| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Department of Animal Science, The University of Tennessee, Knoxville, Tennessee 37996
b University Department of Reproductive and Developmental Sciences, Royal Infirmary of Edinburgh NHS Trust, Edinburgh, Scotland EH397W, United Kingdom
ABSTRACT
The main objective of the study was to investigate the effects of hyperthyroidism on the rat testis interstitium during prepuberty, which is not well understood at present. Male Sprague Dawley rats were injected subcutaneously daily with saline (controls) or tri-iodothyronine (T3, 50 µg/kg body weight; hyperthyroids) from postnatal Day 1. Rats were killed at Days 5, 7, 9, 12, 16, and 21. One testis of each rat was used to determine LH-stimulated (100 ng/ml) testicular androgen secretory capacity in vitro. The other testis was used either for morphometric studies (n = 5) or for immunolocalization of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) to identify steroidogenic cells (n = 3) and 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) to differentially identify adult Leydig cells. Daily T3 injections resulted in significant reductions in body and testis weights. Morphometric analysis revealed that lower testis weights in rats treated with T3 were mainly the result of reductions of total volume of seminiferous cords/tubules. The number of interstitial mesenchymal cells (MCs) was lower (P < 0.05) in T3 rats compared with age-matched controls. The number of fetal Leydig cells (FLCs) was not different between the two groups; however, FLC hypotrophy was detected in T3 rats at Day 16 in contrast to Day 21 in control rats. In both groups, morphologically identifiable adult Leydig cells (ALCs) were observed at Day 12 and thereafter; however, the ALC number per testis in T3 rats was twice as much as those of controls. Positive immunolabeling for 3ß-HSD was first detected in MC/progenitor cells on Day 9 in rats in the T3 group (cells were still spindle-shaped) and on Day 12 in rats in the control group. Testicular testosterone production in vitro was lower (P < 0.05) in T3 rats compared with controls at each age tested and further reductions (<0.05) were observed in T3 rats at Days 16 and 21. Testicular androstenedione production was also lower (P < 0.05) in T3 rats at Days 5 and 7, but increased (P < 0.05) thereafter, than in control rats. These findings support that there are more newly formed ALCs in T3 testes than in those of controls. Moreover, these results demonstrate that hyperthyroidism stimulates premature hypotrophy of FLCs and early differentiation of increased numbers of MCs to ALCs in the prepubertal rat testis, further supporting the view that thyroid hormone has a regulatory role in initiating MC differentiation into ALCs in the prepubertal rat testis.
hormone action, interstitial cells, Leydig cells, testes, testosterone
INTRODUCTION
Fetal Leydig cells appear in the rat testis between 14 and 14.5 days of gestational age [1]. They are present at birth [2–6] and up to 90 days of age [5, 6]. It is documented that fetal Leydig cells undergo cell hypotrophy at the third postnatal week [3, 4, 6] but recent studies have revealed that this is a transient event [6]. It is also interesting to note that, in adult rat testis, Leydig cells show a significant increase in their numbers also at the third postnatal week [3, 4, 6] when the atrophic changes are observed in the fetal Leydig cells. This brings into question whether there is an interdependency of these two Leydig cell types. Further support for this concept is provided by several other studies. Gayton et al. [7] observed that when fetal Leydig cell population was increased in response to hCG treatment, adult Leydig cell differentiation was arrested. Kerr et al. [8] demonstrated premature differentiation of adult Leydig cells in the neonatal rat testis when fetal Leydig cells were destructed by ethane dimethane sulphonate (EDS) treatment. Finally, we recently observed that fetal Leydig cells in postnatal rat testis do not show cell hypotrophy at the third postnatal week under a hypothyroid status and, in such testes, adult Leydig cell differentiation is arrested [4]. It has been also shown that neonatal hyperthyroidism causes early differentiation of adult Leydig cells in the prepubertal rat [9]. Therefore, it is logical to expect precocious hypotrophy of fetal Leydig cells under a hyperthyroid status; however, to date, no such information is available in the literature.
