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Regular Article |
a Department of Public Health and Cell Biology, Section of Histology and Embryology, University of Rome "Tor Vergata," 00173 Rome, Italy
ABSTRACT
In the mouse embryo, at approximately 11.5 days postcoitum (dpc), cells migrate from the mesonephros into the developing testis to contribute to the somatic population of the interstitial compartment (i.e., peritubular myoid cells, Leydig cells, and endothelial cells). Studies from this laboratory have shown that the interstitial population of mesenchymal cells in fetal and newborn mouse testis express the p75 neurotrophin receptor (p75NTR, formerly known as the low-affinity nerve growth factor receptor); part of the cell population progressively congregates around testis cords, later to be replaced by contractile peritubular myoid cells, which express smooth muscle cell markers. In the present study, we show that the migrating cells and the p75NTR-expressing cells are the same population. We also show that the neurotrophin receptor is a useful endogenous marker to follow cell migration within the urogenital ridge and to identify and isolate mesenchymal precursors of myoid cells. A time-course immunolocalization study of the location of p75NTR-bearing cells within the urogenital ridge of mouse embryos between 10.5 and 12.5 dpc showed that the interstitium of the fetal testis was progressively occupied by p75NTR(+) cells. The progressive increase of p75NTR expression within the developing testis was confirmed by immunoblot analysis of proteins isolated from the fetal gonads. Organ cultures of isolated testes or testis-mesonephros grafts confirmed that p75NTR(+) cells do not appear in the testis unless a mesonephros is attached to it. Cells bearing the p75NTR receptor, purified from 12.5-dpc male mouse mesonephroi by immunomagnetic sorting, were able to differentiate in vitro into myoid cells. Immunofluorescence analysis of postnatal testis sections confirmed the presence around the tubules of cells coexpressing p75NTR and
-smooth muscle actin. The ability to identify and purify precursors of myoid cells may be of considerable help for studying the mechanisms regulating their differentiation.
myoid cells, testis
INTRODUCTION
Two separate compartments can be recognized in the developing testis: an interstitial compartment, containing a mesenchymal cell population; and testis cords, containing germ cells and precursors of Sertoli cells. The embryonal development of such structures depends on a complex series of interactions between mesonephros and the adjacent coelomic epithelium [1]. The migration of mesonephric cells into the developing testis plays a pivotal role in testicular morphogenesis, because in the absence of such cell influx, no organized testis cords form [2–5]. A chemical signal is probably attracting mesonephric cells into the testis [4]. The migration of mesonephric cells is essential for male-specific gene expression and Sertoli cell differentiation in XY gonads, and these events are stage specific [6]. Grafting experiments have shown that the sex of the mesonephros as a donor of migrating cells is irrelevant, because a female mesonephros is equally able to support normal cord differentiation [2–4]. If a transgenically labeled mesonephros is grafted to a developing testis, the marker is later found in peritubular myoid cells [2], endothelial cells [4, 7], and cells showing ultrastructural features of steroidogenic Leydig cells [8]. A different origin has been demonstrated for mouse Sertoli cells, because their precursors have been identified in coelomic epithelial cells migrating into the gonad before 11.5 days postcoitum (dpc) [9].
Previous studies from this laboratory have shown that transcripts of the p75 neurotrophin receptor (p75NTR) are present in the developing mouse testis and can be located, by in situ hybridization on 17-dpc testicular sections, in intertubular somatic cells [10]. A comparable picture is obtained by immunohistochemical analysis in the embryonic and early postnatal rat and mouse testis: cells clearly labeled by anti-p75NTR antibody can be seen to occupy the whole intertubular compartment in testicular sections from 14.5-dpc rat embryos and to progressively concentrate around testis cords before birth. During early postnatal development, double-stained sections show that a layer of p75NTR(+) cells surround a more differentiated peritubular layer of desmin-positive myoid cells located immediately around each testis cord [10]. Likewise, in the testis of the 12.5-dpc mouse embryo, intense p75NTR staining is confined to cells in the interstitial compartment [11]; high levels of the receptor persist up to postnatal Day 1, progressively decrease during postnatal development, and completely disappear at puberty [11].
