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Endocrinology, doi:10.1210/en.2007-0677
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Endocrinology Vol. 148, No. 11 5305-5312
Copyright © 2007 by The Endocrine Society

Effects of Maternal Levels of Thyroid Hormone (TH) on the Hypothalamus-Pituitary-Thyroid Set Point: Studies in TH Receptor β Knockout Mice

Manuela Alonso, Charles Goodwin, XiaoHui Liao, David Page, Samuel Refetoff and Roy E. Weiss

Departments of Medicine (M.A., C.G., X.L., D.P., S.R., R.E.W.) and Pediatrics (S.R., R.E.W.), and Committees on Genetics (S.R.) and Molecular Medicine (S.R., R.E.W.), The University of Chicago, Chicago, Illinois 60637

Address all correspondence and requests for reprints to: Roy E. Weiss, M.D., Ph.D., The University of Chicago, MC 3090, Chicago, Illinois 60637. E-mail: rweiss{at}medicine.bsd.uchicago.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A level of thyroid hormone (TH) in agreement with the tissue requirements is essential for vertebrate embryogenesis and fetal maturation. In this study we evaluate the immediate and long-term effects of incongruent intrauterine TH levels between mother and fetus using the TH receptor (TR) β–/– knockout mouse as a model. We took advantage of the fact that the TRβ–/– females have elevated serum TH but are not thyrotoxic due to resistance to TH. We used crosses between heterozygotes with wild-type phenotype (TRβ+/–) males and TRβ–/– females, with a hyperiodothyroninemic (high T4 and T3 levels) intrauterine environment (TH congruent with the TRβ–/– fetus and excessive for the TRβ+/– fetus), and reciprocal crosses between TRβ–/– males and TRβ+/– females, providing a euiodothyroninemic intrauterine environment. We found that TRβ–/– dams had reduced litter sizes and pups with lower birth weight but preserved the mendelian TRβ–/– to TRβ+/– ratio at birth, indicating that the incongruous TH levels did not decrease intrauterine survival of a specific genotype. The results of studies in newborns demonstrate that TRβ+/– pups born to TRβ–/– dams have persistent suppression of serum TSH without a peak. On the other hand, TRβ–/– pups born to TRβ+/– dams have lower serum TSH at birth and a tendency to peak higher, compared with TRβ–/– pups born to TRβ–/– dams. The studies in the adult progeny demonstrate that TRβ+/– mice born to TRβ–/– dams and, thus, exposed to higher intrauterine TH levels, have greater resistance to TH at the level of the pituitary when stimulated with TRH. On the other hand, TRβ–/– mice born to TRβ+/– dams and, thus, deprived of TH in uterine life, were more sensitive to TH when similarly stimulated with TRH. Thus, TH exposure in utero has an effect on the regulatory set point of the hypothalamus-pituitary-thyroid axis, which can be seen early in life and persists into adulthood.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE (TH) is essential for vertebrate embryogenesis and fetal maturation (1). In rat embryos cultured in vitro, addition of serum from animals with either hypothyroidism or hyperthyroidism induced malformations (2). The deleterious effects of hypothyroidism during pregnancy are well known, and include decreased survival and impairment of neuropsychological development of the offspring (3, 4). The consequences of maternal hyperthyroidism are less well understood because it is difficult to distinguish between the effect of TH excess on the mother compared with that on the fetus and the concurrent effects of autoimmunity that produces the high TH levels (5, 6, 7). Furthermore, it has yet to be determined what effect, if any, TH has on the intrauterine environment and the development of the feedback set points of the hypothalamus-pituitary-thyroid (HPT) axis. In contrast to the situation in mature mammals, the effects of TH deficiency or excess during development are generally irreversible. Mice completely deficient in TH receptor (TR) β-gene (homozygous knockout, TRβ–/–) are resistant to TH. They have elevated serum TH and TSH levels but are practically eumetabolic. Heterozygous (TRβ+/–) mice have hormone values that are similar to normal (wild-type, TRβ+/+) mice (8, 9). These mice can be used to assess the immediate and long-term effects exerted by the relative maternal TH excess or deficiency during gestation on the fetus without the concomitant effect of hormone excess or deficiency on the mother, and without the possible effect of antibodies associated with autoimmune thyrotoxicosis.

