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Šošić-Jurjević Et Al Acta Histochemica 2016 PDF
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Šošić-Jurjević Et Al Acta Histochemica 2016 PDF
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Acta Histochemica xxx (2016) xxxxxx
Acta Histochemica
journal homepage: www.elsevier.de/acthis
Svetlana Trifunovic,
Branka So
c ,1 , Vladimir Ajdzanovic 1 , Branko Filipovic,
Natasa Ristic,
Verica Milosevic
Ivana Jaric,
Institute for Biological Research Sinisa Stankovic, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia
a r t i c l e
i n f o
Article history:
Received 7 June 2016
Received in revised form 14 July 2016
Accepted 14 July 2016
Available online xxx
Keywords:
Vitex agnus-castus essential oil
Pituitary
Thyroid
Adrenal
Rats
a b s t r a c t
We previously reported that Vitex agnus-castus L. essential oil (VACEO), when administered to middleaged males, exerts a bone-protective effect, induces silencing of locomotor activities and decreases
pituitary prolactin immunopositivity. To further assess the putative endocrine effects of VACEO, we
examined the pituitary-thyroid and -adrenocortical axes in our model. Sixteen-month-old Wistar rats
were subcutaneously administered 60 mg/kg of VACEO dissolved in sterile olive oil, while the control
group received the same amount of vehicle alone for three weeks. Pituitaries, thyroids and adrenals were
analyzed by qualitative and quantitative histological approaches. Concentration of thyroid stimulating
hormone (TSH), total thyroxine and triiodothyronine (TH), adrenocorticotrophic hormone (ACTH), corticosterone in serum and in adrenal tissue were measured. In VACEO-treated rats, the relative volume
density of pituitary thyrotrophs increased (p < 0.001), while intensity of cytoplasmic TSH immunostaining decreased (p < 0.001), consistent with elevated TSH in serum (p < 0.01). The thyroid tissue was
characterized by a micro-follicular structure, increased relative volume of follicular epithelium (p < 0.05),
decreased volume of luminal colloid (p < 0.001) and increased basolateral expression of sodium-iodide
symporter-immunopositivity (p < 0.05). Serum TH also increased (p < 0.01). The relative volume density
of pituitary corticotrophs decreased (p < 0.05), compatible with decline in circulating ACTH (p < 0.05). Neither tissue nor serum corticosterone levels were affected by VACEO treatment. In conclusion, the observed
changes in TSH and ACTH strongly indicate central endocrine effects of prolonged VACEO treatment. In
this respect, production of ACTH decreased without impact on corticosterone production. Increase in
serum concentration of both TH and TSH are not compatible with a negative feedback loop and suggest
a major change in set-point regulation of the hypothalamic-pituitary-thyroid axis.
2016 Elsevier GmbH. All rights reserved.
1. Introduction
Chasteberry (Vitex agnus-castus L.; VAC; Familia Verbenaceae) is
a small tree or shrub native to the Mediterranean region and Central Asia. Historically, its fruit has been used by the ancient Greeks
and Romans to promote chastity and reduce male sexual desire,
http://dx.doi.org/10.1016/j.acthis.2016.07.007
0065-1281/ 2016 Elsevier GmbH. All rights reserved.
sic-Jurjevi
B., et al., Functional morphology of pituitary -thyroid and -adrenocortical axes in middlePlease cite this article in press as: So
c,
aged male rats treated with Vitex agnus castus essential oil. Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.07.007
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once a day for 3 weeks. Subcutaneous administration was performed into the loose skin over the ank, varying the site of
injection from left to right each day. The control group (C; n = 6)
was administered only a vehicle by the same schedule. The route
of administration, already established by other authors (Katsuyama
et al., 2015; Khalilzadeh et al., 2015), was in our case chosen due to
the intensive odor and the taste of VACEO that would limit its voluntary oral intake. The experiments were repeated, and the same
results (in terms of direction and the level of detected changes)
obtained.
Rats were decapitated 24 h after the last injection. Blood was
collected from the trunk and the sera were stored at 70 C until
assayed.
The animal procedures were in agreement with the EEC Directive (86/609/EEC) 2010/63/EU on the protection of animals used
for experimental and other scientic purposes, and approved by
the Ethical Committee for the Use of Laboratory Animals of the
University of
Institute for Biological Research Sinisa Stankovic,
Belgrade.
2.3. Organ processing
Pituitaries, thyroids and adrenals from each animal were
removed, weighed, and further processed by routine procedures for
histological examinations. In brief, organs were xed in Bouins xative for 48 h and then dehydrated in increasing concentrations of
ethanol and xylene. After embedding in Histowax (Histolab Product
Ab, Gteborg, Sweden), each tissue block was serially sectioned at
5 m thickness on a rotary microtome (RM 2125RT Leica Microsystems, Wetzlar, Germany).
