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a Deparment of Animal Science, The University of Tennessee, Knoxville, Tennessee 37996
b Deparment of Biology, Texas Woman's University, Denton, Texas 76204
c University Department of Reproduction and Development Sciences (Clinical Biochemistry), Royal Infirmary of Edinburgh NHS Trust, Edinburgh, Scotland, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, interstitial cells, Leydig cells, testes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ethane dimethane sulphonate (EDS) is a toxin unique to Leydig cells in several laboratory animals, including the rat [3]. However, the mesenchymal cells in the testis interstitium are not affected by EDS treatment [3]. In the adult rat, lesions appear in Leydig cells within a few hours of EDS administration, and all Leydig cells disappear from the testis interstitium within 48 h [3]. In addition, EDS is cleared from the body within a day. During the first 7–14 days, Leydig cells are absent in the testis interstitium; however, by 21 days, new, small Leydig cells appear in the interstitium [3]. Therefore, the EDS-treated adult rat provides an acceptable model to understand the process of Leydig cell differentiation in the postnatal rat testis.
In the present study, we hypothesized that thyroid hormone is required to induce mesenchymal cell differentiation into Leydig cells in the EDS-treated adult rats. If this hypothesis is true, Leydig cell regeneration should not occur in testes of the EDS-treated hypothyroid rats. In addition, increased numbers of Leydig cells could be expected in the EDS-treated rats if they are hyperthyroid during the period of Leydig cell regeneration. Therefore, in the present investigation, we used EDS-treated rats there were euthyroid, hypothyroid (via PTU treatment and thyroidectomy), and hyperthyroid (daily T3 injections) to test this hypothesis. Thyroidectomized rats were included to verify the hypothyroid effects of PTU on Leydig cell regeneration. The presence of Leydig cells was tested by light microscopy, 3ß-hydroxysteroid dehydrogenase (3ß-HSD) immunocytochemistry, and LH-stimulated testicular androgen secretory capacity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ninety-day-old, thyroid-intact and thyroidectomized male Sprague-Dawley rats were purchased from Harland Industries (Madison, WI) and housed (two animals per cage) under controlled temperature (25°C) and lighting (14L:10D) conditions. The thyroid-intact rats were fed with Agway Prolab (Syracuse, NY) rat formula, and water was provided ad libitum. The thyroidectomized rats were given the same rat diet. However, to maintain normal levels of calcium in the body, thyroidectomized rats (because parathyroids are also removed during surgery) were given drinking water containing 3% (w/v) calcium lactate.
Treatments
Three groups of thyroid-intact rats (control, hypothyroid, and hyperthyroid; n = 32 per group) and one group of thyroidectomized rats (n = 5) were used. All rats received a single i.p. injection of EDS (prepared by Dr. N. Mills; 75 mg/kg body weight) dissolved in a mixture of 0.5 ml of dimethyl sulfoxide and 1.5 ml of water for injection to eliminate existing Leydig cells in the testicular interstitium. Rats in the control group were given daily s.c. injections of 0.025 M NaOH in saline. Rats in the hypothyroid group were provided 0.1% (w/v) PTU (Sigma, St. Louis, MO) in drinking water [1, 4, 5]. Rats in the hyperthyroid group were given daily s.c. injections of T3 (Sigma) at a dosage of 100 µg/kg [2] dissolved in 0.025 M NaOH in saline beginning from Day 1 of EDS administration. Thyroidectomized, EDS-treated rats were not subjected to any further treatment.
