Šošić-Jurjević Et Al Acta Histochemica 2016 PDF

G Model
ACTHIS-51109; No. of Pages 10
ARTICLE IN PRESS
Acta Histochemica xxx (2016) xxxxxx
Contents lists available at ScienceDirect
Acta Histochemica
journal homepage: www.elsevier.de/acthis
Functional morphology of pituitary -thyroid and -adrenocortical axes

in middle-aged male rats treated with Vitex agnus castus essential oil
sic-Jurjevi
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,
Abbreviations: DAB, diaminobenzidine tetrahydrochloride; HPA, adrenal axis;

HPT, thyroid axis; HRP, horseradish peroxidase; NIS, sodium iodide symporter; OD,
optical density; TH, total thyroxine and triiodothyronine; relative volume density,
Vv; VAC, Vitex agnus-castus; VACEO, Vitex agnus-castus L. essential oil; VEGF, vascular endothelial growth factor.
Corresponding author at: Department of Cytology, Institute for Biological
University of Belgrade, Despot Stefan Blvd. 142, 11060,
Research Sinisa Stankovic,
Belgrade, Serbia.
sic-Jurjevi
E-mail address: brankasj@ibiss.bg.ac.rs (B. So

c).
1
These authors equally contributed to the paper.
but this effect is unproven (Blumenthal et al., 2000). However, use

of VAC extract is approved for premenstrual syndrome, mastodynia and climacteric disorders (Blumenthal et al., 2000; Lucks et al.,
2002; Wuttke et al., 2003). More recent experimental research suggests that VAC extract might be also useful for treatment of male
osteoporosis (Sehmisch et al., 2009) or inhibition of prostate cell
proliferation (Weisskopf et al., 2005).
The berry of VAC contains numerous elements conrmed to be
biologically active: avonoids, iridoid glucosides, diterpenoids and
essential oils (Ajdzanovic et al., 2012; Hajd et al., 2007; KuruzmUz et al., 2003; Wuttke et al., 2003). Out of all VAC components,
the bioeffects of essential oil appear to be examined to the lowest
extent.
In Serbian and former Yugoslav folk medicine VAC essential oil
(VACEO) is recommended to treat anxiety, irritability, tachycardia,
insomnia and stomachache (Tucakov, 1990). Besides demonstrated
antibacterial and antifungal activity (Stojkovic et al., 2011), increas-
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
G Model
ARTICLE IN PRESS
B. S osic-Jurjevic et al. / Acta Histochemica xxx (2016) xxxxxx
ing number of studies shows that VACEO decrease anxiety (Setzer,

