Diflerential Immunolocalization of Thyroglobulin (Tg) in the Follicular Epithelium of Rat Thyroid Glands and Its Kinetics under Thyrotropin (TSH) Stimulation. A Quantitative Immunoelectron Microscopic Analysis Using Post-Embedding Immunogold Technique.

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  • Yamamoto Koichi
    Department of Pathology, Yamanashi Medical University
  • Xue Yi
    Department of Pediatrics, Self-Defense Forces Central Hospital
  • Katoh Ryohei
    Department of Pediatrics, Self-Defense Forces Central Hospital
  • Kawaoi Akira
    Department of Pediatrics, Self-Defense Forces Central Hospital

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  • A Quantitative Immunoelectron Microscopic Analysis Using Post-Embedding Immunogold Technique

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Abstract

Using immunoelectron microscopy and a color image analyzer, we quantified the kinetics of synthesis, secretion and reabsorption of thyroglobulin (Tg) in rat thyroid follicular epithelium under thyrotropin (TSH) stimulation. Seven week-old male Wistar rats were injected intravenously with 2IU TSH. Thyroid glands were removed at 5, 30min, 1 and 3 hr after injection, fixed immediately with 1% glutaraldehyde in 4% paraformaldehyde, post-fixed with 1% osmium tetroxide and embedded in LR-White. Immunostaining was performed with anti-rat Tg rabbit antibody and colloidal gold labeled anti-rabbit immunoglobulins. The amount of immunoreactive Tg was evaluated as the density of colloidal gold particles on the follicular lumen and cytoplasmic compartments using the color image analyzer. Area of rough surfaced endoplasmic reticulum (rER) and the mean diameters of subapical vesicles (SV) were measured from electron micrographs magnified ×5, 000 and ×30, 000 respectively using a color image analyzer. Electron microscopy revealed dense, specific labeling of Tg on the follicular lumen and SV, and rather sparse Tg labeling on rER, Golgi apparatus and lysosome in control rats. After TSH stimulation, the density of gold particles on SV increased significantly at 1 and 3 hr after stimulation compared with that of control. The density remained almost unchanged on rER, Golgi apparatus, follicular lumen and colloid droplets (CD). The labeling density on lysosomes gradually increased. Although the density on rER was not changed, the total count of gold particles on rER increased as a result of rER dilation. The diameter and number of SV per cells decreased until 1 hr after stimulation, then gradually increased. These results revealed differential kinetics of Tg in thyroid epithelial cells after TSH stimulation accompanied by morphological changes. The increase in Tg synthesis in rER seemed to be reflected not by the increase in the density of Tg in rER, but by the increase in the area of rER with an unchanged concentration of Tg. A transient decrease in the diameter, number and Tg density in SV suggested that the release of Tg into the follicular lumen followed the mode of regulated secretion.

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