Collection and Processing of Tissue
The EDS-treated rats in the control, hypothyroid, and hyperthyroid groups were killed on Days 2, 7, 14, and 21 (n = 8 per group) and the EDS-treated thyroidectomized rats on Day 21 (n = 5) after the EDS injection. Heart blood was collected from each rat under deep inhalation anesthesia of Metofane (Mallinckroft Veterinary, Inc., Mundelein, IL), and serum was separated and stored at -80°C until assayed for hormone levels by RIA. One testis from each rat in the control, hypothyroid, and hyperthyroid groups was removed and weighed (to obtain fresh testis weight). In addition, the specific gravity was determined by the flotation method [6, 7], and the fresh testis volume was calculated. This testis was used to estimate the LH-stimulated androgen secretory capacity in vitro. The remaining testis of five rats in these three groups (control, hypothyroid, and hyperthyroid) at Days 2, 7, 14, and 21 and one testis from each thyroidectomized rat (n = 5) at Day 21 were fixed by whole-body perfusion technique using 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) as described elsewhere [7]. The fixed testis was removed and weighed (to obtain fixed testis weight), and the specific gravity was determined. The fixed testis volume was also calculated. Tissue cubes (1–2 mm) cut from these testes were postfixed in a 1:1 mixture of 2% osmium tetroxide and 3% potassium ferrocyanide [8], dehydrated in graded ethanols, and embedded in epon-araldyte for morphological and morphometric studies. Separate tissue blocks were also prepared as described elsewhere [7] from each glutaraldehyde-fixed testis to determine the volume changes during tissue processing. The remaining testes of the other three rats in the control, hypothyroid and hyperthyroid groups at Days 2, 7, 14, and 21 and the remaining five testes of the thyroidectomized rats were fixed by immersion in Bouin solution for 6 h, dehydrated in ethanol, and embedded in Paraplast (Oxford Labware, St. Louis, MO) [1, 9].
Morphology and Morphometry
From the polymerized tissue blocks, two tissue sections (thickness, 1 µm) that were 4 µm apart (the first and the fifth section) were cut using an LKB IV ultramicrotome (Pharmacia LKB, Piscataway, NJ) and glass knives. Each pair of these tissue sections was mounted close to each other on a precleaned glass slide (Superfrost Plus; Fisher Scientific, Pittsburgh, PA) stained with methylene blue-azure stain and cover-slipped under Permount (Fisher Scientific). These tissue sections were viewed under an Olympus BH-2 microscope (Olympus, Tokyo, Japan). The different cell types in the testicular interstitium were identified by their morphological characteristics as described elsewhere [1, 10]. Briefly, the profiles of adult Leydig cells in tissue sections appeared as polygonal or slightly elongated profiles, with a cytoplasm that contained little or no lipid droplets. The nucleus of these Leydig cells appeared mostly circular in section, had a characteristic thin rim, and also had several clumps of heterochromatin associated with the nuclear membrane and a prominent nucleolus. Macrophages were approximately circular in section, with a relatively larger diameter than the Leydig cells. Their cytoplasm stained much lighter than those of Leydig cells, and they contained many darkly stained granules and vacuoles of variable sizes. The nucleus of these cells were observed to be relatively darker than those of Leydig cells, owing to the presence of a thicker rim of heterochromatin attached to the nuclear membrane. Mesenchymal cells were observed as spindle-shaped cells in the peritubular region as well as in the central interstitium. Each cell contained an elongated nucleus and little cytoplasm. Myoid cells were seen in sections as thin, elongated cells immediately surrounding the seminiferous tubules. Each cell contained a homogeneously and darkly stained nucleus, and they were intimately adhered to the limiting membrane of the seminiferous tubules. Pericytes were located near the small blood vessels, were crescentic in shape, and had a homogeneous, darkly stained nucleus with a thin layer of cytoplasm. Vascular endothelial cells lined the lumen of blood vessels; they were elongated cells with a faintly stained nucleus and cytoplasm.
Each pair of the two tissue sections described earlier was used to estimate the numerical density of testicular interstitial cell types, namely Leydig cells, macrophages, mesenchymal cells, myoid cells, pericytes, and endothelial cells, using the disector method [11] as described elsewhere [9]. A total of 40 disectors per tissue block and 10 tissue blocks per animal were scored. The numerical density (NV) of each cell type in fresh testicular tissue was determined using the following formula as described elsewhere [7]:
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The area of the tissue section represented by the image on the videoscreen was determined by projecting an image of a slide micrometer on the screen under the same magnification as that of the microscope. The total number of each cell type per testis was determined by multiplying the NV by the fresh testis volume.