We hypothesized that a main sequela of "incongruent" exposure to TH in utero would be a perturbation of the developing HPT axis and its feedback controls. "Incongruent" refers to discrepant TH levels in the dam relative to the expected level for the fetal genotype: high for TRβ+/– fetus carried by a TRβ–/– dam and low for a TRβ–/– fetus carried by a TRβ+/– dam. By the same token, fetuses carried by mothers with a genotype presenting the same TH levels are "congruent." TH levels in blood are regulated by a feedback mechanism through which TH inhibits the production and secretion of TRH by the hypothalamus, and TSH by the thyrotrophs in the pituitary. Maturation of this axis is a complex process extending from midgestation through the neonatal period, with continuing adjustments of the set point during childhood and adolescence (10).

In this study we evaluate the effects on newborn and, subsequently, adult mice exposed to TH levels in utero that are not congruent to their requirements. We used crosses between TRβ+/– males and TRβ–/– females, the progeny of which develop in a hyperiodothyroninemic intrauterine environment (appropriate TH level for the TRβ–/– fetus and excessive for the TRβ+/– fetus), and reciprocal crosses between TRβ–/– males and TRβ+/– females, providing a euiodothyroninemic intrauterine environment (TH deficient for the TRβ–/– fetus and appropriate for the TRβ+/– fetus). In addition, TRβ+/+ females crossed with TRβ+/+ males were studied as controls. Results show early effects on the HPT axis that were detected even at 3 months.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
The TRβ knockout mouse (TRβ–/–) was produced as described in Ref. 8 . All mice studied were from a C57BL/6 background. Heterozygous, TRβ+/– mice, were interbred to generate litters containing TRβ+/–, TRβ–/–, and TRβ+/+ progeny. These mice were then used to generate all the mice analyzed using the mating strategies described in Mating strategies. Mice were evaluated at birth [postnatal d (P) 0], and P3, P7, P14, and P21 for the newborn studies, whereas mice aged 60–80 d were used for the adult studies. Only data from male offspring are presented because TSH values in adult females are lower and often at or below the limit of detection. Blood was obtained from the tail vein under light anesthesia on mice P14 and older, whereas intracardiac puncture was used for this purpose in P0, P3, and P7 mice. Data are reported as the mean ± SE, with five to 25 mice of the same genotype in each group depending on the age. Serum was separated by centrifugation and stored at –20 C until analyzed in the same assay for each experiment. Mice were housed in a controlled environment at 19 C and under 12-h alternating darkness and artificial light cycles. All blood samples were obtained between 1000 and 1300 h. All animal experiments were performed at The University of Chicago according to protocols approved by the Institutional Animal Care and Use Committee. Mouse DNA extraction and genotyping were performed as previously described (11, 12).

Mating strategies
The influence of high maternal TH was studied using crosses between TRβ+/– males and TRβ–/– females, in which progeny develop in a hyperiodothyroninemic intrauterine environment for TRβ+/– but not for TRβ–/– fetuses. Reciprocal crosses were made between TRβ–/– males and TRβ+/– females, providing a euiodothyroninemic intrauterine environment for TRβ+/– and TH deficient for TRβ–/– fetuses. In addition, TRβ+/+ females crossed with TRβ+/+ males were studied as controls. The groups analyzed are described in Table 1Go.


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  		TABLE 1. Mouse groups

 
Measurements of TH and TSH concentrations in serum
Serum TSH in newborns was measured in 10 µl serum using a sensitive, heterologous, disequilibrium double-antibody precipitation RIA (13), and results were expressed in mU/liter. Serum TSH in adult mice was measured in 50 µl serum using the same assay but separate standard curves using as a diluent 50 rather than 10 µl TSH-deficient mouse serum. All TSH samples were individually analyzed for each mouse. Serum total T4 and T3 concentrations were measured by a solid-phase RIA (Coat-a-Count; Diagnostic Products Corp., Los Angeles, CA) adapted for mice, using 25 and 50 µl serum, respectively. For P3 mice, serum samples were pooled from four pups of the same genotype and gender within a litter, and four groups of pooled serum were analyzed, whereas samples from P7, P14, P21, and adult mice were individually analyzed for T4 and T3 concentrations.

