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Endocrinology Vol. 139, No. 4 1853-1862
Copyright © 1998 by The Endocrine Society

ARTICLES

Rat Testicular N-Cadherin: Its Complementary Deoxyribonucleic Acid Cloning and Regulation1

Sanny S. W. Chung, Meng-yun Mo, Bruno Silvestrini, Will M. Lee and C. Yan Cheng

Population Council (S.S.W.C., M.-y.M., C.Y.C.), Center for Biomedical Research, New York, New York 10021; Department of Zoology (S.S.W.C., W.M.L.), The University of Hong Kong, Hong Kong, China; and Institute of Pharmacology and Pharmacognosy (B.S.), University of Rome "La Sapienza," 00185 Rome, Italy

Address all correspondence and reprint requests to: Dr. C. Yan Cheng, Population Council, 1230 York Avenue, New York, New York 10021. E-mail: yan{at}popcbr.rockefeller.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 References
 
Using primer sets specific for mouse N-cadherin and rat testicular RNA for RT-PCR, a full-length complementary DNA (cDNA) coding for rat testicular N-cadherin was isolated. The deduced amino acid sequence of rat N-cadherin yielded a 883-amino acid polypeptide that displayed a 98.6% identity with the mouse homolog. N-Cadherin was found to be expressed by Sertoli and germ cells in the rat testis by RT-PCR. Using Sertoli-germ cell cocultures, it was found that the N-cadherin expression increased with time in culture. To assess whether this is due to a soluble factor(s) released from germ cells that affects Sertoli cell N-cadherin expression, germ cell-conditioned media (GCCM) were fractionated by preparative anion-exchange HPLC, and the resulting fractions were divided into 14 pools. Pool 4 was found to contain a factor(s) that induced a dose-dependent stimulation on Sertoli cell N- cadherin expression with a maximal stimulation at 2 µg protein/dish/4.5 x 106 Sertoli cells. At higher doses between 12 and 32 µg protein/dish, this pool relinquished its effect on Sertoli cell N-cadherin expression suggestive of a biphasic effect. This biphasic effect was confirmed using increasing doses of crude GCCM on Sertoli cell cultures. Since nonviable germ cells failed to stimulate Sertoli cell N-cadherin expression, it illustrates the observed stimulatory effect by GCCM is likely to be mediated via a soluble factor(s) releasing from viable germ cells. These results reveal the presence of a stimulatory factor(s) in GCCM that can modulate Sertoli cell N-cadherin expression in vitro. Since N-cadherin plays a crucial role in facilitating invasive capacity of metastatic tumor cells, the observation of germ cell-released factor(s) in affecting Sertoli cell N-cadherin expression may suggest its possible role in facilitating germ cell migration during spermatogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 References
 
SPERMATOGENESIS and testicular development involve complex and intriguing cellular interactions (for reviews, see Refs. 1–6). The cellular composition and architecture of the mammalian testis change dramatically during development (for reviews, see Refs. 7–9). As spermatogenesis commences, developing germ cells differentiate and traverse from the basal lamina to the adluminal compartment in the seminiferous epithelium where mature spermatozoa can be released into tubular lumen. This likely involves the regulation of numerous intercellular junctions between Sertoli cells and spermatogenic cells (3, 9, 10) via a cycle of attachment and detachment through proteases and protease inhibitors. Furthermore, cell adhesion must be controlled spatially and temporally, both by intrinsic cellular factors and by signals arising from cell surface receptors.

Recently, three members of the cadherin family of Ca2+-dependent cell adhesion molecules (N-cadherin, E-cadherin, and P-cadherin) are shown to be present in the mouse (6, 11, 12, 13), rat (6, 12, 13, 14), and human (15) testis. Cadherins are members of a large family of transmembrane glycoproteins that mediate Ca2+-dependent, homotypic cell-cell adhesion. Through their homophilic binding interactions, they play a crucial role in the maintenance of normal tissue architecture and conferring adhesion specificities of cells (16, 17, 18). Moreover, they have been implicated as modulators of cell polarity, cell sorting, and cell migration (19, 20). Cadherins are thus considered to be important regulators of morphogenesis. Being a transmembrane component of the adherens junction, they are composed of three segments, an extracellular domain responsible for cell-cell interaction, a single-pass transmembrane domain, and a highly conserved cytoplasmic domain that associates with actin filaments via intracellular attachment proteins such as catenins (21, 22) and thus serves to connect the outside of the cell to the cytoskeleton elements. It was shown that N-cadherin plays an important role in regulating cell-cell interactions in the seminiferous epithelium (6, 11, 15). Antibodies directed against N-cadherin blocked Sertoli cell-spermatogenic cell adhesion in vitro (23), illustrating that cadherin plays a pivotal role in the testis.

