Cell Tissue Res (1990) 261 : 54%554

andT'L ue Resemr.h 9 Springer-Verlag 1990

Evidence for the presence of nerve growth factor (NGF) and NGF receptors in human testis Klaus SeidP and Adolf-Friedrich Holstein 2 1 Institute for Hormone and Fertility Research (IHF), Hamburg; z Department of Microscopical Anatomy, University of Hamburg, Hamburg, Federal Republic of Germany Accepted May 31, 1990

Summary. Nerve growth factor ( N G F ) affects morphology and function of isolated and cultured seminiferous tubules from h u m a n testis. Quantitative determination of this neurotrophic protein revealed an a m o u n t of 5.4 ng per g h u m a n testis, suggesting a crucial function of N G F in spermatogenic tissue. With the use of immunohistochemical methods target cells for N G F were identified within the lamina propria. N G F receptors were also visualized on sympathetic nerve fibers crossing the interstitial c o m p a r t m e n t a m o n g adjacent tubules and spatially correlated blood vessels. N G F receptors could be demonstrated on isolated lamina-propria cells even after 2 weeks of culture. Most of the N G F receptor-bearing cells differed from myoid cells of the lamina propria expressing desmin, a m a r k e r for smooth muscle. However, some N G F receptor-expressing cells were found sharing morphological and structural similarities with myoid cells. The present data indicate the existence of a N G F - r e s p o n s i v e lamina-propria cell that influences the tubular wall and also the seminiferous epithelium. Key words: Nerve growth factor receptor Testis Cell culture - Cell plasticity L a m i n a - p r o p r i a cells H u m a n

The neurotrophic protein nerve growth factor ( N G F ) m a y be considered as a prototype for macromolecules essential for the development and the maintenance of growth and functional activities of neurons. All these effects are initiated by binding of N G F to its receptors. N G F receptors were long regarded as specific markers of sympathetic and sensory neurons. However, they were also found to be expressed on non-neuronal cells of dorsal root ganglia in chick embryos ( Z i m m e r m a n n and Send offprint requests to: Dr. Klaus Seidl, Institute for Hormone and Fertility Research, Grandweg 64, D-2000 Hamburg 54, Federal Republic of Germany

Sutter 1983). Recently, N G F protein and N G F m R N A were detected in germ cells of mouse and rat testes (O1son et al. 1987; Ayer-LeLievre et al. 1988). Furthermore, R N A blot analysis revealed the presence of m R N A encoding the N G F receptor in mouse testis (Ayer-LeLievre et al. 1988). These results suggest a role for N G F in the testis. In isolated and cultured tubules of h u m a n testis N G F affects m o r p h o l o g y and function of Sertoli cells and lamina-propria cells (Seidl and Holstein 1990). These observations justify the search for the N G F protein and the N G F target cells in h u m a n testis.

Materials and methods Human testicular material was obtained during surgery from patients undergoing orchiectomy for prostatic carcinoma. For immunohistochemistry, tissues were either frozen in liquid nitrogen or fixed in Bouin's fixative; for cell culture studies, testes were stored on ice until further treatment.

Chemicals, reagents, and additioes

Dulbecco's modified Eagle's medium (DME) with Hank' salts, glutamine and fetal calf serum (FCS) were purchased from Flow Laboratories (Bonn, FRG); gentamycin from Merck (Darmstadt, FRG), and collagenase (type II) from Worthington (Flow Laboratories; Bonn, FRG). Gelatin, bovine serum albumin (BSA), glycine, chlorophenol red, and DNase were obtained from Sigma (Munich, FRG); extracellular matrix (ECM) from Collaborative Research (Atlanta; Heidelberg, FRG), and phenylmethylsulfonyl fluoride, aprotinin, Triton X-100, chlorophenol red-]?-D-galactopyranoside, monoclonal anti-NGF antibody (clone 27/21) and anti-mouse-/%NGF, conjugated with /~-galactosidase from Boehringer Mannheim (Mannheimn, FRG). All further immunologic reagents including monoclonal antibodies against NGF receptors and against desmin were purchased from Amersham Buchler (Braunschweig, FRG); the 96-well culture plates were from Falcon (Becton-Dickinson; Heidelberg, FRG), and the microplates for the enzyme immunoassay from Costar (Tecnomara; Fernwald, FRG). All chemicals were of analytical grade.

