gene-induced disorders of structure, function and thyroglobulin synthesis in congenital goitre (cog/cog) in mice Mutant

L. R.

Adkison, S. Taylor and W. G. Beamer

The Jackson

Laboratory, Bar Harbor, Maine 04609, U.S.A. (Dr L. R. Adkison is now at Mercer University School of Medicine, Georgia 31210, U.S.A.) received

1550 S.

College Street, Macon,

11 October 1989

ABSTRACT

We have investigated thyroid structure and function in mice homozygous for the chromosome 15 mutation, congenital goitre (cog). Abnormal thyroidal hypertrophy and reduced iodine uptake in cog/cog mice were observed as early as day 18 of gestation, corresponding to the onset of thyroid function. Growth continued unabated in mutants throughout the 10-month period of observation. By 2 months of age, thyroid cell hypertrophy obliterated nearly all follicular lumina in cog/cog glands and by 10 months mean mutant thyroid mass exceeded that of age-matched littermates. Twenty-fold serum concentrations of thyrotrophin were significantly increased at all ages examined. While wild type (+/+) and heterozygote (+/cog) mice are indistinguishable from each other, thyroids of homozygote mutants (cog/cog) and the +/cog type are easily discernible from thyroids of the +/+ type by microscopic and thyroglobulin (Tg) analyses. Thyrofollicular cells of both cog/cog and +/cog genotypes contain large vesicles of accumulated, non\x=req-\ glycosylated proteinaceous material not observed in

cells from was

+/+ mice. Autoradiography showed 125I into Tg within recognizable

incorporated only

INTRODUCTION

Young adult mice homozygous for the autosomal recessive mutation, congenital goitre (cog/cog), are characterized by hypothyroidism and goitre (Beamer, Maltais, DeBaets & Eicher, 1987). At both the light and electron microscopy levels, the young adult mutant thyroid has a markedly disordered structure and hypertrophied cells (Beamer et al. 1987; Mayerhofer, Amador, Beamer & Bartke, 1988). The cog mutation is maintained in the AKR.L-Hstrain and maps sufficiently close to the mouse thyroglobulin locus (Tgn) on chromosome 15 to

2b/l

follicular lumina of thyroids from +/cog mice. Serum concentrations of tri-iodothyronine are depressed during development in cog/cog mice. Serum concentrations of thyroxine are depressed during postnatal development but increase progressively to normal concentrations by 10 months of age. Our analyses indicate that full size Tg is produced in thyroid cells from cog/cog mice, though in a greatly reduced quantity, and that Tgs which are several sizes smaller than normal are also produced in both homozygote and heterozygote thyroids. In addition, we observed altered electrophoretic mobilities of 19S Tg and either the absence or greatly reduced quantities of one reduced 12S Tg form. Tg mRNA is apparently normal size in the mutant. However, there is a five- to tenfold reduction in the amount of polyadenylated Tg mRNA available that correlates with the decreased amounts of full size Tg seen in the mutant. Collectively, these findings suggest that the compensatory goitre of cog/ cog mice results from disordered processing of Tg mRNA transcribed from the cog mutant gene.

Journal of Endocrinology

(1990) 126, 51\p=n-\58

suggest that cog may be allelic Rowe, 1987). The Tgn codes for

Tgn (Taylor & single protein of 330 kDa that forms dimers of native thyroglobulin (Tg; Malthiery & Lissitzky, 1987). Basche, Beamer & Schneider (1989) analysed Tg extracted from young adult cog/cog and normal littermate thyroids by specific antibody and sucrose density centrifugation methodology. Evidence for both normal (12S; 330 kDa) and abnormal (3-8S) Tg subunit protein was obtained. Taylor & Rowe (1987) searched for, but did not find, DNA restriction fragment length poly¬ morphisms; however, Tg mRNA was apparently of normal size.

to a

foregoing observations are consistent with the causing a defect in Tg synthesis. To gain further insight into the nature and cause of this mutant gene-induced thyroid disorder, we have extended these observations with morphological and The

cog mutation

biochemical data from fetal to mature adult ages that establish: (1) the compensatory nature of the cog/cog, (2) heterozygous defects in + /cog thyroids, and (3) a quantitative defect in polyadenylated (poly(A)) mRNA in cog/cog.

