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Endocrinology, doi:10.1210/en.2004-0060
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Endocrinology Vol. 145, No. 7 3338-3345
Copyright © 2004 by The Endocrine Society

Thyroid Hormone Affects Secretory Activity and Uncoupling Protein-3 Expression in Rat Harderian Gland

Gabriella Chieffi Baccari, Rossella Monteforte, Pieter de Lange, Franca Raucci, Paola Farina and Antonia Lanni

Dipartimento di Scienze della Vita, Seconda Università di Napoli, 81100 Caserta, Italy

Address all correspondence and requests for reprints to: Drs. G. Chieffi Baccari and A. Lanni, Dipartimento di Scienze della Vita, Seconda Università di Napoli, Via Vivaldi 43, 81100 Caserta, Italy. E-mail: gabriella.chieffi{at}unina2.it or antonia.lanni{at}unina2.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effects of T3 administration on the rat Harderian gland were examined at morphological, biochemical, and molecular levels. T3 induced hypertrophy of the two cell types (A and B) present in the glandular epithelium. In type A cells, the hypertrophy was mainly due to an increase in the size of the lipid compartment. The acinar lumina were filled with lipoproteic substances, and the cells often showed an olocrine secretory pattern. In type B cells, the hypertrophy largely consisted of a marked proliferation of mitochondria endowed with tightly packed cristae, the mitochondrial number being nearly doubled (from 62 to 101/100 µm2). Although the average area of individual mitochondria decreased by about 50%, the total area of the mitochondrial compartment increased by about 80% (from 11 to 19/100 µm2). This could be ascribed to T3-induced mitochondrial proliferation. The morphological and morphometric data correlated well with our biochemical results, which indicated that mitochondrial respiratory activity is increased in hyperthyroid rats. T3, by influencing the metabolic function of the mitochondrial compartment, induces lipogenesis and the release of secretory product by type A cells. Mitochondrial uncoupling proteins 2 and 3 were expressed at both mRNA and protein levels in the euthyroid rat Harderian gland. T3 treatment increased the mRNA levels of both uncoupling protein 2 (UCP2) and UCP3, but the protein level only of UCP3. A possible role for these proteins in the Harderian gland is discussed.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HARDERIAN GLAND (HG) is an orbital gland found in the majority of land vertebrates (for reviews, see Refs.1 and 2). In rodents, this tubulo-alveolar gland is well developed and consists of two portions: a smaller lobule, occupying the upper portion of the eye socket, and a lower, larger, more diffuse lobule occupying the floor of the optic cavity. Despite its large size and considerable phylogenetic age, the functional role of the HG is far from settled. It probably serves to lubricate the eyeball and the nictitating membrane, but other functions have been suggested, including roles in thermoregulation (3, 4) or photoprotection, as part of the retinal-pineal axis (5), and as a source of either pheromones (6, 7) or growth factors (for review, see Ref.2).

The rat HG synthesizes lipids, which pour into the conjunctival sac (8), and also porphyrins, which accumulate as solid luminal accretions (9). Due to the large amounts of porphyrins it produces, the rodent HG is exposed to permanent, powerful oxidative stress (10, 11). The gland also contains serotonin, melatonin, and several other hydroxy- and metoxyindoles (12, 13).

HG activity is influenced by both exogenous (light and temperature) and endogenous factors (such as hormones). Although androgens are known to affect HG morphology and secretory activity, less is known about the influence of thyroid hormones, in particular triiodo-L-thyronine (T3) (for review, see Ref.2). The growth of the rat HG is ontogenetically correlated to an increase in T4 secretion (14). Moreover, thyroidectomy causes HG regression (15), whereas treatment of neonatal animals with T4 accelerates the development of the gland (16). In adult male rats and hamsters, treatment with T3 leads to a reduction in the HG porphyrin concentration (17, 18). The presence of 5'-deiodinase activity, the enzyme that produces T3 via peripheral 5'-deiodination of T4, in both rat and hamster HG (19, 20, 21, 22) is further evidence that this gland is a target for thyroid hormones. Indeed, Di Matteo and collaborators (23) showed that thyroid hormones induce the release of secretory granules from the HG of hypophysectomized frog. However, no detailed investigation of the effects of T3 on the morpho-functional characteristics of the HG has yet been published.

