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a Department of Cell Biology and Histology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
b Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada M5G 1L6
c Institute for Hormone and Fertility Research, University of Hamburg, 22529 Hamburg, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TGF), and a new marker for Leydig cell maturation, relaxin-like factor (RLF).
LH receptor immunoreactivity was found in Leydig cell-depleted testes at 3 and 8 days after EDS administration. The positive (precursor) cells had a mesenchymal-like morphology. The number of LH receptor-positive cells 8 days after EDS administration was 15 ± 4 per 500 Sertoli cell nuclei. Fifteen days after EDS administration, the first new Leydig cells could be observed. These cells stained positively with both the antibodies against the LH receptor and 3ß-HSD, while some cells also stained positively for TGF.
After EDS administration, RLF mRNA disappeared from the testis and reappeared again at the time of the appearance of the first Leydig cells. Concomitant with the increase in the number of Leydig cells, the number of RLF-expressing cells increased.
The observations of the present study give further support to the hypothesis that Leydig cell development in the prepubertal testis, and in the adult testis following EDS administration, takes place along the same cell lineage and suggest, therefore, that the adult EDS-treated rat can serve as a model for studying the adult-type Leydig cell development that normally occurs in the prepubertal rat testis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The presence of LH receptors on Leydig cells in the prepubertal testis has been demonstrated by binding studies (e.g., [6]) and Northern blot analyses [7]. It has been suggested that LH stimulates Leydig cell proliferation directly, or indirectly by regulating the production of growth-promoting factors that act in an autocrine manner. Factors that are highly mitogenic, such as transforming growth factor (TGF) and insulin-like growth factor-1 (IGF-I), have been localized in Leydig cells in vivo [8–10]. Furthermore, in vitro studies have demonstrated that these growth factors promote DNA synthesis in adult-type Leydig cells isolated from 21-day-old rat testes [11, 12].
In the adult rat testis, Leydig cell proliferation is negligible [13]. However, it is possible to induce the formation of new Leydig cells under experimental conditions, such as administration of the Leydig cell toxicant ethane dimethyl sulphonate (EDS). This cytotoxic agent specifically destroys Leydig cells in the adult rat testis, a process that is followed by a complete regeneration of the Leydig cell population [14–23]. The development of the new Leydig cell population after EDS is the result of both differentiation of Leydig cell precursors and proliferation of the newly formed Leydig cells, a process that shows homology to the development of the adult-type Leydig population in the prepubertal testis. Peak values in Leydig cells labeled with [3H]thymidine and cells in mitosis are found at Days 21 and 22 after EDS, respectively [24–26]. It is therefore tempting to speculate that the same factors that are involved in the stimulation of Leydig cell proliferation in the prepubertal testis also stimulate Leydig cell proliferation after EDS administration.
Recently, another marker for Leydig cell maturation has been identified in the testis. This relaxin-like factor (RLF), also known as the Leydig cell insulin-like (Ley-I-L) peptide, a new member of the insulin/relaxin/insulin-like growth factor family, is predominantly expressed in Leydig cells [27]. In the mouse testis, intense immunohistochemical RLF staining can be observed from Day 15 after birth onwards, at the time when the adult-type Leydig cell population starts to develop [28]. Although the function of RLF is unknown, a chemically synthesized human RLF peptide has been shown to interact with relaxin receptors [29].
In an attempt to obtain more information about the process of Leydig cell regeneration after EDS administration and the factors involved in the stimulation of Leydig cell proliferation, we have identified Leydig cells and their precursors by the presence of the steroidogenic enzyme 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and LH receptors. We have further investigated the presence of TGF and RLF during the formation of the new Leydig cell population after EDS, using highly specific monoclonal and polyclonal antibodies and in situ hybridization techniques.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The 3ß-HSD polyclonal antibody was a gift from Dr. V. Luu-The (Laval University, PQ, Canada). The antibody was obtained after immunization of rabbits with 3ß-HSD purified from human placenta [30]. This antibody has been used previously for the localization of 3ß-HSD in the human placenta and ovary as well as in guinea pig and rat gonads and adrenals [30–33].
