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Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, Edinburgh EH16 4SB, United Kingdom
Address all correspondence to: Dr. R. A. Anderson, Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Chancellor’s Building, Little France Crescent, Edinburgh EH16 4SB, United Kingdom. E-mail: r.a.anderson{at}hrsu.mrc.ac.uk.
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Abstract |
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Introduction |
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A number of paracrine factors controlling migration, differentiation, and proliferation of primordial germ cells and Sertoli cells, including members of the TGFß family, have been described in the rodent (11, 12, 13, 14). Neurotrophins are members of the nerve growth factor (NGF) family and are related to TGFß. They regulate neuronal survival and differentiation in nervous tissue (15, 16) and include NGF, brain-derived neurotrophic factor (BDNF), neurotrophin (NT) 3, NT4 (also known as NT5), and NT6 (16). Many of the effects of the neurotrophins are mediated via high-affinity tyrosine kinase (Trk) receptors, which have specificity for the various neurotrophins (17). Three members of the Trk receptor family have been described: TrkA, the receptor for NGF; TrkB, the receptor for BDNF and NT4; and TrkC, the receptor for NT3. Truncated forms of the TrkB and TrkC receptors, which lack the intracellular tyrosine kinase domains, have also been described, although their function is unknown (16, 17, 18). All neurotrophins are also recognized by a more widely expressed receptor known as p75, which is a member of the TNF receptor family (19).
Neurotrophins also may function in nonneuronal tissues (20), including the developing gonads of both sexes. For example, several members of the neurotrophin family and their receptors have been identified in the developing rodent testis (21, 22, 23, 24), and roles in testicular cord formation (23) have been suggested in addition to later involvement in the function and interaction of germ cells and Sertoli cells (25, 26, 27). Preliminary data suggest that members of the neurotrophin family and their receptors may also be expressed in the developing human testis (24). Recent studies have shown that NT4 and its cognate high-affinity receptor TrkB may be of particular importance in the interaction between germ cells and somatic cells in the developing ovary of both rodent and human (28, 29). To determine whether neurotrophins could influence development and maturation of the human fetal testis, we have examined the cell-specific expression and distribution of several members of the neurotrophin family and their receptors during the second trimester, with particular emphasis on NT4 and TrkB.
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Materials and Methods |
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Human fetal testes (13–19 wk gestation) were obtained after medical termination of pregnancy. Women gave consent according to national guidelines (30), and the study was approved by the Lothian Paediatrics/Reproductive Medicine Research Ethics Sub-Committee. Termination of pregnancy was induced by treatment with mifepristone (200 mg orally), followed by 1 mg prostaglandin E1 analog (Gemeprost, Beacon Pharmaceuticals, Tunbridge Wells, UK) administered every 3 h per vaginum. None of the terminations were for reasons of fetal abnormality, and all fetuses appeared morphologically normal. Gestational age was determined by ultrasound examination before termination and confirmed by subsequent direct measurement of foot length. A total of 15 specimens were used for this study (13 wk, two specimens; 14 wk, three; 15 wk, two; 16 wk, two; 17 wk, four; and 19 wk, two specimens).
Testes were dissected free and either fixed for immunohistochemical analysis or snap frozen and stored at -70 C. Fixation was carried out in Bouin’s fixative for 5 h, followed by transfer to 70% ethanol, before processing into paraffin, using standard methods.
Isolation of RNA and RT-PCR
Total RNA was extracted from snap-frozen samples of fetal testis using the RNeasy mini kit (Quiagen, Crawley, UK). RNA was treated with DNAase (Life Technologies, Inc., Paisley, UK), and reverse transcription was performed using a first-strand cDNA synthesis kit (Roche Diagnostics, Lewes, UK) on aliquots containing 1 µg. Subsequently, PCR was performed as described previously (29). Two control tubes were run in parallel, one in which water replaced the RNA and a second omitting reverse transcriptase to ensure there was no genomic DNA contamination. Primers specific for human neurotrophins and their receptors were used and designed to span an intron in the case of the Trks, to prevent amplification of any contaminating DNA (Table 1
). Primers for the constitutively expressed gene GAPDH were used to confirm the integrity of the RNA and efficacy of the PCR reaction. The identity of all PCR products was confirmed by direct sequencing using an Applied Biosystems (Foster City, CA) 373A automated sequencer.
