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Investigation of post-transcriptional events of the thyroglobulin in the thyroid gland of the hypothyroid growth-retarded mouse DW/J-grt

Ji-Ming CHENG1), Ming DING1), Tomomi MIYAMOTO1), Osmu FUJIMORI2), and Takashi AGUI1)
1)Center for Experimental Animal Science, and 2)Second Department of Anatomy, Nagoya City University Medical School, Nagoya, Aichi 468-8601, Japan
Running title: Investigation of thyroglobulin in DW/J-grt mouse
Key words: thyroglobulin, dwarfism, growth-retarded mouse, hypothyroidism, thyroid
Address corresponding: Takashi Agui, Ph.D., Center for Experimental Animal Science, Nagoya City University Medical School, Nagoya, Aichi 468-8601, Japan. E-mail: t.agui@med.nagoya-cu.ac.jp

SUMMARY
The DW/J-grt mouse is a mutant model exhibiting hypothyroidism and dwarfism. The phenotype of the DW/J-grt mouse is similar to that of the DW/J-dw mouse. However, the grt locus was previously mapped to mouse chromosome (Chr) 5 and shown to be different from the dw locus (Chr 16). Here, we investigated the morphology of the thyroid gland and functions of the thyroid gland in DW/J-grt mouse, particularly, the synthesis, transportation, secretion, and iodine incorporation of thyroglobulin (Tg). Morphological analysis showed hypogenesis of follicles in the DW/J-grt thyroid. The investigation of the Tg elucidated that DW/J-grt mouse showed no significant difference from normal mouse with respect to the expression, synthesis, transportation and secretion. The uptake of iodine in the thyroid gland of DW/J-grt mouse is markedly lower when comparing with normal mouse, but the incorporation ratio of iodine into Tg was not different between DW/J-grt and normal mice. These results suggest that responsible gene for the hypothyroidism of DW/J-grt mouse is not attributed to the Tg gene nor the genes that support the post-transcriptional events of Tg.
Key words: thyroglobulin, dwarfism, growth-retarded mouse, hypothyroidism, thyroid

Introduction
The DW/J-grt mouse was derived from the control mouse colony for Snells dwarf (DW/J-dw) mouse, as a mutant exhibiting retarded growth with high serum level of thyrotropin (TSH) and low serum level of thyroid hormone, both tri-iodothyronine (T3) and thyroxine (T4) [1]. Both the DW/J-grt and DW/J-dw mice show dwarfism. However, it was reported that the DW/J-grt mouse is different from the DW/J-dw mouse in several aspects. The most important thing is that the primary defect of the former is the thyroid [2], whereas the latter is the pituitary [3]. We previously mapped the grt locus to mouse chromosome (Chr) 5 at 59 cM using backcross progenies [4], indicating that the grt locus is different from the dw locus (Chr 16) [3].
We have recently identified a mutation in the thyroglobulin (Tg) gene as responsible for hypothyroidism of the WIC-rdw rat [5], exhibiting similar phenotype to the DW/J-grt mouse, namely, dwarfism, low serum T3 and T4, and high serum TSH. In human, mutation of the Tg gene is major cause of congenital hypothyroidism [6, 7, 8], and mutation of the Tg gene is also reported as responsible gene for hypothyroidism in cog/cog mouse [9, 10]. Tg is the major thyroid secretory glycoprotein and is exported through the secretory pathway to the lumen of the thyroid follicles, which is a prerequisite for the thyroid hormone synthesis [11]. However, a mutation of the Tg gene causes storage of the Tg in endoplasmic reticulum, leading to hypotyroidism in above patients and animal models.
Since the Tg locus was mapped to mouse chr 15 [12], different from the grt locus (Chr 5) [4], it is unlikely that the mutation of the Tg gene is responsible for the grt. However, the possibility that anomaly of transportation or secretion of the Tg results in the hypothyroidism in the DW/J-grt mouse cannot be excluded. In this study we, therefore, investigated transcription and post-transcriptional events of the Tg, including synthesis, transportation, secretion and iodine incorporation of Tg in the DW/J-grt mouse thyroid.

Materials and Methods
Animals
DW/J-grt mice were maintained in our facility under specific pathogen-free conditions. Animal breeding rooms were maintained at 23 2 and 50 10% relative humidity with a 12-hour light-dark cycle (lights on from 8:00 a.m. to 8:00 p.m.). Research was conducted according to the Guidelines for the Care and Use of Laboratory Animals of Nagoya City University Medical School. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Nagoya City University Medical School.

