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Biology of Reproduction 59, 344-350 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Development of the Adult-Type Leydig Cell Population in the Rat Is Affected by Neonatal Thyroid Hormone Levels1

Katja J. Teerds2,c, Dirk G. de Rooijc,d, Frank H. de Jonge, , and Laura H. van Haasterd

c Department of Cell Biology and Histology, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, The Netherlands d Department of Cell Biology, Medical School, Utrecht University, 3584 CX Utrecht, The Netherlands e Department of Endocrinology and Reproduction, Medical School, Erasmus University Rotterdam, 3000 DR Rotterdam, The Netherlands


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have investigated the effects of neonatal-prepubertal changes in thyroid hormone levels on the early phases of adult-type Leydig cell development in the rat testis. Hypothyroidism was induced by adding 6-propyl-2-thiouracil (PTU) to the drinking water, while hyperthyroidism was induced by daily injections with triiodothyronine (T3).

The proliferative activity of the Leydig cells in PTU-treated animals was not different from that in age-matched controls through the age of 16 days. Nevertheless, the percentage of Leydig cells (i.e., the proportion of Leydig cells among the total interstitial cell population) was approximately 83% and 67% lower at the ages of 12 and 16 days, respectively. The proliferative activity of the Leydig cells in the T3-treated animals compared to the controls was increased approximately 3-fold at the ages of 12 and 16 days. The percentage of Leydig cells in T3-treated animals was also considerably increased at these two ages (400% and 725%, respectively). Concomitantly, the percentage of peritubular cells was decreased, suggesting that the increase in the percentage of Leydig cells may at least partially be the result of differentiation of peritubularly located precursor cells. Plasma testosterone levels fluctuated considerably at these ages.

Hence, injection of T3 during the neonatal-prepubertal period not only affects Sertoli cell proliferation and differentiation but also directly or indirectly affects the onset of the formation of the adult-type Leydig cell population and its function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The gonadotropins LH and FSH are required for normal growth and differentiation of Leydig cells and Sertoli cells in the testis [1, 2]. Apart from gonadotropins, changes in thyroid hormone levels can also affect testicular development. A transient deficiency of thyroid hormone during neonatal-prepubertal life, induced by treatment with 6-propyl-2-thiouracil (PTU), causes an increase in testicular size and sperm production in adult rats and mice [3–8]. These changes are the result of a delay in Sertoli cell differentiation and a prolongation of Sertoli cell proliferation leading to an approximately 80% increase in Sertoli cell and germ cell numbers [6, 8–10].

The effects of an elevation of thyroid hormone levels during neonatal-prepubertal life are opposite to those of hypothyroidism. Daily administration of the thyroid hormone triiodothyronine (T3) stimulates Sertoli cell differentiation; and, as a consequence, the period during which Sertoli cells are mitotically active is significantly shortened [10]. As a result of this treatment, the final number of Sertoli cells in the adult testis is reduced by approximately 50%, and concomitantly the number of germinal cells is also decreased [10].

All these data implicate a direct effect of thyroid hormone on Sertoli cells. Indeed, Palmero and colleagues [11, 12] and Jannini and coworkers [13] have shown that Sertoli cells contain thyroid hormone receptors and that the function of immature Sertoli cells can be regulated by thyroid hormone. T3, for instance, inhibits Sertoli cell aromatase activity both in vivo and in vitro [14, 15], affects testosterone metabolism [16], and stimulates the production of IGF-I [17].

There exists extensive literature on the effects of thyroid hormone levels on the proliferation, differentiation, and function of Sertoli cells. However, relatively little is known about the effects of neonatal hypothyroidism and hyperthyroidism on the formation of the adult-type Leydig cell population. Hypothyroidism during the neonatal period has been reported to cause Leydig cell hyperplasia in adult rats [18, 19], presumably as a result of an increased proliferation of the adult-type Leydig cells during prepuberty [20]. Whether hyperthyroidism has opposite effects on adult-type Leydig cell proliferation, as it has on Sertoli cell proliferation and differentiation in the neonatal-prepubertal rat [10], is not known. We have, therefore, investigated the effects of neonatal hyperthyroidism on the early stages of adult-type Leydig cell development in the rat testis through 16 days of age by counting interstitial cell numbers. We have compared these results with the effects of hypothyroidism. In addition, the proliferative activity of the interstitial cells was determined as well as some functional aspects of the Leydig cells, such as the presence of the steroidogenic enzyme 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and testosterone production.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies

The polyclonal antibody against 3ß-HSD was a gift from Drs. F. Labrie and V. Luu-The (Laval University, Quebec, PQ, Canada). The antibody was obtained after immunization of rabbits with 3ß-HSD purified from human placenta [21]. 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 [21–24]. A biotinylated goat anti-rabbit immunoglobulin G antibody (ABC-peroxidase staining kit Elite; Vector Labs, Burlingame, CA) was used as a second antibody.