TRH stimulation tests
Male adult progeny (60–80 d old) were given daily ip injections of 0.2 or 0.8 µg L-T3/100 g body weight (BW) for 4 d, and another group of mice was injected with the vehicle alone as a control. Approximately 18 h after the last L-T3 injection, a TRH stimulation test was performed by injection of 0.275 µg TRH ip. Blood samples were obtained from the tail before and 15 min after the TRH injection, and serum TSH levels were measured. Blood was also collected before L-T3 treatment to assess basal hormone levels.

Statistical analysis
Values are reported as mean ± SE. The number of mice is indicated. ANOVA was performed, and if significant effects were seen, P values were calculated by the Student’s t test. Values of the respective limits of assays sensitivities were assigned to samples with undetectable TSH and T4 concentration.

mRNA measurements
mRNA content in tissue was measured by real-time PCR as previously described (14).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Litter size, mendelian ratio, and birth weight of pups born to hyperiodothyroninemic (TRβ–/–) and euiodothyroninemic (TRβ+/–, TRβ+/+) dams
TRβ–/– females have higher TH and TSH levels than TRβ+/+ and TRβ+/– females (TT4 11.5 ± 1.1, 4.3 ± 0.1 and 4.5 ± 0.1 µg/dl; TT3 178 ± 13, 93 ± 4.7 and 94 ± 3.2 ng/dl; and TSH 141 ± 10, 10 ± 0.1 and 10 ± 0.4 mU/liter; P ≤ 0.01). Thus, by controlled mating we can study TRβ–/– dams carrying TRβ+/– fetuses (excessive intrauterine TH exposure) and TRβ–/– fetuses (intrauterine exposure to congruent TH level). In contrast, TRβ+/– dams carry TRβ+/– fetuses (congruent intrauterine exposure to TH) and TRβ–/– fetuses (intrauterine exposure to reduced amount of TH). Both surviving and dead pups were genotyped by PCR and were found at normal mendelian ratios (Table 2Go). However, both litter size and birth weight of pups born to TRβ–/– dams (regardless of gender and genotype) were reduced compared with those born to TRβ+/– and TRβ+/+ dams.


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  		TABLE 2. Effect of TH excess/shortage on the fetus: mendelian ratio, litter size, and pups weight at birth

 
Postnatal profiles of serum TH and TSH concentrations in the offspring of hyperiodothyroninemic (TRβ–/–) and euiodothyroninemic (TRβ+/–, TRβ+/+) dams (Fig. 1Go)
To examine the thyroid function of newborn pups exposed to different levels of TH in utero, the circulating serum levels of TH and TSH of offspring born to TRβ+/+, TRβ+/–, and TRβ–/– dams were measured during postnatal development at 0, 3, 7, 14, and 21 d of age. Only data from male pups are presented, though comparable serum TH and TSH profiles were also seen in female mice (data not shown). In TRβ+/+ pups and TRβ+/– pups born to TRβ+/– dams (groups 1 and 2), TSH levels were high at birth, peaked at P3, and then decreased by P21 to levels similar to those seen in adulthood (Fig 1AGo). Although TRβ+/– pups born to TRβ–/– dams (group 4) had TSH levels at birth similar to those of the preceding two groups, they did not display the expected TSH peak, but rather gradually reached normal levels by P21. In TRβ–/– pups (groups 3 and 5), TSH concentrations became significantly higher compared with TRβ+/+ pups (group 1) at d 7 and peaked at d 14 before reaching near-adult values by P21. TRβ–/– pups born to TRβ+/– dams (group 3) had significantly lower serum TSH values at birth (Fig. 1BGo, inset), and tended to have higher peak serum TSH at P14 compared with TRβ–/– pups born to TRβ–/– dams (group 5) (Fig. 1BGo).