Preparation of tissue sections
Thyroid glnds were removed from animals under with sodium pentobarbitone anesthesia and fixed in Bouins fluid overnight at room temperature. Tissues were then dehydrated in graded ethanol, immersed in xylene and embedded in paraffin wax. Sections were cut, mounted on glass slides, and stained with hematoxylin and eosin (HE), according to the routine techniques.

Reverse transcription-polymerase chain reaction (RT-PCR) of the Tg mRNA
Total RNA was extracted from the thyroids of C57BL/6 and DW/J-grt mice with age of 8-week old. Expression of the Tg gene was estimated by RT-PCR with a primer pair designed according to the cDNA sequence of murine Tg gene [9]; the sense primer was 5'-GAACAGCATGGGGATTCAAA-3'(nucleotide 6072 to 6091) and the antisense primer was 5'-CCCCAGTCTGTAGTTAGCAG-3' (nucleotide 6992 to 7011). The PCR conditions were: 94 for 1 min, 58 for 1 min, and 72 for 2 min with various cycles.

Measurement of the de novo synthesis , transportation and secretion of the Tg
Thyroid glands from C57BL/6, DW/J-grt/+, and DW/J-grt/grt mice were cleaned of connective tissue, chopped into 1-mm cubes, and washed three times with ice-cold Dulbecco's phosphate-buffered saline (PBS). Thyroid glands prepared from 3 mice were pooled and incubated at 37 for 60 min in 1 ml of Dulbecco's modified Eagle's medium (DMEM) lacking methionine and cysteine, and then labeled with [35S] amino acid (ExpressTM Protein Labelling Mix, [35S]-EasyTagTM, NEN Life Science Products, Inc., Boston, MA, U.S.A.) for 45 min at 37. After washing three times with ice-cold Dulbeccos PBS, labeled thyroid samples were chased for various periods in normal DMEM. At the end of the chase periods, samples were treated with 50 mM iodoacetamide in ice-cold PBS for 10 min to alkylate sulfhydryls. The samples were lysed with 100l/gland of sodium dodecyl sulfate (SDS)-lysis buffer containing 125 mM tris(hydroxymethyl)-aminoethane (Tris)-HCl (pH 6.8), 2% SDS, and a cocktail of protease inhibitors (4g/ml chymostatin, 200g/ml bacitracin, 20 g/ml leupeptin, and 10g/ml phenylmethylsulfonyl fluoride (PMSF)) and then centrifuged at 15,000 xg for 45 min at 4. After 4l of supernatants were digested with 0.3 mU/ml endoglycosidase H (Boehringer Mannheim, Mannheim, Germany) in a buffer containing 250 mM sodium citrate (pH 5.3), 2.5% SDS, 50 mM ethylenediamine tetraacetic acid (EDTA), and 5% 2-mercaptoethanol (2-ME) for 15 min at room temperature, samples were applied to SDS- polyacrylamide gel electrophoresis (PAGE). Gels were dried and exposed to X-ray films overnight at -80.

Measurement of iodine accumulation in thyroid glands
Ten-week-old mice were intraperitoneally injected with 0.82Ci of 125INaI (NEN Life Science Products, Inc., Boston, MA, U.S.A.). Four hr after injection, thyroids were removed, and radioactivity was counted by a -counter (ARC-360, Aloka Co. LTD, Tokyo, Japan).

Estimation of iodine incorporation into Tg
Thyroid glands with accumulation of 125Ifollowing in vivo injection of 125INaI were homogenized with 100 l/gland of lysis buffer containing 0.1 M NaCl, 25 mM Tris-HCl (pH 6.8), 5 mM EDTA, 0.1% Triton X-100, and a cocktail of proteinase inhibitors (4g/ml chymostatin, 200g/ml bacitracin, 20 g/ml leupeptin and 10g/ml PMSF). Homogenates were centrifuged at 15,000 xg for 45 min. Aliquots (4l) of the supernatants were applied to SDS-PAGE, stained with Coomassie brilliant blue R250, bands for thyroglobulin were cut off from the gel, and the radioactivity was counted.

Statistical analysis
Values are presented as means S.E.M. They were analysed for statistical significance by Student's t-test and P<0.05 was considered to be significant.

