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a Department of Public Health and Cell Biology, Section of Histology, and
b Department of Surgery, School of Medicine, University of Rome "Tor Vergata", 00173 Rome, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-smooth muscle isoactin, suggesting a role for the so-called neurotrophic system in the differentiation of testicular myoid cells and epididymal smooth muscle cells. In the testis of an 18-wk gestational-age human fetus, immunohistochemical analysis has shown intense immunoreactivity of mesenchymal cells to antibodies for neurotrophin receptors p75, trk A, and trk C, and their ligands NGF and NT-3. In addition, we found that in the human fetal testis, the interstitial cells that are differentiating into peritubular myoid cells are associated with a dense network of nerve fibers. Our data suggest that neurotrophins and their receptors are involved in a multifunctional system that regulates cell differentiation and innervation in the developing testis and epididymis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A body of evidence is emerging showing that neurotrophins exert specific effects on non-neuronal cells as well [13, 22–25] and are involved in morphogenetic signalling processes during development of non-neuronal tissues [26–29]. High levels of p75 are expressed and developmentally regulated in a broad range of adult and embryonic tissues outside the nervous system [30, 31] and are frequently observed at sites of mesenchymal-epithelial interactions. Trk receptors, originally reported to be restricted to the nervous system, have been detected in several adult tissues [32–34] as well as in the mouse embryo, during gastrulation and organogenesis [27]. These receptors have been detected also in the early human fetus, in a variety of different tissues [35, 36]. The presence of high levels of NGF in tissues not showing a correspondingly extensive innervation by NGF-responsive fibers, such as male mouse submandibular gland [15] and the reproductive organs of adult mammals, such as mouse testis and epididymis [37], human testis [38], guinea pig and rabbit prostate [39], and bull seminal vesicles [40], strongly suggest that NGF has extraneuronal activities. Moreover, NGF stimulates the onset of meiosis in the seminiferous epithelium [41]. Gene expression studies have shown the presence of the p75 transcript in Sertoli cells [42] and in spermatocytes and spermatids of adult mouse and rat testis [31]. An NGF-mediated paracrine regulation has been shown in the human prostate, in which NGF produced by stromal cells has been shown to bind to trk A present on the epithelial components of the gland [13]. In view of the findings showing extensive expression of neurotrophins and their receptors in the adult testis and male reproductive tract, and of the data supporting a role for these molecules in the differentiation of a variety of non-neuronal tissues, we have investigated whether the neurotrophin-receptor system is also involved in the regulation of testicular and epididymal morphogenesis. Using immunohistochemical analysis with specific antibodies, we have studied the presence and distribution of all members of the nerve growth factor family of neurotrophins and of their receptors during embryonic and postnatal development of the mouse testis and epididymis, and in fetal human testis. The data reported in the present paper show developmental changes in the expression of neurotrophins and their receptors, and suggest that these molecules are involved in mesenchymal tissue differentiation during testicular and epididymal morphogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Urogenital ridges, testes, and epididymides were collected from CD1 mice at embryonal ages 12.5 to 18.5 days postcoitum, at postnatal ages 1, 5, 10 and 20 days; and from adult CD1 mice approximately 90 days old (Charles River, Lecco, Italy). The morning the vaginal plug was found was considered to be embryonal age 0.5 days postcoitum (dpc), and the day of birth was recorded as day 1 of postnatal age (P1). The animals were killed by cervical dislocation. After the uterus was removed, the embryos were isolated, decapitated, and dissected. The sex of the embryos was assessed by the morphological aspect of the gonads. At 12.5 dpc, a testis can be distinguished from an ovary for its larger blood supply, the presence of cords of somatic and germ cells, and its larger size. Human fetal testes were obtained from spontaneous abortion of a karyotypically normal fetus at 18 wk of gestation. The age of fetus was based on the date of last menstruation and on the crown-rump length measured directly (160 mm). The procedure for recovering human fetal testes has been approved by the University ethical committee.