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Riboprobes were generated using a PCR strategy to incorporate SP6 or T7 phage promoter sequences into NT4 PCR product (29). Probes were labeled with digoxigenin using a commercially available kit (Maxiscript, Ambion Inc., Huntingdon, UK). Briefly, DNA templates (300 ng) were incubated for 60 min at 37 C with transcription buffer; 10 U ribonuclease inhibitor; 0.5 mM each of rATP, rCTP, rGTP; 0.33 mM rUTP; 0.17 mM digoxigenin-11-UTP; and 30 U of appropriate RNA polymerase in a final volume of 20 µl. After the addition of 2 U DNAase I (RNAase-free), the probes were incubated at 37 C for an additional 15 min before being purified through Chromaspin columns (DEPC-100, Clontech, Palo Alto, CA).
In situ hybridization was carried out as described previously (29). Briefly, 5-µm sections were cut and mounted before immersion in 0.4 M HCl for 20 min. Slides were then washed and treated with 1.5 µg/ml proteinase K at 37 C for 10 min in 0.1 M Tris/HCl and 0.05 M EDTA (pH 8.0), transferred to 0.2% glycine at 4 C for 10 min, acetylated with 0.25% acetic anhydride in 0.08 M triethanolamine (pH 8.0) for 10 min, and washed in 4x sodium saline citrate (SSC) for 5 min. Sections were incubated with prehybridization buffer at 50 C for 2 h and then hybridized at 50 C overnight with hybridization buffer.
On the next day, sections were washed in 4x SSC for 10 min, then incubated in RNAase A at 37 C for 30 min, washed twice in 2x SSC for 5 min, transferred to 0.1x SSC with 30% formamide at 37 C for 30 min, and washed in Tris-buffered saline (TBS, pH 7.4) for 5 min. For detection of digoxigenin, labeled sections were then incubated sequentially at room temperature, with 2x 5 min washes in TBS between steps, in: 1) 3% H2O2 in TBS for 30 min; 2) normal rabbit serum (NRS), 1:5 dilution in TBS containing 5 drops/ml avidin block (Vector Laboratories, Peterborough, UK) for 30 min; 3) biotin block (Vector Laboratories) 8 drops/ml in TBS for 30 min; 4) sheep antidigoxigenin (1:100 in NRS/TBS; Roche Diagnostics) for 2 h; 5) biotinylated rabbit antisheep IgG (1:500 in NRS/TBS; Vector Laboratories) for 30 min; 6) ABComplex-horseradish peroxidase (HRP) (DAKO Corp., Cambridge, UK) for 30 min; and 7) diaminobenzidine liquid substrate-chromagen system (DAKO Corp.) for approximately 2 min. Sections were then counterstained in hematoxylin, dehydrated, and coverslip mounted with Pertex mounting medium (Cellpath, Newtown, UK).
Immunohistochemistry
Immunohistochemistry was performed to localize the expression of NGF, NT3, NT4, the receptors TrkB and p75, and bromodeoxyuridine (BrdU), as described previously (29). Briefly, 5-µm sections were mounted, dewaxed, and rehydrated. Antigen retrieval was used for TrkB only by immersion of slides in boiling 0.01 M citrate buffer (pH 6.0) for 2.5 min, then leaving them to stand in the buffer for 10 min. In all cases, endogenous peroxidase activity was inhibited by incubation in 3% H2O2 in methanol for 30 min. After a wash in water, slides were transferred into TBS [0.05 M Tris and 0.85% NaCl (pH 7.4)] for 5 min and blocked for 30 min in the appropriate diluted serum. Sections were then blocked with avidin (0.01 M, 15 min) and biotin (0.001 M, 15 min) (both from Vector Laboratories), with washes in TBS in between. The following primary antibodies were used: NGF, NT4, NT3 (all rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), p75 (mouse monoclonal; Neomarkers, Fremont, CA), anti-BrdU (mouse monoclonal; Roche Diagnostics). These were applied at dilutions of 1:30, 1:100, 1:50, 1:25, and 1:30, respectively. Three antibodies against TrkB were used [rabbit polyclonal (Oncogene, San Diego, CA), rabbit polyclonal (Santa Cruz Biotechnology), and chicken polyclonal (Promega, Southampton, UK)] in serial dilutions of 1:25, 1:50, and 1:100 in the appropriate serum at 4 C overnight.