Immunocytochemistry for 3ß-HSD
Bouin-fixed and Paraplast-embedded testis tissue was used to immunolocalize 3ß-HSD enzyme using a polyclonal antibody raised in rabbits against human placental 3ß-HSD enzyme [12]. Tissue sections (thickness, 5 µm) prepared from these tissue blocks were adhered to ProbOn Plus (Fisher Scientific) glass slides and allowed to dry overnight. On the following day, the sections were dewaxed in xylene, rehydrated in graded ethanol, brought into water, and washed in PBS (pH 7.4). The endogenous peroxidase activity in these tissue sections was inactivated by incubating them in 3% H2O2 in absolute methanol for 30 min at room temperature. Then, the sections were protein blocked by incubating them in a solution containing 10% normal goat serum and 1% BSA (Fraction V, Sigma) in PBS at room temperature for 4 h. The primary antibody, diluted 1:2000 in protein-blocking solution, was applied to the sections and incubated overnight at 4°C. Control sections were incubated in preimmune serum instead of primary antibody. After extensive washing to remove the unbound fraction, the bound antibody was visualized by the Biotin-Streptavidin method using a commercially available, supersensitive detection kit (StrAviGen; BioGenex, San Raman, CA) that has diaminobenzidine tetrahydrochloride as the chromogen. Sections were counterstained with Mayer hematoxylin, dehydrated in a series of increasing concentration of ethyl alcohol, and cover-slipped under Permount.
Testicular Steroidogenesis In Vitro
The fresh testis removed from each rat was weighed, decapsulated, and incubated in Krebs-Ringer bicarbonate buffer aerated for 10 min and supplemented with 2% (w/v) glucose [1, 9, 13] and maximum stimulatory dose of LH (Ovine LH, 100 ng/ml, from the National Pituitary Hormone Distribution Program) [1, 9, 14, 15]. Incubations were performed in 20-ml glass scintillation vials containing 2 ml of buffer in a shaking water bath (90 oscillations/min) at 34°C for 3 h [1, 9, 13]. At the end of the incubation period, the medium was removed, centrifugation performed at 300 x g for 10 min, and the supernatant stored at -80°C until assay for androstenedione and testosterone levels.
RIA for Hormones
Testosterone and androstenedione concentrations in the blood sera and testis incubation media as well as the total T3 content in blood sera were quantified by commercially available RIA kits (Coat-A-Count; DPC, Los Angeles, CA). The sensitivity of the assays for both testosterone and androstenedione was 0.04 ng/ml. In our study, the intra-assay coefficient of variation for both these assays was less than 10%. The assay used to determine the total serum T3 content carried a sensitivity of 7 ng/dl, and the intra-assay coefficient of variation in this assay was less than 8%. The cross-reactivity of the antibody used in the assays for testosterone and androstenedione was less than 2.8% and 1.5% respectively, for any other steroid. The cross-reactivity of the T3 antibody with thyroxine was 0.5%.
Statistical Analysis
Significant differences (P < 0.05) among the means of the evaluated parameters in the same treatment group (i.e., either control, hypothyroid, or hyperthyroid) at different time points or in different treatment groups (i.e., in control, hypothyroid, and hyperthyroid) at the same time point were determined by ANOVA followed by Duncan Multiple Range Test using the linear model of the Statistical Analysis Systems program (Statistical Analysis System Institute, Inc., Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In control rats, body weights did not change during the first week of treatment, but they increased significantly during the second week and remained constant thereafter (Table 1). In the hypothyroid and hyperthyroid groups, the lowest body weights were seen on Day 14 after EDS administration. Comparison of control, hypothyroid, and hyperthyroid rats at each time point following EDS injection revealed that both the hypothyroid and hyperthyroid rats had lower body weights than the control rats on Day 7 and thereafter. The lowest body weight in the hyperthyroid rats was seen on Day 14 after EDS treatment.