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Quantitative determination of NGF Quantitative determination of NGF was performed using a two-site enzyme immunoassay (EIA) (Korsching and Thoenen 1983) with minor modifications. The tissues were homogenized (glass/glass, 20 strokes) in 100 mM TRIS-HC1 (pH 7.0) containing 0.4 M NaC1, 2% gelatin, 2% BSA, 4 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, aprotinin (40 kallikrein-inhibitor units/ml) and 0.1% NAN3. After centrifugation of the homogenates (105 g, 15 min, 20~ C) the supernatants were diluted 1:2 with 0.2% Triton X-100, and the samples then used for the EIA. The assays were performed in microplates (Fast binder), coated with monoclonal anti-NGF antibody using 100 gl coating buffer (50 mM Na2CO3/NaHCO3 pH 9.6, 1 gg/ml anti-NGF antibody and 0.1% NAN3). After 2 h of incubation at 37~ C in a waterbath with constant shaking at 50 rpm, the wells were washed 3 times with 200 gl washing buffer (50 mM TRIS-HC1 pH 7.0, 0.2 M NaC1, 1% gelatin, 0.1% Triton X-100 and 0.1% NAN3). 100 gl of sample or mouse NGF standard solution (range: 0.5~00 pg/assay) in EIA buffer (washing buffer + 1% BSA) were incubated overnight (16-20 h) at 25~ C, with constant shaking at 50 rpm. Subsequently, the wells were rinsed 3 times with washing buffer, and 100 gl EIA buffer containing antimouse-/~-NGF, conjugated with/~-galactosidase (0.2 units/ml) and 10% normal rat serum, was added to each well. After 2 h of incubation at 37~ C and 50 rpm the wells were rinsed again twice with washing buffer and once with substrate buffer (50 mM sodium phosphate buffer containing 1 mM MgC12, pH 7.3). The enzyme reaction was started with 100 gl chlorophenol red-/?-D-galactopyranoside (3.3 mM) in substrate buffer and terminated after 6-10 h at 37~ and 50 rpm by the addition of 100 gl 0.15 M glycineNaOH (pH 10.3). Chlorophenol red, the end product of the enzyme reaction, was measured against the substrate solution at 574 nm. The detection limit of this assay was 0.25 pg/assay (0.01 fmoles/ assay). Nonspecific binding, determined for each sample by coating the wells with mouse IgG fractions in the same way as with the specific antibody, was subtracted from total binding. Values were also corrected by adding NGF to the non-centrifuged tissue homogenate as an internal standard. All samples were measured in quadruplicate.

Immunohistochemistry For frozen sections testicular tissue was placed in liquid nitrogen immediately after orchiectomy; for paraffin sections the tissues were fixed in Bouin's fixative. Tissue sections were exposed to ME20.4 (10 gg/ml), a monoclonal anti-NGF receptor antibody (IgG). After overnight binding at 4~ C, the sections were washed and incubated for 1 h at room temperature with biotinylated goat anti-mouse IgG (1:200). Subsequently, the sections were washed again and treated with a complex of streptavidin and biotinylated horseradish peroxidase (1 : 100). After several washes, 3,3'-diaminobenzidine (0.05%) and H202 (0.03%) in 0.05 M TRIS-HC1 (pH 7.6) were applied for 10 min. The sections were then rinsed in water, dehydrated and mounted. If not otherwise indicated, 0.01 M phosphate-buffered saline (PBS) (pH 7.3) containing 1% BSA and 1% normal goat serum was used during the entire procedure.