MATERIALS AND METHODS

Mice The cog mutation was transferred from AKR/J mice to the AKR.L-H-2b/l strain because of the delayed onset of thymic lymphoma in the latter (Johnson, Bedigian, Cherry & Meier, 1980). In studies with fetal thyroids, mice homozygous for cog or + alíeles at the cogenital goitre locus were obtained by pairing either (cog/cog cog/cog) or (+/+X+/+) parents and inspected daily for vaginal plugs (defined as day 0 of pregnancy). Mice utilized in postnatal thyroid studies were obtained from matings of genotypes (cog/ cog + ¡cog) or ( + /cogx +/cog). Thus unless otherwise specified, phenotypically normal littermate controls consisted of +/cog and + / + genotypes.

Histology and morphometric analysis Tracheas with thyroids were fixed in Bouin's solution at gestational and postnatal ages. Thyroids were serially sectioned and stained with eosin and periodic acidSchiff reagent (PAS). Individual thyroid gland volumes were quantified by morphometric analyses utilizing an image analyser (Optomax System IV, Hollis, NH, U.S.A.). Serum concentrations of tri-iodothyronine (T3), thyrotrophin (TSH) and thyroxine (TJ Large groups (10-12 pairs) of cog/cog and +/? mice

of both

aside for collection of agedata and sera for hormone measurements. At 2-month intervals until 10 months of age, 4-6 pairs of cog/cog and + /? mice of each sex were killed with C02 and cardiac blood was collected from the thoracic cavity. The harvested sera were stored at —24 °C until assayed. Serum concentrations of T3 and T4 were estimated with radioimmunoassay (RIA) kits (T3; Mallinckrodt, St Louis, MO, U.S. .: T4; ICN Biomedicals, Inc., Carson, CA, U.S.A.; Beamer et al. 1987). Serum concentrations of TSH were esti¬ mated with the rat TSH RIA (National Pituitary Program, NIAMDD and NICHD, Baltimore, MD, U.S.A.; Chin, Habener, Martorana et al. 1980) utilizing sexes were set

specific thyroid

mass

reference preparation kindly provided by Dr A. F. Parlow (UCLA-Harbor General Hospital,

a mouse TSH

Torrance, CA, U.S.A.).

uptake of ,25I Near-term dams carrying obligate cog/'cog or +/ + offspring were injected on day 17 of gestation with 2pCi I2rI (Na,25I in phosphate-buffered saline; PBS). Twenty-four hours after injection the dams were killed with C02, and the smallest possible piece of the trachea with thyroids in situ was removed from the fetuses with the aid of a Ziess operation/dissection microscope. Thyroid and trachea pieces were weighed and placed in individual glass tubes, and radioactivity was measured with a Nuclear Chicago Model 1185 gamma counter (63% efficiency). Thyroidal radio¬ activity was corrected for tissue background activity by subtracting the c.p.m./mg found in a small piece of Fetal thyroidal

each fetal liver.

Autoradiography of ,25I in young adult cog /cog and + /?

thyroid glands Two pairs of 8-week-old cog/cog and + /? mice were injected, via their tail veins, with 0-25 pCi l25I in PBS.