The TGF monoclonal antibody MF9 was a gift from Dr. J.E. Kudlow (University of Alabama, Birmingham, AL). The antibody was raised against a synthetic peptide that consisted of the carboxyl terminal 17 amino acids of rat TGF (Peninsula Laboratories, Belmont, CA) and was purified from ascites fluid as described by Kobrin et al. [34]. The TGF antibody, which does not cross-react with epidermal growth factor (EGF), has been used previously for the localization of TGF in the immature rat ovary, bovine ovary, and rat pituitary and testis, and has been shown to be highly specific [8, 34–36].
The LH receptor monoclonal antibody (P1B4) was a gift from Dr. J. Wimalasena (Dept. of Obstetrics & Gynecology, University of Tennessee, Knoxville, TN). The antibody was raised against purified rat LH receptors, as described by Indrapichate et al. [37], and has been shown to bind specifically to LH receptors in different tissues [38].
Biotinylated goat-anti-mouse (LH receptor, TGF) and goat-anti-rabbit (3ß-HSD) IgGs were used as secondary antibodies and were both obtained from Vector Laboratories (Vectastain kit elite, Burlingame, CA). EDS is not commercially available and was prepared as described by Jackson and Jackson [39].
Animals
Adult (6-mo-old) male Wistar rats were used. EDS (60 mg/ml in a mixture of dimethylsulfoxide and H20 at 1:3, v:v) was administered in a single i.p. injection at a dose of 75 mg/kg BW. Groups of 3 rats received either a vehicle injection or were treated with EDS. Animals were killed 3, 8, 15, 22, 28, 35, 49, 56, or 64 days after EDS administration after Nembutal (Sanofi Sante BV, Maassluis, The Netherlands) anesthesia and perfusion fixation.
Immunohistochemical Staining
Testicular tissue was perfusion-fixed in 4% buffered formaldehyde for 4–8 h at room temperature followed by 18 h at 4°C. The tissue was dehydrated and embedded in paraffin. The immunostaining technique was performed on 5-µm sections on aminoalkylsilane-coated glass slides. Sections were deparaffinized, and endogenous peroxidase was blocked with 1% H2O2 in methanol for 30 min. The slides were subsequently washed in 0.01 M Tris-buffered-saline (TBS; pH 7.4), incubated with 0.1 M glycine in TBS for 30 min, and rinsed with TBS. The slides were blocked with 10% normal goat serum for 30 min and then incubated overnight at 4°C with the antibodies against the LH receptor at a dilution of 1:3000, TGF at a dilution of 1:100, and 3ß-HSD at a dilution of 1:300, in TBS with 0.05% acetylated BSA (Aurion, Wageningen, The Netherlands). After this incubation, the slides were rinsed with TBS and then incubated for 60 min with a biotinylated goat-anti-rabbit polyclonal antibody (3ß-HSD) or biotinylated goat-anti-mouse antibody (TGF, LH receptor) diluted 1:200 in TBS containing 0.05% acetylated BSA. Slides were again washed in TBS and subsequently incubated for at least 60 min with the components avidin (A) and biotin (B) of the ABC staining kit (Vector Lab). Both components (A and B) were diluted either 1:500 (TGF and 3ß-HSD) or 1:1000 (LH receptor) and prepared at least 15 min before addition to the sections. Slides were washed again in TBS and Tris-HCl (0.05 M, pH 7.6), and bound antibody was visualized using a 0.6 mg/ml solution of 3,3'-diaminobenzidine tetrachloride (Sigma Chemical Co., St. Louis, MO) in Tris-HCl, to which 0.03% H2O2 was added. Pilot experiments had previously demonstrated that the different dilutions of the antibodies used in this study gave optimal staining results. All slides were subsequently stained with Mayer's hematoxylin.