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Testis Weights
Within each treatment group of thyroid-intact rats, testis weights gradually decreased. The lowest values were observed on Day 14 for control and hyperthyroid rats and on Day 21 for hypothyroid rats. Comparison among the three thyroid-intact treatment groups revealed that the testis weights were not different on Days 2 and 7 following EDS treatment, but on Days 14 and 21, the average testicular weight was significantly greater in the hypothyroid rats compared with the age-matched control and hyperthyroid rats. At both Days 14 and 21, the lowest testis weight was seen in the hyperthyroid rats (Table 1).
Qualitative Morphology
On Day 2 after EDS administration, the testicular interstitium was totally devoid of normal Leydig cells in control rats (Fig. 1A) and hypothyroid and hyperthyroid rats (results not shown). However, Leydig cells undergoing extensive degenerative changes were occasionally observed. These were in the form of darkly stained bodies, with vacuolated cytoplasm and fragmented nuclei. Macrophages were abundant in the testicular interstitium (Fig. 1A). Many mesenchymal cells and lesser numbers of macrophages was seen on Day 7, but Leydig cells were still absent (Fig. 1B). Moreover, occasional mast cells, which were easily identified by their pale-staining nucleus and bright, purple-red cytoplasmic granules, were observed in the testicular interstitium of all treatment groups at this stage. Until this stage of the experiment, the general appearance of the testis interstitium was similar in all three thyroid-intact treatment groups. At Day 14, a further increase in mesenchymal cells was evident in these three treatment groups; however, Leydig cells were still absent (Fig. 1C). Leydig cells were present in both the control and hyperthyroid rats on Day 21 after EDS treatment; however, they were absent in the hypothyroid rats even at Day 21 (Fig. 1D). In control rats, they were exclusively confined to the peritubular region of the interstitium (Fig. 1E). In hyperthyroid rats, Leydig cells were more abundant than in the control rats and were primarily found in the peritubular region (Fig. 1F), but a few were also seen in the central part of the testis interstitium, including the perivascular area. Leydig cells in the peritubular region were more elongated in shape, which contrasted with those found in the central interstitium and perivascular area, which had circular or polygonal profiles. No Leydig cells were observed in the thyroidectomized rats on Day 21 (results not shown). Mast cells were commonly seen in the testicular interstitium of all treatment groups at this stage of the experiment.
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Morphometry
Results of the morphometric studies are shown in Table 2. The total number of mesenchymal cells per testis increased significantly on Days 7, 14, and 21 after EDS treatment in control and hypothyroid rats and on Days 7 and 14 in hyperthyroid rats. However, a significant decline was observed on Day 21 in the hyperthyroid rats. The total number of mesenchymal cells per testis was not different among these three treatment groups on Days 2 and 7 after EDS treatment. At Days 14 and 21, the mesenchymal cell number per testis was highest in the hypothyroid rats and lowest in the hyperthyroid rats. Comparison within each treatment group showed that in all three groups, the total number of macrophages per testis decreased dramatically from Days 2 to 7 after EDS treatment, and then decreased slowly thereafter. Comparison among the treatment groups at each time point after EDS treatment showed that hyperthyroid rats contained significantly fewer macrophages on both Days 14 and 21 compared with age-matched control and hypothyroid rats. Leydig cells were absent in all three treatment groups (control, hypothyroid, and hyperthyroid) on Days 2, 7, and 14 after EDS treatment, and also at Day 21 in the hypothyroid and thyroidectomized rats. The number of Leydig cells per testis in hyperthyroid rats on Day 21 was twofold greater than in control rats. The total number of myoid cells, pericytes, and endothelial cells was not different in the three treatment groups of thyroid-intact rats at any stage of the investigation (results not shown).