In the present paper, we show that the cells which migrate from the mesonephros to the gonad can be identified by the presence on their surface of p75NTR (also known as the low-affinity nerve growth factor [NGF] receptor), that the presence of such antigen can be exploited to isolate these cells, and that p75NTR(+) cells are the precursors of myoid cells (because they differentiate both in vitro and in vivo into such cell type). The availability of pure mesenchymal precursors of myoid cells has great potential for studying the mechanisms involved in the migration and differentiation of such a population.
MATERIALS AND METHODS
Animals
Outbred CD1 mice (Charles River, Calco, Italy) were housed and mated under standard laboratory conditions. Embryos at various developmental stages were obtained by killing the mothers via cervical dislocation at various postcoital times. Midday of the day on which the vaginal plug was found was considered to be Day 0.5 of pregnancy. For experiments in which a more accurate embryo staging was required (i.e., grafting experiments and time-course neurotrophin receptor immunolocalization studies), two additional criteria were used: hind-limb bud morphology [12], and number of tail somites posterior to the hind limb [9, 13]. At stages before sexual dimorphism was apparent, sex of the embryos was determined by examining sex chromatin in amniotic cells [14].
Antibodies
Monoclonal rat anti-mouse p75NTR (MAB357) was obtained from Chemicon International (Temecula, CA) and used for immunomagnetic separation of unfixed cells; for fixed cells or tissues and for immunoblot analysis, polyclonal rabbit anti-mouse p75NTR (AB1554; Chemicon) was used. The immunohistochemical specificity of these antibodies has been well characterized [15]. Directly fluoresceinated monoclonal mouse anti--smooth muscle actin, monoclonal mouse anti--smooth muscle actin, and anti-ß-actin antibodies (F3777, A2547, and A5441, respectively) were from Sigma (St. Louis, MO). Secondary antibodies were Cy3-labelled goat anti-rabbit immunoglobulin (Ig) G (AP132C; Chemicon), fluorescein isothiocyanate (FITC)-labeled sheep anti-mouse IgG (F2266; Sigma), and horseradish peroxidase-conjugated anti-rabbit and anti-mouse Ig (NA934 and NA931, respectively; Amersham Pharmacia Biotech, Little Chalfont, UK).
Immunoblot Analysis
Urogenital ridges were collected from mouse embryos at 11.5, 12.5, and 13.5 dpc. Mesonephroi were removed using a very fine needle, and testes were homogenized in lysis buffer (50 mM Hepes [pH 7.4], 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mg/ml SDS, 15 mM MgCl2, 1 mM EGTA, 2 mM PMSF, 0.5 µg/ml of leupeptin, 0.7 µg/ml of pepstatin, 0.2 U/ml of aprotinin, and 50 mM benzamidine). The homogenized tissues were centrifuged at 13 000 x g for 30 min, and the insoluble material was removed. Protein concentration in the supernatant was evaluated by the Bradford assay. Prestained molecular weight standards (Amersham Pharmacia Biotech) and 25 µg of the extracted proteins were separated by SDS-PAGE on a 7.5% polyacrylamide gel. The gel was then transferred to an enhanced chemiluminescence (ECL) nitrocellulose membrane (Amersham Pharmacia Biotech) using a transblot apparatus (Bio-Rad, Richmond, CA) at 13 V overnight at 4°C. Filters were first incubated for 2 h with a solution of 10% fat-free milk and 0.1% Tween 20 in PBS, then incubated with polyclonal rabbit anti-mouse p75NTR (1:2000) or monoclonal mouse anti-ß-actin (1:1000) primary antibodies for 1 h at room temperature (RT). Preimmune sera were used for control filters. After several washes in PBS-0.1% Tween 20, horseradish peroxidase-conjugated secondary antibodies (1:10 000) were added for 1 h at RT. Immunoreactive bands were detected using an ECL Western blotting system (Amersham Pharmacia Biotech) according to the manufacturer's specifications.