Control experiments were carried out, and representative sections are shown in Figure 2, G and H. In these control experiments, the primary antibodies were either omitted from the procedure (data not shown), or normal rabbit serum (3ß-HSD) (Fig. 2G) or normal mouse serum (TGF, LH receptor; Fig. 2H) were employed instead of the first antibodies.
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Cell Counts
Leydig cell precursors were counted in randomly chosen sections through whole testes of rats 8 days after EDS administration. At least two different sections were counted per testis, using a square lattice grid inserted in the eyepiece of the microscope. Only those nuclei were counted that were located within the square lattice grid. Identification of the mesenchymal-like precursor cells was based on their nuclear morphology, as has been described by several groups of investigators [2, 15, 20], and the presence of immunohistochemical staining for the presence of LH receptors. Positive cells had fusiform nuclei and were located throughout the interstitial compartment. Nuclei of Sertoli cells were also counted until 500 nuclei were scored. Sertoli cell nuclei were identified morphologically by the shape of the nucleus and the presence of a characteristic round nucleolus. Only those Sertoli cell nuclei were included in the cell count in which a nucleolus was present in the nuclear cross-section. The number of mesenchymal-like cells that stained positively with the antibody against the LH receptor was expressed per 500 Sertoli cell nuclei (henceforth called number of Sertoli cells) as previously described [24, 40].
In Situ Hybridization
Rat testes were fixed in buffered formalin and were paraffin-embedded as described above. Ten-micrometer paraffin sections were dewaxed in xylol twice for 10 min each time and transferred through descending grades of ethanol to diethylpyrocarbonate-treated water for 5 min. Endogenous alkaline phosphatase activity was blocked by treatment with 0.2 N HCl for 20 min and 0.3% Triton X-100 in PBS for 15 min, both at room temperature. This was followed by preincubation in proteinase K buffer (100 mM Tris HCl, pH 7.5, 50 mM EDTA) for 30 min at 37°C. Sections were then incubated in 10 µg/ml proteinase K in the same buffer at 37°C for 30 min. The reaction was stopped by transfer of the slides to 0.2% ice-cold glycine solution for 1 min, and sections were post-fixed in 3% cold paraformaldehyde for 5 min. Slides were then rinsed in water for 5 min at room temperature, equilibrated in acetylation buffer (freshly prepared 0.1 M triethanolamine hydrochloride, pH 8.0) for 3 min, and treated with 0.5% acetic anhydride in acetylation buffer for 10 min under vigorous shaking. Slides were then transferred to double-strength SSC (single-strength SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0) for 5 min and dried for 1 h at 50°C before prehybridization.
The prehybridization solution comprised 20 mM Tris HCl (pH 7.5), 0.3 M NaCl, 1 mM EDTA, 100 mM dithiothreitol, 50% deionized formamide, single-strength Denhardt's solution, 100 µg/ml poly(A), 0.5 mg/ml denatured herring sperm DNA, and 0.5 mg/ml calf thymus tRNA. Sections were prehybridized in this solution for 2 h at 52°C. Sections were then covered with hybridization solution, which consisted of the prehybridization solution to which dextran sulfate (final concentration of 10%, dissolved in 50% formamide) and 10 ng digoxigenin-labeled cRNA probe (see below) per 20 µl of hybridization solution were added. Hybridization proceeded overnight at 52°C in a moist chamber. Slides were rinsed in double-strength SSC at room temperature for 15 min, then stringently washed in post-hybridization buffer (0.3 M NaCl, 50% formamide, 20 mM Tris HCl [pH 7.5], 1 mM EDTA, 10 mM dithiothreitol) at 60°C for 10 min. Ribonuclease (RNase) A digestion was then carried out to remove unspecifically bound single-stranded cRNA probe. Sections were incubated for 30 min at 37°C in RNase buffer (0.5 M NaCl, 10 mM Tris HCl pH 7.5, 1 mM EDTA) containing 100 µg/ml RNase A, and rinsed in RNase buffer without the enzyme for a further 30 min. This was followed by additional washes in double-strength SSC at room temperature for 3 min, in 0.1-strength SSC at 52°C for 15 min, and finally in 0.1-strength SSC at room temperature to equilibrate the sections. In order to visualize the digoxigenin label, sections were rinsed in buffer A (0.1 M Tris HCl pH 7.5, 0.15 M NaCl) at room temperature for 15 