Recent studies have shown that high levels of N-cadherin expression in the most invasive carcinoma cells were inversely correlated with their expression of E-cadherin, suggesting the possible role of N-cadherin in facilitating invasion and metastasis (24, 25). In addition, the cadherin-catenin complex was speculated to be implicated not only in cell-cell adhesion, but also in signal transduction pathways (19, 26, 27). The high levels of cadherin expression inhibit ß-catenin-signaling activity (28). Therefore, it seems likely that N- cadherin-mediated cell adhesion in testis may be linked in some way to these signaling events and testicular development. Since the rat N-cadherin has not been cloned, we thought it pertinent to determine the rat N-cadherin nucleotide sequence and to examine whether germ cells can affect Sertoli cell N-cadherin expression via cell-cell contact or released factor(s) in vitro. In addition, we have partially fractionated these modulatory factor(s) by HPLC for preliminary characterization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 References
 
Animals
Male Sprague-Dawley rats weighing between 250 and 300 g and male rats at 20 days of age were obtained from Charles River (Kingston, MA). Rats were killed by CO2 asphyxiation, and testes were removed immediately for isolation of testicular cells. The usage of animals for this study was approved by the Rockefeller University Animal Care and Use Committee (Protocol No. 94132).

Preparation of primary cultures of Sertoli cells
Primary cultures of Sertoli cells were prepared from 20-day-old Sprague-Dawley rats as previously described (29, 30). Sertoli cells at 4.5 x 106 cells/9 ml were plated in 100-mm Petri dishes in serum-free Ham’s F12 nutrient mixture (F12) and DMEM (F12/DMEM) (1:1, vol/vol) containing sodium bicarbonate (1.2 g/liter), HEPES (15 mM), gentamicin (20 mg/liter), insulin (10 µg/ml), transferrin (5 µg/ml), bacitracin (5 µg/ml), and epidermal growth factor (2.5 ng/ml). Cells were maintained in a humidified atmosphere of 95% air-5% CO2 at 35 C for 36 h. Thereafter, cells were hypotonically treated with 20 mM Tris, pH 7.4, for 2.5 min to lyse contaminating germ cells (31). These cells, with a purity of greater than 90%, were then washed twice, used in coculture experiments with immature germ cells, or cultured with crude germ cell-conditioned media (GCCM) or fractionated GCCM proteins.

Preparation of germ cells and GCCM
For GCCM preparation, germ cells were isolated from adult Sprague-Dawley rats (250–300 g body wt) by a mechanical procedure without the use of trypsin or collagenase/dispase (32). Total germ cells were cultured at 22.5 x 106 cells/9 ml in 100-mm Petri dishes in F12/DMEM containing gentamicin (20 µg/ml), sodium lactate (6 mM), sodium pyruvate (2 mM), and bacitracin (10 µg/ml). Cells were then incubated in a humidified atmosphere of 95% air-5% CO2 at 35 C for 18 h. The purity of these germ cell preparations was greater than 90% when assessed by DNA flow cytometry (32). Inasmuch as elongate spermatids and spermatozoa were removed from the cell preparation by glass wool filtration, proteins in the GCCM were largely contributed by round spermatids, spermatocytes, and spermatogonia. The viability of germ cells was greater than 95% at the end of the 18-h incubation when examined by the erythrosin red dye exclusion test (33). Thereafter, media were collected and centrifuged at 300 x g for 60 min in a Sorvall RC-3B refrigerated centrifuge (Sorvall, Inc., Newtown, CT) at 4 C to remove any cellular debris and stored at -20 C until used. For dose-responsive analysis of total GCCM on Sertoli cell N-cadherin expression, crude GCCM was concentrated and equilibrated against F12/DMEM before its inclusion in Sertoli cell cultures. To examine the effects of different GCCM proteins on Sertoli cell N-cadherin expression, GCCM were concentrated and equilibrated against 20 mM Tris, pH 7.4, in a Millipore tangential ultrafiltration unit (Millipore Corp., Bedford, MA) equipped with eight Minitan plates with a molecular mass cut-off at 10 kDa before its fractionation onto a preparative anion-exchange HPLC column.

Fractionation of GCCM by anion-exchange HPLC
About 1–5 liters of GCCM were concentrated and equilibrated against solvent A (20 mM Tris, pH 7.4, at 22 C) and fractionated onto an anion-exchange preparative HPLC column (HR 16/10, 16 x 100 mm, i.d.) as previously described (32, 34, 35). Bound proteins were eluted using a flow rate of 4 ml/min with a linear gradient of 0–80% solvent B (20 mM Tris, 600 mM NaCl, pH 7.4 at 22 C) over a period of 90 min. Fractions of 4 ml each were collected, and the eluted proteins were monitored by UV absorbance at 280 nm. Aliquots of 50 µl from selected fractions were resolved by SDS-PAGE onto 12.5% T (total acrylamide concentration, gm/100 ml, acrylamide + methylenebisacrylamide) SDS-polyacrylamide gels, and proteins were visualized by silver staining. Thereafter, fractions according to different groups of proteins were pooled, concentrated, and equilibrated against F12/DMEM. These pools were then used to culture with Sertoli cells to examine their effect on Sertoli cell N-cadherin expression.