Immunohistochemical Staining
The following affinity-purified rabbit polyclonal antibodies against trk receptors were obtained from Santa Cruz Biotechnology (Santa Cruz, CA): anti-trk A (cat. no. sc-118), recognizing amino acids 763–777 adjacent to the carboxyl terminus of human receptor [43]; anti-trk B (cat. no. sc-12), directed against amino acids 794–808 of mouse trk B [32]; anti-truncated trk B antibody (cat. no. sc-119), raised against a peptide that is specifically present within the carboxyl terminal domain of the [TK-] trk B isoform [44]; and anti-trk C (cat. no. sc-117), directed against amino acids 798 to 812 at the carboxyl terminus of the porcine full-length trk C receptor [45]. Rabbit anti-mouse p75 receptor polyclonal antibody [46] (Chemicon International Inc., Temecula, CA), was used (at a 1:200 dilution) to detect the low-affinity p75 receptor in mouse sections. Sections were also probed with the following Santa Cruz affinity-purified polyclonal anti-neurotrophin antibodies raised against specific peptides: anti-NGF, human reactive (cat. no. sc-548); anti-NGF, mouse reactive (cat. no. sc-548); anti-BDNF (cat. no. sc-546); anti-NT-3 (cat. no. sc-547); and anti-NT-4 (cat. no. sc-545). Optimal antibody concentration for immunohistochemistry of trk receptors and neurotrophins was determined by serial dilution to be 0.5 µg/ml in all instances. According to the manufacturer, each antibody specifically recognizes its antigen without cross-reacting with other molecules of the same family. Furthermore, the immunohistochemical specificity of these antibodies has been extensively characterized elsewhere [29, 47, 48].
The p75 neurotrophin receptor in the human fetal testis was identified by using a monoclonal antibody (at 2 µg/ml) raised against the human receptor (clone ME20.4; Amersham, Buckinghamshire, UK) [49]. This antibody has been recognized as highly specific for the human p75 receptor [38]. The distribution of nerve fibers in human fetal testis was detected by using a monoclonal antibody to neurofilament polypeptide 68 kDa (clone NR4) [50] obtained from Boehringer Mannheim (Indianapolis, IN), used at 7 µg/ml. Anti-laminin mouse monoclonal antibody, raised against rat glomerular basement membrane, was supplied as purified immunoglobulins by the Developmental Studies Hybridoma Bank (Iowa City, IA) and used at 5 µg/ml. -Smooth muscle isoactin was detected by means of a monoclonal antibody (clone 1A4) supplied as an ascites fluid by Sigma Chemical Co. (St. Louis, MO) and used at a 1:500 dilution.
The immunohistochemistry of neurotrophin receptors (p75 and trks), neurofilament protein, -smooth muscle actin, and laminin was performed on cryostat sections (7 µm thick) of testes and epididymides that had been directly embedded in OCT compound (Tissue-Tek; Miles, Inc., Elkhart, IN) without fixation. The sections were collected on gelatin-coated slides, air-dried, and fixed in methanol at -20°C for 10 min. Sections for the detection of neurotrophins were cut from testes and epididymides that had been fixed in Zamboni's fixative [51] for 18 h at 4°C, rinsed in Tris-buffered saline (TBS), soaked in 30% sucrose solution in TBS overnight, and then embedded and frozen in OCT. Before treatment with antibodies, the sections were postfixed in the same fixative for 10 min and then washed three times (15 min each) in TBS-glycine (50 mM Tris, pH 7.5, 150 mM NaCl, 20 mM glycine); the sections were then incubated for 1 h at room temperature (RT) in 5% fetal calf serum in PBS to block aspecific binding. Subsequently, the slides were incubated overnight in a humidified chamber at 4°C with the primary antibodies diluted in 0.1% BSA in PBS, then extensively washed in PBS, and probed for 1 h at RT with cyanine 3-labeled goat anti-rabbit (Chemicon International Inc.) or with fluorescein isothiocyanate (FITC)-labeled sheep anti-mouse secondary antibodies (Sigma Chemical Co.). For double immunostaining, the sections were incubated with two combined primary antibodies and subsequently with the appropriate secondary antibodies, following the procedure described above. At the end of the procedure, the slides were mounted in 60% glycerol in 0.1 M Tris buffer, pH 9.3, and analyzed in a fluorescence microscope equipped with phase contrast optics.