min, blocked by incubation with 20% normal sheep serum in buffer A for 30 min, and incubated for 2 h at room temperature with an alkaline phosphatase-conjugated sheep anti-digoxigenin antibody (Boehringer, Mannheim, Germany) diluted 1:500 in buffer A to which 1% normal sheep serum and 0.3% Triton X-100 were added. Slides were rinsed twice in buffer A for 15 min, and then in buffer B (0.1 M Tris HCl pH 9.5, 0.05 M MgCl2) for 15 min, before the color detection substrate solution was applied (0.3% Triton X-100, 0.175 µg/ml 5-bromo-4-chloro-3-indolyl phosphate [BCIP; Boehringer], 337.5 µg/ml nitroblue tetrazolium [Boehringer], 2.4 mg/ml levamisole) in buffer B. Sections were incubated overnight in the dark at room temperature, and the reaction was stopped by rinsing the slides in Tris (10 mM, pH 7.5) EDTA (1 mM) buffer for 10 min at room temperature. Sections were not counterstained but directly mounted in aqueous mounting medium (Faramount, Dako Diagnostika, Hamburg, Germany).
The rat Leydig cell-specific RLF cDNA was cloned from a rat testis cDNA library made in the bacteriophage lambda gt11 (Clontech, Palo Alto, CA) using the mouse RLF sequence [41] as probe (unpublished; sequence deposited in the EMBL database). The full-length rat RLF cDNA was cloned into the plasmid vector pBluescript II SK+ (Stratagene, La Jolla, CA), and linearized with either PstI or HindIII to create antisense or sense cRNA probes using the T7 or T3 RNA polymerases, respectively. The sense (control) and antisense cRNA probes were prepared by in vitro transcription using digoxigenin-UTP (Boehringer) exactly as recommended by the manufacturer. Template DNA was not removed from the reaction before application in the in situ hybridization.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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immunoreactivity and the absence of RLF mRNA-expressing cells in the interstitium, at 3 and 8 days after EDS administration (Fig. 1, A–C). However, it remained possible to detect interstitial cells with a mesenchymal-like morphology that stained positively with the antibody against the LH receptor at 3 and 8 days after EDS (Fig. 1C). These mesenchymal-like cells that showed LH receptor immunoreactivity were presumed to be Leydig cell precursors. The number of LH receptor-positive cells at these two times after EDS was considerably lower than in the control testes, in which the majority of the positive cells were Leydig cells (Fig. 2F).
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The quality of the histology of the formaldehyde-fixed, paraffin-embedded testicular tissues used for LH receptor immunohistochemistry did not allow a detailed morphometric analysis. However, in order to obtain an impression of the relative numbers of precursor cells in the interstitium, we counted the number of cells that stained positively with the LH receptor antibody at 8 days after EDS, when the wave of precursor cell proliferation had decreased again to control levels [24]. We counted 15 ± 4 positive precursor cells per 500 Sertoli cells. Cells immunopositive for LH receptor were found throughout the interstitial space. The number of LH receptor-positive cells was too low to be able to assign a specific peritubular or perivascular localization to the cells.
In contrast to our previous studies (e.g., [24]) EDS administration and the subsequent testosterone deprivation had detrimental effects on the process of spermatogenesis in the strain of Wistar rats used in the present study. In most seminiferous tubules, spermiogenesis was severely affected by Day 15 after EDS, resulting in the absence of elongated spermatids and in reduced numbers of round spermatids, while in some tubules the number of spermatocytes also seemed to be reduced (Fig. 1E). Around this time, the first morphologically recognizable Leydig cells appeared in the interstitial compartment ([18, 24]; Fig. 1, D–F), as became also apparent by the presence of some 3ß-HSD- and TGF-positive cells (Fig. 1, D and E). LH receptor immunoreactivity could also be detected in these newly formed Leydig cells (Fig. 1F). At this time, the first RLF mRNA-expressing cells were also observed in the interstitial compartment (Fig. 3E), although these cells were sometimes difficult to distinguish because of the high background in the in situ hybridized sections (Fig. 3F). Concomitantly with the appearance of regenerating Leydig cells, mast cells appeared in the interstitial compartment.
