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Regular Article |
a Center for Reproductive Biology, School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4231
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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- and ß-receptors were expressed in isolated testes during all developmental periods examined. Immunoreactivity for PDGF was present throughout the testicular compartment at E14, restricted primarily to testicular cords at E16, and present in cells of the testicular cords with a stronger immunoreactivity in certain interstitial cell types of P0 testis. PDGFR ß-receptor immunoreactivity was primarily localized to the mesonephros of E14 organs and the testicular interstitium of E16 and P0 testes. Tyrphostins did not affect apoptotic cell number in the testis. PDGF had no effect on cell growth in P0 testis cultures. The results show that PDGFs and PDGFRs are expressed in embryonic testis during cord formation in a tissue-specific manner. Inhibition of PDGF actions does not inhibit cord formation but does alter normal cord development and morphology. The observations provide insight into the factors involved in male sex differentiation and embryonic testis development.
developmental biology, early development, growth factors, Sertoli cells, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Organ culture experiments have provided information about the importance of mesonephric cell migration and epithelial-mesenchymal interactions during cord formation. Mesonephric cell migration that takes place between E12.5 and E16.5 is a testis-specific event not present in the embryonic ovary [7]. The migration is not dependent on the sex of the animal from which the mesonephros is taken [6, 7]. Mesonephric cells labeled with a traceable marker were found to migrate into the undifferentiated testis and become peritubular cells around the cords and selected interstitial cells [6]. It is likely that mesonephric cell migration is under the influence of chemotatic factors from Sertoli cells and is prompted upon initial Sertoli cell differentiation [7].
The identity of the chemotactic factor(s) that induces mesonephric cell migration have recently been investigated. Migrating cells from the mesonephros can be identified by expression of the low-affinity neurotropin receptor p75/LNGFR [8]. Previous studies have demonstrated that p75/LNGFR is expressed in a tissue- and sex-specific manner in embryonic rat gonad and mesonephros. Before cord formation, the receptor is localized in the mesonephros. After cord formation (i.e., E14), the receptor localizes to the testis cords, whereas no expression is observed in the ovary until E16.5 [9]. The neurotropin ligand NT3 is localized in testicular cords, while its high-affinity receptor Trk C is expressed in peritubular myoid cells [9]. Localization showed that although neurotropins (i.e., NT3 and nerve growth factor) are primarily expressed inside the cords by Sertoli cells, the high-affinity neurotropin receptors (Trk C and Trk A) are present either in mesonephric cells before cord formation or in the interstitium after cord formation [10]. Interestingly, blocking neurotropin actions with specific inhibitors caused inhibition of cord formation, suggesting that neurotropins produced by Sertoli cells play a role in migration of mesonephric cells (e.g., peritubular myoid cell precursor) and cord formation [10]. Observations suggest that neurotropins play a role in testicular cord formation. It is proposed that other paracrine factors may also be involved.
Platelet-derived growth factors (PDGFs) are paracrine growth factors that mediate epithelial-mesenchymal interactions in various tissues during normal and abnormal processes such as embryo development, wound healing, and tissue fibrosis. Some of the cellular responses that PDGF induces are cell proliferation, migration, and differentiation [11]. PDGF is composed of two polypeptide chains named A-chain and B-chain that are encoded by homologous but distinct genes and can combine to form homodimers (AA or BB) or a heterodimer (AB). PDGF receptors are members of the receptor tyrosine kinase family of receptors. Two receptor types have been identified: the PDGF -receptor, which binds the A- and B-chains with high affinity, and the PDGF ß-receptor, which binds the B-chain with high affinity. Therefore, PDGF-AA induces the -receptor, and PDGF-AB induces the -receptor and the ß-receptor, while PDGF-BB induces all three receptor subtypes. More recently, PDGF-C and PDGF-D have been described as new family members that bind to and activate - and ß-receptors, respectively [12, 13].