Sertoli-germ cell cocultures
To study the effects of germ cells on Sertoli cells or vice versa, immature germ cells, largely spermatogonia and spermatocytes, were isolated from 15-day-old Sprague-Dawley rats by a mechanical procedure without the use of trypsin or collagenase/dispase as previously described (32). These cells were layered carefully on highly purified Sertoli cell monolayers using a germ (4.5 x 106 cells/9 ml) to Sertoli (4.5 x 106 cells/9 ml) cell ratio of 1:1 and cocultured in 100-mm dishes in a humidified atmosphere of 95% air-5% CO2 at 35 C. Thereafter, cells were terminated at different time points (0-, 2-, 4-, and 24-h) for RNA extraction. To examine the effect of crude GCCM on Sertoli cell N-cadherin expression, total GCCM proteins (~90 µg protein/dish) were incubated with Sertoli cells and terminated at specified time points. In experiments where the effect of partially purified GCCM proteins on Sertoli cell N-cadherin expression was examined, different pools of fractions (~8 µg protein/dish) or increasing concentrations of proteins from the Mono Q run were cultured with Sertoli cells for 18 h. Each set of experiments contained duplicate cultures, and each experiment was repeated at least twice.

Detection of N-cadherin messenger RNAs (mRNAs) in testicular cells and their changes by sequential use of RT-PCR
To detect changes of Sertoli cell N-cadherin expression in Sertoli-germ cell cocultures, RT-PCR was performed as described previously (36, 37). The two N-cadherin primers used for PCR were: 5'- GCCACCATATGACTCCCTCTTAGT-3' (sense primer corresponding to nucleotides 2547–2570) and 5'-CAGAAAACTAATTCCAATCTGAAA-3' (antisense primer corresponding to nucleotides 2977–3000), which generated a N-cadherin PCR product of 454 bp. The two ß-actin primers used for PCR were: 5'-TCACCGAGGCCCCTCTGAACCCTA-3' (sense primer corresponding to nucleotides 314–337) and 5'-GGCAGTAATCTCCTTCTGCATCCT-3' (antisense primer corresponding to nucleotides 931–954) (38), which yielded a 641-bp ß-actin product. Briefly, total RNA was extracted from Sertoli-germ cell/Sertoli cell-GCCM coculture using RNA STAT-60 (Tel-Test "B", Inc., Friendswood, TX) as previously described (39). The concentration of RNA was quantified by spectrophotometry at 260 nm. To quantify and compare the expression of N-cadherin mRNA from various samples in PCR, N-cadherin was coamplified with ß-actin, which was used as an internal control to monitor the sample-to-sample variation in RT and PCR. In a series of preliminary experiments, increasing concentrations of template complementary DNA (cDNA) were used to verify the linearity of the assay with respect to the amount of products. The amount of PCR products was also examined over a range of 10–45 amplification cycles for a given amount of template cDNA to select the optimal amplification cycles in the linear range. In all experiments, the amplification of the template RT-cDNA, as well as that of the ß-actin RT-cDNA, was in the linear range as verified in a series of preliminary experiments. The total RNA of each sample (1 µg/sample) and 1 µg of oligo(dT)-15 primer were denatured at 70 C for 5 min, annealed in ice for 5 min, and reversed transcribed at 42 C for 60 min using 15 U of mouse Moloney leukemia virus-reverse transcriptase (Promega, Madison, WI) in the presence of 28 U of RNasin (Promega), 2.5 µl of deoxynucleoside triphosphates [10 mM each of dATP, dGTP, deoxycytidine triphosphate and deoxythymidine triphosphate], 2.5 µl of dithiothreitol (100 mM), and sterile water in a final reaction volume of 25 µl. RT reaction was terminated by heating the samples at 95 C for 5 min. To eliminate intersample variations, a master reagent tube was prepared that included all the required reagents for distribution into the experimental tubes. About one sixth of the RT-reaction product was used as a template for PCR. This RT-reaction product, together with 0.8 µg each of the sense and antisense N-cadherin primers (in experiments where ß-actin was coamplified with N-cadherin, 50 ng of the sense and antisense ß-actin primers were used), 5 µl of 10 x PCR buffer, 8 µl of deoxynucleoside triphosphates (200 µM each of dATP, dGTP, deoxycytidine triphosphate, and deoxythymidine triphosphate), 1.25 U of Taq DNA polymerase (Promega), and sterile water were used for PCR in a final reaction volume of 50 µl. Three drops of Nujol mineral oil (Perkin-Elmer Corp., Norwalk, CT) were added into each tube to prevent sample evaporation. For PCR using radiolabeled primers, about 0.4 µg of the sense N-cadherin was 5'-end labeled with [{gamma}-32P]-ATP (specific activity, 6000 Ci/mmol, Amersham, Arlington Heights, IL) using T4 polynucleotide kinase. About 1 x 106 cpm was used per PCR reaction tube. This radiolabeled primer, together with 0.3 µg of the sense and 0.4 µg of the antisense primers, was used for amplification as described above. In experiments where ß-actin was coamplified with N-cadherin, the radiolabeled sense ß-actin (~1 x 106 cpm) oligo was used in conjunction with 16 ng of the sense and 18 ng of the antisense primers. Initial denaturation was performed at 94 C for 5 min. Thereafter, the following cycling parameters were performed in a Perkin-Elmer/Applied Biosystems (Foster City, CA) DNA Thermal Cycler: denaturation at 94 C for 1 min, annealing at 62 C for 2 min, and extension at 72 C for 3 min. A total of 18–20 cycles were performed, followed by a 15-min extension at 72 C. Under these conditions, the production of all PCR products was in the linear range. An aliquot of 10 µl from each amplified sample tube was resolved onto 5% T polyacrylamide gels in 0.5 x Tris-borate-EDTA buffer, and the PCR products were visualized by ethidium bromide staining. Gels were then dried and exposed to Kodak X-OMAT AR film (Eastman Kodak, Rochester, NY). Autoradiograms from at least three separated experiments were densitometrically scanned at 600 nm using a Pharmacia (Piscataway, NJ) UltraScan XL Laser Densitometer (LKB 2222–020), normalized against ß-actin, and used for statistical analysis by Student’s t test or ANOVA.