The monoclonal antibody against bromodeoxyuridine (BrdU) was obtained from Beckton and Dickinson (Mountain View, CA). A rabbit anti-mouse immunoglobulin G (Nordic, Tilburg, The Netherlands) was used as a second antibody.

Animals and Treatment

Pregnant Wistar rats were obtained from the Central Animal Facilities of the University of Utrecht (The Netherlands). The animals were examined for litters twice a day. On the day of birth (Day 1), the litters were designated at random to one of the three treatment groups (control, PTU or T3). The drinking water of mothers and pups of the PTU group contained 0.1% PTU (Merck, Schuchardt, Germany). Male pups of the T3 group received daily s.c. injections of 100 µg T3/kg BW. T3 (Sigma, St. Louis, MO) was dissolved in 0.025 N NaOH in saline. Controls received daily injections of 0.025 N NaOH in saline.

Animals were killed at Days 5, 9, 12, and 16 after birth by cervical dislocation. Each experimental group consisted of 4–5 animals. Three to four cross sections per testis, at least 100 µm apart, were counted. All experiments were approved by the ethical committee for laboratory animal welfare of the Faculty of Medicine.

Histology and Immunohistochemistry

One hour before the animals were killed, each received an s.c. injection of BrdU 150 mg/kg BW (Sigma). BrdU is a compound that is specifically incorporated by cells in the S-phase of the cell cycle and that gives an indication of the proliferative activity in a tissue. The testes were fixed in Carnoy's fluid for 1.5 h and were subsequently embedded in glycol methacrylate-butanediolmonoacrylate (BDH Laboratory Supplies, Poole, Dorset, UK; Klinipath BV, Zevenaar, The Netherlands) at a ratio of 85:15. Sections of 3 µm were stained using the periodic acid-Schiff technique and were incubated with a monoclonal antibody against BrdU, as described extensively by Van de Kant and de Rooij [25].

Testes of some 16-day-old rats were embedded in paraffin, since material embedded in glycol methacrylate-butanediolmonoacrylate is not suitable for immunohistochemical detection of 3ß-HSD. Paraffin embedded testes were incubated with a polyclonal antibody against 3ß-HSD as described previously by Teerds and Dorrington [26]. Briefly, after deparaffinization, endogenous peroxidase activity was blocked with 1% H2O2 in methanol for 30 min. Subsequently the slides were incubated with 0.1 M glycine in Tris-buffered saline (TBS). The slides were blocked with 5% normal goat serum and incubated overnight at 4°C with the polyclonal antibody against 3ß-HSD (diluted 1:300) in TBS with 0.05% acetylated BSA (Aurion, Wageningen, The Netherlands). During the next step, the slides were incubated with a biotinylated goat anti-rabbit polyclonal antibody (diluted 1:100), and then incubated with the components avidin (A) and biotin (B) of the ABC staining kit. Both components were diluted 1:500, and the solution was prepared 15 min before addition to the sections. Slides were washed with TBS, and bound antibody was visualized using a 0.6 mg/ml solution of 3,3'-diaminobenzidine tetrachloride (Sigma) in Tris buffer (0.05 M, pH 7.4). These sections were counterstained with Gill's hematoxylin.

In control experiments, the monoclonal BrdU or the polyclonal 3ß-HSD antibodies were omitted from the procedure. Alternatively, normal mouse (BrdU) or rabbit serum (3ß-HSD) were employed instead of the first antibodies. A representative control section is shown in Figure. 3.



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  		FIG. 3. Immunohistochemical localization of 3ß-HSD in 16-day-old rat testes. 3ß-HSD-positive Leydig cells are indicated by arrows. A) Control section incubated with preimmune serum instead of primary antibody. B) Testis of a control animal. C) Testis of a PTU-treated rat. D) Testis of an animal that received daily injections of T3. Original magnification x235 (reproduced at 79%); bar = 54 µm.