The presence of PDGF and its receptors has been described in perinatal, pubertal, and adult rat testis [14]. In prenatal and early postnatal animals, the primary expression site for PDGF is Sertoli cells, whereas peritubular myoid cells express PDGF receptors. In adult animals, both receptors and ligands are primarily expressed in Leydig cells. Since PDGF induces peritubular cell chemotaxis, a role for PDGF in myoid cell proliferation and migration has been proposed during testicular development [14]. The objective of the present study was to examine the presence and action of PDGF and PDGF receptors in the embryonic testis at the time of cord formation. The hypothesis tested was that PDGF and its receptor are expressed in testis at the time of cord development and that interruption of PDGF action interferes with normal cord formation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Timed pregnant Sprague-Dawley rats were bred in the institutional vivarium at Washington State University. Institutional Animal Care and Use Committee guidelines were followed in all procedures. On Embryonic Day 13 (E13; sperm-positive plug date was considered to be E0), gonads were dissected with the mesonephros attached. The organs were cultured in drops of medium on Millicell CM filters (Millipore, Bedford, MA) floating on the surface of 0.4 ml of CMRL 1066 media (Gibco BRL, Gaithersburg, MD) supplemented with penicillin-streptomycin, insulin (1 µg/ml), and transferrin (10 µg/ml). PDGF receptor tyrosine kinase-specific inhibitors, tyrphostins AG1295 and AG1296 (Calbiochem, La Jolla, CA), were dissolved in vehicle (DMSO) and used to treat one of the gonads, and the contralateral gonad served as control (final concentration of DMSO, 0.1%). Control organs were also treated with 0.1% DMSO. The medium was changed on the second day of culture. E13 gonads and mesonephros were typically maintained for 3 days in culture, at which point cords were developed in the paired controls [15]. Images of whole organs were obtained using a digital image analysis system (Diagnostic Instruments Inc., Sterling Heights, MI). Organ cultures involved at least three separate experiments, and each experimental group consisted of five to six male embryos.
Genomic DNA Isolation and Polymerase Chain Reaction for SRY
Polymerase chain reaction (PCR) for SRY was conducted on each embryo as previously described [15] to determine the sex of E13 embryos. Briefly, embryonic tails were collected to isolate genomic DNA by standard procedures. The tissue was homogenized in a digestion buffer (400 mM NaCl, 50 mM Tris pH 7.5, 100 mM EDTA, 0.5% SDS), and treated with proteinase K (0.57 mg/ml) for at least 4 h at 55°C. The samples were centrifuged, and the supernatant was collected. The supernatant was mixed with an equal volume of 100% ethanol, and DNA was precipitated. The pellet was washed with 500 µl of 70% ethanol. The pellet was air-dried, resuspended in 500 µl buffer (100 mM Tris, pH 7.5, and 1 mM EDTA) and incubated at 65°C for 15 min. The PCR was performed using 1 µl of genomic DNA with primers for SRY. The SRY primers had the following sequences: 5'-CGGGATCCATGTCAAGCGCCCCATGAATGCATTTATG-3' and 5'-GCGGAATTCACTTTAGCCCTCCGATGAGGCTGATAT-3'. The PCR was performed using an annealing temperature of 55°C for 30 cycles to yield a product of 240 base pairs (bp).
Hematoxylin and Eosin Staining, Morphometry, and Cell Apoptosis
After the whole organs were imaged, they were fixed in Bouin solution for 2 h and stored in 70% ethanol until embedded in paraffin. Paraffin-embedded tissues were serially sectioned. The tissue sections closest to the largest cross-sectional area of the organs were used in the analyses.
Hematoxylin and eosin staining and morphometry One of the serial sections from each experiment was stained with hematoxylin and eosin using standard procedures. Stained sections were imaged by light microscopy to assess the cord number per testis and cord diameters. To obtain cord numbers per testis, the cords were counted in an entire cross-sectional area of the testis for all of the organs. The average cord number for each experiment was calculated. Cord diameters were measured for all cords in three randomly selected microscopic areas of each testis under 400x magnification. The average cord diameter for each experiment was determined and used as one replicate for each experiment to obtain a mean ± SD for statistical analysis.