RNA extraction and Southern blot
Total RNA was isolated from primary cultures of Sertoli cells, germ cells, or animal tissues by RNA STAT-60 as previously described (39). To determine the cellular source of N-cadherin in the testis, N-cadherin mRNA in Sertoli and germ cells was examined by RT-PCR and Southern blot. After electrophoresis of the PCR products, gel was denatured under alkaline condition, neutralized, and electroblotted onto a Nytran membrane in 1 x TAE buffer (40 mM Tris, 2 mM EDTA, pH 8.5, with acetic acid at 22 C). cDNAs were cross-linked onto the membrane under UV, prehybridized for 4 h at 42 C in 6 x SSPE buffer (0.9 M NaCl, 60 mM sodium phosphate, 6 mM EDTA, pH 7.4, with NaOH at 22 C)/5 x Denhardt’s buffer [0.2% Ficoll (type 70, Mr 70,000), 0.2% polyvinylpyrrolidone, and 0.2% BSA]/0.5% deionized formamide. Thereafter, the blot was hybridized overnight at 42 C in 6 x SSPE/1% SDS containing a {gamma}-32P-labeled N-cadherin oligonucleotide (antisense, 5'-TGTCGGCCAGCTTCTTGAAG-3' corresponding to nucleotides 2679–2698). The blot was then washed once with 6 x SSPE/1%SDS for 15 min at room temperature and twice for 30 min at 58 C with 1 x SSPE/1% SDS and exposed to x-ray film.

Gene cloning and sequencing of rat N-cadherin
The cDNA cloning and sequencing strategy of the full-length rat N-cadherin was essentially as previously described (29, 40, 41). Briefly, three overlapping fragments designated fragment 1 (corresponding to nucleotide 1–1171), fragment 2 (corresponding to nucleotide 1092–2125), and fragment 3 (corresponding to nucleotide 2043–3000) were synthesized by PCR from a rat Sertoli cell cDNA expression library (29). The specific primers were designated according to the known mouse N-cadherin full-length cDNA (18) and as follows: for fragment 1, 5'-ATGTGCCGGATAGCGGGAGG-3' (sense primer corresponding to nucleotides 1–20) and 5'-CTTCTCCGTAGAAAGTCATG-3' (antisense primer corresponding to nucleotides 1152–1171), which yielded a PCR product of 1171 bp; for fragment 2, 5'-CACAGCCACAGCCGTCAT-3' (sense primer corresponding to nucleotides 1092–1109) and 5'-AGTCTCCATTGGAGTCAC-3' (antisense primer corresponding to nucleotides (2108–2125), which yielded a PCR product of 1034 bp; for fragment 3, 5'-TATCACAGATTCAGGGAATC-3' (sense primer corresponding to nucleotides 2043–2062) and 5'-CAGAAAACTAATTCCAATCTGAAA-3' (antisense primer corresponding to nucleotides (2977–3000), which yielded a PCR product of 958 bp. The cDNAs with the expected size were gel purified, subcloned into pGEM-T vector (Promega), and sequenced by dideoxynucleotide chain termination method using Sequenase (Amersham, Arlington Heights, IL).