Inhibition of adult Leydig cell differentiation in the prepubertal rat testis under thyroid hormone deficiency is also suggestive of a regulatory role by the thyroid hormone on mesenchymal cell differentiation into adult-type Leydig cells in the prepubertal rat testis. Although it has been previously shown that there is early differentiation of adult Leydig cells in rats under neonatal hyperthyroidism [9], it is not known whether hyperthyroidism could induce early atrophy of fetal Leydig cells, more mesenchymal cell differentiation, or both, to produce more adult Leydig cells. In addition, other structural and functional changes in the testis interstitium of the prepubertal rat under hyperthyroid conditions are not known at present. Moreover, although the effects of hyperthyroidism on Sertoli cells are documented to some extent in prepubertal humans [10], to our knowledge, no data are available on Leydig cells in prepubertal humans or any other species.
MATERIALS AND METHODS
Animals
Female Sprague Dawley rats in midpregnancy were obtained from Harland Industries (Madison, WI). They were housed one to a cage under controlled temperature (25°C) and lighting (14L:10D) conditions. All rats were fed with Agway Prolab rat formula (Syracuse, NY) and drinking water was provided ad libitum. Rats were observed twice daily (morning and evening) for litters and the day of birth of pups was considered as Day 1 of their age.
Treatments
Twelve groups of male rat pups (n = 8 rats per group) were used. Each rat in 6 of these groups received a daily s.c. injection of T3 (50 µg/kg body weight) dissolved in 0.025 N NaOH in saline, beginning from postnatal Day 1. The remaining 6 groups of rat pups served as controls and were given daily subcutaneous injections of 0.025 N NaOH in saline. Both control and T3 rats were killed at Days 5, 7, 9, 12, 16, and 21. The animal protocols used in this study were approved by The University of Tennessee Animal Care and Concerns Committee.
Collection and Preparation of Tissues
Under deep inhalation anesthesia of Metofane (Mallincroft Veterinary Inc., Mundelein, IL), heart blood was collected from rats in both groups at Days 5, 7, 9, 12, 16, and 21 (n = 8 rats/group), and sera were prepared and stored at -80°C until assay.
In 5-, 7-, and 9-day-old rats (n = 8 rats per group) both testes were removed while the animals were anesthesized with Metofane (Mallincroft). One testis of each of these younger rats (n = 8 testes per group) was utilized to determine LH-stimulated in vitro steroid secretion capacity (description follows). The remaining testis of 5 rats from each group was weighed to obtain the fresh testis weight and the specific gravity (determined by floatation techniques [11, 12]) to obtain the fresh testis volume. This step was followed by fixing these testes by immersion in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Small cuts were made on the testis capsule to facilitate penetration of fixative into testis parenchyma. After 2–3 h of fixation the testes were cut in to blocks (1–2 mm) and allowed to further fix in the same fixative overnight at 4°C. The remaining testis in the other three rats of each group was fixed by immersion in Bouins solution for 5–6 h, washed with 70% ethanol for several days until the yellow color/picric acid disappeared from the ethanol, processed, and embedded in Paraplast (melting point = 50°C) to be used for immunocytochemical studies. In rats of the 12-, 16-, and 21-day age groups (n = 8 rats per group), 1 testis (n = 8 testes per group) was used to determine the LH-stimulated androgen secretory capacity in vitro following determination of the testis volume, similar to the procedures used in the younger rats of this study. The ipsilateral testis of five rats in each treatment group was fixed by a whole body perfusion technique using 2.5% glutaraldehyde in cacodylate buffer (pH 7.4) as described previously [4, 6, 12]. The fixed testis was removed, weight (fixed testis weight) and specific gravity were determined, and then processed and embedded in epon-araldite according to previously published reports [4, 6, 12, 13]. The ipsilateral testis of the other three rats in each treatment group was fixed and processed for immunocytochemistry similar to the procedure described earlier for the testes of younger rats.