In this study we examined the effects of T3 on lipid secretion by the rat HG by means of morphometric, histochemical, and ultrastructural analyses. Further, because T3 has marked influences over energy metabolism in most tissues, we studied the effect of an animal’s thyroid state on the mitochondrial compartment, correlating mitochondrial activity with a morphological analysis. As HG cells show a remarkable lipid-handling activity and are exposed to a high level of oxidative stress, we thought it interesting to look for evidence of the presence of mitochondrial uncoupling proteins (UCPs) and at the influence that T3 might exert over their expressions. UCPs are homologous proteins constituting a subfamily of mitochondrial anion carriers. UCP1 (cloned in 1985, and called UCP until 1997) is exclusively located in brown adipose tissue, which, by uncoupling this tissue’s mitochondria, leads to physiologically important, hormonally regulated heat production in response to cold stress or diet (24). Since 1997, other genes, encoding proteins closely related to UCP1 and whose function is still a matter for debate, have been discovered. UCP2 and UCP3 seemed particularly interesting because of their putative roles in both lipid handling and oxidative stress (24), and because the expressions of these proteins in several tissues are under the influence of T3 (25, 26).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and T3 determination
Male Wistar rats, weighing 300–350 g, were kept under regulated conditions of temperature (28 C) and lighting (12-h light, 12-h dark cycles). They received commercial food pellets (Mil-Rat, Morini, Italy) and water ad libitum. Hyperthyroidism was induced in some rats (n = 14) by seven daily ip injections of 15 µg T3/100 g body weight (Sigma-Aldrich Corp., St. Louis, MO). Control rats (n = 14) received saline injections. At the end of this treatment, rats were anesthetized by an i.p. injection of chloral hydrate (40 mg/100 g body weight), then decapitated. The trunk blood was collected, and the serum was separated and stored at –20 C for later T3 determination. The HGs were dissected out, cleaned, weighed, and immediately processed for the determination of respiratory activity. Some glands were rapidly immersed in liquid nitrogen either for UCP mRNA and UCP protein determinations or for cryostat sections. Pieces of glands were quickly immersed in fixative for light or electron microscopy, as described below. Total T3 levels were determined in 50-µl samples of serum using reagents and protocols supplied by BD Biosciences (Franklin Lakes, NJ). All experiments were performed in accordance with local and national guidelines governing animal experiments.

Respiratory parameters determination
HGs were homogenized, using a Potter Elvehjem homogenizer, in an isolation medium consisting of 220 mM mannitol, 70 mM sucrose, 20 mM Tris-HCl, and 1 mM EDTA, pH 7.4. Mitochondrial respiration was determined polarographically at 30 C in a respiratory medium consisting of 80 mM KCl, 50 mM HEPES, 5 mM phosphate buffer, 10 nM sodium succinate, 3.75 µM rotenone, and 1% free fatty acid BSA, pH 7.0. State 3 respiration was initiated by the addition of 300 µM ADP, and the method described by Estabrook (27) was used to calculate the rates of state 4 and 3 respiration and the respiratory control ratio. The protein concentration was determined by the method of Hartree (28).

Histology, histochemistry, and ultrastructure
Pieces of HGs were rapidly immersed in Bouin’s fluid and phosphate-buffered 4% formalin solution (pH 7.4). Paraffin sections (5 µm thick) were stained with Tri-Chrome Mallory stain. A histochemical test for proteins was carried out using the mercury-bromophenol blue method, whereas mucosubstances were detected by the periodic acid-Schiff (PAS). For lipid detection, formol-calcium-fixed frozen sections (5–10 µm) were stained with Sudan Black B.

For electron microscopy, pieces of HG (3 mm3) were promptly immersed and left for 3 h in Karnovsky’s fixative in cachodylate buffer (pH 7.4), then postfixed for 2 h in cachodylate buffer containing 1% osmium tetroxide. The samples were dehydrated through a graded ethanol series, and finally embedded in Epon 812. Semithin sections (1 µm) were stained with 1% toluidine blue. Ultrathin sections stained with 4% uranyl acetate, followed by 1% lead citrate were examined using a Philips 301 transmission electron microscope (Philips Electronic Instruments, Rahway, NJ).