2009), induce pain relief (Khalilzadeh et al., 2015) and improve
menopausal symptoms (Lucks et al., 2002). We demonstrated that
VACEO obtained from rape berries stimulates activity of thyroid
C cells and decreases bone turnover in middle-aged male rats
(Pantelic et al., 2013). In the same model we identied sedative effect of VACEO (Ignjatovic et al., 2012). Several authors
described that VAC extract and its components act as dopamine,
estrogen and/or opioid receptor agonists, thus exerting the biological actions, including well-documented inhibition of pituitary
prolactin secretion (Dobetsberger and Buchbauer, 2011; Meier
et al., 2000; Webster et al., 2011; Wuttke et al., 2003). We also
detected decreased percentage of lactotrophs per anterior pituitary unit volume in middle-aged males treated with VAC essential
oil (Ignjatovic et al., 2012). Therefore, we hypothesized pituitarymediated endocrine effects of prolonged treatment with VACEO.
The rationale link between the potential of VACEO application as a sedative and the endocrine processes, underlying
various psycho-emotional states, is not established yet. Of all
hypothalamic-pituitary-endocrine end organs, disturbance in thyroid (HPT) and adrenal (HPA) axes have been implicated most often
in anxiety and depressive disorders (Musselman and Nemeroff,
1996). The incidence of these disorders is dramatically elevated
with advancing age (Hatzinger et al., 2000; Mariotti et al., 1995).
Keeping in mind the previous data pertinent to putative
pituitary-mediated endocrine effects of prolonged treatment with
VACEO, as well as the importance of stability of HPT and HPA axes
for healthy ageing, in the present study we further tested if VACEO
alter functional morphology of pituitary -thyroid or -adrenocortical
axes in our middle-aged model.
2. Materials and methods
2.1. Plant material and isolation of essential oil
Ripe VAC berries were identied and collected by Prof. Dr. Dra
gan Grubisic (Institute for Biological Research Sinisa Stankovic,
University of Belgrade, Serbia) in Igalo, Montenegro. A voucher
specimen (No VAC23987) was deposited at the Institute for Bio Material was air-dried. The oil
logical Research Sinisa Stankovic.
was isolated by hydrodistillation using a Clevenger-type apparatus for 3 h exactly as previously reported (Stojkovic et al., 2011).
The yield of the oil was 0.72%. Identication of the components of the essential oil was based on GC and GC/MS analyses
(Stojkovic et al., 2011). The essential oil composition remain
stable during time, and the main components of the VACEO
obtained from ripe berries (>1%) at the beginning of the experiment
were: (i) monoterpenes 1,8-cineole (eucalyptol; 16.3%), sabinene
(13.4%), -pinene (9.4%), limonene (6.8%), -terpinyl acetate (4.6%),
trans--Caryophyllene (4.1%), terpinen-4-ol (2.7%), -terpineol
(2.5%), p-cymene monoterpenes (2.3%), and -myrcene (1.9%), (ii)
sesquiterpenes trans--farnesene (9.3%) and caryophyllene oxide
(4.6%), and (iii) diterpenes (E,Z)-geranyl linalool (2.1%), sclarene
diterpenes (2.0%) and abietatriene (1.2%).
2.2. Animal experiments
Male Wistar rats were used for this study. The animals were
housed in the unit for experimental animals at the Institute for
Belgrade, Serbia. Rats were
Biological Research Sinisa Stankovic,
maintained in a constant temperature (22 2 C) and light/dark
(12/12 h) cycle, with free access to food and water.
At the age of 16 months, rats were divided in two groups: the
rst group was subcutaneously administered VACEO (60 mg/kg of
b.w., n = 6) dissolved in sterile olive oil (nal volume was 0.3 ml)
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
(Ajdzanovic et al., 2009; So

c et al., 2015). In brief, after
initial tissue deparafnization, endogenous peroxidase activity was
blocked by incubation with 0.3% hydrogen peroxide in methanol
for 15 min. Reduction of non-specic background staining was
achieved by incubation with normal porcine serum (Dakopatts,
Glostrup, Denmark; 1:10) for 45 min.
Pituitary thyrotrophs or corticotrophs were identied by incubation with polyclonal rabbit antisera (donation from Dr. A.F.
sic-Jurjevi
c,
G Model
ARTICLE IN PRESS
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)
Relative pituitary Absolute thyroid Relative thyroid

weight
weight (mg)
weight
(mg 100/b.w)
(mg 100/b.w)
Absolute adrenal Relative adrenal

weight (mg)
weight
(mg 100/b.w)
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
The values are means SD (n = 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.
Parlow, National Institute of Health, Bethesda, MD, USA; validated

by Dr Parlows laboratory) directed against rat thyroid-stimulating
hormone beta subunit (TSH; 1:500; NIDDK-anti-rTSH-IC-1,
AFP-1274789) or adrenocorticotrophic hormone (ACTH; 1:1000;
AFP-156102789), respectively, overnight at 4 C.
Rabbit polyclonal antisera directed against human vascular
endothelial growth factor (VEGF; Abcam, Cambridge, UK; 1:100;
abcam ab46154, Lot No. GR247559-1; Cambridge, MA, USA) was
applied (overnight at 4 C) for detection of VEGF immunopositivity in all examined endocrine glands. This antibody is appropriate
for immunohistochemistry (frozen and parafn sections), as previously validated by abcam , while the species reactivity included
mouse, rat and human.
For functional characterization of thyroid tissue by IHC (Grard
et al., 2003; Faggiano et al., 2007), the rabbit antisera directed
against rat sodium iodide symporter (NIS, Acris antibodies GmbH,