Results
Morphological investigation
Since it was suggested that the primary defect of the DW/J-grt mouse was dysfunction of the thyroid [1], morphological analysis of the thyroid was performed. As shown in Fig. 1, the DW/J-grt thyroid (Fig. 1C) was significantly smaller than the C57BL/6 (Fig. 1A) and DW/J-+/grt (Fig. 1B) thyroids, and two third of the glands was replaced by adipose tissue. Moreover, histological structures in the DW/J-grt thyroid showed hypogenesis of the follicles with less colloid material in the follicular lumina, and the size of colloid lumina and follicular epithelial cells was smaller than that of the C57BL/6 thyroid. In DW/J-+/grt thyroids, tissue structures were similar to those of the C57BL/6 thyroids, although some parts of the glands were replaced by adipose tissue. Histological result of DW/J-+/grt thyroids indicated that although hypogenesis occurred at an intermediate level between wild-type and grt/grt thyroids possibly due to the gene dosage effect, a number of follicles showed normal structure, suggesting that they developed normally. This is consistent with the fact that the grt inheritance is autosomal recessive.

The mRNA expression of the Tg gene
The mRNA expression of the Tg was examined by RT-PCR in the thyroid glands of the C57BL/6 and DW/J-grt mice. RT-PCR was performed with different cycles. As shown in Fig. 2, intensities of the bands for the PCR products were the same between the C57BL/6 and DW/J-grt mice, indicating that the transcription of Tg in DW/J-grt mice was normal.

Assessment of synthesis, transportation and secretion of the Tg
Synthesis, transportation and secretion of 35S-labeled Tg after chase in normal medium was examined.35S-labeled Tg was digested with endoglycosidase H after homogenization of thyroids with SDS-lysis buffer, and then applied to SDS-PAGE. In this assay, Tg transported to the Golgi apparatus is resistant to the endoglycosidase treatment, whereas Tg resident in the endoplasmic reticulum is sensitive. As shown in Fig. 3, significant level of the endoglycosidase-resistant Tg was evident in DW/J-grt thyroid samples pulse-chased for 1 hr. Further, endoglycosidase-sensitive Tg was significantly reduced in DW/J-grt thyroid samples pulse-chased for 3 hr. These data suggest that de novo synthesis of Tg and transportation of Tg from the endoplasmic reticulum to the Golgi apparatus did not differ among the wild-type, heterozygous, and grt/grt thyroids. Transportation to the Golgi apparatus in the DW/J-grt thyroid seemed rather faster than that in other two geno-type thyroids.
Secretion of the labeled Tg after chase was also examined (Fig. 4). The amount of Tg secreted into the medium from DW/J-grt thyroids was not different from that of wild-type and heterozygous thyroids.

Accumulation of iodine in thyroid glands
An important function of the thyroid is modulating the homeostasis of iodine in the body, and the iodination of the thyroglobulin leading to thyroid hormone synthesis at the end-stage of the secretary pathway [10]. To assess this function of the thyroid, we examined accumulation of iodine in the thyroids of C57BL/6, DW/J-+/grt and DW/J-grt mice. As shown in Fig. 5, accumulation of iodine in the DW/J-+/grt thyroid after injection of 125I NaI intraperitoneally was approximately half of the wild-type thyroids and accumulation of iodine in the DW/J-grt thyroid was much less than in the wild-type and heterozygous thyroids. This seems reflect the gene dosage effect of grt and wild-type alleles. Morphological investigation revealed that follicles are smaller in the DW/J-grt thyroid than in the normal mouse, and are not well ordered (Fig. 1). Forthermore, the part of glands was replaced by adipose tissue (Fig. 1). The reduction of accumulation of the iodine in the DW/J-grt mouse thyroid may be the consequence of the decrease in the number of follicles or deficiency in the function of follicles. Next, we examined incorporation ratio of iodine into the Tg. By estimation of the incorporation ratio of iodine accumulated in the thyroids to iodine incorporated into Tg, it was elucidated that approximately half of the accumulated iodine in the thyroids was incorporated into Tg in all three geno-type thyroids (Fig. 6). This result suggests that the accumulated iodine was incorporated normally into Tg even in the DW/J-grt thyroids, although the absolute amount of incorporated iodine was obviously very low.

