Sheep Thyroid Research Articles

The uptake of [ 35S]methimazole by sheep thyroid slices in vitro

The uptake of [ 35S]methimazole by sheep thyroid slices has been shown to be activated in the presence of iodide. The total uptake ( Q) of [ 35S]methimazole was shown to be the sum of a saturable process and a non-saturable process. The constants Q max, K s and P in the two-term equation were determined using a published statistical method and a Fortran IV computer programme. Diiodotyrosine (DIT) at a 0.1 mM concentration stimulated the saturable uptake of [ 35S]methimazole appreciably in the absence of iodide, whilst thyroid-stimulating hormone (TSH) inhibited uptake in the presence of iodide and was of no effect in the absence of iodide. Propylthiouracil (PTU) inhibited the saturable uptake of [ 35S]methimazole whilst perchlorate had no effect.

Calcitonin Distribution within Pig, Sheep and Cow Thyroid

The distribution of calcitonin within the pig, sheep and cow thyroid was examined by measuring the concentration of the hormone in serial sagittal and transverse 5-mm sections of whole thyroids. It was observed that calcitonin in the pig thyroid was concentrated in the anterior to right lateral parts of the thyroid. Calcitonin was localized in the central region of the sheep thyroid with a relatively lower concentration in the peripheral parts of the gland. In the cow thyroid a uniform distribution of calcitonin was observed. The results of this study indicate a species specific and characteristic distribution of calcitonin within the thyroid of the pig, sheep and cow.

Molecular weight of the thyroglobulin messenger RNA of sheep thyroid gland

Poly(A)+ mRNA from sheep thyroid total or thyroglobulin-specific polysomes obtained by immunological precipitation, was purified by two cycles of chromatography on oligo(dT)-cellulose. Upon electrophoresis in 98 % formamide-polyacrylamide gels, the purified RNA showed a major species of M r 2.8×10 6. The correlation found between the very high concentration of this species and its thyroglobulin messenger activity in the reticulocyte lysate protein synthesis system demonstrates that the thyroglobulin mRNA contains enough bases to code for the thyroglobulin peptide chain (M r 300 000).

Thyroglobulin messenger ribonucleic acid translation in vitro.

Total polysomes were obtained by homogenization of sheep thyroid glands in Tris‐Cl‐buffered sucrose, pH 7.5, treatment with 2% Triton X‐100 and centrifugation of the magnesium‐treated 27000 ×g supernatant through 1 M sucrose. Purification of thyroglobulin‐specific polysomes was performed by indirect immunoprecipitation with rabbit antibodies to sheep thyroglobulin and donkey antibodies to rabbit immunoglobulin G. Thyroglobulin polysomes represented 30% of total polysomes. Synthesis of thyroglobulin peptides in a reticulocyte cell‐free system, programmed with total RNA from immunoprecipitated polysomes, was 3 times higher than when total RNA from unfractionated polysomes was used. The size of the poly(A)‐containing thyroglobulin mRNA was estimated by the capacity of RNA fractions obtained by sucrose density gradient centrifugation of total or immunopurified polysomal RNA to hybridize with 3H‐labeled poly(U) and to stimulate thyroglobulin peptide synthesis in a reticulocyte lysate. 65% of the thyroglobulin messenger activity was located in a 33–36‐S RNA peak and 35% in zones corresponding to 30‐S and 26‐S RNA species.The thyroglobulin‐specific immunoprecipitable peptides synthesized in the reticulocyte lysate from the 33–36‐S RNA were shown by polyacrylamide gel electrophoresis in denaturing conditions to contain a major component co‐migrating with the constituent peptide chain of sheep thyroglobulin (Mr 330000). Other components migrated with Mr of about 210000 and 120000.