Statistical analysis
To assess whether the effect of germ cells, GCCM, or fractions of GCCM after preparative anion-exchange HPLC on Sertoli cell N- cadherin steady-state mRNA level is statistically significant, as shown in Figs. 3DGo, 4Go, D and F, and 5B, statistical analyses were performed by ANOVA with Tukey’s HSD (honestly significant difference) test using the GB-STAT Statistical Analysis Software Package (V 3.0) from Dynamic Microsystems, Inc. (Silver Spring, MD). Using Tukey’s test for ANOVA, results of individual samples were compared with controls and samples subjected to the same treatment within the same group as well as between groups; thus, this test makes use of a single value against which all differences are compared. This comparison is essentially important for data presented in Fig. 3DGo, since this excludes the possibility that the changes of Sertoli cell N-cadherin expression induced by GCCM (SC+GCCM) or GC (SC+GC) are not due to a time-dependent change in N-cadherin expression in Sertoli cells alone (SC).



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  		Figure 3. Changes of the N-cadherin steady-state mRNA level in Sertoli cells, Sertoli-germ cell cocultures, and in Sertoli cells cultured with GCCM. Primary cultures of Sertoli cells (A), Sertoli-germ cell cocultures (B), and Sertoli cells cultured with GCCM (C) were terminated at specified time points (0-, 2-, 4- and 24-h) for RNA extraction. 0 h indicated that RNA STAT-60 was added to Sertoli cells before the addition of germ cells and was used to indicate the basal N-cadherin steady-state mRNA level. RT-PCR was used to detect the changes of N-cadherin expression in these samples, which were coamplified with ß-actin as described in Materials and Methods. D, Densitometric scanning of autoradiograms such as those shown in panels A–C that were normalized against ß-actin. Each bar represents the mean ±SD of two experiments. Each time point has replicate cultures in each experiment. ns, Not significantly different by ANOVA. **, Significantly different by ANOVA, P < 0.01.

 


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  		Figure 4. Partial purification of authentic germ cell factor(s) that modulate Sertoli cell N-cadherin expression. A, Anion-exchange HPLC fractionation of Sertoli cell N-cadherin stimulator(s) from 1 liter of GCCM. A total of 14 protein peaks were noted. B, An aliquot of 50 µl from selected fractions as shown in panel A was resolved by SDS-PAGE onto 12.5% T SDS-polyacrylamide gels, and proteins were visualized by silver staining. C, An aliquot of 0.8 µg protein from pools of Mono Q fractions as shown in panel A was screened for its effect on Sertoli cell N-cadherin expression, and pool 4 (fractions 21–22) was shown to possess the biological activity when compared with controls where Sertoli cells were cultured in the absence of GCCM. D, When the expression of Sertoli cell N-cadherin for each treatment group was normalized against ß-actin from two separate experiments using pools of fractions from two different batches of GCCM after HPLC separation, as described in Materials and Methods, only pool 4 was found to enhance Sertoli cell N-cadherin expression significantly. E, Fractions 21–22 from pool 4 were cultured with Sertoli cells at 0.4–32 µg protein/4.5 x 106 Sertoli cells/9 ml in 100-mm dishes. The pooled sample exhibited a biphasic stimulatory activity on Sertoli cell N-cadherin expression. F, Densitometric scanning of autoradiograms such as the one shown in panel E but normalized against ß-actin from two separate experiments. Each data point is mean ±SD of two experiments. Each experiment had replicate cultures for each treatment group. ns, Not significantly different from control; *, significantly different by ANOVA, P < 0.05; **, significantly different by ANOVA, P < 0.01.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
References
 
N-Cadherin is expressed in Sertoli and germ cells
RT-PCR was used to assess whether N-cadherin is expressed in both Sertoli and germ cells. A 454-bp PCR product corresponding to the expected size of N-cadherin mRNA was found in the testis, brain, Sertoli, and germ cells. To verify that germ cells indeed express N-cadherin mRNA, these PCR products were electroblotted onto a Nytran membrane and visualized using a {gamma}-32P-labeled N-cadherin oligonucleotide internal to the 454-bp PCR product by Southern blot. Figure 1Go indicates that the testis, brain, and Sertoli cells yielded a 454-bp radioactive band together with three different batches of germ cells. This illustrates that both Sertoli and germ cells express N-cadherin in the seminiferous epithelium. The authenticity of the RT-PCR products from Sertoli and germ cells was verified by direct nucleotide sequencing of this 454-bp product.



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  		Figure 1. Cellular origin of N-cadherin mRNA in the rat testis. Southern blot analysis of the PCR products derived from testis (lane T), brain (lane B), Sertoli cells (lane SC), and three batches of germ cells (lane GC). About 1 µg total RNA from T (lane 1), B (lane 2), SC (lane 3), and GC (lanes 5, 6) and 3 µg RNA from GC (lane 4) were used for RT. About one sixth of the RT product from each sample was served as template for PCR as described in Materials and Methods.