Microscopy and Morphometry
From the polymerized epon-araldite-embedded testis tissue blocks, serial sections of 1 µm in thickness were cut using a LKB IV ultramicrotome (Pharmacia LKB, Piscataway, NJ) and glass knives. Two sections, which were 4 µm apart (i.e., the first and the fifth of the serial sections) were collected, mounted adjacent to each other on a precleaned glass slide (Superfrost Plus; Fisher Scientific, Pittsburgh, PA), stained with methylene blue azure II stain, and coverslipped under Permount (Fisher Scientific, Fair Lawn, NJ). These sections were viewed under an Olympus BH-2 microscope (Olympus, Tokyo, Japan) for morphological and morphometric studies. Numerical density of testicular interstitial cell types was obtained by using the disector method [14] as previously described [6]. Six tissue blocks per rat from 5- and 7-day-old rats, 8 tissue blocks per rat from 9-day-old rats, and 10 tissue blocks per rat from 12-, 16-, and 21-day-old rats were used. The area of the disector was calculated (length x width) as 4230 µm2. Different cell types in the testicular interstitium were identified by their characteristic morphology as described in previous studies [3, 4, 6]. The total number of each cell type per testis was calculated by multiplying the numerical density of each cell type by fresh testis volume.
The volume density of testicular components (defined as volume of the component per unit volume of testis) was obtained by the point counting method [15] as described previously [13] using a 40x objective lens and an 8x ocular lens with a test grid containing 121 test points. The absolute volume of each testicular component was obtained by multiplying the volume density of that component by the fresh testis volume. The average volume of each interstitial cell type was calculated by dividing the total volume of that cell type by its total number.
Immunocytochemistry for 3ß-Hydroxysteroid Dehydrogenase (3ß-HSD) and 11ß-Hydroxysteroid Dehydrogenase-1 (11ß-HSD1)
Bouins-fixed and Paraplast-embedded tissues were used to immunolocalize 3ß-HSD and 11ß-HSD1 enzymes. Sections (5 µm) were cut from these tissue blocks and adhered to Probe On Plus (Fisher Scientific) glass slides and left at room temperature overnight. On the following day, these sections were dewaxed by three changes in xylene, rehydrated by passing them through a series of decreasing concentrations of ethanol, and brought to deionized water. These sections were then washed in PBS (pH 7.6). Both antigen-retrieved and normal slides were used to immunolocalize 3ß-HSD (marker for all steroid-secreting cells, including Leydig cells) and 11ß-HSD1 enzymes. Briefly, the sections were submerged in 0.01 M citrate buffer (pH 6) and microwaved at full power (650 watts, 4 times, 5 min each). After keeping the sections undisturbed for an additional 20 min in the same buffer, the sections were washed in PBS. In all sections, the endogenous peroxidase activity was blocked by incubating the sections in 3% H2O2 in absolute methanol for 20 min at room temperature. The nonspecific binding was prevented by incubating the sections in a solution containing 10% normal goat serum and 1% bovine serum albumin (Fraction V, Sigma) for 3 h at room temperature. Test and control sections were incubated overnight at 4°C in primary antibody or in normal rabbit serum diluted in protein blocking solution, respectively. The polyclonal antibody used in this study to immunolocalize 3ß-HSD was a rabbit immunoglobulin G (IgG) raised against purified human placental 3ß-HSD protein [16] and has previously been used in immunostaining of 3ß-HSD in rat testis [2, 17]. An antibody dilution of 1:2000 was seen as optimal after preliminary studies using dilutions ranging from 1:500 to 1:5000. The polyclonal antibody used in immunostaining of 11ß-HSD1 was elucidated in rabbit against rat liver 11ß-HSD1 [18] and has been demonstrated to be effective in immunolocalization of this antigen in adult-type Leydig cells in rat testes [4, 18]. In our study, this antibody was diluted 1:4000 in blocking solution before use. After extensive washing to remove unbound fraction, the bound antibody was detected by the biotin-streptavidin method using a commercially available highly sensitive detection kit (BioGenex, San Raman, CA) according to the manufacturer's instructions. In this kit, diaminobenzidine (DAB) was used as a chromogen. These sections were counterstained with Mayers hematoxylin, dehydrated in graded alcohol, and cover-slipped under Permount.
LH-Stimulated Testicular Androgen Secretory Capacity In Vitro
The fresh testis removed from each rat (n = 8 per group) was weighed, decapsulated, and incubated in Krebs-Ringer bicarbonate buffer (pH 7.4, aerated for 10 min) containing 2% glucose and a maximum stimulatory dose of LH (100 ng/ml, [19, 20]) at 34°C in a shaking water bath (90 oscillations per min) for 3 h as described before [4, 6, 21]. At the end of the incubation period, the medium was separated, centrifuged at 300 x g for 10 min, and the supernatant was stored at -80°C until analysis for testosterone and androstenedione content by radioimmunoassay.
