Using our animal models of thyroid dysfunction (TD), we could show that sex differences of features of hyper- and hypothyroidism occur throughout mouse life. Thereby, hyperthyroidism was likely to be associated with persistent, exaggerated or new manifestation of sex differences, whereas disappearance of sex effects was observed under hypothyroid conditions. Moreover, phenotypic traits in male and female mice were dominated either by sex (body temperature, muscular strength, locomotor activity), thyroid function (heart rate, muscle endurance and coordination) or age (body weight) and resulted in distinct effects of TD in different organs and functional systems.
Regulation of body temperature, muscular strength and locomotor activity under TD is dominated by sex
Rectal body temperature differed between younger male and female mice under all TH conditions  and persisted in adult and old age with higher body temperature in female than male mice. This was irrespective of thyroid status and suggests a strong sex-dependent effect (Fig. 4a, b). Non-invasive methods of rectal and surface body temperature measurements correlate very well with mice core body temperature . However, a drawback of these methods is that only certain time points can be measured and differences in small time frames may therefore be missed. As reported previously in a study investigating the influence of sex hormones on body temperature via radiotelemetry , sex differences were most obvious at daytime and in the proestrous and estrous periods with higher values in 3-months-old females than males. Ovariectomy in these mice resulted in similar body temperature pattern during proestrous and estrous as in male mice, indicating that differences in these time frames were regulated by sex hormones. However, throughout metestrus and diestrus stages, body temperature was comparable between ovariectomized and control female mice suggesting that sex hormones are not the only factors contributing to body temperature regulation in female mice. As no influence of THs was addressed in this study, we conclude from our measurements, that TH excess and deprivation likely have stronger influence on basal body temperature in general, rather than on time points during estrous cycle.
Other examples for the predominant effect of sex on phenotypic traits were muscular strength and locomotor activity. As already described for younger mice , a major sex difference was found in the chimney test, which particularly assesses strength and coordination in adult and old mice at eu-, hyper- and hypothyroid mice. Only few old animals were able to pass the test. Hence, conclusions can be derived from adult age only and showed that female mice had a much higher ability to climb up the tube at all TH conditions, while only few male mice were capable to pass the chimney test even at euthyroid state (Fig. 6a). This finding suggests that female mice may have a better movement coordination and muscle strength and are more fatigue resistant than male mice. Sex difference in muscle function has been described in human studies, showing less fatigue and faster recovery in female than male muscles  and this observation was also confirmed in a mice study . Interestingly, TD had a stronger impact on females than male mice, with seemingly decrease of sex difference, e.g. in the chimney test in hypothyroid adult mice. While the observed interplay between TH and sex hormones may affect muscle and/or neurological function of the central and peripheral nervous system [25, 26], it may also be influenced by BW and mouse size. As females are lighter and smaller than males at all TH conditions and ages, they may be in a more favourable position to climb up the tube. Thus, when assessing motor function beside muscle strength and endurance, BW and size are likely considerable contributing factors.
Changes in activity were assessed by the open field test and were quantified by measuring the total distance travelled in 5 min. Significant sex difference was noted with higher activity of female mice at different TH conditions and persisted in adult, but differences decreased with old age (, Fig. 6c, d). The observed findings are in line with other studies confirming a higher locomotor activity and increased voluntary exercise in female mice [22, 27], indicating an influence of sex hormones on activity of female mice, irrespective of TH status.
Thyroid function dominates the influence on heart rate, muscle endurance and motor coordination
Another very important issue was the influence of THs and sex on heart rate, as tachycardia or bradycardia are well-known manifestations of TD. Sex difference for heart rate was present in younger euthyroid mice but disappeared by TH excess or deprivation , suggesting that alteration of TH status might mask impact of sex hormones. In adult and old mice, no sex impact was found for heart rate, irrespective of TH status (Fig. 4c, d). Thus, TH status dominated the effects of sex and age on heart rate. Of note, measurements of heart rate were performed in a non-invasive ECG, which although mice were pre-trained to the restrainer, may have resulted in increased stress and could have masked small differences in heart rate.
The impact of TD, sex and age on muscle endurance and motor coordination was assessed by the rotarod test. In our previous study, we found that TD impaired performance in young male and female mice with diminished sex difference under hyper- and hypothyroid conditions while euthyroid female mice generally show better results in the rotarod test . In adult and old mice, the sex difference also disappeared under TH excess or deprivation (Fig. 5a–f). Interestingly, this was due to either a weaker performance of female mice (hyperthyroid) or improvement of endurance and motor coordination in males (hypothyroid). Reduced physical function and dexterity has been described in hyperthyroid patients , as well as an influence of hypothyroidism on myofiber composition and function . However, to our best knowledge, comprehensive analysis of different motor tests in mice with TD has not yet been performed. Thus, interpretation of our results remains speculative and might not only be a result of changes in muscle function, but could also be dependent on the change of BW due to TD.