 
Cloning, nucleotide sequence, and primary structure analysis of rat testicular N-cadherin
The strategy for cloning and sequencing of the full-length rat N-cadherin cDNA clone and its partial restriction map are shown in Fig. 2AGo. The nucleotide sequence of the N-cadherin cDNA and its deduced amino acid sequence are shown in Fig. 2BGo. This 3-kb N-cadherin cDNA has a 36-bp 5'-untranslated region and a 283-bp 3'-untranslated region at the 3'-end (Fig. 2BGo). The open reading frame coded for a 883-amino acid polypeptide with a 23-amino acid signal peptide. The mature protein of rat testicular N-cadherin has a calculated Mr of 96,307. At the nucleotide sequence level, rat N-cadherin displayed a 94% identity with the mouse N-cadherin. At the amino acid level, rat N-cadherin displayed a 98.6% homology with the mouse homolog (Fig. 2CGo).



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  		Figure 2. Nucleotide sequence analysis of rat testicular N-cadherin full-length cDNA and comparison of its deduced amino acid sequence with mouse N-cadherin. A, Sequencing strategy of the full-length rat N-cadherin cDNA. Closed box indicates predicted open reading frame of the clone and orientation of the PCR product. Selected restriction sites are shown above the diagram. Arrows represent the strand and the extent of DNA sequenced. Three overlapping fragments of rat N-cadherin were synthesized by PCR as described in Materials and Methods. B, The nucleotide sequence and the predicted amino acid sequence of rat testicular N-cadherin. The sequence was derived from three overlapping cDNA clones. The upper right-hand numbers designate the nucleotide positions. Negative values indicate nucleotide sequence of the 5'-end noncoding region. The lower left-hand numbers show the deduced amino acid positions of the protein. The signal peptide begins with the ATG codon for Met and consists of 23 amino acids. The arrowhead indicates the predicted cleavage site. Nucleotides 2,719–3,000 represent the 3'-end untranslated region. C, Comparison of the deduced amino acid sequence of the rat testicular N-cadherin and mouse N-cadherin. Rat and mouse N-cadherin display 98.6% identity at the amino acid level. Shaded boxes represent amino acids that are different between the two species. Arrowhead indicates the cleavage site that generates the mature protein.

 
Expression of N-cadherin in Sertoli cells is regulated by germ cells
Since there is a significant increase in the germ cell to Sertoli cell ratio during testicular development, we sought to examine whether the increase in N-cadherin expression during testicular development, as shown in a previous study (14), reflected a positive modulation of Sertoli cell N- cadherin expression by germ cells. Sertoli cells at 4.5 x 106 cells per 100-mm dish/9 ml were cultured with an equal number of immature germ cells (mainly spermatogonia, and spermatocytes), or crude GCCM (~90 µg protein/dish) for different time points and compared with Sertoli cells cultured alone. It was found that the steady-state N-cadherin mRNA level in Sertoli cells at 2–24 h was similar to time 0 (Fig. 3AGo). However, there was a time-dependent increase in N-cadherin expression in the Sertoli-germ cell cocultures (Fig. 3BGo). Interestingly, GCCM appeared to stimulate Sertoli cell N-cadherin expression (Fig. 3CGo). The results presented in Fig. 3Go, A–C, were then densitometrically scanned and normalized against ß-actin from two separate experiments (Fig. 3DGo), which illustrates that the increase in the testicular N-cadherin steady-state mRNA level during maturation may be the result of an up-regulation by germ cells through secretory biological factor(s).

Fractionation of biological factor(s) in GCCM that affects Sertoli cell N-cadherin expression
To further characterize factor(s) in GCCM that modulated Sertoli cell N-cadherin expression, as shown in Fig. 3Go, we fractionated GCCM by HPLC for bioassays. Using about 1–5 liters of GCCM containing 20–100 mg proteins for preparative anion-exchange HPLC, a total of 14 protein peaks were noted when the eluent was monitored by UV absorbance at 280 nm (Fig. 4AGo). Aliquots of 50 µl from selected fractions were resolved by SDS-PAGE under reducing conditions, and the proteins in the SDS-polyacrylamide gels were visualized by silver staining (Fig. 4BGo). Different pools (1 through 14) of fractions were prepared. When aliquots of 8 µg protein/dish were used to culture with Sertoli cells, a prominent N- cadherin expression-stimulatory activity was found in pool 4 (fraction 21–22) when analyzed by RT-PCR (Fig. 4CGo). When the expression of N-cadherin was normalized against ß-actin from two separate experiments, pool 4 was shown to stimulate Sertoli cell N-cadherin expression (Fig. 4DGo). These results illustrate the presence of a putative Sertoli cell-modulatory factor(s) in GCCM that can affect N-cadherin expression. We next examined whether pool 4 shown in Fig. 4CGo can yield a dose-dependent stimulatory effect on Sertoli cell N-cadherin steady-state mRNA level. When different concentrations of GCCM proteins in pool 4 from 0.4–32 µg protein/dish were incubated with 4.5 x 106 Sertoli cells/9 ml/100-mm dish for 18 h, a biphasic effect on Sertoli cell N-cadherin expression was noted (Fig. 4Go, E and F). This pool induced a dose-dependent stimulation with a maximal stimulation at 2 µg protein/dish (Fig. 4Go, E and F). Thereafter, increasing concentrations of pool 4 relinquished its effect on Sertoli cell N-cadherin expression (Fig. 4Go, E and F).