Morphometric and statistical analyses
The morphological parameters were quantified with the aid of an image analyzer. For morphometric analysis (type A and type B cell percentages, average type A and type B cell areas) digitized transverse sections were viewed under a Nikon Eclipse E600 light microscope (Nikon, Melville, NY) fitted with a JVC TK-C1381 photocamera connected to a Pentium III computer. Five randomly chosen sections (Tri-Chrome Mallory stained) from each animal were analyzed using Lucia 4.1 ScMeas on Mutech software. A total of 100 cells from each animal (n = 8) were counted.

Ultrastructural parameters (mitochondria number per 100 µm2, mitochondrial total area per 100 µm2, and mitochondrial individual area) were measured on electron micrographs scanned by a SCAPSCAN 1236 scanner (Agfa, Ridgefield Park, NJ). The area measurements were accomplished by computer mouse-directed tracing of the digitized images on a video display screen using Image J software. A total of 1000 mitochondria from each animal (n = 5) in each experimental group were included in the measurements. The mitochondrial number in each animal was counted in 20 different cells.

The values obtained for each parameter were compared by ANOVA, followed by Duncan’s test (for multigroup comparison) or by unpaired t test (for between-group comparison). All data are expressed as the mean ± SD. The level of significance was set at P < 0.05.

RNA isolation
Total RNA was isolated using a protocol similar to that described by Chomczynski and Sacchi (29). Briefly, less than 100 mg HG were ground to powder in liquid nitrogen, after which at least 5 ml lysis buffer were added before the mixture was homogenized further using a Polytron (Brinkmann Instruments, West Orange, NY), making sure that the viscosity of the solution was kept to a minimum (to ensure effective inactivation of endogenous ribonuclease activity during the subsequent phenol extractions).

RT-PCR assays
One microgram of total RNA was reverse transcribed using 1 pmol oligo(deoxythymidine) primers (15 nucleotides; Sigma Genosys Corp., Cambridge, UK), 2.0 U Superscript reverse transcriptase, 0.5 U ribonuclease inhibitor, and 1 mM deoxy-NTPs in reverse transcriptase buffer (all from HT Biotechnology, Cambridge, UK). The total volume was adjusted to 20 µl with distilled H2O. The reaction was carried out for 1 h at 40 C. One quarter of the reverse transcriptase reaction mixture was used directly for the PCR in a total volume of 20 µl, containing 0.2 U SuperTaq polymerase, 0.2 mM deoxy-NTPs, SuperTaq PCR buffer (all from HT Biotechnology), and 300 nM of the relevant oligonucleotide primers (Sigma Genosys Corp.). These primers had the following sequences: UCP2 sense, 5'-AACAGTTCTACACCAAGGGC-3'; UCP2 antisense, 5'-AGCATGGTAAGGGCACAGTG-3' (GenBank accession no. NM-011671), generating a fragment of 471 bp; UCP3 sense, 5'-ATGGATGCCTACAGAACCAT-3'; and UCP3 antisense, 5'-CTGGGCCACCATCCTCAGCA-3' (GenBank accession no. U92069), generating a fragment of 312 bp. As an internal control, the same cDNAs were amplified using ß-actin oligonucleotide primers with the following sequences: ß-actin sense, 5'-TTGTAACCAACTGGGACGATATGG-3'; and ß-actin antisense, 5'-GATCTTGATCTTCATGGTGCTAGG-3' (GenBank accession no. J00691), generating a fragment of 764 bp. Parallel amplifications (20, 25, and 30 cycles) of a given cDNA were used to determine the optimum number of cycles. After 30 cycles, a readily detectable signal within the linear range was observed. For the actual analysis, samples were heated for 5 min at 94 C, then 30 cycles were carried out, each consisting of 1 min at 94 C, 1.5 min at 50 C, and 1.5 min at 72 C. This was followed by a final 10-min extension at 72 C. One half (UCP2 and UCP3) or one quarter (ß-actin) of the PCR products was separated on a 2% agarose gel containing ethanol bromide, and the products were readily visualized. Gels were scanned, and reverse image signal intensities were quantified by means of a Bio-Rad (Hercules, CA) Molecular Imager FX using the supplied software.

