Deiodinase-mediated thyroid hormone inactivation minimizes thyroid hormone signaling in the early development of fetal skeleton

Abstract

Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To study this, pregnant mice were made hypothyroid and fetuses harvested at embryonic days (E) 12.5, 14.5, 16.5 and 18.5. Despite a marked reduction in fetal tissue concentration of both T4 and T3, bone development, as assessed at the distal epiphyseal growth plate of the femur and vertebra, was largely preserved up to E16.5. Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrophic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocytes, in addition to a slight delay in endochondral and intramembranous ossification. This suggests that up to E16.5, thyroid hormone signaling in bone is kept to a minimum. In fact, measuring the expression level of the activating and inactivating iodothyronine deiodinases (D2 and D3) helped understand how this is achieved. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly ( approximately 10-fold) at E18.5, and even more at 14 days after birth (P14). In contrast, D2 mRNA expression increased significantly by E18.5 and markedly ( approximately 2.5-fold) by P14. The reciprocal expression levels of D2 and D3 genes during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time suggest that coordinated reciprocal deiodinase expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development.

Fig. 1

Concentrations of total T4 and T3 in the serum of dams and pups and in the whole body homogenates of the fetuses. (A and B) Maternal serum levels of T4 and T3, respectively. (C and D) Fetal whole body concentrations of T4 and T3, respectively. (E and F) Post-natal serum levels of T4 and T3, respectively. Maternal serum levels and fetal concentrations of T4 and T3 were determined on the following gestational days: 12.5, 14.5, 16.5 and 18.5. Post-natal serum levels were determined at 2, 4, 7, 14, 21 and 35 days after birth. Hypothyroidism (HYPO) was induced by adding sodium perchlorate and methimazole to the drinking water of the dams. Euthyroid (EUT) dams, fetuses and pups are untreated animals. Values represent the mean±SEM (n = 4–12). *p<0.05, ** p<0.01 and ***p<0.001 vs. EUT by Student t-test.

Fig. 2

Effect of hypothyroidism on body length and weight during pre- and post-natal development. (A) Crown–rump length, (B) weight and (C, D and E) mean length of humerus, femur and tibia, respectively, from E12.5 to P35. Results are expressed as mean±SEM of 13–45 samples. *p<0.05, **p<0.01 and ***p<0.001 vs. EUT by Student t-test.

Fig. 3

Intramembranous ossification during pre- and post-natal development. Analysis of intramembranous ossification by alizarin red and alcian blue staining in the (A) fetal skull (1,2× magnification) and in the (B) skull of mice on the 14th post-natal day (0,8× magnification). (C) Diagram of the anatomy of the skull bones, sutures and fontanelles. EUT: euthyroid mice; HYPO: hypothyroid mice; E: embryonic age; P: post-natal days; PI: posterolateral fontanelle; Sq: squamosal suture; arrow: delayed closure of sutures; asterisk: delayed closure of fontanelles.

Fig. 4

Endochondral ossification during pre- and post-natal development. Analysis of endochondral ossification by alizarin red and alcian blue staining in the (A) fetal hind limb and in the (B) femur of mice during the post-natal development. EUT: euthyroid mice; HYPO: hypothyroid mice; E: embryonic age; P: post-natal days; arrow: secondary ossification. Scale bar: 1 mm for all.

Fig. 5

Effect of hypothyroidism on the vertebral morphology during pre-natal development (A and B) Mouse sample of fetus at embryonic day (E) 12.5, (C and D) E14.5, (E and F) E16.5 and (G and H) E18.5. Samples from (A, C, E and G) euthyroid (EUT) and (B, D, F and H) hypothyroid (HYPO) fetuses. as: anterior sclerotome; ps: posterior sclerotome; n: notochord; V: vertebral body; PZ: proliferative zone; HZ: hypertrophic zone; O: ossification center; arrow: cartilage extracellular matrix. Scale bar: 50 µm for all.

Fig. 6

Effect of hypothyroidism on the femoral morphology during pre-natal development. (A and B) Mouse sample of fetus at embryonic day (E) 12.5, (C and D) E14.5, (E–H) E16.5 and (I–L) E18.5. Samples from (A, C, E, G, I and K) euthyroid (EUT) and (B, D, F, H, J and L) hypothyroid (HYPO) fetuses. K and L are details from I and J, respectively. Bl: blastema of the femur. HZ: hypertrophic zone; DEp: distal epiphysis; Dia: diaphysis; PZ: proliferative zone; M: metaphysis; PhZ: pre-hypertrophic zone; arrow head: cortical bone; arrow: cartilage extracellular matrix. Scale bar: 100 µm (A, B, C, D, I and J) and 50 µm (E, F, G, H, K and L).

Fig. 7

Effect of hypothyroidism on the femoral morphology during post-natal development (A–D) Pups on the 7th post-natal day (P). (E–H) P14 pups. (I–L) P35 mice. The squares at G, H, K and L indicate the area of the detail in each picture. PZ: proliferative zone; HZ: hypertrophic zone; EGP: epiphyseal growth plate; arrow: developed secondary ossification center; arrow head: beginning of secondary ossification. Scale bar: 100 µm (A–D; G, H, K and L) and 200 µm (E, F, I and J).

Fig. 8

Relative mRNA expression of collagen type 1 (Col1), osteocalcin (OC), collagen type X (Col10), TRαl and TRβ1 in the femur of fetuses. mRNA expression of (A) Col1, (B) OC, (C) Col10, (D) TRα1, and (E) TRβ1. The mRNA expression was analyzed by real-time PCR and the relative gene expression was determined by designating EUT group expression at E14.5 to 1. E: embryonic days. Values are the mean±SEM of 3–5 samples. *p<0.05, ** p<0.01, ***p<0.001 vs. EUT by Student t-test.

Fig. 9

D2 and D3 mRNA relative expression pattern in the femur during pre and post-natal development (A) Fetuses at embryonic days 14.5, 16.5 and 18.5 and (B) mice at the post-natal days (P) 4, 7, 14 and 35. The mRNA expression was analyzed by real-time PCR and the relative gene expression was determined by designating the EUT group expression at E14.5 to 1 for each gene, separately. The values are the mean±SEM (n = 5). ^ap<0.05, E14.5 vs. E16.5; ^bp<0.05, E14.5 vs. E18.5; ^cp<0.01, E18.5 vs. all fetal ages; ^dp<0.05, P4 vs. P7; ^ep<0.01, P14 vs. all post-natal ages.

Fig. 10

Effect of thyroid status on D2 and D3 mRNA expression and activity in the femur of mice during pre-natal development. (A) D2 mRNA expression. (B) D2 activity. (C) D3 mRNA expression. (C) D3 activity. The mRNA expression was analyzed by real-time PCR and the relative gene expression was determined by designating the EUT group expression at E14.5 to 1 (A and C). D2 and D3 enzyme activities were measured in the bones of E 18.5 fetus. E: embryonic days. Values represent the mean ± SEM of determinations on three independent tissue preparations. *p<0.05, **p<0.01 vs. EUT by Student t-test.