Cell culture and immunocytochemistry Human testicular tissue was decapsulated and cut into pieces of 1 cm 3. Interstitial tissue was removed by digestion in DME containing 0.1% collagenase (37~ C, 1 h). The resulting fragments of isolated tubules were washed 3 times with DME. Aggregates of a mixed population of Sertoli cells, peritubular cells and germ cells were obtained by sequential 0.1% collagenase digestions in DME containing 10 gg/ml DNase. After each of 3 4 periods of digestion (37~ C, 15 min) the tubular fragments were disrupted by repetitive pipetting (10 times); the aggregates were allowed to settle and the

supernatant was discarded. After the last digestion the aggregates (10-200 cells per aggregate) were washed twice in DME. Microwell plates (96 wells, 6 mm diameter) were precoated for 1 h with 50 gl extracellular matrix (ECM, diluted 1 : 10 with DME) at 4~ C and washed twice with sterile water. The pretreated culture dishes were washed with DME and received 50 gl culture medium (DME supplemented with 2 mM glutamine, 44.4 mM NaHCO3, gentamycin (20 gg/ml) and 10% (v/v) heat-inactivated FCS) and 50 gl of cell suspension (200 aggregates). Medium was changed after 12 h and every 4th day of incubation at 34.5~ C (5% CO2/95% air). After 3, 7 and 14 days, respectively, the cells were washed twice with 0.1 M PBS and fixed in methanol containing 5% acetic acid at - 2 0 ~ C for 10 min. Following rehydration in 0.1% PBS immunocytochemistry was performed with monoclonal anti-desmin antibodies or monoclonal ME20.4-IgG as described above.

Results

Presence of NGF protein in human testis U s i n g a highly specific a n d sensitive (detection limit: 0.01 fmoles/assay) two-site e n z y m e i m m u n o a s s a y for q u a n t i t a t i v e d e t e r m i n a t i o n o f / ? - N G F , this n e u r o t r o p h i c p r o t e i n was detected in h u m a n testis (Table 1). T h e testes c o n t a i n e d 5.44 n g / % N G F per g wet weight, whereas n o specific N G F was detected in h u m a n b l o o d , i m p l y i n g t h a t all m e a s u r e d N G F was o f testicular origin.

Immunohistochemical identification of the NGF receptor in human testis I m m u n o h i s t o c h e m i c a l studies o n h u m a n testicular tissue revealed specific b i n d i n g o f the m o n o c l o n a l a n t i - N G F receptor a n t i b o d y (ME20.4) to cells o f the l a m i n a propria (Fig. 1 a - c , 2a). I n frozen sections positive s t a i n i n g was seen t h r o u g h o u t the l a m i n a p r o p r i a b u t was m o r e i n t e n s e in its o u t e r layers (Fig. 1 a, b). I n p a r a f f i n sections f r o m testicular m a t e r i a l treated with B o u i n ' s fixative, o n l y a few seminiferous t u b u l e s showed positive s t a i n i n g in the o u t e r layers o f the l a m i n a p r o p r i a (Fig. 1 c). N G F receptors were also visualized a r o u n d b l o o d vessels (Fig. 1 a) a n d o n s y m p a t h e t i c nerve b u n dles crossing the interstitial space (Fig. 2b). N o s t a i n i n g was observed w i t h i n the t u b u l a r cords.

Plasticity of cultured lamina-propria cells T h e ability o f h u m a n l a m i n a - p r o p r i a cells to express N G F - r e c e p t o r p r o t e i n s was r e t a i n e d after the i s o l a t i o n o f these cells f r o m their n o r m a l in-vivo e n v i r o n m e n t a n d c u l t u r i n g for 7 or 14 days (Fig. 3 ~ g ) . T h e cells s h o w i n g Table 1. NGF content in human testes

Testis weight (g/pair of organs) NGF (ng/pair of organs) NGF (ng/g of tissue)

38.5 284.9 5.44 • 0.68

Numbers in parentheses represent numbers of patients tested. a Values are expressed as mean _+SEM