Figure 1
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  		FIG. 1. Developmental serum profiles of TSH and TH levels in the offspring of euiodothyroninemic and hyperiodothyroninemic dams during the first 3 postnatal weeks. Serum TSH (A and B), total T4 (C and D), and total T3 (E and F) levels. Bar graph insets: serum TSH at P0 (B) and serum T4 at P3 (C and D). Values are the mean ± SE. TSH determinations were performed in individual animals using four to 27 pups evaluated at birth (0 d), and 3, 7, 14, and 21 d of age. For T4 and T3 determinations, serum samples were pooled from four pups of the same genotype and gender within a litter (n = 4 pools) at P3. At 7, 14, and 21 d of age, the serum samples were individually analyzed (n = 4–8). Significant differences P ≤ 0.05; *, compared with group 1; #, group 2 compared with group 4; +, group 3 compared with group 5.

 
Total T4 and T3 levels for all groups of mice were low at P3 and increased until they peaked at P14, reaching adult levels by P21 (Fig. 1Go, C–F). At P3, pups born to TRβ–/– dams, regardless of genotype (groups 4 and 5; TT4 0.8 ± 0.1 and 3.2 ± 0.5 µg/dl, respectively), had higher T4 than those pups born to TRβ+/– dams (groups 2 and 3; TT4 0.47 ± 0.07 and 1 ± 0.29 µg/dl, respectively; P ≤ 0.05) (Fig. 1Go, C and D, insets). Furthermore, TRβ–/– pups (groups 3 and 5) had higher T4 at P3 than their TRβ+/– littermates (groups 2 and 4). By P7, TRβ+/– pups born to TRβ–/– dams (group 4; TT4 1 ± 0.1 µg/dl) had the same T4 levels as TRβ+/+ pups and TRβ+/– pups born to TRβ+/– dams (groups 1 and 2; TT4 1 ± 0.2 and 1.1 ± 0.3 µg/dl, respectively). On the other hand, by P7, TRβ–/– pups born to TRβ+/– dams had higher T4 than TRβ–/– pups born to TRβ–/– dams (groups 3 and 5; TT4 4 ± 0.4 and 2 ± 1.4 µg/dl, respectively; P ≤ 0.01).

The resistance to TH (RTH) phenotype of higher total T4, T3, and TSH is already fully manifested in the TRβ–/– pups by P7. In TRβ–/–, as in TRβ+/+ and TRβ+/– pups, the T4 and T3 peaks occurred on P14, except at levels 2- to 3-fold higher. In contrast, the TSH peak at P7 was delayed until P14 in TRβ–/– pups and reached a much higher level (Table 3Go).


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  		TABLE 3. Developmental TH and TSH landmarks of pups exposed to different TH levels in utero

 
Sensitivity of the HPT axis to TH feedback regulation in the adult male offspring of hyperiodothyroninemic (TRβ–/–) and euiodothyroninemic (TRβ+/+, TRβ+/–) dams
The adult TRβ–/– progeny, regardless of intrauterine thyroid environment, had the same baseline thyroid function tests that were consistent with RTH (Fig. 2Go). Although the T3 levels were the same for both TRβ+/+ and TRβ+/– mice, TRβ+/– mice had significantly higher T4 (groups 2 and 4; TT4 4.3 ± 0.1 and 3.8 ± 0.1 µg/dl, respectively) than the TRβ+/+ progeny (group 1; TT4 3.3 ± 0.1 µg/dl; P ≤ 0.01). Furthermore, the T4 of TRβ+/– mice born to TRβ+/– dams (group 2) was significantly higher (P ≤ 0.05) than the T4 of TRβ+/– mice born to TRβ–/– dams (group 4), whereas their TSH was significantly lower (TSH 13.7 ± 1.2 and 20.4 ± 2.2 mU/liter, respectively; P ≤ 0.01).


Figure 2
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  		FIG. 2. Serum TSH and TH levels in adult offspring of euiodothyroninemic and hyperiodothyroninemic dams. Serum TSH (A), total T4 (B), and total T3 (C) levels. Determination of TSH, and total T3 and T4 were performed on serum samples obtained at baseline in adult male offspring (60–80 d old). Values are the means ± SE. The number of animals in each group is in parentheses.

