The growth-retarded (grt) mouse, originally isolated from a closed colony of Snell's dwarf mouse (DW/J strain), shows growth retardation that is inherited in a recessive manner. We have already reported that this strain exhibits severe primary hypothyroidism with significantly reduced plasma levels of thyroxine (T4), dramatically elevated plasma titers of thyroid-stimulating hormone (TSH) and an increase in the number of immunoreactive TSH cells in the pituitary gland. The thyroid gland of the grt mouse exhibits characteristically numerous smaller follicles with poor colloid accumulation. In order to elucidate the possible site of the defect in the grt mouse, and in particular to clarify the discrepancy between elevated plasma TSH titers and reduced T4 levels, we examined the bioactivity of TSH recovered from the plasma sample of the grt animal and the responses of the grt thyroid gland to exogenous TSH and other thyroid hormone secretagogues. Plasma samples from the grt mice invariably exhibited significant levels of TSH bioactivity following injection into normal test mice. Thus, the reduced responsiveness to TSH exhibited by the grt mice is not due to the reduced bioactivity of TSH. Administration of exogenous TSH to the grt mice failed to elevate the plasma T4 and triiodothyronine (T3) levels in vivo or to stimulate free T4 and free T3 releases from the grt thyroid gland in vitro. The thyroid gland of the grt mouse exhibited a markedly diminished response of adenylate cyclase to exogenous TSH as compared to the gland of euthyroid littermates. Production of cAMP in the grt mouse was significantly increased following stimulation of the thyroid glands with forskolin, cholera toxin, prostaglandin (PG) E1 and isoproterenol. These results strongly suggest a defect in TSH responsiveness, particularly in TSH-TSH receptor-Gs protein-adenylate cyclase signalling system including the expression and the function of TSH receptor and the TSH receptor-Gs protein coupling, in the thyroid gland of the grt mice.
Strains of mice that exhibit autosomally inherited dwarfism include Snell (dw; Snell, 1929), Ames (df; Shaible and Gowen, 1961) and little (lit; Eicher and Beamer, 1976), which are all forms of primary hypopituitarism, and hypothyroid (hyt; Beamer et al., 1981) and congenital goiter (cog; Beamer et al., 1987), both of which are forms of primary hypothyroidism. The lit strain shows an isolated growth hormone (GH) deficiency (Lin et al., 1993), whereas dw and df mice exhibit prolactin (PRL) and thyroid-stimulating hormone (TSH) deficiencies together with GH deficiency (Camper et al., 1990; Ingraham et al., 1990; Karin et al., 1990).
In contrast to these mice, two distinct mutants of primary hypothyroidism are associated with thyroid hormone deficiency. In hyt mice, a point mutation of transmembrane domain IV of the Gs protein-linked TSH receptor results in the hyporesponsiveness of the thyroid gland to TSH (Stein et al., 1991, 1994). In cog mice, a mutation in the acetylcholinesterase-like domain of thyroglobulin causes congenital goiter with hypothyroidism (Kim et al., 1998).
The grt (growth-retarded) mouse was first reported as a mutant spontaneously derived from the Snell's dwarf (DW/J) mouse with characteristic growth pause followed by the delayed onset of pubertal growth (Yoshida et al., 1994). In this mutant mouse, plasma titers of thyroxine (T4) were significantly lowered while those of TSH were greatly elevated (Yoshida et al., 1994; Tomita et al., 1995; Kobayashi et al., 1997). The results indicate that a defect exists within the thyroid gland, or more specifically, in the signalling process of the gland of the grt mouse.
In order to clarify possible sites of the defect in the signalling pathway in the thyroid gland of the grt mouse, we therefore investigated the mode of the release of triiodothyronine (T3) and T4 by the thyroid gland in vivo and in vitro in response to TSH and other thyroid stimulators in the grt mouse.
MATERIALS AND METHODS
Normal and grt mice were prepared by mating homozygous or heterozygous female mice with homozygous male siblings. The animals were maintained under conditions of controlled temperature (23±1°C), relative humidity (60±5%) and lighting (0800–2000), and were kept on laboratory chow (CRF-1, Charles River Co., Japan) and tap water ad libitum.
Determinations of plasma thyroid hormones and TSH
Blood samples were collected from the mice by orbital puncture, and stored at –30°C until use. The plasma levels of T3, T4 and TSH were assayed using radioimmunoassay systems for T3 and T4 (Ortho-Clinical Diagnostics), and rat-TSH (Amersham, RPA554), respectively.
Histology of the thyroid gland
The thyroid glands were carefully isolated from 5-week-old male mice. Following dissections under dissecting microscope the glands were fixed in Bouin's solution for 24 hr. They were then dehydrated through a series of graded ethanol solutions, embedded in paraffin (Paraplast, Oxford, UK) and sectioned at a thickness of 4 μm. The sections were stained with periodic acid-Schiff (PAS) and hematoxylin, and observed under a light microscope.
