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From the * Department of Animal Sciences, Human
& Animal Physiology Group, Wageningen University, Wageningen, The
Netherlands; and the Department of
Endocrinology and Metabolism, Utrecht University, Utrecht, The Netherlands,
and the Center for Reproductive Medicine, Academic Medical Center, Amsterdam,
The Netherlands.
| Correspondence to: Dr Katja J. Teerds, Department of Animal Sciences, Human & Animal Physiology Group, Wageningen University, Marijkeweg 40, 6709 PG Wageningen, The Netherlands (e-mail: katja.teerds{at}wur.nl). |
| Received for publication February 14, 2008; accepted for publication May 8, 2008. |
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Abstract |
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Key words: Thyroxine, Leydig cell development, Sertoli cell differentiation, hyperthyroid
,
leukemia-inhibiting factor receptor, and c-kit, and the absence of LH
receptors and steroidogenic enzyme expression, are thought to differentiate
into 3β-hydroxysteroid dehydrogenase (3β-HSD)/LH
receptor–positive Leydig cell progenitors between days 10 and 12 after
birth (Ge et al, 2006;
Teerds et al, 2007).
Subsequently, these cells undergo further proliferation and differentiate into
immature adult-type Leydig cells between days 28 and 35 after birth. Around
this age the proliferative activity of the cells is decreasing rapidly, and
the immature-type Leydig cells gradually develop into mature terminally
differentiated adult-type Leydig cells. By the end of puberty the development
of the adult population is completed
(Haider, 2004). Apart from gonadotrophins, endogenously produced (growth) factors as well as thyroid hormones are of importance in the regulation of these processes (Haider, 2004). In the past decade and a half, considerable attention has been paid to the effects of thyroid hormones on testicular development. Transient neonatal-prepubertal hypothyroidism, for instance, was shown to induce an increase in testicular size in adult rats and mice. These changes were caused by a prolongation of Sertoli cell proliferation and a delay in Sertoli cell differentiation leading to approximately 80% and 140% increases in Sertoli cell and germ cell numbers, respectively (Cooke et al, 1993; Hess et al, 1993; Van Haaster et al, 1993; Simorangkir et al, 1995). In parallel to the effects on Sertoli cell development, adult-type Leydig cell progenitor formation was delayed under these conditions, whereas Leydig cell proliferation was prolonged. This led to an approximately 70% increase in the size of the Leydig cell population in the adult testis (Hardy et al, 1993; Mendis-Handagama et al, 1998; Teerds et al, 1998).
The effects of elevated thyroid hormone levels during the neonatal-prepubertal period appear to be opposite to those of hypothyroidism. Daily administration of the thyroid hormone tri-iodothyronine (T3) has been shown to stimulate Sertoli cell differentiation, leading to a premature cessation of Sertoli cell proliferation (Van Haaster et al, 1993). As a consequence the final number of Sertoli cells in the adult testis was reduced by approximately 50% and concomitantly the total number of germ cells was also decreased (Van Haaster et al, 1993). The formation of Leydig cell progenitors and thus the development of the adult-type Leydig cell population was advanced under these conditions (Teerds et al, 1998; Ariyaratne et al, 2000). In all these studies hyperthyroidism was induced by daily injections of T3 to neonatal-prepubertal animals, in doses ranging from 5 to 10 µg/100 g body weight. When T3 is given in such high doses, it is likely to affect the physical condition of these young animals. Indeed, we previously reported that after 5 days of treatment the rats began to display a fast, light tremor of the paws (Teerds et al, 1998). When treatment was continued the effects of T3 became increasingly severe and, therefore, depending on the dose of T3 injected daily these experiments were usually terminated by the time the animals had reached the age of 16 to 21 days (Teerds et al, 1998; Ariyaratne et al, 2000; Mendis-Handagama et al, 2007). These side effects are by themselves not surprising because a daily dose as low as 0.5 µg T3/100 g body weight already results in thyroid-stimulating hormone (TSH) concentrations below the detection limit of the radioimmunoassay (RIA), indicative of hyperthyroidism (Escobar-Morreale et al, 1997).