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Immunocytochemistry
At Days 2 and 7 after EDS treatment, 3ß-HSD-positive cells (Leydig cell progenitors and Leydig cells) were absent in the testis interstitium of rats in all experimental groups. A few peritubular mesenchymal cells showed positive immunolabeling for 3ß-HSD on Day 14 in hyperthyroid rats (Fig. 2A), but none showed positive immunolabeling in the control and hypothyroid rats (results not shown). At Day 21, hyperthyroid rats contained many 3ß-HSD-positive cells at the peritubular region and a few away from the peritubular region (Fig. 2B). The 3ß-HSD-positive cells found away from the peritubular region were rounder in configuration compared with those found in the peritubular region. Although 3ß-HSD-positive cells were observed in control rats at Day 21 (Fig. 2C), they were few in number compared with those in hyperthyroid rats of the same age (Fig. 2B). These 3ß-HSD-positive cells in control rats at Day 21 were found exclusively in the peritubular region. However, 3ß-HSD-positive cells were not seen in hypothyroid rats at any stage of the investigation (Fig. 2D) or in thyroidectomized rats at Day 21.
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Hormones
Serum testosterone levels (Fig. 3A) were less than the detection limit (0.04 ng/ml) of the assay in control rats from Days 2–14, in hypothyroid rats from Days 2–21, and in hyperthyroid rats at Days 2 and 7 after EDS treatment. A low level of testosterone was seen in hyperthyroid rats at Day 14. At Day 21, a sharp rise in serum testosterone levels was observed in both the control and hyperthyroid rats; however, the concentration in the hyperthyroid rats was twice that in control rats at Day 21. Serum androstenedione levels (Fig. 3C) could not be detected in all three groups of thyroid-intact rats at Days 2, 7, and 14. At Day 21, a negligible amount was detected in control rats, and a significantly higher amount was seen in the hyperthyroid rats.
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The LH-stimulated testicular testosterone secretory capacity in vitro was in agreement with the presence or absence of Leydig cells in the testis interstitium of each treatment group, and it followed a pattern similar to that of the serum testosterone levels, but with larger values on Days 14 and 21 (Fig. 3B). Androstenedione levels in the incubation media were detected only in control rats on Day 21 and in hyperthyroid rats on Days 14 and 21. On Day 21, hyperthyroid rats produced threefold as much androstenedione as controls rats of the same age (Fig. 3D).
Serum levels of T3 continued to increase in control rats, whereas they decreased significantly in hypothyroid rats with time after EDS injection. Serum T3 levels in hypothyroid rats were significantly reduced on Day 7 and thereafter compared with control rats. In hyperthyroid rats, serum T3 levels remained elevated throughout the experiment, although at Days 7 and 14, these levels were lower than those at Days 2 and 21 (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study demonstrates that the number of mesenchymal cells per testis shows a twofold increase at Day 7 following EDS treatment, and that it continues to increase to Day 21 in control and hypothyroid rats and Day 14 in hyperthyroid rats. These results compare favorably with the findings of previously published, autoradiographic studies that demonstrated a wave of mesenchymal cell proliferation in mature rats following EDS administration [19, 20]. The increase in mesenchymal cell numbers after EDS treatment can be viewed as a mechanism to provide adequate precursor cells with which to repopulate the testis interstitium with Leydig cells. No difference was found among the total numbers of mesenchymal cells per testis at Day 7 in the three groups of thyroid-intact rats, so it seems logical to suggest that the rate of increase in the mesenchymal cell number per testis in these rats is approximately the same at this early stage. By contrast, at Day 14, the hypothyroid rats contained significantly higher numbers of mesenchymal cell, and the hyperthyroid rats contained significantly fewer mesenchymal cells than control rats. Because these changes in the mesenchymal cell numbers occurred in the absence of mesenchymal cell differentiation into Leydig cells at this early stage, these findings can be attributed to differences in the thyroid hormone status on the rate of mesenchymal cell proliferation in these rats. Similar patterns of changes in mesenchymal cell numbers have been observed during prepubertal development of the rat testis, in which hypothyroidism resulted in increased numbers of mesenchymal cells [1, 21] and hyperthyroidism suppressed their multiplication [22]. Alternatively, mesenchymal cells may have differentiated into another interstitial cell type that we have not quantified in the present study, either as a result of the thyroid hormone status or as a reaction to EDS treatment (or both). In addition, the mesenchymal cells may have been engulfed by the macrophages, which are abundant in the testis interstitium (compared with published values for untreated rats [9, 15]) following EDS treatment. On Day 21, the hyperthyroid rats contained significantly fewer mesenchymal cells per testis than at Day 14. This observation, together with the finding of a doubling of the number of Leydig cells in the hyperthyroid rats on Day 21, implies the differentiation of increased numbers of mesenchymal cells into Leydig cells in this treatment group compared with control rats. Moreover, the continuous increase in mesenchymal cell numbers and the absence of Leydig cells in hypothyroid rats suggest that hypothyroidism inhibits the differentiation of adult Leydig cells from the mesenchymal cells and, as a result, mesenchymal cell numbers continue to increase in the hypothyroid rats.