Assay of TSH bioactivity
Plasma samples were obtained from grt/grt and +/grt mice by orbital puncture, and stored at –30°C until use. Each plasma sample was either undiluted or diluted with 9 volumes of 0.9% NaCl solution containing 0.2% bovine serum albumin, and 1 ml of the test solution was injected intraperitoneally (i.p.) to each normal test mouse of 3 months of age. Bovine TSH (bTSH; Sigma T8931, 25mIU/g body weight) was similarly administered i.p. as positive controls. Test animals were bled by orbital puncture just before and 6 hr after the administration of test solutions. Plasma levels of T3 and T4 of test animals were measured as described previously.
In vivo responsiveness of the thyroid gland to TSH in grt and normal mice
The grt/grt and the +/grt mice of 3 months of age and both sexes were injected i.p. with saline containing bTSH at a dose of 1.56, 6.25, or 25.0 mIU / g body weight. Control animals were treated with saline. Blood samples were collected by orbital puncture for determinations of plasma T3 and T4 levels just before and 6 hr after the administration of either TSH or saline. The levels of T3 and T4 were measured using radioimmunoassay systems as noted previously.
Effects of TSH on free T4 and T3 release from the thyroid glands of grt and normal mice in vitro
The mice used in this and the following experiments ranged in age from 3 to 6 months. All animals were killed by cervical dislocation. For incubations, two thyroid lobes from +/+, +/grt, and grt/grt mice were rapidly removed, weighed and kept in glass vials containing 0.5 ml of ice-cold modified Eagle's medium (MEM) with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid, 25 mM NaHCO3, and 2 mM L-glutamine (pH 7.4). After preincubation for 30 min, the tissue was transferred to the vial containing MEM with or without 20 mIU bTSH/ml and incubated for an additional 8 hr. All incubations were done at 37°C in a shaking incubator, and all vials were capped after gassing with a 5% CO2-95% O2 mixture. Each vial was thereafter gassed in this way every 2 hr. Free T3 and T4 levels in the medium were assayed using radioimmunoassay systems for free T3 and T4 (Ortho-Clinical Diagnostics), respectively. Results were obtained with two lobes from all phenotypes per point and expressed as picomoles free T3 and T4 per milligram thyroid wet weight.
Effects of TSH, cholera toxin, forskolin, prostaglandin (PG) E1 and isoproterenol on cAMP production in the thyroid glands of grt and normal mice in vitro
Preparations of the thyroid glands were made under the same conditions as those described previously. After preincubation for 30 min, the tissue was transferred to vials containing MEM with 1 mM 3-isobutyl-1-methylxanthine (IBMX, Wako Chemicals, 095-03413) and one of the test materials [forskolin (Wako Chemicals, 067-02191), cholera toxin (Research Biochemicals International, C-158), PGE1 (Sigma, P5515), isoproterenol (Sigma, I6504) or bTSH] and were then incubated for an additional 30 min. All incubations were performed at 37°C under a 5% CO2-95% O2 mixture. Thyroid glands were washed with assay buffer, boiled, homogenized in ice-cold assay buffer, and centrifuged at 9,000g at 4°C. The concentration of cAMP in the supernatants was measured using an enzyme immunoassay kit (Amersham, RPN255). A preliminary cAMP determination was used to establish the optimal dilutions for all test groups. Results are expressed as picomoles cAMP per milligram thyroid wet weight.
Characteristics of the thyroid gland in the grt mouse
Figure 1 shows typical photomicrographs of PAS-stained sections of the thyroid gland from 5-week-old normal (A) and grt (B) mice. The glands of normal animals exhibited well-developed follicles and accumulation of PAS-positive colloids in the follicles (Fig. 1A). By contrast, in glands recovered from grt mice, there were greater numbers of smaller follicles per unit area and much poorer accumulation of PAS-positive colloids in the follicles (Fig. 1B). No lymphocytes were detected in the glands of either grt or normal mice.
Plasma concentrations of thyroid hormones were significantly lower in grt mice than in normal euthyroid animals, although plasma titers of TSH were greatly elevated in the grt mouse (Table 1).
Plasma T4, T3 and thyroid-stimulating hormone (TSH) values in the grt/grt and normal (+/+ or +/grt) mice.