In case of paired adrenal glands, one randomly chosen was used
for histological analyses, while the other was immediately frozen
and stored at 70 C, to be further used for determination of tissue
corticosterone level.
The relative organ weight was calculated as an absolute organ
weight (mg) divided by the animals nal body weight (g) 100.
2.4. Histological staining procedures
For qualitative and quantitative histological analyses, sections
taken from the anterior, central and posterior parts of the pituitary,
thyroid and adrenal glands were histochemically and/or immunohistochemically stained.
Thyroid and adrenal sections were stained by routine hematoxylin and eosin (HE) and/or Novelli (acid fuchsin-light green)
sic
methods, following previously described procedures (So
Jurjevic et al., 2015).
Novelli staining was applied in order to get insight into the organ
vasculature. In brief, sections were incubated in hot 1 N HCl (60 C,
3 min), followed by staining in 1% acid fuchsin (Fluka Chemie AG,
Buchs, Switzerland; 30 s) and 1% light green (Sigma-Aldrich, St.
Louis, MO, USA; 3 min), respectively. In between, the slides were
washed in distilled water. As the result, acid fuchsin-stained purple
erythrocytes within the blood vessels and capillary network were
clearly visible at the green background of the thyroid or adrenal
tissue.
Immunohistochemical (IHC) staining of the pituitary, thyroid
and adrenal sections was performed as previously described
sic-Jurjevi
sic-Jurjevi
B., et al., Functional morphology of pituitary -thyroid and -adrenocortical axes in middlePlease cite this article in press as: So
c,
aged male rats treated with Vitex agnus castus essential oil. Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.07.007
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Table 1
Effects of Vitex agnus castus essential oil (VACEO) treatment on body mass and organ weights in middle-aged rats.
C
VACEO
Body mass
(g)
Absolute
pituitary
weight (mg)
Absolute liver
weight (mg)
Relative liver
weight
(g 100/b.w.)
650.0 2.90
664.0 70.0
15.0 2.0
16.0 4.0
2.3 0.3
2.3 0.6
30.8 3.2
35.2 3.7
11.2 0.6
11.8 0.6
3.4 0.2
3.6 0.3
33.0 2.6
27.0 2.0
4.2 0.6
4.1 0.4
3.9 0.7
4.6 0.6
Fig. 1. Representative micrographs of immunohistochemical staining for TSH in anterior pituitary of (A) control and (B) Vitex agnus castus essential oil (VACEO)-treated
middle-aged rat; (C) the relative volume density (VV , %) of pituitary TSH immunopositive cells per anterior pituitary unit, (D) optical density (OD) for TSH cell immunostaining and (E) concentration of TSH (ng/ml) in serum of control and VACEO- treated rats; n = 6, **p < 0.01, ***p < 0.001.
sic-Jurjevi
B., et al., Functional morphology of pituitary -thyroid and -adrenocortical axes in middlePlease cite this article in press as: So
c,
aged male rats treated with Vitex agnus castus essential oil. Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.07.007
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Fig. 2. Representative micrographs of immunohistochemical staining for VEGF in anterior pituitary of (A) control and (B) Vitex agnus castus essential oil (VACEO)-treated
middle-aged rat; (C) the relative volume density (VV , %) of pituitary VEGF-immunopositive cells per anterior pituitary unit, (D) optical density (OD) for VEGF immunostaining
in positive cells; n = 6.
sic-Jurjevi
B., et al., Functional morphology of pituitary -thyroid and -adrenocortical axes in middlePlease cite this article in press as: So
c,
aged male rats treated with Vitex agnus castus essential oil. Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.07.007
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Fig. 3. Representative micrographs of hematoxylin and eosin stained thyroid sections in (A) control and (B) Vitex agnus castus essential oil (VACEO)-treated middle-aged
rat, as well as of Novelli stained thyroid sections in (C) control and (D) VACEO-treated rat.
run, with intra-assay CVs for TSH of 6.1%, for ACTH of 9.6%, for T4
of 4.7%, for T3 of 3.6%, and for corticosterone of 8.0%.
3. Results
The tissue homogenates were prepared with one randomly chosen adrenal gland from each animal (n = 6 for each experimental
group) in TSE buffer pH 7.9 (1 mg tissue: 1 l of buffer) on ice, using
the Ultra Turrax (IKA Analytical, Staufen, Germany). The buffer
contained 15 mmol/l TRIS (Sigma-Aldrich, Saint Louis, MO, USA),
250 mmol/l D (+)-Sucrose (VWR BDH Prolabo, Leuven, Belgium)
and 5 mmol/l EDTA (Sigma-Aldrich, Saint Louis, MO, USA). The
tissue homogenates were centrifuged at 105,000 x g for 1 h. Corticosterone concentration in the supernatant was determined by
enzyme immunoassay (R&D Systems Inc., Minneapolis, USA). All
samples were assayed in duplicate together in one run, with intraassay CV of 8%. The adrenal content of corticosterone is expressed
as ng corticosterone/mg wet adrenal weight.
