Effects of intracerbroventricularly (ICV) administered octreotide on gonadotrophic cells (FSH and LH) of adult Wistar female rats were examined by immunocytochemical and morphometric methods. The animals received ICV three 1.0 mg doses of octreotide dissolved in 10 mL saline every second day. The controls were treated with equivalent volume of physiological saline by the same schedule. FSH- and LH-producing cells were examined using peroxidase-antiperoxidase immunocytochemical procedure. Morphometric and stereologic examinations were performed to evaluate changes in the number, volume and volume densities of gonadotrophic cells. In females treated with octreotide, the gonadotrophic cells were smaller and often pycnotic, while the number of FSH- and LH-immunopositive cells per unit area (mm2) was significantly reduced. Octreotide also induced a significant reduction of the FSH- and LH-immunoreactive cells volume, as well as of their volume densities. On the basis of these results it can be concluded that octeotride, centrally administered to adult female rats provokes changes in immunocytochemical and morphometric features of both types of gonadotrophic cells.Ispitivani su efekti oktreotida davanog intracerebroventrikularno (ICV) odraslim ženkama pacova na gonadotropne ćelije (FSH i LH). Životinje su tretirane sa ukupno tri doze od 1 mg oktreotida rastvorenog u 10 mL fiziološkog rastvora svakog drugog dana. Kontrole su na isti način primile odgovarajuću zapreminu fiziološ kog rastvora. FSH i LH ćelije su ispitivane imunocitohemijskim postupkom peroksidaza-antiperoksidaza. Da bi se ustanovile promene u broju, zapremini i zapreminskoj gustini gonadotropnih ćelija primenjen je morfometrijski kao i stereološki pristup. Kod ženki tretiranih oktreotidom, gonadotropne ćelije su bile manje i često piknotič ne. Broj imunopozitivnih FSH i LH ćelija po jedinici površine (mm2) je bio statistički značajno smanjen. Oktreotid je, takodje, izazvao značajno smanjenje zapremine FSH i LH imunoreaktivnih ćelija, kao i njihove zapreminske gustine. Na osnovu ovih rezultata, može se zaključiti da je oktreotid, dat ICV odraslim ženkama pacova doveo do promena imunocitohemijskih i morfometrijskih osobina oba ispitivana tipa gonadotropnih ćelija

Filipović, Branko R.

Lovren, Mirjana B.

Manojlović-Stojanoski, Milica N.

Milošević, Verica

Nestorović, Nataša

Ristić, Nataša

Sekulić, Milka I.

Šošić-Jurjević, Branka

Digital Repository of Archived Publications - Institute for Biological Research Sinisa Stankovic (RADaR)