Incorporation of [ 14]arachidonate in pig thyroid lipids and prostaglandins

In pig and sheep thyroid, the arachidonate content of phosphatidylinositol (14.3–17.5%) is much higher than in the other phospholipids. In the thyroid, phosphatidylinositol seems to play an important role in the biosynthesis of prostaglandins. In neutral thyroid lipids, the arachidonate content is much higher in diacylglycerols (15.8%) than in monoacylglycerols (2.9%) or triacylglycerols (4.2%). However, the most important pool of esterified arachidonate is triacylglycerols (1030 nmol of arachidonate/g tissue). Arachidonate represents a very small part of total free fatty acids measured in the presence of albumin and indomethacin (0.65% or 16.4 nmol/g tissue). Thyrotropin (50 munits/ml) causes after 1 h a 2-fold increase in the level of free arachidonate (37.3 nmol/g tissue). Pig thyroid slices rapidly take up [ 14C] arachidonate and incorporate it into neutral lipids and phospholipids. Specific activity of phosphatidylinositol is 3-fold higher than that of phosphatidylcholine after an hour incubation. Specific activity of diacylglycerols is 8-fold higher than that of triacylglycerols. Thyrotropin (50 munits/ml) causes a significant decrease (32–33%) of incorporation of radioactivity in lipids as compared with standard incubation. This result is compatible with the isotopic dilution of the labeled [ 14C]arachidonate by nonradioactive arachidonate liberated in the incubation medium under thyrotropin action. Slices and homogenates of pig thyroid weakly convert [ 14C] arachidonate to prostaglandins E 2 and F 2α (1–2%). Thyrotropin (50 munits/ml) always diminishes the conversion of radioactivity to prostaglandins as compared with standard incubation. This result is compatible with the above-mentioned hypothesis.

Detection of a soluble serotonin-binding protein in the mammalian myenteric plexus and other peripheral sites of serotonin storage.

Abstract—Groups of neurons intrinsic to the mammalian myenteric plexus have been shown to have both tryptophan hydroxylase and a specific uptake mechanism for serotonin. They are probably serotonergic. A soluble protein with a high binding affinity for serotonin, similar to a protein previously found in rat brain by TAMIR & HUANG (1974), has now been found in the myenteric plexus of both rabbit and guinea pig. Partial purification of the protein from the rabbit's myenteric plexus by ammonium sulfate fractionation increased the ratio of specific to nonspecific serotonin binding almost 3‐fold. Two dissociation constants for serotonin binding were obtained by equilibrium dialysis: 6.7 × 10−10 M and 4.8 × 10−7 M. The protein was similar to the soluble serotonin‐binding protein of CNS: the indole derivatives 5, 6‐ and 5, 7‐dihydroxytryptamine, and 6‐hydroxytryptamine inhibited serotonin binding by 50% at 10−7 M; norepinephrine was a poor inhibitor of serotonin binding; most of the serotonin‐protein complex had a very high molecular weight and did not penetrate a 6.5% acrylamide gel. The appearance of the serotonin binding protein during development of the intestine in fetal rabbit correlates closely with the development of a serotonin uptake mechanism by nerves of this tissue and precedes the ingrowth of the adrenergic innervation. In‐vitro administration of 6‐hydroxydopamine to adult animals has no effect on the binding capacity for serotonin. Binding activity in denervated preparations is only 1/5 that of innervated tissue. It is concluded that the serotonin‐binding protein, which has been found associated with serotonergic pathways in the CNS, is found associated with serotonergic neurons in the periphery as well. Since a similar serotonin‐binding protein is also found in sheep thyroid, which stores but does not take up serotonin, the protein may be a component of the serotonin storage mechanism.

UPTAKE OF MACROMOLECULES BY THE THYROID

ABSTRACT The uptake by thyroid tissue of radioactively labelled macromolecules was studied in vitro to elucidate the mechanism of endocytosis of thyroglobulin by the follicular cell. Minced sheep thyroid was incubated with [125I]polyvinylpyrrolidone (PVP*), [3H]inulin, [125I]thyroglobulin or [125I]fibrinogen. Tissue radioactivity comprised a "weakly bound" component, removed by prolonged washing and a residual "firmly bound" component. TSH, 5 mU/ml, increased the magnitude of both these components. The TSH effect on PVP* uptake demonstrated hormone and tissue specificity and was reproduced by dbcAMP. The quantity of PVP* firmly bound rose with time of incubation with a rapid initial uptake followed by a slower "linear" increase. Vinblastine markedly reduced the "linear" rate of uptake of firmly bound radioactivity. The release of non-protein bound 125I into the medium during incubation with [125I]fibrinogen was stimulated by TSH. The rapid initial uptake of firmly bound radioactivity probably represents firm adsorption, the subsequent linear rate endocytosis.

