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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
Morphological and biochemical changes in the Harderian gland of hypothyroid rats
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
* Author for correspondence (e-mail: gabriella.chieffi{at}unina2.it)
Accepted 11 December 2007
 |
Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: Harderian gland, ultrastructure, hormone action, hypothyroidism
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Chieffi et al., 1996).
Numerous functions have been attributed to this gland, including lubrication
of the eye and nictitating membrane, thermoregulation
(Thiessen, 1988;
Shanas and Terkel, 1996) and
photoprotection (Hugo et al.,
1987; Spike et al.,
1990). Furthermore, it is part of the retinal–pineal axis
(Hoffman et al., 1985) as well
as a source of either pheromones or growth factors
(Seyama et al., 1992;
Shanas and Terkel, 1996). In
some mammals, the HG activity is influenced by endogenous factors (such as
prolactin, thyroid hormones and steroid hormones) and exogenous factors (such
as light and temperature) (Hoffman et al.,
1989a; Buzzell and
Menendez-Pelaez, 1992; Buzzell
et al., 1992; Menendez-Pelaez
et al., 1993; Buzzell et al.,
1994). During the 1940s, the interrelationship between the thyroid
gland and the HG was suggested by several researchers
(Smelser, 1943;
Boas and Bates, 1954;
Boas and Scow, 1954) who used
glandular mass as their main study end point. These studies also suggested
species differences: guinea pigs reacted to thyroidectomy with HG hypertrophy
(Smelser, 1943) whereas the
HGs of thyroidectomised rats decreased in mass
(Boas and Bates, 1954). Decades
later, thyroxine (T4) injections were found to hasten the appearance of new
porphyrins in the HG of neonatal rats
(Wetterberg et al., 1970).
Recently, we demonstrated that 3,5,3'-triiodothyronine (T3)
administration to adult rats induces HG hypertrophy
(Chieffi Baccari et al.,
2004). T4 injections lead to an increase in porphyrin content in
female hamsters, an effect that is reversed under conditions of thyroid
hormone deficiency (Hoffman,
1971; Hoffman et al.,
1989a; Hoffman et al.,
1989b; Hoffman et al.,
1990). In addition, hypothyroidism induces a consistent increase
in N-acetyl transferase (NAT) activity in male hamster HG
(Buzzell et al., 1989) as well
as in porphyrin levels (Hoffman et al.,
1990). Furthermore, Hoffman and co-workers
(Hoffman et al., 1990) have
demonstrated that thyroid hormones act directly on the hamster HG rather than
indirectly through modification of thyroid-stimulating hormone (TSH) synthesis
and/or release. Nuclear receptors for T3 have been described in the HG of male
and female golden hamsters (Vilchis and
Pérez-Palacios, 1989). The presence of type II thyroxine
5'-deiodinase (5'D), an enzyme that converts thyroxine to
triiodothyronine, in both rat and hamster HGs, provided further evidence that
this gland is a target for thyroid hormones
(Delgado et al., 1988;
Osuna et al., 1990). Following
thyroidectomy, the activity of 5'D in rat HG increases and exhibits a
marked circadian rhythm (Guerrero et al.,
1987). However, the effects of hypothyroid status on the
morphofunctional characteristics of rat HG remain to be elucidated.
In this study, we investigated the effects of experimentally induced
hypothyroidism on the morphology of rat HG by histochemistry and
ultrastructural analyses. In addition, since we found that some morphological
signs are indicative of cell death by apoptosis, we compared both
mitochondrial and cytosolic expression of cytochrome c [the principal
initiator molecule released from mitochondria to cytosol to initiate the
apoptotic program in cells (Green and
Reed, 1998)] in euthyroid and hypothyroid rat HGs.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). Control
rats (N=8) received saline injections. At the end of the treatment,
rats were first anesthetised by an i.p. injection of chloral hydrate (40
mg–1 body mass) and then decapitated. The trunk blood was
collected, and the serum was separated and stored at –20°C for later
T3 and TSH determinations. The HGs were dissected out, weighed and rapidly
immersed in liquid nitrogen for cytochrome c determination or for
cryostat sectioning. 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, USA). Serum TSH levels were measured by
radioimmunoassay (RIA) using materials and protocols supplied by Amersham
(Milan, Italy). Experiments were performed in accordance with local and
national guidelines governing animal experiments.