Radioimunoassay of Hormones
Commercially available radioimmunoassay kits (DPC, San Francisco, CA) were used to determine testosterone and androstenedione concentrations in the incubation media and T3 levels in blood sera. The sensitivity of assays for both androgens was 0.04 ng/ml. In our study, the intra-assay coefficients of variation were less than 10% for these two hormones. The cross-reactivity of antibodies in testosterone and androstenedione assay kits were less than 2.8% and 1.5% for any other steroids, respectively. The sensitivity of the assay for T3 was 7 ng/dl and the intra-assay coefficients of variation in this assay was less than 8%. The cross-reactivity of the antibody used in the assay kit for total T3 with thyroxine was 0.5%.
Statistical Analysis
Significant differences (P < 0.05) between the means of each parameter tested for T3 and control rats at each age were determined by Student's t-test. Differences within a treatment group at different ages were determined by ANOVA followed by Duncan's Multiple Range Test.
RESULTS
Body and Testis Weights
Body weights in both control and T3-treated rats increased with age (Table 1). Comparison of body weights between the two treatment groups revealed that the average body weights of T3-injected rats were significantly lower than those of controls at all ages tested. A gradual increase in testis weights with increasing age was observed in both treatment groups (Table 1). Comparison of testis weights in T3 and control groups at each time point revealed that the average testis weight in T3 rats at Day 5 was slightly but significantly larger than that of control rats. The average testis weight was not significantly different between control and T3 rats at Day 7, but was significantly smaller than those of controls at Days 9, 12, 16, and 21. However, if testicular weight was expressed in terms of body weight, no differences were observed between control and hyperthyroid rats (Table 1).
|
Testicular Morphology
No apparent differences in testicular morphology were observed between T3 and control rats at Days 5 and 7. At Day 9, myoid cells in the T3 group appeared more distinct than those of controls. Fetal Leydig cells in control testes from Days 5–16 and T3 testes from Days 5–12 appeared as large polygonal profiles (Fig. 1, A–C). However, a majority of the fetal Leydig cell profiles in T3 rats at Day 16 appeared smaller than those of controls at Day 16 (Fig. 1, C and D) and younger. Moreover, fetal Leydig cells in both control and T3 rats at Day 21 (Fig. 1, E and F) appeared smaller than those at early ages (Fig. 1, A and B).
|
Adult Leydig cells were first detected in both treatment groups at Day 12 (Fig. 1, C and D); however, it appeared that there were more adult Leydig cells in T3-injected rats compared with controls. At Day 12, many of the adult Leydig cells in control rats contained only a thin rim of cytoplasm but a prominent round nucleus (Fig. 1C), but the same-aged T3 rats contained many adult Leydig cells with more cytoplasm in them (Fig. 1D). The number and size of adult Leydig cells rapidly increased with advancing age in both groups, but were more profound in T3 rats. Mitotic divisions of adult Leydig cells were observed as soon as they were detected on Day 12 in both treatment groups (Fig. 1, G and H) and these were more abundant in T3 rats.
Morphometry
Number of cells per testis for each interstitial cell type is presented in Table 2. The total number of fetal Leydig cells per testis in both T3 and control groups was not significantly different within a group at any age studied. Numbers of adult Leydig cells per testis significantly increased at each age from their first detection at Day 12. The number per testis of all the other interstitial cell types (i.e., macrophages, mesenchymal cells, myoid cells, endothelial cells, and pericytes) also increased gradually in the two treatment groups from Day 5 through Day 21. Comparison of rats in T3 and control groups at each age revealed that the number of adult Leydig cells per testis was significantly higher in T3 rats compared with control rats at all ages, beginning from Day 12. The total number of mesenchymal cells per testis was significantly reduced in T3-injected rats than controls at all ages studied. The number of macrophages per testis was not different between the two treatment groups except at Day 21, at which the T3 group contained more of these cells. In T3 rats there were more myoid cells per testis at Day 5, the number was not different at Day 7, and a lesser number of them was seen at Day 9 and thereafter compared with their control counterparts. Significant reductions were seen in the number of endothelial cells and pericytes per testis at Days 16 and 21 in T3 animals compared with their age-matched controls.