Specificity of Immunolabeling
Several control experiments were performed in order to check the specificity of the immunoreactions. To test for nonspecific binding of the primary antibodies, control sections were either incubated with nonspecific rabbit or mouse immunoglobulins at concentrations equal to those of the primary antibodies, or processed with the primary antibody omitted. In such conditions, staining was completely abolished in all instances. As another control, in the case of neurotrophin and neurotrophin receptor immunohistochemistry, the corresponding immunizing peptides (all from Santa Cruz) were used for competition experiments. In these cases, the undiluted antibody (100 µg/ml) was preincubated for 2 h at RT with the same volume of the corresponding peptide (200 µg/ml) and then diluted before immunolabeling.
Western Blotting and Immunoprecipitation Analysis
PC12 cells, used as a control for p75 and trk A receptor expression, were purchased from the American Type Culture Collection (Manassas, VA). They were grown in RPMI 1640 (Gibco BRL, Gaithersburg, MD) supplemented with 10% heat-inactivated horse serum and 5% fetal bovine serum, and then cultured for 2 days in the presence of 50 ng/ml mouse NGF (Collaborative Research, Bedford, MA) before use. Detergent lysates of P1 testis and epididymis as well as of PC12 cells, adult mouse brain, kidney, and cerebellum, were prepared in radioimmunoprecipitation assay (RIPA) buffer (PBS pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM PMSF, 0.2 U/ml aprotinin, and 1 mM sodium orthovanadate). Insoluble material was removed by centrifugation (10 000 x g for 10 min) and protein concentration in the supernatants was determined. For Western blot analysis, extracted proteins, prestained molecular weight standards (Amersham), recombinant BDNF, NT-3, and NT-4 (Alexis, San Diego, CA) and mouse NGF (Collaborative Research) were separated by SDS-PAGE on 7.5% or 12% acrylamide gels and transferred to ECL nitrocellulose membrane (Amersham) using a transblot apparatus (Bio-Rad, Richmond, CA) at 100 V for 2 h at 4°C. Filters were first blocked for 1 h in 10% fat-free milk in PBS and then probed with the primary antibody (0.5 µg/ml in all instances) for 1 h at RT. For controls, primary antibody was either replaced with nonimmune IgG at the same concentration or used in the presence of the immune peptide. The filters were washed in PBS-0.1% Tween 20 and incubated for 1 h at RT with a horseradish peroxidase-conjugated anti-rabbit antibody (Amersham) at 1:10 000 dilution. Immunoreactive bands were detected using enhanced chemiluminescence Western blotting system (ECL; Amersham) following the manufacturer's instructions. Immunoprecipitation of the trk A receptor was performed using protein extracts incubated with the anti-trk A antibody (1 µg/ml) for 16 h at 4°C. The immune complexes were collected with protein A-Sepharose beads and washed in RIPA buffer. The samples were then solubilized by boiling in sample buffer, and subjected to electrophoresis and Western blot analysis as above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The expression of neurotrophins (NGF, BDNF, NT-3, and NT-4) and their receptors (p75 and trk A, B, and C) during mouse testis development was investigated at 12.5 dpc, the stage at which testis cords start to form, and at various intervals throughout embryonic and postnatal development. Immunolocalization of the p75 receptor was combined with double-immunofluorescence detection of -smooth muscle isoactin, a marker of terminal differentiation in myoid cells [52]. The analysis of the cryosections led to the detection of a specific expression pattern for p75 and truncated trk B; no trk A, full-length trk B, or trk C immunoreactivity was detected in these specimens. In the 12.5-dpc fetal testis, intense p75 staining was seen on the entire population of