Herford, Germany; 1:1000; Catalog No. EUD4101, Lot No. LN2074A;
validated by Acris antibodies GmbH), and the rabbit antisera
directed against human thyroglobulin (Tg; Dakopatts, Glostrup,
Denmark; 1:500; Catalog No. A0251, Lot No. 00010709) that was
not species specic (Grard et al., 2004), were applied overnight
at 4 C. Swine anti-rabbit IgG- horseradish peroxidase (HRP;
Dakopatts, Glostrup, Denmark; 1:100; Code No. P 0399, Lot No.
20011615) was applied as a secondary antiserum for 1 h. Visualizations were performed using diaminobenzidine tetrahydrochloride
(DAB; Dakopatts, Glostrup, Denmark) at concentrations suggested
by the manufacturer.
The sections were counter-stained with hematoxylin and
mounted in DPX medium (Sigma-Aldrich, Barcelona, Spain). Dig-
sic-Jurjevi
c,
G Model
4
ARTICLE IN PRESS
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.
ital images were made on a DM RB Photomicroscope with a DFC

320 CCD Camera (Leica, Wetzlar, Germany).
All incubation steps were performed at room temperature in a
dark humid chamber. For washes and antibody dilutions, 0.1 mol/l
phosphate saline buffer (PBS; pH 7.4) was applied. Representative
sections, which were processed in the same way as described above,
using rabbit non immune sera instead of the primary antibodies in
the incubation (at the same concentration as the primary antibody),
were used for the control purposes.
2.5. Quantitative analyses of digital immunohistochemistry
images
Immunostaining intensity was determined by using the
Windows-based ImageJ program (ImageJ, Version 1.50f), as previously described (Ajdzanovic et al., 2016). As a rst step, we implemented the spectral deconvolution method of DAB/Hematoxylin
color spectra, by using optimized optical density (OD) vectors of
the color deconvolution plugin for separation of the DAB color spectra. To determine the OD for the RGB channel of Hematoxylin and
DAB, we followed the protocol as previously described by Varghese
et al. (2014). Since the OD is proportional to the concentration of
the stain, the amount of stain present will be a factor determining the optical density at a wavelength specic to the stain, as per
the Lambert-Beer law (Jhne, 1997). The OD for each channel is
dened as: OD = log (Max intensity/Mean intensity), wherein Max
intensity is 255 for 8-bit images. For quantication of immunostaining intensity, 20 unbiasedly captured images (3371 2696 pixels;
2040 objective magnication; the microscopy platform has
been described above) per pituitary or thyroid per animal were
analyzed.
2.6. Stereological analyses

Stereological analyses were carried out by point-counting
sic-Jurjevi
method (Weibel, 1979) as previously described (So

c et al.,
2015). Briey, four to ve 5 m-thick sections from the anterior, central and posterior parts of each animals pituitary (TSH-,
ACTH- or VEGF-IHC-stained) or thyroid (HE-stained and NIS- IHCstained) were analyzed. The measurements were carried out using
a newCAST stereological software package (Visiopharm Integrator
System, version 3.2.7.0; Visiopharm, Denmark), at objective magnication of 20x. The counting area was dened using a mask tool;
test grid with uniformly spaced test points and lines was provided
by the new-CAST software. Relative volume density (VV ) was calculated as the ratio of the number of points hitting examined tissue
component (Pp) divided by the number of points hitting the reference space (Pt): VV (%) = Pp/Pt 100. Relative volume densities
(VV ) were calculated for each tissue component, representing VV
of the thyrotrophs or corticotrophs per anterior pituitary unit volume; for the thyroid, VV of the epithelium, colloid and interstitium,
as well as VV of the basolateral NIS- IHC-positive epithelium were
determined.
2.7. Hormone analyses

Serum TSH was measured with a rat RIA kit (Immunodiagnostics
systems GmbH, Frankfurt am Main, Germany). ACTH was determined by IMMULITE kit (DPC, Los Angeles, USA). Total T4 and T3
were quantied by ECLIA (Roche Diagnostics GmbH, Mannheim,
Germany) and corticosterone by EIA (R&D Systems Inc., Minneapolis, USA) kits. All samples were assayed in duplicate together in one
sic-Jurjevi
c,
G Model
ARTICLE IN PRESS
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%.
mean SD. Values of p < 0.05 were considered statistically signicant.
2.8. Measurement of corticosterone content in adrenal

tissue
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
3.1. Body mass and organ weights