Sections were then washed and incubated for 30 min with a biotinylated secondary antibody diluted 1:500 in the appropriate serum. For NGF, NT4, and NT3, a biotinylated antirabbit secondary antibody was used; for p75 and anti-BrdU (both DAKO Corp.), biotinylated antimouse was applied; and for TrkB, both biotinylated antirabbit (DAKO Corp.) and antichicken (Jackson Laboratories, Bar Harbor, ME) secondary antibodies were used. After washes in TBS, sections were incubated with avidin biotin HRP-linked complex (DAKO Corp.) according to the manufacturer’s instructions, and bound antibody was visualized using 3,3'-diaminobenzidine tetra-hydrochloride (DAKO Corp.). Primary antibodies were omitted as negative controls, except for NGF, in which the primary antibody was preabsorbed with the blocking peptide (Santa Cruz Biotechnology).
All sections were counterstained with hematoxylin, dehydrated, mounted, and visualized by light microscopy. Images were captured using an Olympus Provis microscope (Olympus Optical Co., London, UK) equipped with a Kodak DCS330 camera (Eastman Kodak, Rochester, NY) and assembled using Photoshop 6.0 (Adobe, Mountain View, CA).
Immunoblotting
Immunoblotting was carried out according to the protocol described previously (29). Briefly, fetal testes were homogenized in denaturing buffer, and samples (20 µg protein) were diluted with an equal volume of reducing loading buffer and boiled for 5 min. Proteins were separated by SDS-PAGE on a 4–20% gradient Tris-glycine gel using Tris-glycine SDS running buffer (both from Novex, Invitrogen, Amsterdam, The Netherlands) in parallel with prestained protein molecular weight markers (Bio-Rad, Hercules, CA) and blotted onto polyvinylidene difluoride membranes (Amersham Life Sciences, Buckinghamshire, UK) overnight using a wet-blot apparatus (Bio-Rad).
Thereafter, membranes were soaked for 5 min in methanol and washed briefly several times in TBS. They were then blocked for 8 h at room temperature in 0.02 M TBS (pH7.6) containing 3% weight/volume BSA (Sigma) and 5% powdered milk. Membranes were washed in TBS with 0.1% Tween 20, and then incubated for overnight with the primary antibody. Antibodies to NT4 (rabbit polyclonal; Santa Cruz Biotechnology), full-length TrkB (rabbit polyclonal; Oncogene), and p75 (mouse monoclonal; Neomarkers) were applied at dilutions of 1:500, 1:50, and 1:500, respectively, in TBS with 0.1% Tween 20 and 1% BSA at 4 C. Primary antibody was omitted as a negative control. Bound antibody was detected using either antirabbit or antimouse HRP-linked secondary antibodies (1:4000; Amersham Life Sciences) and the enhanced chemiluminescence visualization system (Amersham Life Sciences) according to the manufacturer’s instructions.
Testis culture
Testes from three fetuses of 14, 16, and 19 wk gestation were dissected free of adherent tissues using sterile technique, bisected longitudinally, and then cut into slices approximately 0.5 mm thick under a dissecting microscope. Samples of fresh tissue were fixed for histological analysis. The remaining tissue fragments were cultured on 0.4 µm pore Millicell CM filters (Millipore, Bedford MA) in a 24-well plate (Transwell, Costar, High Wycombe, UK). To each well 0.4 ml of medium was added, enough to just cover the tissue fragments. Any remaining wells were partially filled with medium to maintain humidity in the culture vessel.
The medium comprised MEM
(Life Technologies, Inc., Paisley, UK) containing 3 mg/ml BSA, antibiotics (100 IU/ml penicillin, 100 µg/ml streptomycin sulfate, and 0.125 µg/ml amphotericin), 5 µg/ml insulin, 5 µg/ml transferrin, and 5 µg/ml sodium selenite, 2 mmol/liter glutamine, and 2 mmol/liter pyruvate (all chemicals supplied by Sigma). To some wells the Trk inhibitor K252a (Calbiochem, Nottingham, UK) was added, at a concentration of 100 nmol/liter. Dimethylsulfoxide was used as the solvent for K252a and was added to control wells at the appropriate dilution. The cultures were maintained at 37 C in a humidified incubator, under 5% CO2, in air for 48 h. After 24 h, the medium was removed and replaced with fresh medium containing 30 µmol/liter BrdU (Sigma) to label proliferating cells. At the end of the culture period, tissue fragments were fixed for 1 h in Bouin’s fluid, and then transferred to 70% ethanol before embedding in paraffin wax for histological analysis. The tissue was sectioned at 5 µm thickness and immunostained for BrdU, using the protocol described above.