The mice were killed with C02 10 min later and the thyroids dissected out and placed in ice-cold 3% (v/v) glutaraldehyde : 1 % (w/v) paraformaldehyde for autoradiographic analyses at the ultrastructural level. After routine processing of tissue fragments for electron microscopy, thin sections were mounted on colloidoncoated glass slides, and coated with Ilford L4 emulsion (Polysciences Inc., Warrington, PA, U.S.A.). After a 7day exposure, slides were developed in Microdol X (Kodak, Rochester, NY, U.S.A.), and the films floated onto a water bath. Copper grids (300 mesh) were inverted over sections and the film picked up onto clean parafilm. After removing the colloidon by dipping into amyl acetate, the grids were stained with uranyl acetate and lead citrate, and screened for evi¬ dence of silver grains in various parts of the thyroid follicular structures in normal and cog/cog glands.

Thyroglobulin extraction Thyroids were collected from 8-week-old mice, frozen in liquid nitrogen, and stored at 70 °C. Thyroid extracts were prepared by homogenizing frozen thy¬ roids in PBS containing phenyl-methyl-sulphonylfluoride (PMSF; 4 mmol/1), followed by centrifugation —

at 850 g for

10 min at 4 °C to remove cellular debris and

particles. The supernate was precipitated with an equal volume of saturated ammonium sulphate solution. Following centrifugation, the precipitate was redissolved in PBS + PMSF and dialysed against several changes of PBS. These preparations were stored in

PBS + PMSF+ 50%

&ThoreIl, 1984).

(v/v) glycerol (Ericsson, Larsson

Electrophoresis and protein analysis Thyroglobulin protein extracts from + / +, + /cog and cog/cog mice were separated by polyacrylamide gel electrophoresis. Denaturing gels (5% non-gradient polyacrylamide or 3-17% gradient polyacrylamide, w/v) contained 0-375 mol Tris-HCl/1 (pH 8-8) and 0-1 %

(w/v) sodium dodecyl sulphate (SDS). Non-denaturing gels contained 009 mol Tris/1,008 mol boric acid/1 (pH 8-3) and 00026mol EDTA/1. Running buffer was 0-025 mol Tris-HCl/1 (pH 8-3), 0-192 mol glycine/1 and 01% SDS. Purified bovine and porcine Tg (Sigma, St Louis, MO, U.S.A.) were used along with standard molecular size markers (Sigma).

Anti-thyroglobulin A polyclonal anti-Tg was produced in rabbits for use in protein analyses. A pool of thyroids from +/ + mice was homogenized in PBS. The Tg-enriched frac¬ tion was precipitated by ammonium sulphate, and the Tg (identified by purified bovine Tg standard) of 12 Svedberg units (S) was separated from this fraction by SDS-polyacrylamide gel electrophoresis. The 12S Tg was electro-eluted from gel fragments, mixed with Freund's adjuvant, and injected (1 mg/immunization) into multiple sites s.c. Preimmune and immune sera were titrated against bovine Tg by Western blot analyses. Immune serum (diluted more than 1 : 2500) recognized Tg from thyroid extracts but gave no reac¬ tion to extracts of thymus, heart or liver. Preimmune serum was

without detectable reaction to any tissue

extracts.

3mol lithium chloride/1 and 001 mol vanadyl ribonucleoside complex/1, homogenized, and then soni¬ cated for 1 min. RNA was then extracted as described by Auffray & Rougeon (1980). Poly(A) RNA was isolated from total cellular RNA using Hybond

affinity paper (Amersham, Arlington Heights, IL, U.S.A.) as directed by the manufacturer. The quantities of poly(A) mRNA were estimated in replicate analyses by binding poly(A) to affinity sub¬ strate paper, releasing the bound poly(A) mRNA from the paper at 70 °C, and precipitating in messenger

ammonium acetate and ethanol. Concentration was determined by spectrophotometric analysis at 260 nm. Statistics

analysed by /-test and by analysis of vari¬ Differences between means were compared by ance. Duncan's new multiple range test. Data

were

RESULTS

The existence of deficient thyroid function in cog/cog mice was readily demonstrable in fetal tissue. As summarized in Table 1, morphometric analyses of thyroids from 18-day-old fetuses revealed a significant (P