|
The volume density of the seminiferous cords/tubules and the testis interstitium and its components are shown in Table 3, but will not be further discussed. The absolute volume of the seminiferous cords/tubules gradually increased with age in both treatment groups. At Day 5, T3 rats had more seminiferous cords in volume per testis than the age-matched controls. From Days 7–12 there were no differences between the two groups, but at Days 16 and 27, T3 rats had significantly lower volumes of seminiferous cords/tubules than the controls of the same age. The absolute volumes of the testis interstitium and its components, except the fetal Leydig cells, increased with age in both treatment groups (Table 4). Comparisons between the two treatment groups at each age revealed that the absolute volume of the testis interstitium was not significantly different between the two treatment groups at any age studied. The absolute volume of adult Leydig cells per testis was significantly higher in T3 rats at each age that they were present, but the absolute volume of fetal Leydig cells per testis was different only at Day 16; a lower value was observed with T3 rats compared with the age-matched controls. The absolute volumes of mesenchymal cells and myoid cells per testis were lower in T3 rats compared with controls at all ages tested.
|
|
The average volume of a fetal Leydig cell in control rats remained unchanged from Day 5 through Day 16, but a 50% reduction was seen at Day 21 (Table 5). In T3 rats the average volume of a fetal Leydig cell remained unchanged until Day 12; however, at Days 16 and 21 they were 50% smaller than those in previous ages. The average volume of an adult Leydig cell and a macrophage increased with advancing age in both treatment groups. Comparisons between the two treatment groups revealed that the average volume of each of these cell types was not significantly different at many ages studied except fetal Leydig cells at Days 16 and 21, mesenchymal cells at Day 21, and myoid cells at Day 21.
|
Immunocytochemistry
Fetal Leydig cells were the only testicular interstitial cell type that was positive for 3ß-HSD in control rats at Days 5, 7, and 9; and in T3 rats at Days 5 and 7 (results not shown). Although fibroblast-like progenitor cells (first stage of the adult Leydig cell development) positive for 3ß-HSD were still absent in control rats on Day 9 (Fig. 2A), they were observed in T3 rats (Fig. 2B). However, on Day 12 and thereafter, 3ß-HSD-positive progenitor cells (elongated in shape), adult Leydig cells, and fetal Leydig cells were easily identified in both treatment groups (Fig. 2, C–F). Visual comparison of testes sections in control and T3 rats showed the presence of more 3ß-HSD-positive adult Leydig cells in hyperthyroid rats than in control rats (compare Fig. 2C with Fig. 2D, and Fig. 2E with Fig. 2F). Cells positive for 11ß-HSD1 were not detected in the testis interstitium of either control and T3 rats at Days 5, 7, 9, 12, and 16. On Day 21, few Leydig cells, weakly positive for 11ß-HSD1 enzyme, were observed in both treatment groups (results not shown).
|
Testicular Steroidogenesis In Vitro
LH-stimulated testicular testosterone secretory capacity in control rats was not significantly changed from Day 5 through Day 16 (Fig. 3A). On Day 21, a slight but significant decrease in testosterone production was observed in control rats (Fig. 3A). By contrast, testicular testosterone secretory capacity significantly declined with advancing age in T3-treated rats, which was extremely low at Days 16 and 21 (Fig. 3A). Comparison between the two treatment groups at each age showed that the testosterone secretory capacity per testis in T3 rats was significantly lower than those of age-matched controls. LH-stimulated androstenedione production per testis in vitro in control rats remained unchanged until Day 12 and increased significantly thereafter. In T3 rats, androstenedione production per testis continued to increase beginning from postnatal Day 9. Comparison of the two groups revealed that the testicular androstenedione-producing capacity in T3 animals was significantly less on Days 5 and 7 than it was in the age-matched controls; however, it was significantly higher on Day 9 and thereafter, compared with controls.
|
Serum T3
Serum T3 levels in control rats showed low levels from Day 5 through Day 9, a gradual but progressive increase was seen from Day 9 to Day 21, and the value on Day 21 was twofold greater compared with that at Days 5–7. Serum T3 levels in T3-treated rats were maintained severalfold higher than those of controls at every age tested (Fig. 3C).