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By Day 22 after EDS administration, the number of newly formed Leydig cells had increased significantly, and these cells all stained positively with the antibodies against 3ß-HSD and LH receptors (Fig. 1, G and I) and expressed RLF mRNA (Fig. 3G). Surprisingly, not all newly formed Leydig cells stained positively for TGF (Fig. 1H). With the reappearance of testosterone-producing Leydig cells, the process of spermatogenesis also started to improve (Fig. 1, G–I).
The Leydig cell population continued to increase in size. By Day 35 after EDS administration, all Leydig cells had become positive for TGF (data not shown). Around Day 56 after EDS, the Leydig cell population had almost completely regenerated; all Leydig cells stained positively when incubated with the antibodies against 3ß-HSD, TGF, or LH receptor (Fig. 2, A–C) and expressed RLF mRNA (Fig. 3I). No difference in immunoreactivity between the regenerated Leydig cells and the Leydig cells in the adult control testis could be observed (Fig. 2, D–F), nor any difference in the expression of RLF mRNA (Fig. 3, A and I). In most seminiferous tubules, the process of spermatogenesis had been restored completely by Day 56 after EDS administration (Fig. 2, A–C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and RLF can be detected in the rat testis after the administration of EDS. Previous studies had demonstrated that Leydig cell precursors in the adult rat testis were able to respond to administration of the LH analogue hCG by differentiating into new Leydig cells [23, 42–47], suggesting that these cells must possess LH/CG receptors. Northern blot analyses of poly(A)+ RNA isolated from testes of EDS-treated hypophysectomized rats indeed revealed the presence of the transcript encoding the full-length LH receptor in precursor cells [47]. The four other LH receptor mRNA transcripts that presumably code for truncated forms of this receptor, as reported previously in the (pre)pubertal and adult rat testis [7, 48–51], were also expressed in Leydig cell precursors [47]. These observations were further confirmed by in situ hybridization studies that showed a clear signal in interstitial cells 3 days after EDS administration [52].
Nevertheless, it was impossible to demonstrate significant [125I]hCG binding in interstitial cell preparations isolated during the first 2 wk after EDS administration [19, 53, 54]. Low levels of specific binding were detected for the first time in isolated precursor cell fractions from Day 15 after EDS onwards [55]. The lower sensitivity of the [125I]hCG binding assay in comparison to the method of detecting of LH receptor mRNA may explain this apparent discrepancy. Using the more sensitive method of immunohistochemistry to detect LH receptors, we were able to demonstrate the presence of receptor protein in Leydig cell precursors. Three and 8 days after EDS administration, when all Leydig cells had disappeared (e.g., [14, 18]), cells could be detected that stained positively with the antibody against the LH receptor. These cells had a mesenchymal-like morphology, supporting previous morphological and cell kinetic observations by Kerr et al. [15] and Teerds et al. [24] in the adult testis following EDS, and by Hardy et al. [2] in the prepubertal testis, that these cells are the precursors of the Leydig cells. Pulse-chase studies using [3H]thymidine incorporation as a marker for cell proliferation showed that mesenchymal-like cells that had incorporated the label during the first few days after EDS administration developed into new Leydig cells [24]. Similar experiments carried out in the prepubertal rat also demonstrated that proliferating mesenchymal-like cells are the precursors of the adult-type Leydig cells [2].
The present study is the first that has identified the precursor of the Leydig cell in the adult testis immunohistochemically within the first few days after EDS administration. A next step would be to further characterize this precursor cell immunocytochemically at the transmission electron microscopical level; however, this is beyond the scope of the present study.