Detection of apoptotic cells by TUNEL One of the serial sections from each experiment was used to assess the apoptotic cell number to determine the effect of treatments on apoptosis. To detect apoptotic cells, the Apoptosis Detection System (Promega, Madison, WI) was used as originally described [16]. This system measures fragmented DNA from apoptotic cells by catalytically incorporating fluorescein-12-dUTP at the 3' OH DNA end using the enzyme terminal deoxynucleotidyl transferase, which forms a polymeric tail using the principal of the TUNEL assay. The fluorescent cells in the entire testis cross-section were counted using fluorescence microscopy. The surface area of the sections in which the cell counts were obtained was measured using the NIH Image Program (http:rsb.info.nih.gov/nih.image/). The average number of fluorescent cells per 105 pixels of testis area from one experiment was used as a replicate in statistical analysis.
RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
Total RNA was obtained using Tri Reagent (Sigma, St. Louis, MO). Briefly, testes were dissected out from embryos or rats on E13, E14, and E16, and postnatal Day (P) 0, and P20. Testes and mesonephros from E13 embryos were dissected, and individual testes were separated from mesonephros. At least three individual testes from each developmental period were separately lysed in Tri Reagent (1 ml/50–100 mg tissue). After 0.2 ml of chloroform/1 ml Tri Reagent was added, the mixture was centrifuged at 12 000 x g for 15 min at 4°C, and the colorless upper aqueous phase was transferred to a fresh tube. Isopropanol (0.5 ml/1 ml of solution) was added to pellet the RNA. Reverse transcription (RT) was performed using Moloney murine leukemia virus-reverse transcriptase and specific 3' primers for PDGF-A, PDGF-B, PDGF -receptor, PDGF ß-receptor, and cyclophilin (1B15) under standard conditions [10].
Polymerase chain reaction (PCR) procedures were performed at 60°C annealing temperature for 30 cycles as previously described [10]. All RT-PCR reactions were conducted at least two times using at least three separate RNA preparations. The primer sequences and product sizes are as follows: PDGF-A, 5'-CGCAGGAAGAGAAGTATTGAGG-3' and 5'-GTCACACTGAACAAACGGACAC-3' (510 bp); PDGF-B, 5'-CACAGAGACTCCGTAGACGAAG-3' and 5'-GAGGGGTCACTACTGTCTCACG-3' (415 bp); PDGF -receptor, 5'-AGGAGGAGAAGTTCTCAGGAGC-3' and 5'-TACTTCAGTGTCTGGATCCGTG-3' (497 bp); PDGF ß-receptor, 5'-CAAGTTCAGCTCCAGTGATGTG-3' and 5'-TCCGAAGAGTAATCTGTCACC-3' (638 bp); cyclophilin, 5'-CTGCTGGGGAAGAGGAGAGGAGAAC-3' and 5'-GAGTGGTGGGCAGGTGTCTT-3' (105 bp). The identity of all PCR products was confirmed using restriction enzyme digests.
PDGF and PDGF ß-Receptor Immunohistochemistry
Rat testes at E14, E16, and P0 were fixed in Histochoice (Amresco, Solon, OH), embedded in paraffin, and sectioned. The sections (3–5 µm in thickness) were used for PDGF or PDGF ß-receptor immunohistochemistry according to standard procedures [17] with modifications as previously described [18]. Briefly, the sections were deparaffinized, rehydrated, and microwaved in 0.01 M citrate buffer (pH 6) on the high setting until the solution began to boil and then for 10 min on the medium-low setting. The sections then were blocked with 1% BSA for 30 min at room temperature. Immunohistochemistry used the following antibodies. The PDGF antibody (Sigma) was an anti-PDGF developed in goat using natural human PDGF as the immunogen and was cross-reactive with PDGF-AA, PDGF-AB, and PDGF-BB. The PDGF ß-receptor antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was an anti-PDGF ß-receptor antibody raised against an epitope mapping to the carboxy terminus of the PDGF ß-receptor of mouse origin and was cross-reactive with mouse and rat receptors. Both antibodies were diluted 1:100 in 1% BSA. The sections were incubated with antibodies overnight at room temperature. Immunoreactivities were visualized by successive incubation with biotinylated rabbit anti-goat immunoglobulin (Ig) G (secondary antibody), streptavidin-horseradish peroxidase (HRP), and 3,3' diaminobenzidine. Sections were counterstained with Harris hematoxylin. Negative controls for PDGF or PDGF ß-receptor immunohistochemistry were obtained by substituting primary antibodies with normal goat IgG or the secondary antibody with blocking solution. In an additional negative control for PDGF ß-receptor, sections were incubated with anti-PDGF ß-receptor antibody plus a seven to ten times excess of synthetic blocking peptide (Santa Cruz Biotechnology). The experiments were repeated three times. In each experiment, serial sections of two separate cross-sections consisting of two to five testes from each stage were used. One section was used for each negative control.