 
In addition to baseline TSH measurements, TRH stimulation tests were performed to assess subtle abnormalities in the pituitary-thyroid axis. In animals not given L-T3, the fold change (Fig. 3AGo) from baseline in response to TRH in TRβ–/– mice (groups 3 and 5) and in TRβ+/– pups born to TRβ–/– dams (group 4) was significantly less than in TRβ+/+ mice (group 1), regardless of their maternal exposure to TH. However, the absolute TSH value after TRH stimulation (in the absence of L-T3; Fig. 3BGo) in TRβ–/– mice (groups 3 and 5) was the same as in TRβ+/+ mice (group 1) but was significantly lower in TRβ+/– mice (groups 2 and 4; P ≤ 0.05).


Figure 3
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  		FIG. 3. Effect of L-T3 on the TSH response to TRH in adult offspring of euiodothyroninemic and hyperiodothyroninemic dams. TRH stimulation was performed in male progeny (60–80 d old). Mice from the five groups were treated for 4 d with two different doses of L-T3 (0.2 or 0.8 µg L-T3/100 g BW) or with the vehicle only. TSH was measured before and 15 min after administration of TRH. A, Fold change of TSH in response to TRH in mice not given L-T3. The responses in TRβ–/– mice (groups 3 and 5) were independent of the maternal genotype, but the TRβ+/– mice from TRβ–/– dams (group 4) had a blunted response compared with TRβ+/– mice from TRβ+/– dams. B, TSH response to TRH in mice not given L-T3 (basal response). The TSH response to TRH was significantly lower in the TRβ+/–mice (groups 3 and 5) compared with the other groups (1, 2, and 4; P ≤ 0.05). C, TSH responses to TRH in mice given L-T3, expressed as percentage of the basal response. Note the paradoxical increase in TRβ+/– mice born to TRβ–/– dams (group 4) receiving 0.2 µg L-T3 and the greater suppressive effect in TRβ–/– mice born to TRβ+/– mothers (group 3). The number of animals in each group is in parentheses.

 
In a different study, mice from each group were treated on different occasions with either 0.2 or 0.8 µg L-T3/100 g BW for 4 d before performing TRH stimulation tests (Fig. 3CGo). The doses of L-T3 are based on our previous work in mice showing that the 0.2 µg/100-g BW dose would only partially suppress the TSH response to TRH in TRβ+/+ mice, whereas the higher, 0.8 µg/100-g BW dose produces almost full suppression (11, 15). With 0.2 µg L-T3 treatment, TRβ+/+ mice and TRβ+/– mice born to TRβ+/– dams had TSH responses reduced by 19 and 15% (groups 1 and 2, respectively). However, there was a paradoxical increase of 36% (P ≤ 0.001) in the TSH response of TRβ+/– mice born to TRβ–/– dams (group 4). This was not observed with the higher dose (0.8 µg/100 g BW), in which TRβ+/+ mice and all TRβ+/– mice, regardless of the intrauterine TH milieu, suppressed to the same degree (groups 1, 2, and 4; 66, 75, and 61%, respectively). Therefore, on provocative testing it appears that TRβ+/– mice born to TRβ–/– dams (group 4) are more resistant to TH at the level of the pituitary than TRβ+/+ mice and TRβ+/– mice born to TRβ+/– dams (groups 1 and 2).

TRβ–/– mice born to TRβ+/– dams were more sensitive to the lower dose (0.2 µg/100 g BW) of L-T3 than TRβ–/– mice born to TRβ–/– dams (group 3, –37% vs. group 5, –15%; P ≤ 0.01; Fig. 3CGo). Indeed, TRβ–/– mice born to TRβ+/– dams had maximal suppression with the low dose as is evidenced by the fact that the higher dose (0.8 µg/100 g BW) of L-T3 did not result in a greater suppression of TSH (–37 and –38%, respectively). TRβ–/– mice, regardless of their mother’s genotype, suppressed to the same degree with the 0.8 µg/100 g BW L-T3 (groups 3 and 5, –38 and –47%, respectively), but much less than the TRβ+/+ mice and TRβ+/– mice (groups 1, 2, and 4; –66, –75, and –61%, respectively; P ≤ 0.01; Fig. 3CGo).