Bioactivity of grt TSH in stimulating the thyroid gland
In order to clarify whether or not the TSH molecules of grt mice are biologically active in inducing production and secretion of thyroid hormones in mice, 3-month-old normal mice were injected i.p. with aliquots of plasma obtained from grt/grt mice or +/grt animals and plasma levels of T4 and T3 in the treated animals were determined 6 hr after plasma administration. Bovine TSH caused secretions of significantly greater amounts of T4 and T3 from normal thyroid glands, while administrations of saline or test plasma from +/grt mice failed to induce additional production of thyroid hormones in the treated animals (Fig. 2). When the plasma samples from grt/grt mice were given, significantly higher levels of T4 or T3 were detected in the systemic circulation of the treated animals (Fig. 2). It was thus revealed that TSH in the plasma of grt/grt mice had an ample biological activity in provoking the thyroid glands of the treated mice to produce and secrete significantly high levels of T4 or T3. No sex differences were found in the concentrations of thyroid hormones among treated animals nor plasma test samples and therefore the data from male and female animals were calculated altogether.
Responses of the thyroid glands in vivo and in vitro to exogenous TSH in grt mice
In order to examine whether the thyroid gland of the grt/grt mouse is able to produce thyroid hormones in response to exogenous stimulating hormone, various doses of bTSH were given i.p. to 3-month-old grt mice and plasma titers of T4 and T3 were determined 6 hr after the injection. Thyroid glands of phenotypically normal (+/grt) mice exhibited distinctive dose-responsive curves for the release of T4 and T3 in response to bTSH, while the glands of the growth-retarded (grt/grt) mutants thoroughly failed to respond to any doses of exogenous bTSH (Fig. 3).
When the excised thyroid glands from +/+, +/grt and grt/grt mice were incubated in vitro with bTSH for 8 hr, no significant amounts of T4 or T3 were released into the medium from the grt/grt glands, although significantly greater amounts of T4 and T3 were released into the medium from +/+ and +/grt glands than from corresponding controls (Fig. 4). It was proved that the thyroid glands from the growth-retarded mutants failed to respond amply to TSH in vitro.
Effects of TSH, cholera toxin, forskolin, PGE1 and isoproterenol on cAMP production by the grt thyroid glands in vitro
To evaluate the effects of TSH and other thyroid hormone secretagogues on cAMP production, isolated thyroid lobes from +/+, +/grt and grt/grt mice were incubated in vitro in the presence or absence of TSH, cholera toxin or forskolin. Bovine TSH produced 60- to 100-fold increases in cAMP accumulation by the +/+ and +/grt thyroid glands, while it failed to cause the production of cAMP from the grt/grt glands (Fig. 5). Cholera toxin and forskolin were significantly effective in producing cAMP from the thyroid glands of either +/+, +/grt or grt/grt mice, although the grt thyroids were apparently less responsive to forskolin in producing cAMP than pheno-typically normal (+/+ and +/grt) glands (Fig. 5). PGE1 and isoproterenol similarly stimulated the production of cAMP by the thyroids of +/+, +/grt or grt/grt mice (Fig. 6).
Our hormonal assay showed that plasma TSH titers were dramatically elevated in the grt mice, although plasma T4 and T3 concentrations were much lower than those in normal mice at 3 months of age (Table 1). There was no significant difference in the weight of the thyroid gland per unit body weight between the normal mice and the grt mice (0.84±0.03 mg/10 g body weight vs. 0.90±0.04 mg/10 g body weight, respectively) of the same age. Histology revealed smaller follicles with poor colloid accumulation in the thyroid glands of the grt mice (Fig. 1). However, the presence of PAS-positive materials in the follicles exhibited the production of thyroglobulin in the grt mice. In addition to the present result, it was shown that TSH cells in the pituitary glands of grt mice showed a reduced intensity of immunostaining and marked increase in number (Kobayashi et al., 1997), the result being similar to the case of hypertrophied TSH cells (thyroidectomy cells) (Moriarty et al., 1976). Furthermore, the grt mice responded adequately to T4 replacement (Yoshida et al., 1994, Kobayashi et al., 1997). These findings support the idea that the mutant gene induces a defect in the TSH responsiveness of the thyroid gland, resulting in a congenital type of non-goitrous primary hypothyroidism.
The congenital type of primary hypothyroidism is almost always characterized by the presence of goiter in mice (Beamer et al., 1987), cats (Jones et al., 1992) and experimentally 6-n-propil-2-thiouracil-treated rats (Kikuyama et al., 1974). The development of goiter in cog/cog mice is due to a defect in thyroglobulin synthesis (Kim et al., 1998). On the other hand, non-goitrous animal models have been reported for several animals, such as hyt/hyt mice (Beamer et al., 1980), dfc/dfc cats (Tanase et al., 1991), and rdw/rdw rats (Umezu et al., 1998). The hypothyroidism in the hyt/hyt mice reflects the hyporesponsiveness of the thyroid gland to TSH (Stein et al., 1991).
Our studies indicated that grt mice produced biologically active TSH-like molecules that were capable of stimulating the thyroid gland in a mouse bioassay (Fig. 2). The result showed that the TSH molecules in grt mice had normal biological activities and were not related to the site of the inherited defect in these mice. Furthermore, these results suggest that the increased numbers of immunoreactive TSH cells in the anterior pituitary gland of grt mice (Kobayashi et al., 1997) may reflect a normal physiological feedback response of the diminished T4 and T3 production by the grt thyroid glands.