As expected from the results of many previous studies [3], we also observed apoptotic degeneration and removal of Leydig cells from the testis interstitium within 2 days after EDS administration. Therefore, this process clearly was not affected by the thyroid status of the rat. Similar to previously published reports [17, 18, 23–31], no Leydig cells were detected in the present study during the first 2 wk following EDS administration, but Leydig cells were seen at Day 21. Evidence has also been presented previously [24, 27] for a mesenchymal origin for the newly formed Leydig cells in the EDS-treated rat testis, and their site of differentiation has been described as peritubular and perivascular. Although in the present study we observed a mesenchymal origin for the new generation of Leydig cells, they were confined to the peritubular position. Therefore, we suggest that the Leydig cells found away from the peritubular region represented either the Leydig cells that were formed later during this process or the cells that were displaced from a peritubular position following the initial differentiation stage. This view is supported by the observed differences in the shapes of the Leydig cells and the 3ß-HSD-positive cells in the peritubular and nonperitubular regions of the testis interstitium. Based on these findings, we suggest that the peritubular mesenchymal cells are the principal, if not the only, precursor cells for the new generation of Leydig cells in the EDS-treated adult rats. Similarly, peritubular mesenchymal cells have been observed as the precursor cells for the adult Leydig cells in prepubertal rat testis [2, 19, 23, 32], except for one report that suggests the mesenchymal cells in the central interstitium as the Leydig cell precursors [33]. It is difficult for us to accept this suggestion, however, which is contradictory to the findings of many others in the field, especially because this statement has not been justified in the said study [33] or in a follow-up study using appropriate markers to identify these precursor cells [34].
The observations of a doubling of the number of Leydig cells per testis in hyperthyroid rats compared with control rats at Day 21 and the absence of Leydig cells in the hypothyroid rats were intriguing. To our knowledge, these findings demonstrate for the first time the importance of thyroid hormone in the process of Leydig cell regeneration from mesenchymal cells in the adult rat testis and also following EDS administration. Despite the lack of Leydig cells in any of the thyroid-intact treatment groups up to and on Day 14, 3ß-HSD-positive progenitor cells were identified in the peritubular region of the testis interstitium in hyperthyroid rats on Day 14. At this stage, they were mostly elongated in shape; interestingly, elongated, spindle-shaped cells containing 3ß-HSD-positive cells in the testis interstitium of neonatal rats were described by Lording and de Kretser [32] more than two decades ago. This observation in the hyperthyroid rats of the present investigation implies that T3 treatment caused accelerated differentiation of Leydig cells in these rats in addition to production of increased numbers of Leydig cells. The absence of 3ß-HSD-positive cells in the hypothyroid and thyroidectomized rats confirms that the failure of Leydig cell differentiation in the hypothyroid rats was, indeed, due to the thyroid hormone deficiency and not to any other effects of PTU. These findings are reminiscent of the findings on thyroid-hormone deficiency and arrest in adult Leydig cell differentiation in the prepubertal rat [1, 2, 22]. Therefore, we cannot agree with the observation that hypothyroidism induces proliferation and increased numbers of adult Leydig cells in the neonatal rat testis as published in a recent report [35].