Introduction
Somatostatin or somatotropin release inhibiting
hormone (SRIH) was originally detected by Krulich et
al. (1) during the studies of the distribution of growth
hormone-releasing factor in rat hypothalamus. In
1973, it was characterized by Brazeau et al. (2) and
since then attracted much attention because of its
wide variety of biological functions in nervous and
other tissues. At the periphery, SRIH is a modulator of
endocrine and exocrine functions and regulates diffe-
rentiation and proliferation of normal and tumour cells
(3’6). Within the nervous system, SRIH acts as a neu-
romodulator with physiological effects on neuroen-
docrine, motor and cognitive functions (3, 7, 8). Be-
cause of its ability to inhibit many functions of various
organs, its therapeutic value in clinical conditions
involving the hyperfunction of these organs, was expe-
cted. There are two important native bioactive soma-
tostatins: a tetradecapeptide ’ somatostatin 14 (SRIH-
14) and an amino-terminal-exteded form somatostatin
28 (SRIH-28). Both of them have a short duration of
action (a half-life in the circulation of less than three
minutes) which made their clinical use difficult. Thus,
octreotide, a synthetic somatostatin analogue consis-
ting of 8 amino acids, was introduced for clinical use.
It was shown to inhibit the release of growth hormone
(GH), glucagon and insulin in monkeys 45, 11, and
1.3 times more powerfully, respectively, than SRIH-14.
Furtheremore, its elimination half-life after subcuta-
neous administration is two hours (9).
In the pituitary gland, SRIH dramatically inhibits
GH secretion and also blocks the release of other ade-
nohypophyseal hormones such as thyrotrophin (TSH;
2, 10) prolactin (PRL), and in some cases, adrenocor-
ticotropin (ACTH) (11, 12). However, the effects of
SRIHs on gonadotrophin release are still controversial.
Several authors reported that SRIH does not affect
gonadotrophin secretion (3, 13, 14). On the other
hand, some others found that somatostatin induced
reduction of gonadotrophin-producing tumours (15)
Jugoslov Med Biohem 2004; 23 (2) 149
UC 577,1; 61                                                                                                               ISSN 0354-3447
Jugoslov Med Biohem 23: 149–153, 2004 Originalni nau~ni rad
Original paper
Address of correspondence
Nata{a Nestorovi}, M.Sc.
Institute for Biological Research
29 Novembra 142
11060 Belgrade
Serbia and Montenegro 
Telephone: # (381–11)–207–8323 ext. 123
Fax: # (381–11)–761–433
E-mail: rnata¤ibiss.bg.ac.yu
EFFECTS OF INTRACEREBROVENTRICULARLY ADMINISTERED 
OCTREOTIDE ON GONADOTROPHIC CELLS IN FEMALE RATS
Nata{a Nestorovi}, Mirjana Lovren, Milka Sekuli}, Nata{a Negi}, Branka [o{i}-Jurjevi}, 
Milica Manojlovi}-Stojanoski, Branko Filipovi}, Verica Milo{evi}
Sini{a Stankovi} Institute of Biological Research, Belgrade, Serbia and Montenegro
Summary: Effects of intracerbroventricularly (ICV) administered octreotide on gonadotrophic cells (FSH
and LH) of adult Wistar female rats were examined by immunocytochemical and morphometric methods. The
animals received ICV three 1.0 mg doses of octreotide dissolved in 10 mL saline every second day. The controls
were treated with equivalent volume of physiological saline by the same schedule. FSH- and LH-producing cells
were examined using peroxidase-antiperoxidase immunocytochemical procedure. Morphometric and stereologic
examinations were performed to evaluate changes in the number, volume and volume densities of gonadotrophic
cells. In females treated with octreotide, the gonadotrophic cells were smaller and often pycnotic, while the num-
ber of FSH- and LH-immunopositive cells per unit area (mm2) was significantly reduced. Octreotide also induced
a significant reduction of the FSH- and LH-immunoreactive cells volume, as well as of their volume densities. On
the basis of these results it can be concluded that octeotride, centrally administered to adult female rats provokes
changes in immunocytochemical and morphometric features of both types of gonadotrophic cells.
Key words: rat females, somatostatin, octreotide, gonadotrophic cells, immunocytochemistry, stereology
and their secretion (16). This prompted us to examine
the effects of ICV administered octreotide on immuno-
cytochemical and morphometric properities of gona-
dotrophic cells in female rats.
Material and Methods
Animals
Adult virgin Wistar female rats maintained at 22
± 2 °C with a 12/12 h light/dark cycle and free access
to water and food, were used.
Surgical procedures were performed under ether
anaesthesia. A headset was implanted into the rats
and used for intracerebroventricular (ICV) injections. A
minimum recovery time of five days was allowed be-
fore the experiments. The headset consisted of Sila-
stic-sealed 20-gauge cannula (17), implanted into the
lateral cerebral ventricle, 1.0 mm posterior and 1.5
mm lateral to the bregma and 3 mm below the corti-
cal surface. A small stainless cerebroventricular steel
cannula and a screw were cemented in the skull with
dental acrylic resin (Stigmall; ICN Galenika).
Treatment protocol 