Congenital goitre in sheep: isolation of the iodoproteins which replace thyroglobulin.

Immunological and chromatographic studies of proteins from the congenital goitre of South Australian Merino sheep revealed that normal thyroglobulin is absent from the thyroid glands of these sheep. However, a thyroglobulin-immunoreactive iodoprotein was isolated by affinity chromatography on agarose gel to which antibody against thyroglobulin had been covalently bound. Sucrose-gradient ultracentrifugation indicated that this iodoprotein had a sedimentation coefficient of 8S and a molecular weight of approximately 175000. This iodoprotein is therefore about one quarter the size of normal thyroglobulin (19S; 660000) and is similar in size to the subunit of thyroglobulin (3-8S; 165000) although this has usually been described as non-iodinated except when derived by reductive fission. In addition the goitre extract contained iodoproteins which had the immunological properties of serum albumin and immunoglobulin G. Determination of the iodine and iodoamino acid content of the hydrolysed iodoproteins revealed that they contained iodothyronines which were able to contribute to the production of thyroid hormones although the total iodothyronine content of the goitrous gland was less than that of the normal sheep thyroid gland.

The effect of prolonged Kale feeding on the thyroid glands of sheep

Four groups of sheep were fed the following rations: Group I Kale ad lib., Group II Kale ad lib.+0·22 kg. blood meal/flaked maize mixture, Group III Kale ad lib.+0·22 kg. blood meal/flaked maize mixture and 10 mg. Kl weekly by mouth, Group IV 0·9 kg. hay+0·22 kg. blood meal/flaked maize mixture. They were fed these rations for 17 weeks when the sheep were killed. The thyroids were removed, weighed and pieces were taken for histological examination. The kale had a goitrogenic effect which was overcome by the iodine supplementation. The group receiving kale showed only a three-fold increase in thyroid weight whereas the group receiving the blood meal/flaked maize mixture in addition to the kale showed a six-fold increase in weight. The mixture had no goitrogenic effect when fed without kale to the control group. The mixture appears to increase the goitrogenicity of the kale. The histology shows considerable hypertrophy of follicular epithelial cells and a diminution in colloid and this accounts for the weight incrase of the glands.

Inhibition by Iodide of the Activation of the Thyroid Cyclic 3′,5′-AMP System

The action of iodide on the cyclic AMP system of dog thyroid slices has been studied. Iodide inhibits the enhancement of cyclic AMP accumulation in the presence of TSH. Such an effect is also observed in horse, beef and sheep thyroid slices, but not in dog kidney slices stimulated by parathyroid hormone or in rat parotid slices stimulated by isoproterenol. The effect in dog thyroid slices is suppressed by 1mM NaClO4, 1mM methimazole and 1mM propylthiouracil. Similar data have been obtained for prostaglandin E1 stimulation. Effects of thyrotropin mediated by cyclic AMP, i.e., activation of iodothyronine secretion, 1-14C-glucose oxidation, and lactate formation, were also inhibited by iodide but not by iodide and methimazole. Similar activations when caused by dbcAMP were not inhibited by iodide. The data suggest a model in which an intracellular agent resulting from the oxidation of iodide acts on the thyroid cyclic AMP system.

Radioiodine in animal thyroid glands from 1966-1972.