Biphasic effect of the biological factor(s) in GCCM that affects Sertoli cell N-cadherin expression
To further confirm the biphasic effect of pool 4 on Sertoli cell N-cadherin steady-state mRNA level as shown in Fig. 4Go, E and F, increasing concentrations of crude GCCM at 0.4–50 µg protein/dish were incubated with 4.5 x 106 Sertoli cells/9 ml/100-mm dish for 18 h, and a biphasic effect on Sertoli cell N-cadherin expression was also noted. This pool induced a dose-dependent stimulation with a maximal stimulation at 8 µg protein/dish (Fig. 5AGo). Thereafter, increasing concentrations of crude GCCM proteins relinquished their effect on Sertoli cell N-cadherin expression (Fig. 5Go, A and B). To assess whether Sertoli-germ cell contact can also modulate Sertoli cell N-cadherin expression, nonviable germ cells were cocultured with Sertoli cells at a Sertoli-germ cell ratio of 1:5 for 2, 4, and 24 h and compared with cocultures at 0 h. Nonviable germ cells were obtained by freezing germ cells isolated from adult rat testes, which consisted of largely spermatocytes, round spermatids, and some spermatogonia (32) at -20 C for 24 h, and thawing at room temperature to obtain a cell population with a viability of less than 2% when monitored by erythrosine red dye exclusion test (32). These nonviable germ cells were washed for three times in F12/DMEM by centrifugation (15,000 x g, 5 min each) to remove cytosolic proteins before their use. It was found that nonviable germ cells cocultured with Sertoli cells failed to stimulate Sertoli cell N-cadherin expression (data not shown), illustrating that the effects of GCCM on Sertoli cell N-cadherin expression shown in Figs. 3–5GoGoGo are likely mediated by a soluble factor(s) released by germ cells rather than by cell-cell contacts.



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  		Figure 5. Biphasic effect of the biological factor(s) in GCCM that affects Sertoli cell N-cadherin expression. A, Increasing concentrations of crude GCCM (0.4–50 µg protein/dish) incubated with Sertoli cells yielding a biphasic effect on Sertoli cell N-cadherin expression. B, Densitometric scanning of autoradiograms such as the one shown in panel A but normalized against ß-actin. Each bar represents the mean ±SD of two experiments. Each experiment had replicate cultures for each treatment group. ns, Not significantly different by ANOVA; *, significantly different by ANOVA, P < 0.05.

 
Discussion
The traverse of developing germ cells from the basal to the adluminal compartment in the seminiferous epithelium throughout spermatogenesis is known to involve cell adhesion molecules via cycles of disruption and regeneration of intercellular junctions (for reviews, see Refs. 1–6). Cadherins, a group of cell adhesion molecules previously shown to be present in the rat testis, are found to be involved in cell-cell interactions in the testis (6, 12, 13, 14, 23). Other studies have demonstrated the cadherin-catenin complex may be involved in signal transduction since ß-catenin, a cytoplasmic protein tightly associated with the intracellular domain of cadherin, is a component of a signal transduction pathway that can be activated by an extracellular ligand, the wingless/Wnt (42, 43, 44, 45, 46). Activation of this pathway induces translocation of ß-catenin to the nucleus, suggesting ß-catenin may be involved in regulating gene expression (27, 47). ß-Catenin has also been shown to bind LEF-1, a DNA-binding transcription factor, and affects LEF-1-induced DNA bending (48, 49, 50). In addition, ß-catenin also binds to the product of the adenomatous polyposis coli (APC) tumor suppressor gene (51, 52). Interestingly, APC protein is found in punctate clusters near the ends of microtubules that protrude into actively migrating membrane structures (53), suggesting a role for APC protein in cell migration. On the other hand, ß-catenin may play a role in regulating cell adhesion because it is a substrate for several tyrosine kinases that suppress cell adhesion (54, 55). ß-Catenin also forms complexes with epidermal growth factor receptor (56). Thus, it is likely that the action of N-cadherin/catenin can be influenced by extrinsic signals arising from the classic growth factor pathways. Furthermore, ß-catenin signaling can be regulated by cadherins as high levels of cadherin expression inhibit ß-catenin- signaling activity (28). The level of cadherin expression can also set a threshold over which ß-catenin/armadillo must accumulate to transduce the signal. Alternatively, cadherins can play a more active role in signaling, similar to the putative Wnt receptor, by controlling the release of ß-catenin/armadillo protein into the cytoplasm (21). Since the ß-catenin-APC protein interactions are critical for regulating cell migration during epithelial tubulogenesis (57), if germ cells can indeed affect ß-catenin function via N-cadherin through a secretory factor(s), this may suggest the presence of a potential regulatory mechanism by which germ cells facilitate their migration in the epithelium through the cadherin-ß-catenin cytoplasmic signaling machinery. Even though immunohistochemistry study revealed the presence of ß-catenin at the site of Sertoli-germ cell junctions, it could not be consistently localized and demonstrated in the adult testis (58). As such, these results seemingly suggest that testicular N-cadherin may anchor to cells via a non-catenin protein and exerts its action independent of catenin. However, it is also likely that the anti-catenin antibody used in this early study (58) only recognized some but not all epitopes of catenin. Needless to say, these results illustrate that N-cadherin, which is expressed by both Sertoli and germ cells, may somehow modulate cellular migration in the epithelium.