Cell Counts

Identification of the different types of interstitial cells was based on their nuclear morphology, localization in the interstitial tissue, and staining characteristics of the cytoplasm of these cells. The method of identification of different types of interstitial cells according to their localization was based upon descriptions by De Kretser and Kerr [27], Hardy et al. [18], and Teerds et al. [28]. Briefly, nuclei of Leydig cells, macrophages, perivascular cells (lymphatic endothelial cells and pericytes), peritubular cells, and mesenchymal cells were counted. Leydig cells were identified by their oval-to-round nucleus in combination with the specific blue-purple staining of their cytoplasm. This was typical for Leydig cells and was not found in any other cell type in the interstitial tissue, e.g., mesenchymal cells. Furthermore, Leydig cells stained positively with the antibody against 3ß-HSD. The nuclei of the macrophages were usually somewhat smaller and more irregularly shaped than the Leydig cell nuclei, while the distribution of heterochromatin was different from that in Leydig cells. The cytoplasm of the macrophages stained pink (periodic acid-Schiff-positive). The pericytes had fusiform nuclei and were always located in direct apposition to the vascular wall, as was the case for the perivascularly located lymphatic endothelial cells. These two cell types were counted together and called perivascular cells. The nuclei of the peritubular cells were directly apposed to the basal lamina of the seminiferous tubules. It was not possible to discriminate between peritubularly located lymphatic endothelial cells, peritubularly located mesenchymal cells, and myoid cells. Therefore, these cell types were counted together and referred to as peritubular cells. Mesenchymal cells that were located in the central regions of the interstitial space and did not touch the peritubular or the perivascular cells were scored separately.

Cross sections through the whole testis were made in which areas were chosen at random. The nuclei of cells in these areas were counted using a square lattice grid inserted in the eyepiece of the microscope. At least three different sections, 100 µm or more apart, were counted until 1000 interstitial cell nuclei were scored. The percentage of each cell type was calculated per 1000 interstitial cells. In this way, an indication of changes within the interstitial cell population was obtained, e.g., of differentiation of peritubular and mesenchymal cells into Leydig cells. This method of cell counting does not give an indication of absolute increases in interstitial cell numbers per testis.

Furthermore, the BrdU labeling index for each of the interstitial cell types was determined by calculation from the total numbers of each cell type counted (labeled plus unlabeled) and the number of cells labeled with BrdU.

Average nuclear diameters of Leydig cells were measured as described previously [29]. Nuclei were measured only if the optical middle of the nucleus, where the diameter was maximal, could be found at some focal level within the section. At least 20 Leydig cell nuclei per testis were measured in each of the different treatment groups. There appeared to be no significant difference between the diameter of Leydig cell nuclei among the different groups of animals.

RIAs

After decapitation of the animals, trunk blood was collected. Plasma was stored at -20°C until further processing. Because of the small serum volume of the younger animals, samples (n = 2–11) within each age group of 5-, 9-, and 12-day-old animals were pooled, while serum samples of 16-day-old animals were measured separately. Testosterone concentrations in plasma were assayed by RIA as described by Verjans et al. [30]; intra- and interassay coefficients of variation were lower than 8% and 12%, respectively.

Statistical Analysis

Statistical analysis of the data was carried out using the Mann Whitney-U test. Values were considered to be significantly different from the control group when p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Body and Testes Weights

Body and testes weights increased at the same rate in control and PTU-treated rats through the age of 12 days. On Day 16 after birth, body and testes weights of the PTU-treated rats were significantly reduced when compared to those of the controls, as reported previously by Van Haaster et al. [31], using the same groups of animals.

The first symptoms of hyperthyroidism became apparent in the T3-injected animals at the age of 5 days, as they displayed a fast, light tremor of the paws. The body weights of these rats were significantly reduced from Day 12 after birth through Day 16. The testes weights in control and T3-treated animals increased at the same rate throughout the experiment, as shown previously by Van Haaster et al. [10], using the same group of animals. Since the effects of T3 treatment became increasingly severe, we stopped this treatment at the age of 16 days. Because the aim of this study was to compare the effects of hyperthyroidism on early adult-type Leydig cell development with those of hypothyroidism, PTU treatment was also not continued after the age of 16 days.