Preparation of mitochondria and Western immunoblot analysis
HG mitochondria were isolated after homogenization in an isolation medium consisting of 220 mM mannitol, 70 mM sucrose, 20 mM Tris-HCl, 1 mM EDTA, 5 mM EGTA, and 5 mM MgCl2, pH 7.4 (all from Sigma-Aldrich Corp.). After this homogenization, samples were centrifuged at 700 x g, and supernatants were collected and transferred into new tubes for subsequent centrifugation at 10,000 x g. The mitochondrial pellet obtained was washed twice, then resuspended in a minimal volume of isolation medium and kept on ice. For Western immunoblot analysis, mitochondria were prepared in the isolation medium described above supplemented with the following protease inhibitors: 1 mM benzamidine, 4 µg/ml aprotinin, 1 µg/ml pepstatin, 2 µg/ml leupeptin, 5 µg/ml bestatin, 50 µg/ml N-tosyl-L-phenylalanine-chloromethyl ketone, and 0.1 mM phenylmethylsulfonylfluoride (all from Sigma-Aldrich Corp.). Mitochondria were lysed by resuspending them in sodium dodecyl sulfate loading buffer, as described by Laemmli (30), then heated for 3 min at 95 C. Thirty-microgram protein aliquots from the mitochondrial lysates were loaded in each lane, and separated on a 13% SDS-PAGE gel. Analysis of each mitochondrial sample was performed on two separate gels. Proteins were detected by a chemiluminescence protein detection method based on the protocol supplied with a commercially available kit (NEN Life Science Products, Boston, MA) using a polyclonal antibody against either UCP2 (Santa Cruz Biotechnology, Santa Cruz, CA) or UCP3 (Chemicon International, Temecula, CA) and an antirabbit antibody (primary and secondary antibodies, respectively). The mitochondrial sample protein concentration was determined by the method described by Hartree (28). UCP2 and UCP3 levels were determined and quantified in two separate preparations, each containing two glands from two different rats.

Immunohistochemistry
For the assessment of cell proliferation, Bouin-fixed paraffin sections were incubated overnight with mouse anti-proliferating cell nuclear antigen (anti-PCNA) monoclonal antibody (Dako A/S, Glostrup, Denmark) diluted 1:200 in 0.1 M PBS at pH 7.4 with 0.1% BSA (antibody diluent). After washing in PBS, the sections were incubated for 1 h with biotinylated goat antimouse IgG (Dako) diluted 1:500, followed by incubation for 1 h with peroxidase-conjugated streptavidin (Roche, Mannheim, Germany) diluted 1:500.

For detection of UCP2, formalin-fixed paraffin sections were incubated overnight with goat polyclonal UCP2 antiserum (Santa Cruz Biotechnology) diluted 1:50 in antibody diluent. After washing in PBS, the sections were incubated for 1 h at room temperature with rabbit antigoat Ig (Sigma-Aldrich Corp.) diluted 1:200.

For detection of UCP3, formalin-fixed paraffin sections were incubated overnight with rabbit antihuman UCP3 (Chemicon International) diluted 1:50 in antibody diluent. After washing in PBS, the sections were incubated for 1 h at room temperature with biotinylated swine antirabbit immunoglobulin (Dako) diluted 1:500, followed by incubation for 1 h with peroxidase-conjugated streptavidin (Roche) diluted 1:500.

The antigens were visualized using 3,3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich Corp.) and 0.3% H2O2 in PBS solution. For the negative controls, the primary antibody was omitted. The specificity of each antibody was determined by liquid phase preabsorption for 18 h at 4 C with the respective synthetic antigens.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
T3 treatment produced rats with significantly higher T3 levels (2.7± 0.1 nmol/liter) than euthyroid rats (0.92 ± 0.07 nmol/liter). HG weight was higher in T3-treated rats (0.30 ± 0.03 g) than in saline-injected rats (0.22 ± 0.02 g).