After recovery from the surgery, the rats were
divided into two groups each consisting of five ani-
mals. Females of the first group received ICV three 1.0
mg doses of octreotide (Sandoz, Switz.) dissolved in 10
mL saline every second day. The second group ser-
ving as a control received the equivalent volume of
physiological saline by the same schedule. All animals
were killed by decapitation under deep ether anaes-
thesia five days after the last injection. 
Immunocytochemistry
The pituitaries were excised, fixed in Bouin's so-
lution, dehydrated and embedded in paraffin wax. Pi-
tuitary sections (5 mm thick) were immunocytochemi-
cally stained. For immunocytochemistry, series of se-
ven sections of the pituitary cut through three tissue
levels (dorsal, middle and ventral portion) of the pars
distalis were used. The pituitary gonadotrophs were
localized by the peroxidase-antiperoxidase (PAP) met-
hod. Non-specific background staining was reduced
by incubation with non-immune porcine serum (1:10;
45 min). Anti-rat bLH serum (diluted with PBS 1:500)
and anti-rat bFSH (diluted with PBS 1:300) kindly pro-
vided by Dr. A.F. Parlow, NIH, Bethesda, MD, USA,
served as primary antibodies (45 min, ambient tem-
perature). Subsequently, the sections were treated
with secondary antibody (1:200 swine-anti-rabbit IgG;
45 min; a product of DAKO A/S, Glostrup, Denmark)
and rabbit-antiperoxidase serum (1:100; 45 min; a
products of DAKO A/S, Glostrup, Denmark). The anti-
gen-antibody complex was visualized by incubating
the sections with chromogen substrate, 0.05% 3,3-
diaminobenzidine (DAB; Serva, Heidelberg, Germany)
and 0.03% H2O2. The incubated sections were coun-
terstained with haematoxylin. Control sections were
incubated with normal porcine serum without primary
antisera. 
Morphometry
Immunocytochemically stained sections of pitui-
taries cut through three tissue levels of the pars dista-
lis were used for morphometric examinations of anti-
rat bFSH and anti-rat bLH reactive cells with visible
nuclei. The cell volume of gonadotrophic cells (Vc)
and their volume densities (VV) were estimated under
the light microscope at 1000 Vv magnification on 5 mm
thick sections, using the M42 multipurpose test sys-
tem (18). The volumes of FSH- and LH-positive cells
were expressed in mm3 and their volume densities as
percentages of total pituitary cells in mm3. At the same
time, the number of immunoreactive cells per unit
area (mm3) in each section was analyzed.
Results
Gonadotrophic cells (FSH and LH) of control
female rats were strongly immunostained, present thro-
ughout pituitary pars distalis, occasionally alone or in
clusters and often in close contact with blood capillaries
(Figs. 1a, b). They were large, polygonal, oval or poly-
hedral in shape, with prominent, often eccentrically
located nuclei. In the group ICV treated with octreotide,
gonadotrophic cells were smaller comparing to the
controls and often pycnotic (Figs. 1b, c).
In pituitaries of octreotide-treated female rats,
the number of FSH- and LH-immunoreactive cells per
unit area (mm2) was decreased by 21.4% and 17.2%,
respectively, and the difference in comparison with the
control was statistically significant (p<0.005) (Figure
2a). Stereological analysis showed that octreotide
treatment resulted in a significant volume decrease in
both types of gonadotrophic cells (Figure 2b). The
volume of immunopositive FSH cells was reduced by
15.6% (p<0.01), while the immunolabelled LH cell
volume was decreased by 20.6% (p<0.005). In
octreotide-treated females the percentage of FSH-
and LH-positive cells per volume unit (mm3) of total
pituitary gland tissue, i.e. the volume density, was sig-
nificantly reduced and the extent of this reduction was
about equal in both types of gonadotrophic cells
(Figure 2c). Namely, volume density of FSH-immu-
nopositive cells was decreased by 30.6% (p<0.001)
and that of LH-reactive cells by 30.1%.
Discussion
The pituitary gland is a major target for the phys-
iological actions of somatostatins. Although some
150 Jugoslov Med Biohem 2004; 23 (2)
Jugoslov Med Biohem 2004; 23 (2) 151
Figure 1.   Parts of pars distalis of the adenohypophysis
in control and octreotide-treated rat females. 
a) Immunoreactive bFSH and (b) bLH cells in the controls;
c) Small FSH-immunopositive cells and 
(d) immunoreactive LH cells after octreotide treatment.
Arrow indicates small pycnotic FSH-positive cell. 
Objective (Magnification 63×). 
Figure 2.   a) The number (No) of immunoreactive FSH
and LH cells per unit area (mm2) in control (C) and
octreotide-treated rat females; b) Cellular (⎯Vc ) volume
(mm3) of FSH- and LH-immunopositive cells in control 
and octreotide-treated animals; c) Volume density (⎯VV ) 
of immunoreactive FSH and LH cells expressed in 
percentages (%) of total adenohypophyseal cells in mm3 
in control (C) and octreotide-treated rats. All values 
are presented as means ± SD. *p<0.01,
**p<0.005, ***p<0.001. 
400
300
200
100
0
FSH LH
FSH LH
a)
b)
No/mm2
⎯Vc (mm3)
C
Octreodide
* * * *
C
Octreodide
1200
800
400
0
* * *
FSH LH c)
C
Octreodide
12
8
4
0
* * *
* * *
a
b
c
d