Testicular Cell Culture and Growth Assay
To generate a testicular cell culture from P0 testis, the tunica was removed and the testis was digested with 0.125% trypsin, 0.1% EDTA, and 0.02 mg/ml DNase (Sigma) in Hanks balanced salt solution (HBSS) (Gibco, Grand Island, NY) for 15 min at 37°C. The trypsin was inactivated with 10% calf serum. The samples were triturated with a pipette tip and washed twice in 1 ml HBSS. The cells were suspended, and 4 x 104 cells were plated in 24-well culture plates containing 1 ml F12 medium (Gibco) supplemented with 10% bovine calf serum (BCS) (HyClone, Logan, UT) overnight or until reaching approximately 25% confluency. Peritubular cells were isolated from P20 testis using a protocol previously described [19]. Approximately 4 x 104 cells were plated in 24-well culture plates in 1 ml F12 media supplemented with 10% BCS for 24–48 h when the cells were approximately 25% confluent. For both P0 testis and P20 peritubular myoid cells, culture media were replaced with Dulbecco modified Eagle medium with 0.1% BCS for 48 h. Previous studies have shown that epidermal growth factor (EGF) and BCS can stimulate P0 testis cell growth, so these were used in the current study as positive controls. The cells were treated for 24 h with 0–250 ng/ml concentrations of PDGF-BB (R&D Systems, Inc., Minneapolis, MN), 100 ng/ml EGF (Sigma), or 10% BCS as positive controls. The medium was removed after the 24-h treatment period, and the cells were cultured in medium containing [3H]thymidine (10 µCi/ml) for 6 h. After the 6-h incubation, the culture medium was discarded, and the cells were processed using the [3H]thymidine assay. Briefly, a solution of 0.5 M NaH2P04 (pH 7.3; 500 µl) was added to each well, and the cells were sonicated. Half of the sonicated cells were placed on DE-81 filters (Whatman, Maidstone, England) on a manifold, and a vacuum was applied. After three washes with the phosphate buffer, the filters were dried, placed in counting vials with 5 ml of scintillation fluid, and counted. The remaining sonicate was used for DNA assays to normalize for the number of cells per well [15].
Statistical Analysis
The data from testis morphometry, apoptotic cell counts, and cell growth assays were analyzed using GraphPad Prism (GraphPad Software, Inc., San Diego, CA). The values were expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA. The difference between the mean was determined using the Tukey multiple comparison. A statistically significant difference was confirmed at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effects of tyrphostins on cord formation were assessed using E13 organ cultures. One gonad was treated, and the contralateral gonad served as a control. In initial experiments, doses of tyrphostins at 5, 10, 15, and 25 µM were tested, and the optimum doses were determined to be 15 and 10 µM for AG1295 and AG1296, respectively (data not shown). The 15 µM AG1295 or the 10 µM AG1296 was used in the experiments presented. During a 3-day culture, the organs were treated daily and imaged. Both control and treated testes formed cords at the end of the culture. However, whereas control organs formed cords with a normal appearance, organs treated with 15 µM AG1295 or 10 µM AG1296 formed abnormal cords with a swollen appearance (Fig. 1). Images of whole treated organs also showed that the number of cords per testis was reduced and that the cords had an enlarged appearance. These data suggest that although inhibition of PDGF did not block cord formation, it interfered with normal cord development.