The iodine-131 content of thyroid glands from Australian sheep and New Zealand sheep and cattle has been measured at weekly to monthly intervals from 1966 to 1972. In Australian sheep thyroid 131I levels rose sharply from 2 to 4 weeks after the start of each series of Pacific bomb tests. Maximum levels were reached 6 to 18 weeks after tests commenced. The highest average levels reached ranged from 1,000 picocuries per gramme (pCi/gm) in 1966 to 65 pCi/gm in 1972. Values for New Zealand animals were similar to those found in Australian sheep. Radioactivity detected on several occasions between Pacific tests was probably due to Chinese atmospheric tests.

Iodinated thyrolipids: their possible role in homonogenesis.

ABSTRACT A total of 42 human thyroid glands (nodular goitre 9, adenoma with cystic degeneration 6; toxic goitre 10; carcinoma 14; and normal thyroid gland 3) were examined in vitro for iodination of an unidentified polar non-phosphatide lipid fraction (fraction II). The radioiodine incorporation in fraction II was 49.3 %, 43.6 %, 32.8 %, 20.7 %, and 22.0 % respectively in normal, nodular goitre, adenoma with cystic degeneration, toxic goitre and thyroid carcinoma. In vitro studies with surviving sheep thyroid slices did not show any relation between the iodination of fraction II and thyroxine formation over a period of 120 min. However, a highly significant correlation (r-value= 0.96375) was observed between the iodination of fraction II and thyroxine formation in vivo in the rat thyroid gland over a period of 24 h. We have previously postulated that iodination of fraction II may be interrelated to thyroxine formation. In the light of this hypothesis and the above results we suggest that the iodination of fraction II and thyroxine formation in the thyroid gland may be interrelated, the degree of iodination of fraction II being modulated by the amount of thyroxine formed within the thyroid gland.

Immunochemical characterization of thyroglobulin-related components synthesized in cell-free systems

Various sheep thyroid subcellular fractions enriched either in rough microsomes or in Golgi structures (agranular microsomes) and isolated total polyribosomes have been incubated in the presence of various labeled precursors: 14C-labeled amino acids and nucleotides of 14C-labeled monosaccharides. Digitonin extracts of the microsomal fractions contained an acid-insoluble 14C-labeled material, a part of which can always be specifically bound by anti-19-S sheep thyroglobulin antibodies: 20 to 50% in the rough microsomal extracts labeled with 14C-labeled amino acids, [ 14C]mannose or N-[ 14 C] acetylglucosamine ; 45 to 70% in the agranular microsome extracts labeled with [ 14C]galactose, N-[ 14 C] acetylglucosamine or [ 14C]sialic acid. Polyribosomes incubated with 14C-labeled amino acids secreted into the medium an acid-insoluble labeled material containing up to 30% of thyroglobulin immunoreactive material. Sucrose gradient centrifugation showed that 14C-labeled proteins extracted from rough microsomes were essentially located in a 6–8-S peak accompanied, in the case of labeling with 14C-labeled amino acids, by a 2–4-S peak. Proteins synthesized by polyribosomes were in a 3–5-S peak. In agranular microsomal extracts a 17- to 19-S species was predominant. The proportion of 14C-labeled thyroglobulin immunologically detectable increased with increasing sedimentation coefficient (5–10% in the 2–4-S zone, up to 90% in the 17–19-S zone), whatever labeled precursor was used. In the case of rough microsomal extracts, an evolution has been observed in vitro: storage of the dialyzed extracts at −20°C or incubation in conditions favoring disulfide interchange generally increased the percent of immunologically-precipitable material, whereas 10- to 12-S and 17- to 19-S species often appeared. A parallelism between the acquisition of immunoreactive determinants, the evolution of the three-dimensional structure and the incorporation of carbohydrates, during the first steps of synthesis and secretion of the thyroglobulin is suggested and discussed.