The number of precursor cells that stained positively with the LH receptor antibody was low. This was not quite unexpected, since we have previously shown that the majority of the regenerating Leydig cells did not develop from differentiating precursor cells but were formed as a result of proliferation of the newly formed Leydig cells [24]. When one compares the number of newly formed Leydig cells (expressed per 500 Sertoli cells) at Day 14 after EDS with the number of LH receptor-positive cells 8 days after EDS, there appears to be a remarkable similarity in cell numbers (8 ± 3 [24] and 15 ± 4, respectively).
The newly formed Leydig cells observed at Day 15 after EDS administration stained positively with both the antibody against the LH receptor and that against 3ß-HSD. The latter observation confirms enzyme histochemical data by Molenaar et al. [18]. RLF mRNA expression was also found for the first time in these Leydig cells. We did not observe RLF expression at 3 and 8 days after EDS in Leydig cell precursors. Because of the high background levels in the in situ hybridized sections, however, we cannot completely rule out the presence of low levels of RLF expression in the precursor cells. Nevertheless, this does not seem to be likely, since Balvers et al. [28] showed that in adult hpg mice, in which adult-type Leydig cells remain in an undifferentiated/precursor state, RLF expression was absent.
The regeneration of the Leydig cell population was completed between Days 56 and 64 after EDS [14, 15, 20, 21]; at this stage, all Leydig cells stained positively with the antibodies against 3ß-HSD and the LH receptor, and expressed RLF mRNA.
RLF is a novel peptide factor that shows structural homology to the insulin-IGF-relaxin family of hormonal factors. Although RLF is expressed at very high levels in Leydig cells of all mammalian species investigated so far, its function is unknown (reviewed by Ivell, [27]). In prepubertal mice, an up-regulation of RLF mRNA levels is observed concomitantly with the development of the adult-type Leydig cell population, implying a relation between adult-type Leydig cell maturation and RLF expression. These observations are supported by immunohistochemical data that localized RLF specifically in maturing Leydig cells [28]. In adult hpg mice, in which Leydig cells are present in an undifferentiated/precursor state, no detectable expression of RLF mRNA was found. When these animals are injected twice daily with hCG, differentiation of adult-type Leydig cells is induced, and concomitantly, RLF mRNA levels are up-regulated, supporting the hypothesis that RLF is a marker for Leydig cell maturation [28]. The present study offers further support for this, since RLF expression first becomes apparent in the EDS-treated rat testis at the time of the development of a new Leydig cell population.
We were unable to investigate the presence of RLF protein in precursor cells and maturing Leydig cells by immunohistochemistry. The antibodies we have used in previous studies were raised in rats and therefore cannot be applied on rat testicular sections.
The newly formed Leydig cells after EDS administration proliferate actively [24–26]. The factors involved in the regulation of this increased proliferative activity are largely unknown, although increased plasma LH/hCG levels have been shown to enhance Leydig cell proliferation in the prepubertal as well as the adult rat [5, 23, 42, 45, 56]. Several studies have demonstrated that plasma LH levels were elevated up to 14 days after EDS because of the absence of testosterone-producing Leydig cells. By 21 days after EDS administration, at the time when the wave of Leydig cell proliferation was initiated, plasma LH levels had returned to the pretreatment range [17, 20, 21, 24–26]. This suggests that other factors, possibly together with LH, are responsible for the increased Leydig cell proliferation.
This situation is more or less comparable to that in the prepubertal rat, in which the proliferation of immature adult-type Leydig cells was also maximally stimulated when LH levels were not significantly elevated [57]. In vitro studies have demonstrated that growth factors such as TGF and IGF-I, which are locally produced in the testis [8–10, 58], stimulated DNA synthesis in Leydig cells isolated from 21-day-old rats [11, 12].
