Variable suppression of serum thyroxine in female mice of different inbred strains by triiodothyronine administered in drinking water

Abstract

Background: Recombinant-inbred mouse strains differ in their susceptibility to Graves'-like hyperthyroidism induced by immunization with adenovirus expressing the human thyrotropin (TSH) receptor. Because one genetic component contributing to this susceptibility is altered thyroid sensitivity to TSH receptor agonist stimulation, we wished to quantify thyroid responsiveness to TSH. For such studies, it is necessary to suppress endogenous TSH by administering L-3,5,3′-triiodothyronine (L-T3), with the subsequent decrease in serum thyroxine (T4) reflecting endogenous TSH suppression. Our two objectives were to assess in different inbred strains of mice (i) the extent of serum T4 suppression after L-T3 administration and (ii) the magnitude of serum T4 increase induced by TSH.

Methods: Mice were tail-bled to establish baseline-serum T4 before L-T3 administration. We initially employed a protocol of L-T3-supplemented drinking water for 7 days. In subsequent experiments, we injected L-T3 intraperitoneally (i.p.) daily for 3 days. Mice were then injected i.p. with bovine TSH (10 mU) and euthanized 5 hours later. Serum T4 was assayed before L-T3 administration, and before and after TSH injection. In some experiments, serum T3 and estradiol were measured in pooled sera.

Results: Oral L-T3 (3 or 5 µg/mL) suppressed serum T4 levels by 26%-64% in female BALB/c mice but >95% in males. T4 suppression in female B6 mice ranged from 0% to 90%. In C3H mice, L-T3 at 3 µg/mL was ineffective but 5 µg/mL achieved >80% serum T4 reduction. Unlike inbred mice, in outbred CF1 mice the same protocol was more effective: 83% in females and 100% suppression in males. The degree of T4 suppression was unrelated to baseline T4, T3, or estradiol, but was related to mouse weight and postmortem T3, with greater suppression in larger mice (outbred CF1 animals and inbred males). Among females with serum T4 suppression >80%, the increase in serum T4 after TSH injection was greater for BALB/c and C3H versus B6 mice. Moreover, the T4 increment was higher in female than in male BALB/c.

Conclusions: Our data provide important, practical information for future in vivo studies in inbred mice: we recommend that responses to TSH be performed in female animals injected with L-T3 i.p. to suppress baseline T4.

FIG. 1.

Schedules for L-3,5,3′-triiodothyronine (L-T3) administration to suppress baseline thyroxine (T4) before injecting bovine thyrotropin (TSH) (10 mU) to induce T4 secretion. (A) L-T3 administered in drinking water according to East-Palmer et al. (9). (B) L-T3 injected intraperitoneally (i.p.) as described by Maia et al. (11).

FIG. 2.

Suppression of T4 by L-T3 in drinking water (A–D) or injected i.p. (E). Mice studied were BALB/c, B6, and C3H/He (C3H), all inbred, and outbred CF1 mice. As described in the Materials and Methods section, suppression was calculated as the decrease from baseline T4 expressed as a percentage of baseline T4. The data are shown as a bar graph, where the bars indicate the mean suppression values for each experiment; error bars are standard error of the mean (where n = 3–5) or the standard deviation (where n = 2). The number of mice in each experimental group is shown below their respective bars; females are indicated by F, and males by M. All mice were given drinking water with 3 μg/mL L-T3 except for two BALB/c and five C3H animals that were provided with 5 μg/mL (as indicated).

FIG. 3.

The magnitude of suppression induced by L-T3 in water is related to mouse weight and postmortem T3 (A–C) but not to baseline T4, T3, or estradiol (D–F). From the mean suppression values for BALB/c, C57BL/6, C3H/He, and CF1 mice (bar graphs in Fig. 2), the data were subdivided into two groups: group I, <80% suppression; group II, >80% suppression. Number of animals in each group: group I, n = 8; group II, n = 5. Values significantly higher for group II versus group I: **p < 0.002 and *p < 0.02 (t-test) and ^#p = 0.019 (rank sum test).

FIG. 4.

T4 release after L-T3-induced suppression >90% (A, C, E) and <80% (B, D, F) for inbred (BALB/c, B6, and C3H) and outbred (CF1) mice. (A, B) Baseline T3 before T3, serum T4 before (first bar) and after (second bar) TSH injection; (E, F) net increase in T4 after TSH injection (for calculation, see the Materials and Methods section). L-T3 administration by water (w) or i.p. injection is indicated; the number of mice in each experiment is in parentheses. Values significantly different: (C, D) **p < 0.001; *p = 0.003 (paired t-tests); (E) ^#p < 0.036 BALB(F) versus B6(F) (rank sum test); **p = 0.002 BALB(F) versus BALB(M) (t-test).