We determined and compared the in vivo and in vitro responsiveness of the thyroid glands of grt and normal mice, as measured by changes in the plasma T3 and T4 levels or in the concentrations of free T4 and T3 in medium after administration of exogenous bTSH. The results showed that the thyroid glands of the grt mice were unresponsive to bTSH both in vivo and in vitro (Figs. 3, 4). This mutant mouse could therefore be classified as exhibiting primary, rather than secondary or tertiary, hypothyroidism.
TSH signal transduction occurs through several intracellular messenger systems (Kopp et al., 1998). The primary pathway generates cAMP by activation of adenylate cyclase through a Gs protein coupled to the TSH receptor. Since TSH molecules of the grt mouse themselves are not the cause for the reduced thyroid gland responsiveness to TSH, we investigated the TSH-TSH receptor-Gs protein-adenylate cyclase system. As we have demonstrated, in the grt mice the response of cAMP production to exogenous TSH is small (Fig. 5). However, this deficit in cAMP production can be overcome by addition of forskolin or cholera toxin (Fig. 5). Forskolin stimulates cAMP accumulation by direct action on the catalytic subunit of adenylate cyclase (Seamon et al., 1981). Therefore, the site of the defect in the grt mouse is not in the adenylate cyclase of the thyroid gland. Cholera toxin inhibits the intrinsic GTPase activity of Gs α by ADP-ribosylation, a regulator of adenylate cyclase (Cassel et al., 1977). Since we have demonstrated that the Gs protein can be activated by cholera toxin in stimulating cAMP production, the grt defect does not lie at the level of the Gs protein or this protein's interaction with adenylate cyclase. We also observed that PGE1 and isoproterenol were effective in stimulating the grt thyroid gland to produce cAMP (Fig. 6). PGE1 and isoproterenol work through receptors that are independent of the TSH receptor in the thyroid gland. The normal response to PGE1 and isoproterenol that was observed indicates that there is no generalized uncoupling of other cell surface receptors to Gs proteins. Therefore, no mutation seems to be present in the portion of the Gs α subunit protein that interacts with thyroid gland surface receptors for PG, ß-adrenergic agents, or TSH. Taken together, these results indicate that the grt mice have a severe defect in the TSH receptor, altered receptor levels and binding affinity, or alteration in the communication and association of the TSH receptor with the Gs protein α subunit. Each of these defects could result in reduced production of cAMP in response to TSH.
Another example of a non-goitrous primary hypothyroidism is found in the hyt/hyt mouse (Beamer et al., 1981). Genetic methods have been used to map the hyt defect to mouse chromosome 12 (Beamer et al., 1981). Biochemical studies on the hyt/hyt mouse have revealed that the cAMP levels of the thyroid glands are not stimulated by TSH, but are stimulated by other agonists that are capable of activating the Gs protein-coupled adenylate cyclase pathway and raised the possibility that a defect lies at the level of the TSH receptor (Stein et al., 1991). Recently, a Pro 556 to Leu mutation in the fourth transmembrane domain of the TSH receptor in the hyt mouse has been identified (Stein et al., 1994). The mutant receptor does not bind TSH and is nonfunctional (Gu et al., 1995).
The grt mice, like hyt mice, exhibit defects in TSH signal transduction that are related to the adenylate cyclase-cAMP system. However, partial linkage analysis utilizing micro-satellite polymorphism has demonstrated that the grt gene is not identical with the hyt gene, i.e. TSH receptor (Yoshida et al., 1994). Recently, the grt locus has been shown to be on chromosome (Chr) 5 (Agui et al., 1997), which verifies that the grt gene is different from the dw (Chr 16), df (Chr 11), lit (Chr 6), hyt (Chr 12) and cog (Chr 15) genes. Although the genetic defect in the two types of hypothyroid (grt and hyt) mice may be different, the consequence of the defect in these two mutants appears to be the reduced cAMP generation in response to stimulation by TSH, which could explain the impairment in T4 and T3 release from the thyroid glands. Therefore, further biochemical and molecular studies are required for the elucidation of the grt gene, particularly of the function of the TSH receptor, the TSH receptor-Gs protein coupling, the transcription factors for TSH receptor expression and other parts of the subsequent signalling system in the thyroid gland.
The authors express their sincere thanks to Dr. T. Yoshida (Star dust Square, Studio & Acupuncture practice) for supplying the grt mice. We are also grateful to Dr. T. Ohishi (Saitama Children's Clinical Center) and Ortho-Clinical Diagnostics K.K. for their help in the measurement of total T3, and T4, and free T3 and T4 by radioimmuno-assay.