Results on body mass and organ weights are provided in Table 1.
No signicant changes were detected in VACEO-treated animals in
comparison with the values obtained for control middle-aged male
rats.
3.2. VACEO-induced changes in the pituitary-thyroid axis of
middle-aged rats
The thyrotrophs were mainly located in the medioventral portion of the pituitary pars distalis, grouped in small clusters or as
single cells (Fig. 1A). TSH immunostaining was prominent in the
cytoplasm of these cells (Fig. 1A). After VACEO administration, the
relative volume density of TSH- positive cells per anterior pituitary unit volume increased by 73% (p < 0.001) in comparison with
the control value (Fig. 1B, C). At the same time, intensity of cytoplasmic TSH immunostaining decreased by 23% (p < 0.001; Fig. 1D),
indicating elevated secretion of thyroid-stimulating hormone. In
line with this, concentration of TSH in sera was 45% (p < 0.01) higher
in comparison with the value obtained for control rats (Fig. 1E).
sic-Jurjevi
c,
G Model
6
ARTICLE IN PRESS
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.
We further assessed if pituitary VEGF IHC expression, a key

paracrine factor of angiogenesis and vascular permeability, was
affected by VACEO treatment. In the anterior pituitary, VEGFimmunopositive cells were mainly localized around the numerous
dilated blood vessels. The VEGF-immunoreactivity was diffusely
distributed throughout the cytoplasm and obviously present in
different types of anterior pituitary cells both in the control and
in VACEO-treated rats (Fig. 2A, B). The relative volume density of
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
c,
G Model
ARTICLE IN PRESS
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
c,
G Model
8
ARTICLE IN PRESS
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).
cytoplasm of the thyrocytes in most follicles (Fig. 4A). However, in

micro-follicles of VACEO-treated animals Tg-positivity of luminal
colloid was at the same level as the epithelial one (Fig. 4B). NISimmunopositivity was localized in the cytoplasm (weak intensity)
and/or at the basolateral membrane (high intensity) of the thyrocytes (Fig. 4C), and the letter was more prominent upon VACEO
treatment (Fig. 4D).
Stereological measurements revealed that the relative VV of

the epithelium increased (by 14%, p < 0.05; Fig. 5A), while the relative VV of the colloid decreased (by 68%, p < 0.001; Fig. 5C) in
the thyroids of VACEO-treated rats. In addition, the relative Vv of
basolateral NIS-immunopositive cells was 33% (p < 0.05; Fig. 5D)
increased upon VACEO treatment. Serum concentrations of total T4
and T3 after VACEO treatment increased by 23% (p < 0.01; Fig. 5E)
sic-Jurjevi
c,
G Model
ARTICLE IN PRESS
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
c,
G Model
ARTICLE IN PRESS
10
hyperthyroidism and ACTH suppression followed with unchanged