Cell-free peptide synthesis and carbohydrate incorporation by various thyroid particles

Various sheep thyroid particles were prepared and used in cell-free systems to study either peptide synthesis or carbohydrate incorporations of endogenous acceptors including thyroglobulin precursors. Rough microsomes ( i.e. microsomes enriched in granular vesicles), total polyribosomes and free polyribosomes were purified from a post-mitochondrial supernatant in Tris-HCl, MgCl 2 and sucrose and were incubated, in conditions allowing peptide synthesis, with either 14C-labeled amino acids, UDP-N-[ 14 C]acetylglucosamine or GDP-[ 14C]mannose. Amino acid-incorporating efficiencies of rough microsomes, prepared according to several slightly different procedures, and of polysomes were compared on an RNA basis and found roughly similar. Only microsomes, not polyribosomes, were able to incorporate N- acetylglucosamine and mannose from their nucleotides. Several facts are consistent with a membranous extra-ribosomal sugar incorporation to completed and released peptide chains: the lack of significant inhibition by cycloheximide or puromycin, the almost total extractability by sodium deoxycholate of the rough microsomal carbohydrate labels, contrary to only about 20% of the peptide label, and finally the efficiency of rough microsomes to incorporate N- acetylglucosamine and mannose in incomplete systems ( i.e. without peptide synthesis). Golgi-rich agranular microsomes were purified from a post-mitochondrial supernatant in sodium phosphates, Dextran and sucrose and were incubated in conditions avoiding membrane rupture, with either UDP-N-[ 14 C]acetylglucosamine , UDP-[ 14C]galactose or CMP-N- acetyl-[ 14C]neuraminic acid, to study incorporation of relatively peripheral sugars in the more complex heterosaccharide units of thyroglobulin. Sucrose gradient ultracentrifugation of recently prepared digitonin extracts from variously labeled rough and agranular microsomes, and of an amino acid-labeled polyribosomal supernatant, exhibited quite distinctive patterns, suggestive of multistep events.

Role of inositol cyclic phosphate in stimulated tissues.

THYROID-STIMULATING hormone markedly stimulates phosphatidylinositol turnover in sheep thyroid gland. The enzyme catalysing the degradation of phosphatidylinositol in animal tissues can split the phospholipid, with the formation of a diglyceride and a new cyclic ester, D-myoinositol-1: 2-cyclic phosphate as well as D-myoinositol-1-phosphate2,3. Increased phosphatidylinositol turnover has been described in a variety of conditions in which cells are stimulated to secrete or into other hyperactivity. The physiological possibilities of this degradation are obvious, and it was suggested2,3 that the catabolism of phosphatidylinositol and the release of inositol cyclic phosphate might be more directly relevant to the cell stimulation than to the biosynthesis involved in the turnover. Such an hypothesis is attractive, for an accumulation of phosphatidylinositol has never been demonstrated during stimulated conditions. In addition Ca2+ is an essential requirement for the formation of inositol cyclic phosphate as well as playing an important part in secretory processes4.

Intracellular distribution of amines and calcitonin in the sheep thyroid gland

The distribution of amines (dopamine, histamine, 5-hydroxytryptamine, and norepinephrine) and calcitonin in the sheep thyroid gland was studied by quantitative analyses of these substances in subcellular fractions examined with the electron microscope. The fractions were prepared by centrifugation in continuous sucrose gradients. Histamine was mainly found in the supernatant fraction and dopamine in the gradient pellet together with mast cell granules. This indicates a release of histamine, but not of dopamine, from the mast cell granules, which contain both these amines. Norepinephrine was mainly recovered in the supernatant fraction and adjacent fraction. 5-Hydroxytryptamine and calcitonin were concentrated in one fraction. This fraction contained a high concentration of dense, argentaffin granules, indistinguishable from those found in sections of parafollicular cells. These observations show that 5-hydroxytryptamine and the polypeptide calcitonin are both stored almost exclusively in the cytoplasmic granules of the parafollicular cells.

Antigenic difference between informofers and protein bound to polyribosomal mRNA from rat liver.