studies indicate that SRIH does not affect FSH and LH
secretion (3, 13, 14), our results clearly demonstrate
that centrally used somatostatin analogue octreotide
acted inhibitory on immunocytochemical and mor-
phometric characteristics of both types of gonado-
trophic cells. FSH- and LH-immunoreactive cells were
often pycnotic after three bolus ICV injections of
octreotide, what is a clear sign of its inhibitory influ-
ence. The number of gonadotrophic cells per unit area
of pituitary pars distalis was lower in octreotide-treat-
ed females rats than in the controls that received phy-
siological saline by the same route. Furthermore, ste-
reological analysis showed a significant decrease in
volume and volume densities of FSH- and LH cells
after octreotide treatment. 
The inhibitory action of somatostatin was also
detected under in vitro conditions. It suppressed LH
secretion in cultured human foetal cells (19) and sig-
nificantly inhibited LH but not FSH release in the pre-
sence of luteinizing hormone releasing hormone
(LHRH) in the culture of rat pituitary cells (20). It
should also be mentioned, that octreotide efficiently
lowered high LH levels in women with polycystic ovary
syndrome (PCOS; 21). We have previously demon-
strated in vivo inhibition of pituitary gonadotrophs by
somatostatin. Intracerebroventricularly administered
SRIH-14 and -28 acted inhibitory on pituitary FSH and
LH cells of adult rat females, as judged by morpho-
metric parameters (22). Furthermore, both these
somatostatins, ICV administered to male rats, not only
induced a decrease in LH cells size, but also reduced
LH serum levels (23). 
The biological effects of somatostatin are medi-
ated through its five distinct receptor subtypes (SSTR
1’5). The presence of all five SSTR's mRNA in every
type of hormone-secreting cells (GH, TSH, PRL, LH,
FSH and ACTH) of rat pituitary gland was documen-
ted by O'Carroll and Krempels (24). According to
these authors the majority of LH cells was colocalized
with SSTR-2 mRNA, while FSH cells expressed low but
similar amounts of all five SSTR mRNAs. In a parallel
study, Day et al. (25) detected colocalization of SSTR-
2 and -5 mRNAs in FSH-secreting cells, while among
these two, SSTR-5 was dominant. Octreotide primari-
ly interacts with SSTR-2 and SSTR-5 and binds with a
moderate affinity at SSTR-3 (26). Therefore, this so-
matostatin analogue can affect both types of gona-
dotrophic cells. The ability of octreotide to reduce the
size and secretion of gonadotropinomas (15, 16) and
to lower pathologically high LH levels in women with
PCOS may be of a very important clinical relevance.
In conclusion, our results indicate that ICV admi-
nistered somatostatin analogue octreotide exerts sig-
nificant inhibitory effects on the immunocytochemical
and morphometric characteristics of both types of
gonadotrophs.
Acknowledgement. This work was supported by
the Ministry for Science, Technology and De-
velopment of Serbia, Contract #1710. The authors
are grateful to Dr. A.F. Parlow of the National Institute
of Health, Bethesda, MD, USA, for a kind donation of
antisera. 
152 Jugoslov Med Biohem 2004; 23 (2)
EFEKTI INTRACEREBROVENTRIKULARNOG TRETMANA 
OKTREOTIDOM NA GONADOTROPNE ]ELIJE @ENKI PACOVA
Nata{a Nestorovi}, Mirjana Lovren, Milka Sekuli}, Nata{a Negi}, Branka [o{i}-Jurjevi}, 
Milica Manojlovi}-Stojanoski, Branko Filipovi}, Verica Milo{evi}
Institut za biolo{ka istra`ivanja »Sini{a Stankovi}«, Beograd, Srbija i Crna Gora
Kratak sadr`aj: Ispitivani su efekti oktreotida davanog intracerebroventrikularno (ICV) odraslim `enkama
pacova na gonadotropne }elije (FSH i LH). @ivotinje su tretirane sa ukupno tri doze od 1 mg oktreotida rastvorenog
u 10 mL fiziolo{kog rastvora svakog drugog dana. Kontrole su na isti na~in primile odgovaraju}u zapreminu fizio-
lo{kog rastvora. FSH i LH }elije su ispitivane imunocitohemijskim postupkom peroksidaza-antiperoksidaza. Da bi
se ustanovile promene u broju, zapremini i zapreminskoj gustini gonadotropnih }elija primenjen je morfometrij-
ski kao i stereolo{ki pristup. Kod `enki tretiranih oktreotidom, gonadotropne }elije su bile manje i ~esto pikno-
ti~ne. Broj imunopozitivnih FSH i LH }elija po jedinici povr{ine (mm2) je bio statisti~ki zna~ajno smanjen. Oktreo-
tid je, takodje, izazvao zna~ajno smanjenje zapremine FSH i LH imunoreaktivnih }elija, kao i njihove zapreminske
gustine. Na osnovu ovih rezultata, mo`e se zaklju~iti da je oktreotid, dat ICV odraslim `enkama pacova doveo do
promena imunocitohemijskih i morfometrijskih osobina oba ispitivana tipa gonadotropnih }elija.
Klju~ne re~i: `enke pacova, somatostatin, oktreotid, gonadotropne }elije, imunocitohemija, stereologija
Jugoslov Med Biohem 2004; 23 (2) 153
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Received: November 14, 2003
Accepted: December 18, 2003


Effects of intracerebroventricularly administered octreotide on gonadotrophic cells in female rats

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Effects of intracerebroventricularly administered octreotide on gonadotrophic cells in female rats

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