At this juncture, it is difficult to suggest whether thyroid hormone acts directly on mesenchymal cells or indirectly via the seminiferous tubules as local factors that stimulate Leydig cell differentiation, as has been observed in the unilaterally cryptorchid EDS rats [31, 36]. Results of some previous studies suggest that thyroid-hormone receptors are present only in the immature testis, not in the adult testis, and are confined exclusively to the seminiferous tubules [37, 38]. However, data are now available to support the presence of thyroid-hormone receptors in cells of the rat testis interstitium, including the mesenchymal cells [39–41]. Therefore, a direct effect of thyroid hormone on mesenchymal cells to initiate their differentiation into Leydig cells is still a possibility.
Testis has a relatively high concentration of resident macrophages [42], and the estimated total number per adult rat testis is approximately 7 million cells [9, 15]. Therefore, the number of macrophages per testis on Day 2 after EDS treatment as obtained in the present study is approximately threefold greater than the published values for untreated adult rats [9, 15]. An increase number of macrophages in the testis interstitium 2 days following EDS treatment has been previously documented by several investigators [17, 26, 43, 44]. However, the mechanism by which the number of testicular macrophages increases within a such a short time is not clear from the present study or from the previous ones. We did not detect any mitotic figures in macrophages in the present investigation; however, division of existing macrophages in the testis is one possibility [44]. Although differentiation of mesenchymal cells into macrophages has been suggested [45] based on the histological origins of the connective tissue cell types, this is not a valid explanation, because macrophages derive from colony-forming units similar to blood cells and not from the mesenchymal cells [46]. A possibility also exists that the increased number of macrophages is due to the migration of this cell type from other areas of the body, to clean up the excessive tissue debris resulting from Leydig cell apoptosis following EDS treatment. In addition, it is logical to expect elevated numbers of macrophages in the testis interstitium before the differentiation of a new population of Leydig cells takes place, because macrophages are essential for this process to occur [47, 48]. Removal of testicular macrophages by intratesticular injection of dichloromethylene diphosphonate containing liposomes prevents regeneration of Leydig cells following EDS administration to mature rats [47, 48].
The present study also showed significant reductions in the number of macrophages per testis in all three treatment groups of thyroid-intact rats on Day 7 after EDS, although these values were still higher than the previously published, pretreated values [9, 15]. In the present investigation, the number of macrophages per testis reached, or was close to, pretreatment values by Day 14 in hyperthyroid rats and by Day 21 in the other two groups. Although the mechanism underlying this phenomenon is not clear, it is logical to speculate that the newly formed Leydig cells may be capable of down-regulating macrophage numbers with time to maintain the proper ratio between these two cell types in the testis interstitium. The gradual decline in macrophage numbers with time observed in the present study is in agreement with the observations of Kerr et al. [24] but is different from the results of Wang et al. [49], who reported a transient decline of macrophage numbers in EDS-treated rats around Day 21. This discrepancy may be attributed to the differing procedures used to identify and quantify macrophages in these studies. The current morphometric study and that of Kerr et al. [24] used plastic-embedded material to identify macrophages morphologically, whereas Wang et al. [49] counted macrophages in 5-µm-thick frozen sections after immunocytochemical staining for specific antigens.
Serum concentrations of T3 in hyperthyroid and hypothyroid rats in the present investigation were in agreement with their hyperthyroid and hypothyroid conditions, respectively. Our data on serum testosterone levels in control rats were comparable to the results published in previous reports [18, 25, 26, 50] and involved undetectable serum levels of testosterone during the first and second weeks following EDS treatment in these three treatment groups and a sharp rise during the third week. The absence of such an increase of serum testosterone levels in hypothyroid rats and the detection of twice as much testosterone in hyperthyroid rats compared with control rats are in agreement with the inhibition of Leydig cell development in the hypothyroid rats and the abundance of Leydig cells in the hyperthyroid rats compared with control rats. Testicular androstenedione secretory capacity in vitro was measured in the present investigation to monitor the differentiation of new Leydig cells. To our knowledge, androstenedione concentrations in serum of adult rats following EDS administration have not been published before. Nevertheless, results of the present investigation show that serum androstenedione levels follow a pattern similar to that of testosterone, but at a much lower concentration. The LH-stimulated testicular androgen production in vitro demonstrated higher levels of androgens, but in a pattern similar to those of serum hormones. These results further demonstrate the stimulatory effects of T3 and the inhibitory action of PTU on Leydig cell differentiation; to our knowledge, other studies are not available with which compare these findings.
