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First published online February 1, 2008
Journal of Experimental Biology 211, 606-612 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.015115

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Morphological and biochemical changes in the Harderian gland of hypothyroid rats

Rossella Monteforte¹, Alessandra Santillo¹, Antonia Lanni¹, Salvatore D'Aniello² and Gabriella Chieffi Baccari^1,*

¹ Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, via Vivaldi, 43, 81100-Caserta, Italy
² Department de Genetica, Facultat de Biologia, Universitat de Barcelona, Spain

View larger version (120K): [in this window] [in a new window]   Fig. 1. Paraffin and cryostat sections of euthyroid and hypothyroid rat Harderian glands (HGs). (A) The glandular cells of euthyroid rat HG contain dense basophilic secretory granules and basal nuclei. L, lumen. (B) Glandular cells from hypothyroid rat HG show pale secretory granules. The acinar lumina (L) are filled with secretory granules mixed with nuclei and cytoplasmic fragments. A and B are Mallory stained. (C) The acinar cells from euthyroid rat HG are positive for the mercury–Bromophenol Blue reaction for protein. (D) In hypothyroid rat HG some glandular cells are weakly positive for the mercury–Bromophenol Blue reaction (blue stained), whereas the others are negative (brown stained). Pycnotic nuclei (arrows) can be observed. Acini coalescing can also be observed (asterisks). (E) The glandular epithelium of euthyroid rat HG is weakly positive for the AB/PAS reaction. L, lumen. (F) Glandular cells from hypothyroid rat HG are positive for AB/PAS reaction. The lumina (L) are filled with strongly positive AB/PAS secretion. (G) Cryostat section from euthyroid rat HG. Large Sudan Black-positive vacuoles (arrowheads) and porphyrin accretions (arrows) are present in glandular cells. (H) Cryostat section of hypothyroid rat HG. A few small vacuoles stained with Sudan Black are observed outside the acini (arrows). Scale bars, 16 µm.

View larger version (138K): [in this window] [in a new window]   Fig. 2. Electron micrographs of type A cells from euthyroid rat Harderian gland (HG). (A) Large cytoplasmic vacuoles filled with moderately electron dense material occupy all the cytoplasm. The basal nucleus (N) has a prominent nucleolus. Scale bar, 7 µm. (B) Mitochondria with numerous tubular cristae and glycogen granules (arrows) are observed throughout the cytoplasm. Scale bar, 1 µm.

View larger version (120K): [in this window] [in a new window]   Fig. 3. Electron micrographs of type A cells from hypothyroid rat Harderian gland (HG). (A) Heterogeneous vacuoles almost devoid of secretory products can be seen throughout the cytoplasm. Scale bar, 5.4 µm. (B) Vacuolisations (arrows) are evident in the mitochondria, which are condensed. V, secretory vacuoles. Scale bar, 1.6 µm.

View larger version (99K): [in this window] [in a new window]   Fig. 4. Electron micrographs of type A cells from hypothyroid rat Harderian gland (HG). (A) Autophagic vacuoles (asterisks) can be seen close to smooth endoplasmic reticulum (arrow). Arrowheads indicate glycogen granules; m, mitochondria. Scale bar, 1.2 µm. (B) A cytoplasmic vacuole showing the characteristic lamellar substructures (arrows). (C) A secretory vacuole showing concentric systems of lamellae. Scale bars, 1 µm.

View larger version (131K): [in this window] [in a new window]   Fig. 5. Electron micrographs of type B cells from rat Harderian gland (HG). (A) Type B cells from euthyroid rat HG are characterised by cytoplasmic electron-translucent vacuoles and a basal nucleus (N). Numerous mitochondria (arrows) are observed throughout the cytoplasm. (B) Type B cells from hypothyroid rat HG have a dark nucleus (N) with peripheral condensation of chromatin and a few cytoplasmic vacuoles. Loose smooth endoplasmic reticulum (SER), as well as numerous mitochondria (arrows), can be observed throughout the cytoplasm. Scale bars, 5.4 µm.

View larger version (105K): [in this window] [in a new window]   Fig. 6. Electron micrographs of hypothyroid rat Harderian gland (HG). (A) Type A cells have pycnotic nuclei (N) and heterogeneous secretory vacuoles throughout the cytoplasm. Coalescence of secretory vacuoles (arrow) can be seen. Scale bar, 5.4 µm. (B) Type A cells have a basal pycnotic nucleus and a few secretory vacuoles (V) throughout the amorphous cytoplasm. Arrows indicate mitochondria. Scale bar, 4 µm.

View larger version (48K): [in this window] [in a new window]   Fig. 7. TUNEL reaction in rat Harderian gland (HG). (A) Fluorescein-labelled nuclei (arrows) are observed in hypothyroid rat HG. (B) No fluorescence is present in the nuclei of euthyroid rat HG. Scale bars, 16 µm.

View larger version (13K): [in this window] [in a new window]   Fig. 8. Western blot detection of cytochrome c protein in mitochondrial and cytosolic fractions of euthyroid and hypothyroid rat Harderian gland (HG). In the euthyroid HG, the intensity of the cytochrome c band (14 kDa) in the mitochondrial pellet was significantly (P<0.01) higher than that in cytosolic fraction. Conversely, in hypothyroid HG, the cytochrome c band was significantly more intense (P<0.01) in the cytosolic fraction than in the mitochondrial pellet. Each mitochondrial and cytosolic preparations contained three glands from three different rats. C, cytosolic fraction; M, mitochondrial pellet.