2.9. Statistical analyses
Statistical analyses of all obtained results were performed using
GraphPad Prism v.6 for Windows (San Diego, CA, USA). Normality
of distribution was conrmed by Kolmogorov-Smirnov test and the
equality of variance by F test. Unpaired Students t-test was used to
evaluate differences between groups. All results were expressed as
sic-Jurjevi
B., et al., Functional morphology of pituitary -thyroid and -adrenocortical axes in middlePlease cite this article in press as: So
c,
aged male rats treated with Vitex agnus castus essential oil. Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.07.007
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Fig. 4. Representative micrographs of Tg (A,B), NIS (C,D) and VEGF (E,F) immunopositivity observed in thyroid sections of (A,C,E) control and (B,D,F) Vitex agnus castus
essential oil (VACEO)-treated middle-aged rat.
VEGF-immunopositive cells, as well as the immunostaining intensity were not signicantly affected by the treatment (Fig. 2C, D).
Thyroid tissue of VACEO-treated animals was characterized by
micro-follicular structure in comparison with the controls (Fig. 3A,
B). The surrounding capillary network of the follicles was not
notably affected (Fig. 3C, D).
In the thyroids of control rats Tg-positivity was expressed in the
follicular epithelium and in the luminal colloid, being darker in the
sic-Jurjevi
B., et al., Functional morphology of pituitary -thyroid and -adrenocortical axes in middlePlease cite this article in press as: So
c,
aged male rats treated with Vitex agnus castus essential oil. Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.07.007
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Fig. 5. The relative volume density (Vv; %) of thyroid (A) epithelium, (B) interstitium, (C) colloid and (D) basolateral NIS-immunopositive follicular epithelium, as well as
serum (E) T4 and (F) T3 concentrations (nmol/l) in control C and Vitex agnus castus essential oil VACEO-treated middle-aged rats; n = 6, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 6. Representative micrographs of immunohistochemical staining for ACTH in anterior pituitary of (A) control and (B) Vitex agnus castus essential oil (VACEO)-treated
middle-aged rats; (C) relative volume density (VV, %) of pituitary ACTH immunopositive cells per anterior pituitary unit, (D) optical density (OD) for ACTH cell immunostaining
and (E) concentration of ACTH (pg/ml) in serum of control C and VACEO- treated rats; n = 6, **p < 0.01, ***p < 0.001.
sic-Jurjevi
B., et al., Functional morphology of pituitary -thyroid and -adrenocortical axes in middlePlease cite this article in press as: So
c,
aged male rats treated with Vitex agnus castus essential oil. Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.07.007
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Fig. 7. Representative micrographs of hematoxylin and eosin (HE) stained adrenocortical zona fasciculata in (A) control and (B) Vitex agnus castus essential oil (VACEO)treated middle-aged rat, as well as of Novelli-stained adrenocortical zona fasciculata in (C) control and (D) VACEO-treated rats (arrowheads vacuolization, arrows capillary
network), and micrographs of VEGF-immunostained adrenocortical sections in (E) control and (F) VACEO-treated rats (ZG zona glomerulosa, ZF zona fasciculata, ZR zona
reticularis).
sic-Jurjevi
B., et al., Functional morphology of pituitary -thyroid and -adrenocortical axes in middlePlease cite this article in press as: So
c,
aged male rats treated with Vitex agnus castus essential oil. Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.07.007
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Fig. 8. Corticosterone levels in (A) adrenal tissue and (B) serum in control C and Vitex agnus castus essential oil (VACEO)-treated middle-aged rats; n = 6.
and 40% (p < 0.01; Fig. 5F), respectively, in comparison to the control
values.
3.3. VACEO-induced changes in pituitary-adrenal axis of
middle-aged rats
The pituitary corticotrophs were evenly distributed throughout the pituitary pars distalis, in small groups or as single cells.
ACTH IHC positivity was uniformly distributed in the cytoplasm
of these irregularly-shaped cells (Fig. 6A, B). VACEO administration
decreased the relative volume density of corticotrophs by 18% per
pituitary unit volume (p < 0.05; Fig. 6C), while cytoplasmic ACTH
immunostaining intensity was not signicantly changed (Fig. 6D).
Concentration of ACTH in serum decreased (by 52%, p < 0.05; Fig. 6E)
in comparison with the values obtained for the controls.