corticosterone levels in middle-aged male rats. From the standpoint
of HPA axis in ageing males, VACEO application may be benecial,
but the observed stimulation of HPT system prompts us to be cautious. Further data obtained from both human and animal studies
are needed to decide about remedial potential of VACEO.
Conict of interest
The authors declare that they have no conict of interest.
Acknowledgements
This research was supported by grant from Ministry of Education, Science and Technological Development of the Republic of
Serbia (No. 173009). Vladimir Ajdzanovic and Verica Milosevic are
participating to the COST Action FA 1403 POSITIVE (Interindividual
variation in response to consumption of plant food bioactives and
determinants involved), supported by COST (European Cooperation
in Science and Technology).
References
sic-Jurjevi
V., So
B., Filipovic,
B., Trifunovic,
S., Manojlovic-Stojanoski,
Ajdzanovic,
c,
M., Milosevic,
V., 2009. Genistein-induced histomorphometric and
M., Sekulic,
hormone secreting changes in the adrenal cortex in middle-aged rats. Exp.
Biol. Med. (Maywood) 234, 148156.
sic-Jurjevi
V., Spasojevic,
I., Pantelic,
J., So
B., Filipovic,
B., Milosevic,
Ajdzanovic,
c,
V., Severs, W., 2012. Vitex agnus-castus L. essential oil increases human
erythrocyte membrane uidity. J. Med. Biochem. 31, 222227.
sic-Jurjevi
V.Z., Jaric,
I.M., Z ivanovic,
J.B., Filipovic,
B.R., So
B.T., Ristic,
Ajdzanovic,
c,
S.D., Milosevic,
V.L., 2016. Histological parameters of the
N.M., Stankovic,
adrenal cortex after testosterone application in a rat model of the andropause.
Histol. Histopathol., http://dx.doi.org/10.14670/HH-11-749.
Al-Gahtany, M., Horvath, E., Kovacs, K., 2003. Pituitary hyperplasia. Hormones
(Athens) 2, 149158.
Baquedano, M.S., Ciaccio, M., Dujovne, N., Herzovich, V., Longueira, Y., Warman,
D.M., Rivarola, M.A., Belgorosky, A., 2010. Two novel mutations of the TSH-beta
subunit gene underlying congenital central hypothyroidism undetectable in
neonatal TSH screening. J. Clin. Endocrinol. Metab. 95, E98103.
Blumenthal, M., Goldberg, A., Brinckmann, J., 2000. Herbal Medicine, Expanded
Commission E Monographs, Integrative Medicine Communications, 1st ed.
American Botanical Council, Newton, MA.
Dobetsberger, C., Buchbauer, G., 2011. Actions of essential oils on the central
nervous system: an updated review. Flavour. Fragr. J. 26, 300316.
Faggiano, A., Caillou, B., Lacroix, L., Talbot, M., Filetti, S., Bidart, J.M., Schlumberger,
M., 2007. Functional characterization of human thyroid tissue with
immunohistochemistry. Thyroid 17, 203211.
Grard, A.C., Daumerie, C., Mestdagh, C., Gohy, S., De Burbure, C., Costagliola, S.,
Miot, F., Nollevaux, M.C., Denef, J.F., Rahier, J., Franc, B., De Vijdler, J.J., Colin,
I.M., Many, M.C., 2003. Correlation between the loss of thyroglobulin
iodination and the expression of thyroid-specic proteins involved in iodine
metabolism in thyroid carcinomas. J. Clin. Endocrinol. Metab. 88, 49774983.
Grard, A.C., Denef, J.F., Colin, I.M., van den Hove, M.F., 2004. Evidence for
processing of compact insoluble thyroglobulin globules in relation with
follicular cell functional activity in the human and the mouse thyroid. Eur. J.
Endocrinol. 150, 7380.
Hajd, Z., Hohmann, J., Forgo, P., Martinek, T., Dervarics, M., Zupk, I., Falkay, G.,
Cossuta, D., Mth, I., 2007. Diterpenoids and avonoids from the fruits of
Vitex agnus-castus and antioxidant activity of the fruit extracts and their
constituents. Phytother. Res. 21, 391394.
Hatzinger, M., Wotjak, C.T., Naruo, T., Simchen, R., Keck, M.E., Landgraf, R.,
Holsboer, F., Neumann, I.D., 2000. Endogenous vasopressin contributes to
hypothalamic-pituitary-adrenocortical alterations in aged rats. J. Endocrinol.
164, 197205.
sic-Jurjevi
D., Tovilovic,
G., So
B., Filipovic,
B., Janac,
B., Milosevic,
V.,
Ignjatovic,
c,
M., 2012. Bioactivity of the essential oil from berries of Vitex agnus
Tomic,
castus in middle aged male rats. Dig. J. Nanomater. Bios. 7, 17271734.
Jhne, B., 1997. Practical Handbook on Image Processing for Scientic Applications.
CRC Press, Boca Raton, Florida, USA, pp. 7682.