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Ultrastructure
For electron microscopy, pieces of HGs (3 mm3) were promptly
immersed and left for 3 h in Karnovsky's fixative in cacodylate buffer (pH
7.4) and then postfixed for 2 h in cacodylate buffer containing 1% osmium
tetroxide. The samples were dehydrated through a graded ethanol series and
finally embedded in Epon 812. Ultrathin sections, stained with 4% uranyl
acetate and then with 1% lead citrate, were examined using a Philips 301
transmission electron microscope (Philips Electronic Instruments, Rahway,
NJ).
DNA nick end labelling of tissue sections
The terminal uridine deoxynucleotidyl transferase dUTP nick end labeling
(TUNEL) reaction was performed on paraffin sections (5 µm thick) of HG
using an in situ cell death detection kit (Roche Applied Science,
Mannheim, Germany). Specifically, sections were pretreated with the
permeabilisation solution (0.1% Triton X-100, 0.1% sodium citrate). Then, they
were rinsed twice in PBS and incubated with a mixture containing terminal
deoxynucleotidyl transferase (TdT) (1:20) and fluorescein-labelled dUTP, for
60 min at 37°C in a dark humidified chamber. After washing in PBS, the
slides were examined under a fluorescence microscope.
Preparation of mitochondrial and cytosolic fractions
HG mitochondria were isolated after homogenisation in an isolation medium
consisting of 220 mmol l–1 mannitol, 70 mmol
l–1 sucrose, 20 mmol l–1 Tris–HCl, 1
mmol l–1 EDTA, 5 mmol l–1 EGTA, and 5 mmol
l–1 MgCl2, pH 7.4 (all from Sigma-Aldrich Corp., St Louis,
MO, USA), supplemented with a protease inhibitor cocktail (Roche Applied
Science, Mannheim, Germany). After homogenisation, samples were centrifuged at
700 g for 10 min and supernatants were collected and
transferred into new tubes for subsequent centrifugation at 17000
g. The obtained mitochondrial pellet was washed twice and then
resuspended in a minimal volume of isolation medium and kept on ice. The
supernatant contained the cytosolic fraction
(Singh et al., 2003).
Protein concentrations of mitochondrial and cytosolic fractions were estimated using a modified Bradford assay (Bio-Rad, Melville, NY, USA).
Western immunoblot analysis
Proteins from both mitochondrial and cytosolic fractions (30 µg each)
were boiled in Laemmli buffer for 5 min. Afterwards, the samples were
subjected to SDS–PAGE (13% polyacrylamide) under reducing conditions.