Age modifies sex differences in body weight under TH excess and deprivation
In our initial study, a sex-dependent influence was noted with faster weight gain in hyperthyroid male compared to female mice . In contrast, this sex dependency reversed with age and close to the human situation, adult and old male mice lost BW under T4 excess whereas female mice still gained weight (Fig. 3b, e). Similarly, TH deprivation was accompanied by BW loss in both, male and female mice at adult and old ages, in contrast to unchanged BW in young hypothyroid mice . Hence, both observations implicate that age is a critical factor in BW change under TD in mice, suggesting a switch in metabolism. BW is influenced by energy intake and energy expenditure, the latter depending on physical activity and body temperature. Body temperature itself is a balance between heat production and dissipation . Mice subjected to normal laboratory conditions of 23 °C need approximately 50% of daily food intake to maintain their body temperature whereas clothed humans only need 2%  underlining the species-specific traits of metabolism and energy expenditure. Thus, availability of food and amount of food intake is strongly correlated with metabolism and activity [32,33,34]. Surprisingly, TH excess in young mice resulted in higher food intake in females than males . This sex difference disappeared in hyperthyroid adult and old mice and does not explain the observed differences in BW change of male mice (Additional file 2: Figure S1). Similarly, no significant differences were found for food intake in hypothyroid male and female mice compared to euthyroid controls, suggesting again that food intake is not responsible for BW loss with TH deprivation. Thus, we hypothesize that age-dependent differences in energy expenditure and/or body composition may account for the observed sex-specific BW changes in mice in different life stages.
Interplay between sex, age and TH status on phenotypical traits of TD is not mirrored in serum TH concentrations
The observed phenotypic traits of TD in male and female mice are not conclusively explained by serum TH concentrations. An obvious difference in response to T4 treatment was observed in all age groups. If subjected to chronic T4 treatment, female but not male mice of all ages had twofold higher TT4, FT4 and FT3 serum concentrations (, Fig. 2). This has already been observed by us in an earlier study using young mice  and by others using T3 , but was not expected to appear in older mice. Different mechanisms may be considered to contribute to this observation. First, T4 injected in the peritoneum needs to be absorbed via blood or lymphatic vessels . Subsequently, within the circulation, the majority of T4 is bound to thyroxin-binding globulin (TBG), transthyretin (TTR) or albumin. Thus, sex dependency in TT4 concentrations could result from differences in binding protein amount or capacity. In mice, however, a higher serum TBG concentration was found in male compared to female mice under euthyroid conditions  and was confirmed in our young study on hepatic transcript level . Chronic T4 treatment strongly represses TBG gene expression in both sexes  and might therefore unlikely cause different TBG serum concentrations in hyperthyroid male and female mice. However, sex differences on TTR or albumin concentrations might occur under TH excess independent of TBG, but were not investigated in this study.
Strikingly, despite the influence of sex on TT4 serum concentrations and although only a minority of THs circulate in an unbound form, the same sex effect was noted for FT4 and FT3 serum concentrations. Thus, one may speculate that elevated FT4 concentrations in female mice could have resulted from oversaturation of binding proteins, but a conclusive cause remains unclear.
Second, FT4 and FT3 need to be transported into the organs, which are facilitated by specific TH transporters . TH transporter expression on the cell surface can vary with sex, which was shown by us for brown adipose tissue, but less so for liver or heart on a transcript level  and could contribute to the observed sex impact on serum concentrations.
Third, as FT3 serum concentrations depend on T4 conversion by deiodinases , a sex impact on deiodination also has to be considered. Deiodinase 1 (Dio1) was previously reported to be differently active in young male and female mice. Thereby, renal Dio1 was found to be more active in female mice, while hepatic Dio1 showed higher activity in male than female mice . A subsequent study investigated the effect of age on sexual dimorphism of Dio1, but showed no differences in hepatic Dio1 activity at 12 months of age, while sex differences persisted for renal Dio1 activity . Thus, higher renal Dio1 activity, though not investigated in our study, might have influenced FT3 serum values in female mice.
In contrast, TH deprivation induced by combined MMI/ClO4−/LoI treatment, resulted in very low TT4 and unchanged FT3 serum concentrations in all age groups. Thereby, a more severe degree of hypothyroidism occurred in old females as demonstrated by lower FT4 and higher TSH concentrations compared to male mice (Fig. 2).