissues, e.g. the submucosa of the intestines, may regulate the growth of the overlying epithelium in a paracrine fashion. Another example of mesenchymal-epithelial interaction is known from chondroossification (50). Epithelial expression of TGFß2 is found primarily in regions of active morphogenesis, involving epithelial-mesenchymal interactions (50). In conclusion, the widespread epithelial expression of TGFß2 mRNA was correlated with epithelial differentiation per se.

The participation of TGFß2 in multiple developmental processes, such as epithelial-mesenchymal interactions, cell growth, extracellular matrix production, and tissue remodeling, was recently shown by Sanford et al. (51) while studying TGFß2 null mice. The tissues affected included cardiac tissue, lung, craniofacial system, limb, spinal column, eye, inner ear, and the urogenital tract. Although preliminary, the urogenital defects included testicular ectopia and testicular unilateral hypoplasia. Interestingly, there was no phenotypic overlap with TGFß1 and TGFß3 null mice, indicating the particular importance of TGFß2 in the testis.

Based on the observations obtained in the present study, we suppose that TGFß2 is an important paracrine mediator of the mesenchymal-epithelial interaction in rat testicular cells and might play an important role during testicular development.


   Acknowledgments
 
We gratefully acknowledge the excellent technical assistance of Elke Völck-Badouin and Andrea Dersch.


   Footnotes
 
1 This work was supported by the Deutsche Forschungs-gemeinschaft Au 48/13–2 and Au 48/13–3. Back

2 The EMBL/DDBJ/GenBank accession number for rat TGFß2 is AJ132718. Back

Received May 11, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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