Histology and histochemistry
The rat HG is a tubulo-alveolar gland surrounded by a connective tissue capsule. Two cell types made up the glandular epithelium; type A cells contained large cytoplasmic vacuoles filled with secretory material, whereas type B cells contained small unstained vacuoles (Fig. 1AGo). Whether the A and B cell types are different forms of the same cell in different stages of activity (or different stages of a secretory cycle) or whether they are independent cell types is not clear (see Discussion). The average area of both A and B cells in euthyroid rat HG was about 200 ± 20 µm2. Type A cells occurred more frequently than type B cells (~3:1). In both cell types, the basal nuclei were euchromatic and usually contained prominent nucleoli. Myoepithelial cells were observed in close association with the basal surface of the glandular epithelium (Fig. 1AGo). The glandular epithelium was intensely positive for the Sudan Black reaction (Fig. 2AGo) and weakly positive for PAS. Material intensely positive for bromophenol blue was detected in the lumina (not shown).



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  		FIG. 1. Semithin sections of euthyroid (A) and hyperthyroid (B and C) rat HGs. A, Two cell types are present in the glandular acini of the euthyroid rat. Cells of type A (A) display large cytoplasmic vacuoles containing secretory material, whereas those of type B (B) contain smaller, clear vacuoles. Basal nuclei with evident nucleoli are present in both cell types. A myoepithelial cell (arrow) can be seen at the base of an acinus. B, Type A cells (A) exhibit a cytoplasm filled with secretory vacuoles that are released by exocytosis. The basal nuclei (arrows) are picnotic. The lumen (L) is filled with secretory product. B, B cells. C, Olocrine secretion in T3-treated rat HG. Toluidine Blue stain. Scale bars, 8 µm.

 


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  		FIG. 2. Cryostat sections from euthyroid (A) and hyperthyroid (B) rat HGs. Note the presence of more numerous, larger lipid droplets (arrows) in hyperthyroid than euthyroid rat acini. Sudan Black B stain. Scale bars, 15 µm.

 
The morphology of the hyperthyroid rat HG differed considerably from that in the euthyroid rat. Type A cells displayed vacuoles filled with secretory product (Fig. 1Go, B and C). The basal nuclei were mostly picnotic. The acini exhibited large lumina filled with secretory material (Fig. 1BGo). Merocrine (Fig. 1BGo) and sometimes olocrine (Fig. 1CGo) secretions were observed. The average area of A cells (318 ± 20 µm2) and that of B cells (262 ± 15 µm2) were significantly (P < 0.01) higher than those in euthyroid rats. In the glandular epithelium of hyperthyroid rats, we observed a higher number of B cells than in the euthyroid rat HG; the ratio of A/B cells was 2:1 (rather than 3:1). To investigate whether thyroid hormone induces B cell proliferation, PCNA immunohistochemistry was performed. PCNA, a protein expressed in the nucleus of cells in the S phase of the cellular cycle, is essential for DNA replication and elongation (31, 32). Very few cells (not significant) in either eu- or hyperthyroid rat HG were positive (not shown). Both glandular epithelium and lumina of the hyperthyroid rat HG were strongly positive for Sudan Black (Fig. 2BGo), positive for both PAS, and intensely positive for the bromophenol blue reaction for proteins (not shown).

Ultrastructure
In euthyroid rats, type A cells exhibited a euchromatic basal nucleus with peripheral condensation of chromatin and evident nucleoli (Fig. 3Go). A large number of vacuoles containing a moderately electron-dense substance as well as an extensive smooth endoplasmic reticulum (SER) and mitochondria were seen within the cytoplasm of this type of cell. Type B cells also exhibited an euchromatic basal nucleus, but less numerous, smaller cytoplasmic vacuoles than type A cells (Fig. 3Go). The secretory vacuoles contained granular osmiophilic material, whereas the cytoplasm was rich in both smooth endoplasmic reticulum and mitochondria.



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  		FIG. 3. Electron micrograph of euthyroid rat HG. The type A cell (A) displays large cytoplasmic vacuoles filled with moderately electron-dense material among tubules of SER. The basal nucleus (N) shows prominent nucleoli. Type B cell (B) displays small unstained vacuoles containing granular osmiophilic material. Numerous mitochondria and a basal nucleus (N) characterize this cell type. Scale bar, 7.1 µm.