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To better assess the morphologic alterations of the cords caused by tyrphostins, sections of the cultured organs were further analyzed after hematoxylin and eosin staining using light microscopy. Control organ sections had "regular" cords with distinct borders (Fig. 2, A, C, E, and G), whereas organ sections treated with 15 µM AG1295 (Fig. 2, B and D) or 10 µM AG1296 (Fig. 2, F and H), had enlarged (swollen) testicular cords that were also fused together. The alteration by tyrphostin treatments resulted in a decrease in cord number per testis and an increase in cord diameter (Fig. 3). The cord number per testis was reduced approximately 40% by both 15 µM AG1295 and 10 µM AG1296. Both treatments significantly reduced cord number per testis section. In addition, cord diameter was increased approximately 60% by both 15 µM AG1295 and 10 µM AG1296. The differences were statistically significant (Fig. 3). These results demonstrate that the PDGF receptor tyrphostin signaling antagonists alter normal cord morphology, and the results provide a quantitative measurement of the alterations.
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Cell Apoptosis
To assess whether cell apoptosis was involved in alteration of cord morphology by tyrphostins, TUNEL analysis was used. Apoptotic cell number in testes treated with tyrphostins were slightly elevated (Fig. 4). However, differences were not statistically different between treated and control testes. The data suggest that treatment-induced changes in the cord morphology are not due to changes in cell apoptosis in the developing testis.
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PDGF and PDGF ß-Receptor Immunohistochemistry
Immunohistochemistry was conducted to examine localization of PDGF and its receptor, PDGF ß-receptor. The experiments focused on the PDGF ß-receptor because of its demonstrated role in the testis. In E14 testis, immunoreactivity to PDGF was diffuse, and its staining intensity was low. Immunoreactivity to PDGF was slightly more intense in the testis than in the mesonephros. In the mesonephros, mesonephric ducts were distinctly immunoreactive to PDGF (Fig. 5A). In contrast, PDGF ß-receptor immunoreactivity was stronger in the mesonephros than in the testis. In the mesonephros, mesonephric ducts were devoid of the staining (Fig. 5B). In certain areas, interstitial tissues extending from the mesonephros to the testis were also immunoreactive. In E16 testis and mesonephros, PDGF immunoreactivity was more intense in the testis. The staining was slightly more intense inside the cords than in interstitial regions (Fig. 5C). In contrast, PDGF ß-receptor immunoreactivity primarily localized to the interstitium, whereas cords showed weak or no immunoreactivity (Fig. 5D). In P0 testis, PDGF immunoreactivity was present primarily inside the cords with minimal interstitium immunoreactivity, except in selected cells that appeared to be Leydig cells (Fig. 5E). Immunoreactivity for PDGF ß-receptor was present in the interstitium but not in the cords (Fig. 5F). For all stages, the negative control sections used nonimmune goat IgG instead of PDGF antibody, or PDGF ß-receptor antibody plus blocking peptide. Minimal specific staining was found in the negative control sections. Typical examples for PDGF (Fig. 5G) and PDGF ß-receptor (Fig. 5H) controls are shown. Additional negative controls, leaving out secondary antibody or substituting PDGF ß-receptor antibody with goat nonimmune IgG, showed no immunoreactivity (data not shown). The data demonstrate that PDGF localizes primarily to the testis and the receptor localizes primarily to the mesonephros or the interstitium. In addition, protein levels progressively increase as the organ develops.