mesenchymal cells spread through the interstitial tissue outlining the developing testis cords, and immunoreactivity was totally absent in the cord cells (Fig. 1A). During embryonic development, a large number of mesenchymal cells can be easily identified in the interstitial compartment by phase contrast microscopy, on the basis of their elongated shape and distribution in strands [53]. High levels of p75 expression persisted around testicular cords up to P1 (Fig. 1B); double-immunostaining analysis showed coexpression of p75 (Fig. 1B) and -smooth muscle isoactin (Fig. 1E) in the inner layers of the mesenchyme, where differentiation of the myoid cell phenotype takes place. During postnatal development, p75 staining intensity gradually decreased (P10; Fig. 1C), becoming undetectable at puberty (P20; Fig. 1D); at the same time, as differentiation of myoid cells proceeded, -smooth muscle isoactin staining became heavier (Fig. 1, F and G). These findings suggest that myoid cells differentiate from p75-expressing mesenchymal cells and that the expression of this receptor might in some way be involved in this process. The presence of p75 receptor was confirmed by Western blot of P1 total testis lysate. As shown in Figure 2A, the same antibody used for immunohistochemistry recognized a 75-kDa protein and additional bands representing different forms of the molecule [54, 55]. A qualitatively identical pattern was detected in PC12 cells used as positive control. Truncated trk B immunoreactivity was present during embryonic and postnatal development in the peritubular compartment and appeared to be developmentally regulated as well (Fig. 1, H–J). Truncated trk B staining appeared at a later stage of testicular morphogenesis (14.5 dpc, Fig. 1H) than p75 staining (12.5 dpc). Truncated trk B staining gradually decreased after birth, becoming detectable in only a few peritubular cells at puberty (P20; Fig. 1J) and undetectable in the adult testis (not shown). Western blot analysis of the expression of truncated trk B receptor showed a prominent 95-kDa band corresponding to the size of the truncated receptor as well as other bands of the variably glycosylated receptor [44], in both brain (used as a positive control) and P1 testis (Fig. 2B).
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Neurotrophin expression in the developing testis was located by immunofluorescence and validated by Western blotting. Testis sections showed high levels of immunoreactive NT-3 during fetal and postnatal development, with no evidence of specific staining for any other neurotrophin. NT-3 immunoreactivity was detected in the cytoplasm of peritubular mesenchymal cells from 14.5-dpc to P20 mice (Fig. 3, A–C). The most intense labeling was detected in the testis of newborn (P1) animals (Fig. 3B); NT-3 staining, as it occurs with neurotrophin receptors p75 and truncated trk B, gradually decreased during postnatal development. NT-3 immunoreactivity was confined to a few peritubular cells at puberty (P20; Fig. 3C) and disappeared in the adult testis (not shown). The presence of NT-3 in the developing testis was validated by Western blot analysis of whole P1 testis lysate (Fig. 4A), which showed a doublet of proteins running at a position consistent with the molecular mass of NT-3 (13.5 kDa). One of the bands corresponded to recombinant NT-3 molecular size, while the other—running slightly above it—might derive from a process of glycosylation or from the presence of heterogeneously cleaved NT-3 precursor [56]. Control immunoblot analysis (Fig. 4B) demonstrated that the antibody detected the expected NT-3 bands in kidney and cerebellum lysates and did not cross-react with mouse NGF and recombinant BDNF and NT-4. The same experiment showed that when the anti-NT-3 antibody was preincubated with the immune peptide, immunoreactivity was essentially abolished (Fig. 4C).