NUCLEAR RNA with DNA-like base composition (heterogeneous nuclear RNA, Hn-RNA) is complexed with globular protein particles called informofers1–4. When mRNA is liberated from polysomes by EDTA or incubation with puromycin, it is isolated as a ribonucleoprotein complex (mRNP)5–8. Polyacrylamide gel electrophoresis has been used to study whether the informofer protein and the protein of polysomal mRNP are completely or partly identical9–11. The protein bound to haemoglobin mRNA is different from the informofer protein, but in rat liver and sheep thyroid polysomes a protein was found with characteristics similar to the informofer protein6,12,13. We have used a new immunological procedure to show that the proteins of the two ribonucleoprotein complexes in the rat liver are immunologically different.

Protein Synthesis in Isolated Thyroid Mitochondria

Amino acid incorporation into sheep thyroid mitochondria was reassessed since the only previous results on the subject showed some contradictions with most data obtained with rat liver mitochondria. The incorporation of leucine or of a mixture of amino acids into thyroid mitochondria was linear up to 90 min. Leucine concentration in the incubation medium was an important rate limiting factor of thyroid mitochondrial protein synthesis. Chloramphenicol, puromycin and ethidium bromide were strong inhibitors, while RNAase, actinomycin D, rifampicin and cycloheximide did not exert any inhibitory effect on amino acid incorporation. Addition of various inhibitors and uncouplers of mitochondrial metabolism further showed that optimal mitochondrial protein synthesis required intact electron transport, and oxidative phophorylation, and exogenous substrate and phosphate, while the endogenous pool of adenine nucleotides seemed to be selfsufficient.(Endocrinology91: 817, 1972)

Studies of compensatory growth in sheep

SUMMARYA number of observations have been made on 5- to 6-month old wether Hampshire heep undergoing uninterrupted growth (36·2% gain in shrunk wool-free live weight over 58 days), undernutrition (a 21·7% fall in body weight over 27 days) and then followed by compensatory growth (a gain of 62·2% in live weight over 52 days).Sheep being underfed had increased, and compensating sheep had decreased, apparent digestibility coefficients of feed dry matter compared to control sheep. Sheep undergoing compensatory growth drank more water, ingested more food per unit body weight, laid down less body fat, more protein and retained more water in their bodies than did control sheep. Tritiated water allowed estimation of total body water with an accuracy comparable to other reports in the literature.Changes in the weight and in RNA-P and DNA-P content of anterior pituitary, thyroid and adrenal glands were measured. Sheep thyroid glands were examined histologically to assess relative TSH outputs and no significant differences detected.Rat bio-assay of cavernous sinus plasma for somatotrophin and ACTH activity was made on these sheep. Somatotrophin was detected, but experimental groups did not statistically significantly differ in relative potency (TJ.S.P. units/ml fresh plasma). Compensating sheep had. significantly lower plasma somatotrophin potency per unit empty body-weight values than did the underfed sheep.No ACTH activity was detected in the sheep plasma of the several groups.During severe undernutrition the anterior pituitary gland decreased in size, but not in cell numbers, and still elaborated somatotrophin. Decrease in body size tends to increase the ratio of circulating somatotrophin per unit body size and thus may facilitate, in a permissive manner, the enhanced growth rate of animals undergoing compensatory growth.Restitution and gain of body size by compensatory growth is accompanied by hypertrophy of the anterior pituitary gland. This gland, after compensatory growth, showed evidence of enhanced synthesizing capacity.

Reactivation of reduced bovine pancreatic ribonuclease by subcellular fractions from calf thyroid glands.

Calf thyroid subcellular fractions have been used to study the reactivation of chemically reduced bovine pancreatic ribonuclease. Both the microsomal and the high speed supernatant fractions of this tissue accelerate the reactivation of the reduced ribonuclease. Similar reactivation is observed with the microsomal fractions prepared from other tissues of various species, but not with their supernatant fractions. Supernatant fractions prepared from rat, rabbit, guinea pig and sheep thyroid glands accelerate also the reactivation of the reduced ribonuclease. It is suggested that the calf thyroid supernatant system which accelerates the reactivation of reduced ribonuclease is protein in nature and localized in the colloid. (Endocrinology 88: 742, 1971)