DISCUSSION
Our preliminary studies demonstrated that daily injection of T3 at a dosage of 100 µg/kg body weight starting from Day 1 of age as used in some earlier short-term studies [9, 22] caused severe hyperthyroidism and death of pups around Day 12. Therefore, in this study, we used 50 µg/kg body weight of T3 to produce hyperthyroidism without compromising the general health of the rats.
The results of the study demonstrate that hyperthyroidism induced by T3 injection during the neonatal period affects body and testicular weights. Reductions in body weight of T3-injected animals as early as Day 5 of age indicate acute effects of hyperthyroidism on general growth of these rats, despite using a significantly lower dose of T3 compared with previously published studies [9, 22–24]. However, these differences at each age disappeared when testis and body weights were expressed as a ratio, highlighting that smaller rats have smaller testicles, at least in this instance.
It is an established fact that the final testicular size and its sperm-producing capacity depend on the testicular Sertoli cell content [25, 26]. Postnatal proliferation of Sertoli cells in the rat takes place during the first 3 wk of life [27]. Reduced testis size in rats after T3 injections has been shown to be the result of inhibited proliferation and premature differentiation of Sertoli cells in the postnatal rat resulting in dramatic reduction in the Sertoli cell number per testis [22]. Morphometric results of the present study agree with this view because reduced testis weights of T3 rats was associated with the reduction in the volume of seminiferous cords/tubules in T3 rats.
The continuous increase of interstitial cell types including mesenchymal cells, macrophages, myoid cells, endothelial cells, and pericytes in control animals with advancing age can be explained by the expansion of the interstitial compartment of the testis associated with continuous growth of the testis and agrees with previous studies [3, 4, 6]. This explanation is also valid for the observed increase in these cell numbers in T3-treated rats. The observation of similar cell numbers for macrophages, endothelial cells, and pericytes in the two treatment groups compare favorably with the results of similar total interstitial volumes in both treatment groups. The significantly reduced myoid cell numbers in T3 rats than those of control rats on Days 9–21 can be explained by the significant reduction in the total volume of seminiferous cords/tubules in T3-treated rats. The significantly reduced number of mesenchymal cells in the testis interstitium of the hyperthyroid rats compared with control rats compare favorably with the observation that T3 rats have more adult Leydig cells than the control rats, and support the hypothesis that T3 treatment has resulted in more mesenchymal cell differentiation into Leydig cells.
Hypothyroidism in the neonatal rat results in exactly the opposite for mesenchymal and adult Leydig cell numbers [4]. It was interesting to note that mitotic activity in testicular mesenchymal cells in the developing rat testis was observed under both euthyroid and hyperthyroid conditions. Because thyroid hormone receptor mRNA has been detected in these cells in a previous study [28], it is possible that the effects of T3 on testicular mesenchymal cell mitotic activity is direct. These observations taken together imply that thyroid hormone induces Leydig cell development in the prepubertal rat testis not only by stimulating the differentiation of mesenchymal cells, but also by increasing the number of precursor cells.
The total number of fetal Leydig cells per testis in control and T3 rats was unchanged during the tested period in both control and thyroid hormone-treated rats. This is in agreement with previously published reports [3, 4, 6]. Moreover, the results of the present study on the average volume of a fetal Leydig cell in control rats are similar to those previously published reports [3, 4, 6]. Although the reason or reasons for fetal Leydig cell atrophy at Day 21 in control rats is not clear, this has been interpreted as a part of the normal developmental process in the postnatal rat testis [4]. Therefore, the absence of fetal Leydig cell hypotrophy on Day 21 of age in the postnatal rat testis under hypothyroid conditions [4] suggests an involvement of thyroid hormone in the event of fetal Leydig cell atrophy in control rats. In our investigation, the discovery of the precocious atrophy of fetal Leydig cells (i.e., at Day 16) in T3 rats demonstrates an opposite effect of hyperthyroidism on fetal Leydig cells in comparison to hypothyroidism.