(1) (1) (5)a

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Fig. 1a-e. Immunohistochemical evidence for the presence of NGF receptors in the human testis (frozen sections : a, b paraffin section: e) : a Distribution of immunoreactive material around the seminiferous epithelium and a blood vessel (arrow); note a more intense staining of the outer layers of the tubular wall. b Immunoreaction for NGF receptors within the lamina propria (counterstained with

hemalum), phase-contrast micrograph, e In paraffin sections from testicular material treated with Bouin's fixative, NGF receptors could be visualized only in the outer layer of the lamina propria. Without the primary antibody, no peroxidase staining was obtained, x 280

Fig. 2a, b. NGF receptor immunoreaction a on a lamina-propria cell between two adjacent tubules; b on sympathetic nerve bundles crossing the interstitial space, x 550

positive staining with ME20.4 differed in their shape and m o r p h o l o g y from cells of the lamina propria expressing desmin, a marker for smooth muscle (Fig. 3 a c). After 3 days of culture most of the myoid cells had long processes, elongated nuclei and were heavily stained with anti-desmin antibodies. Seven days after seeding, the desmin-containing myoid cells displayed different phenotypes. One subpopulation consisted of thin cellular elements exhibiting elongated nuclei and long, thin and compact processes, some of which contained growth

cone-like structures (Fig. 3 b). Another type of desminexpressing lamina-propria cell was elongated and broad in shape and contained a round or ovoid nucleus with a centrally located nucleolus (Fig. 3 c). This latter type was still observed after 14 days of culture, whereas the process-bearing cell (Fig. 3 a, b) occurred only rarely at this time. The N G F receptor-bearing Cells exhibited multiple processes, round nuclei and centrally located nucleoli after a culture period of 7 days (Fig. 3 d). After a further week these ceils had grown in size and gradually

552

Fig. 3a-g. a Isolated human lamina-propria cells stained with antibodies against desmin after 3 days of culture. Myoid cells growing out from cell aggregates, b, c Desmin-containing cells after 7 days of culture. Note the plasticity of cultured lamina-propria cells expressing different phenotypes. One type of cell was thin and elongated in shape (b) and contained elongated nuclei and long processes with growth cone-like structures (arrow). Other desmin-expressing cells were elongated and broad, and displayed ovoid nuclei

(c). d-g Isolated lamina-propria cells stained with antibodies against NGF receptors after 7 (d) and 14 days of culture (e-g). The NGF receptor-bearing cells had numerous processes and round nuclei after 7 days of culture (d). During the subsequent week they grew in size and formed either a network (e) or aggregates around central Sertoli cells. (f). Some NGF receptor-containing cells exhibited long processes with growth cone-like structures (g). a-e, g x280; f x90

b e c a m e a r r a n g e d in a n e t w o r k (Fig. 3 e) or at the periphery o f Sertoli-cell aggregates (Fig. 3f). As shown in Fig. 3 e n o t all cells within this n e t w o r k expressed N G F receptors a l t h o u g h they a p p e a r e d to have similar m o r phological phenotypes. Some cells were f o u n d bearing long a n d c o m p a c t processes with g r o w t h cone-like structures (l~ig. 3 g). These processes were heavily stained with ME20.4.

Discussion Nerve g r o w t h factor is considered to be a t r o p h i c protein essential for the survival o f sympathetic and sensory neurons during o n t o g e n y (Levi-Montalcini and Angeletti 1968; T h o e n e n a n d Barde 1980). Recently, it was s h o w n that germ cells o f m o u s e and rat testes synthesize N G F (Olson et al. 1987; Ayer-LeLievre et al. 1988). Further-