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PDGF Ligand and Receptor mRNA Expression in Testis During Development
To test the hypothesis that PDGF and its receptor genes are expressed in the testis at the time of cord formation and testis development, RT-PCR for PDGF-A, PDGF-B, PDGF -receptor, and PDGF ß-receptor was conducted. Messenger RNA for PDGF-A, PDGF-B, PDGF -receptor, and PDGF ß-receptor was expressed in E13, E14, E16, P0, and P20 testes. In addition, E13 cultured testis also expressed all four genes examined (Fig. 6). The identities of amplified products were determined by restriction enzyme mapping. Multiple bands were present for PDGF-B and the PDGF -receptor, and both digested as expected. It is speculated that these bands are due to splice variants, but the reason for their occurrence will require further analysis. These data show that the PDGF ligand and receptor genes are expressed in the testis during cord formation and may play a role in this process.
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Effect of PDGF on Testicular Cell Growth
To determine the potential effects of PDGF on growth of testicular cells, a growth assay using [3H]thymidine incorporation was performed. All of the doses of PDGF (10–250 ng/ml) caused a slight increase in cell growth (Fig. 7A). However, the increase was not statistically significant (P > 0.05). Peritubular myoid cells from P20 rat testis were treated with 10–100 ng/ml PDGF to test the bioactivity of the PDGF preparation used. All of the doses of PDGF tested increased peritubular cell growth (Fig. 7B). Previous studies have shown that EGF and 10% BCS can stimulate growth of cells from P0 rat testis, and in the present study, they were used as positive controls and were found to stimulate cell growth. The data suggest the PDGF used was bioactive and that the P0 testicular cells have a limited capacity to proliferate in response to PDGF. Because of the small size of the E14 testis, the cost of E14 testis cell growth analysis is prohibitive.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The data presented support the hypothesis and suggest that the paracrine growth factor PDGF has a role in testis cord formation. However, unlike the effects of inhibition of neurotropin actions [9, 10], inhibition of PDGF action did not completely block cord formation. Organs treated with tyrphostins that are specific inhibitors of PDGF developed testicular cords, but the cords were swollen and apparently fused. This finding indicates that the actions of PDGF may not be as critical as the actions of neurotropins or may be compensated by other paracrine factors.
The doses of tyrphostins used in this study were within the range that specifically inhibit the PDGF signaling pathway. In initial experiments, data showed that 10–15 µM concentrations were optimum for both antagonists. While a concentration of 5 µM was not effective in altering cord morphology, a concentration of 25 µM was toxic to the organs in culture (data not shown). The tyrphostins AG1295 and AG1296 have both been used as inhibitors of PDGF actions in various cell types. AG1295 (10 µM) inhibits PDGF-BB-induced PDGF ß-receptor autophosphorylation and cell proliferation in human and porcine smooth muscle cells in a specific and reversible manner [21]. Similarly, 5 µM AG1295 specifically inhibits PDGF-BB-stimulated PDGF receptor autophosphorylation and phosphorylation of downstream targets such as Shc, ERK 1/2, and Elk-1 in human mesangial cells [22]. As for AG1296, 10–100 µM of AG1296 inhibits PDGF receptor autophosphorylation and PDGF-induced cell growth in vitro and in vivo while having no apparent effect on EGF receptor autophosphorylation and EGF-induced mitogenesis in pulmonary myofibroblasts [23]. Similarly, 1–100 µM AG1296 inhibits receptor autophosphorylation, PDGF-induced tyrosine phosphorylation of PI3K (phosphotidylinositol 3-kinase) and MAPK (mitogen-activated protein kinase), cell growth, cell cycle progression, and cyclin-E-associated cyclin-dependent kinase activity, while having no effect on similar parameters induced by the EGF signaling system in mouse fibroblasts [24]. AG1296 was found not to influence ligand binding or receptor dimerization, but it did inhibit autophosphorylation of PDGF ß-receptor at tyrosine 857 in a canine kidney epithelial cell line [25]. Based on the dose curve data as well as doses used in previous studies, doses of AG1295 (15 µM) and AG1296 (10 µM) used in the present study are well within the dose range that is specific for PDGF receptor tyrosine kinase activity.