Organ Cultures
Urogenital ridges were isolated by stereomicroscopical dissection at approximately 0600 h from 11.25-dpc embryos (15–16 tail somites). The ridges were collected in Hepes-buffered minimum essential medium (MEM; Sigma) containing 1 mg/ml of BSA, and testes were separated from mesonephroi using a very fine needle. The absence of residual mesonephric tissue was carefully assessed by stereomicroscopical observation, and gonads carrying any visible shred of mesonephros were not used. For grafting experiments, gonads and mesonephroi belonging to different embryos were used. Each isolated gonad was placed side by side to a mesonephros carefully deprived of its own gonad, and after 24 h of culture, fusion was checked. Organs were cultured on a 1% agar-coated, stainless-steel grid placed in the well of a Falcon in vitro fertilization dish (Falcon 3653; Becton Dickinson Labware, Bedford, MA), in Dulbecco's modified Eagle's medium (DMEM; Sigma) with 10% fetal calf serum (FCS; Gibco Life Technologies, Gaithersburg, MD), 2 mM glutamine, 0.5 mM pyruvate, 0.1 mM ß-mercaptoethanol, 100 U/ml of penicillin, and 0.05 mg/ml of streptomycin (all from Sigma). Cultures were maintained for 4 days at 37°C in an atmosphere of 5% CO2 in air.
Preparation of Mesonephric Cell Suspension and Immunomagnetic Cell Sorting
Mesonephroi isolated from 12.5-dpc male mice were incubated for 5 min at RT with continuous agitation in trypsin-EDTA (T3924; Sigma), washed, and transferred in a Hank's-based digestion buffer containing 1 mg/ml of BSA, 2 mg/ml of collagenase type XI (1900 U/mg; Sigma), and 2 mg/ml of DNAse grade II (Boehringer Mannheim GmbH, Mannheim, Germany) for 15 min at 37°C. Undigested fragments were removed by sedimentation at unit gravity for 3 min, and the supernatant cell suspension was pelleted, washed and resuspended in MEM/BSA, and finally, filtered through a 20-µm Millipore nylon screen (NY2002500; Millipore Corporation, Bedford, MA) to obtain a single cell suspension. Cell concentration was evaluated in a Neubauer chamber and brought to 107 cells/ml with MEM/BSA. Monoclonal rat anti-p75NTR antibody was added (5 µg/ml), and the cells were incubated for 1 h at 4°C. Cells were then washed and resuspended in 80 µl of the same medium, to which 20 µl of magnetic microbead-conjugated goat anti-rat IgG (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were added. After 15 min of incubation at 4°C, p75NTR(+) cells were sorted out by use of a magnetic field device (MiniMACS system; Miltenyi Biotec) according to the manufacturer's specifications.
Culture of Immunomagnetically Sorted Cells
Round (13-mm) coverglasses treated overnight with 5 N HCl, washed with double-distilled H2O, heat sterilized, and transferred to the four wells of Nunc multidish plates (Nunc, Roskilde, Denmark) were coated for 1 h at RT with 1:20 Matrigel (Becton Dickinson). The solution was then removed, and the wells were washed and filled with 0.5 ml of DMEM and supplemented as reported in the Organ Culture section. Immunomagnetically sorted cells were added to the wells (2 x 105 cells/well) and cultured for various times at 37°C in a humidified atmosphere of 5% CO2 in air.
Immunohistochemistry and Immunocytochemistry
Urogenital ridges were collected from mouse embryos at various postcoital ages between 10.5 and 12.5 dpc, and testes were collected on postnatal Day 5. Tissues were embedded in OCT (Sakura Finetek, Torrance, CA), frozen on nitrogen vapors, and stored at -80°C until use. Eight-micrometer cryostat sections, collected on gelatin-coated slides, were fixed in methanol for 10 min at -20°C. Sections were then incubated (30 min at RT) with 10% FCS in PBS to block antibody aspecific binding and immunostained with polyclonal rabbit anti-p75NTR antibody (1:400 in PBS/BSA for 30 min at 37°C). Control sections were incubated with nonspecific rabbit immunoglobulins in the same conditions as the primary antibody. The anti-p75NTR antibody was revealed by incubating the sections with Cy3-labelled goat anti-rabbit IgG (2.5 µg/ml for 45 min at RT). Postnatal testicular sections were double-stained with directly labeled monoclonal -smooth muscle actin (-SMA; 1:500) as well. To visualize the entire cell population, cell nuclei were routinely stained with Hoechst 33258 (0.5 µg/ml added to the secondary antibody solution). Slides were mounted with Möwiol [16] (Polysciences, Inc., Warrington, PA) and examined by epifluorescence with a Zeiss (Oberkochen, Germany) Axioplan 2 microscope.