Within the adrenal cortex of control middle-aged rats,
corticosterone-producing zona fasciculata (zf) was characterized
by long, straight cords of voluminous polygonal cells that were
intercalated by blood vessels. Within these cells occasional vacuolization was noticed (Fig. 7A). VACEO treatment induced discrete
changes within the zf of middle-aged rats. Namely, the cytoplasmic
vacuolization was more pronounced within cellular parenchyma,
while the capillary network was less prominent (Fig. 7B, D). On the
other hand, the intensity of VEGF-IHC staining throughout the all
adrenal cortex, including zf that was in our focus, was not signicantly altered (Fig. 7F).
Corticosterone levels in adrenal tissue (Fig. 8A) and serum
(Fig. 8B) were not signicantly changed after VACEO application.
4. Discussion
In this study we showed for the rst time that VACEO, when
applied in a bioactive dose during prolonged time, interferes with
the pituitary -thyroid and -adrenocortical axes in middle-aged
male rats. The observed morpho-functional perspective of VACEO
endocrine effects is complex and most likely due to the modulation
of their central regulation.
We previously reported that the dose and composition of VACEO
applied in this experiment had bone-protective (Pantelic et al.,
2013) and sedative (Ignjatovic et al., 2012) effects. Similar regime
of treatment with VACEO of comparable composition exerted analgesic effects in young-aged rats (Khalilzadeh et al., 2015). The same
authors demonstrated that even high doses (5 g/kg) of VACEO had
been well tolerated, followed with the absolute rate of survival
(Khalilzadeh et al., 2015). In humans, 32.5 mg of daily ingested
VACEO during prolonged period of time signicantly reduced various climacteric complains (Lucks et al., 2002).
Protracted VACEO treatment stimulated pituitary thyrotroph
cell population and elevated concentration of TSH in serum. At
the level of thyroid, histological analysis revealed micro-follicular
structure, enhanced colloidal Tg-positivity and basolateral expression of NIS. The obtained data indicate increased presence of
secretory active follicles (Suzuki and Kohn, 2006), as well as TSHinduced active iodine uptake (Grard et al., 2003; Riedel et al.,
2001), respectively. Both serum T4 and T3 were increased in this
experiment, clearly indicating central hyperthyroidism and altered
set-point of TSH regulation by TH.
VACEO treatment decreased percentage of corticotrophs per
anterior pituitary unit volume and lowered circulating concentration of ACTH. However, discrete histological changes and
unaffected tissue and serum corticosterone levels indicate that
functioning of adrenocortical zf was not changed. Based on these
data, we assume that VACEO application induced the central
inputs on pituitary ACTH production. In line with this, diterpene
Ginkgolide B was found to attenuate the expression of CRH gene and
CRH secretion (Marcilhac et al., 1998), crucial ACTH driving force.
However, adrenal zf did not respond to low serum ACTH. Keeping in
mind the rise in corticosterone concentrations in our middle-aged
model (Ajdzanovic et al., 2009), this may be explained by increased
sensitivity of adrenal cortex to ACTH stimulation with advancing
age (Sonntag et al., 1987).
The algorithm of effects of VACEO, mainly consisting of
monoterpenes, sesquiterpenes and diterpenes, appears to be
complex and multifactorial, including different receptor systems
(dopaminergic, opioidergic, GABAergic, and/or other) and their
interaction (Dobetsberger and Buchbauer, 2011). If VACEO acted
only as dopaminergic agonist, its suppressive effect on anterior pituitary hormone-producing cells, including corticotrophs
(Pivonello et al., 2004) and thyrotrophs (Baquedano et al., 2010)
is expected herein. Likewise, we previously detected suppressive
effects on PRL (Ignjatovic et al., 2012) and now on ACTH production
upon VACEO application. However, VACEO induced hyperplasia of
pituitary thyrotrophs and elevation of serum TSH and TH, indicating additional molecular targets of VACEO action. Keeping in
mind that pituitary weight was not affected by VACEO treatment,
and that pituitary thyrotrophs are a small population of mainly
bi- or poly-hormonal cells (Villalobos et al., 2004), we assume
that trandifferentiation from lactotrophs, corticotrophs, or other
hormone-producing type contributed signicantly to the detected
change. TRH is considered most important for initiation of both thyrotroph proliferation and transdifferentiation (Al-Gahtany et al.,
2003). As ACTH and endorphin production in the pituitary
are entwined, it is rationally to believe that possible decrease in
endogenous opiate production followed with TRH release (Jordan
et al., 1986) also gures in this respect. Further studies of hypothalamic pituitary networks, including hormonal and paracrine
regulators, other than herein unchanged VEGF, are needed to dene
molecular mechanisms of VACEO actions.
In conclusion, to the best of our knowledge this is the rst report
on effects of VACEO on HPT and HPA axes. We observed central
sic-Jurjevi
B., et al., Functional morphology of pituitary -thyroid and -adrenocortical axes in middlePlease cite this article in press as: So
c,
aged male rats treated with Vitex agnus castus essential oil. Acta Histochemica (2016), http://dx.doi.org/10.1016/j.acthis.2016.07.007
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