Testicular Morphology

In the rat testis, Leydig cells undergo two clearly defined periods of proliferation and differentiation. The first population of Leydig cells is formed during fetal life and gradually decreases in size after birth. In the neonatal testis, these fetal-type Leydig cells are located in small clusters surrounded by fibroblast-like cells [32, 33]. The second population of Leydig cells develops during the (pre)pubertal period, the first of these adult-type Leydig cells being found around Day 10 after birth [34, 35]. Since it is not always easy to discriminate between fetal-type and adult-type Leydig cells, no attempts were undertaken to count these cell types separately.

We observed that in the 5-day-old rat testis the Leydig cells were indeed localized in characteristic clusters. At this age, independent of the treatment, both the percentage of Leydig cells as well as the percentage of BrdU-labeled Leydig cells were low (Fig. 1, A and B). No differences between the controls and the treated groups of animals could be detected. The first significant differences were observed at the age of 9 days when the percentage of Leydig cells in the control animals was slightly but significantly higher than in the PTU-treated animals (Fig. 1A). By 12 days of age, a sharp increase occurred in the percentage of Leydig cells as well as in the percentage of BrdU-labeled Leydig cells in the T3-treated group when compared to the control group (Fig. 1, A and B). These differences were even more pronounced at Day 16 of age (Fig. 1, A and B).



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  		FIG. 1. Percentages of Leydig, peritubular, perivascular, and mesenchymal cells (calculated per 1000 interstitial cells) (A, C, E, G), and percentages of BrdU-labeled Leydig, peritubular, perivascular, and mesenchymal cells (B, D, F, H) in control (filled bars), PTU-treated (coarsely hatched bars), and T3-treated (finely hatched bars) rats. Treatment was started immediately after birth, and animals were killed 5, 9, 12, or 16 days after birth. The rats received an injection of BrdU 1 h before being killed. *p < 0.05 compared with controls (Mann-Whitney U test). Values are means ± SD (n = 4–5).

Both labeled and unlabeled Leydig cells were often found in the vicinity of the seminiferous tubules. An example of BrdU-labeled interstitial cells is given in Figure 2, which shows the histology of a representative testicular section of a control rat at the age of 16 days.



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  		FIG. 2. Representative histological section of a 16-day-old control rat testis stained with periodic acid-Schiff and hematoxylin. The rat received an injection of BrdU 1 h before being killed. Sections were incubated with an antibody against BrdU, and labeling was visualized using silver enhancement (for details see Materials and Methods). (Labeled) Leydig cells are indicated by arrows and (labeled) peritubular cells by arrowheads. The format of the nuclear profile of the peritubular cells depends on the way a cross-section was cut through the nucleus. Original magnification x475; bar = 21 µm.

The effects of elevated or decreased thyroid hormone levels on the number of Leydig cells became even more pronounced when sections of testicular tissue were stained immunohistochemically with an antibody against the steroidogenic enzyme 3ß-HSD, a marker for Leydig cells. As is clearly shown in Figure 3, many more 3ß-HSD positive Leydig cells were present in the interstitium of T3-treated animals when compared to the age-matched controls or the PTU-treated animals.

The effects of the different treatments on the seminiferous tubules in these animals have been described in detail elsewhere [10, 31].

At the ages of 12 and 16 days, when the percentage of Leydig cells increased rapidly in the T3-treated groups of rats, the percentage of peritubular cells underwent a marked decrease compared to those in the control group (Fig. 1C). No significant differences could be detected in the percentages of labeled peritubular cells between the controls and the PTU- or T3-treated groups at the four ages shown in Figure 1D.

Furthermore, no significant changes could be detected in the percentages of perivascular and mesenchymal cells between the control animals and the PTU- or T3-treated groups at any of the ages studied (Fig. 1, E and G). The same held true for the percentages of BrdU-labeled perivascular and mesenchymal cells.

Serum Hormone Levels

Testosterone levels were measured in plasma from T3-treated rats and controls. Plasma testosterone levels in both controls and T3-treated rats were highly variable. Only at 16 days of age could a significant difference be observed between controls and treated animals (Fig. 4).