Collectively, these data suggest that the feedback regulation of the pituitary-thyroid axis of TRβ+/– mice born to TRβ–/– dams (group 4) and TRβ–/– mice born to TRβ+/– dams (group 3) is altered and that the relative intrauterine excess or shortage of TH has an effect on the regulatory set point of the pituitary-thyroid axis that persists in adulthood.

Measurement of TR{alpha}1 and TR{alpha}2 mRNA in pituitaries and hypothalami in the adult male offspring of hyperiodothyroninemic (TRβ–/–) and euiodothyroninemic (TRβ+/+, TRβ+/–) dams at baseline
Previous studies from our laboratory and by others in various tissues of mice and humans with TRβ gene deletion have shown that there are no compensatory increases in TR{alpha}1 and 2 (9, 14, 16). This included pituitary TR{alpha}1 and 2 mRNAs (14). Nevertheless, we measured the mRNA content by real-time PCR in the untreated adult male progeny and these mRNAs in all genotypes and found no differences in TR{alpha}2. More specifically, TR{alpha}1, expressed as fold-difference ± SD compared with TRβ+/+ mice, values were 0.60 ± 0.22 and 0.99 ± 0.22 for pituitary and 0.88 ± 0.11 and 0.85 ± 0.07 for hypothalami of TRβ+/– and TRβ–/– mice, respectively, born to TRβ–/– mice. When mice were born to TRβ+/– dams, values of TR{alpha}1 expression were 0.57 ± 0.25 and 0.75 ± 0.09 for pituitary and 0.89 ± 0.04 and 0.77 ± 0.06 for hypothalami of TRβ+/– and TRβ–/– mice, respectively. There are small differences in the expression of TR{alpha}1 mRNA at the level of the hypothalamus in the TRβ+/– born to TRβ–/– dams and at the level of the pituitary in TRβ–/– mice born to TRβ+/– dams that, although statistically significant, are likely not physiologically relevant.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we demonstrate that exposure in utero to TH has an effect on the regulatory set point of the HPT axis that can be seen early in life and persists into adulthood. These findings suggest that differences in thyroid function among adults may correlate with the phenotype of the mother and, therefore, the level of TH exposure in utero.

Transplacental passage of maternal TH is responsible for the TH supply to the fetus before the development of a functioning thyroid gland (4). At 16–18 wk in the human, there is a progressive increase in TSH associated with increases in total and free T4 levels between 20 wk and term (17, 18). There are at least three factors that influence TH action in the fetus:

  1. The level of free hormone, which is dependent on the production of T4 binding globulin by the fetal liver in response to maternal estrogen and T4.
  2. Maturation of the TH-dependent negative feedback control of pituitary TSH and hypothalamic TRH (10, 19). It is the maturation of this axis that we propose may "lock" the feedback set point and is responsible for the variation in adult thyroid function.
  3. The ontogeny of TR isoforms that mediate the action of TH have unique developmental and tissue-specific patterns of expression.

TRs are expressed during development before the appearance of the thyroid gland anlage and, therefore, may play ligand-independent roles during the early development. In fact, unliganded TR{alpha}1 represses genes involved in cochlear development (20). In the rat, TR{alpha}1 accounts for virtually all T3-binding activity in fetal liver during embryonic d 19. In mammals, including rat and mouse, the prominence of TR{alpha}1 in fetal liver and brain raises the possibility that this receptor isoform may perform specialized functions in the fetus and that TRβ1 serves other functions at later stages of development (21).