These two growth factors are produced not only in the prepubertal testis but in the adult testis as well by Sertoli cells and peritubular/myoid cells [58–62]. Since Leydig cells possess receptors for epidermal growth factor (EGF), to which TGF can bind [63], and receptors for IGF-I [9, 64], it may very well be possible that locally produced TGF and IGF-I are involved in the stimulation of Leydig cell proliferation during the regeneration period following EDS administration. Studies by Drummond and colleagues [65] have demonstrated that interstitial fluid collected from testes between Days 21 and 28 after EDS administration, at the time when Leydig cell proliferation was stimulated maximally, was highly mitogenic to BALB/c 3T3 cells. The presence of TGF in the newly formed Leydig cells, at the time when they start their wave of proliferation [24, 26], gives further support to this hypothesis.
Although Sertoli cells and peritubular/myoid cells have been shown to secrete TGF in vitro, immunolabeling for this growth factor could not be detected on paraffin sections in the present study. Hence, the production of TGF by Sertoli cells in vivo is either rather low, or the growth factor is rapidly secreted, as has been discussed elsewhere [8].
The pattern of appearance of TGF was identical to what has been found in the immature testis during prepubertal/pubertal development [8, 12]. This suggests that the development of new Leydig cells in the adult testis following EDS administration and the formation of the adult-type Leydig cell population in the prepubertal/pubertal testis may take place along the same cell lineage (reviewed in [45, 46]).
The observation that Leydig cells remain positive for TGF even after proliferation has ceased suggests that this growth factor may affect other cell functions as well, such as steroid production (reviewed by Saez and Lejeune [66]).
Taken together, the present study demonstrates that after EDS administration, Leydig cell precursors can be identified morphologically and immunohistochemically, using an antibody against the LH receptor. The appearance of 3ß-HSD, TGF, and RLF during Leydig cell maturation is identical to what has been observed previously in the prepubertal rat testis. These observations give further support to the hypothesis that the regeneration of the Leydig cell population after EDS administration can serve as a model for adult-type Leydig cell development in the prepubertal testis.
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ACKNOWLEDGMENTS |
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antibody, and Dr. J. Wimalasena (Dept. Obstetrics & Gynecology, University of Tennessee, Knoxville, TN) for the LH receptor antibody. We are grateful to Mr. J.C. van Oudheusden for skillful technical assistance, and to Mr. H. Halsema and Foto Breeman (Culemborg, The Netherlands) for their help in preparing the plates of photomicrographs. |
FOOTNOTES |
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2 Correspondence: Katja J. Teerds, Department of Biochemistry, Cell Biology and Histology, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.176, 3508 TD Utrecht, The Netherlands. FAX: 31 30 2516853; k.teerds{at}pobox.accu.uu.nl
Accepted: February 2, 1999.
Received: November 2, 1998.
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S.M.L. Chamindrani Mendis-Handagama and H.B. Siril Ariyaratne Differentiation of the Adult Leydig Cell Population in the Postnatal Testis Biol Reprod, September 1, 2001; 65(3): 660 - 671. [Abstract] [Full Text] [PDF]
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H.B. S. Ariyaratne, N. Mills, J. I. Mason, and S.M.L. C. Mendis-Handagama Effects of Thyroid Hormone on Leydig Cell Regeneration in the Adult Rat Following Ethane Dimethane Sulphonate Treatment Biol Reprod, October 1, 2000; 63(4): 1115 - 1123. [Abstract] [Full Text]
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H.B. Siril Ariyaratne, J. Ian Mason, and S.M.L. C. Mendis-Handagama Effects of Thyroid and Luteinizing Hormones on the Onset of Precursor Cell Differentiation into Leydig Progenitor Cells in the Prepubertal Rat Testis Biol Reprod, September 1, 2000; 63(3): 898 - 904. [Abstract] [Full Text]
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H.B. Siril Ariyaratne, S.M.L. Chamindrani Mendis-Handagama, D. Buchanan Hales, and J. Ian Mason Studies on the Onset of Leydig Precursor Cell Differentiation in the Prepubertal Rat Testis Biol Reprod, July 1, 2000; 63(1): 165 - 171. [Abstract] [Full Text]
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