Sections of uncultured control and cultured tissue were analyzed to investigate the effects of culture and of K252a on the number and proliferation of the various cell types present. Analysis was carried out blind using the Area Fraction Probe in the Stereologer software program (Systems Planning and Analysis Inc., Alexandria, VA), as described previously (31). Tissue was serially sectioned, and sections greater than 20 µm apart were counted, ensuring the same cells were not counted more than once. The counting was performed using a 121-point grid in the eyepiece of the microscope. Only cells whose nuclei lay beneath the intersections on the grid were counted. The number of germ cells, Sertoli cells, peritubular cells, and interstitial cells present per randomly chosen grid were recorded, with the number of each cell type that was immunostained for BrdU. Between 25 and 73 grids were counted for each testis piece, as determined by the program. The number of points lying outside the tissue in any grid was also recorded and the total cell numbers counted were corrected for this. The average number of each cell type present per grid was calculated for each experimental condition, and data were analyzed using paired t tests.
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Results |
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Expression of mRNAs was detected by RT-PCR using RNA extracted from fetal testes (Fig. 1). RT-PCR for the constitutively expressed gene GAPDH was used to confirm the integrity of the RNA. Human fetal brain and placenta were used as positive controls (data not shown). mRNA for the neurotrophins NGF (Fig. 1a), NT3 (Fig. 1b), NT4 (Fig. 1c), and BDNF (Fig. 1d) and the receptors TrkA (Fig. 1f), TrkB (Fig. 1h), TrkC (Fig. 1g), and p75 (Fig. 1j) was detected in all specimens at all gestations examined. Both full-length and the truncated forms of TrkB were detected (Fig. 1 h and i). Products of 228- and 204-bp size were detected for TrkC (Fig. 1 g), representing spliced variants of the gene (32) confirmed by sequencing. In some samples at all gestations an additional PCR product of 300 bp was noted and the sequence found to be unrelated to the tyrosine kinase receptor family.
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Cell-specific patterns of expression of NT4 were further investigated by nonradioactive in situ hybridization on fixed tissue sections. NT4 mRNA expression was predominantly localized to the peritubular cells (Fig. 2, A and B). NT4 mRNA was also detected in Sertoli cells but no mRNA appeared to be expressed in gonocytes (Fig. 2B). A low level of expression was detected in the interstitium, with the exception of the endothelial cells of small blood vessels, which showed more clearly positive expression. There was no apparent change in the pattern or intensity of expression of NT4 mRNA over the gestational range examined (14–19 wk). Tissue sections incubated with the sense riboprobe showed no staining (Fig. 2A, inset) confirming the specificity of the probe.
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Expression of NT4, NGF, NT3, and p75 proteins was detected by immunohistochemistry in all specimens examined across the gestational range of 14–19 wk. Expression of NT4 was confirmed by immunolocalization of the protein to the cytoplasm of the peritubular cells and Sertoli cells within the seminiferous tubules; gonocytes were immunonegative (Fig. 2
, C and D). Very little immunostaining was noted in the interstitial cells, but endothelial cells within small blood vessels were immunopositive for NT4 (data not shown). The most intense immunopositive reaction for NGF was in Sertoli cells (Fig. 2E), although some gonocytes also appeared immunopositive. Weak staining was noted in the interstitial cells (Fig. 2E). NT3 protein was also immunolocalized mainly to the Sertoli cells, with some interstitial staining (Fig. 2F). Very few gonocytes stained positively for NT3, and no protein was detected in the peritubular cells.
Immunolocalization of p75 was most intense in the peritubular cell compartment (Fig. 2, G and H), but it was also present in the interstitial cells. Neither Sertoli cells nor gonocytes showed any p75 immunoreactivity. Despite the use of three different antibodies, specific staining for TrkB receptor protein was not detectable.
No change in pattern or intensity of staining for any of the proteins detected was noted over the gestational range examined. Control experiments conducted by omitting the application of the primary antibody or using a specific blocking peptide, in the case of NGF, showed no staining (Fig. 2, C and E, insets).
Immunoblotting
The presence of NT4, TrkB, and p75 proteins in the fetal testis was confirmed by immunoblotting; a positive control of rat cerebral cortex was run on all gels (Fig. 3). The immunoblot of NT4 protein expression detected a single band of 21 kDa molecular mass in all samples of fetal testis (Fig. 3A). For detection of TrkB, the antibody used was directed against the full-length protein. Two immunoreactive bands of 110 kDa and 80 kDa molecular mass were detected (Fig. 3B). These bands are reported to represent the glycosylated and unglycosylated forms, respectively, of the TrkB protein (33). Two immunoreactive bands were also detected for the p75 receptor, at 75 kDa and 65 kDa, in all samples of fetal testis (Fig. 3C). In all cases, the sizes of the proteins detected were identical to those in the positive control tissue. These immunoblots were repeated three times using different samples; no consistent changes with gestation were noted. A negative control was also performed for all three proteins by omitting the primary antibody, and in all cases immunoreactivity was abolished.