Analysis of mitochondrial and cytosolic samples was performed on two separate
gels. After electrophoresis, proteins were transferred to a nitrocellulose
membrane. Each membrane was treated for 1 h with blocking solution (TBS/T: 5%
non-fat powdered milk in 25 mmol l–1 Tris, pH 7.4; 200 mmol
l–1 NaCl; 0.5% Triton X-100) and then it was incubated
overnight at 4°C with a rabbit anti-human polyclonal antibody against
cytochrome c (Cell Signalling Technology, Inc., Danvers MA, USA)
diluted 1:1000. After washing with TBS/T and TBS, membranes were incubated
with the horseradish-peroxidase-conjugated secondary antibody (1:4000) for 1 h
at room temperature. The reactions were detected using a chemiluminescence
protein detection method based on the protocol supplied with a commercially
available kit (NEN Life Science Products, Boston, MA, USA). In both
mitochondrial and cytosolic fractions, cytochrome c levels were
determined using two preparations containing three glands from three different
rats. The amount of cytochrome c protein was quantified by employing
a Bio-Rad Molecular Imager FX using the supplied software (Bio-Rad
Laboratories, Milan, Italy). The values obtained were compared by ANOVA
followed by an unpaired t-test (for between-group comparison). All
data were expressed as the mean ± s.d. The level of significance was
set at P<0.01.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Histology and histochemistry
The rat HG is a tubulo-alveolar gland surrounded by a connective tissue
capsule. In the HG of euthyroid rats, the glandular epithelium had pyramidal
cells with basal nuclei and large lumina
(Fig. 1A). There are no
morphological differences between the sexes in rat HG (for reviews, see
Payne, 1994;
Chieffi et al., 1996). We
found that the morphology of the HG of hypothyroid rats
(Fig. 1B) differed considerably
from that of euthyroid rats (Fig.
1A). Indeed, the HG of hypothyroid rats was characterised by
glandular cells that displayed pale secretory granules in the cytoplasm.
Furthermore, the acinar lumina were filled with secretory granules mixed with
nuclei and cytoplasmic fragments of glandular cells
(Fig. 1B).
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;
Chieffi et al., 1996).
Electron micrographs revealed marked differences in the shape of glandular
cells between euthyroid and hypothyroid rats. In euthyroid rats, type A cells
(Fig. 2A) had a large number of
cytoplasmic vacuoles containing a moderately electron-dense substance and
basal nuclei with evident nucleoli. Numerous mitochondria with tubular cristae
and glycogen granules were observed (Fig.
2B). In hypothyroid rats, type A cells had heterogeneously shaped
cytoplasmic vacuoles (Fig. 3A).
They were almost devoid of secretory products. Mitochondria with a few cristae
often showed a condensed configuration and vacuolisation
(Fig. 3B). A few glycogen
granules were seen throughout the cytoplasm
(Fig. 4A). Occasionally,
bodies, interpreted as autophagic vacuoles, were observed
(Fig. 4A). Some granules had a
characteristic lamellar substructure (Fig.
4B) and they often formed patterns resembling myelin forms
(Fig. 4C).
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As shown in Fig. 6, some cells (types A and B) of the hypothyroid rat HG displayed strong signs of degeneration, including condensation of chromatin and pycnosis, profiles indicative of coalescence of secretory vacuoles (Fig. 6A), amorphous cytoplasm, and alteration of mitochondria (Fig. 6B).
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Cytochrome c in mitochondrial and cytosolic fractions
Cytochrome c expression in mitochondrial and cytosolic fractions
of euthyroid and hypothyroid rat HGs was compared. In the euthyroid state, the
intensity of the cytochrome c band in the mitochondrial pellet was
significantly (P<0.01) higher than that in cytosolic fraction
(Fig. 8). Conversely, under
hypothyroid conditions, the cytochrome c band was significantly more
intense (P<0.01) in the cytosolic fraction than in the
mitochondrial pellet (Fig.
8).
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|
DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Leonard and Visser, 1986).
IOPA inhibits all three types of deiodinase enzymes; however, whereas its
effect is strong on type II and III, it is comparatively weak on type I
(Kaplan and Utiger, 1978;
St Germain, 1994). In the
present study, the most notable histological effect of hypothyroidism in the
rat HG was the decrease in cytoplasmic lipoproteic secretory granules and the
increase in secretion in the lumina. The release of granules, together with
cellular debris and some pycnotic nuclei in the lumina, suggests a holocrine
type of secretion. This phenomenon was rarely observed in the HG of euthyroid
animals in which merocrine secretion is prevalent
(Woodhouse and Rhodin, 1963;
Abe et al., 1980;
Sakai, 1981;
Satoh et al., 1992). Hence,
the decreased weight of the HG in the hypothyroid condition could be ascribed
to the massive release of glandular secretion. Consistently, we have
previously demonstrated that the increased mass of the HG in hyperthyroid rats
is due to the activation of the secretory activity induced by T3, which,
indeed, provokes an accumulation of lipoproteic secretion into the cells and
lumina (Chieffi Baccari et al.,
2004).