Cultured organs were embedded in OCT compound, frozen on nitrogen vapors, and stored at -80°C. Eight-micrometer cryostat sections were treated according to the above protocol.
Alkaline phosphatase activity was detected by incubating sections for 15 min in a freshly made aqueous solution obtained by mixing 960 µl of Fast Red TR salt (1 mg/ml; Sigma) with 40 µl of Naphthol AS-BI (4 mg/ml, pH 9.5; Sigma) [17].
Cultures of immunomagnetically sorted cells were analyzed for the expression of p75NTR and -SMA following the above immunostaining protocol. Unlabeled anti--SMA antibody was used at 1:400 and revealed with FITC-labeled sheep anti-mouse IgG (8 µg/ml for 45 min at RT).
RESULTS
Location of p75NTR(+) Cells Within the Urogenital Ridge as a Function of Time
A time-course immunolocalization study of p75NTR expression within the developing urogenital ridge showed that p75NTR immunoreactive cells progressively appear in the fetal testis (Fig. 1). At 10.5 dpc (approximately seven tail somites), a cluster of p75NTR-bearing cells was present in the mesonephros, around the mesonephric tubules, whereas no staining was detected over the thin gonadal bud (Fig. 1b). At 11.25 dpc (15–16 tail somites), anti-p75NTR-labeled cells had moved to a mesonephric region abutting the gonadal bud, in which no staining was present (Fig. 1d). At these early stages of development (i.e., 10.5 and 11.25 dpc), gonads were positively identified on the sections by alkaline phosphatase staining of germ cells (Fig. 1, a and c). Six hours later (11.5 dpc, 18 tail somites), the gonadal ridge was more clearly outlined, and p75NTR immunoreactive cells were seen crowding at the interface between the mesonephros and gonad. In addition, columns of p75NTR(+) cells were, in most samples, present within the gonadal region proximal to the mesonephros (Fig. 1e). At 11.75 dpc, clusters and trabeculae of p75NTR immunoreactive cells were always clearly visible within the gonadal ridge (Fig. 1f). At 12.5 dpc, when testis cords have become quite evident in the male gonadal ridge, p75NTR(+) cells were seen to completely fill the stromal compartment (Fig. 1g).
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Identification of p75NTR Expression in the Developing Testis by Immunoblot Analysis
The histochemical finding of p75NTR immunoreactive cells in the developing testis and their progressive increase in number was validated by immunoblot analysis of protein extracted from 11.5-, 12.5-, and 13.5-dpc fetal testes and separated by SDS-PAGE. As shown in Figure 2, a 75-kDa immunoreactive band is recognized by the same antibody as used for immunohistochemistry; consistent with morphological observations, the band is only faintly visible at 11.5 dpc but becomes more intense with testicular development. The PC12 cells used as a positive control showed a qualitatively identical pattern. The ß-actin immunoreactive band, which was quantitatively similar in all the lanes, confirmed that equal amounts of protein were present in each sample.
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Organ Cultures
To confirm that the appearance of p75NTR(+) cells in the testis during urogenital ridge development was due to a cell influx from the mesonephros, gonads from CD1 male mice were cultured for 4 days with or without a grafted mesonephros (Fig. 3). When testes from 11.25-dpc embryos (15–16 tail somites) were perfectly cleared of their mesonephros and cultured for 4 days (six different experiments), no p75NTR(+) cells appeared in the organ (Fig. 3c). When a mesonephros and a testis from 11.25-dpc embryos were cultured in close apposition (five different experiments), they generally fused within 24 h, and p75NTR(+) cells appeared in the interstitial compartment of the testis (Fig. 3e), as occurred in control cultures of intact urogenital ridges (six different experiments; Fig. 3a). In all these experiments, testes were positively identified on sections of the cultured organs by alkaline phosphatase staining of germ cells (data not shown).
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Immunomagnetic Selection of p75NTR(+) Cells
The presence of the neurotrophin receptor on mesenchymal cells of the urogenital ridge was exploited to isolate such cell populations from the mesonephros. The enzymatic digestion of mesonephroi collected from 12.5-dpc male mouse embryos yielded a heterogeneous cell population in which only 68% ± 5.1% (average of nine experiments ± SD) of the cells were labeled by the anti-p75 neurotrophin receptor antibody; the nature of p75NTR-negative cells was not studied. After immunomagnetic sorting (six experiments), a cell population was obtained that was greatly enriched in p75NTR(+) cells (96% ± 3.5%; Fig. 4, a and b), with a 73% ± 4.9% recovery.