To evaluate the effects on newborn and adult mice exposed to different levels of maternal TH, we used the TRβ–/– knockout mice as a model. These mice provide a recessive model for the human disorder of RTH (22), with elevated TH and TSH levels (9, 12). With controlled matings, we studied euiodothyroninemic TRβ+/– dams and euthyroid hyperiodothyroninemic TRβ–/– dams, each of which carry both TRβ+/– and TRβ–/– fetuses. It has been established that the amount of TH in serum and tissues of rodent embryos and pups is dependent and proportional to that circulating in the mother (23, 24). This is also true in humans (6). Therefore, fetuses harboring the same phenotype as the mother would be exposed to levels of TH that are congruent to their requirements. In contrast, those with a different phenotype would be exposed either to excessive or insufficient TH levels. We acknowledge that TRβ–/– mice, although resistant to TH action, are not identical to the common form of RTH in humans (25). In these mice, the RTH is caused by disruption of both alleles of the TRβ, with no resulting dominant negative effect of the mutant allele. Thus, although this model does not mimic the garden variety RTH in humans, it is a proper model to use in the study of the effect of maternal TH on otherwise normal fetuses because receptor deficiency is not complicated by a dominant negative effect of a mutant allele. Importantly, in the current mouse model, there is no dominant negative effect involving TR{alpha}. It should also be noted that, although both man and mouse have hemochorial placentas, there might be differences in the way these species handle maternal TH levels. However, there are many findings that suggest the conclusions from experiments performed in mice may be relevant to our understanding of human fetal thyroid physiology.

First, we studied the effect of maternal TH levels not congruent to the phenotype of the fetus by evaluating the mendelian ratio of born genotype, litter size, and pup’s weight at birth. It has been shown that women with high TH levels, but euthyroid due to RTH, have an increased rate of miscarriages, which appears to involve predominantly unaffected fetuses. In addition, women with RTH give birth to unaffected infants with suppressed TSH and lower weight, indicating that the high maternal TH levels produce fetal thyrotoxicosis (26). We found that the mendelian ratio genotype at birth was preserved in both matings, indicating that the embryos, whose TH needs were different than those of their mother’s, did not have increased intrauterine mortality. However, TRβ–/– dams had smaller litter sizes and pups of lower birth weight. Although TRβ–/– mice have been described as fertile (8, 9), their reproductive function has not been studied in detail. Further studies need to determine if the smaller litter sizes and lower pup’s birth weight are due to impaired embryogenesis of fetuses exposed to high maternal TH or due to receptor insufficiency unrelated to TH concentration.

Second, to examine the thyroid function of newborns exposed to incongruent intrauterine TH levels, the circulating serum concentrations of TH and TSH were determined during the first 3 wk of life of the offspring of TRβ+/+, TRβ+/–, and TRβ–/– dams. T3 and T4 levels are low at birth and increase until they peak on P14. Thereafter, both serum T4 and T3 levels decrease to reach near-adult levels on P21 (27, 28, 29). On the other hand, TSH is relatively high at birth in all genotypes, except the TRβ–/– mice, which peaks at P3 and then decreases by P21 to levels similar to those seen in adulthood. We have found abnormal TH and TSH patterns in the newborns with TH needs presumed different from that of their mother’s on the basis of genotype. TRβ+/– pups born to TRβ–/– dams have persistent suppression of serum TSH without a peak associated with higher levels of T4 at P3 (group 4 compared with group 2). Similarly, normal human infants carried by RTH mothers are born with suppressed TSH levels (26). On the other hand, TRβ–/– pups born to TRβ+/– dams have lower serum TSH at birth and a tendency to peak higher, compared with TRβ–/– pups born to TRβ–/– dams. In those pups, the perinatal production of TSH is understimulated as a result of a mechanism that still remains to be explained. The RTH phenotype of TRβ–/– mice, consisting of higher serum TH and TSH levels, is already fully manifested by P7 and their thyroid glands have displayed an increase in the number and size of colloid-containing follicles (9). As in TRβ+/+ and TRβ+/– mice, the serum TH peak of TRβ–/– mice occurs at P14, though at much higher levels, whereas their TSH peak was delayed until P14 as well, and, likewise, at much higher levels, decreasing later to near-adult levels by P21. This delay in TSH peak may reflect the higher TH levels needed to negatively regulate TSH production and release at the level of the resistant pituitary and/or hypothalamus. The physiological set point for TSH synthesis and release is determined by a balance between a positive input from hypothalamic TRH and a strong negative influence by TH. Together, these opposing influences likely determine the amount of TSH synthesized and secreted. Also of note is that decrease in T4 levels in the TRβ–/– pups born to TRβ–/– dams occurs between d 3 and 7. This decrease, not associated with a change in the TSH or T3 concentrations, suggests that the pups in group 5 have a more rapid turnover of T4. Although statistically significant, it may not be physiologically relevant.