Identical results obtained on Leydig cell regeneration in hypothyroid and thyroidectomized rats following EDS treatment strongly suggest that observed differences between the control and hypothyroid rats on Leydig cell differentiation are not due to a direct action of PTU on testicular interstitium. Rather, they are caused by the hypothyroid effects of PTU. A similar conclusion has been reached by Cooke and Meisami [4] regarding the effect of PTU on immature Sertoli cells in the prepubertal rat testis.
In summary, morphometric, immunocytochemical, and hormonal findings of the present study reveal that the regeneration of Leydig cells in the EDS-treated adult rat testis is arrested by hypothyroidism and stimulated by hyperthyroidism. That the lowest number of mesenchymal cells and the highest number of Leydig cells per testis occurred in hyperthyroid rats, and that the highest number of mesenchymal cells and the absence of Leydig cells occurred in the hypothyroid rats, add strong support to the hypothesis that thyroid hormone is essential for the differentiation of mesenchymal cells to Leydig cells in the postnatal rat testis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by CMH-Grants from COE R180101-08 UT Minkel; JIM-MRC. 
2 Correspondence. FAX: 865 974 2215; mendisc{at}utk.edu
Accepted: May 18, 2000.
Received: February 16, 2000.
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 M.M. Ben Saad and D.L. Maurel Reciprocal Interaction Between Seasonal Testis and Thyroid Activity in Zembra Island Wild Rabbits (Oryctolagus cuniculus): Effects of Castration, Thyroidectomy, Temperature, and Photoperiod Biol Reprod, April 1, 2004; 70(4): 1001 - 1009. [Abstract] [Full Text] [PDF]
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H. Zhu, H. Ma, H. Ni, X.-H. Ma, N. Mills, and Z.-M. Yang Expression and Regulation of Lipocalin-Type Prostaglandin D Synthase in Rat Testis and Epididymis Biol Reprod, April 1, 2004; 70(4): 1088 - 1095. [Abstract] [Full Text] [PDF]
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F.-p. Zhang, T. Pakarainen, F. Zhu, M. Poutanen, and I. Huhtaniemi Molecular Characterization of Postnatal Development of Testicular Steroidogenesis in Luteinizing Hormone Receptor Knockout Mice Endocrinology, March 1, 2004; 145(3): 1453 - 1463. [Abstract] [Full Text] [PDF]
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A. Salva, M. P. Hardy, X.-f. Wu, C. M. Sottas, D. T. MacLaughlin, P. K. Donahoe, and M. M. Lee Mullerian-Inhibiting Substance Inhibits Rat Leydig Cell Regeneration after Ethylene Dimethanesulphonate Ablation Biol Reprod, March 1, 2004; 70(3): 600 - 607. [Abstract] [Full Text] [PDF]
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 M. G. Wade, S. Parent, K. W. Finnson, W. Foster, E. Younglai, A. McMahon, D. G. Cyr, and C. Hughes Thyroid Toxicity Due to Subchronic Exposure to a Complex Mixture of 16 Organochlorines, Lead, and Cadmium Toxicol. Sci., June 1, 2002; 67(2): 207 - 218. [Abstract] [Full Text] [PDF]
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I.-s. Kim, H.B. Siril Ariyaratne, and S.M.L. C. Mendis-Handagama Changes in the Testis Interstitium of Brown Norway Rats with Aging and Effects of Luteinizing and Thyroid Hormones on the Aged Testes in Enhancing the Steroidogenic Potential Biol Reprod, May 1, 2002; 66(5): 1359 - 1366. [Abstract] [Full Text] [PDF]
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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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