In the present investigation we have expanded the above-mentioned in vivo studies on the effects of transient neonatal-prepubertal hyperthyroidism on gonadal development by using a more physiological approach and by expanding the hyperthyroid condition from the fetal period up to adulthood. First of all we selected thyroxine (T4) instead of T3 as the thyroid hormone of choice, because most thyroid hormone released from the thyroid consists of T4 and is converted to the active thyroid hormone, T3, within its target cells. Second, T4 was not injected but dams were exposed to thyroid hormone via the addition of T4 to their diet, resulting in a more gradual exposure to the hormone throughout the day compared to 1 daily injection. The diet was prepared in such a way that the estimated daily uptake of T4 was 6 µg/100 g body weight. A gradually released daily dose as low as 2 µg/100 g body weight of T4 has been shown to induce a hyperthyroid condition in pubertal female rats (Escobar-Morreale et al, 1997). Third, in order to expose pups to elevated thyroid hormone levels in utero, dams were put on the experimental diet 2 weeks before mating. After parturition and weaning the diet was continued and pups were sacrificed at different ages, after which the initiation of spermatogenesis and the development of the adult-type Leydig cell population were analyzed and related to the endocrine condition of the animals in terms of the levels of various hormones.
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Methods |
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Animals and Treatment
The animal experiments and protocols were approved by the Animal Welfare
Committee of Wageningen University. Wistar WU (HsdCpbWU) rats were obtained
from Harlan (Horst, The Netherlands) at the age of 7 weeks (females) or 10
weeks (males). The female rats were housed individually after arrival. Room
temperature (20.5°C to 21.5°C), humidity (55% to 65%) and light regime
(lights on from 0300 to 1700 hours; light intensity, 60 to 80 lux),
respectively, were controlled. From the age of 9 weeks onward, the female rats
were fed an iodide-poor diet based on AIN 1993 requirements (Research Diet
Services, Wijk bij Duurstede, The Netherlands;
Reeves et al, 1993;
Schröder-Van der Elst et al,
1998). This diet was supplemented with 7 µg iodide per 100 g
dry weight diet (control diet) to fulfill the normal iodine requirements of
rats. At the age of 10 weeks the rats were put on the experimental diets: the
control diet or the control diet supplemented with T4, resulting in
an approximate intake of 15 µg T4 per 100 g body weight. At the
age of 12 weeks, the female rats were mated. Pups were weaned at the age of 28
days. The offspring were group-housed at least up to the age of 49 days after
birth.
Groups of 7 to 12 pups were sacrificed at the age of 7, 9, 12, 15, 21, 28, 35, 42, 49, or 64 days postpartum, respectively. The experimental diets were continued throughout pregnancy until sacrifice of the pups. Two hours before the rats were sacrificed, each pup received a subcutaneous injection of BrdU (150 mg/kg BW) in saline. BrdU is a compound that is specifically incorporated in the DNA of cells in the S-phase of the cell cycle and that gives an indication of the proliferative activity of the cells in a tissue. Rats were anesthetized using carbon dioxide and oxygen (2:1). Blood was collected by a small incision in the tail of the dams or by heart puncture (5 IU heparin per mL blood) (Fluttert et al, 2000). Rats were killed by decapitation and organs were collected. Plasma was stored at –20°C until further analysis.
Histology and Immunohistochemistry
The testes were successively fixed in Methacarnoy fluid for 2 hours and
alcohol (100%) for 30 minutes. After fixation, testes were stored in alcohol
(70%) until they were dehydrated and embedded in paraffin. Five-micrometer
sections were cut and mounted on glass slides at 100-µm intervals (3 to 4
sections per slide).