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After 48 h of culture, there was a small increase in the number of gonocytes in comparison with the time 0 samples (P = 0.02) (Fig. 4A) and a decrease in the number of interstitial cells (P = 0.05). The numbers of Sertoli cells and peritubular cells did not change significantly during this period of culture. Consistent with this increase in gonocyte number, an average of 12% of gonocytes were immunostained with BrdU, compared with less than 2% of Sertoli cells and peritubular cells.
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). There was also a significant fall in the number of peritubular cells (P = 0.006) and a small but statistically significant (P = 0.04) reduction in the number of Sertoli cells (Fig. 4A). In contrast, there was an increase in the number of interstitial cells, but this did not reach statistical significance. The number of proliferating (i.e. BrdU immunopositive) gonocytes was reduced by an average of 58%, from 2.4 ± 0.6 to 0.9 ± 0.2 per grid (Fig. 4, B–D), very similar to the reduction in the number of gonocytes (54%). The number of proliferating peritubular cells was also reduced, by a mean of 71%, from 1.9 ± 0.8 to 0.5 ± 0.1 per grid, compared with a 30% reduction in the number of peritubular cells. Thus, there was a mean fall of 26% in the proportion of gonocytes that were BrdU immunopositive, compared with a fall of 71% for peritubular cells. However these decreases did not reach statistical significance. There was also a reduction in the number of proliferating Sertoli cells, from 0.8 ± 0.5 to 0.3 ± 0.0 per grid, which again did not reach statistical significance. |
Discussion |
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Neurotrophins are small, secreted proteins related to the TGFß superfamily, originally identified on the basis of their role in the regulation of neuronal survival. However, neurotrophins also may regulate cell migration (35) and differentiation (36). In addition to signaling through high-affinity Trk receptors, neurotrophins also signal through the p75 receptor, and the pattern of expression of the different receptor types influences the response (37, 38). Further interaction is suggested by the demonstration that the immature form of NGF, termed proNGF, has a greater affinity for p75 than the mature form of NGF (39). Thus, neurotrophin signaling is a complex interaction between the ligands and the Trk/p75 receptor compliment present. In addition to the full-length TrkB, we have identified mRNA for truncated TrkB in the developing testis. The truncated form lacks the tyrosine kinase domain and is predominantly expressed in nonneuronal cells (33, 40). Although the function of truncated receptors remains elusive, it has been postulated that these isoforms have a role as cell adhesion molecules acting as a selective barrier preventing the diffusion of neurotrophin and promoting elimination by internalization (41, 42).
There are increasing data regarding the expression of neurotrophins and their receptors in the developing rodent testis but very little data in the human. In the rodent, testis expression has been detected from very early in development (21, 23). The present data suggest a cell-specific distribution of the neurotrophins, although some cell types, such as Sertoli cells, expressed more than one neurotrophin. Expression of multiple neurotrophins may be required for normal development of some neuronal cell types (43). We were unable to localize TrkB using immunohistochemistry although, using RT-PCR and Western blotting analysis, expression of both full-length and truncated TrkB mRNA and the full-length protein was demonstrated. Previous reports of predominant localization of p75 in the peritubular cells in the rat (22, 23, 24) are in agreement with our findings in the human. This pattern of expression shows some parallels with that seen in the rodent and human fetal ovary (28, 29) in which p75 is localized to the stromal cells surrounding clusters of replicating germ cells.