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Differentiation of p75NTR(+) Cells in Culture
Immunoselected cells were cultured for up to 3 days on coverglasses coated with a thin film of Matrigel, a reconstituted basement membrane. The expression of p75NTR and of -SMA, a marker of cell differentiation along the myoid phenotype, was evaluated at various culture times. At the beginning of the culture, no expression of -SMA was detected in the immunoselected p75NTR-expressing cells. After 18 h of culture, the cell population appeared to be heterogeneously stained for the two antigens: most of the cells were expressing the -SMA differentiation marker, and a minority were either double positive for p75NTR and -SMA or were still labeled for the p75NTR antigen only (Fig. 4, c and d). After 48 h of culture, nearly all of the cells expressed high levels of -SMA (Fig. 4e). Similar results were seen at 72 h of culture (data not shown).
Location of p75NTR(+) Cells in Postnatal Testis
Postnatal Day 5 mouse testicular sections were double immunostained with antibodies against p75NTR and -SMA. As shown in Figure 5, cells expressing the receptor (stained red) were seen to occupy the interstitial compartment, whereas a continuous layer of -SMA(+) cells (stained green) surrounded the seminiferous tubules. Cells coexpressing the two markers (stained yellow) were present as a thin, discontinuous layer interposed between the interstitial p75NTR(+) cells and the peritubular -SMA(+) cells.
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DISCUSSION
Neurotrophins are a family of related polypeptide growth factors that includes NGF, brain-derived neurotrophic factor, neurotrophin (NT)-3, and NT-4/5 [18]. These factors interact with two types of cell surface receptors: the p75NTR, formerly known as the low-affinity NGF receptor; and the trk family of tyrosine kinase receptors (i.e., trkA, trkB, and trkC). Each neurotrophin preferentially binds one of the trk members, whereas all neurotrophins are capable of binding the p75NTR. Although in most cases neurotrophin signal transduction requires that the two receptors interact to form a heterodimer, p75NTR may have, by itself and without interacting with trk receptors, biological functions in cell migration [19], cell invasiveness [20], and programmed cell death [21–24].
Neurotrophins and their receptors have been most extensively studied for their roles in nervous system development and neuron survival and differentiation [25–27]. The expression of neurotrophins and their receptors is, however, well documented in several nonneuronal tissues, in which they appear to play an autocrine or paracrine role for cell migration and tissue organization and maintenance, both during morphogenesis and in the adult [28, 29]. A putative involvement of the p75NTR as a morphogenetic inducer is supported by several observations. During kidney development in the rat, neurotrophins and their receptors are expressed at sites of mesenchymal/epithelial interactions and appear to be involved in tubulogenesis [30, 31]. Expression of p75NTR has also been reported at sites of similar cell interactions that affect tooth morphogenesis [32, 33] and inner ear development in chick and rat embryos [34]. The p75NTR immunoreactivity and mRNA transcripts have been observed in mesenchymal cells surrounding tracheal and bronchiole epithelium during rat lung development [35]. It should be emphasized that, in all cases, expression of the receptor begins well before such embryonal structures become innervated.
In the mouse, the gonadal ridge begins to differentiate between 10.5 and 11.5 dpc; at that time, when the mesonephros appears as a well defined structure and the gonad is just budding as a thickening on its ventromedial side, we have observed by immunolocalization studies that stromal cells in the mesonephric compartment express the p75NTR (Fig. 1). At these early stages of gonadal development, we have not been able to detect the expression of any neurotrophin (or of any trk receptor) by mesonephric or gonadal cells of the urogenital ridge [11]. The only other members of the neurotrophic system (in addition to p75NTR) that have been detected in the prenatal mouse testis are a truncated form of the trkB receptor and neurotrophin NT-3, which are coexpressed by mesenchymal cells of the developing testis at E14.5 (i.e., 
3 days after their arrival in the gonad) [11]. A similar situation exists in the developing rat testis, in which immunohistochemical detection of trkC on mesenchymal cells follows by 2 days the appearance of p75NTR-bearing cells in the interstitium [36]. The possibility cannot be excluded that in the mouse, at these early stages of development, the levels of neurotrophic factors are too low to be detected by the assay systems used, but in these cells, p75NTR may also possibly be responding to some novel ligand different from the known neurotrophins. An additional, intriguing possibility is that the presence of p75NTR on mesenchymal cells, in the absence of a trk receptor, might be used as a death receptor [21–24], possibly to eliminate cells that make errors in migration.