553 more, we observed that this neurotrophic factor stabilizes morphology and function of Sertoli cells and cells of the lamina propria in cultured seminiferous tubules from human testes (Seidl and Holstein 1990). The data obtained in this study demonstrate the presence of NGF in human testes (5.44 ng/g testis). Interestingly, human and mouse testicular NGF levels are in the same range; 10 ng N G F per g of tissue was found in mouse testis (Ayer-LeLievre et al. 1988), and evidence from our own studies revealed no significant difference between human and mouse testicular levels (data not shown). These results suggest a basic functional principle for NGF in spermatogenic tissue of vertebrate species. The effect of N G F on cultured tubules led to the search for the NGF target cells in the testis. In the peripheral nervous system NGF is known to have multiple effects on neurons including axonal sprouting and directional neuritic growth toward an NGF gradient established by non-neuronal cells (Levi-Montalcini and Angeletti 1968; Varon and Adler 1981). The biological action of NGF is initiated by the binding of the dimeric protein to cell-surface receptors of the neuron (Banerjee et al. 1968; Herrup and Shooter 1973). The Sertoli cell is, like the neuron, a highly asymmetrical element with long and multiple processes, which partly or completely surround the developing germ cells (Fawcett 1975; Russell et al. 1983; Weber et al. 1983; Wong and Russell 1983; Aumfiller et al. 1988). Moreover, the cytoskeleton and microtubule patterns in neuronal and Sertoli cells have similar morphological, structural and functional properties (Fawcett 1975; Vogl et al. 1983a, b; Neely and Boekelheide 1988). Despite the similarities between Sertoli cells and neurons, we did not observe NGF receptors on Sertoli cells using immunohistochemical methods. NGF receptors were identified on cells of the lamina propria, around blood vessels and on sympathetic nerve bundles crossing the interstitial space between neighbouring tubules. N G F receptors were demonstrated in human testicular tissue by means of the monoclonal antibody ME20.4, which was prepared using WM245 melanoma cells as immunogen (Ross et al. 1984). This antibody is specific for the human N G F receptor. Positive staining has been observed in peripheral nerves, neurofibromas, pheochromocytomas, submandibular gland, melanomas, nevi and in the A875 melanoma cell line, all of human origin (Ross et al. 1984). ME20.4 shows no cross reactivity with NGF receptors of other vertebrate species, for example, those of rat pheochromocytoma (PC12) cells or chick embryonic sensory neurons (Ross et al. 1984). Accordingly, there was no reactivity with mouse testicular tissue or with isolated sympathetic and sensory neurons from mouse and embryonic chicken (data not shown). In frozen sections, N G F receptors were seen throughout the lamina propria but the expression of this protein was more intense in the outer layers. However, only the outer lamina-propria layers of a few seminiferous tubules were slightly stained in paraffin sections from testicular material fixed in Bouin's solution. This indicates a loss of antigen due to the fixation procedure and, therefore, only high levels of NGF receptors in the outer layers could be visualized with immunoperoxidase staining. Although in frozen sections the processes

of lamina-propria cells appeared to be stained with ME20.4 antibody we cannot exclude the possibility that at least some of the stained structures were of neuronal origin passing very close to the myoid cells. The existence of sympathetic nerve fibers invading areas of interstitial testicular tissue along the blood vessels has been documented by many authors (Kuntz 1919; Baumgarten and Holstein 1967, 1968, 1971). Since the density of the sympathetic innervation is closely correlated with the amount of NGF protein and NGF mRNA (Korsching and Thoenen 1983; Heumann et al. 1984), a correlation between the presence of sympathetic nerves and NGF in the testis might be suggested. However, as shown by Kunz (1919) and Baumgarten and Holstein (1967) and confirmed in the present study, no sympathetic fibers cross the lamina propria and invade the seminiferous epithelium. Nevertheless, an attractive (tropic) effect of intratubular NGF on extratubular nerve fibers cannot be excluded with certainty. It is important to note that sympathetic nerve fibers within the lamina propria must be postganglionic elements. It is therefore very unlikely that the NGF effects on cultured seminiferous tubules from human testis (Seidl and Holstein 1990) were mediated by disconnected sympathetic axons persisting in the tubular wall after isolation. In isolated and cultured cells we were able to visualize the NGF receptor even after 2 weeks of culture. These data suggest the existence of a target cell for NGF in the tubular wall. Interestingly, in our cell culture system most of the NGF-receptor-bearing cells clearly differed in size, shape and morphology from myoid cells of the lamina propria, which express desmin, a marker for smooth muscle. This diversity might suggest two different types of interacting cells within the lamina propria. However, some NGF-receptor expressing cells share similarities with myoid cells with regard to their shape and the structural properties of their processes. In organ cultures, the myoid phenotype of lamina-propria cells was retained only when NGF was added to the culture medium (Seidl and Holstein 1990). Therefore, we suggest that NGF is at least one of the signal molecules responsible for maintaining the myoid character of lamina-propria cells. Further studies will be focussed on the question of whether the expression of NGF receptors in cells of the lamina propria is related to the degree of differentiation of myoid cells. The maintenance of a well-structured epithelium in tubules from human testis exposed to NGF is based on the stabilization of the natural phenotype of the myoid cells of the lamina propria and the clearly polarized Sertoli cells. We were unable to detect NGF receptors on Sertoli cells. Consequently, the effects on Sertoli cells must be mediated by an NGF-dependent release of unknown agents from the NGF-target cells within the lamina propria. Several lines of evidence suggest a close structural and functional relationship between mesenchymal and epithelial cells. It was suggested that mesenchymal cells influence the function of adjacent epithelial cells by specific inducer substances (Grobstein 1967). Modulation of the epithelial-like Sertoli cells by specific proteins derived from the mesenchymaMike lamina-propria cells has been reported by many investigators (Tung and Fritz 1980; Hutson and Stocco 1981; Skinner and