In order to determine the proliferation of the developing adult-type Leydig cell population, testis sections were incubated with a monoclonal antibody against BrdU and then with a polyclonal antibody against 3β-HSD. In short, sections were deparaffinized and transferred to a 1% periodic acid solution at 55°C for 30 minutes. Sections were then rinsed, incubated with 5% normal horse serum in 0.01 M phosphate-buffered saline (PBS; pH 7.4) for 10 minutes, rinsed with PBS, and then incubated with a monoclonal anti-BrdU antibody diluted 1:100 in PBS plus 0.05% BSA-c for 60 minutes at room temperature. Sections were again rinsed in PBS, incubated with a biotinylated horse anti-mouse antibody diluted 1:100 in PBS plus 0.05% BSA-c for 60 minutes, rinsed, and incubated with Vectastain ABC reagent (diluted 1:1000 in PBS plus 0.05% BSA-c) for 60 minutes. Sections were again rinsed in 3.3'-diaminobensidine (DAB) and then incubated with DAB (0.06 mg/mL PBS plus 0.03% H2O2). Next the sections were rinsed in PBS, incubated with 10% normal goat serum in PBS for 30 minutes, and incubated overnight with the polyclonal rabbit anti-3β-HSD antibody diluted 1:300 in PBS plus 0.05% BSA-c at 4°C. Next the sections were rinsed in PBS, incubated with an alkaline phosphatase labeled secondary goat anti-rabbit antibody in PBS plus 0.05% BSA-c for 60 minutes, and rinsed in PBS, after which alkaline phosphatase activity was demonstrated using naphthol AS MX phosphate (1 mg naphthol AS MX phosphate, 50 µl N,N-dimethylformamide in 8 mL Tris-HCl 0.1 M [pH 8.5]), Fast Blue BB Base (2 mg Fast Blue BB Base with 50 µL 2M HCl and 50 µL 4% NaNO2), and levamisole (5 mg). Slides were rinsed, briefly counterstained with Mayer hematoxylin, and mounted with gelatin.
Fetal-type Leydig cells, Leydig cell progenitors, and immature and mature adult-type Leydig cells were counted in randomly chosen sections. At least 3 different sections were analyzed per testis, using a square lattice grid inserted in the eyepiece of the microscope. Only those Leydig cell nuclei were counted that were located within the borders of the square lattice grid. Fetal-type, progenitor, immature, and mature adult-type Leydig cells were identified by the presence of 3β-HSD immunoreactivity (blue-stained cytoplasm). Only those 3β-HSD–positive cells were counted in which the nucleus was present. Proliferating Leydig cells were identified by the presence of BrdU staining (brown) of the nucleus. Per testis, 1500 Leydig cells (3β-HSD–labeled and 3β-HSD plus BrdU–labeled) were counted and the percentage of Leydig cells in the S phase of the cell cycle was calculated. In the case of young animals in which less than 1500 3β-HSD–positive cells were present, all Leydig cells in 4 testicular cross-sections were counted.
In order to determine the progression of spermatogenesis under hyperthyroid conditions, sections of testes of 15-, 21- and 28-day-old rats were stained using the periodic acid–Schiff technique. For each seminiferous tubule the most advanced type of germ cell was determined in 3 randomly chosen testis sections. In total at least 50 tubule cross-sections were analyzed at random per animal. In the same testis sections the diameters of 15 circular seminiferous tubule cross-sections were measured per section (3 sections per animal) and the mean tubule diameter was calculated. The percentage of tubule cross-sections containing a clear (>100 µm2) lumen was also determined by randomly scoring at least 50 tubule cross-sections per animal.
RIAs
RIAs for T4 (DSL-3200), T3 (DSL-3100), testosterone
(DSL-4100), and leptin (RL-83K) were performed according to the manufacturer's
protocol. Plasma LH, FSH, TSH, and prolactin levels were determined by
validated in-house double-antibody RIAs for rat serum analysis
(Mattheij et al, 1995; Palm et
al,
2001a,b)
using materials supplied by the National Institute of Diabetes and Digestive
and Kidney Diseases (NIDDK; Bethesda, Maryland). For LH, rLH-I-9 was used as
label and anti-rLH-S-11 as antiserum; for FSH, rFSH-I-7 as label and
anti-rFSH-S-11 as antiserum; for TSH, rTSH-I-9 as label and anti-TSH-RIA-6 as
antiserum; and for prolactin, rPRL-I-5 as label and anti-rPRL-415 as
antiserum. SACcel (donkey anti-rabbit) was used as a secondary antibody. The
levels of hormones are expressed in terms of NIDDK standards. The detection
limits of the assays were 5 ng/mL for T4; 0.25 ng/mL for
T3; 0.5 ng/mL for leptin; 0.03 ng/mL for LH; 0.4 ng/mL for FSH; and
0.1 ng/mL for testosterone, TSH, and prolactin. The intra-assay and interassay
variations were determined using several pools of rat serum and were less than
11% for all purchased RIAs and less than 9.5% for all in-house RIAs.