Interestingly, although the rat HG secretes predominantly lipoproteic
substances, histochemical tests revealed the presence of mucosubstances in the
acinar lumina of hypothyroid rat HG, suggesting that under such conditions a
biochemical modification of HG secretion takes place. An increase in the
glycosaminoglycan content has been demonstrated in guinea pig HG after
thyrotropin injections (Winand and Kohn,
1973). Therefore, mucosubstances accumulation in the HG of
hypothyroid rats could be an effect of increased TSH serum levels.
The ultrastructural changes observed in hypothyroid rat HG could be
attributed to the alterations in glandular activity elicited by thyroid
hormone deficiency, thereby leading to eventual cell death. The electron
microscopy revealed that in the final stages of cellular damage some cells had
pycnotic nuclei and alterations in the cytoplasmic architecture without any
accompanying swelling and rupture of nuclear and cytoplasmic membranes,
indicative of necrosis. It is well known that cytochrome c is
released from mitochondria in response to apoptotic stimuli
(Green and Reed, 1998). We
found cytochrome c translocation from mitochondria to the cytosol in
hypothyroid rat HG. Hence, the alteration in mitochondrial morphology observed
in hypothyroid rat HG may be due to the translocation of apoptogenic
molecules. Consistently, as evidenced by TUNEL, DNA fragmentation in the gland
of hypothyroid rat indicates the occurrence of apoptosis.
Interestingly, besides the occurrence of apoptosis in the hypothyroid rat
HG, we also observed autophagic activity. Indeed, autophagic vacuoles and
lipofuscins were detected and interpreted as the end result of autophagy. Our
interpretation was based on recent studies describing an alternative pathways
for active self-destruction called programmed cell death (PCD) type II or
autophagy. Apoptosis, or PCD type I is characterised by condensation of
cytoplasm and preservation of organelles, essentially without autophagic
degradation. Autophagic cell death exhibits autophagic degradation of Golgi
apparatus, polyribosomes, and endoplasmic reticulum, events that normally
precede nuclear destruction (for a review,
Bursch, 2001). Since apoptotic
and autophagic PCD were are not mutually exclusive phenomena they could
actually coexist in the hypothyroid rat HG. Autophagy has been recently
described in hamster HG as a survival mechanism for fighting against cell
damage resulting from physiological oxidative stress
(Tomàs-Zapico et al.,
2005).
Further studies are still needed to clarify the molecular mechanism
underlying programmed cell death in rat HG under conditions of hypothyroidism.
Although it is well known that thyroid hormone deficiency leads to extensive
apoptosis during cerebellar development, the mechanisms by which it occurs
still remain unclear. What is known is that thyroid hormones seem to preserve
the mitochondrial architecture by inhibiting the release of apoptogenic
molecules that can eventually lead to excess apoptosis
(Singh et al., 2003).
Recently, a possible involvement of pronerve growth factor–p75
neurotrophin receptor pathway in hypothyroidism-enhanced apoptosis has been
demonstrated during rat cortical development
(Kumar et al., 2006). In
addition, it has been demonstrated that hypothyroidism increases the level of
p73, a protein involved in apoptosis, during rat liver regeneration
(Alisi et al., 2005).
In conclusion, our findings indicate that the neuroendocrine–thyroid axis plays a pivotal role in preserving the structural integrity of rat HG and, therefore, its secretory activity. Without doubt, further studies are indispensable to clarify the mechanisms responsible for the morphological and biochemical changes in the HG and, consequently, the interplay between this gland and thyroid hormone deficiency.
LIST OF ABBREVIATIONS
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Acknowledgments |
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