 
In hyperthyroid rats, type A cells displayed picnotic nuclei and accumulations of secretory products in large cytoplasmic vacuoles (Fig. 4AGo). Frequently, profiles suggestive of a coalescence of secretory vacuoles were observed. Exocytotic release of droplets occurred (Fig. 4BGo). More numerous mitochondria with dilated intracristal spaces were seen with respect to the untreated control (Fig. 4CGo). Type B cells displayed a euchromatic nucleus with an evident nucleolus (Fig. 5AGo). Secretory vacuoles were heterogeneous in shape; most of them contained granular osmiophilic material, whereas others contained moderately electron-dense substance (Fig. 5AGo). Clumps of glycogen and a large number of mitochondria were present throughout the dense cytoplasm of this cell type (Fig. 5Go). Mitochondria frequently showed a condensate configuration, with dense matrix and swollen cristae (Fig. 5Go, B and C). Mitochondrial divisions were often observed (Fig. 5CGo). Both the mitochondria number (101 ± 17/100 µm2) and the mean mitochondrial total area (19.7 ± 4/100 µm2) in B cells were significantly (P < 0.001) greater in hyperthyroid than in euthyroid rats (respectively, 62 ± 8 and 11.4 ± 3/100 µm2). The average individual mitochondrial area (range, 0.021–0.53 µm2) in the hyperthyroid HG was about half that in the euthyroid HG (range, 0.016–1.13 µm2). Due to the large amount of secretory product in type A cells, morphometric data related to mitochondria were not evaluated in this type of cell.



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  		FIG. 4. Electron micrographs of T3-treated rat HGs. A, Type A cells display picnotic nuclei (N) and large secretory vacuoles that frequently fuse, forming large stores (asterisks). Scale bar, 12 µm. B, Exocytosis in a type A cell (asterisks). Scale bar, 2 µm. C, Mitochondria exhibiting a condensate configuration with ballooned cristae (arrows) are in close association with SER tubules and with lipid droplets (V). Scale bar, 1.4 µm.

 


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  		FIG. 5. Electron micrographs of hyperthyroid rat HGs. A, Type B cell (B) displays a euchromatic nucleus (N) with a prominent nucleolus as well as numerous mitochondria and secretory vacuoles. Some secretory vacuoles contain moderately osmiophilic material (arrows). A, Type A cell. Scale bar, 1.2 µm. B, Detail of a type B cell showing mitochondria with dense matrix and packed cristae, and clumps of glycogen. Scale bar, 0.9 µm. C, Images suggestive of mitochondrial division (arrows). Scale bar, 1.5 µm.

 
Respiratory parameters
Mitochondrial respiratory rates are shown in Table 1Go. With succinate used as substrate, both state 4 and state 3 oxygen consumption were significantly (P < 0.05) greater in hyperthyroid rats than in euthyroid ones (by 24% and 31%, respectively). The respiratory control ratio values were not different between these groups.


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  		TABLE 1. Respiratory parameters in the HG of euthyroid (N) and T3-treated (N + T3) rats

 
UCP mRNA and protein levels
To investigate whether mRNAs for UCPs are present in the HG and to evaluate the effect on them of T3 treatment, we used an RT-PCR-based method (Fig. 6AGo). The mRNAs for UCP-2 and –3 were detectable in the euthyroid HG. T3 treatment increased the levels of mRNAs for UCP-2 and –3 (by ~6- and 8-fold, respectively). Western immunoblot analysis of mitochondrial protein from HG from euthyroid and hyperthyroid rats revealed that UCP2 protein was present in both, and its level was not influenced by the thyroid state (Fig. 6BGo). It should be noted that the UCP2 protein level was about 2-fold lower than that in the euthyroid spleen positive control (Fig. 6BGo). In contrast to our finding with UCP2, the mitochondrial UCP3 protein level was increased 5-fold by hyperthyroidism (Fig. 6BGo).