Jordan, D., Veisseire, M., Borson-Chazot, F., Mornex, R., 1986. In vitro effects of
endogenous opiate peptides on thyrotropin function: inhibition of
thyrotropin-releasing hormone release and absence of effect on thyrotropin
release. Neurosci. Lett. 67, 289294.
Katsuyama, S., Otowa, A., Kamio, S., Sato, K., Yagi, T., Kishikawa, Y., Komatsu, T.,
Bagetta, G., Sakurada, T., Nakamura, H., 2015. Effect of plantar subcutaneous
administration of bergamot essential oil and linalool on formalin-induced
nociceptive behavior in mice. Biomed. Res. 36, 4754.
Khalilzadeh, E., Vafaei Saiah, G., Hasannejad, H., Ghaderi, A., Ghaderi, S., Hamidian,
G., Mahmoudi, R., Eshqi, D., Zangisheh, M., 2015. Antinociceptive effects, acute
toxicity and chemical composition of Vitex agnus-castus essential oil.
Avicenna J. Phytomed. 5, 218230.
Kuruzm-Uz, A., Strch, K., Demirezer, L.O., Zeeck, A., 2003. Glucosides from vitex
agnus-castus. Phytochemistry 63, 959964.
Lucks, B.C., Srensen, J., Veal, L., 2002. Vitex agnus-castus essential oil and
menopausal balance: a self-care survey. Complement. Ther. Nurs. Midwifery 8,
148154.
Marcilhac, A., Dakine, N., Bourhim, N., Guillaume, V., Grino, M., Drieu, K., Oliver, C.,
1998. Effect of chronic administration of Ginkgo biloba extract or Ginkgolide
on the hypothalamic-pituitary-adrenal axis in the rat. Life Sci. 62, 23292340.
Mariotti, S., Franceschi, C., Cossarizza, A., Pinchera, A., 1995. The aging thyroid.
Endocr. Rev. 16, 686715.
Meier, B., Berger, D., Hoberg, E., Sticher, O., Schaffner, W., 2000. Pharmacological
activities of Vitex agnus-castus extracts in vitro. Phytomedicine 7, 373381.
Musselman, D.L., Nemeroff, C.B., 1996. Depression and endocrine disorders: focus
on the thyroid and adrenal system. Br. J. Psychiatry, 123128.
sic-Jurjevi
J., Filipovic,
B., So
B., Ajdzanovic,
V., Trifunovic,
S., Medigovic,
Pantelic,
c,
V., 2013. Vitex agnus-castus essential oil affects thyroid C cells
I., Milosevic,
and bone metabolism in middle-aged male rats. Acta Vet. Beograd 63, 2335.
Pivonello, R., Ferone, D., de Herder, W.W., Kros, J.M., De Caro, M.L., Arvigo, M.,
Annunziato, L., Lombardi, G., Colao, A., Hoand, L.J., Lamberts, S.W., 2004.
Dopamine receptor expression and function in corticotroph pituitary tumors. J.
Clin. Endocrinol. Metab. 89, 24522462.
Riedel, C., Levy, O., Carrasco, N., 2001. Post-transcriptional regulation of the
sodium/iodide symporter by thyrotropin. J. Biol. Chem. 276, 2145821463.
Sehmisch, S., Boeckhoff, J., Wille, J., Seidlova-Wuttke, D., Rack, T., Tezval, M.,
Wuttke, W., Stuermer, K.M., Stuermer, E.K., 2009. Vitex agnus castus as
prophylaxis for osteopenia after orchidectomy in rats compared with estradiol
and testosterone supplementation. Phytother. Res. 23, 851858.
Setzer, W.N., 2009. Essential oils and anxiolytic aromatherapy. Nat. Prod. Commun.
4, 13051316.
Sonntag, W.E., Goliszek, A.G., Brodish, A., Eldridge, J.C., 1987. Diminished diurnal
secretion of adrenocorticotropin (ACTH), but not corticosterone, in old male
rats: possible relation to increased adrenal sensitivity to ACTH in vivo.
Endocrinology 120, 23082315.
sic-Jurjevi
B., Filipovic,
B., Renko, K., Miler, M., Trifunovic,
S., Ajdzanovic, V.,
So
c,
V., 2015. Testosterone and estradiol treatments differently
Khrle, J., Milosevic,
affect pituitary-thyroid axis and liver deiodinase 1 activity in orchidectomized
middle-aged rats. Exp. Gerontol. 72, 8598.
c,
D., Sokovic,
M., Glamoclija, J., Dzamic,
A., Ciri
A., Ristic,
M., Grubisic,
D.,
Stojkovic,
2011. Chemical composition and antimicrobial activity of Vitex agnus-castus L.
fruits and leaves essential oils. Food Chem. 128, 20172022.
Suzuki, K., Kohn, L.D., 2006. Differential regulation of apical and basal iodide
transporters in the thyroid by thyroglobulin. J. Endocrinol. 189, 247255.
Tucakov, J., 1990. Healing with Plants Phytotherapy. Rad, Beograd (Serbian).
Varghese, F., Bukhari, A.B., Malhotra, R., De, A., 2014. IHC Proler: an open source
plugin for the quantitative evaluation and automated scoring of
immunohistochemistry images of human tissue samples. PLoS One 9, e96801.
Villalobos, C., Nnez,