Abnormal cord formation in tyrphostin-treated embryonic testis organ cultures appears to be due to failure of PDGF to act on critical cellular processes. PDGF has been shown to influence many cellular processes such as growth, differentiation, migration, extracellular matrix production, and cell survival [26]. In the testis, PDGF stimulates cell proliferation, Ca2+ mobilization, extracellular matrix production [27], and cell migration [14] of peritubular myoid cells. Based on the observation of abnormal cord formation in tyrphostin-treated testis, it is speculated that tyrphostin causes an alteration in mesonephric cell differentiation that leads to abnormal cord formation.
The anti-apoptotic activity of PDGF has been previously demonstrated [28, 29] and plays a role in the development of various organs [30]. To determine if PDGF receptor inhibitors affect cellular apoptosis and potentially cause abnormal cord formation, the apoptotic cell number in testes was examined. The results demonstrate that although tyrphostin treatment causes a slight increase in apoptotic cell number, the increase is not statistically significant. It appears that apoptosis and/or cell survival is not the causal event in the actions of the PDGF antagonists.
To examine whether PDGF and PDGF receptor genes are present in testis during cord formation, an RT-PCR procedure was conducted. Messenger RNAs for PDGF-A PDGF-B, PDGF -receptor, and PDGF ß-receptor were present in E13, E14, E16, P0, and P20 testes, as well as in E13 testis after a 3-day organ culture. This is the first report showing mRNA expression for PDGFs and their receptors before E20 in the developing testis. This finding extends previous reports showing the presence of PDGF and PDGF receptor mRNA in the E20, P1, P5, and P20 rat testis [14, 31]. The observations demonstrate that gene expression for PDGF ligands and receptors is present during the period of cord formation and testis development.
To examine the presence and localization of PDGF and receptor proteins, immunohistochemistry for PDGF and PDGF ß-receptor was performed. Immunohistochemistry showed that PDGF was primarily present in the testicular compartment at E14 and inside the cords at E16 and P0. PDGF ß-receptor was present primarily in the mesonephric compartment at E14 and in the interstitium at E16 and P0. The observations are consistent with previous work [14] showing that PDGFs are localized inside testicular cords whereas PDGF receptors are localized in the interstitium at E20 and P5. Testicular localization of PDGF and mesonephric and interstitial localization of PDGF ß-receptor demonstrate a possible role for PDGFs in mesenchymal-epithelial interactions [14]. The observations further suggest a role for PDGFs in testicular cord formation in embryonic testis.
To assess the effect of PDGF on testis cell growth, P0 rat testicular cells were used in an [3H]thymidine incorporation assay. The data show that although PDGF slightly stimulates cell growth, the stimulation is not statistically significant. Peritubular myoid cells were previously reported to be responsive to PDGF in terms of cell growth [27]. PDGF significantly stimulated peritubular myoid cell growth, which demonstrated that the PDGF used was bioactive. The lack of a response of cells from P0 testis may be influenced by using a total-cell preparation as compared with an enriched-cell population. Previous studies have shown that EGF and calf serum stimulate P0 testis cell culture growth, and these reagents were used for positive controls in the present study. Further studies are needed to assess the actions and role of PDGF in the embryonic testis.
In summary, PDGF and its receptors are expressed during testis development from E13 through P20. PDGF is localized primarily to the cords, whereas PDGF ß-receptor is localized in the mesonephros and interstitium. Inhibition of PDGF actions with antagonists of PDGF receptor signaling interrupts normal cord morphology and causes swollen and fused cords. This causes a reduction in cord number per testis and an increased cord diameter. Therefore, PDGF and its receptors are expressed in the embryonic testis in a tissue-specific manner and appear to have a role in epithelial-mesenchymal cell interactions during cord formation. Inhibition of PDGF signaling does not inhibit cord formation but does alter normal cord development and morphology. These observations provide insight into paracrine growth factors that are involved in sex determination and embryonic testis development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH grants to M.K.S. 
2 Correspondence. FAX: 509 335 2176; skinner{at}mail.wsu.edu
Accepted: October 24, 2001.
Received: July 23, 2001.
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REFERENCES |
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