Third, we studied the long-term effect of maternal TH levels on the adult male offspring by evaluating the serum TH and TSH levels at baseline and the TSH response to TRH stimulation before and after pretreatment with two doses of L-T3. In adulthood, TRβ–/– progeny, regardless of intrauterine thyroid environment, have the same baseline thyroid function tests compatible with RTH. However, the TRβ+/– progeny born to TRβ–/– dams have elevated T4 but TSH similar to TRβ+/+, indicating mild RTH at the level of the pituitary thyrotrophs. Mice heterozygous for a deletion of one of the TRβ alleles were reported to have had thyroid function tests indistinguishable from TRβ+/+ mice (8, 9). In the current study, using a larger number of animals back crossed to homogeneity in the C57B strain, TRβ+/– mice have slightly higher T4 levels than TRβ+/+ mice, likely as a result of haploinsufficiency. The finding of a significantly lower TSH-fold response to TRH stimulation (at baseline) has not been previously reported in the TRβ+/– mouse born to TRβ–/– dams. The precise reason for this observation, although unknown, is also likely due to haploinsufficiency. The decrease in fold change is seen in the TRβ+/– pups born to TRβ–/– dams compared with the TRβ+/– born to TRβ+/– dams, which is as the TRβ+/+. This suggests that otherwise normal pups, when born to RTH dams, are more resistant to TH. The high intrauterine thyroid levels may influence the set point of the HPT axis. The observations made in the TRβ–/– progeny are in agreement with those previously reported of a reduced percent increase of TSH in response to TRH stimulation at baseline (11, 15). After 0.2 µg T3 treatment, the reduction of TSH response to TRH in TRβ+/+ and TRβ+/– mice born to TRβ+/– was 19 and 15%, respectively. However, there was a paradoxical increase of 36% (P ≤ 0.001) in the TSH response of TRβ+/– mice born to TRβ–/– dams, suggesting a higher RTH as a result of intrauterine exposure to excessive TH. On the other hand, TRβ–/– mice born to TRβ–/– dams had a TSH response diminished by 15% after 0.2 µg T3 treatment, whereas TRβ–/– mice born to TRβ+/– dams were more sensitive (–37%; P ≤ 0.05) as a result of exposure in utero to TH insufficient for their phenotype requirement. In other words, mice exposed to incongruous TH levels in utero have an altered threshold for negative feedback at the level of the pituitary in response to provocative testing. We did not observe in our TRβ–/– mice the paradoxical increase of TSH in response to TRH stimulation after the lower L-T3 dose as we have previously reported (11, 15). However, these results were obtained from intercrossed homozygous mice, TRβ–/–, which in addition to high maternal TH level, had all homozygous embryos, whereas these reported herein were obtained by intercrossed heterozygous and homozygous mice, and, therefore, had both heterozygous and homozygous fetuses, possibly affecting the intrauterine TH milieu. It should be noted that we were able to reproduce the response in TRβ–/– mice born to TRβ–/– parents (intercrossed, data not shown).

The data presented here show for the first time that incongruent intrauterine TH levels have an effect on the set points of the HPT axis. Further studies are required to establish the mechanisms mediating these effects, and to establish whether the interaction between the mother and fetus can have an effect on the mother as well.


   Footnotes
 
Supported in part by Grants RR18372, DK17050, and DK20595 from the National Institutes of Health, The Seymour Abrams Thyroid Research Fund, and the Rabbi Morris Esformes Endowment. Publication cost was defrayed by Provell Pharmaceuticals, LLC, Honeybrook, PA.

Disclosure Statement: The authors have nothing to declare.

First Published Online August 9, 2007

Abbreviations: BW, Body weight; HPT, hypothalamus-pituitary-thyroid; P, postnatal d; RTH, resistance to thyroid hormone; TH, thyroid hormone; TR, thyroid hormone receptor.

Received May 21, 2007.

Accepted for publication August 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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