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  		FIG. 4. Testosterone levels in nM in plasma of T3-treated rats (closed circles) and age-matched controls (open circles). Treatment was started directly after birth, and animals received daily injections of T3 or vehicle for 5, 9, 12, or 16 days before being killed. *p < 0.005 compared with controls (Mann Whitney-U test). Values are means ± SD (n = 4–7).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study show that changes in neonatal-prepubertal thyroid hormone levels profoundly influence the onset of the development of the adult-type Leydig cell population in the immature rat testis. Changes in neonatal-prepubertal thyroid hormone levels have also been shown to affect Sertoli cell differentiation. When thyroid hormone levels are reduced, Sertoli cell differentiation is delayed while the period during which these cells are mitotically active is prolonged [3–8, 31, 36]. In contrast to this, an elevation of thyroid hormone levels stimulates Sertoli cell differentiation and consequently reduces the period during which Sertoli cells are mitotically active [10].

The Leydig cells present in the testis at Day 5 after birth are of fetal origin [32–34, 37, 38]. In controls at this age, the percentage of Leydig cells and their proliferative activity was rather low, confirming earlier observations by Orth [39], Kerr and Knell [32], and Kuopio et al. [33]. An increase (T3 treatment) or decrease (PTU treatment) in thyroid hormone levels and the concomitantly occurring decrease in plasma FSH levels [10, 31] did not affect the percentages of Leydig cells. Nor were the percentages of any of the other interstitial cell types affected.

In the rat testis, the second generation of Leydig cells, the adult-type Leydig cells, starts to develop from Day 10 after birth onwards as a result of differentiation of mesenchymal-like precursor cells [34]. A significant increase in the percentage of Leydig cells occurs first around Day 21 after birth [34], although at this age the proliferative activity of newly formed Leydig cells is still low, reaching a maximum around Day 30 after birth [35].

The percentage of Leydig cells in the T3-treated animals was increased considerably at Days 12 and 16 after birth compared to that of the respective age-matched control groups. This was confirmed by the presence of many 3ß-HSD positive cells. Concomitantly with the rise in the percentage of Leydig cells, the percentage of peritubular cells decreased, suggesting that at least part of the newly formed adult-type Leydig cells had differentiated from peritubularly located precursor cells. At the same time, the proliferative activity of the Leydig cells increased approximately 3-fold compared to that in the age-matched controls. At the age of 16 days, plasma FSH levels are not different from those in the controls [10]. Plasma testosterone levels were highly variable among the different groups of animals but were reduced significantly in the T3-treated animals at the age of 16 days. At this age in the hyperthyroid animals, precursor cells were rapidly differentiating into adult-type Leydig cells, and these newly formed cells exhibited a high proliferative activity. In contrast to fetal Leydig cells, which produce testosterone, the main steroids produced by young adult-type Leydig cells are the 5{alpha}-reduced steroids, dihydrotestosterone and 3{alpha}-androstenediol. This switch in testicular steroid production from testosterone to 5{alpha}-reduced steroids could offer an explanation for the decreased plasma testosterone levels in the T3-treated animals compared to the controls. Furthermore, it is also possible that elevated levels of T3 stimulate the local production of growth factors that are known to inhibit fetal/adult-type Leydig cell steroid synthesis, e.g., transforming growth factor ß (TGFß) [40, 41].

Although no difference in the percentage of labeled Leydig cells was observed between the PTU-treated and the age-matched control groups up to the age of 16 days, the percentage of Leydig cells at the ages of 9, 12, and 16 days was slightly but significantly lower in the PTU groups. These results suggest that in the presence of decreased thyroid hormone levels, the differentiation of mesenchymal-like precursor cells into adult-type Leydig cells and the subsequent wave of proliferation of these newly formed Leydig cells are either inhibited or delayed.

This hypothesis is corroborated further by observations in a pilot study in which we found that the differentiation of precursor cells into Leydig cells increased gradually when PTU treatment was stopped at the age of 26 days. A few days later, this was followed by a rise in adult-type Leydig cell proliferation (unpublished observations). The results of an extensive morphometric analysis and an immunohistochemical localization study for the presence of 11ß-HSD1, a marker for adult-type Leydig cells, give further support to this hypothesis (see companion article [42]).

These observations are in contrast to the study by Hardy and colleagues [20]. The latter authors reported that neonatal hypothyroidism resulted in elevated levels of Leydig cell proliferation as measured by the incorporation of [3H]thymidine in cells in S-phase, leading to an increase in Leydig cell numbers. We did not observe elevated incorporation of BrdU nor did Mendis-Handagama et al. [42] find an increase in adult-type Leydig cell numbers after morphometric analysis. At present, we cannot explain these different observations. The amount of PTU added to the drinking water was identical in all three studies. Furthermore, it is not likely that the differences in labeling indices were caused by the use of different methods for labeling cells in S-phase. The labeling efficiency of these two methods has been shown to be identical [43].