Substantial changes in the expression of neurotrophic system components occur in the postnatal rat and mouse testis, in which peritubular cells progressively lose the p75 receptor [10], whereas truncated trkB and neurotrophin NT-3 persist in the same cells in the mouse until puberty [11]. In the adult, p75NTR (or a truncated form of it) and NGF are expressed by cells of the seminiferous epithelium in the mouse [10, 37], rat [38–40], bovine [41], and human testis [42]. This intricate picture suggests that neurotrophins and their receptors play complex autocrine or paracrine roles in both testicular development and spermatogenesis.
Between 11.5 and 16.5 dpc, cell migration occurs from the mesonephros into the male gonad [2, 4]; such cell influx is essential for testicular morphogenesis [2]. As a first step to assess whether the mesonephric cell population bearing the neurotrophin receptor antigen is involved in such migration, we examined the location of p75NTR-expressing cells as a function of time during the early stages of urogenital development. As shown in Figure 1, at 10.5 and 11.25 dpc, p75NTR immunoreactive cells are strictly limited to the mesonephric compartment and then progressively appear within the gonadal ridge; at 12.5 dpc, when testis cords are well outlined in the developing gonad, the interstitial compartment of the testis is filled with such cells (Fig. 1g). Testicular expression of p75NTR persists throughout the subsequent fetal life and can still be detected during postnatal development (Fig. 5), until at least Day 10 [11].
In most samples, a small number of p75NTR(+) cells were already present in the testis bud at 11.5 dpc (Figs. 1e and 2). In a previous study, mesonephric cells began their migration into the testis at a slightly later time (after 11.5 dpc) [2]. Such a minor discrepancy in migration timing might be explained on the basis of differences in the mouse strains used (i.e., Q vs. CD1) or of differences in embryo staging. In addition, a recent study has shown that normal testis cords form in cultured XY CD1 gonads that have been removed from their mesonephros at 18 tail somites, corresponding to 11.5 dpc [6]. Because cord morphogenesis requires incoming mesonephric cells [2–4, 7, 8], this finding suggests that mesonephric cell migration has already begun by 11.5 dpc in CD1 mice. For this reason, in experiments requiring very accurate staging, such as the time-related immunolocalization of p75NTR(+) cells, to obtain a finer stage identification and to decrease sample variability, we staged the embryos by counting the number of tail somites [9, 13].
To show directly that the appearance of p75NTR(+) cells in the testis bud is strictly dependent on its association with the mesonephros, we organ-cultured male gonadal ridges taken from 15- to 16-tail somite embryos (i.e., 11.25 dpc) with or without a grafted mesonephros. In agreement with findings in previous studies [2, 3, 12], we observed little or no morphological differentiation in the early male gonad cultured in isolation from a mesonephros (data not shown) and no expression of p75NTR in the poorly differentiated testis (Fig. 3c). The appearance of p75NTR(+) cells in the interstitium of the developing testis only occurs in the presence of an attached mesonephros, reflecting the occurrence of a cell influx from the mesonephros to the developing gonad, as reported by other authors [2].
Experiments in the present study show that mesonephric mesenchymal p75NTR(+) cells are able to differentiate in vitro into myoid cells (as discussed below). Recent studies in which a transgenic mouse strain ubiquitously expressing ß-galactosidase was used as the mesonephric cell donor showed that immigrant blue cells colonize the testis and give rise to myoid, endothelial, and Leydig cells [4, 7, 8]. The possibility that precursors of such cell types directly originate within the developing testis, under the influence of diffusible factors released by the mesonephros, can be discounted on the basis of experiments showing that when a permeable filter is interposed between testis-mesonephros cocultures, the former differentiates rather poorly and the organization in cords is no better than that in cultures of isolated testes [2]. The mesonephric cell population bearing the p75NTR, as identified in the present study, appears to correspond to the migrating cell population described in the studies cited above because of its strictly limited localization to the mesonephric compartment at the earliest stages of urogenital ridge formation, its appearance in the testicular compartment at the same time that cell migration into the testis begins, and finally, because of the ability of these cells to differentiate into myoid cells both in vitro and in vivo (as discussed below).