In the present study we observed morphologically identifiable adult Leydig cells (i.e., without immunocytochemistry) at Day 12 and thereafter in both control and T3-injected groups. The earliest detection of morphologically identifiable adult-type Leydig cells in the postnatal rat testis is documented as Day 10 [3]. The present study showed that there were more than double the number of adult Leydig cells in T3-treated rats compared with control rats. These results confirm the previous observations of Teerds et al. [9], which stimulated differentiation and increased mitotic activity of Leydig cells in the rat testis under hyperthyroid conditions. The immunocytochemical observations on 3ß-HSD in our study not only confirmed the earlier differentiation of adult Leydig cells in the hyperthyroid rats but also the presence of more adult Leydig cells in these rats compared with controls, suggesting that thyroid hormone induces Leydig cell differentiation in prepubertal rat testis. Therefore, it is not possible for us to comprehend how hypothyroidism could induce Leydig cell hyperplasia in the prepubertal rat testis as reported by Hardy et al. [28]. Lack of detectable differences in the immunolabeling for 11ß-HSD1 activity between control and T3-treated rats in our study suggests that, despite the stimulated differentiation of adult Leydig cells under hyperthyroid conditions, their maturation is not stimulated by T3 treatment. These findings suggest that Leydig cells require other factors for their maturation process.
Although we did not quantify the Leydig cells in mitotis, we frequently observed dividing Leydig cells in both treatment groups; however, the incidence was more in hyperthyroid rats. This observation suggests that adult Leydig cells are capable of undergoing mitosis immediately after their differentiation from their mesenchymal cell precursors. Further support for this suggestion comes from the observations of Teerds et al. [9] who reported increased bromodeoxyuridine incorporation into Leydig cells in the rat testis at Day 12 of age and thereafter. The presence of 3ß-HSD-positive Leydig progenitor cells together with mitotically dividing adult Leydig cells in the prepubertal rat testis before Day 21 of age implies that establishment of the adult population of Leydig cells in the rat testis results from two simultaneously occurring processes (i.e., mesenchymal cell differentiation and division of newly formed Leydig cells). This finding also contrasts with the suggestion of separate periods of differentiation and division (i.e., differentiation of mesenchymal cells during the third and fourth weeks and division of Leydig cells thereafter) as proposed by Hardy and coworkers [29, 30].
The results of our study on in vitro LH-stimulated testicular androgen production in control and T3 rats closely reflect the cellular changes that take place in the testis with advancing age. Consistent with our earlier observations [4], the testosterone production per testis in control rats remained unchanged from Day 5 through Day 16 while a lower value was detected on Day 21. This observation is simultaneously observed with the atrophy of fetal Leydig cells at this stage of testis development [3, 4, 6]. However, androstenedione production in control rats increased significantly from Day 12 onward as a result of the development of the adult population of Leydig cells; this finding agrees our previous report [4]. In contrast, T3-treated animals showed significantly reduced testosterone production in response to LH at all ages tested in the present study, and a very dramatic decrease at Days 16 and 21. Testosterone is primarily if not exclusively produced by the fetal Leydig cells at these early ages because the developing adult Leydig cells produce little or no testosterone at this time of testis development [31–33]. Therefore, the decline in testicular testosterone secretory capacity with advancing age from Days 5–12 in T3 rats is indicative of a pronounced inhibitory effect of T3 on fetal Leydig cell testosterone-secretory capacity, which occurs in the absence of cell hypotrophy. Further reductions in testicular testosterone-secretory capacity in T3 rats on Days 16 and 21 could be attributed to the significant reductions in the average volume of fetal Leydig cell at these ages, because Leydig cell volume has been shown to be positively correlated with its steroidogenic ability [13]. At Days 5 and 7, fetal Leydig cells were the only source of testicular androgens. Therefore, the reduced androstenedione secretion in T3 rats on these days compared with control rats is further suggestive of an inhibitory effect of T3 on fetal Leydig cell steroidogenesis. The increase observed in the androstenedione secretion per testis from Day 9 onward can be explained by the precocious differentiation of adult Leydig cells from mesenchymal cells under the stimulatory effect of T3.
The results of the study provide strong evidence to suggest that thyroid hormone has a regulatory role in stimulating the differentiation of mesenchymal cells into adult Leydig cells in the prepubertal rat testis. Thyroid hormone also appears to exert an inhibitory effect on fetal Leydig cell steroidogenic function, stimulation of fetal Leydig cell atrophy, and regulation of mesenchymal cell proliferation (i.e., in addition to differentiation) during Leydig cell differentiation in the prepubertal rat testis.