Statistical Analysis
Data are expressed as mean ± standard error of the mean (SEM).
Previous experiments have suggested that testicular development is dependent
on the founders (unpublished data); hence, per age group, only 1 pup per dam
was used. Statistical analysis was carried out using SPSS 12.0.1 for Windows
(SPSS Inc, Chicago, Illinois). Data were tested for normality using the
Shapiro-Wilk test. If normality could be assumed, groups were compared using
Student's t test for equality of means (corrected for equal
variances); if normality could not be assumed a Mann-Whitney U test
was used. Values of P < .05 were considered to be significantly
different.
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Results |
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No differences were noted in body weight between the dams, nor in litter size, gender ratio, or birth weight of their offspring, in any of the experiments. The body weights of the hyperthyroid pups receiving 15 µg T4 per 100 g body weight were slightly, but significantly, decreased on days 15, 28, 42, and 49 after birth when compared to the respective age-matched control groups (Figure 1A). Testis weight, on the contrary, was significantly decreased only on day 15 after birth in the T4-fed animals (Figure 1B).
Leptin, a hormone secreted by adipocytes, positively correlates to body fat mass and indirectly to body weight. From days 15 to 28 and 49 to 64 after birth, plasma leptin levels were approximately 30% to 50% lower in the hyperthyroid animals compared to the age-matched controls (Figure 1C).
Thyroid Hormone and TSH Levels
Previous experiments have shown that about 40% of the orally supplemented
dose of T4 was taken up by the intestine
(Chung and Van Middlesworth,
1967; DiStefano et al,
1992). To assess the thyroid status of the T4-fed
animals, plasma TSH, T4, and T3 concentrations were
determined. TSH levels of the hyperthyroid dams were reduced by about 50% as
compared to the control group at the first day of pregnancy, the day of birth,
and the day of weaning. TSH levels in the hyperthyroid offspring were
significantly reduced from day 15 after birth onwards compared to the
age-matched controls (Figure
2A). Concomitantly, plasma T4 levels were increased 3-
to 5-fold compared with the age-matched control levels
(Figure 2B). The rise in plasma
T3 levels occurred at a slightly later age; from 28 days after
birth onwards plasma T3 levels were increased approximately 2-fold
compared to the age-matched control rats (data not shown).
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Sertoli Cell Development and Spermatogenesis
To evaluate Sertoli cell development under hyperthyroid conditions, Sertoli
cell proliferation, tubule lumen formation, tubule diameter, and the
progression of spermatogenesis were investigated. No differences in
termination of Sertoli cell proliferation were observed between hyperthyroid
and euthyroid control animals. Initiation of seminiferous tubule lumen
formation, a marker for Sertoli cell differentiation
(Van Haaster et al, 1993),
normally occurs between days 15 and 17 after birth. In the present study about
16% of the seminiferous tubules showed an opening at day 15 postpartum in the
euthyroid control group, whereas in the hyperthyroid animals the percentage of
tubules with a lumen tended to be lower; only 5% of the tubules had an opening
at this age. Because of the relatively large variation in the tubule opening
within the groups, this difference did not reach the level of significance. At
the age of 21 days seminiferous tubule lumen formation was almost complete
(
95%) in both groups, and at day 28 postpartum all tubules showed the
presence of a lumen.