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Expression of Neurotrophin Receptors and Neurotrophins in the Developing Mouse Epididymis
Cryostat sections of mouse epididymis at different embryonic and postnatal development stages were immunostained for detection of neurotrophins and their receptors. At 12.5 dpc, mesonephric mesenchyme was p75-positive (Fig. 1A). As morphogenesis of the epididymis from the mesonephric duct proceeded (14.5 dpc), a thick layer of intensely stained mesenchymal cells surrounded the duct (Fig. 5A). At P1, the immunoreaction was generally strong close to the epididymal duct and less intense in the surrounding interstitial tissue (Fig. 5C). After birth, receptor staining intensity started to gradually decrease (Fig. 5E) (P10) until it became undetectable in the adult (Fig. 5F). From 14.5 dpc onward, p75-positive mesenchymal cells closely surrounding the epididymal duct were found to be -smooth muscle isoactin-positive as well (Fig. 5, B and D). During postnatal development, the constant decrease of p75-reactivity correlated with an increase in -smooth muscle isoactin staining (not shown), a pattern similar to that observed during differentiation of testicular myoid cells. The presence of p75 receptor in the developing epididymis was confirmed by Western blot analysis of epididymis detergent lysate from P1 mice (Fig. 2A).
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A series of developing epididymis cryostat sections were stained with antibodies against each of the trk receptors. Positive immunolocalization results were obtained with the anti-truncated trk B receptor antibody, which colocalized with p75-positive mesenchymal cells (P1; Fig. 5G). As in the developing testis, staining became detectable at 14.5 dpc, gradually decreased during postnatal development, and was undetectable in the adult (not shown). Western blot analysis of neonatal epididymis showed the same truncated trk B immunoreactive bands as detected in the testis at the same age (Fig. 2B). Full-length trk B and trk C were not detected in the epididymis at any stage. Intense trk A immunoreactivity was observed on the plasma membrane of the epididymal epithelium during embryonic and postnatal development up to the adult. Trk A staining, first detected on the epithelium of the mesonephric duct and tubules of the 12.5-dpc mouse (Fig. 5H), was constantly observed during embryonic and postnatal growth up to the adult (Fig. 5, I and J). In the adult epididymis, epithelial cell labeling was restricted to the baso-lateral membrane: no labeling was detected on the apical membrane or stereocilia (Fig. 5J). Western blot analysis of anti-trk A immunoprecipitate from lysate of P1 epididymis showed bands at 110 and 140 kDa, corresponding to those present in the PC12 cell lysate used as a positive control (Fig. 2C).
A parallel immunocytochemical evaluation of neurotrophin expression was carried out in the developing epididymis. Beginning with 14.5 dpc, mesenchymal cells, expressing the p75 and truncated trk B receptors, were intensely immunoreactive to NT-3 antibody as well. Staining was detected between 14.5 dpc and P20, showing maximal intensity during the neonatal period (Fig. 3E) and a gradual decrease towards puberty (Fig. 3F). In the embryo, immunoreactive cells were uniformly distributed in the intertubular compartment whereas during postnatal development immunoreactivity was restricted to a few layers of concentrically arranged periductal cells (Fig. 3F). No significant immunoreactivity was detected in the adult (not shown). The presence of NT-3 in the developing epididymis was confirmed by Western blot analysis, which showed immunoreactive bands at a position consistent with NT-3 known molecular weight (Fig. 4A).
Finally, we found intense NT-4-like immunoreactivity within the smooth muscle cell layer of the adult epididymis (Fig. 3H). No cells immunopositive for neurotrophic factor NT-4 were observed at any embryonic or prepubertal stage of development. This finding, awaiting further investigation, suggests that NT-4 might play a role in the physiology of the epididymal duct.