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  		FIG. 6. A, RT-PCR-based measurement of UCP2 and UCP3 mRNA levels in the HG of control (N) and T3-treated (N+T3) rats. ß-Actin mRNA levels were measured as the internal standard. Each lane contains PCR product derived from the appropriate cDNA, for which 1 µg total RNA was used. Each treatment was performed in triplicate, and treatments are indicated above the lanes. B, Western immunoblot analysis of UCP2 and UCP3 levels. Duplicate filters were stained for either UCP2 or UCP3. Each lane contained 30 µg mitochondrial protein from a single rat. Each treatment was performed in duplicate, and treatments are indicated above the lanes. S, Spleen; M, gastrocnemius muscle.

 
Immunohistochemical localization of UCP-2 and –3 proteins
UCP2 was detected in the cytoplasm of both type A and type B cells in euthyroid and hyperthyroid rat HG (Fig. 7Go). UCP3 expression was greater in hyperthyroid rat HG than in euthyroid HG (Fig. 8Go). In hyperthyroid rat HG, UCP3 was localized in the basal cytoplasm of type A cells, but was found throughout the cytoplasm of type B cells (Fig. 8Go), with a more intense UCP3 immunopositivity observed in B cells.



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  		FIG. 7. Immunohistochemistry for UCP2 in HG from euthyroid and T3-treated rats. UCP2 immunopositivity was detected in the glandular cells of both euthyroid (A) and hyperthyroid (C) rats. B, UCP2 antibody-negative control section of euthyroid HG; D, UCP2 antibody-negative control section of hyperthyroid HG. Scale bars, 8 µm.

 


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  		FIG. 8. Immunohistochemistry for UCP3 in HG from euthyroid and hyperthyroid rats. A, UCP3 immunopositivity in euthyroid rat HG; B, UCP3 antibody-negative control section of euthyroid HG; C, UCP3 is highly expressed throughout the cytoplasm of type B cells (arrows) in hyperthyroid HG. A, Type A cells. D, UCP3 antibody-negative control section of hyperthyroid HG. Scale bars, 8 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results indicate that T3 exerts marked effects on several morpho-functional parameters in the rat HG. At the morphological level, it is evident that T3 increases HG weight by inducing hypertrophy of its cells. The immunohistochemistry for PCNA, a molecular marker of mitotic proliferation (31, 32), suggests that there was no HG cell proliferation. In T3-treated rats, both A and B cell types were hypertrophic, but the hypertrophy was associated with different ultrastructural patterns. In type A cells, the hypertrophy mainly involved an increase in the size of the lipid compartment, with lipid droplets coalescing and accumulating in large cytoplasmic vacuoles. Moreover, the basal nuclei appeared picnotic, the acinar lumina were filled with lipoproteic substances, and the glandular cells often exhibited an olocrine-like secretory pattern. In type B cells, on the other hand, the hypertrophy largely consisted of a marked proliferation of mitochondria (endowed with tightly packed cristae).

The ratio of A cells/B cells was also affected by T3; B cells in the hyperthyroid rat HG were more numerous than in the euthyroid equivalent. The ratio of A/B cells decreased from 3:1 in euthyroid rats to 2:1 after T3 treatment. As there was no evidence of B cell proliferation, we suspect that A and B cells may represent different stages of a secretory cycle, with B cells representing the initial stage and A cells the end of the activity cycle. T3, by increasing HG lipogenesis, might induce the release of secretory product by A cells, and therefore change them to B cells, for a new cycle to begin (regeneration). As shown schematically in Fig. 9Go, A and B cells could go through a three-stage activity cycle. A given cell would be in stage 1 (type B cell) immediately after a period of exocytotic discharge, when the cell would contain few lipid vacuoles and numerous nonswollen mitochondria. Stage 2 (intermediate stage) is characterized by the proliferation of swollen mitochondria and lipid vacuoles, and glycogen accumulation. In stage 3 (type A cell), the cytoplasm fills with lipoproteic vacuoles, and an exocytotic release of droplets occurs. This cycle of events fits well with the results reported by Woodhouse and Rhodin (33), who found that in the mouse HG, each of the cell types (A and B cells) appeared to go through an activity cycle involving three stages. The hypothesis that A and B cells represent different stages of a secretory cycle is supported by the presence of secretory vacuoles, characteristic of type A cells, in B cells within hyperthyroid glands.