The progression of the process of spermatogenesis under hyperthyroid conditions was investigated at the ages of 15, 21, and 28 days. At the age of 15 days the most advanced type of germ cells in the control animals was pachytene spermatocytes. In 48 ± 9.8% of the seminiferous tubules investigated, leptotene and zygotene spermatocytes were present. In contrast, in none of the seminiferous tubules studied in hyperthyroid testes were pachytene spermatocytes observed, and the percentage of seminiferous tubules containing leptotene and zygotene spermatocytes was significantly lower compared to the age-matched controls (15.8% ± 14.1%, P = .03). By the age of 21 and 28 days these differences between the control and hyperthyroid animals had disappeared. These results were confirmed by the diameter of the seminiferous tubules. The tubule diameter was significantly smaller in the hyperthyroid animals at the ages of 15 and 21 days (P < .01); tended to be smaller on day 28 (P = .06); and did not differ significantly between the control and hyperthyroid groups from day 35 postpartum onwards (data not shown).
Plasma FSH levels were in line with these results. FSH levels were nearly always significantly lower in the hyperthyroid rats from the age of 12 days onwards when compared to the age-matched controls. Although plasma FSH levels tended to be lower in the hyperthyroid animals at the ages of 35 and 42 days, this did not reach the level of significance (Figure 3).
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Leydig Cell Proliferation
To evaluate Leydig cell proliferation and differentiation, BrdU
incorporation in 3β-HSD–positive Leydig cells was determined. In
the strain of rats used in the present study, the first progenitor-type Leydig
cells are formed around day 13 after birth. The BrdU–3β-HSD
double-positive cells observed in the testis at 7, 9, and 12 days postpartum
were thus of fetal origin. These fetal-type Leydig cells are often localized
in characteristic clusters (reviewed by
Haider, 2004). At the age of
15 days postpartum proliferating progenitor-type Leydig cells were detected,
both in the control rats and in the hyperthyroid animals
(Figure 4). Histologically, the
proliferating progenitor cells were identified by their spindle-shaped to oval
nucleus and their location either in close vicinity of the seminiferous
tubules or blood vessels (reviewed by
Haider, 2004). Surprisingly,
the percentage of proliferating progenitor cells was initially significantly
lower in the hyperthyroid testis, compared to the age-matched controls
(Figure 5). However, by the age
of 21 days this difference had disappeared. By the age of 35 days, when most
progenitor-type Leydig cells have differentiated into immature-type Leydig
cells, Leydig cell proliferation was significantly elevated in the
hyperthyroid rats compared to the controls. Because of the absence of specific
markers for either progenitor-type or immature-type Leydig cells, it was not
possible to discriminate between these 2 Leydig cell types. By the age of 49
days the level of Leydig cell proliferation in the T4-fed animals
had returned to the age-matched control level
(Figure 5).
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Discussion |
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Several in vitro and in vivo studies have shown that the active thyroid
hormone T3 can directly influence Sertoli cell proliferation and
function, such as cellular aromatase activity
(Ulisse et al, 1994;
Palmero et al, 1995), androgen
metabolism (Panno et al, 1994)
and IGF-I production (Palmero et al,
1990). One of the mechanisms by which thyroid hormones could
influence Leydig and Sertoli cell development is through activation of thyroid
hormone receptors (TR; Palmero et al,
1988,
1992). Although both
TR1 and TRβ1 have been demonstrated to be present in
neonatal-prepubertal Sertoli cells, the described effects on proliferation and
function have been shown to be predominantly mediated through TR1
(Jannini et al, 1994;
Holsberger et al, 2005).
Preliminary experiments using lower doses of T4 in the diet, resulting in approximate intakes of 3.5, 5.0, and 7.5 µg T4 per 100 g body weight, did not result in significant changes in any of the parameters investigated other than minor changes in plasma levels of thyroid hormones and TSH. Nevertheless, a trend in several of the investigated parameters could be observed, which was in line with the currently described dose of an intake of 15 µg T4/100 g body weight per day. In comparison, Escobar-Morreale and colleagues (1997) infused a daily dose of 4 to 8 µg T4/100 g body weight in juvenile thyroidectomized rats to study the effects on leptin secretion. The infusion of T4 resulted in a 3- to 4-fold increase in T4 and a 2-fold increase in T3 plasma levels. These increases in thyroid hormone levels are comparable to those in the present study. Based on this relatively small increase in plasma T4 and T3 levels in the present study, it is concluded that dietary administration of 15 µg/100 g body weight per day results in a relatively mild form of hyperthyroidism.