ACKNOWLEDGMENTS
We thank Kreis Weigel and Phil Snow in the photography department of The University of Tennessee Institute of Agriculture for their assistance in preparing the color plates, Dr. M.P. Hardy (Population Council, New York) for sharing with us Dr. Carl Monder's 11ß-HSD1 antibody, and Dr. A.F. Parlow and the National Pituitary Hormone Distribution Program for providing the ovine LH.
FOOTNOTES
First decision: 13 March 2000.
1 Correspondence. FAX: 865 974 2215; mendisc{at}utk.edu 
Accepted: March 29, 2000.
Received: February 15, 2000.
REFERENCES
5, 4 isomerase from placenta: expression in non-steroidogenic cells of a protein that catalyzes the dehydrogenation/isomerization of C21 and C19 steroids. Endocrinology 1990; 126:2493–2498.
-androstan-17ß-ol-3-one to 5-androstane-3ß,17ß diol and 5-androstane-3,17ß diol by rat testicular cells in vitro. J Reprod Fertil 1979; 57:143–147.This article has been cited by other articles:
|
|
 |
|
  E. Rijntjes, H. J. M. Swarts, R. Anand-Ivell, and K. J. Teerds Prenatal induced chronic dietary hypothyroidism delays but does not block adult-type Leydig cell development Am J Physiol Endocrinol Metab, February 1, 2009; 296(2): E305 - E314. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Dakhova, D. O'Day, N. Kinet, N. Yucer, M. Wiese, G. Shetty, and P. Ducy Dickkopf-Like1 Regulates Postpubertal Spermatocyte Apoptosis and Testosterone Production Endocrinology, January 1, 2009; 150(1): 404 - 412. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Wagner, S. M. Wajner, and A. L. Maia The role of thyroid hormone in testicular development and function J. Endocrinol., December 1, 2008; 199(3): 351 - 365. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Rijntjes, A. T. Wientjes, H. J. M. Swarts, D. G. de Rooij, and K. J. Teerds Dietary-Induced Hyperthyroidism Marginally Affects Neonatal Testicular Development J Androl, November 1, 2008; 29(6): 643 - 653. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Anguiano, N. Aranda, G. Delgado, and C. Aceves Epididymis Expresses the Highest 5'-Deiodinase Activity in the Male Reproductive System: Kinetic Characterization, Distribution, and Hormonal Regulation Endocrinology, August 1, 2008; 149(8): 4209 - 4217. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S M L C Mendis-Handagama, H B S Ariyaratne, L Mrkonjich, and R Ivell Expression of insulin-like peptide 3 in the postnatal rat Leydig cell lineage: timing and effects of triiodothyronine-treatment Reproduction, February 1, 2007; 133(2): 479 - 485. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. K. Potter, M. G. Zager, and H. A. Barton Mathematical model for the androgenic regulation of the prostate in intact and castrated adult male rats Am J Physiol Endocrinol Metab, November 1, 2006; 291(5): E952 - E964. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Wang, Y. Chen, Y. Ge, P. Ma, Q. Ma, J. Ma, H. Wang, S. Xue, and D. Han Immunoexpression of Tyro 3 Family Receptors--Tyro 3, Axl, and Mer--and Their Ligand Gas6 in Postnatal Developing Mouse Testis J. Histochem. Cytochem., November 1, 2005; 53(11): 1355 - 1364. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Naciff, K. A. Hess, G. J. Overmann, S. M. Torontali, G. J. Carr, J. P. Tiesman, L. M. Foertsch, B. D. Richardson, J. E. Martinez, and G. P. Daston Gene Expression Changes Induced in the Testis by Transplacental Exposure to High and Low Doses of 17{alpha}-Ethynyl Estradiol, Genistein, or Bisphenol A Toxicol. Sci., August 1, 2005; 86(2): 396 - 416. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. H. Foote Effect of High-Dose Liothyronine on Semen Quality and Recovery Following Withdrawal in Rabbits J Androl, November 1, 2002; 23(6): 839 - 844. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
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]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
