Figure 1.
Contiguous coronal images were collected from wild type (WT), Tpst1-/- (T1), and Tpst2-/- (T2) females (n = 3). The tissue region comprising the right and left sublingual and submandibular glands (A) were traced and an area per slice was calculated and a total volume was determined which was then normalized to body weight in grams as described in Methods (B). Results are expressed as mean ± S.E.M. Statistical differences between groups were tested using unpaired, two-tailed t-tests with equal sample variance and an α ≤ 0.05. Salivary gland volume of Tpst2 -/- mice was significantly smaller than wild type (p = 0.002) and Tpst1-/- (p = 0.04), respectively (indicated by an asterisk). However, the salivary gland volumes of wild type and Tpst1-/- mice were not statistically different (p = 0.57).
Figure 2.
Submandibular gland histology.
Submandibular glands from wild type (A) and Tpst1-/- (B), and Tpst2-/- (C) females were embedded for histology and 5 µm sections were stained with Masson’s trichrome. Images are representative of the 3 animals from the groups analysed in Figure 1. 100 x Objective.
Figure 3.
Salivary gland function in wild type, Tpst1-/-, and Tpst2 -/- mice.
Saliva was collected from age-matched wild type (WT), Tpst1-/- (T1), and Tpst2-/- (T2) males and females as described in Methods. Pilocarpine-induced salivary flow normalized to body weight in grams (A, B) and α-amylase activity normalized to protein concentration (C, D) were measured as described in Methods. All results are represented as the mean ± S.E.M. For wild type and Tpst1-/- males and females, n = 5. For Tpst2-/- males and female, n = 3 and 4, respectively. Statistical differences between groups were tested using unpaired, two-tailed t-tests with equal sample variance and an α ≤ 0.05. An asterisk indicates p < 0.01 compared to wild type values.
Figure 4.
Sulfotyrosine Western blot analysis of salivary glands.
Submandibular, sublingual, and parotid glands from wild type (WT), Tpst1-/- (T1), and Tpst2-/- (T2) mice were collected, homogenized and post-nuclear supernatant were prepared as described in Methods. Non-reduced proteins (10 µg per lane) were resolved in 4-12% Bis-Tris polyacrylamide gels. Proteins were either transferred onto nitrocellulose membranes and probed with PSG2 followed by HRP conjugated secondary antibody (A) or detected with Imperial protein stain (ThermoScientific) (B). Human heparin cofactor II (HCII), a known tyrosine-sulfated protein, served as a positive control.
Figure 5.
Schematic diagram of thyroid hormone rescue protocol and timing of experimental endpoints.
Sexually mature Tpst2+/- males and females were conditioned to control chow or chow supplemented with thyroid powder for 3 weeks. Mating pairs were then established and continued on the control or experimental diet throughout pregnancy. Weanlings were then placed on the same diet until they were sacrificed at 15 weeks of age. All animals were weighed weekly and serum total T4 levels were determined at 5, 10, and 15 weeks of age. Saliva was collected at 12 weeks of age and animals were sacrificed at 15 weeks of age for histological analysis. Groups of 10-13 animals of each genotype (Tpst2+/+, Tpst2+/-, Tpst2-/-) and sex were included in the study.
Figure 6.
Serum total T4 levels were determined at 5, 10, and 15 weeks of age for 4 mice randomly selected from the 10-13 mice in each experimental group. WT = wild type. T2 = Tpst2-/-. Results are expressed as mean ± S.E.M.
Figure 7.
Body weight were measured weekly for all mice in each experimental group (n = 10-13). WT = wild type. T2 = Tpst2-/-. Results are expressed as mean ± S.E.M. Differences between groups was assessed with a two-way repeated-measures ANOVA using Prism 6 software. Statistical differences at each age were tested post-hoc using unpaired, two-tailed t-tests with equal sample variance and an α ≤ 0.05.
Figure 8.
Saliva was collected at 12 weeks of age from the two experimental groups as described in Methods. Pilocarpine-induced salivary flow normalized to body weight in grams (A, B) and α-amylase activity normalized to protein concentration (C, D) were measured as described in Methods. Salivary flow for all mice in each experimental group, whereas α-amylase activity was measured in 4 mice randomly selected from the 10-13 mice in each experimental group. WT = wild type. T2 = Tpst2-/-. Results are expressed as mean ± S.E.M. Statistical differences between groups were tested using unpaired, two-tailed t-tests with equal sample variance and an α ≤ 0.05. An asterisk indicates p < 0.01 compared to wild type values. A lancet indicates p < 0.02 compared to wild type values.
Figure 9.
Submandibular gland histology.
At 15 weeks of age submandibular glands from female (top panels) or males (bottom panels) were harvested and then fixed, wax embedded, sectioned, and stained with Masson’s trichrome as described in Methods. Images are representative of analyses of the 4 mice randomly selected from the >10 mice in each experimental group in Figure 7. 100 x Objective.