554 F r i t z 1985, 1986). Sertoli cells a n d l a m i n a - p r o p r i a cells are s e p a r a t e d b y a b a s e m e n t m e m b r a n e , a c o m p l e x ext r a c e l l u l a r m a t r i x ( H a d l e y a n d D y m 1987), p r o d u c e d b y b o t h cell t y p e s in close c o o p e r a t i v i t y ( S k i n n e r et al. 1985). This e x t r a c e l l u l a r m a t r i x is k n o w n to i n d u c e a n i n - v i t r o p o l a r i z a t i o n o f Sertoli cells ( H a d l e y et al. 1985). Since N G F helps to m a i n t a i n the p h e n o t y p e o f l a m i n a p r o p r i a cells a n d the p o l a r i z e d s t r u c t u r e o f Sertoli cells in c u l t u r e d t u b u l e s (Seidl a n d H o l s t e i n 1990), it is t e m p t ing to s p e c u l a t e t h a t N G F m i g h t be r e s p o n s i b l e for the elaboration of an appropriate extracellular matrix by l a m i n a - p r o p r i a cells, a p r o c e s s t h a t c o n s e q u e n t l y affects Sertoli cell m o r p h o l o g y a n d f u n c t i o n . O u r o b s e r v a t i o n s s h o w t h a t (i) N G F is p r e s e n t in t e s t i c u l a r tissue, (ii) N G F h a s a p h y s i o l o g i c a l effect o n i s o l a t e d a n d c u l t u r e d s e m i n i f e r o u s t u b u l e s (Seidl a n d H o l s t e i n 1990), a n d (iii) N G F r e c e p t o r s a r e localized o n cells o f the l a m i n a p r o p r i a w h i c h are i n f l u e n c e d b y N G F d u r i n g the c u l t u r e p e r i o d (Seidl a n d H o l s t e i n 1990). These results f a v o r the a s s u m p t i o n t h a t N G F p l a y s a n i m p o r t a n t role in the m a i n t e n a n c e o f s t r u c t u r e a n d f u n c t i o n o f s e m i n i f e r o u s tubules. Acknowledgements. The authors thank Mrs. S. Edel and Mrs. M. Reichmann for excellent technical assistance, Drs. R. Hubmann and T. von Kiigelgen, Department of Urology, Allgemeines Krankenhaus St. Georg, Hamburg, for kindly supplying testicular material, and Dr. C.A. McArdle (IHF) for helpful comments on the manuscript. We are particularly grateful to Dr. F. Leidenberger (IHF) for his encouragement and support. This work was also supported by grant Ho 388/6-1 from the Deutsche Forschungsgemeinschaft (DFG).

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