Neurotrophin and Neurotrophin Receptor Immunoreactivity in Fetal Human Testis
Expression of neurotrophins and their receptors in human developing testis from an 18-wk-old fetus was evaluated by immunohistochemistry. Cryosections probed with the human-specific p75 receptor monoclonal antibody ME.20 showed intense immunoreactivity on mesenchymal cells in the interstitial compartment (Fig. 6A). Sections were also immunostained with polyclonal antibodies specific for the various trk receptors. trk A- and trk C-like immunoreactivity was present and displayed identical localization with p75 within the mesenchyme (Fig. 6, B and C). Only the peritubular compartment displayed immunoreactivity, while testis cords were essentially devoid of receptor immunostaining. Additional trk A immunoreactivity was observed on the epithelium of the efferent ductules at the hilum of the testis (data not shown). Staining was routinely negative in control experiments (not shown). No full-length or truncated trk B immunoreactivity was detected in these specimens.
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Neurotrophin expression was immunohistochemically evaluated at the same gestational age using the series of specific polyclonal antibodies described in Materials and Methods. This analysis showed a diffuse pattern of NGF- and NT-3-like immunostaining (Fig. 6, D and F). More specifically, the antibody against an NGF-specific peptide labeled both interstitial mesenchymal cells and somatic cells of testicular cords. NT-3-like staining was confined to the interstitial compartment, where sparse intensely positive cells were interspersed among weakly immunoreactive mesenchymal cells. Preabsorption of neurotrophin antibodies with excess immune peptide abolished the specific staining patterns (Fig. 6, E and G). No immunoreactivity for BDNF and NT-4 was detected.
The ongoing differentiation of the peritubular mesenchymal cell population was investigated by a monoclonal antibody against -smooth muscle isoactin. The results showed that each testicular cord was enveloped by a layer of -smooth muscle isoactin-positive interstitial cells (Fig. 6H), with the interposition of a continuous basement membrane detected by anti-laminin antibodies (Fig. 6I).
Neurotrophins expressed during testicular development might also be involved in innervation of the interstitial compartment. To study such possible correlation, monoclonal antibodies to 68-kDa neurofilament protein were used to visualize the extent of innervation and to locate nerve fibers in the gestational testis. The results showed the presence of a network of nerve fibers on the surface of the testis cords, where the differentiation of myoid cells takes place (Fig. 6J).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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By means of specific polyclonal antibodies, immunohistology and Western blot analysis showed that various neurotrophins and their receptors are differentially expressed in a growth-regulated fashion and in specific tissue compartments during testicular and epididymal development. At a very early stage of gonadal formation in the mouse (12.5 dpc) intense p75 receptor immunoreactivity was observed on the mesenchyme surrounding developing testicular cords and the mesonephric duct; no trk receptor or neurotrophin immunoreactivity was seen at this stage. Although we were not able to detect neurotrophin expression at 12.5 dpc, we cannot exclude the possibility that the level was too low to be demonstrated by immunocytochemistry or that p75 binds a different ligand. A possible morphogenetic role has been frequently assumed for p75 because of its extensive expression at several sites of mesenchymal/epithelial interactions, with a pattern not correlated with innervation. For instance, a correlation between the expression of p75 and morphogenetic events has been demonstrated in the embryonic rat kidney, in which the receptor is transiently expressed at high levels in the condensing mesenchyme [59]. A role for this common neurotrophin receptor has been suggested also during ovarian histogenesis in the rat. During this process, high levels of p75 have been detected in mesenchymal cells that disrupt the epithelial cords and intercalate between pregranulosa cells during follicular formation [28]. Our developmental stage-related double-immunofluorescence study has shown an inverse relationship between the expression of p75 and of -smooth muscle isoactin in the inner layers of mesenchymal cells that surround both developing testis cords and the epididymal duct. These data, suggesting that myoid cells of the testis and epididymal smooth muscle cells differentiate from the p75-positive cells, indicate a potential role for this receptor molecule in the regulation of this process.