Recent studies have suggested a role of N-cadherin in cohesion of tumor cells which, in addition to promoting their interaction with the surrounding stromal cells, facilitates cell invasion and metastasis (24, 25). A study to define the developmental pattern of testicular N-cadherin mRNA has demonstrated an elevated expression of N-cadherin at 42 days of age, which coincides with the release of the first wave of testicular spermatozoa into the tubular lumen (14, 59), suggesting N-cadherin may be involved in spermiation. These data, combined with the demonstration that Sertoli cell N-cadherin expression can be regulated by germ cells via a soluble factor(s), illustrate germ cells are likely to participate in the events leading to their migration.

It was previously shown that the primary amino acid sequence of N-cadherin in residues between 1 and 272 is highly conserved between the chicken and the mouse (16, 17, 18). This stretch of sequence, the known cell binding site of which may be necessary for interactions between two cadherin molecules (16, 17, 18), is also present in the rat N-cadherin. Moreover, the entire rat and mouse N-cadherin primary sequences (18) share greater than 98% identity, suggesting that N-cadherin is a highly conserved molecule.

Studies by immunohistochemistry have shown that N-cadherin is restricted to Sertoli cells and mid-to-late spermatogenic cells and not detected in Leydig or peritubular myoid cells in the interstitium (6). In addition, mRNA for both E- and P-cadherin was not detected in Sertoli cells (14), further emphasizing the importance of N-cadherin in cell-cell interactions in the testis. Our results are consistent with two earlier reports that demonstrated the expression of N-cadherin by Sertoli cells, pachytene spermatocytes, and round spermatids (6, 60). The role of N-cadherin in germ cells is not immediately understood. During testicular maturation, the expression of N-cadherin was found to be spatiotemporally regulated (11). Moreover, the binding of round spermatids to Sertoli cells and the Sertoli-spermatogenic cell adhesion were mediated by N-cadherin (23, 58, 61), since anti-N-cadherin antibody inhibited the binding of round spermatids onto Sertoli cells, suggesting the active involvement of N-cadherin in Sertoli-germ cell adhesion. As such, a factor(s) that can regulate N-cadherin expression will likely be an important molecule to understand Sertoli-germ cell interactions. Even though this factor has not been isolated and fully characterized, this report illustrates unequivocally the presence of such a factor in GCCM.

Other studies have shown that the production of N- cadherin by Sertoli cells was stimulated in a concentration-dependent manner by testosterone in the presence of FSH (61). FSH alone can also induce an increase in Sertoli cell N-cadherin mRNA level in vitro, which can be further enhanced by estradiol (62). The actions of FSH are mediated by specific receptors that are functionally coupled via membrane-associated G proteins to the adenyl cyclase cAMP-generating pathway (63, 64). Studies using cAMP analogs or cAMP-inducing agents were shown to increase Sertoli cell N-cadherin expression, which can be further enhanced by estradiol (62). However, steroids such as estradiol, testosterone, and dihydrotestosterone alone failed to stimulate Sertoli cell N-cadherin mRNA levels in vitro (62). In contrast, these steroids are potent regulators of testicular N-cadherin mRNA levels in vivo (65). For example, estradiol, but not testosterone, dihydrotestosterone, or progesterone, is capable of regulating N-cadherin mRNA levels in the immature mouse testis (65). The germ cell released factor(s) that can affect Sertoli cell N-cadherin expression, as shown in this study, is not likely a steroidal factor(s) since most of the steroids should have been removed in our initial ultrafiltration step during sample preparation. The identity of this factor must await its isolation from GCCM.

In summary, these results illustrate that germ cells may play a more active role than originally anticipated in controlling their migration and possibly differentiation during spermatogenesis via a coupling mechanism between physical adhesion and developmental signaling. The identification and evaluation of these biological factor(s) in GCCM will definitely yield new insights in understanding testicular physiology.


   Footnotes
 
1 Supported in part by grants from the Noopolis Foundation, the Rockefeller Foundation (PS-9528, PS-9601), CONRAD Program (CIG-96–05), NIH (HD-13541), and Hong Kong Research Grant Council (HKU 7235/97M). This work was performed as part of a dissertation submitted to the Hong Kong University Higher Degree Committee by S.S.W.C. for the partial fulfillment for the requirements of the Degree of Doctor of Philosophy. Back

Received September 22, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 References
 

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