L., Garca-Sancho, J., 2004. Anterior pituitary thyrotropes are
multifunctional cells. Am. J. Physiol. Endocrinol. Metab. 287, E11661170.
Webster, D.E., He, Y., Chen, S.N., Pauli, G.F., Farnsworth, N.R., Wang, Z.J., 2011.
Opioidergic mechanisms underlying the actions of Vitex agnus-castus L.
Biochem. Pharmacol. 81, 170177.
Weibel, E.R., 1979. Practical methods for biological morphometry. In: Stereological
Methods. Academic Press, London, pp. 1415.
Weisskopf, M., Schaffner, W., Jundt, G., Sulser, T., Wyler, S., Tullberg-Reinert, H.,
2005. A Vitex agnus-castus extract inhibits cell growth and induces apoptosis
in prostate epithelial cell lines. Planta Med. 71, 910916.
Wuttke, W., Jarry, H., Christoffel, V., Spengler, B., Seidlov-Wuttke, D., 2003. Chaste
tree (Vitex agnus-castus)-pharmacology and clinical indications.
Phytomedicine 10, 348357.
sic-Jurjevi
c,

Šošić-Jurjević Et Al Acta Histochemica 2016 PDF

Caricato da

Informazioni sul documento

Titolo originale

Copyright

Formati disponibili

Condividi questo documento

Condividi o incorpora il documento

Opzioni di condivisione

Hai trovato utile questo documento?

Questo contenuto è inappropriato?

Copyright:

Formati disponibili

Šošić-Jurjević Et Al Acta Histochemica 2016 PDF

Caricato da

Copyright:

Formati disponibili

G Model

ACTHIS-51109; No. of Pages 10

Contents lists available at ScienceDirect

Functional morphology of pituitary -thyroid and -adrenocortical axes

Abbreviations: DAB, diaminobenzidine tetrahydrochloride; HPA, adrenal axis;

E-mail address: brankasj@ibiss.bg.ac.rs (B. So

but this effect is unproven (Blumenthal et al., 2000). However, use

ing number of studies shows that VACEO decrease anxiety (Setzer,

(Ajdzanovic et al., 2009; So

ACTHIS-51109; No. of Pages 10

B. S osic-Jurjevic et al. / Acta Histochemica xxx (2016) xxxxxx

Relative pituitary Absolute thyroid Relative thyroid

Absolute adrenal Relative adrenal

The values are means SD (n = 6).

Parlow, National Institute of Health, Bethesda, MD, USA; validated

against rat sodium iodide symporter (NIS, Acris antibodies GmbH,

ital images were made on a DM RB Photomicroscope with a DFC

2.6. Stereological analyses

method (Weibel, 1979) as previously described (So

2.7. Hormone analyses

mean SD. Values of p < 0.05 were considered statistically signicant.

2.8. Measurement of corticosterone content in adrenal

3.1. Body mass and organ weights

We further assessed if pituitary VEGF IHC expression, a key

cytoplasm of the thyrocytes in most follicles (Fig. 4A). However, in

Stereological measurements revealed that the relative VV of

hyperthyroidism and ACTH suppression followed with unchanged

Villalobos, C., Nnez,

Potrebbero piacerti anche