In several of the hyperthyroid age groups a minor but significant decrease in body weight was observed when compared to the age-matched controls. In another investigation Ooka and Shinkai (1986) induced a hyperthyroid condition by adding T4 (10 mg/L) to the drinking water of rats, resulting in plasma T4 and T3 levels comparable to what was observed in the present study. Ooka and Shinkai also described a slight but significant decrease in body weight after 2 months of treatment (1986). In contrast, daily injections of 4 to 10 µg T3/100 g body weight have much more severe effects on body weight, depending on the time of initiation and on the duration of the treatment (Eayrs, 1967; Moussavi, 1990; Van Haaster et al, 1993; Barsano et al, 1994). Contributing to the lower body weight is the body fat mass. As a consequence of the hyperthyroid condition the metabolic rate of rats is increased, resulting in a lower body fat mass. One of the hormones produced by adipose tissue is leptin, a product of the ob gene, which regulates body weight by signaling the size of the energy stores in adipose tissue. Serum leptin levels have been shown to correlate with body fat mass. When the latter is reduced, leptin levels are lowered as well (Frederich et al, 1995). Indeed, in the present study plasma leptin levels were reduced, suggestive of a lower body fat mass and expanding the observations by the Escobar-Morreale group, who demonstrated that in thyroidectomized young rats made hyperthyroid by infusion of either T3 or T4, leptin levels were significantly reduced (Escobar-Morreale et al, 1997).
Surprisingly, neither Sertoli cell proliferation nor differentiation was influenced by the mild hyperthyroid condition as established in the present study. In contrast, in studies in which T3 was administered at high doses by daily injections from birth to the age of 16 days, Sertoli cell proliferation was terminated prematurely, whereas tubule lumen formation, a marker for Sertoli cell differentiation, was advanced. Furthermore, the initiation and progression of spermatogenesis was advanced under these conditions (Van Haaster et al, 1993; Holsberger et al, 2005). Apparently the injection of high doses of T3 from the day of birth onwards causes a more rapid elevation in plasma T3 levels, resulting in an advancement of Sertoli cell proliferation and differentiation, whereas the induction of hyperthyroidism by the feed does not seem to influence Sertoli cell development. In the present study plasma T3 levels were elevated significantly for the first time by the age of 28 days in the hyperthyroid animals. Plasma T4 levels were significantly elevated for the first time at the age of 15 days. This would suggest that T4 could only influence testis development after the age of 15 days, when Sertoli cells have already nearly ceased to proliferate.
Although mild hyperthyroidism does not seem to affect Sertoli cell
proliferation and differentiation, we did observe a transient delay in the
progression of spermatogenesis at the time when plasma T4 levels
were significantly elevated for the first time. In 15-day-old euthyroid
control rats the most advanced type of germ cells present in the seminiferous
tubules were pachytene spermatocytes, whereas in the hyperthyroid testes the
most advanced germ cell types were leptotene and zygotene spermatocytes,
suggesting that even relatively small changes in thyroid hormone levels can
affect spermatogenesis transiently. This effect on spermatogenesis may be
induced indirectly via the Sertoli cells, or directly via activation of
TR1, which is present not only in Sertoli cell nuclei but also in
spermatogonia and spermatocytes (Buzzard et
al, 2000). TR mRNA and protein levels in the testis are
known to be suppressed under hyperthyroid conditions
(Rao et al, 2003;
Lee et al, 2007). We would
therefore like to hypothesize that in the current experiment TR
expression in the testis is reduced because of the elevated T4
levels, thus temporarily negatively influencing the initiation and progression
of spermatogenesis. The slight delay in the progression in spermatogenesis
could also be explained by a reduction in plasma FSH levels in the
hyperthyroid animals as observed at the ages of 12 and 15 days. Alternatively,
an as yet unidentified factor may be responsible for this transient delay, an
assumption that is supported by the fact that FSH and T4 levels
continue to be low and elevated, respectively, at the age of 21 days in the
hyperthyroid animals, whereas spermatogenesis is no longer different between
control and hyperthyroid rats. In contrast, more severe forms of
hyperthyroidism as induced by daily injections of T3 seem to have
opposite effects on the progression of spermatogenesis
(Van Haaster et al, 1993).