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  		FIG. 9. Schematic representation of the activity cycle of rat HG cells. A and B cell types may go through an activity cycle comprising three stages. In stage 1 (type B cell), which occurs immediately after a period of exocytotic discharge (stage 3), the cell contains few lipid vacuoles and numerous nonswollen mitochondria, and the euchromatic nucleus is basally located. Stage 2 (intermediate stage) is characterized by the proliferation of both swollen mitochondria and lipid vacuoles, and the nucleus shows evident nucleoli. In stage 3 (type A cell), the cytoplasm fills with large secretory vacuoles, and exocytotic release of droplets occurs; the nucleus is picnotic.

 
The mitochondrial compartment was also markedly affected by T3. Our morphometric analysis revealed that the number of mitochondria in the B cells of the hyperthyroid rat HG was nearly increased 2 times (from 62 to 101/100 µm2). In the T3-treated rat HG, although the average area of the individual mitochondrion was decreased by about 50%, the total area of the mitochondrial compartment increased by about 80% (from 11 to 19/100 µm2). This could be ascribed to an increased number of smaller mitochondria, due to division of preexisting mitochondria. The frequent observation of mitochondrial division in this study supports this hypothesis. Mitochondrial proliferation (with an increased number of smaller mitochondria) after T3 administration was previously reported by Goglia and collaborators (34) in rat liver. Particularly intriguing is the configuration of the mitochondria in the hyperthyroid HG. They have a condensed configuration with dilated intracristal spaces, indicating increased phosphorylative activity. The present mitochondrial morphological and morphometric data correlated well with the biochemical results. Indeed, both states 4 and 3 of respiration were increased in the hyperthyroid rats, consistent with either an increased number or an increased activity of mitochondria.

The results concerning the expressions of UCP2 and UCP3 in the HG are also noteworthy. In this paper we are the first to demonstrate expressions of UCP2 and UCP3 in the HG at both mRNA and protein levels. The gland also expressed the mRNAs for UCP4 and UCP5 (which are usually expressed in brain and liver/brain, respectively; data not shown), and to our knowledge this is the first case of a tissue expressing four different UCPs. The relevance of these concomitant expressions is unclear at the moment, and any discussion would merely be speculative. We are particularly interested in the presence of UCP2 and UCP3. First, although UCP2 mRNA is expressed in a large number of tissues, the protein has been detected in only a few tissues (35). There is considerable evidence that UCP2 may play an important role in the control of the redox state of a cell and its oxidative stress (35, 36). This may be particularly relevant in the HG, in which the oxidative stress is considerable (11), and therefore UCP2 could play a protective role in HG cells. UCP2 levels in the HG were not affected by T3. This result indicates that other factors may affect the expression of this protein in the HG, and it will be intriguing in future studies to search for such factors. UCP3 expression, on the other hand, was stimulated by T3. This result fits well with the modifications induced by T3 in the morpho-functional parameters of the HG cells. After T3 administration there were increases in both lipid synthesis and mitochondrial activity. There is increasing evidence of UCP3 involvement in conditions of increased lipid metabolism (24). The role of UCP3 in such conditions has been proposed to lie in the export of fatty acid anions (which are hazardous when they accumulate in the mitochondrial matrix) or in the export of fatty acid peroxides (which are very hazardous for both mitochondria and the cell itself) (37, 38, 39). In our study mitochondrial respiration was increased after T3 administration, and lipids are candidates for supporters of the increased metabolism. In such a hyperthyroid state, in line with the previously mentioned putative role of UCP3, the results reported here may be explained by the need to protect HG cells in a situation of enhanced lipid metabolism and oxidative stress. Finally, if the hypothetical uncoupling activity of UCPs proves to be a fact, then a thermogenic function for them in HG cannot be excluded.


   Acknowledgments
 
We thank Mr. F. Iamunno for technical assistance.


   Footnotes
 
This work was supported by Grant MIUR-COFIN 2002 (protocol 2002058717).

Abbreviations: HG, Harderian gland; PAS, periodic acid-Schiff; PCNA, proliferating cell nuclear antigen; SER, smooth endoplasmic reticulum; UCP, uncoupling protein.

Received January 20, 2004.

Accepted for publication March 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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R. Monteforte, A. Santillo, A. Lanni, S. D'Aniello, and G. C. Baccari
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