Functional roles for the neurotrophins in morphological sex determination, cell migration, and testicular cord formation have been suggested (23, 24). Treatment of organ cultures of E13 rat testis with K252a inhibited cord formation (23). In the present studies, we have also used K252a to investigate the functional activity of neurotrophin signaling in the developing human testis. K252a is an indole carbazole, widely used as a potent and selective inhibitor of the intracellular protein kinase domain of Trk receptors without affecting other serine/threonine kinases at the concentrations used here (23, 44, 45). K252a blocks the activity of all high-affinity Trk receptors, so these data do not permit interpretation in terms of which neurotrophin(s) and receptor(s) might be involved in the effects observed. Although the investigation of selective blockade of specific Trk receptors has been attempted in the investigation of many models, including the developing testis (23), the effects are generally much less marked than those of K252a, consistent with considerable redundancy in neurotrophin signaling, as also demonstrated by the phenotypes of knockout animals (16). In this culture system, the integrity of the tissue was well maintained, and cell proliferation was detected. This was noted to be at a much higher rate in gonocytes that in other cell types. The major effect of K252a on the testicular cords was on gonocyte survival, with relatively little effect on Sertoli cells. Sertoli cells were the primary site of expression of NGF and NT3, and although the site of expression of Trk receptors was not demonstrated in the present study, TrkA has been previously reported to be expressed by rat Sertoli cells between E16 and P0 (21). These results suggest that neurotrophins are critical for gonocyte survival and replication, but their effects may be mediated indirectly via Sertoli cells. This is the classic pattern of Sertoli cell/germ cell interaction, with paracrine signaling between these two cell populations, most clearly exemplified by the mediation of the effects of FSH and androgen. It is also apparent that the relative number of gonocytes and Sertoli cells is maintained during early testicular development despite a 10-fold increase in the number of gonocytes between 6 and 9 wk gestation (6); the present data suggest that neurotrophin signaling may contribute to the regulation of this ratio. Selective gonocyte loss has not previously been reported as a result of neurotrophin blockade, but cord formation is certainly affected, inevitably involving gonocyte survival (23). These data may be compared with the recent demonstration that neurotrophins are involved in germ-cell survival in the human fetal ovary at a comparable stage of development (29), although in the ovary it appears that germ cells are a direct site of neurotrophin action. Although that may also be the case in the testis, direct evidence is thus far lacking.
Whereas K252a resulted in a marked loss of gonocytes, there was also a reduction in the number of peritubular cells. There was also a striking reduction in gonocyte and peritubular-cell proliferation. Because the fall in the number of BrdU immunopositive peritubular cells was much greater than the fall in the total number of that cell type (71% vs. 30%), whereas the comparable figures for gonocytes were similar (58% vs. 54%), these data may suggest a specific effect of neurotrophin on peritubular-cell proliferation in addition to survival, consistent with those cells being the major site of expression of NT4 and the p75 receptor. The importance of neurotrophins and their receptors in rat testicular cord formation (21, 23) may also reflect a major site of action on the peritubular cell population. Because the p75 receptor may promote apoptosis in the absence of Trk receptor signaling (37, 46), relatively unopposed p75 signaling may contribute to the decrease in peritubular cell proliferation and survival in the presence of K252a.
We have recently demonstrated the expression of estrogen receptor (ER) ß within the human fetal testis (47). Sertoli and peritubular cells were demonstrated to express both ERß 1 and 2 isoforms, whereas gonocytes expressed only ERß2, indicating that Sertoli and peritubular cells in particular may be sites of estrogen action. Estrogen and neurotrophins interact in neural development, selectively enhancing neuronal growth and development (48), and ER mRNA may be coexpressed with neurotrophins (49, 50). Some of these effects of estrogen may be mediated by ER other than the classical ER (51). The expression of both neurotrophins and their receptors and ERß in Sertoli cells and peritubular cells suggest that these pathways may interact in a wide variety of cell types.
In conclusion, this study demonstrates that neurotrophins and their receptors are expressed in the developing human testis during the second trimester. Neurotrophins have been demonstrated to be crucial for cellular migration, germ-cell survival, and proliferation (21, 22, 23, 24, 34) in the rodent testis; and the present results suggest that they are likely to be of similar importance in the human, with a major role in the regulation of proliferation and survival of germ cells and peritubular cells.
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Footnotes |
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Received February 6, 2003.
Accepted April 21, 2003.
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and the epidermal growth factor receptor in embryonic rat testis development. Biol Reprod 62:477–490This article has been cited by other articles:
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  S.M. Coutts, N. Fulton, and R.A. Anderson Environmental toxicant-induced germ cell apoptosis in the human fetal testis Hum. Reprod., November 1, 2007; 22(11): 2912 - 2918. [Abstract] [Full Text] [PDF]
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, Uncultured, time 0 samples; 
, control samples cultured for 48 h; 
, tissue treated in culture with K252a neurotrophin receptor inhibitor. Mean ± SEM; *, P < 0.05 vs. control 48 h cultures. B, Number of proliferating (BrdU immunopositive) cells of each type present per grid in the human fetal testis after 48 h of culture.