Whether thyroid hormones can influence Leydig cell progenitor formation
directly or indirectly is less clear. Tagami and colleagues were the first to
report the presence of thyroid hormone receptors in the interstitial
compartment, presumably in the Leydig cells
(Tagami et al, 1990), though
it was not clear at that stage which type of thyroid hormone receptor was
expressed. Several years later, Hardy et al
(1996) demonstrated the
presence of TR1 and TR2 mRNA by Northern blot analysis in
isolated, highly purified progenitor, immature, and mature adult-type Leydig
cells. These observations were confirmed by Buzzard and colleagues
(2000) using RT-PCR and
immunohistochemistry. Direct effects of thyroid hormone on Leydig cell
function in vitro have not been reported so far. Previous in vivo studies
showed that the formation of Leydig cell progenitors and thus the development
of the adult-type Leydig cell population was advanced by daily injections of
T3 (Teerds et al,
1998; Ariyaratne et al,
2000). In contrast, the development of the adult-type Leydig cell
population was not advanced in the present investigation. On the contrary, we
observed a lower level of Leydig cell proliferation in 15-day-old hyperthyroid
animals compared to the euthyroid controls. The pattern of Leydig cell
proliferation continued to be different from that of the euthyroid control
animals. In the control animals a peak in Leydig cell proliferation was
observed at the age of 28 days, followed by a decline, whereas in the
hyperthyroid animals this decline was first observed at the age of 42 days.
Whether this is caused by a direct effect of the elevated thyroid hormone
levels remains to be investigated. Recently it has been shown, however, that
T3 is capable of inhibiting androgen receptor function in vitro by
weakening coactivator binding to the androgen receptor
(Estébanez-Perpiñá et
al, 2007). Hence, thyroid hormones may affect Leydig cell
development and function either directly through binding to the TR1 or
by binding to the androgen receptors present in these cells. The effects of
mild hyperthyroidism on Leydig cell development appear to be more pronounced
than the effects on spermatogenesis.
The results of the present study are in contrast to previous studies in which hyperthyroidism was induced by daily injections, in which T3 was shown to advance Sertoli cell and Leydig cell development as well as spermatogenesis (Van Haaster et al, 1993; Teerds et al, 1998; Ariyaratne et al, 2000). The most important difference between the setup of the present experiment and the previous studies is the fact that we used T4, the precursor of T3, which under normal physiological conditions is present at much higher levels in the circulation than the active thyroid hormone T3, and which has a longer half-life than T3 (Chopra and Sabatino, 2000). Another difference with these previous studies is that in our study the pups had already been exposed to elevated T4 levels in utero. Administration of thyroid hormone through the diet also results in a more continuous exposure compared to the peak exposure when 1 injection is given every day for a certain period of time. The levels of thyroid hormone to which the animals were exposed among these studies are therefore different. The present study suggests that a continuous, relatively mild elevation of thyroid hormone levels has only a minor effect on testicular development compared to the effect on animals exposed to large amounts of thyroid hormone administered in a pulse-like way. Our experiment in rats are in line with the minor effects seen in adult humans, where only limited and reversible effects on semen quality are seen in the presence of mild hyperthyroidism, effects mostly related to a reduced sperm motility (Krassas et al, 2002; Ceccarelli et al, 2006).
Taken together, the results of the present study suggest that a continuous mild elevation of thyroid hormone levels, induced through the addition of T4 to the diet, does not result in an advancement of testicular development as in experiments using high doses of T3 administered in a pulse-like fashion. On the contrary, Leydig cell development as well as the progression of spermatogenesis were initially slightly and transiently delayed.
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Acknowledgments |
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