Discussion
We recently identified that a cause for hypothyroidism in the WIC-rdw rat is a missense mutation of the Tg gene5. Since the hormonal abnormality of the DW/J-grt mouse is similar to that of the WIC-rdw rat i.e. reduced level of both serum T3 and T4 and abnormally elevated level of serum TSH. Mapping study of the grt locus indicated that a mutation of the Tg gene is unlikely to be responsible for the grt phenotype. However, the possibility of impaired Tg transportation or secretion as shown in the WIC-rdw rat was considered. Thus, we investigated in this study post-transcriptional events of the Tg in the DW/J-grt mouse thyroid. In this study it was elucidated that expression of the Tg mRNA, synthesis of Tg, incorporation of iodine into Tg, and both transportation and secretion of Tg were all normal in the DW/J-grt thyroids. Severely reduced accumulation of iodine into the thyroid gland in in vivo experiments (Fig. 5) may be attributed to hypogenesis of the thyroid. Thus, these results excluded the possibility that a defect of putative genes which support synthesis, transportation and secretion of the Tg might be responsible for hypothyroidism of the DW/J-grt mouse.
TSH released from the pituitary controls hormogenesis in the thyroid. Furthermore, it is known that TSH stimulation is necessary for the development of thyroid follicles. The result shown in a previous report2that in vivo administration of TSH to DW/J-grt mice did not restore either the level of serum T4 nor growth may implicate the deficiency in the function of TSH receptor (TSHR). Mutation of the Tshr gene was already known in the hyt mouse13. The hyt or Tshr gene locus was, however, mapped to mouse Chr 12, different from the grt locus (Chr 5). Therefore, it is unlikely that a mutation of the Tshr gene is responsible for the grt mutation. However, future investigation of the function of TSHR including signal transduction may shed light on the identification of a cause for hypothyroidism in the DW/J-grt mouse.

References
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9. Kim, P.S., Hossain, S.A., Park, Y-N., Lee, I., Yoo, S-E., Arvan, P. A single amino acid change in the acetylcholinesterase-like domain of thyroglobulin causes congenital goiter with hypothyroidism in the cog/cog mouse: A model of human endoplasmic reticulum storage diseases. Proc. Natl. Acad. Sci. USA 1998; 95: 9909-9913.
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Figure Legends
Fig. 1. Histological structure of thyroid glands in C57BL/6 (A), DW/J-+/grt (B), and DW/J-grt/grt (C) mice. HE staining. The bar shown in C indicates 80 mm and magnification is all the same in A, B, and C.
Fig. 2. RT-PCR of the Tg mRNA in the C57BL/6 and DW/J-grt mice. The 940 and 581 bp bands represent RT-PCR products for Tg and b-actin, respectively. M represent molecular marker of 174/-Hae digest. Lanes 1 and 2, 3 and 4, and 5 and 6 represent the RT-PCR results of Tg with 28, 30 and 35 cycles, respectively. Lanes 7 and 8 represent the RT-PCR result of b-actin with 25 cycles. Lanes 1, 3, 5 and 7 are results of DW/J-grt mice, and lanes 2, 4, 6 and 8 are results of C57BL/6 mice.
Fig. 3. Transportation of 35S-labeled Tg in pulse-chased thyroid glands. Thyroid glands removed from C57BL/6, DW/J-+/grt, and DW/J-grt/grt mice were labeled with 35S-methionine and cysteine for 45 min and pulse-chased for 1 or 3 hr. Thyroids were homogenized with SDS-lysis buffer, digested with endoglycosidase H, and then applied to SDS-PAGE. Labeled Tg was visualized by autoradiography.
Fig. 4. Secretion of 35S-labeled Tg from pulse-chased thyroid glands. Thyroid glands removed from C57BL/6, DW/J-+/grt, and DW/J-grt/grt mice were labeled with 35S-methionine and cysteine for 45 min, pulse-chased for 1 or 3 hr, and Tg secreted into the medium was analyzed by SDS-PAGE. Labeled Tg was visualized by autoradiography.
Fig. 5. Accumulation of 125I-iodine in the thyroid glands. C57BL/6, DW/J-+/grt, and DW/J-grt/grt mice were intraperitoneally injected with125INaI, then 2 hr after injection thyroid glands were removed, and radioactivity was counted. Data were normalized by wet weights of the thyroids.

Fig. 6. Incorporation ratio of 125Iiodine into Tg. Thyroid glands were removed from mice received an intraperitoneal injection of 125INaI, homogenized with SDS-lysis buffer, centrifuged, and an aliquot of the supernatants was applied to SDS-PAGE. After staining the gels with Coomasie Brilliant Blue R250, radioactivity of the Tg band was counted. The data are shown as a ratio of accumulated iodine in the thyroid to iodine incorporated into Tg.


Nagoya City University
Medical School