A study in a controlled environment of the mechanisms that regulate the differentiation of embryonic mesenchymal cells into myoid cells requires that mesenchymal precursors be isolated to near-purity. Some of the classical cell purification methods (e.g., density gradients) can hardly be applied considering the limited number of cells that can be obtained from small embryonic organs. In preliminary experiments, we tried a preplating purification method based on the presence of cell adhesion molecules on differentiated cells [43–45]; in such experiments, mesonephric cell suspensions were preplated on collagen- or Matrigel-coated substrates to deplete the population of differentiated cells. A modest, nonsignificant enrichment in p75NTR(+) cells was obtained (data not shown), but this was not sufficient to allow further use of the purified population. The identification of p75NTR as a specific cell marker and the availability of antibodies against such a cell surface antigen have been previously exploited as a tool to obtain near-homogeneous neuronal cell populations from heterogeneous cell suspensions through the use of panning [46], fluorescence-activated cell sorting [47], and immunomagnetic sorting [48]. In the present study, by using a simple, commercial magnetic immunoselection method (MiniMACS), we obtained a highly enriched population of mesenchymal cells of mesonephric origin bearing the neurotrophin receptor. This technique allowed us to obtain a near-homogeneous (96% p75NTR+) mesonephric cell suspension with a good cell recovery (73%; Fig. 4, a and b).
The immunoselected cell population cultured on a Matrigel-coated substrate in the presence of 10% FCS differentiated into myoid cells: the peritubular smooth muscle cells (Fig. 4). The possible presence of markers of Leydig and endothelial cell differentiation was not examined. Myoid cell differentiation can be evaluated by the expression of smooth muscle phenotypic markers such as desmin [49, 50], -SMA [51], and integrins [43–45]. At early culture times, -SMA began to be coexpressed with p75NTR in some of the cells (Fig. 4, c and d). As differentiation proceeded, -SMA became clearly visible in most of the cells as thick cables (Fig. 4e), whereas p75NTR disappeared (data not shown), suggesting that p75NTR is a marker of the undifferentiated precursors of the myoid cells. The same conclusion can be drawn by examining postnatal testicular sections (Fig. 5), in which cells expressing both markers are visible as a thin, discontinuous layer between the p75NTR(+) cells of the interstitial compartment and the differentiated -SMA(+) cells surrounding the tubules. Precursors of myoid cells with mesenchymal characters have been described within the myoid cell layer in the early postnatal rat testis [52].
In conclusion, in the present study, we propose p75NTR as a novel marker for mesenchymal cells that migrate from the mesonephros into the developing testis. The identification of a marker for such a cell population might be of great help in further studies of the mechanisms that regulate migration and differentiation of these cells.
ACKNOWLEDGMENTS
The authors wish to thank Mr. Gabriele Rossi for skilled technical assistance and Mr. Graziano Bonelli for preparation of the photographs.
FOOTNOTES
First decision: 18 August 2000.
1 Supported by grants from Ministero dell'Università e della Ricerca Scientifica e Tecnologica. 
2 Correspondence: Gregorio Siracusa, 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; e-mail: g.siracusa@uniroma2.it
Accepted: September 11, 2000.
Received: July 19, 2000.
REFERENCES
6ß1 is localized at specific sites of cell-to-cell contact in rat seminiferous epithelium. Biol Reprod 1995; 52:79–87.[Abstract]-smooth muscle isoactin is a specific differentiation marker. Biol Reprod 1990; 42:351–365.[Abstract]This article has been cited by other articles:
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|
 |
|
  A. Jeanes, D. Wilhelm, M. J Wilson, J. Bowles, P. J McClive, A. H Sinclair, and P. Koopman Evaluation of candidate markers for the peritubular myoid cell lineage in the developing mouse testis Reproduction, October 1, 2005; 130(4): 509 - 516. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 M. Uzumcu, K. A. Dirks, and M. K. Skinner Inhibition of Platelet-Derived Growth Factor Actions in the Embryonic Testis Influences Normal Cord Development and Morphology Biol Reprod, March 1, 2002; 66(3): 745 - 753. [Abstract] [Full Text] [PDF]
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