Beginning with 14.5 dpc, p75-positive mesenchymal cells express the truncated isoform of trk B and neurotrophin NT-3. The delayed expression of NT-3, as compared to the early detection of p75, suggests a possible role for the former neurotrophin in later post-inductive growth and differentiation of the testis and epididymis. Our attempts to detect the specific receptor of NT-3 (trk C) have failed, probably because the levels of this molecule in non-neural tissues are generally low. In fact, although the expression of trk C mRNA has been demonstrated in several non-neural tissues, including the urogenital mesenchyme of the mouse embryo [27], the detection of the corresponding protein by immunological methods has generally failed [27, 60]. The expression of trk C mRNA has been detected in our laboratory in P1 rat testis by reverse transcription-polymerase chain reaction (RT-PCR), using oligonucleotide primers based on the sequence of the extracellular domain of the rat trk C (not shown). Therefore, we cannot exclude the possibility that the lack of trk C immunoreactivity was due to the expression of truncated isoforms of this receptor that could not be recognized by our C-terminal antibody. Further analysis using antibodies against specific isoforms of the receptor as well as Northern blot experiments are needed to define the expression of this molecule in our tissues. Although the expression of trk C remains to be clarified, the presence of p75/NT-3-positivity in cells that are induced to express smooth muscle isoactin during differentiation suggests possible autocrine/paracrine mechanisms based on neurotrophin signalling in testicular myoid cell and epididymal smooth muscle cell differentiation. It is worth noting in this context that the expression of neurotrophin-3 along with full-length and truncated isoforms of neurotrophin receptors has been implicated in in vitro cell migration and proliferation of human and rat smooth muscle cells [61].
By using a specific anti-truncated trk B antibody, a developmentally regulated pattern of immunoreactivity, spatially and temporally correlated with NT-3 expression, was found in the interstitial compartment of the testis and epididymis. Although the functions of the truncated isoforms of neurotrophin receptors are unknown, a role in recruiting the ligand where high concentrations are required or a role as a cell adhesion molecule has been postulated [62, 63].
In the present study, we found trk A immunoreactivity to be strictly limited to mesonephric tubules and duct, and to the epithelium of the developing and adult epididymis. Several reports have described the expression of trk A, using a histochemical approach, in a wide variety of epithelial cells including prostatic epithelial cells [13], epidermis [24], and collecting tubules of the kidney [48]. In these tissues, the observation that the same epithelial cells or the surrounding stromal cells are capable of producing NGF or other neurotrophins has suggested that the neurotrophin/receptor system may play a crucial role in survival or behavior of the epithelial component. For instance, an NGF-mediated paracrine regulation has been shown in the prostate, in which NGF produced by stromal cells binds to trk A expressed by the epithelial component [13]. In the adult epididymis, evidence for the expression of NGF is limited to the detection of the NGF mRNA by Northern blot analysis [37]. Our data, showing the presence of NT-3 in the developing epididymis and of NT-4-like immunoreactivity in the smooth muscle cells in the adult suggest that both neurotrophins might exert local paracrine effects on stromal or/and epithelial components.