Sertoli cells of both immature rats and piglets have been shown to possess high-affinity thyroid hormone binding sites [11, 12, 14, 16, 17]. Thyroid hormone has been demonstrated to influence Sertoli cell functions in vitro, such as androgen metabolism, androgen binding protein mRNA levels, production of IGF-I, and aromatase activity [14, 16, 17]. Two different thyroid hormone receptor genes, {alpha} and ß and their isoforms, have been described so far. By using Northern blot analysis, Jannini et al. [13] have shown that in the testis only the thyroid hormone receptor {alpha}1 gene is expressed. In situ hybridization studies localized the thyroid hormone receptor {alpha}1 mRNA exclusively within the seminiferous tubules, most likely in the Sertoli cells. No hybridization signal was detected in the interstitial compartment. In contrast to this, Jana and Bhattacharya [44] observed specific binding of 125I-T3 in nuclei from Leydig cells isolated from goat testes. Treatment of goat Leydig cells with T3 resulted in an increase in protein synthesis before stimulation of androgen release, suggesting a direct effect of thyroid hormone on Leydig cell function [44]. However, the Leydig cell cultures used in these studies were only 75% pure and may thus have been contaminated with thyroid hormone receptor-expressing Sertoli cells. More recently, Hardy et al. [20] observed the presence of thyroid hormone receptor {alpha}1 and {alpha}2 mRNA in purified testicular mesenchymal cells isolated from 21-day-old rats and immature Leydig cells isolated from 35-day-old animals. These authors reported Sertoli cell contamination to be minor and not responsible for the positive Northern blot signals. Hence, changes in thyroid hormone levels during the neonatal-prepubertal period of Leydig cell development may directly affect Leydig cells and their precursors or may indirectly affect these cells through Sertoli cell derived factor(s).

Sertoli cells produce growth factors that have been implicated in affecting Leydig cell proliferation, such as TGF{alpha} [45–47] and TGFß [48–50], IGF I [51], and interleukin 1 [52]. It may very well be possible that the synthesis and secretion of some of these factors is influenced by changes in T3 levels and thus could affect the development of the adult-type Leydig cell population. It is not likely that changes in plasma thyroid-stimulating hormone (TSH) levels, which occur as a result of PTU or T3-treatment, affect Leydig cell development, since Leydig cells do not contain TSH receptors [53, 54].

It is intriguing that apparently there seems to be a relationship between the stage of differentiation of the Sertoli cells [10, 31] and the onset of adult-type Leydig cell development. Hypothyroidism causes a delay in the differentiation of precursors into Leydig cells, while precursor cell differentiation and the onset of the wave of adult-type Leydig cell proliferation are advanced in the case of hyperthyroidism. On the basis of the observations in the present study and the previously reported effects of PTU and T3 treatment on Sertoli cell development, we postulate that the thyroid hormone levels during the neonatal-prepubertal period are not only crucially important for Sertoli cell differentiation [10, 31] but also directly or indirectly affect the onset of the formation of the adult-type Leydig cell population and its function.


   ACKNOWLEDGMENTS
 
The authors thank Drs. V. Luu-The and F. Labrie (Medical Research Council Group in Molecular Endocrinology, CHUL Research Center, and Laval University, Quebec, PQ, Canada) for the 3ß-HSD polyclonal antibody. We are grateful to Ms. H.L. Roepers-Gajadien and Ms. M. de Boer-Brouwer for skillful technical assistance, Ms. H. de Waal and Mr. H. Halsema for preparing the figures, and Foto Breeman (Culemborg, the Netherlands) for preparing the photomicrographs.


   FOOTNOTES
 
1 K.J.T. is a recipient of a senior fellowship of the Royal Netherlands Academy of Arts and Sciences. Back

2 Correspondence: Katja J. Teerds, Department of Cell Biology and Histology, Faculty of Veterinary Medicine, P.O. Box 80.157, 3508 TD Utrecht, The Netherlands. FAX: 31 30 516853; k.teerds{at}pobox.ruu.nl Back

Accepted: March 24, 1998.

Received: October 20, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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