In this paper, we have identified potential autocrine/paracrine mechanisms for neurotrophins in the developing human testis as well. No evidence of this kind has been previously reported. Our immunohistochemical findings show that the human fetal testis at 18 wk of gestation displays in the interstitial compartment a diffuse pattern of immunoreactivity to antibodies against neurotrophin receptors p75, trk A, and trk C, as well as against neurotrophins NGF and NT-3. The localization of the p75 receptor and NT-3 is exactly coincident with that found in the mouse embryo, while the additional immunoreactivity to antibodies against NGF and the high-affinity receptors trk A and C reflects a difference between rodents and humans. The expression of high-affinity receptors trk A and C identifies potential autocrine or paracrine neurotrophin signalling during differentiation of specific cell types of the interstitial compartment. In this regard, the presence of -smooth muscle isoactin in the interstitial cells at the same stage of development suggests that neurotrophins and their receptors could play a role in the differentiation of myoid cells. Several previous reports have shown that during embryogenesis neurotrophin receptors and their ligands are involved in a multifunctional system that controls morphogenesis and innervation. A wide range of non-neural tissues express neurotrophin receptors in patterns that do not seem correlated with innervation, such as the expression of trk receptors during gastrulation in the mouse embryo [27] or during human kidney development [29]. On the other hand, it must be considered that in other tissues their expression in the mesenchyme peaks at the time when these tissues are undergoing extensive innervation. It is commonly accepted that seminiferous tubules are not innervated and that very few nerve fibers reach the interstitial compartment of the testis, where they associate with its vasculature. Since the immunohistochemical results of the present study had indicated the presence of high levels of neurotrophins and their receptors in the fetal human testis, we have investigated the distribution of nerve fibers by means of a monoclonal antibody that specifically identifies the 68-kDa neurofilament protein. Such investigation demonstrated the presence of nerve fibers that reach the surface of the testicular cords and become associated in a characteristic pattern with the peritubular environment, where myoid cells are differentiating. While the existing data in the literature have established that testicular innervation is essentially confined to the vasculature, our original observations show a close interaction between nerve fibers and myoid cells in the fetal human testis and suggest the presence of a neuronal control on seminiferous tubules contraction. Further investigations are in progress to identify the nerve fibers associated with the myoid cells of the developing testis and to study their localization during postnatal development and in the adult testis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Mario A. Russo, Department of Public Health and Cell Biology, Section of Histology, University of Rome "Tor Vergata", Via di Tor Vergata 135, 00133, Rome, Italy. FAX: 39 06 72596172; russo{at}med.uniroma2.it
Accepted: June 3, 1999.
Received: February 18, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  J. Tomaszewski, A. Joseph, D. Archambeault, and H. H.-C. Yao Essential roles of inhibin beta A in mouse epididymal coiling PNAS, July 3, 2007; 104(27): 11322 - 11327. [Abstract] [Full Text] [PDF]
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 A. Puglianiello, L. Campagnolo, D. Farini, D. Cipollone, M. A. Russo, and G. Siracusa Expression and role of PDGF-BB and PDGFR-{beta} during testis morphogenesis in the mouse embryo J. Cell Sci., March 1, 2004; 117(7): 1151 - 1160. [Abstract] [Full Text] [PDF]
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 L. L. L. Robinson, J. Townsend, and R. A. Anderson The Human Fetal Testis Is a Site of Expression of Neurotrophins and Their Receptors: Regulation of the Germ Cell and Peritubular Cell Population J. Clin. Endocrinol. Metab., August 1, 2003; 88(8): 3943 - 3951. [Abstract] [Full Text] [PDF]
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R. A. Anderson, L. L. L. Robinson, J. Brooks, and N. Spears Neurotropins and Their Receptors Are Expressed in the Human Fetal Ovary J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 890 - 897. [Abstract] [Full Text] [PDF]
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 W. Lambert, R. Agarwal, W. Howe, A. F. Clark, and R. J. Wordinger Neurotrophin and Neurotrophin Receptor Expression by Cells of the Human Lamina Cribrosa Invest. Ophthalmol. Vis. Sci., September 1, 2001; 42(10): 2315 - 2323. [Abstract] [Full Text] [PDF]
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L. Campagnolo, M.A. Russo, A. Puglianiello, A. Favale, and G. Siracusa Mesenchymal Cell Precursors of Peritubular Smooth Muscle Cells of the Mouse Testis Can Be Identified by the Presence of the p75 Neurotrophin Receptor Biol Reprod, February 1, 2001; 64(2): 464 - 472. [Abstract] [Full Text]
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A. S. Cupp, G. H. Kim, and M. K. Skinner Expression and Action of Neurotropin-3 and Nerve Growth Factor in Embryonic and Early Postnatal Rat Testis Development Biol Reprod, December 1, 2000; 63(6): 1617 - 1628. [Abstract] [Full Text]
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